Chitosan production from agro-wastes by Aspergillus tubingensis: A sustainable approach for beef burger quality improvement

Chitosan production from agro-wastes by Aspergillus tubingensis: A sustainable approach for beef burger quality improvement | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chitosan production from agro-wastes by Aspergillus tubingensis: A sustainable approach for beef burger quality improvement Shimaa A. Amin, Khadiga A. Abou-Taleb, Basma T. Abd-Elhalim, Dina Y. Abdelghani, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4397361/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Chitosan is commonly obtained by deacetylation of chitin from crustacean shell wastes such as shrimp and crab, but unfortunately, these sources appear limited for their unavailability. So, an alternative abundant chitin source is in need as fungi mycelia. Results In this study, chitosan production through submerged (SMF) and solid-state fermentation (SSF) of Aspergillus tubingensis utilizing various agro-wastes and by-products like whey, blackstrap molasses, beet waste, rice (husk& straw), wheat bran and sawdust as a sole carbon source was investigated. The best waste was beet waste (33.12% and 38.61% of chitin and chitosan yields for SSF and whey (whey (24.22% and 35.90% of chitin and chitosan yields) for SmF, as compared to the control of sucrose (17.11% and 20.38% of chitin and chitosan yields) after 8–12 and 8 days of incubation, respectively. The fungal polymer had antibacterial activity against Staphylococcus aureus DSMZ20231, Salmonella Typhimurium ATCC14028, Bacillus cereus DSMZ345, Pseudomonas fluorescens NRRL800, and Escherichia coli ATCC69373 at a significant concentration of 1.0% were assayed using Kirby-Bauer disc diffusion method. Staph. aureus DSMZ20231& S. Typhimurium ATCC14028 were more susceptible to chitosan, which gave the largest zone inhibition of 47 and 43 mm, respectively. Moreover, the polymer's antibacterial activity was tested in the broth medium during different incubation periods (12-48h). results indicated that all tested pathogenic strains exhibited complete inhibition after 24 h incubation except B. cereus DSMZ345 was the most stable up to 48h. Application of fungal chitosan (1.0%) for a laboratory-manufactured beef burger led to improve quality analysis of the texture, physical or technological and chemical properties than control without chitosan. At the same time, beef burger supplemented with chitosan was significantly accepted overall by the panelist than the control. Therefore, fungal chitosan extended the beef burger's shelf life and maintained its quality indices during freezing storage. Conclusions Fungal chitosan is a beneficial natural antimicrobial, and applying it to beef burgers improves microbiological, technological, and chemical quality while also extending shelf life by up to 8 weeks, making it a viable alternative to chemical protective additives. Future research on this fungal chitosan will focus on large-scale meat or poultry preservation or additive applications. Antibacterial activity Aspergillus tubingensis Bacterial pathogens Beef burger Biochitosan Food preservatives Shelf-life Solid-state fermentation Submerged fermentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Food industries especially meat products are categorized as highly perishable products owing to the richness of unsaturated fatty acids, oxidative stability, and high risk of microbial spoilage which lead to various unfavourable changes in colour flavour, and texture. For quality enhancement and freshness maintenance, numerous food chemical additives were used. These chemicals have antimicrobial and antioxidant behaviour utilized in the meat industry like sorbic acid, citric acid, propionic acid, nitrite salts, benzoic acid, and various collections of enzymes [ 1 , 2 ]. On the contrary, these chemicals represent a high-risk factor for human health that is responsible for cancers, allergic reactions, and gastrointestinal disorders [ 3 ]. So, there is a necessary need for natural antimicrobials and antioxidant replacements such as chitosan, pediocin, polylysine, and nisin to ensure biosafety and increase the shelf life [ 4 , 5 ]. Chitosan is a linear polysaccharide composed of randomly distributed β-(1–4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit) [ 6 ]. Chitosan is industrially obtained using alkali solutions by partial deacetylation of chitin, which is obtained from the exoskeletons of insects and shell waste of shrimps, crustaceans, lobsters, krill, squid’s backbones, and crabs. Unfortunately, these resources are not abundant and available all the time in high quantities according to seasons and fishing industry sites. In addition, the extensive steps for obtaining chitosan from marine crustaceans are demineralization, deproteinization, discolouration, and deacetylation. This meant that the production of one kilogram of chitosan, consumed 6.3 kg of HCl and 1.8 kg of NaOH is required [ 7 ], so the consumption of such a high quantity of chemicals results in environmental pollution. Besides, the produced chitosan is not resilient and inconsistent with poor quality [ 8 ]. So, new chitosan resources that are abundant with low cost and have the ability of optimise from is for need. On the contrary, fungi biomass can be obtained continuously by the fermentation process that does not have any seasonal and transportation limitations. In addition, less production time with uniform properties [ 9 ]. In addition, fungal mycelia do not require any additional treatments like crustacean wastes which have a high level of inorganic materials and need demineralization treatment during the processing. The cultivation of fungal cells has fulfilled the previous consideration for chitosan production by varying fermentation conditions [ 10 ]. In addition, fungi are easy to handle, harvest, and control in order to produce high-quality chitosan. Various fungal species including Absidia coerulea , Aspergillus glauca , A. niger , A. terreus , Mucor rouxii , Gongronella butleri , Cunninghamella blakesleeanus , Rhizopus delemar , R. oryzae , Mortierella isabelina , and Lentinus edodes have been investigated for chitin and chitosan production [ 11 , 12 ]. The fermentation approach become affordable because of using waste streams in an energy-efficient way as the microbial cells work as factories with low temperatures [ 13 ]. Fungal biomass cultivation can undergo using two main approaches, solid-state (SSF) and submerged substrate (SMF) fermentation utilizing agricultural and/or food industrial wastes and by-products as alternatives for expensive fermentation media to minimize chitosan production costs as it could be considered a green synthesis economic approach [ 11 ]. Owing to the multi-dimensional, and high functionality structure of chitosan it has widespread application in food, paper, wastewater treatment, pharmaceutical, biomedical, agricultural, biotechnological, and food-related industries [ 8 , 14 ]. Utilizing chitosan as a biological coating to improve the shelf life of meat products can be considered a healthy method of meat preservation [ 15 , 16 ]. Meat and meat products are excellent sources of nutrients for humans; however, they also provide a favourable environment for microbial growth. To prevent the microbiological contamination of livestock foods, synthetic preservatives, including nitrites, nitrates, and sorbates, have been widely used in the food industry due to their low cost and strong antibacterial activity. The use of synthetic chemical preservatives is recently being considered by customers due to concerns related to negative health issues. Therefore, the demand for natural substances as food preservatives has increased with the use of plant-derived and animal-derived products, and microbial metabolites. These natural preservatives inhibit the growth of spoilage microorganisms or food-borne pathogens by increasing the permeability of microbial cell membranes, interrupting of protein synthesis, and cell metabolism [ 17 ]. Owing to chitosan’s exquisite properties like antimicrobial, film-forming, gas barrier, antioxidant, nontoxicity, biocompatibility, biodegradability activity, high absorption potential, and low moisture barrier make it a pioneer compound for different food products [ 18 ]. This study aimed to synthesize fungal A. tubingensis chitosan under submerged and solid-state fermentation using various agro-wastes and by-products, as well as to investigate the time kinetics of fungal growth and chitin and chitosan formation. Use fungal chitosan as a food additive to improve the antibacterial, sensory, chemical, and physical qualities of beef burgers, as well as the shelf life. Methods Fungal strain and pathogenic bacterial strains The pure culture of Aspergillus tubingensis was used for chitosan synthesis. It was obtained from the Agricultural Microbiology Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. Activation and preparation of the strain with Czapek's agar medium at 28 ± 2°C for 48 h. Preserve and maintain at 5°C in a refrigerator. Five pathogenic bacterial strains of Staphylococcus aureus DSMZ 20231, Salmonella typhimurium ATCC 14028, Bacillus cereus DSMZ 345, Pseudomonas fluorescens NRRL 800 and Escherichia coli ATCC 69373 were used to evaluate antibacterial activity. These strains were obtained from the Microbial Culture Collection Center (Cairo MIRCEN), Faculty of Agriculture, Ain Shams University, Cairo, Egypt. These strains were maintained on Luria-Bertani agar (LB) medium [ 19 ]. Media used Czapek's agar medium [ 20 ] was used for cultivating Aspergillus tubingensis . It contains as follows (g/L): sucrose, 30.0; sodium nitrate, 2.0; dipotassium phosphate, 1.0; magnesium sulphate, 0.50; potassium chloride, 0.50; ferrous sulphate, 0.010 and agar, 20. Luria-Bertani agar medium [ 19 ] was used for pathogenic bacterial strains maintenance. This was contained (g/L): peptone, 10; yeast extract, 5; sodium chloride 5 and agar, 20. Mueller Hinton agar medium [ 21 ] was used for antibacterial activity assay, which contained (g/L): meat infusion, 6.0; casein hydrolysate, 7.5; starch, 1.5 and agar, 15. Vogel Johnson agar [ 19 ] for Staphylococcus aureus enumeration. Its composition is as follows (g/L): Tryptone, 10; yeast extract, 5; Mannitol, 10; Dipotassium hydrogen phosphate, 5; Lithium chloride, 5; Glycine, 10; Phenol red, 0.025 and agar, 20. Before use, a sterile solution of 1% potassium tellurite was added to a sterile medium at a rate of 20 ml /L. Bacillus cereus base agar [ 20 ] for Bacillus cereus enumeration. Its composition is as follows (g/L): peptone, 1; mannitol, 10; sodium chloride, 2; magnesium sulphate, 0.1; disodium hydrogen phosphate, 2.5; potassium dihydrogen phosphate, 0.25; sodium pyruvate, 10; bromothymol blue, 0.12 and agar, 20. Before use, a sterile egg yolk solution was added to a sterile medium at a rate of 25 mL/ L. Salmonella Shigella (SS) agar [ 19 ] was used as a selective medium for detecting and enumerating Salmonella spp. It contains as follows (g/L): protease peptone, 5; lactose, 10; bile salt, 8.5; sodium citrate, 8.5; sodium thiosulfate, 8.5; ferric sulfate, 1.0; brilliant green, 0.33; neutral red, 0.025; beef extract, 5.0 and agar, 15. King’s B medium [ 22 ] was used for enumerating Pseudomonas spp. was composed (g/L) of proteose peptone, 20; K 2 HPO 4 , 1.5; MgSO 4 .7H 2 O, 1.5; glycerol, 10 ml and agar, 15. MacConkey agar medium (OXOID CM0115) [ 20 ] was used in the cultivation of pathogenic bacteria of Escherichia coli . The weight of the medium powder (51.5 g) was suspended in 1000 mL of distilled water and boiled for full dissolving. The medium was then dispensed in flasks and sterilized. All these media were adjusted to pH 7.2 ± 0.2 and autoclaved for 15 min at 121°C. The liquid media used in this study is the same as previously shown without adding agar. All media were sterilized at 121°C by autoclaving for 15 min. Spore suspension as standard fungal inoculum preparation The fungal strain was cultivated on Czapek's agar slants until sporulation for 48 h at 28 ± 2°C. The spore suspension was prepared by adding 5 mL of sterile distilled water to the agar slant and gently scraping the surface of the culture with a sterile inoculation loop. The spore suspension from the slant was pooled to count using the Neubauer hemocytometer slide [ 23 ]. One millilitre of spore suspension contained 1x 10 7 spores/ mL. Agro-wastes and by-products Solid agro-waste such as beet waste, rice husk, rice straw, sawdust, and wheat bran, and liquid residues such as whey and blackstrap molasses were utilized in this study. As mentioned by Abd-Elhalem, El-Sawy [ 24 ], the solid wastes were prepared by washing in cold water and then warm water, followed by drying overnight at 50°C, grounding and sieving to eliminate large particles. Blackstrap sugarcane molasses water-diluted by a ratio of 1:1 and pH value adjusted to be 4.0 using H 2 SO 4 (0.1 N), then heated at 100°C for 1 h and then neutralized with CaCO 3 and kept overnight to discard undesirable metals. The suspended solids and fibrous particles were removed by centrifugation (PRO-HOSPITAL.8) at 6,000 rpm for 15 min. This clarified molasses was stored at 4°C [ 25 ]. Whey acidity was adjusted to pH 4.5 and heated at 121°C for 15 min for denaturing proteins, then centrifuged at 10000 rpm for 15 min. Filtration for removing the precipitates, and then the supernatant was adjusted to pH 6.3 and maintained at 4°C [ 26 ]. The total carbon content of the solid materials of beet waste, rice husk, rice straw, sawdust, and wheat bran were 50.0, 48.3, 46.5, 37.8, and 39.3%, respectively, according to reports of the Central Laboratory, Horticulture Research Institute, Agriculture Research Center, Giza, Egypt [ 24 ]. Whereas, the blackstrap sugarcane molasses and whey were found their contain total sugar being 42.4% and 4.3%, respectively as previously determined by Abou-Taleb, Mashhoor [ 27 ]. Fermentation processes for chitosan production Submerged fermentation (SmF) technique It was carried out in plugged Erlenmeyer flasks (250 mL) containing 50 mL of Czapek's broth medium in which sucrose was replaced by a similar concentration of each of the tested carbon sources (by-products of whey and blackstrap molasses) and inoculated with 3% (v/v) of the tested strains. The flasks were incubated on a rotary shaker (XuyMeu) at 28°C and 150 rpm of agitation speed [ 28 ]. Samples were drawn every 2 days under aseptic conditions. The collected pellicle samples were used to measure biomass and chitin and recovery of chitosan, as mentioned below. Solid-state fermentation (SSF) technique Solid agro-industrial wastes were supplied for a solid-state fermentation medium with respect to heat transfer restrictions with suitable porosity and moisture in the solid substrate medium of fermentation. Dry substrates were weighed by 30 g in 500 mL Erlenmeyer flasks, and then the moisture content was adjusted (as the moisture content of 8–10%, which is not enough for the growth of fungi) by adding Czapek's broth medium without sucrose (as a basal minerals medium) to 50%. The flasks were hand-shaken to homogenize the solid medium and autoclaved at 121°C for 20 min. One millilitre of spore suspension (1x10 7 spores/mL) was inoculated into the sterilized media with shaking to distribute the spores under sterilized conditions. The flasks were cotton-plugged and remained static during incubation for 12 days at 28°C [ 28 ]. Samples were drawn every 2 days under aseptic conditions. The collected mat samples were used to measure biomass and chitin and recovery of chitosan, as mentioned below. Biomass (cell dry weight) determination The cell dry weight was measured by harvesting the cells after filtration of the culture using filter paper (Whatman® qualitative filter paper, Grade 1, circles, diam. 45 mm, Sigma-Aldrich) and subsequent washing thrice with distilled water. The cells were dried at 80°C till constant weight was attained [ 29 ]. Chitin and chitosan recovery According to the method mentioned by Dhillon, Kaur [ 11 ], Maghsoodi and Yaghmaei [ 28 ], After the fungus cultivation, the culture was filtered through a Whatman No.1 to separate the biomass from the growth medium. The biomass cake was oven-dried at 55 − 50°C overnight, then weighed. Mix 1 g of the dried biomass with 30 mL of KOH (1N) with a ratio of 1:30, then stir vigorously for 15 min. Heat the mixture at 121°C for 20 min (Autoclaving), then centrifuge at 6000 rpm for 17 min to separate insoluble cell wall parts involving chitosan. The deposit was discarded and washed repeatedly with distilled water to neutralize the pH. The washed deposit was overnight oven-dried at 40°C. Mixed at 1:30 ratio with 30 ml of 2% acetic acid, then heated at 95°C for 6 h. Then centrifuge the mixture at 6000 rpm for 17 min, transfer the supernatant to mix with a 2N KOH at a volume ratio of 1: 1. Then centrifuge at 6000 rpm for 17 min. Discard the supernatant while keeping the precipitate to distilled water washing for pH neutralization. The last yellowish sediment was washed with a 1: 1 ethanol-acetone solution and oven-dried at 40°C overnight. The chitin and chitosan dry weighed were measured. The yields [ 30 ] and chitosan productivity [ 31 ] were calculated as follows: Chitin yield (Y) = \(\left(\frac{\text{C}\text{h}\text{i}\text{t}\text{i}\text{n} \text{d}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \left(\text{g}\right)}{\text{C}\text{e}\text{l}\text{l} \text{m}\text{a}\text{s}\text{s} \text{f}\text{o}\text{r}\text{m}\text{a}\text{t}\text{i}\text{o}\text{n} \left(\text{g}\right)}\right) \text{x} 100\) Chitosan yield (Y) = \(\left(\frac{\text{C}\text{h}\text{i}\text{t}\text{o}\text{s}\text{a}\text{n} \text{d}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \left(\text{g}\right)}{\text{C}\text{h}\text{i}\text{t}\text{i}\text{n} \text{d}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} \left(\text{g}\right)}\right) \text{x} 100\) Chitosan productivity (g/d) = \(\left(\frac{\mathbf{C}\mathbf{h}\mathbf{i}\mathbf{t}\mathbf{o}\mathbf{s}\mathbf{a}\mathbf{n} \mathbf{d}\mathbf{r}\mathbf{y} \mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \left(\mathbf{g}\right)}{\mathbf{T}\mathbf{i}\mathbf{m}\mathbf{e} \left(\mathbf{d}\right)}\right)\) Fungal chitosan antibacterial activity Preparation of pathogenic bacterial inoculum The tested pathogenic bacterial inoculum was pre-cultured in Mueller Hinton broth medium for 24 h in a rotary shaker at 37°C until the growth gave turbidity of McFarland barium sulfate standard 0.5. The inoculum of each strain was standardized by measurement of the optical density using a spectrophotometer at 625 nm which ranged from 0.08–0.12. the standard inoculum was adjusted at a concentration of 10 8 cells/mL [ 32 ]. Chitosan antibacterial activity using the Kirby-Bauer disc diffusion method Seven trials of fungal chitosan ranging from 0.03 to 1.2% (prepared in 1.0% acetic acid at pH 5.5) were used to study their antibacterial activities on a solid medium using Kirby-Bauer disc diffusion method as standardized by the Clinical and Laboratory Standards Institute [ 33 ]. Plates containing Mueller Hinton agar medium were inoculated with a standardized inoculum of the test pathogenic bacteria (dip a sterile cotton swab into the suspension) by streak method. Then, the saturated filter paper discs (Filter Paper Whatman No. 1, 11 mm in diameter) with 10 µL of chitosan were placed on agar surfaces. Acetic acid (1.00%) and Ciprofloxacin (5.00 µg) were used as negative and positive controls, respectively. Plates were incubated at 37°C for 24 h. Then, the diameter of the inhibition zone was measured (mm). Chitosan antibacterial activity test by the inhibition of microbial growth in the broth medium Chitosan concentrations at 0.05 and 1.0% were added to Mueller Hinton broth medium in order to study their inhibition of pathogenic bacterial growth in the broth medium [ 34 ]. The propagation was carried out in Erlenmeyer flasks (250 mL in volume) containing 50 mL Mueller Hinton broth medium. Flasks were inoculated with 1 mL pathogenic bacterial standard inoculum and shaken on a rotary shaker (150 rpm) for 72 h at 37°C. About 5 ml were taken periodically every 12 h. The number of cell forming units (CFU/ mL) was determined using poured plate count on specific media for each pathogenic bacterial strain [ 35 ]. Plates were incubated at 37°C for 24 h and then enumerated for viable bacteria. Beef burger preparation The beef burger was manufactured in the lab. condition according to the method of Jiménez-Colmenero [ 36 ]. The beef burger mixture contains (g): minced meat 226.875, onion juice 1.25, garlic 0.625, salt 5.0, spices 3.75, ice water 12.5, and 1.0% chitosan. The beef burgers were prepared using a manual-burger piston (15 cm diameter and 1.3 cm height) with about 150 g beef mixture for each slide. The uncooked and cooked samples were subsequently examined for chemical, physical, and sensory evaluation, as mentioned below. Chemical analysis of uncooked and cooked beef burger The chemical analysis of the cooked samples was determined through the procedures described by Latimer and George [ 37 ] the total solids (TS), moisture, ash, fats, crude protein, and total carbohydrates were calculated using difference [ 38 ]. Total carbohydrates = \(\left[\mathbf{T}\mathbf{o}\mathbf{t}\mathbf{a}\mathbf{l} \mathbf{S}\mathbf{o}\mathbf{l}\mathbf{i}\mathbf{d}\mathbf{s} \left(\mathbf{T}\mathbf{S}\right) – (\mathbf{F}\mathbf{a}\mathbf{t}+ \mathbf{P}\mathbf{r}\mathbf{o}\mathbf{t}\mathbf{e}\mathbf{i}\mathbf{n} + \mathbf{A}\mathbf{s}\mathbf{h})\right]\) Cooked beef burger quality analysis Physical analysis Texture profile analysis (TPA) of cooked samples, the different texture characteristics (hardness, adhesiveness, cohesiveness, springiness, and gumminess) were measured at 23°C using an Instron Universal Testing Machine model 1195, Stable Micro System (SMS) Ltd., Godalming, UK, loaded with Dimension Software SMS software [ 39 ]. This analysis was performed at the Research Institute, Agriculture Research Center, Giza. Cooking loss was measured [ 40 ]. A total of 20 g of beef burger samples was shaped to form a circular loop. The weight of burgers was determined before and after being cooked on an electric grill. Cooking loss (%) = \(\left(\frac{\mathbf{U}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \left(\mathbf{g}\right)- \mathbf{C}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \left(\mathbf{g}\right)}{\mathbf{U}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \left(\mathbf{g}\right)}\right) \mathbf{x} 100\) The beef burger shrinkage (%) was measured [ 41 ]. A difference between the uncooked burger diameter and cooked burger diameter was regarded as the percentage of shrinkage and calculated as follows: Shrinkage (%) = \(\left(\frac{\left(\mathbf{D}\mathbf{i}\mathbf{a}\mathbf{m}\mathbf{e}\mathbf{t}\mathbf{e}\mathbf{r} \mathbf{o}\mathbf{f} \mathbf{u}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \right(\mathbf{m}\mathbf{m}) - \mathbf{D}\mathbf{i}\mathbf{a}\mathbf{m}\mathbf{e}\mathbf{t}\mathbf{e}\mathbf{r} \mathbf{o}\mathbf{f} \mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r}(\mathbf{m}\mathbf{m}\left)\right)}{\mathbf{D}\mathbf{i}\mathbf{a}\mathbf{m}\mathbf{e}\mathbf{t}\mathbf{e}\mathbf{r} \mathbf{o}\mathbf{f} \mathbf{u}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \left(\mathbf{m}\mathbf{m}\right)}\right) \mathbf{x} 100\) The reduction in beef burger thickness (RBT) was calculated as follows: RBT (%) = \(\left(\frac{\mathbf{C}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \mathbf{t}\mathbf{h}\mathbf{i}\mathbf{c}\mathbf{k}\mathbf{n}\mathbf{e}\mathbf{s}\mathbf{s} - \mathbf{U}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \mathbf{t}\mathbf{h}\mathbf{i}\mathbf{c}\mathbf{k}\mathbf{n}\mathbf{e}\mathbf{s}\mathbf{s})}{\mathbf{C}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} \mathbf{t}\mathbf{h}\mathbf{i}\mathbf{c}\mathbf{k}\mathbf{n}\mathbf{e}\mathbf{s}\mathbf{s}}\right) \mathbf{x} 100\) Chemical analysis The moisture retention (MR) value represents the amount of moisture retained in the cooked product per 100 g of sample and was determined [ 42 ] according to the equation below: Moisture retention (%) = \(\frac{\mathbf{M}\mathbf{o}\mathbf{i}\mathbf{s}\mathbf{t}\mathbf{u}\mathbf{r}\mathbf{e} \mathbf{o}\mathbf{f} \mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r} }{\mathbf{M}\mathbf{o}\mathbf{i}\mathbf{s}\mathbf{t}\mathbf{u}\mathbf{r}\mathbf{e} \mathbf{o}\mathbf{f} \mathbf{u}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r}} \mathbf{x} 100\) The fat retention (FR) value represents the amount of fat retained in the product after cooking. Fat retention was calculated [ 43 ] by using the equation as follows: FR (%) = \(\left(\frac{\mathbf{C}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \left(\mathbf{g}\right) \mathbf{X} \mathbf{F}\mathbf{a}\mathbf{t} \mathbf{i}\mathbf{n} \mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r}\left(\mathbf{\%}\right)}{\mathbf{U}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t} \left(\mathbf{g}\right) \mathbf{X} \mathbf{F}\mathbf{a}\mathbf{t} \mathbf{i}\mathbf{n} \mathbf{u}\mathbf{n}\mathbf{c}\mathbf{o}\mathbf{o}\mathbf{k}\mathbf{e}\mathbf{d} \mathbf{b}\mathbf{e}\mathbf{e}\mathbf{f} \mathbf{b}\mathbf{u}\mathbf{r}\mathbf{g}\mathbf{e}\mathbf{r}\left(\mathbf{\%}\right)}\right) \mathbf{x} 100\) Sensory properties evaluation of beef burger In order to evaluate the consuming quality of the beef burger, sensory parameters analysis (appearance, texture, aroma, taste, and overall acceptability) was made. The evaluation was performed by 20 members (12 females and 8 males, with an age range between 10 and 61)) of the panel of graduate judges, post-graduate students and staff members of the Faculty of Agriculture, Ain Shams University and families in Egypt, using a 9-point hedonic scale from 0 (lowest) to 9 (highest) and the method according to Coda, Lanera [ 44 ]. The scorecard used for the Hedonic rating test, as previously designed [ 45 ], is shown in Table S1 . Storage (Shelf-life) periods of beef burger The beef burger supplemented with fungal chitosan was maintained under freezing conditions for 8 weeks. The microbiological analysis was evaluated as mentioned below during zero, 4, 6, and 8 weeks storage periods. Microbiological evaluation of laboratory-manufactured beef burger Laboratory-manufactured beef burgers were microbiologically analyzed by aseptic transfer of 10 g of homogenized burger into 90 mL of peptone water (1.0%). Serial dilutions were prepared in the same diluents. Different microbial groups ( Staph. aureus , E. coli and Pseudomonas sp.) were enumerated using the poured plates technique [ 35 ] on specific media as previously stated. The plates were supplemented with the Vogel Johnson agar, Bacillus cereus agar base, Salmonella Shigella (SS) agar and Violet red bile (VRB) agar, respectively. after incubation at 37°C for 24 h the count of bacteria expressed as Log CFU/mL. As well as Salmonella spp. of in laboratory beef burger samples were detected on Salmonella Shigella agar plates. Statistical analysis In triplicate, the experimental values were analyzed using analysis of variance (ANOVA) throughout the IBM® SPSS® Statistics software (version 19) and represented as the mean ± standard division of the mean. According to Starkings [ 46 ], the Duncan's Multiple Range Test at the 5% level was deemed significant; hence the significance of differences between means was determined at the 95% confidence level ( p -value ≤ 0.05 value). Results Chitosan production from A. tubingensis using different fermentation Submerged fermentation (SmF) The influence of liquid by-products whey and blackstrap molasses as carbon sources using the SmF approach vs. sucrose as control is shown in Table 1 and Fig. 1 . Table 1 reveals that after 8 days of incubation, the sucrose-containing control medium had the highest significant (p < 0.05) values of fungal biomass, chitin, and chitosan, with 1.52 g/L of cell dry weight, 0.26 g chitin/g biomass, and 0.053 g chitosan/g biomass, respectively. The incubation period was extended to 12 days in a medium supplemented with whey or blackstrap molasses, yielding 3.22 or 1.70 g cell dry weight / L medium, 0.78 or 0.41 g chitin/g biomass, and 0.28 or 0.12 g chitosan /g biomass, respectively. According to these results, whey by-product was preferred for chitosan synthesis from the tested strain over blackstrap molasses and sucrose (control), which increased approximately 1.3-fold and 5.3-fold, respectively. Figure 1 shows the calculated and displayed chitin and chitosan yields and chitosan productivity. After 12 days, the highest peaks of chitin and chitosan yields and chitosan productivity were recorded in a medium supplemented with whey (24.22%, 35.90%, and 0.023 g/L/d, respectively), which was higher than the results obtained in a medium supplemented with blackstrap molasses (24.12%, 29.27%, and 0.010 g/L/d, respectively). During 8 to 10 days of incubation, the maximum yield of chitin (17.11 and 17.22%) and chitosan (20.38 and 19.23%) with chitosan productivity of 0.0066 and 0.005 were obtained in the control medium containing sucrose. Table 1 Cell mass dry weight, chitin, and chitosan production VS. incubation periods for A. tubingensis using agro by-products and wastes as a carbon source by submerged and solid-state fermentation Fermentation methods By-Products Parameters Fermentation time (days) and wastes 2 4 6 8 10 12 14 Submerged Sucrose (Control) CDW (g/L) 0.17 f 0.45 e 1.07 d 1.52 a 1.51 a 1.48 b 1.23 c Chitin DW (g/ g biomass) 0.02 f 0.06 e 0.13 d 0.26 a 0.26 a 0.24 b 0.20 c Chitosan DW(g/ g biomass) 0.001 f 0.006 e 0.014 d 0.053 a 0.050 a 0.043 b 0.037 c Whey CDW (g/L) 0.26 g 0.73 f 1.19 e 1.70 d 2.39 c 3.22 a 2.91 b Chitin DW (g/ g biomass) 0.06 e 0.09 e 0.21 d 0.32 c 0.51 b 0.78 a 0.53 b Chitosan DW(g/ g biomass) 0.004 f 0.009 e 0.06 d 0.10 c 0.15 b 0.28 a 0.16 b Blackstrap molasses CDW (g/L) 0.06 g 0.13 f 0.21 e 0.35 d 0.68 c 1.70 a 1.66 b Chitin DW (g/ g biomass) 0.002 e 0.007 d 0.02 c 0.05 c 0.18 b 0.41 a 0.40 a Chitosan DW(g/ g biomass) 0.0001 f 0.0006 e 0.003 d 0.010 c 0.046 b 0.12 a 0.100 a Solid-state Rice husk CDW (g/L) 0.09 f 0.26 e 0.32 d 0.78c 1.54a 1.31b ND Chitin DW (g/ g biomass) 0.003 e 0.009 e 0.024d 0.083c 0.24a 0.18b ND Chitosan DW(g/ g biomass) 0.0002 f 0.0008 e 0.002d 0.007c 0.043a 0.028b ND Rice straw CDW (g/L) 0.34f 0.63e 1.51d 2.39 c 3.25a 2.86b ND Chitin DW (g/ g biomass) 0.02d 0.05d 0.22c 0.61 b 0.75a 0.66ab ND Chitosan DW(g/ g biomass) 0.0006f 0.003e 0.038d 0.11 c 0.15a 0.13b ND Beet waste CDW (g/L) 0.85f 2.01e 3.00d 4.77 a 4.27b 3.71c ND Chitin DW (g/ g biomass) 0.18e 0.42d 0.78c 1.58a 1.47b 0.82c ND Chitosan DW(g/ g biomass) 0.02e 0.08d 0.19c 0.61a 0.47ab 0.21c ND Sawdust CDW (g/L) 0.17e 0.29d 0.35c 0.62b 1.38a 1.35a ND Chitin DW (g/ g biomass) 0.004e 0.007d 0.02c 0.05b 0.21a 0.20a ND Chitosan DW(g/ g biomass) 0.0000e 0.0002d 0.001c 0.009b 0.027a 0.025a ND Wheat bran CDW (g/L) 0.81f 1.89e 2.28d 4.23a 4.01b 3.23c ND Chitin DW (g/ g biomass) 0.14f 0.34e 0.61d 1.26a 1.13b 0.84c ND Chitosan DW(g/ g biomass) 0.015f 0.061e 0.14d 0.36a 0.32ab 0.21c ND CDW = cell dry weight, Chitin DW = chitin dry weight, Chitosan DW = chitosan dry weight and ND = Not detected. a,b The values in small letters in the same row with differing superscripts indicate a significant difference ( p ≤ 0.05). Solid-state fermentation (SSF) The data in Table 1 and Fig. 2 show that when using solid substrates (rice husk, rice straw, beet waste, sawdust, and wheat bran) through SSF, the values of biomass and chitin and chitosan yields were significantly increased with increasing fermentation periods up to 10 days, with the exception of presented beet waste and wheat bran up to 8 days. Table 1 shows that beet waste and wheat brane had the highest significance in terms of biomass (4.77 and 4.23 g cell dry weight/100g substrate), chitin (1.58 and 1.26 g /g biomass), and chitosan (0.61 and 0.36 g /g/biomass), respectively, followed by rice straw (3.25 g cell dry weight /100 g substrate, 0.75 g chitin dry weight / g biomass, and 0.15 g chitosan dry weight /g biomass, respectively). While the lowest values of biomass (1.54 and 1.38 g cell dry weight /100 g substrate), chitin dry weight (0.24 and 0.21 g /g biomass), and chitosan dry weight (0.043 and 0.027 g /g biomass) were obtained on rice husk and sawdust after 10 days of incubation, respectively. The maximum percentage of chitin (33.12 and 29.79%) and chitosan (38.61 and 28.57%) yields and chitosan productivity (0.076 and 0.045 g/g/d) were achieved after 8 days on beet waste and wheat bran, respectively, as shown in Fig. 2 . On the other hand, sawdust waste had the lowest chitin and chitosan yields (15.22 and 12.86%) and chitosan productivity (0.0027 g/g/d). Based on preliminary data, it was important to emphasize that chitosan synthesis from A. tubingensis using solid agro-industrial waste (beet waste) under the SSF approach was shown to be more advantageous than liquid by-products using the SmF technique. As a result, the beet waste was chosen for chitosan synthesis by the tested strain using SSF in the following investigations. Fungal chitosan as an antibacterial agent Chitosan antibacterial activity using Kirby-Bauer disc diffusion method As indicated in Table 2 , seven concentrations of fungal chitosan (0.03 to 1.2%) were tested against Gram-positive and -negative bacteria compared to ciprofloxacin (the antibiotic applied as a positive control) and acetic acid (a chemical killer used as a negative control). The results showed no significant difference between chitosan at 1.0% and ciprofloxacin against all tested pathogenic strains except B. cereus DSMZ 345, Ps. fluorescens NRRL 800, and E. coli ATCC 69373. Ciprofloxacin inhibited the tested bacteria strongly, with inhibition zone diameters ranging from 35 to 49 mm. In contrast, acetic acid had a limited effect, resulting in zone diameters ranging from 7 to 10 mm. At 0.03%, chitosan did not affect the antibacterial agent against the tested pathogenic bacterial strains. Moreover, chitosan at 0.05 to 1.2% inhibited the growth of every tested pathogenic bacterial strain with varying inhibition zone diameters. For all tested pathogenic bacterial strains, the zone diameter increased with increasing concentration until reaching the maximum value at 1.00%. Staph. aureus DSMZ 20231 exhibited the largest inhibition zone diameter, measuring 47 mm, but B. cereus DSMZ 345 was more resistant to chitosan at 1.0% (the smallest zone = 19 mm). The minimum inhibitory concentration (MIC) of chitosan on the solid culture technique was 0.05% against all tested pathogenic strains, with diameters ranging from 13 to 22 mm. Even though B. cereus DSMZ 345 was more resistant to chitosan at the tested concentrations and had a MIC value of 0.2% (zone dimeter = 15 mm). Table 2 Fungal chitosan antimicrobial activity at various concentrations against Gram-positive and negative bacterial strains Acetic acid (1.00%) is a negative control, and ciprofloxacin (5.00 μg) is a positive control. a,b The values in small letters in the same column with differing superscripts indicate a significant difference (p ≤ 0.05), ± Standard division. Chitosan antibacterial activity test by the inhibition of microbial growth in the broth medium Figure 3 shows that adding chitosan (0.05 and 1.00%) to growth media reduced the growth of tested strains during the first 12 hours of growth. All tested strains' growth increased gradually throughout incubation, peaking after 48 hours. Chitosan concentrations of 0.05% and 1.00% reduced growth at rates of 0.23 to 0.49 CFU/ h and 0.29 to 0.56 CFU/ h, respectively. Chitosan at 1% recorded higher growth reduction rate values, with S. Typhimurium ATCC 14028 having the highest value. Additionally, after 24 h of incubation with both chitosan concentrations, all tested pathogenic strains exhibited complete inhibition. On the contrary, B. cereus DSMZ 345 was the most stable bacterium for chitosan inhibition effect. Both 0.05 and 1.00% chitosan did not inhibit the growth of B. cereus DSMZ 345 during the incubation period (48 h). Application of fungal chitosan for laboratory-manufactured beef burger Chemical analysis of uncooked and cooked beef burger The chemical composition of uncooked and cooked beef burgers is shown in Table 3 . Moisture, ash, fat, protein, TS and TC content of uncooked samples ranged between 60.40–61.00%, 1.36–1.38%, 20.10–20.00%, 15.54–15.56%, 39.60–39.00%, and 2.60–2.10%, respectively. The contents for cooked beef burgers were in the range of 58.86–59.60%, 1.33–1.38%, 18.67–19.89%, 15.42–15.49%, 41.14–40.40%, and 5.70–3.60%, respectively. Table 3 Chemical analysis of uncooked and cooked beef burger Chemical analysis (%) Treatments Moisture Ash Fat Protein TS TC Uncooked Control 60.40 a 1.36 b 20.10 a 15.54 a 39.60 2.60 Chitosan 1.0% 61.00 a 1.38 a 20.00 a 15.56 a 39.00 2.10 Cooked Control 58.86 bc 1.33 b 18.67 c 15.42 a 41.14 5.70 Chitosan 1.0% 59.60 b 1.38 a 19.89 b 15.49 a 40.40 3.60 TS, Total solids; TC, Total carbohydrates. a,b The values in small letters in the same column with differing superscripts indicate a significant difference (p ≤ 0.05). Beef burger quality analysis The quality analysis of beef burgers including texture, physical (cooking) and chemical properties, has been presented in Table 4 . The texture properties of hardness, Adhesiveness, cohesiveness, springiness, gumminess and chewiness of the control and treated with chitosan samples were between 204.6-225.2 mm, 175.31-200.66 mJ, 0.47 − 0.43, 1.19–1.46 mm, 96.3–96.6 N, and 114.23-140.99 mJ, respectively. All parameters in a sample treated with chitosan had higher values than the control except the cohesiveness ratio was slightly decreased. The cooking (physical) properties of cooking loss, shrinkage and RBT of the control and treated with chitosan samples were between 18.02–16.8%, 14.3–10.6%, and 32.3–29.1%, respectively. The chemical properties of moisture retention and fat retention of the control and treated chitosan samples were between 97.45–97.70%, and 92.89–99.45%, respectively. Table 4 Cooked beef burger quality (texture, physical and chemical) analysis Treatments Texture analysis Hardness Adhesiveness Cohesiveness Springiness Gumminess Chewiness (mm) (mJ) Ratio (mm) (N) (mJ) Control 204.6 175.31 0.47 1.19 96.3 114.23 Chitosan 1.0% 225.2 200.66 0.43 1.46 96.6 140.99 Physical analysis (%) Cooking loss Shrinkage RBT Control 18.02 14.3 32.3 Chitosan 1.0% 16.8 10.6 29.1 Chemical analysis (%) Moisture retention Fat retention Control 97.45 92.89 Chitosan 1.0% 97.70 99.45 RBT = Reduction in Beef Burger Thickness. Sensory properties evaluation of beef burger About 20 of varied ages panelists were requested to evaluate the cooking laboratory-manufactured beef burger with 0 (control non-treated with chitosan) and 1.0% chitosan. These treatments' consumer panel data were analyzed and presented in Fig. 4 . Addition of 1.0% chitosan affected significantly appearance (7.9), aroma (8.2), color (8), taste (8.1), taste (8.1), and overall acceptability (8.1). Whereas the beef burger without chitosan (control) showed lower values and recorded significant differences with beef burger supplemented with 1% chitosan. Storage (Shelf-life) periods of beef burger All manufactured beef burgers treated with 0% chitosan (control non-treated) or 1.0% chitosan (treated) were stored in frozen condition for 8 weeks in order to study their efficiency against pathogenic bacteria. Data presented in Table 5 exposed that all fresh burger samples (control and treated with 1.0% chitosan) at zero time were highly contaminated with the tested pathogenic bacteria. E. coli achieved the uppermost values of 4.2 and 4.21 Log CFU/g at 0 and 1.0% chitosan, respectively, followed by Pseudomonas spp. and Staph. aureus of 3.45 and 3.40 Log CFU/ g, and 3.30 and 3.28 Log CFU/g, respectively. In addition, Salmonella ssp. growth on Salmonella Shigella agar plates at zero time only, but no growth occurred during the storage periods. Results also indicated that the number of pathogenic bacteria significantly decreased with an increase in the storage periods up to 8 weeks. The reduced number of the tested pathogenic bacteria in treated samples with 1.0% chitosan was more significant than in the control sample with 0% chitosan. Chitosan reduces the growth of pathogenic bacteria with diverse levels. E. coli was the most susceptible to chitosan, which achieved the highest growth reduction percentage (78.1%), followed by Pseudomonas spp. and Staph. aureus being 70.6% for the former and 66.76% for the latter by the end of the storage period. All beef burger without chitosan shows attenuation in a bacterial count throughout the storage period under a frozen state to reach the minimum after 8 weeks. The percentage of growth reduction was 18.8%, 24.63%, and 28.18% for E. coli , Pseudomonas spp., and Staph. aureus , respectively. Table 5 Microbiological evaluation of laboratory-manufactured beef burgers during storage periods Samples Storage period weeks Log count of bacteria (CFU / g) Detection of Salmonella spp. Staph. aureus % growth reduction E. coli % growth reduction Pseudomonas spp. % growth reduction Non-Treatment (control) 0 3.30 ± 0.16 Cf − 4.20 ± 0.51 cF - 3.45 ± 0.28 dG − + 4 2.48 ± 0.32 bE 24.84 3.69 ± 0.35 bE 12.14 3.00 ± 0.12 bcE 13.04 nil 6 2.50 ± 0.11 bE 24.24 3.70 ± 0.31 bE 11.90 2.96 ± 0.47 bE 14.20 nil 8 2.37 ± 0.27 aD 28.18 3.41 ± 0.18 aD 18.80 2.60 ± 0.15 aD 24.63 nil Treatment with 1.0% chitosan 0 3.28 ± 0.62 dF − 4.21 ± 0.25 dF − 3.40 ± 0.39 dF − + 4 1.51 ± 0.25 cC 53.96 1.70 ± 0.18 cC 59.62 1.90 ± 0.33 cC 44.12 nil 6 1.19 ± 0.17 bAB 63.72 1.10 ± 0.29 bB 73.87 1.45 ± 0.19 bB 57.35 nil 8 1.09 ± 0.15 Aa 66.76 0.92 ± 0.41 aA 78.10 1.00 ± 0.24 aA 70.60 nil % growth reduction = Xt-X0*100/ X0, + growth,− no growth. a,b The values in small letters in the same column with differing superscripts indicate a significant difference ( p ≤ 0.05) between each treatment individually with a microbe, A,B The values in capital letters in the same column with differing superscripts indicate a significant difference ( p ≤ 0.05) between non-treatment and treatment with 1.0% chitosan, ± Standard division. Discussion Chitosan is an amino polysaccharide, and it is commercially produced from chitin by alkali deacetylation [ 47 , 48 ]. It was assumed chitosan production using fungi is more favourable than the crustaceans as they are not available with high-cost chitosan purification steps. Many Fungi strains were investigated for chitosan production utilizing agro-wastes and by-products in a trial to minimize production costs and, in the same line, increase chitosan production. For this target, they used wheat straw to cultivate Lentinus edodes and obtained a chitosan production yield of about 50 mg/g cell [ 49 ]. Chitosan production with Absidia coerulea on potato peels [ 50 ]. Also, they used apple pomace for chitosan production by Gongronella butleri , which provided a high yield of about 217 mg/g cells [ 51 ]. The chitosan production from Agaricus sp., Pleurotus sp., and Ganoderma sp. using sawdust and rice straw agro-wastes [ 52 ]. A chitosan production from R. arrhizus using corn-steep liquor with a yield of 29.3 mg/g cells [ 53 ]. The cultivated A. terreus on apple waste extract, produced a biomass concentration of 30 g/L and chitosan of 0.14 g/g cells after 5 days of incubation [ 54 ]. In the current study, agro-wastes and by-products were utilized with two different techniques SSF and SMF, for chitosan production by A. tubingensis . For the SMF technique, whey and blackstrap sugarcane molasses were the utilized by-products, and it was found that whey was the best source compared to blackstrap molasses. Whey was responsible for the fungal cell dry weight and chitosan increase by 1.89-fold and 2.33-fold, respectively, after 12 days of incubation. For the SSF approach, the beet waste followed by wheat bran were the best sources as the fungal cell dry weight was increased by 3.14-fold and 2.78-fold, and chitosan by 9.81-fold and 6.79-fold, respectively after 8 days of incubation when compared with control in SMF of sucrose. This SSF approach is assumed to be better than the SMF for chitosan production by A. tubingensis . In agreement with these findings, Maghsoodi and Yaghmaei [ 28 ] reported that the yield of chitosan using SSF (w/w) was higher than that of SMF (w/v) owing to that the filamentous fungi are most exploited because of their ability to grow on the complete solid substrate while the low amount of mycelia produced in SMF, so there has been considerable interest in producing chitosan using the SSF process. In the same way, the quality and amount of chitosan extracted from the fungal mycelia depend on the fungal cultivation method, as shaking had deleterious effects [ 54 ]. In particular, shear forces can damage conidiophores leading to decreased spore production. Beet waste was the compromised source for A. tubingensis chitosan production with SSF compared to sucrose (control). Chitosan was examined as an antimicrobial agent with a broad spectrum, affecting Gram-positive and negative bacterial pathogens. Chitosan is a bioactive compound with bactericidal or bacteriostatic effects, often with no diversity between activities [ 55 ]. Data indicated that fungal chitosan had a positive impact on Gram-positive because it had a mode of action related to the electrostatic reaction that occurs between the positive charge of chitosan and the negative charge of the bacterial cell wall [ 56 ]. The cell wall of Gram-positive bacteria had a wall of teichoic acids and lipoteichoic acids in peptidoglycan layers. Both compounds had a negatively charged anionic backbone [ 57 , 58 ]. The teichoic acids can provide arranged uniform high-density negative charges in the cell wall, thereby inhibiting the passage of ions across the membrane. The positively charged chitosan interacts electrostatically with the negatively charged teichoic acid in peptidoglycans, leading to the destruction of the cell membrane, leakage of intracellular components and the entrance of chitosan into the microbial cells [ 59 ] or the leakage of proteins and other intracellular constituents by chitosan [ 60 ]. Other studies exhibited the electrostatic reaction promoting changes in the properties of membrane wall permeability, so it motivates internal osmotic imbalances and thus prevents the growth of microorganisms [ 61 ]. The surface of Gram-negative bacteria comprises negative charges from the phosphate and pyrophosphate groups of lipopolysaccharide in the outer layer of the outer membrane. Lipopolysaccharide gave a high negative charge due to neutralization by positive charges from chitosan, resulting in disruption of the outer membrane., enabling chitosan to penetrate the cell membrane, thus leading to bacterial cell death [ 62 ]. Many researchers stated that increasing chitosan concentrations enlarges the zone of inhibition of tested pathogens such as Staph. aureus, S. Typimurium and B. cereus . Collected data proved that Gram-positive bacteria were more sensitive to chitosan than Gram-negative bacteria [ 63 ]. Chitosan was more active against Gram-positive bacteria such as Bacillus spp., Listeria monocytogenes , Streptococcus pyogenes and Staph. aureus (MERS strain) as found in another research [ 64 ]. MIC of chitosan varied according to the tasted microorganism as well as the chitosan futures. In some studies, the minimum inhibitory concentration (MIC) of chitosan ranged from 0.05 to 0.1% according to the bacterial strain [ 65 ]. The chitosan nanoform lowered the MIC about 21–41 times against E. coli 25922, S. choleraesuis ATCC 50020, and Staph. aureus 25923[ 66 ]. Both chitosan and trimethyl-chitosan (TMC) affected the growth of E. coli and Staph. aureus and reduced the maximum absorbance (at 620 nm) after 12 h incubation for E. coli using 0.5-2.0 g/L chitosan or TMC. The same trend was observed with Staph. aureus using 1.0% chitosan. Moreover, increasing chitosan concentration did not significantly drop bacterial growth [ 64 , 67 ]. Chitosan in low concentration binds to the bacterial cell wall's negative charge and causes cell membrane injury, leading to cell death. In contrast, the protonated chitosan's high concentration may coat the cell surface and avoid intracellular leakage. In addition, the negative charge of the cell wall varied, rendering the growth phase and affecting the bacterial susceptibility to chitosan. E. coli O157-H7 and Staph. aureus CCRC12657 cells in the middle of exponential growth (after 10 h of growth) were more sensitive to chitosan than cells in stationary growth [ 60 ]. Minced meat and beef burgers are semi-perishable, rich in nutrients and moisture, supporting microbial growth and food spoilage agents. Microbial growth caused an undesirable change in food odder, texture, colour, and chemicals and restricted product shelf-life [ 68 ]. The grown need a bioactive compound with antibacterial activity such as chitosan, biodegradable, non-toxic, edible, and effective against various spoilage bacteria and molds [ 67 ]. Different studies stated that chitosan had a strong antibacterial effect against pathogenic and spoilage bacteria in meat products by destroying the cell membrane's permeability leading to bacterial death [ 69 ] Venkatachalam and Lekjing [ 70 ] reported that chitosan reduced lactic acid bacteria (LAB), psychrophilic group and Enterobacteriaceae count in pork patties, and their efficiency augmented when chitosan was supplemented with clove oil and nisin also, the shelf-life of pork patties increased twofold by using chitosan and mixture of chitosan, clove oil, and nisin in pork patties. Significant effect of 1.5% chitosan and 2% thyme oil mixture against LAB, Enterobacteriaceae, Pseudomonas spp., and molds during storage under modified atmosphere packaging. The mix also extended the shelf life of chicken-pepper kebab to reach 14 days instead of 6 days in chicken-pepper kebab without chitosan and thyme oil mixture [ 71 ]. In addition to the efficiency of chitosan as an antimicrobial in meat and meat products, chitosan enhanced the quality of colour, overall acceptability, odder and texture of meat products[ 72 ]. The shelf life of 1% chitosan with fresh pork sausages increased from 7 to 15 days at a chilled temperature [ 73 ]. The effects of chitosan films on pork sausage microbial count, demonstrating a significant (p-value < 0.05) antimicrobial impact of the films against molds and yeasts, lactic acid bacteria, and the total mesophilic viable count (TMVC) [ 74 ]. Results indicated that the moisture values had no significant differences for uncooked treatments but they decreased in cooked treatments, which might be due to the increase in solid material content [ 75 ]. Chitosan beef burgers showed higher significance in ash values than control, it might be due to a high mineral content in chitosan beef. Fish burgers formulated with mashed pumpkin and mashed potato showed higher moisture and lower protein, fat and ash contents than the control groups [ 76 ]. Cooked beef patties with added PM showed higher ash content compared to C samples (p < 0.05), probably due to the high mineral content of the pumpkin mix. The protein values in all treatments had no significant differences in both uncooked and cooked samples [ 75 ]. The fat values were highly significant in uncooked samples and slightly decreased in cooked samples due to heat treatment using an electric grill due to melting the fat in cooked samples. All parameters of texture (hardness, Adhesiveness, springiness, gumminess and chewiness) in a sample treated with chitosan had higher values than the control except the cohesiveness ratio was slightly decreased. The hardness and chewiness values of pork burgers formulated with albedo-fibre powder were increased with the increasing fibre amount, while springiness and cohesiveness of the samples did not show significant differences and gumminess was increased with only the addition of 5% powder [ 77 ]. The textural parameters of the products could show differences according to the natural structure the amount of the non-meat ingredient and the amount of replaced meat in the formulation[ 75 ]. Results clearly showed that the cooking weight loss and shrinkage of samples decreased in samples supplemented with 1.0% chitosan than in control samples. It might be due to chitosan demonstrating barrier capabilities to prevent water removal during the cooking process by making water molecular hydrogen bonds that make them more effective in avoiding weight loss in the final product[ 78 , 79 ]. Conclusions Data could be summarized that fungal polymer chitosan was produced from A. tubingensis on beet waste as the only carbon source during solid-state fermentation after 8 days of incubation, achieving 32.91%. At a polymer concentration of 1.0%, this polymer inhibited Staph. aureus DSMZ 20231, S. Typhimurium ATCC 14028, B. cereus DSMZ 345, Ps. fluorescens NRRL 800, and E. coli ATCC 69373. The fungal polymer was applied in the food industry as a food additive on a laboratory scale for the preservation and extension of the shelf life of beef burgers to 8 weeks. In addition, adding polymer to beef burgers improved the physio-chemical and sensory features of the product. As a consequence, future studies will focus on applying the fungal polymer chitosan as a food-packing ingredient. Declarations Acknowledgements The authors would like to thank the Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Cairo and Food Technology Research Institute, Agriculture Research Center, Giza, Egypt, for supporting the current experiments. Author Contributions Conceptualization, Kh.A.A., R.F.A., B.T.A., D.Y.A. and S.A.A.; methodology, Kh.A.A., R.F.A., B.T.A, D.Y.A and Sh.A.A.; software, Kh.A.A. and B.T.A.; validation, R.F.A., Sh.A.A. and D.Y.A.; formal analysis, Kh.A.A., R.F.A.,B.T.A., D.Y.A. and Sh.A.A.; investigation, Kh.A.A.; resources, Sh.A.A. and D.Y.A.; data curation, Kh.A.A., R.F.A., B.T.A.; writing—original draft preparation, Kh.A.A., R.F.A., B.T.A, D.Y.A and Sh.A.A.; writing—review and editing, Kh.A.A., R.F.A., B.T.A, D.Y.A and Sh.A.A.; All authors have read and agreed to the published version of the manuscript. Funding No fund. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Not applicable. Competing interests The authors claim to have no conflicts of interest. Consent for publication Not applicable. References Silva MM, Lidon F. Food preservatives–An overview on applications and side effects. Emir J Food Agric. 2016:366-73.https://doi.org/10.9755/ejfa.2016-04-351 Javanmardi F, Rahmani J, Ghiasi F, Hashemi Gahruie H, Mousavi Khaneghah A. The association between the preservative agents in foods and the risk of breast cancer. Nutr Cancer. 2019;71(8):1229-40.https://doi.org/10.1080/01635581.2019.1608266 Bakhtiary F, Sayevand HR, Mousavi Khaneghah A, Haslberger AG, Hosseini H. Antibacterial efficacy of essential oils and sodium nitrite in vacuum processed beef fillet. 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Meat Science. 2014;97(2):270-6.https://doi.org/10.1016/j.meatsci.2014.02.010 Amoli PI, Hadidi M, Hasiri Z, Rouhafza A, Jelyani AZ, Hadian Z, et al. Incorporation of low molecular weight chitosan in a low-fat beef burger: Assessment of technological quality and oxidative stability. Foods. 2021;10(8):1959.https://doi.org/10.3390/foods10081959 do Amaral DS, Cardelle-Cobas A, do Nascimento BM, Monteiro MJ, Madruga MS, Pintado MME. Development of a low fat fresh pork sausage based on chitosan with health claims: Impact on the quality, functionality and shelf-life. Food Funct. 2015;6(8):2768-78.https://doi.org/10.1039/C5FO00303B Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4397361","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":314980365,"identity":"4c824561-0b12-4b3b-8a6a-3e95543d0579","order_by":0,"name":"Shimaa A. 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Abd-Elhalim","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Basma","middleName":"T.","lastName":"Abd-Elhalim","suffix":""},{"id":314980372,"identity":"418c3845-9d34-402a-a32d-3002acb2a8f7","order_by":3,"name":"Dina Y. Abdelghani","email":"","orcid":"","institution":"Food Technology Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Dina","middleName":"Y.","lastName":"Abdelghani","suffix":""},{"id":314980375,"identity":"2593d169-601e-4736-b0a1-2e7a54406578","order_by":4,"name":"Rania F. Ahmed","email":"","orcid":"","institution":"Ain Shams University","correspondingAuthor":false,"prefix":"","firstName":"Rania","middleName":"F.","lastName":"Ahmed","suffix":""}],"badges":[],"createdAt":"2024-05-09 23:54:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4397361/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4397361/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58573616,"identity":"5905c185-3637-4fbb-b434-0bccfe8c585b","added_by":"auto","created_at":"2024-06-18 11:38:49","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":405731,"visible":true,"origin":"","legend":"\u003cp\u003eChitin and chitosan yields and chitosan productivity using agro by-products under submerged fermentation\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4397361/v1/9cc635cc7453953e6f782840.jpeg"},{"id":58574181,"identity":"ad900ced-be02-4c7f-a0d6-e9c2677f5be3","added_by":"auto","created_at":"2024-06-18 11:46:49","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":608091,"visible":true,"origin":"","legend":"\u003cp\u003eChitin and chitosan yields and chitosan productivity using agro-wastes under solid-state fermentation\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4397361/v1/0e365b2d2b7f842723737b0d.jpeg"},{"id":58573612,"identity":"0093f6c5-372f-4a5c-8072-cb8133180706","added_by":"auto","created_at":"2024-06-18 11:38:49","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":391689,"visible":true,"origin":"","legend":"\u003cp\u003eFungal Chitosan at concentrations of 0.05 and 1.00% as antibacterial activity test by inhibiting pathogenic bacterial growth in the broth medium\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4397361/v1/114e092b5841126dd37475ef.jpeg"},{"id":58573615,"identity":"1daeac63-1a2c-4bd4-9a75-36e0c7013594","added_by":"auto","created_at":"2024-06-18 11:38:49","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":182688,"visible":true,"origin":"","legend":"\u003cp\u003eSensory properties evaluation score or percent % of beef burgers without chitosan and supplemented with 1.0% fungal chitosan.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4397361/v1/756fa26bd7b56039bc51419f.jpeg"},{"id":106971375,"identity":"96ef65ce-f187-4c0c-af1b-d381153cdbbc","added_by":"auto","created_at":"2026-04-15 10:18:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3292969,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4397361/v1/7b938b07-eef8-48a9-9fd6-59f278493b53.pdf"},{"id":58573614,"identity":"8117f440-490f-4d9d-be9a-76746bc59a26","added_by":"auto","created_at":"2024-06-18 11:38:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":648902,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4397361/v1/44bc9622c4a4f8baf1773309.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chitosan production from agro-wastes by Aspergillus tubingensis: A sustainable approach for beef burger quality improvement","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFood industries especially meat products are categorized as highly perishable products owing to the richness of unsaturated fatty acids, oxidative stability, and high risk of microbial spoilage which lead to various unfavourable changes in colour flavour, and texture. For quality enhancement and freshness maintenance, numerous food chemical additives were used. These chemicals have antimicrobial and antioxidant behaviour utilized in the meat industry like sorbic acid, citric acid, propionic acid, nitrite salts, benzoic acid, and various collections of enzymes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. On the contrary, these chemicals represent a high-risk factor for human health that is responsible for cancers, allergic reactions, and gastrointestinal disorders [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. So, there is a necessary need for natural antimicrobials and antioxidant replacements such as chitosan, pediocin, polylysine, and nisin to ensure biosafety and increase the shelf life [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Chitosan is a linear polysaccharide composed of randomly distributed β-(1\u0026ndash;4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylated unit) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Chitosan is industrially obtained using alkali solutions by partial deacetylation of chitin, which is obtained from the exoskeletons of insects and shell waste of shrimps, crustaceans, lobsters, krill, squid\u0026rsquo;s backbones, and crabs. Unfortunately, these resources are not abundant and available all the time in high quantities according to seasons and fishing industry sites. In addition, the extensive steps for obtaining chitosan from marine crustaceans are demineralization, deproteinization, discolouration, and deacetylation. This meant that the production of one kilogram of chitosan, consumed 6.3 kg of HCl and 1.8 kg of NaOH is required [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], so the consumption of such a high quantity of chemicals results in environmental pollution. Besides, the produced chitosan is not resilient and inconsistent with poor quality [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. So, new chitosan resources that are abundant with low cost and have the ability of optimise from is for need. On the contrary, fungi biomass can be obtained continuously by the fermentation process that does not have any seasonal and transportation limitations. In addition, less production time with uniform properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, fungal mycelia do not require any additional treatments like crustacean wastes which have a high level of inorganic materials and need demineralization treatment during the processing. The cultivation of fungal cells has fulfilled the previous consideration for chitosan production by varying fermentation conditions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition, fungi are easy to handle, harvest, and control in order to produce high-quality chitosan. Various fungal species including \u003cem\u003eAbsidia coerulea\u003c/em\u003e, \u003cem\u003eAspergillus glauca\u003c/em\u003e, \u003cem\u003eA. niger\u003c/em\u003e, \u003cem\u003eA. terreus\u003c/em\u003e, \u003cem\u003eMucor rouxii\u003c/em\u003e, \u003cem\u003eGongronella butleri\u003c/em\u003e, \u003cem\u003eCunninghamella blakesleeanus\u003c/em\u003e, \u003cem\u003eRhizopus delemar\u003c/em\u003e, \u003cem\u003eR. oryzae\u003c/em\u003e, \u003cem\u003eMortierella isabelina\u003c/em\u003e, and \u003cem\u003eLentinus edodes\u003c/em\u003e have been investigated for chitin and chitosan production [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The fermentation approach become affordable because of using waste streams in an energy-efficient way as the microbial cells work as factories with low temperatures [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Fungal biomass cultivation can undergo using two main approaches, solid-state (SSF) and submerged substrate (SMF) fermentation utilizing agricultural and/or food industrial wastes and by-products as alternatives for expensive fermentation media to minimize chitosan production costs as it could be considered a green synthesis economic approach [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Owing to the multi-dimensional, and high functionality structure of chitosan it has widespread application in food, paper, wastewater treatment, pharmaceutical, biomedical, agricultural, biotechnological, and food-related industries [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Utilizing chitosan as a biological coating to improve the shelf life of meat products can be considered a healthy method of meat preservation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Meat and meat products are excellent sources of nutrients for humans; however, they also provide a favourable environment for microbial growth. To prevent the microbiological contamination of livestock foods, synthetic preservatives, including nitrites, nitrates, and sorbates, have been widely used in the food industry due to their low cost and strong antibacterial activity. The use of synthetic chemical preservatives is recently being considered by customers due to concerns related to negative health issues. Therefore, the demand for natural substances as food preservatives has increased with the use of plant-derived and animal-derived products, and microbial metabolites. These natural preservatives inhibit the growth of spoilage microorganisms or food-borne pathogens by increasing the permeability of microbial cell membranes, interrupting of protein synthesis, and cell metabolism [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Owing to chitosan\u0026rsquo;s exquisite properties like antimicrobial, film-forming, gas barrier, antioxidant, nontoxicity, biocompatibility, biodegradability activity, high absorption potential, and low moisture barrier make it a pioneer compound for different food products [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This study aimed to synthesize fungal \u003cem\u003eA. tubingensis\u003c/em\u003e chitosan under submerged and solid-state fermentation using various agro-wastes and by-products, as well as to investigate the time kinetics of fungal growth and chitin and chitosan formation. Use fungal chitosan as a food additive to improve the antibacterial, sensory, chemical, and physical qualities of beef burgers, as well as the shelf life.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFungal strain and pathogenic bacterial strains\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe pure culture of \u003cem\u003eAspergillus tubingensis\u003c/em\u003e was used for chitosan synthesis. It was obtained from the Agricultural Microbiology Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. Activation and preparation of the strain with Czapek's agar medium at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 48 h. Preserve and maintain at 5\u0026deg;C in a refrigerator.\u003c/p\u003e \u003cp\u003eFive pathogenic bacterial strains of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e DSMZ 20231, \u003cem\u003eSalmonella typhimurium\u003c/em\u003e ATCC 14028, \u003cem\u003eBacillus cereus\u003c/em\u003e DSMZ 345, \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e NRRL 800 and \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 69373 were used to evaluate antibacterial activity. These strains were obtained from the Microbial Culture Collection Center (Cairo MIRCEN), Faculty of Agriculture, Ain Shams University, Cairo, Egypt. These strains were maintained on Luria-Bertani agar (LB) medium [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMedia used\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCzapek's agar medium [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was used for cultivating \u003cem\u003eAspergillus tubingensis\u003c/em\u003e. It contains as follows (g/L): sucrose, 30.0; sodium nitrate, 2.0; dipotassium phosphate, 1.0; magnesium sulphate, 0.50; potassium chloride, 0.50; ferrous sulphate, 0.010 and agar, 20. Luria-Bertani agar medium [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] was used for pathogenic bacterial strains maintenance. This was contained (g/L): peptone, 10; yeast extract, 5; sodium chloride 5 and agar, 20. Mueller Hinton agar medium [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] was used for antibacterial activity assay, which contained (g/L): meat infusion, 6.0; casein hydrolysate, 7.5; starch, 1.5 and agar, 15. Vogel Johnson agar [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] for \u003cem\u003eStaphylococcus aureus\u003c/em\u003e enumeration. Its composition is as follows (g/L): Tryptone, 10; yeast extract, 5; Mannitol, 10; Dipotassium hydrogen phosphate, 5; Lithium chloride, 5; Glycine, 10; Phenol red, 0.025 and agar, 20. Before use, a sterile solution of 1% potassium tellurite was added to a sterile medium at a rate of 20 ml /L. \u003cem\u003eBacillus cereus\u003c/em\u003e base agar [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] for \u003cem\u003eBacillus cereus\u003c/em\u003e enumeration. Its composition is as follows (g/L): peptone, 1; mannitol, 10; sodium chloride, 2; magnesium sulphate, 0.1; disodium hydrogen phosphate, 2.5; potassium dihydrogen phosphate, 0.25; sodium pyruvate, 10; bromothymol blue, 0.12 and agar, 20. Before use, a sterile egg yolk solution was added to a sterile medium at a rate of 25 mL/ L. Salmonella Shigella (SS) agar [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] was used as a selective medium for detecting and enumerating \u003cem\u003eSalmonella\u003c/em\u003e spp. It contains as follows (g/L): protease peptone, 5; lactose, 10; bile salt, 8.5; sodium citrate, 8.5; sodium thiosulfate, 8.5; ferric sulfate, 1.0; brilliant green, 0.33; neutral red, 0.025; beef extract, 5.0 and agar, 15. King\u0026rsquo;s B medium [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] was used for enumerating \u003cem\u003ePseudomonas\u003c/em\u003e spp. was composed (g/L) of proteose peptone, 20; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 1.5; MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 1.5; glycerol, 10 ml and agar, 15. MacConkey agar medium (OXOID CM0115) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] was used in the cultivation of pathogenic bacteria of \u003cem\u003eEscherichia coli\u003c/em\u003e. The weight of the medium powder (51.5 g) was suspended in 1000 mL of distilled water and boiled for full dissolving. The medium was then dispensed in flasks and sterilized.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAll these media were adjusted to pH 7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 and autoclaved for 15 min at 121\u0026deg;C. The liquid media used in this study is the same as previously shown without adding agar. All media were sterilized at 121\u0026deg;C by autoclaving for 15 min.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSpore suspension as standard fungal inoculum preparation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe fungal strain was cultivated on Czapek's agar slants until sporulation for 48 h at 28\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. The spore suspension was prepared by adding 5 mL of sterile distilled water to the agar slant and gently scraping the surface of the culture with a sterile inoculation loop. The spore suspension from the slant was pooled to count using the Neubauer hemocytometer slide [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. One millilitre of spore suspension contained 1x 10\u003csup\u003e7\u003c/sup\u003e spores/ mL.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAgro-wastes and by-products\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSolid agro-waste such as beet waste, rice husk, rice straw, sawdust, and wheat bran, and liquid residues such as whey and blackstrap molasses were utilized in this study. As mentioned by Abd-Elhalem, El-Sawy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], the solid wastes were prepared by washing in cold water and then warm water, followed by drying overnight at 50\u0026deg;C, grounding and sieving to eliminate large particles. Blackstrap sugarcane molasses water-diluted by a ratio of 1:1 and pH value adjusted to be 4.0 using H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e (0.1 N), then heated at 100\u0026deg;C for 1 h and then neutralized with CaCO\u003csub\u003e3\u003c/sub\u003e and kept overnight to discard undesirable metals. The suspended solids and fibrous particles were removed by centrifugation (PRO-HOSPITAL.8) at 6,000 rpm for 15 min. This clarified molasses was stored at 4\u0026deg;C [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Whey acidity was adjusted to pH 4.5 and heated at 121\u0026deg;C for 15 min for denaturing proteins, then centrifuged at 10000 rpm for 15 min. Filtration for removing the precipitates, and then the supernatant was adjusted to pH 6.3 and maintained at 4\u0026deg;C [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The total carbon content of the solid materials of beet waste, rice husk, rice straw, sawdust, and wheat bran were 50.0, 48.3, 46.5, 37.8, and 39.3%, respectively, according to reports of the Central Laboratory, Horticulture Research Institute, Agriculture Research Center, Giza, Egypt [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Whereas, the blackstrap sugarcane molasses and whey were found their contain total sugar being 42.4% and 4.3%, respectively as previously determined by Abou-Taleb, Mashhoor [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFermentation processes for chitosan production\u003c/h2\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eSubmerged fermentation (SmF) technique\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIt was carried out in plugged Erlenmeyer flasks (250 mL) containing 50 mL of Czapek's broth medium in which sucrose was replaced by a similar concentration of each of the tested carbon sources (by-products of whey and blackstrap molasses) and inoculated with 3% (v/v) of the tested strains. The flasks were incubated on a rotary shaker (XuyMeu) at 28\u0026deg;C and 150 rpm of agitation speed [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Samples were drawn every 2 days under aseptic conditions. The collected pellicle samples were used to measure biomass and chitin and recovery of chitosan, as mentioned below.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSolid-state fermentation (SSF) technique\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSolid agro-industrial wastes were supplied for a solid-state fermentation medium with respect to heat transfer restrictions with suitable porosity and moisture in the solid substrate medium of fermentation. Dry substrates were weighed by 30 g in 500 mL Erlenmeyer flasks, and then the moisture content was adjusted (as the moisture content of 8\u0026ndash;10%, which is not enough for the growth of fungi) by adding Czapek's broth medium without sucrose (as a basal minerals medium) to 50%. The flasks were hand-shaken to homogenize the solid medium and autoclaved at 121\u0026deg;C for 20 min. One millilitre of spore suspension (1x10\u003csup\u003e7\u003c/sup\u003e spores/mL) was inoculated into the sterilized media with shaking to distribute the spores under sterilized conditions. The flasks were cotton-plugged and remained static during incubation for 12 days at 28\u0026deg;C [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Samples were drawn every 2 days under aseptic conditions. The collected mat samples were used to measure biomass and chitin and recovery of chitosan, as mentioned below.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eBiomass (cell dry weight) determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe cell dry weight was measured by harvesting the cells after filtration of the culture using filter paper (Whatman\u0026reg; qualitative filter paper, Grade 1, circles, diam. 45 mm, Sigma-Aldrich) and subsequent washing thrice with distilled water. The cells were dried at 80\u0026deg;C till constant weight was attained [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChitin and chitosan recovery\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eAccording to the method mentioned by Dhillon, Kaur [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], Maghsoodi and Yaghmaei [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], After the fungus cultivation, the culture was filtered through a Whatman No.1 to separate the biomass from the growth medium. The biomass cake was oven-dried at 55\u0026thinsp;\u0026minus;\u0026thinsp;50\u0026deg;C overnight, then weighed. Mix 1 g of the dried biomass with 30 mL of KOH (1N) with a ratio of 1:30, then stir vigorously for 15 min. Heat the mixture at 121\u0026deg;C for 20 min (Autoclaving), then centrifuge at 6000 rpm for 17 min to separate insoluble cell wall parts involving chitosan. The deposit was discarded and washed repeatedly with distilled water to neutralize the pH. The washed deposit was overnight oven-dried at 40\u0026deg;C. Mixed at 1:30 ratio with 30 ml of 2% acetic acid, then heated at 95\u0026deg;C for 6 h. Then centrifuge the mixture at 6000 rpm for 17 min, transfer the supernatant to mix with a 2N KOH at a volume ratio of 1: 1. Then centrifuge at 6000 rpm for 17 min. Discard the supernatant while keeping the precipitate to distilled water washing for pH neutralization. The last yellowish sediment was washed with a 1: 1 ethanol-acetone solution and oven-dried at 40\u0026deg;C overnight. The chitin and chitosan dry weighed were measured. The yields [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and chitosan productivity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] were calculated as follows:\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eChitin yield (Y) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(\\frac{\\text{C}\\text{h}\\text{i}\\text{t}\\text{i}\\text{n} \\text{d}\\text{r}\\text{y} \\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t} \\left(\\text{g}\\right)}{\\text{C}\\text{e}\\text{l}\\text{l} \\text{m}\\text{a}\\text{s}\\text{s} \\text{f}\\text{o}\\text{r}\\text{m}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\left(\\text{g}\\right)}\\right) \\text{x} 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eChitosan yield (Y) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(\\frac{\\text{C}\\text{h}\\text{i}\\text{t}\\text{o}\\text{s}\\text{a}\\text{n} \\text{d}\\text{r}\\text{y} \\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t} \\left(\\text{g}\\right)}{\\text{C}\\text{h}\\text{i}\\text{t}\\text{i}\\text{n} \\text{d}\\text{r}\\text{y} \\text{w}\\text{e}\\text{i}\\text{g}\\text{h}\\text{t} \\left(\\text{g}\\right)}\\right) \\text{x} 100\\)\u003c/span\u003e\u003c/span\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eChitosan productivity (g/d) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(\\frac{\\mathbf{C}\\mathbf{h}\\mathbf{i}\\mathbf{t}\\mathbf{o}\\mathbf{s}\\mathbf{a}\\mathbf{n} \\mathbf{d}\\mathbf{r}\\mathbf{y} \\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t} \\left(\\mathbf{g}\\right)}{\\mathbf{T}\\mathbf{i}\\mathbf{m}\\mathbf{e} \\left(\\mathbf{d}\\right)}\\right)\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFungal chitosan antibacterial activity\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003ePreparation of pathogenic bacterial inoculum\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe tested pathogenic bacterial inoculum was pre-cultured in Mueller Hinton broth medium for 24 h in a rotary shaker at 37\u0026deg;C until the growth gave turbidity of McFarland barium sulfate standard 0.5. The inoculum of each strain was standardized by measurement of the optical density using a spectrophotometer at 625 nm which ranged from 0.08\u0026ndash;0.12. the standard inoculum was adjusted at a concentration of 10\u003csup\u003e8\u003c/sup\u003e cells/mL [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChitosan antibacterial activity using the Kirby-Bauer disc diffusion method\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSeven trials of fungal chitosan ranging from 0.03 to 1.2% (prepared in 1.0% acetic acid at pH 5.5) were used to study their antibacterial activities on a solid medium using Kirby-Bauer disc diffusion method as standardized by the Clinical and Laboratory Standards Institute [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Plates containing Mueller Hinton agar medium were inoculated with a standardized inoculum of the test pathogenic bacteria (dip a sterile cotton swab into the suspension) by streak method. Then, the saturated filter paper discs (Filter Paper Whatman No. 1, 11 mm in diameter) with 10 \u0026micro;L of chitosan were placed on agar surfaces. Acetic acid (1.00%) and Ciprofloxacin (5.00 \u0026micro;g) were used as negative and positive controls, respectively. Plates were incubated at 37\u0026deg;C for 24 h. Then, the diameter of the inhibition zone was measured (mm).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChitosan antibacterial activity test by the inhibition of microbial growth in the broth medium\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eChitosan concentrations at 0.05 and 1.0% were added to Mueller Hinton broth medium in order to study their inhibition of pathogenic bacterial growth in the broth medium [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The propagation was carried out in Erlenmeyer flasks (250 mL in volume) containing 50 mL Mueller Hinton broth medium. Flasks were inoculated with 1 mL pathogenic bacterial standard inoculum and shaken on a rotary shaker (150 rpm) for 72 h at 37\u0026deg;C. About 5 ml were taken periodically every 12 h. The number of cell forming units (CFU/ mL) was determined using poured plate count on specific media for each pathogenic bacterial strain [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Plates were incubated at 37\u0026deg;C for 24 h and then enumerated for viable bacteria.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBeef burger preparation\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe beef burger was manufactured in the lab. condition according to the method of Jim\u0026eacute;nez-Colmenero [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The beef burger mixture contains (g): minced meat 226.875, onion juice 1.25, garlic 0.625, salt 5.0, spices 3.75, ice water 12.5, and 1.0% chitosan. The beef burgers were prepared using a manual-burger piston (15 cm diameter and 1.3 cm height) with about 150 g beef mixture for each slide. The uncooked and cooked samples were subsequently examined for chemical, physical, and sensory evaluation, as mentioned below.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eChemical analysis of uncooked and cooked beef burger\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe chemical analysis of the cooked samples was determined through the procedures described by Latimer and George [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] the total solids (TS), moisture, ash, fats, crude protein, and total carbohydrates were calculated using difference [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTotal carbohydrates = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left[\\mathbf{T}\\mathbf{o}\\mathbf{t}\\mathbf{a}\\mathbf{l} \\mathbf{S}\\mathbf{o}\\mathbf{l}\\mathbf{i}\\mathbf{d}\\mathbf{s} \\left(\\mathbf{T}\\mathbf{S}\\right) \u0026ndash; (\\mathbf{F}\\mathbf{a}\\mathbf{t}+ \\mathbf{P}\\mathbf{r}\\mathbf{o}\\mathbf{t}\\mathbf{e}\\mathbf{i}\\mathbf{n} + \\mathbf{A}\\mathbf{s}\\mathbf{h})\\right]\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCooked beef burger quality analysis\u003c/h2\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003ePhysical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTexture profile analysis (TPA) of cooked samples, the different texture characteristics (hardness, adhesiveness, cohesiveness, springiness, and gumminess) were measured at 23\u0026deg;C using an Instron Universal Testing Machine model 1195, Stable Micro System (SMS) Ltd., Godalming, UK, loaded with Dimension Software SMS software [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This analysis was performed at the Research Institute, Agriculture Research Center, Giza.\u003c/p\u003e \u003cp\u003eCooking loss was measured [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. A total of 20 g of beef burger samples was shaped to form a circular loop. The weight of burgers was determined before and after being cooked on an electric grill.\u003c/p\u003e \u003cp\u003eCooking loss (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(\\frac{\\mathbf{U}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t} \\left(\\mathbf{g}\\right)- \\mathbf{C}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t} \\left(\\mathbf{g}\\right)}{\\mathbf{U}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t} \\left(\\mathbf{g}\\right)}\\right) \\mathbf{x} 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe beef burger shrinkage (%) was measured [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. A difference between the uncooked burger diameter and cooked burger diameter was regarded as the percentage of shrinkage and calculated as follows:\u003c/p\u003e \u003cp\u003eShrinkage (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(\\frac{\\left(\\mathbf{D}\\mathbf{i}\\mathbf{a}\\mathbf{m}\\mathbf{e}\\mathbf{t}\\mathbf{e}\\mathbf{r} \\mathbf{o}\\mathbf{f} \\mathbf{u}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\right(\\mathbf{m}\\mathbf{m}) - \\mathbf{D}\\mathbf{i}\\mathbf{a}\\mathbf{m}\\mathbf{e}\\mathbf{t}\\mathbf{e}\\mathbf{r} \\mathbf{o}\\mathbf{f} \\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r}(\\mathbf{m}\\mathbf{m}\\left)\\right)}{\\mathbf{D}\\mathbf{i}\\mathbf{a}\\mathbf{m}\\mathbf{e}\\mathbf{t}\\mathbf{e}\\mathbf{r} \\mathbf{o}\\mathbf{f} \\mathbf{u}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\left(\\mathbf{m}\\mathbf{m}\\right)}\\right) \\mathbf{x} 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe reduction in beef burger thickness (RBT) was calculated as follows:\u003c/p\u003e \u003cp\u003eRBT (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(\\frac{\\mathbf{C}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\mathbf{t}\\mathbf{h}\\mathbf{i}\\mathbf{c}\\mathbf{k}\\mathbf{n}\\mathbf{e}\\mathbf{s}\\mathbf{s} - \\mathbf{U}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\mathbf{t}\\mathbf{h}\\mathbf{i}\\mathbf{c}\\mathbf{k}\\mathbf{n}\\mathbf{e}\\mathbf{s}\\mathbf{s})}{\\mathbf{C}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} \\mathbf{t}\\mathbf{h}\\mathbf{i}\\mathbf{c}\\mathbf{k}\\mathbf{n}\\mathbf{e}\\mathbf{s}\\mathbf{s}}\\right) \\mathbf{x} 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eChemical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe moisture retention (MR) value represents the amount of moisture retained in the cooked product per 100 g of sample and was determined [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] according to the equation below:\u003c/p\u003e \u003cp\u003eMoisture retention (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{\\mathbf{M}\\mathbf{o}\\mathbf{i}\\mathbf{s}\\mathbf{t}\\mathbf{u}\\mathbf{r}\\mathbf{e} \\mathbf{o}\\mathbf{f} \\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r} }{\\mathbf{M}\\mathbf{o}\\mathbf{i}\\mathbf{s}\\mathbf{t}\\mathbf{u}\\mathbf{r}\\mathbf{e} \\mathbf{o}\\mathbf{f} \\mathbf{u}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r}} \\mathbf{x} 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe fat retention (FR) value represents the amount of fat retained in the product after cooking. Fat retention was calculated [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] by using the equation as follows:\u003c/p\u003e \u003cp\u003eFR (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left(\\frac{\\mathbf{C}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t} \\left(\\mathbf{g}\\right) \\mathbf{X} \\mathbf{F}\\mathbf{a}\\mathbf{t} \\mathbf{i}\\mathbf{n} \\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r}\\left(\\mathbf{\\%}\\right)}{\\mathbf{U}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t} \\left(\\mathbf{g}\\right) \\mathbf{X} \\mathbf{F}\\mathbf{a}\\mathbf{t} \\mathbf{i}\\mathbf{n} \\mathbf{u}\\mathbf{n}\\mathbf{c}\\mathbf{o}\\mathbf{o}\\mathbf{k}\\mathbf{e}\\mathbf{d} \\mathbf{b}\\mathbf{e}\\mathbf{e}\\mathbf{f} \\mathbf{b}\\mathbf{u}\\mathbf{r}\\mathbf{g}\\mathbf{e}\\mathbf{r}\\left(\\mathbf{\\%}\\right)}\\right) \\mathbf{x} 100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSensory properties evaluation of beef burger\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn order to evaluate the consuming quality of the beef burger, sensory parameters analysis (appearance, texture, aroma, taste, and overall acceptability) was made. The evaluation was performed by 20 members (12 females and 8 males, with an age range between 10 and 61)) of the panel of graduate judges, post-graduate students and staff members of the Faculty of Agriculture, Ain Shams University and families in Egypt, using a 9-point hedonic scale from 0 (lowest) to 9 (highest) and the method according to Coda, Lanera [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The scorecard used for the Hedonic rating test, as previously designed [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStorage (Shelf-life) periods of beef burger\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe beef burger supplemented with fungal chitosan was maintained under freezing conditions for 8 weeks. The microbiological analysis was evaluated as mentioned below during zero, 4, 6, and 8 weeks storage periods.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMicrobiological evaluation of laboratory-manufactured beef burger\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLaboratory-manufactured beef burgers were microbiologically analyzed by aseptic transfer of 10 g of homogenized burger into 90 mL of peptone water (1.0%). Serial dilutions were prepared in the same diluents. Different microbial groups (\u003cem\u003eStaph. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e sp.) were enumerated using the poured plates technique [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] on specific media as previously stated. The plates were supplemented with the Vogel Johnson agar, \u003cem\u003eBacillus cereus\u003c/em\u003e agar base, Salmonella Shigella (SS) agar and Violet red bile (VRB) agar, respectively. after incubation at 37\u0026deg;C for 24 h the count of bacteria expressed as Log CFU/mL. As well as \u003cem\u003eSalmonella\u003c/em\u003e spp. of in laboratory beef burger samples were detected on Salmonella Shigella agar plates.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn triplicate, the experimental values were analyzed using analysis of variance (ANOVA) throughout the IBM\u0026reg; SPSS\u0026reg; Statistics software (version 19) and represented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard division of the mean. According to Starkings [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], the Duncan's Multiple Range Test at the 5% level was deemed significant; hence the significance of differences between means was determined at the 95% confidence level (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 value).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eChitosan production from \u003cem\u003eA. tubingensis\u003c/em\u003e using different fermentation\u003c/h2\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSubmerged fermentation (SmF)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe influence of liquid by-products whey and blackstrap molasses as carbon sources using the SmF approach vs. sucrose as control is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e reveals that after 8 days of incubation, the sucrose-containing control medium had the highest significant \u003cem\u003e(p\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) values of fungal biomass, chitin, and chitosan, with 1.52 g/L of cell dry weight, 0.26 g chitin/g biomass, and 0.053 g chitosan/g biomass, respectively. The incubation period was extended to 12 days in a medium supplemented with whey or blackstrap molasses, yielding 3.22 or 1.70 g cell dry weight / L medium, 0.78 or 0.41 g chitin/g biomass, and 0.28 or 0.12 g chitosan /g biomass, respectively. According to these results, whey by-product was preferred for chitosan synthesis from the tested strain over blackstrap molasses and sucrose (control), which increased approximately 1.3-fold and 5.3-fold, respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the calculated and displayed chitin and chitosan yields and chitosan productivity. After 12 days, the highest peaks of chitin and chitosan yields and chitosan productivity were recorded in a medium supplemented with whey (24.22%, 35.90%, and 0.023 g/L/d, respectively), which was higher than the results obtained in a medium supplemented with blackstrap molasses (24.12%, 29.27%, and 0.010 g/L/d, respectively). During 8 to 10 days of incubation, the maximum yield of chitin (17.11 and 17.22%) and chitosan (20.38 and 19.23%) with chitosan productivity of 0.0066 and 0.005 were obtained in the control medium containing sucrose.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCell mass dry weight, chitin, and chitosan production \u003cem\u003eVS.\u003c/em\u003e incubation periods for \u003cem\u003eA. tubingensis\u003c/em\u003e using agro by-products and wastes as a carbon source by submerged and solid-state fermentation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFermentation methods\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBy-Products\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c10\" namest=\"c4\"\u003e \u003cp\u003eFermentation time (days)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eand wastes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eSubmerged\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSucrose (Control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.45\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.07\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.52\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.23\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.13\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.24\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.20\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.006\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.014\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.053\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.050\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.043\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.037\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eWhey\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.26\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.73\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.19\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.70\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.39\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.21\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.78\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.53\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.009\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.06\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.10\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBlackstrap molasses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.13\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.21\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.35\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.68\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.66\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.002\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.007\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.41\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0001\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0006\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.003\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.010\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.046\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.100\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"14\" rowspan=\"15\"\u003e \u003cp\u003eSolid-state\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRice husk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.32\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.78c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.54a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.31b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.009\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.024d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.083c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.24a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.18b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0002\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0008\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.002d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.007c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.043a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.028b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRice straw\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.63e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.51d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.39\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.25a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.86b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.22c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.75a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.66ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0006f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.038d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.11\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.15a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.13b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eBeet waste\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.85f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.01e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.00d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.27b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.71c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.18e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.78c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.58a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.47b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.82c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.08d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.61a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.47ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.21c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSawdust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.29d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.35c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.62b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.38a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.35a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.004e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.007d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.02c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.05b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.21a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.20a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0000e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0002d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.001c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.009b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.027a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.025a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eWheat bran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCDW (g/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.81f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.89e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.28d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.23a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4.01b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.23c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitin DW (g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.14f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.34e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.61d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.26a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.13b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.84c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChitosan DW(g/ g biomass)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.015f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.061e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.14d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.36a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.32ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0.21c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003eCDW\u0026thinsp;=\u0026thinsp;cell dry weight, Chitin DW\u0026thinsp;=\u0026thinsp;chitin dry weight, Chitosan DW\u0026thinsp;=\u0026thinsp;chitosan dry weight and ND\u0026thinsp;=\u0026thinsp;Not detected. \u003csup\u003ea,b\u003c/sup\u003e The values in small letters in the same row with differing superscripts indicate a significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eSolid-state fermentation (SSF)\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe data in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that when using solid substrates (rice husk, rice straw, beet waste, sawdust, and wheat bran) through SSF, the values of biomass and chitin and chitosan yields were significantly increased with increasing fermentation periods up to 10 days, with the exception of presented beet waste and wheat bran up to 8 days. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows that beet waste and wheat brane had the highest significance in terms of biomass (4.77 and 4.23 g cell dry weight/100g substrate), chitin (1.58 and 1.26 g /g biomass), and chitosan (0.61 and 0.36 g /g/biomass), respectively, followed by rice straw (3.25 g cell dry weight /100 g substrate, 0.75 g chitin dry weight / g biomass, and 0.15 g chitosan dry weight /g biomass, respectively). While the lowest values of biomass (1.54 and 1.38 g cell dry weight /100 g substrate), chitin dry weight (0.24 and 0.21 g /g biomass), and chitosan dry weight (0.043 and 0.027 g /g biomass) were obtained on rice husk and sawdust after 10 days of incubation, respectively. The maximum percentage of chitin (33.12 and 29.79%) and chitosan (38.61 and 28.57%) yields and chitosan productivity (0.076 and 0.045 g/g/d) were achieved after 8 days on beet waste and wheat bran, respectively, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. On the other hand, sawdust waste had the lowest chitin and chitosan yields (15.22 and 12.86%) and chitosan productivity (0.0027 g/g/d). Based on preliminary data, it was important to emphasize that chitosan synthesis from \u003cem\u003eA. tubingensis\u003c/em\u003e using solid agro-industrial waste (beet waste) under the SSF approach was shown to be more advantageous than liquid by-products using the SmF technique. As a result, the beet waste was chosen for chitosan synthesis by the tested strain using SSF in the following investigations.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eFungal chitosan as an antibacterial agent\u003c/h2\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003eChitosan antibacterial activity using Kirby-Bauer disc diffusion method\u003c/h2\u003e \u003cp\u003eAs indicated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, seven concentrations of fungal chitosan (0.03 to 1.2%) were tested against Gram-positive and -negative bacteria compared to ciprofloxacin (the antibiotic applied as a positive control) and acetic acid (a chemical killer used as a negative control). The results showed no significant difference between chitosan at 1.0% and ciprofloxacin against all tested pathogenic strains except \u003cem\u003eB. cereus\u003c/em\u003e DSMZ 345, \u003cem\u003ePs. fluorescens\u003c/em\u003e NRRL 800, and \u003cem\u003eE. coli\u003c/em\u003e ATCC 69373. Ciprofloxacin inhibited the tested bacteria strongly, with inhibition zone diameters ranging from 35 to 49 mm. In contrast, acetic acid had a limited effect, resulting in zone diameters ranging from 7 to 10 mm. At 0.03%, chitosan did not affect the antibacterial agent against the tested pathogenic bacterial strains. Moreover, chitosan at 0.05 to 1.2% inhibited the growth of every tested pathogenic bacterial strain with varying inhibition zone diameters. For all tested pathogenic bacterial strains, the zone diameter increased with increasing concentration until reaching the maximum value at 1.00%. \u003cem\u003eStaph. aureus\u003c/em\u003e DSMZ 20231 exhibited the largest inhibition zone diameter, measuring 47 mm, but \u003cem\u003eB. cereus\u003c/em\u003e DSMZ 345 was more resistant to chitosan at 1.0% (the smallest zone\u0026thinsp;=\u0026thinsp;19 mm). The minimum inhibitory concentration (MIC) of chitosan on the solid culture technique was 0.05% against all tested pathogenic strains, with diameters ranging from 13 to 22 mm. Even though \u003cem\u003eB. cereus\u003c/em\u003e DSMZ 345 was more resistant to chitosan at the tested concentrations and had a MIC value of 0.2% (zone dimeter\u0026thinsp;=\u0026thinsp;15 mm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eFungal chitosan antimicrobial activity at various concentrations against Gram-positive and negative bacterial strains\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eAcetic acid (1.00%) is a negative control, and ciprofloxacin (5.00 μg) is a positive control. \u003csup\u003ea,b\u003c/sup\u003e The values in small letters in the same column with differing superscripts indicate a significant difference (p ≤ 0.05), ± Standard division.\u003c/p\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eChitosan antibacterial activity test by the inhibition of microbial growth in the broth medium\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that adding chitosan (0.05 and 1.00%) to growth media reduced the growth of tested strains during the first 12 hours of growth. All tested strains' growth increased gradually throughout incubation, peaking after 48 hours. Chitosan concentrations of 0.05% and 1.00% reduced growth at rates of 0.23 to 0.49 CFU/ h and 0.29 to 0.56 CFU/ h, respectively. Chitosan at 1% recorded higher growth reduction rate values, with \u003cem\u003eS. Typhimurium\u003c/em\u003e ATCC 14028 having the highest value. Additionally, after 24 h of incubation with both chitosan concentrations, all tested pathogenic strains exhibited complete inhibition. On the contrary, \u003cem\u003eB. cereus\u003c/em\u003e DSMZ 345 was the most stable bacterium for chitosan inhibition effect. Both 0.05 and 1.00% chitosan did not inhibit the growth of \u003cem\u003eB. cereus\u003c/em\u003e DSMZ 345 during the incubation period (48 h).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eApplication of fungal chitosan for laboratory-manufactured beef burger\u003c/h2\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eChemical analysis of uncooked and cooked beef burger\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe chemical composition of uncooked and cooked beef burgers is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Moisture, ash, fat, protein, TS and TC content of uncooked samples ranged between 60.40\u0026ndash;61.00%, 1.36\u0026ndash;1.38%, 20.10\u0026ndash;20.00%, 15.54\u0026ndash;15.56%, 39.60\u0026ndash;39.00%, and 2.60\u0026ndash;2.10%, respectively. The contents for cooked beef burgers were in the range of 58.86\u0026ndash;59.60%, 1.33\u0026ndash;1.38%, 18.67\u0026ndash;19.89%, 15.42\u0026ndash;15.49%, 41.14\u0026ndash;40.40%, and 5.70\u0026ndash;3.60%, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChemical analysis of uncooked and cooked beef burger\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e \u003cp\u003eChemical analysis (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMoisture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eFat\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eUncooked\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.40\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.36\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e39.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eChitosan 1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20.00\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e39.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCooked\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58.86\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.67\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e41.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e5.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eChitosan 1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e19.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e40.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eTS, Total solids; TC, Total carbohydrates. \u003csup\u003ea,b\u003c/sup\u003e The values in small letters in the same column with differing superscripts indicate a significant difference (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eBeef burger quality analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe quality analysis of beef burgers including texture, physical (cooking) and chemical properties, has been presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The texture properties of hardness, Adhesiveness, cohesiveness, springiness, gumminess and chewiness of the control and treated with chitosan samples were between 204.6-225.2 mm, 175.31-200.66 mJ, 0.47\u0026thinsp;\u0026minus;\u0026thinsp;0.43, 1.19\u0026ndash;1.46 mm, 96.3\u0026ndash;96.6 N, and 114.23-140.99 mJ, respectively. All parameters in a sample treated with chitosan had higher values than the control except the cohesiveness ratio was slightly decreased. The cooking (physical) properties of cooking loss, shrinkage and RBT of the control and treated with chitosan samples were between 18.02\u0026ndash;16.8%, 14.3\u0026ndash;10.6%, and 32.3\u0026ndash;29.1%, respectively. The chemical properties of moisture retention and fat retention of the control and treated chitosan samples were between 97.45\u0026ndash;97.70%, and 92.89\u0026ndash;99.45%, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCooked beef burger quality (texture, physical and chemical) analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatments\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eTexture analysis\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHardness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAdhesiveness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCohesiveness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSpringiness\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGumminess\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eChewiness\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(mJ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e(mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e(N)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(mJ)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e204.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e175.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e114.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan 1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e225.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e140.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003ePhysical analysis (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCooking loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShrinkage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRBT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e18.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan 1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e16.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e29.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eChemical analysis (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMoisture retention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFat retention\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e97.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan 1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e97.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eRBT\u0026thinsp;=\u0026thinsp;Reduction in Beef Burger Thickness.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eSensory properties evaluation of beef burger\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAbout 20 of varied ages panelists were requested to evaluate the cooking laboratory-manufactured beef burger with 0 (control non-treated with chitosan) and 1.0% chitosan. These treatments' consumer panel data were analyzed and presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Addition of 1.0% chitosan affected significantly appearance (7.9), aroma (8.2), color (8), taste (8.1), taste (8.1), and overall acceptability (8.1). Whereas the beef burger without chitosan (control) showed lower values and recorded significant differences with beef burger supplemented with 1% chitosan.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eStorage (Shelf-life) periods of beef burger\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll manufactured beef burgers treated with 0% chitosan (control non-treated) or 1.0% chitosan (treated) were stored in frozen condition for 8 weeks in order to study their efficiency against pathogenic bacteria. Data presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e exposed that all fresh burger samples (control and treated with 1.0% chitosan) at zero time were highly contaminated with the tested pathogenic bacteria. \u003cem\u003eE. coli\u003c/em\u003e achieved the uppermost values of 4.2 and 4.21 Log CFU/g at 0 and 1.0% chitosan, respectively, followed by \u003cem\u003ePseudomonas\u003c/em\u003e spp. and \u003cem\u003eStaph. aureus\u003c/em\u003e of 3.45 and 3.40 Log CFU/ g, and 3.30 and 3.28 Log CFU/g, respectively. In addition, \u003cem\u003eSalmonella\u003c/em\u003e ssp. growth on Salmonella Shigella agar plates at zero time only, but no growth occurred during the storage periods. Results also indicated that the number of pathogenic bacteria significantly decreased with an increase in the storage periods up to 8 weeks. The reduced number of the tested pathogenic bacteria in treated samples with 1.0% chitosan was more significant than in the control sample with 0% chitosan. Chitosan reduces the growth of pathogenic bacteria with diverse levels. \u003cem\u003eE. coli\u003c/em\u003e was the most susceptible to chitosan, which achieved the highest growth reduction percentage (78.1%), followed by \u003cem\u003ePseudomonas\u003c/em\u003e spp. and \u003cem\u003eStaph. aureus\u003c/em\u003e being 70.6% for the former and 66.76% for the latter by the end of the storage period. All beef burger without chitosan shows attenuation in a bacterial count throughout the storage period under a frozen state to reach the minimum after 8 weeks. The percentage of growth reduction was 18.8%, 24.63%, and 28.18% for \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e spp., and \u003cem\u003eStaph. aureus\u003c/em\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMicrobiological evaluation of laboratory-manufactured beef burgers during storage periods\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStorage period weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c8\" namest=\"c3\"\u003e \u003cp\u003eLog count of bacteria (CFU / g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDetection of\u003c/p\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eStaph. aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% growth reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e% growth reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e% growth reduction\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eNon-Treatment\u003c/p\u003e \u003cp\u003e(control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eCf\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ecF\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003edG\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ebE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003ebE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ebcE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003enil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ebE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ebE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003ebE\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003enil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eaD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eaD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eaD\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003enil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTreatment with 1.0% chitosan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003csup\u003edF\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003edF\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003edF\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e53.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e59.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ecC\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e44.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003enil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ebAB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e63.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e73.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ebB\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e57.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003enil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003eAa\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e78.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eaA\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e70.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003enil\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003e% growth reduction\u0026thinsp;=\u0026thinsp;Xt-X0*100/ X0, + growth,\u0026minus; no growth. \u003csup\u003ea,b\u003c/sup\u003e The values in small letters in the same column with differing superscripts indicate a significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) between each treatment individually with a microbe, \u003csup\u003eA,B\u003c/sup\u003e The values in capital letters in the same column with differing superscripts indicate a significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) between non-treatment and treatment with 1.0% chitosan, \u003cem\u003e\u0026plusmn;\u003c/em\u003e Standard division.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eChitosan is an amino polysaccharide, and it is commercially produced from chitin by alkali deacetylation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. It was assumed chitosan production using fungi is more favourable than the crustaceans as they are not available with high-cost chitosan purification steps. Many Fungi strains were investigated for chitosan production utilizing agro-wastes and by-products in a trial to minimize production costs and, in the same line, increase chitosan production. For this target, they used wheat straw to cultivate \u003cem\u003eLentinus edodes\u003c/em\u003e and obtained a chitosan production yield of about 50 mg/g cell [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Chitosan production with \u003cem\u003eAbsidia coerulea\u003c/em\u003e on potato peels [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Also, they used apple pomace for chitosan production by \u003cem\u003eGongronella butleri\u003c/em\u003e, which provided a high yield of about 217 mg/g cells [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The chitosan production from \u003cem\u003eAgaricus\u003c/em\u003e sp., \u003cem\u003ePleurotus\u003c/em\u003e sp., and \u003cem\u003eGanoderma\u003c/em\u003e sp. using sawdust and rice straw agro-wastes [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. A chitosan production from \u003cem\u003eR. arrhizus\u003c/em\u003e using corn-steep liquor with a yield of 29.3 mg/g cells [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The cultivated \u003cem\u003eA. terreus\u003c/em\u003e on apple waste extract, produced a biomass concentration of 30 g/L and chitosan of 0.14 g/g cells after 5 days of incubation [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In the current study, agro-wastes and by-products were utilized with two different techniques SSF and SMF, for chitosan production by \u003cem\u003eA. tubingensis\u003c/em\u003e. For the SMF technique, whey and blackstrap sugarcane molasses were the utilized by-products, and it was found that whey was the best source compared to blackstrap molasses. Whey was responsible for the fungal cell dry weight and chitosan increase by 1.89-fold and 2.33-fold, respectively, after 12 days of incubation. For the SSF approach, the beet waste followed by wheat bran were the best sources as the fungal cell dry weight was increased by 3.14-fold and 2.78-fold, and chitosan by 9.81-fold and 6.79-fold, respectively after 8 days of incubation when compared with control in SMF of sucrose. This SSF approach is assumed to be better than the SMF for chitosan production by \u003cem\u003eA. tubingensis\u003c/em\u003e. In agreement with these findings, Maghsoodi and Yaghmaei [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] reported that the yield of chitosan using SSF (w/w) was higher than that of SMF (w/v) owing to that the filamentous fungi are most exploited because of their ability to grow on the complete solid substrate while the low amount of mycelia produced in SMF, so there has been considerable interest in producing chitosan using the SSF process. In the same way, the quality and amount of chitosan extracted from the fungal mycelia depend on the fungal cultivation method, as shaking had deleterious effects [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In particular, shear forces can damage conidiophores leading to decreased spore production. Beet waste was the compromised source for \u003cem\u003eA. tubingensis\u003c/em\u003e chitosan production with SSF compared to sucrose (control).\u003c/p\u003e\u003cp\u003eChitosan was examined as an antimicrobial agent with a broad spectrum, affecting Gram-positive and negative bacterial pathogens. Chitosan is a bioactive compound with bactericidal or bacteriostatic effects, often with no diversity between activities [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Data indicated that fungal chitosan had a positive impact on Gram-positive because it had a mode of action related to the electrostatic reaction that occurs between the positive charge of chitosan and the negative charge of the bacterial cell wall [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The cell wall of Gram-positive bacteria had a wall of teichoic acids and lipoteichoic acids in peptidoglycan layers. Both compounds had a negatively charged anionic backbone [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The teichoic acids can provide arranged uniform high-density negative charges in the cell wall, thereby inhibiting the passage of ions across the membrane. The positively charged chitosan interacts electrostatically with the negatively charged teichoic acid in peptidoglycans, leading to the destruction of the cell membrane, leakage of intracellular components and the entrance of chitosan into the microbial cells [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] or the leakage of proteins and other intracellular constituents by chitosan [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Other studies exhibited the electrostatic reaction promoting changes in the properties of membrane wall permeability, so it motivates internal osmotic imbalances and thus prevents the growth of microorganisms [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The surface of Gram-negative bacteria comprises negative charges from the phosphate and pyrophosphate groups of lipopolysaccharide in the outer layer of the outer membrane. Lipopolysaccharide gave a high negative charge due to neutralization by positive charges from chitosan, resulting in disruption of the outer membrane., enabling chitosan to penetrate the cell membrane, thus leading to bacterial cell death [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Many researchers stated that increasing chitosan concentrations enlarges the zone of inhibition of tested pathogens such as \u003cem\u003eStaph. aureus, S. Typimurium\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e. Collected data proved that Gram-positive bacteria were more sensitive to chitosan than Gram-negative bacteria [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Chitosan was more active against Gram-positive bacteria such as \u003cem\u003eBacillus\u003c/em\u003e spp., \u003cem\u003eListeria monocytogenes\u003c/em\u003e, \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e and \u003cem\u003eStaph. aureus\u003c/em\u003e (MERS strain) as found in another research [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. MIC of chitosan varied according to the tasted microorganism as well as the chitosan futures. In some studies, the minimum inhibitory concentration (MIC) of chitosan ranged from 0.05 to 0.1% according to the bacterial strain [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The chitosan nanoform lowered the MIC about 21\u0026ndash;41 times against \u003cem\u003eE. coli\u003c/em\u003e 25922, \u003cem\u003eS. choleraesuis\u003c/em\u003e ATCC 50020, and \u003cem\u003eStaph. aureus\u003c/em\u003e 25923[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Both chitosan and trimethyl-chitosan (TMC) affected the growth of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eStaph. aureus\u003c/em\u003e and reduced the maximum absorbance (at 620 nm) after 12 h incubation for \u003cem\u003eE. coli\u003c/em\u003e using 0.5-2.0 g/L chitosan or TMC. The same trend was observed with \u003cem\u003eStaph. aureus\u003c/em\u003e using 1.0% chitosan. Moreover, increasing chitosan concentration did not significantly drop bacterial growth [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Chitosan in low concentration binds to the bacterial cell wall's negative charge and causes cell membrane injury, leading to cell death. In contrast, the protonated chitosan's high concentration may coat the cell surface and avoid intracellular leakage. In addition, the negative charge of the cell wall varied, rendering the growth phase and affecting the bacterial susceptibility to chitosan. \u003cem\u003eE. coli\u003c/em\u003e O157-H7 and \u003cem\u003eStaph. aureus\u003c/em\u003e CCRC12657 cells in the middle of exponential growth (after 10 h of growth) were more sensitive to chitosan than cells in stationary growth [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMinced meat and beef burgers are semi-perishable, rich in nutrients and moisture, supporting microbial growth and food spoilage agents. Microbial growth caused an undesirable change in food odder, texture, colour, and chemicals and restricted product shelf-life [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The grown need a bioactive compound with antibacterial activity such as chitosan, biodegradable, non-toxic, edible, and effective against various spoilage bacteria and molds [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Different studies stated that chitosan had a strong antibacterial effect against pathogenic and spoilage bacteria in meat products by destroying the cell membrane's permeability leading to bacterial death [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] Venkatachalam and Lekjing [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] reported that chitosan reduced lactic acid bacteria (LAB), psychrophilic group and \u003cem\u003eEnterobacteriaceae\u003c/em\u003e count in pork patties, and their efficiency augmented when chitosan was supplemented with clove oil and nisin also, the shelf-life of pork patties increased twofold by using chitosan and mixture of chitosan, clove oil, and nisin in pork patties. Significant effect of 1.5% chitosan and 2% thyme oil mixture against LAB, \u003cem\u003eEnterobacteriaceae, Pseudomonas\u003c/em\u003e spp., and molds during storage under modified atmosphere packaging. The mix also extended the shelf life of chicken-pepper kebab to reach 14 days instead of 6 days in chicken-pepper kebab without chitosan and thyme oil mixture [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. In addition to the efficiency of chitosan as an antimicrobial in meat and meat products, chitosan enhanced the quality of colour, overall acceptability, odder and texture of meat products[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. The shelf life of 1% chitosan with fresh pork sausages increased from 7 to 15 days at a chilled temperature [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. The effects of chitosan films on pork sausage microbial count, demonstrating a significant (p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) antimicrobial impact of the films against molds and yeasts, lactic acid bacteria, and the total mesophilic viable count (TMVC) [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eResults indicated that the moisture values had no significant differences for uncooked treatments but they decreased in cooked treatments, which might be due to the increase in solid material content [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Chitosan beef burgers showed higher significance in ash values than control, it might be due to a high mineral content in chitosan beef. Fish burgers formulated with mashed pumpkin and mashed potato showed higher moisture and lower protein, fat and ash contents than the control groups [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Cooked beef patties with added PM showed higher ash content compared to C samples (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), probably due to the high mineral content of the pumpkin mix. The protein values in all treatments had no significant differences in both uncooked and cooked samples [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The fat values were highly significant in uncooked samples and slightly decreased in cooked samples due to heat treatment using an electric grill due to melting the fat in cooked samples. All parameters of texture (hardness, Adhesiveness, springiness, gumminess and chewiness) in a sample treated with chitosan had higher values than the control except the cohesiveness ratio was slightly decreased. The hardness and chewiness values of pork burgers formulated with albedo-fibre powder were increased with the increasing fibre amount, while springiness and cohesiveness of the samples did not show significant differences and gumminess was increased with only the addition of 5% powder [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. The textural parameters of the products could show differences according to the natural structure the amount of the non-meat ingredient and the amount of replaced meat in the formulation[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Results clearly showed that the cooking weight loss and shrinkage of samples decreased in samples supplemented with 1.0% chitosan than in control samples. It might be due to chitosan demonstrating barrier capabilities to prevent water removal during the cooking process by making water molecular hydrogen bonds that make them more effective in avoiding weight loss in the final product[\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData could be summarized that fungal polymer chitosan was produced from \u003cem\u003eA. tubingensis\u003c/em\u003e on beet waste as the only carbon source during solid-state fermentation after 8 days of incubation, achieving 32.91%. At a polymer concentration of 1.0%, this polymer inhibited \u003cem\u003eStaph. aureus\u003c/em\u003e DSMZ 20231, \u003cem\u003eS. Typhimurium\u003c/em\u003e ATCC 14028, \u003cem\u003eB. cereus\u003c/em\u003e DSMZ 345, \u003cem\u003ePs. fluorescens\u003c/em\u003e NRRL 800, and \u003cem\u003eE. coli\u003c/em\u003e ATCC 69373. The fungal polymer was applied in the food industry as a food additive on a laboratory scale for the preservation and extension of the shelf life of beef burgers to 8 weeks. In addition, adding polymer to beef burgers improved the physio-chemical and sensory features of the product. As a consequence, future studies will focus on applying the fungal polymer chitosan as a food-packing ingredient.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the Department of Agricultural Microbiology, Faculty of Agriculture, Ain Shams University, Cairo and Food Technology Research Institute, Agriculture Research Center, Giza, Egypt, for supporting the current experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Kh.A.A., R.F.A., B.T.A., D.Y.A. and S.A.A.; methodology, Kh.A.A., R.F.A., B.T.A, D.Y.A and Sh.A.A.; software, Kh.A.A. and B.T.A.; validation, R.F.A., Sh.A.A. and D.Y.A.; formal analysis, Kh.A.A., R.F.A.,B.T.A., D.Y.A. and Sh.A.A.; investigation, Kh.A.A.; resources, Sh.A.A. and D.Y.A.; data curation, Kh.A.A., R.F.A., B.T.A.; writing\u0026mdash;original draft preparation, Kh.A.A., R.F.A., B.T.A, D.Y.A and Sh.A.A.; writing\u0026mdash;review and editing, Kh.A.A., R.F.A., B.T.A, D.Y.A and Sh.A.A.; All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo fund.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors claim to have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSilva MM, Lidon F. Food preservatives\u0026ndash;An overview on applications and side effects. Emir J Food Agric. 2016:366-73.https://doi.org/10.9755/ejfa.2016-04-351\u003c/li\u003e\n\u003cli\u003eJavanmardi F, Rahmani J, Ghiasi F, Hashemi Gahruie H, Mousavi Khaneghah A. The association between the preservative agents in foods and the risk of breast cancer. Nutr Cancer. 2019;71(8):1229-40.https://doi.org/10.1080/01635581.2019.1608266\u003c/li\u003e\n\u003cli\u003eBakhtiary F, Sayevand HR, Mousavi Khaneghah A, Haslberger AG, Hosseini H. Antibacterial efficacy of essential oils and sodium nitrite in vacuum processed beef fillet. Appl Food Biotechnol. 2018;5(1):1-10.https://doi.org/10.22037/afb.v5i1.17118\u003c/li\u003e\n\u003cli\u003eJafarzadeh S, Jafari SM, Salehabadi A, Nafchi AM, Kumar USU, Khalil HA. Biodegradable green packaging with antimicrobial functions based on the bioactive compounds from tropical plants and their by-products. 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Incorporation of low molecular weight chitosan in a low-fat beef burger: Assessment of technological quality and oxidative stability. Foods. 2021;10(8):1959.https://doi.org/10.3390/foods10081959\u003c/li\u003e\n\u003cli\u003edo Amaral DS, Cardelle-Cobas A, do Nascimento BM, Monteiro MJ, Madruga MS, Pintado MME. Development of a low fat fresh pork sausage based on chitosan with health claims: Impact on the quality, functionality and shelf-life. Food Funct. 2015;6(8):2768-78.https://doi.org/10.1039/C5FO00303B\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antibacterial activity, Aspergillus tubingensis, Bacterial pathogens, Beef burger, Biochitosan, Food preservatives, Shelf-life, Solid-state fermentation, Submerged fermentation","lastPublishedDoi":"10.21203/rs.3.rs-4397361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4397361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChitosan is commonly obtained by deacetylation of chitin from crustacean shell wastes such as shrimp and crab, but unfortunately, these sources appear limited for their unavailability. So, an alternative abundant chitin source is in need as fungi mycelia.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, chitosan production through submerged (SMF) and solid-state fermentation (SSF) of \u003cem\u003eAspergillus tubingensis\u003c/em\u003e utilizing various agro-wastes and by-products like whey, blackstrap molasses, beet waste, rice (husk\u0026amp; straw), wheat bran and sawdust as a sole carbon source was investigated. The best waste was beet waste (33.12% and 38.61% of chitin and chitosan yields for SSF and whey (whey (24.22% and 35.90% of chitin and chitosan yields) for SmF, as compared to the control of sucrose (17.11% and 20.38% of chitin and chitosan yields) after 8\u0026ndash;12 and 8 days of incubation, respectively. The fungal polymer had antibacterial activity against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e DSMZ20231,\u003cem\u003eSalmonella Typhimurium\u003c/em\u003e ATCC14028,\u003cem\u003eBacillus cereus\u003c/em\u003e DSMZ345, \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e NRRL800, and \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC69373 at a significant concentration of 1.0% were assayed using Kirby-Bauer disc diffusion method. \u003cem\u003eStaph. aureus\u003c/em\u003e DSMZ20231\u0026amp; \u003cem\u003eS. Typhimurium\u003c/em\u003e ATCC14028 were more susceptible to chitosan, which gave the largest zone inhibition of 47 and 43 mm, respectively. Moreover, the polymer's antibacterial activity was tested in the broth medium during different incubation periods (12-48h). results indicated that all tested pathogenic strains exhibited complete inhibition after 24 h incubation except \u003cem\u003eB. cereus\u003c/em\u003e DSMZ345 was the most stable up to 48h. Application of fungal chitosan (1.0%) for a laboratory-manufactured beef burger led to improve quality analysis of the texture, physical or technological and chemical properties than control without chitosan. At the same time, beef burger supplemented with chitosan was significantly accepted overall by the panelist than the control. Therefore, fungal chitosan extended the beef burger's shelf life and maintained its quality indices during freezing storage.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eFungal chitosan is a beneficial natural antimicrobial, and applying it to beef burgers improves microbiological, technological, and chemical quality while also extending shelf life by up to 8 weeks, making it a viable alternative to chemical protective additives. Future research on this fungal chitosan will focus on large-scale meat or poultry preservation or additive applications.\u003c/p\u003e","manuscriptTitle":"Chitosan production from agro-wastes by Aspergillus tubingensis: A sustainable approach for beef burger quality improvement","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 11:38:44","doi":"10.21203/rs.3.rs-4397361/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0e9ba8c-6041-481f-9dad-020eabbe1571","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-15T09:59:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-18 11:38:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4397361","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4397361","identity":"rs-4397361","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}