Biosynthesis of Lactic Acid by Mutagenetic Lactic Acid Bacteria Strains Isolated From Processed Milk

Biosynthesis of Lactic Acid by Mutagenetic Lactic Acid Bacteria Strains Isolated From Processed Milk | 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 Biosynthesis of Lactic Acid by Mutagenetic Lactic Acid Bacteria Strains Isolated From Processed Milk Michael Bamitale OSHO, Precious Jesuferanmi AWOSILE, Gbemisola Mary ALABA This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3963690/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 Lactic acid, a valuable weak natural organic acid extensively utilized in various industries has been conventionally synthesized through chemical processes. However, the growing demand for sustainable and eco-friendly production methods has stimulated interest in microbial synthesis. The study was to explore the potential of multi-strain lactic acid bacteria isolated from processed liquid milk to synthesize lactic acid. Microorganisms were isolated and screened on deMan Rogosa Sharpe (MRS) agar and were identified according to morphological and biochemical characteristics. Mutagenesis of positive isolate using chemical and physical means, quantitative and qualitative production using High-Performance Liquid Chromatography (HPLC) were investigated. The isolate was discovered to have a clear zone on MRS agar, which allowed for its identification as Lactobacillus sp. It was then chosen for mutagenesis using ethidium bromide (EB) and UV light. Thirteen mutants were identified, and three of those were examined to see if they could generate lactic acid using various substrates. HPLC confirmed mutants and parent strain to produce significant homo-fermentative lactic acid in cheese whey substrate. The parent strain gave a significant yield of 2004.87ugml -1 as compared to UV and EB mutant strains with 1457.67ugml -1 and 239.10ugml -1 respectively. Optimum lactic acid yields were produced at 37°C, pH 4.5, and 150 rpm 16 h fermentation period. This study showed that mutagenesis did not influence optimum lactic acid production. The yield improvement that occurred via mutations might have diverted the metabolism from lactic acid production towards mixed acid fermentation, hence produce reduced levels of lactic acid. Applied & Industrial Microbiology Lactic Acid Synthesis Mutagenetic LAB Strains Processed Milk Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Lactic acid (2-hydroxypropanoic acid, CH 3 -CH(OH)-COOH), a valuable organic acid, has gained significant attention for its various industrial applications. Traditionally, lactic acid has been produced through chemical processes, which are associated with environmental concerns and limited sustainability. As a result, there is a growing interest in microbial synthesis of lactic acid using lactic acid bacteria (LAB) (Sun et al ., 2017). Lactic acid bacteria are known for their ability to convert sugars into lactic acid through fermentation. Isolation and screening of LAB from different sources, including bovine milk, have been carried out to identify strains with high lactic acid production capabilities. Bovine milk is a promising source of LAB due to its abundance and the natural presence of lactic acid bacteria which possess the unique capability to convert sugars into lactic acid through fermentation (Sun et al ., 2017; Johansen et al ., 2020). Genetic engineering techniques, such as targeted gene disruption and gene knockout, have been employed to introduce specific genetic modifications in LAB. These techniques utilize recombinant DNA technology to manipulate LAB genomes. For instance, the disruption of lactate dehydrogenase genes using genetic engineering tools has been reported to enhance lactic acid production in LAB strains (Zhang et al ., 2022). In summary, mutagenesis in LAB involves the use of chemical mutagens, physical mutagens, and genetic engineering techniques to induce genetic mutations and improve lactic acid production. These approaches have been successfully employed to modify LAB strains and enhance their capabilities in various industrial applications. Genetic engineering techniques have been employed to enhance the metabolic efficiency of lactic acid bacteria for increased lactic acid production. Genetic modifications targeting key enzymes and metabolic pathways involved in lactic acid synthesis have shown promising results. These modifications can lead to improved substrate utilization and lactic acid yield (Yadav et al ., 2021). Multi-strain lactic acid bacteria consortia have demonstrated the potential for enhanced lactic acid production. Chen et al . (2019) investigated the utilization of strain combinations, including Lactobacillus plantarum , for effective utilization of rice straw hydrolysates and observed improved lactic acid production compared to individual strains. The selection and improvement of multi-strain lactic acid bacteria involve various strategies, such as genetic engineering, strain combination, and optimization of fermentation conditions. Genetic engineering techniques can be employed to enhance the metabolic pathways of LAB, thereby increasing lactic acid production (Yadav et al ., 2021). Combining different strains of LAB can lead to synergistic effects, resulting in higher productivity (Chen et a l., 2019). Additionally, optimizing fermentation conditions, including temperature, pH, and nutrient availability, can further enhance lactic acid synthesis (Dong et al ., 2018). Optimization of fermentation conditions is crucial for maximizing lactic acid yield. Factors such as temperature, pH, and nutrient availability play significant roles in the efficiency of lactic acid fermentation. Dong et al ., (2018) optimized fermentation conditions for lactic acid production from Jerusalem artichoke tuber hydrolysate by Lactobacillus paracasei LA104, resulting in increased lactic acid yield. The industrial applications of lactic acid have further emphasized the need for sustainable and efficient production methods. Lactic acid serves as a food additive, pH regulator, and precursor for biodegradable polymers. Nguyen et al ., (2022) highlighted the industrial applications of lactic acid and the importance of optimizing fermentation conditions for improved production. Overall, the literature demonstrates the significance of microbial synthesis of lactic acid using LAB. Isolation of LAB from bovine milk, genetic engineering techniques, utilization of multi-strain consortia, and optimization of fermentation conditions all contribute to improving lactic acid production. These approaches promote sustainable and eco-friendly production methods and offer practical solutions for industrial-scale lactic acid production. The study on microbial synthesis of lactic acid by improved multi-strain lactic acid bacteria from processed liquid milk is justified by its potential to provide sustainable and environmentally friendly methods for lactic acid production, optimize resource utilization, improve process efficiency and yield, and cater to industrial applications. The most effective technique to obtain genetically stable and practical strains of microbes for industrially significant products has always been to isolate and screen them from natural sources. Furthermore, because fermentation uses renewable raw resources as opposed to petrochemicals, it is becoming a more significant activity. The findings from this study contributed to advancing the field of microbial synthesis and provide practical solutions for the production of lactic acid. The objective of this study was to explore the potential of multi-strain lactic acid bacteria isolated from processed liquid milk and screened isolates for desirable traits and production of pure isomeric form of lactic acid. Processed liquid milk serves as an abundant source of LAB, and these bacteria have evolved to efficiently ferment lactose into lactic acid. By enhancing the performance of these natural lactic acid producers, overall lactic acid production yield and efficiency can be improved (Johansen et al ., 2020). Materials And Methods SAMPLE COLLECTION: Three brands of commercial evaporated tin milk samples were obtained from supermarket for the study and composite of these samples was used. For the production of lactic acid, two fermentation broth, corn steep liquor and cheese whey were obtained commercially from Iwo Road market in Ibadan North West Local Government, Oyo State. ISOLATION AND SCREENING OF LAB For the enrichment and isolation of LAB, aliquots of the commercial milk was serially diluted to a dilution factor of 10 − 6 and 0.1ml was then spread evenly on MRS agar plates, LAPT medium and MacConkey agar plates. The plates were incubated for 44 h at 37 ˚C. For those that developed growth on the agar plates, they were then sub-cultured into MRS agar plates, LAPT medium and MacConkey agar plates to obtain a pure culture and then incubated for 24 h at 37 o C. It was then kept on nutrient agar slant. Screening of LAB was carried out as bacterial colonies were picked and streaked on fresh MRS agar plates and supplemented with sodium azide (0.02 gl -1 ), bromocresol purple (0.012 gL -1 ) and 2% (w/v) sucrose, placed in anaerobic jar and then incubated for 44 h at 37 o C (Yoganand et al., 2012 ). PHYSICAL & CHEMICAL MUTAGENS Bacteria in the logarithmic growth phase (17 h culture) were diluted and plated onto MRS agar supplemented with 4 g of sucrose and 0.012 g of bromocresol purple. For 30, 60, and 90 s, UV light at a distance of 2 cm was applied to the Petri plates. There was also a control group that included 10 − 1 , 10 − 2 , and 10 − 3 dilution cultures in it, but they were not subjected to UV light. All cultures were incubated at 37 ˚C for 60 h while being completely darkened by wrapping in aluminium foil. A modified version of the Gawel et al., ( 2002 ) protocol was employed. The logarithmic growth phase (17 h culture) of bacteria was extracted from MRS broth and centrifuged at 10,000 rpm for 15 mins at 37°C in a cold centrifuge. The bacteria was then twice washed with a cold, sterile 0.9 g NaCl solution using a vortex. The procedure was done twice. Two millilitres of each cell suspension containing 0.25, 0.5, and 0.75 mgmL -1 of ethidium bromide were added, and the mixture was agitated for 30 mins at 200 rpm on an orbital shaker set at 37˚C. After centrifuging the treated cells for 15 mins at 10,000 rpm for 15 min at 37 ˚C, they were twice washed with MRS broth and 0.9% NaCl solution. MRS broth was added to the washed and treated cell suspension and 100 \(\mu l\) (0.1 mL) of the cell was dispensed on a fresh MRS agar plates and spread on the agar using a spreader then incubated at 37 ˚C for 60 h. Control culture was not exposed to ethidium bromide. MOLECULAR CHRACTERIZATION EFFECT OF INCUBATION PERIOD ON LACTIC ACID PRODUCTION The time course of lactic acid production by Wild Strain isolate (WS), Physical mutant isolate and Chemical mutant isolate was evaluated by taking the concentration value at every 6 h interval for 36 h incubation. Incubation periods under consideration were between 12 h and 18 h. The organisms were incubated for 36 h at 37 o C. PRODUCTION OF LACTIC ACID FROM FERMENTATION BROTH For the production of lactic acid, two fermentation broth, corn steep liquor and cheese whey were used. The pH of each broth was taken using the pH meter. Broths were acidic pH 3.5-4.0 and were therefore adjusted to 4.5 with 0.1 N NaOH. Each broth (280 mL) were prepared in a conical flask and a mixture of the two broths in equal proportion and then 40 ml of each broth were dispensed into100 mL conical flasks with broths in seven conical flasks each and then sterilized before it was inoculated with 200 \(\mu l\) of isolates which include a wild strain, three physical mutagen (30 s, 60 s and 90 s) and three chemical mutagen (0.25, 0.5 and 0.75mgmL -1 ) from nutrient broth. After inoculation, 0 h titration was done and the titre value was obtained. The samples were then placed in a water bath shaker at 37 o C for 36 h at a speed of 150 rpm and at every 6 h interval, the titre value was obtained and this process was repeated at each interval. From the result we calculated the optimum value from all the broth and the kind of sample inoculated into the broth. The whole process was only repeated for cheese whey substrate with only 3 samples (wild strain, 90 s as physical mutagen and 0.5 mgmL -1 as chemical mutagen). In this process, the samples were incubated in the water bath shaker for 10 h at 37 o C at a speed of 150 rpm and at every 2 h interval the titre value was obtained and recorded. Using the formula 0.9 V where ‘V’ denotes the volume in mL of 0.1 M NaOH needed to neutralize 10 mL of lactic acid solution, the concentration of lactic acid produced by the LAB was estimated using the volume of NaOH. QUALITATIVE ANALYSIS OF LACTIC ACID USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY A modified version of the Ramanjooloo et al. , (2014) procedure was applied to the qualitative determination process. In order to ascertain whether a homolactate bacteria was indeed isolated and the impact of various mutagenesis techniques on the lactic acid output, the fermentation end-products were qualitatively determined. At the sixth hour which had the optimum and suitable production, a sample was taken from each to be centrifuged for qualitative analysis. After centrifuging, the supernatant was dispensed in a sterile cryoviale and stored in a refrigerator before it was sent for the analysis. HPLC Conditions Mobile Phase A: 10 mM KH 2 PO 4 in water pH: 3.0 (98% v/v) Mobile Phase B: Acetonitrile (2% v/v) FR: 1.0 mL/min SP: Waters X bridge Shield RP (150 mm x 4.6 mm I.D, 3.5 um) Column Temp: 40 degC Injection Vol.: 50 uL P = ~ 120 Bar Standard Preparation of 2000 ugmL -1 Stock solution: Equivalent of 50 mg (58 mg of Lactic Acid solution) was weighed and diluted with mobile phase A in a 25 mL volumetric flask to the mark with same diluent. Calibration standards of 20-1000 ugmL -1 were prepared from the stock solution for HPLC analysis. Sample Preparation: Samples were centrifuged at 4000 rpm for 10 mins. The supernatant was diluted by 4 using mobile phase A. HPLC - Agilent 1100 series with an online degasser, quaternary pump, auto liquid sampler, thermostated column compartment and a variable UV wavelength detector running on Chemstation Software. Results ISOLATION AND IDENTIFICATION OF ISOLATES It was discovered through the isolation and identification of the isolates that the processed liquid milk sample showed a yellow zone on the MRS agar plate supplemented with 2% (w/v) sucrose, bromocresol purple (0.012 g/l), and sodium azide (0.02 g/l). Colonies on MRS agar plates enriched were chosen following 44 h of incubation at 37˚C. EFFECT OF PHYSICAL & CHEMICAL MUTAGENS ON ISOLATES The effect of the UV light exposure on the isolate showed change in the morphology and growth of the organism but still exhibited a little yellow zone on the plate. It also reduced the survival rate of the isolate such that there was no much growth on the plate. Further analysis showed that it inhibited the ability of the organism to hydrolyze protein and also produce or stop the activity of the enzyme lactate dehydrogenase. The effect of the ethidium bromide exposure on the isolate showed the isolate struggling to survive on the plates after each exposure at different concentration such that the colonies that grew were few. There was no visible change in the morphology but still exhibited a yellow/cream zone. Further analysis showed that the chemical inhibited the production of enzyme lactate dehydrogenase by the organism and also stops the enzyme from performing its activity. MOLECULAR CHRACTERIZATION EFFECT OF INCUBATION PERIOD ON LACTIC ACID PRODUCTION Fig1, Fig 2, Fig 5 and Fig 8 revealed that the optimum lactic acid production was at 18 h at 36 h incubation period. At 36 h incubation period, there were increments in lactic acid production in cheese whey from 12 h up to 18 h of incubation. Activity decreased slowly up thereafter till 36 h. So the maximal incubation period of the studied organism for lactic acid production was 18 h. PRODUCTION OF LACTIC ACID FROM WILD STRAIN ISOLATE After the 36 h incubation of the Wild Strain (WS) in cheese whey, corn steep liquor and the mixture, the production of lactic acid was proven to be relatively stable and high (4.5 mgml -1 ) in cheese whey (S1) between 6-18 h before it declined at 18 h while in corn steep liquor (S2) it increased at 6 h with a concentration of 3.69 mgml -1 and declined at the remaining period of incubation. For the mixture (S3), it was relatively stable at the incubation period from 6-18 h and increased at the 18 h (3.69 mgml -1 ) before it became stable again and finally declined at 30 h incubation period. PRODUCTION OF LACTIC ACID FROM PHYSICAL MUTANT ISOLATE For physical mutant in cheese whey, U30 had a relatively stable production of lactic acid between 6 h to 24 h and declined at 30 h- 36 h. U60 had a stable production of lactic acid within the incubation period of 6 h to 12 h and then declined from incubation period of 12 h to 36 h. At incubation period of 12 h, U90 had a very high production of lactic acid (4.76 mgml -1 ). According to the graph, U90 had the most lactic acid generation in the cheese whey. U30 produced more lactic acid in corn steep liquor over the first 18 h of incubation (from 2.52 and 2.97 to 3.06 mgml -1 ), but this production subsequently decreased over the course of the next 18 h. U60 had a concentration of 2.79 at incubation period of 6 h and declined or reduced for the rest of the incubation period. U90 also had a little increase in the production from 1.87 mgml -1 at 6 h to 2.7 mgml -1 at 12 h and later reduced for the rest of the incubation period. Therefore from the values, U30 had the highest production of lactic acid in corn steep liquor. In the mixture, among the three mutants, U30 had the highest production of lactic acid (3.78 mgml -1 ) at incubation period of 12 h. PRODUCTION OF LACTIC ACID FROM CHEMICAL MUTANTS For the chemical mutants in cheese whey, E50 had the highest production of lactic acid (5.04 mgml -1 ) among the three mutants at 18 h incubation period and reduced for the rest of the incubation period. The other two mutants were relatively stable and low. In corn steep liquor, E50 produced the highest concentration of lactic acid (3.24 mgml -1 ) among the three mutants at 18 h incubation period and decreased for the remaining period. E25 increased in its production (2.88 mgml -1 ) a little at 6 h incubation period and kept reducing and increasing for the remaining period while E75 also had an increase in the production of lactic acid (3.06 mgml -1 ) at 18 h incubation period and reduced after 18 h till 36 h incubation period. In mixture, the three mutants produced lactic acid at a relative stable concentration of 3.6mg/ml and reduced or declined after 18 h incubation period. Among the three, E25 produced the highest concentration of lactic acid 3.70 mgml -1 at 12 h of 36 h incubation period. In conclusion, during the 36 h incubation period, only cheese whey enabled optimum production of lactic acid with the following isolates; WS, U90 and E50. OPTIMIZATION OF LACTIC ACID From the results shown in Fig1, Fig 2 and Fig 5, it was found that lactic acid production under optimized condition of the isolate; OIS, U90 and E50 yielded 4.5 mgml -1 , 4.76 mgml -1 and 5.04 mgml -1 respectively at 12 -18 h of liquid-state fermentation which is higher than that obtained in other liquid-state fermentation so far reported on cheese why during the 36 h incubation period. QUALITATIVE DETERMINATION OF LACTIC ACID USING HPLC HPLC is for confirmation of the presence of the compound for the study and the qualitatively analysis (to know the amount present). The isolates were subjected to HPLC analysis confirming the presence and the amount of the compound, lactic acid in the three isolates. The HPLC analysis on the isolate; Wild Strain Isolate, Physical mutant (at 90 s exposure) and chemical mutant (0.5 mg/ml) (WS, U90 and E50) respectively showed that WS yielded 2004.87 ugml -1 which was the optimum lactic acid production with a retention time of 2.622 min and area cover of 364.3 5 mAU. U90 yielded 1457.67 ugml -1 lactic acid production with a retention time of 2.64 min and area cover of 263.53 mAU. E50 yielded 239.10 ugml -1 lactic acid production with a retention time of 2.53 min and area cover of 39.01 mAU which turned out to be the lowest production. Discussion Since sodium azide is a strong inhibitor of iron-porphyrin, it effectively stopped the growth of most fungi and non-LAB organisms. Because LAB are not iron-porphyrin synthetes, they can develop when sodium azide is present. The negative catalase test result is also due to this Lactobacillus trait. The most successful method for visual detection of LAB was determined to be bromocresol purple (0.012 gL -1 ). Bromocresol blue is a pH indicator that turns yellow when lactic acid is produced. The amount of lactic acid that a bacterial colony produces is indicated by the yellow circle surrounding it. Adnan and Tan (2007) state that mesophilic bacteria reach their highest reproductive and activity temperature at 37˚C, which is why this temperature was selected for the incubation period. According to Kotzamanidis et al. (2002), bacterial cells undergo autolysis as a result of rising lactate concentrations, which lowers cell biomass. The reason the lactate content kept rising even soon after is that the released lactate dehydrogenase enzymes are still active. This study showed that mutagenesis of the isolate by ultraviolet ray and ethidium bromide did not result in the increase of lactic acid production compared to the production from parental strain while Banjo et al ., (2018) reported that the mutagenesis of A. flavus by ultraviolet ray and ethidium bromide resulted in an increased ascorbic acid yield of 6.99 gL -1 and 7.28 gL -1 production respectively compared to a yield of 3.92 gL -1 from the parental strain of A. flavus. The HPLC analysis also showed the effect of the exposure of the strain chemically and physically on their ability to produce more lactic acid. For the effect of the UV light on the strain which was the physical mutagenesis, there are two possible things that could have occurred such that the exposure led to the inhibition of production of the enzyme lactate dehydrogenase or the enzyme was produced and it inhibited the ability of the organism to hydrolyze the protein or possibly killed the organism during the process. For the effect of the ethidium bromide on the strain which was the chemical mutagenesis, there are also two possible actions that could have occurred during the production such that the chemical stopped the production of the enzyme lactate dehydrogenase from the organism which aids the lactic acid production or the enzyme was produced but was inhibited by the chemical from performing its activity. If it was said that the organism died, there will be no production of the enzyme or lactic acid. Conclusion In conclusion, the result of this study showed that the wild strain isolate yielded the optimum production of lactic acid than the chemical and physical mutant isolate. Thus, it does not agree with the work of Khanam and Prasuna (2014), who reported that ethidium bromide increased enzyme production. The result also revealed that cheese whey was a more favourable substrate for the production of lactic acid than corn steep liquor in fermentation. Declarations Conflict of Interest Statement I am enclosing herewith a manuscript entitled “Biosynthesis of Lactic Acid by Mutagenetic Lactic Acid Bacteria Strains Isolated From Processed Milk” submitted to Folia Microbiologica. With the submission of this manuscript we would like to undertake that the above mentioned manuscript has not been published elsewhere, accepted for publication elsewhere or under editorial review for publication elsewhere. We also disclose that there is no potential sources of conflict of interest as may be relevant to the manuscript. References Adnan AFM, Tan IKP (2007) Isolation of lactic acid bacteria from Malaysian foods of the isolates for industrial potential. Biores Technol 98: 1380-1385. https://doi.org/10.1016/j.biortech.2006.05.034 Banjo TT, Kareem SO, Banjo TO, Abayomi OS (2018) Strain improvement of Aspergillus flavus for enhanced ascorbic acid production by physical and chemical mutagenesis. 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Adv Biosci Biotechnol (3): 398-407. http://dx.doi.org/10.4236/abb.2012.34057 Zhao J, Zhang B, Meng Y, Han Y, Wang Y (2020) Improvement of lactic acid production in Lactobacillus brevis by mutagenesis and optimization of fermentation conditions. J Biotechnol 10(5): 1-11. Plates Plate 1 is available in the Supplementary Files section Additional Declarations The authors declare no competing interests. Supplementary Files Plate1.png Plate 1: Morphological growth of LAB on MRS agar supplemented with sodium azide, bromocresol purple and sucrose 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. 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09:57:28","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-3963690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3963690/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51372871,"identity":"20eaefe7-a6fd-4750-af05-e8853650a8cc","added_by":"auto","created_at":"2024-02-20 12:41:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23955,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of wild strain isolate (WS) in cheese whey (S1), corn step liquor (S2) and mixture (S3) in the production of lactic acid mutants, U30 had the highest production of lactic acid (3.78 mgml\u003csup\u003e-1\u003c/sup\u003e) at incubation period of 12 h.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/8d97cd12e0eb4eacb3260cf7.png"},{"id":51372327,"identity":"f2d2e4d9-3c6e-4358-81b3-681327ab5bb9","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":35685,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of physical mutants in cheese whey on lactic acid production\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/f9c0bb574f2ff80cb5d13847.png"},{"id":51372325,"identity":"55abe9cc-a126-47ff-bd4c-cc658c42604a","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32948,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of physical mutants in corn steep liquor on lactic acid production\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/a3aedd1358b2421e857e2345.png"},{"id":51372329,"identity":"5a5e2142-c43b-4a53-959f-01d788d804f1","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30252,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of physical mutants in mixture on lactic acid production\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/2790e35f3ab3733abfc3b9cf.png"},{"id":51372333,"identity":"2de16f61-2fe6-44c4-bb98-c631c4da5fa0","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":27218,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of chemical mutants in cheese whey on lactic acid production\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/a83cab67c4d805420bc72e28.png"},{"id":51372873,"identity":"dabfad44-bc22-4f41-b0d5-fd54fea78e2a","added_by":"auto","created_at":"2024-02-20 12:41:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":30826,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of chemical mutant isolate in corn steep liquor on lactic acid production\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/2617ca0ea3543beb2e6e80ec.png"},{"id":51372334,"identity":"4795a9af-789f-4634-967b-5d59ac013818","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":29022,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of chemical mutant isolate in mixture on lactic acid production\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/1e372f9e00d993f84b44bdb9.png"},{"id":51372331,"identity":"8a9e6ae3-0d53-4ad2-b6f3-627db5d67f09","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":56589,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of lactic acid standard assay\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/b4954d745794edcb4c69fe3f.png"},{"id":51372336,"identity":"5f2a5813-90fc-479a-8bb5-4df853e5aa27","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":37358,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of lactic acid assay of Wild strain isolate from Cheese Whey\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/088b18581d0b34b8eb5cc850.png"},{"id":51372335,"identity":"f3e4fb68-bddf-4947-9a37-75a801e7d484","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":45472,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of lactic acid assay of Physical mutant isolate from Cheese Whey\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/edfb8207e8c3fef6b5d3b23e.png"},{"id":51372330,"identity":"8367ed5c-0564-475e-a34e-7113517e63ba","added_by":"auto","created_at":"2024-02-20 12:33:47","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":42420,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of lactic acid assay of Chemical mutant isolate from Cheese Whey\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/1b98d1c73268b809f640b1e3.png"},{"id":51373065,"identity":"eb43e69d-f54d-47df-9ff1-da9fe860607b","added_by":"auto","created_at":"2024-02-20 12:49:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":557728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/0f0ec8e8-6f70-4eaf-9d9b-070f440c5ec7.pdf"},{"id":51372872,"identity":"a54d8a89-bef7-4b0f-9d57-4e08c255de59","added_by":"auto","created_at":"2024-02-20 12:41:47","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":880061,"visible":true,"origin":"","legend":"\u003cp\u003ePlate 1: Morphological growth of LAB on MRS agar supplemented with sodium azide, bromocresol purple and sucrose\u003c/p\u003e","description":"","filename":"Plate1.png","url":"https://assets-eu.researchsquare.com/files/rs-3963690/v1/4bce34bf2bea1cc781256860.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eBiosynthesis of Lactic Acid by Mutagenetic Lactic Acid Bacteria Strains Isolated From Processed Milk\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLactic acid (2-hydroxypropanoic acid, CH\u003csub\u003e3\u003c/sub\u003e-CH(OH)-COOH), a valuable organic acid, has gained significant attention for its various industrial applications. Traditionally, lactic acid has been produced through chemical processes, which are associated with environmental concerns and limited sustainability. As a result, there is a growing interest in microbial synthesis of lactic acid using lactic acid bacteria (LAB) (Sun \u003cem\u003eet al\u003c/em\u003e., 2017). Lactic acid bacteria are known for their ability to convert sugars into lactic acid through fermentation. Isolation and screening of LAB from different sources, including bovine milk, have been carried out to identify strains with high lactic acid production capabilities. Bovine milk is a promising source of LAB due to its abundance and the natural presence of lactic acid bacteria which possess the unique capability to convert sugars into lactic acid through fermentation (Sun \u003cem\u003eet al\u003c/em\u003e., 2017; Johansen \u003cem\u003eet al\u003c/em\u003e., 2020).\u003c/p\u003e\n\u003cp\u003eGenetic engineering techniques, such as targeted gene disruption and gene knockout, have been employed to introduce specific genetic modifications in LAB. These techniques utilize recombinant DNA technology to manipulate LAB genomes. For instance, the disruption of lactate dehydrogenase genes using genetic engineering tools has been reported to enhance lactic acid production in LAB strains (Zhang \u003cem\u003eet al\u003c/em\u003e., 2022).\u003c/p\u003e\n\u003cp\u003eIn summary, mutagenesis in LAB involves the use of chemical mutagens, physical mutagens, and genetic engineering techniques to induce genetic mutations and improve lactic acid production. These approaches have been successfully employed to modify LAB strains and enhance their capabilities in various industrial applications.\u003c/p\u003e\n\u003cp\u003eGenetic engineering techniques have been employed to enhance the metabolic efficiency of lactic acid bacteria for increased lactic acid production. Genetic modifications targeting key enzymes and metabolic pathways involved in lactic acid synthesis have shown promising results. These modifications can lead to improved substrate utilization and lactic acid yield (Yadav \u003cem\u003eet al\u003c/em\u003e., 2021). Multi-strain lactic acid bacteria consortia have demonstrated the potential for enhanced lactic acid production. Chen \u003cem\u003eet al\u003c/em\u003e. (2019) investigated the utilization of strain combinations, including \u003cem\u003eLactobacillus plantarum\u003c/em\u003e, for effective utilization of rice straw hydrolysates and observed improved lactic acid production compared to individual strains. The selection and improvement of multi-strain lactic acid bacteria involve various strategies, such as genetic engineering, strain combination, and optimization of fermentation conditions. Genetic engineering techniques can be employed to enhance the metabolic pathways of LAB, thereby increasing lactic acid production (Yadav \u003cem\u003eet al\u003c/em\u003e., 2021). Combining different strains of LAB can lead to synergistic effects, resulting in higher productivity (Chen \u003cem\u003eet a\u003c/em\u003el., 2019). Additionally, optimizing fermentation conditions, including temperature, pH, and nutrient availability, can further enhance lactic acid synthesis (Dong \u003cem\u003eet al\u003c/em\u003e., 2018).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOptimization of fermentation conditions is crucial for maximizing lactic acid yield. Factors such as temperature, pH, and nutrient availability play significant roles in the efficiency of lactic acid fermentation. Dong \u003cem\u003eet al\u003c/em\u003e., (2018) optimized fermentation conditions for lactic acid production from Jerusalem artichoke tuber hydrolysate by \u003cem\u003eLactobacillus paracasei\u003c/em\u003e LA104, resulting in increased lactic acid yield. The industrial applications of lactic acid have further emphasized the need for sustainable and efficient production methods. Lactic acid serves as a food additive, pH regulator, and precursor for biodegradable polymers. Nguyen \u003cem\u003eet al\u003c/em\u003e., (2022) highlighted the industrial applications of lactic acid and the importance of optimizing fermentation conditions for improved production.\u003c/p\u003e\n\u003cp\u003eOverall, the literature demonstrates the significance of microbial synthesis of lactic acid using LAB. Isolation of LAB from bovine milk, genetic engineering techniques, utilization of multi-strain consortia, and optimization of fermentation conditions all contribute to improving lactic acid production. These approaches promote sustainable and eco-friendly production methods and offer practical solutions for industrial-scale lactic acid production.\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study on microbial synthesis of lactic acid by improved multi-strain lactic acid bacteria from processed liquid milk is justified by its potential to provide sustainable and environmentally friendly methods for lactic acid production, optimize resource utilization, improve process efficiency and yield, and cater to industrial applications. The most effective technique to obtain genetically stable and practical strains of microbes for industrially significant products has always been to isolate and screen them from natural sources. Furthermore, because fermentation uses renewable raw resources as opposed to petrochemicals, it is becoming a more significant activity. The findings from this study contributed to advancing the field of microbial synthesis and provide practical solutions for the production of lactic acid.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe objective of this study was to explore the potential of multi-strain lactic acid bacteria isolated from processed liquid milk and screened isolates for desirable traits and production of pure isomeric form of lactic acid. Processed liquid milk serves as an abundant source of LAB, and these bacteria have evolved to efficiently ferment lactose into lactic acid. By enhancing the performance of these natural lactic acid producers, overall lactic acid production yield and efficiency can be improved (Johansen \u003cem\u003eet al\u003c/em\u003e., 2020).\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eSAMPLE COLLECTION:\u003c/p\u003e\u003cp\u003eThree brands of commercial evaporated tin milk samples were obtained from supermarket for the study and composite of these samples was used. For the production of lactic acid, two fermentation broth, corn steep liquor and cheese whey were obtained commercially from Iwo Road market in Ibadan North West Local Government, Oyo State.\u003c/p\u003e\u003cp\u003eISOLATION AND SCREENING OF LAB\u003c/p\u003e\u003cp\u003eFor the enrichment and isolation of LAB, aliquots of the commercial milk was serially diluted to a dilution factor of 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e and 0.1ml was then spread evenly on MRS agar plates, LAPT medium and MacConkey agar plates. The plates were incubated for 44 h at 37 ˚C. For those that developed growth on the agar plates, they were then sub-cultured into MRS agar plates, LAPT medium and MacConkey agar plates to obtain a pure culture and then incubated for 24 h at 37 \u003csup\u003eo\u003c/sup\u003eC. It was then kept on nutrient agar slant. Screening of LAB was carried out as bacterial colonies were picked and streaked on fresh MRS agar plates and supplemented with sodium azide (0.02 gl\u003csup\u003e-1\u003c/sup\u003e), bromocresol purple (0.012 gL\u003csup\u003e-1\u003c/sup\u003e) and 2% (w/v) sucrose, placed in anaerobic jar and then incubated for 44 h at 37 \u003csup\u003eo\u003c/sup\u003eC (Yoganand et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePHYSICAL \u0026amp; CHEMICAL MUTAGENS\u003c/p\u003e\u003cp\u003eBacteria in the logarithmic growth phase (17 h culture) were diluted and plated onto MRS agar supplemented with 4 g of sucrose and 0.012 g of bromocresol purple. For 30, 60, and 90 s, UV light at a distance of 2 cm was applied to the Petri plates. There was also a control group that included 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e dilution cultures in it, but they were not subjected to UV light. All cultures were incubated at 37 ˚C for 60 h while being completely darkened by wrapping in aluminium foil.\u003c/p\u003e\u003cp\u003eA modified version of the Gawel et al., (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) protocol was employed. The logarithmic growth phase (17 h culture) of bacteria was extracted from MRS broth and centrifuged at 10,000 rpm for 15 mins at 37\u0026deg;C in a cold centrifuge. The bacteria was then twice washed with a cold, sterile 0.9 g NaCl solution using a vortex. The procedure was done twice. Two millilitres of each cell suspension containing 0.25, 0.5, and 0.75 mgmL\u003csup\u003e-1\u003c/sup\u003e of ethidium bromide were added, and the mixture was agitated for 30 mins at 200 rpm on an orbital shaker set at 37˚C. After centrifuging the treated cells for 15 mins at 10,000 rpm for 15 min at 37 ˚C, they were twice washed with MRS broth and 0.9% NaCl solution. MRS broth was added to the washed and treated cell suspension and 100\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu l\\)\u003c/span\u003e\u003c/span\u003e (0.1 mL) of the cell was dispensed on a fresh MRS agar plates and spread on the agar using a spreader then incubated at 37 ˚C for 60 h. Control culture was not exposed to ethidium bromide.\u003c/p\u003e\u003cp\u003eMOLECULAR CHRACTERIZATION\u003c/p\u003e\u003cp\u003eEFFECT OF INCUBATION PERIOD ON LACTIC ACID PRODUCTION\u003c/p\u003e\u003cp\u003eThe time course of lactic acid production by Wild Strain isolate (WS), Physical mutant isolate and Chemical mutant isolate was evaluated by taking the concentration value at every 6 h interval for 36 h incubation. Incubation periods under consideration were between 12 h and 18 h. The organisms were incubated for 36 h at 37 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\u003cp\u003ePRODUCTION OF LACTIC ACID FROM FERMENTATION BROTH\u003c/p\u003e\u003cp\u003eFor the production of lactic acid, two fermentation broth, corn steep liquor and cheese whey were used. The pH of each broth was taken using the pH meter. Broths were acidic pH 3.5-4.0 and were therefore adjusted to 4.5 with 0.1 N NaOH. Each broth (280 mL) were prepared in a conical flask and a mixture of the two broths in equal proportion and then 40 ml of each broth were dispensed into100 mL conical flasks with broths in seven conical flasks each and then sterilized before it was inoculated with 200\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu l\\)\u003c/span\u003e\u003c/span\u003e of isolates which include a wild strain, three physical mutagen (30 s, 60 s and 90 s) and three chemical mutagen (0.25, 0.5 and 0.75mgmL\u003csup\u003e-1\u003c/sup\u003e) from nutrient broth. After inoculation, 0 h titration was done and the titre value was obtained. The samples were then placed in a water bath shaker at 37 \u003csup\u003eo\u003c/sup\u003eC for 36 h at a speed of 150 rpm and at every 6 h interval, the titre value was obtained and this process was repeated at each interval. From the result we calculated the optimum value from all the broth and the kind of sample inoculated into the broth.\u003c/p\u003e\u003cp\u003eThe whole process was only repeated for cheese whey substrate with only 3 samples (wild strain, 90 s as physical mutagen and 0.5 mgmL\u003csup\u003e-1\u003c/sup\u003e as chemical mutagen). In this process, the samples were incubated in the water bath shaker for 10 h at 37 \u003csup\u003eo\u003c/sup\u003eC at a speed of 150 rpm and at every 2 h interval the titre value was obtained and recorded.\u003c/p\u003e\u003cp\u003eUsing the formula 0.9 V where \u0026lsquo;V\u0026rsquo; denotes the volume in mL of 0.1 M NaOH needed to neutralize 10 mL of lactic acid solution, the concentration of lactic acid produced by the LAB was estimated using the volume of NaOH.\u003c/p\u003e\u003cp\u003eQUALITATIVE ANALYSIS OF LACTIC ACID USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY\u003c/p\u003e\u003cp\u003eA modified version of the Ramanjooloo \u003cem\u003eet al.\u003c/em\u003e, (2014) procedure was applied to the qualitative determination process. In order to ascertain whether a homolactate bacteria was indeed isolated and the impact of various mutagenesis techniques on the lactic acid output, the fermentation end-products were qualitatively determined. At the sixth hour which had the optimum and suitable production, a sample was taken from each to be centrifuged for qualitative analysis. After centrifuging, the supernatant was dispensed in a sterile cryoviale and stored in a refrigerator before it was sent for the analysis.\u003c/p\u003e\u003cp\u003e\u003cem\u003eHPLC Conditions\u003c/em\u003e\u003c/p\u003e\u003cp\u003eMobile Phase A: 10 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e in water pH: 3.0 (98% v/v)\u003c/p\u003e\u003cp\u003eMobile Phase B: Acetonitrile (2% v/v)\u003c/p\u003e\u003cp\u003eFR: 1.0 mL/min\u003c/p\u003e\u003cp\u003eSP: Waters X bridge Shield RP (150 mm x 4.6 mm I.D, 3.5 um)\u003c/p\u003e\u003cp\u003eColumn Temp: 40 degC\u003c/p\u003e\u003cp\u003eInjection Vol.: 50 uL\u003c/p\u003e\u003cp\u003eP\u0026thinsp;=\u0026thinsp;~\u0026thinsp;120 Bar\u003c/p\u003e\u003cp\u003eStandard Preparation of 2000 ugmL\u003csup\u003e-1\u003c/sup\u003e Stock solution: Equivalent of 50 mg (58 mg of Lactic Acid solution) was weighed and diluted with mobile phase A in a 25 mL volumetric flask to the mark with same diluent.\u003c/p\u003e\u003cp\u003eCalibration standards of 20-1000 ugmL\u003csup\u003e-1\u003c/sup\u003e were prepared from the stock solution for HPLC analysis.\u003c/p\u003e\u003cp\u003eSample Preparation: Samples were centrifuged at 4000 rpm for 10 mins. The supernatant was diluted by 4 using mobile phase A.\u003c/p\u003e\u003cp\u003eHPLC - Agilent 1100 series with an online degasser, quaternary pump, auto liquid sampler, thermostated column compartment and a variable UV wavelength detector running on Chemstation Software.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eISOLATION AND IDENTIFICATION OF ISOLATES\u003c/p\u003e\u003cp\u003eIt was discovered through the isolation and identification of the isolates that the processed liquid milk sample showed a yellow zone on the MRS agar plate supplemented with 2% (w/v) sucrose, bromocresol purple (0.012 g/l), and sodium azide (0.02 g/l). Colonies on MRS agar plates enriched were chosen following 44 h of incubation at 37˚C.\u003c/p\u003e\u003cp\u003eEFFECT OF PHYSICAL \u0026amp; CHEMICAL MUTAGENS ON ISOLATES\u003c/p\u003e\u003cp\u003eThe effect of the UV light exposure on the isolate showed change in the morphology and growth of the organism but still exhibited a little yellow zone on the plate. It also reduced the survival rate of the isolate such that there was no much growth on the plate. Further analysis showed that it inhibited the ability of the organism to hydrolyze protein and also produce or stop the activity of the enzyme lactate dehydrogenase. The effect of the ethidium bromide exposure on the isolate showed the isolate struggling to survive on the plates after each exposure at different concentration such that the colonies that grew were few. There was no visible change in the morphology but still exhibited a yellow/cream zone. Further analysis showed that the chemical inhibited the production of enzyme lactate dehydrogenase by the organism and also stops the enzyme from performing its activity.\u003c/p\u003e\n\u003cp\u003eMOLECULAR CHRACTERIZATION\u003c/p\u003e\n\u003cp\u003eEFFECT OF INCUBATION PERIOD ON LACTIC ACID PRODUCTION\u003c/p\u003e\n\u003cp\u003eFig1, Fig 2, Fig 5 and Fig 8 revealed that the optimum lactic acid production was at 18 h at 36 h incubation period. At 36 h incubation period, there were increments in lactic acid production in cheese whey from 12 h up to 18 h of incubation. Activity decreased slowly up thereafter till 36 h. So the maximal incubation period of the studied organism for lactic acid production was 18 h.\u003c/p\u003e\n\u003cp\u003ePRODUCTION OF LACTIC ACID FROM WILD STRAIN ISOLATE\u003c/p\u003e\n\u003cp\u003eAfter the 36 h incubation of the Wild Strain (WS) in cheese whey, corn steep liquor and the mixture, the production of lactic acid was proven to be relatively stable and high (4.5 mgml\u003csup\u003e-1\u003c/sup\u003e) in cheese whey (S1) between 6-18 h before it declined at 18 h while in corn steep liquor (S2) it increased at 6 h with a concentration of 3.69 mgml\u003csup\u003e-1\u003c/sup\u003e and declined at the remaining period of incubation. For the mixture (S3), it was relatively stable at the incubation period from 6-18 h and increased at the 18 h (3.69 mgml\u003csup\u003e-1\u003c/sup\u003e) before it became stable again and finally declined at 30 h incubation period.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePRODUCTION OF LACTIC ACID FROM PHYSICAL MUTANT ISOLATE\u003c/p\u003e\n\u003cp\u003eFor physical mutant in cheese whey, U30 had a relatively stable production of lactic acid between 6 h to 24 h and declined at 30 h- 36 h. U60 had a stable production of lactic acid within the incubation period of 6 h to 12 h and then declined from incubation period of 12 h to 36 h. At incubation period of 12 h, U90 had a very high production of lactic acid (4.76 mgml\u003csup\u003e-1\u003c/sup\u003e). According to the graph, U90 had the most lactic acid generation in the cheese whey. U30 produced more lactic acid in corn steep liquor over the first 18 h of incubation (from 2.52 and 2.97 to 3.06 mgml\u003csup\u003e-1\u003c/sup\u003e), but this production subsequently decreased over the course of the next 18 h. U60 had a concentration of 2.79 at incubation period of 6 h and declined or reduced for the rest of the incubation period. U90 also had a little increase in the production from 1.87 mgml\u003csup\u003e-1\u003c/sup\u003e at 6 h to 2.7 mgml\u003csup\u003e-1\u003c/sup\u003e at 12 h and later reduced for the rest of the incubation period. Therefore from the values, U30 had the highest production of lactic acid in corn steep liquor. In the mixture, among the three mutants, U30 had the highest production of lactic acid (3.78 mgml\u003csup\u003e-1\u003c/sup\u003e) at incubation period of 12 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePRODUCTION OF LACTIC ACID FROM CHEMICAL MUTANTS\u003c/p\u003e\n\u003cp\u003eFor the chemical mutants in cheese whey, E50 had the highest production of lactic acid (5.04 mgml\u003csup\u003e-1\u003c/sup\u003e) among the three mutants at 18 h incubation period and reduced for the rest of the incubation period. The other two mutants were relatively stable and low. In corn steep liquor, E50 produced the highest concentration of lactic acid (3.24 mgml\u003csup\u003e-1\u003c/sup\u003e) among the three mutants at 18 h incubation period and decreased for the remaining period. E25 increased in its production (2.88 mgml\u003csup\u003e-1\u003c/sup\u003e) a little at 6 h incubation period and kept reducing and increasing for the remaining period while E75 also had an increase in the production of lactic acid (3.06 mgml\u003csup\u003e-1\u003c/sup\u003e) at 18 h incubation period and reduced after 18 h till 36 h incubation period. In mixture, the three mutants produced lactic acid at a relative stable concentration of 3.6mg/ml and reduced or declined after 18 h incubation period. Among the three, E25 produced the highest concentration of lactic acid 3.70 mgml\u003csup\u003e-1\u003c/sup\u003e at 12 h of 36 h incubation period.\u003c/p\u003e\n\u003cp\u003eIn conclusion, during the 36 h incubation period, only cheese whey enabled optimum production of lactic acid with the following isolates; WS, U90 and E50.\u003c/p\u003e\n\u003cp\u003eOPTIMIZATION OF LACTIC ACID\u003c/p\u003e\n\u003cp\u003eFrom the results shown in Fig1, Fig 2 and Fig 5, it was found that lactic acid production under optimized condition of the isolate; OIS, U90 and E50 yielded 4.5 mgml\u003csup\u003e-1\u003c/sup\u003e, 4.76 mgml\u003csup\u003e-1\u003c/sup\u003e and 5.04 mgml\u003csup\u003e-1\u003c/sup\u003e respectively at 12 -18 h of liquid-state fermentation which is higher than that obtained in other liquid-state fermentation so far reported on cheese why during the 36 h incubation period.\u003c/p\u003e\n\u003cp\u003eQUALITATIVE DETERMINATION OF LACTIC ACID USING HPLC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPLC is for confirmation of the presence of the compound for the study and the qualitatively analysis (to know the amount present). The isolates were subjected to HPLC analysis confirming the presence and the amount of the compound, lactic acid in the three isolates. The HPLC analysis on the isolate; Wild Strain Isolate, Physical mutant (at 90 s exposure) and chemical mutant (0.5 mg/ml) \u0026nbsp;(WS, U90 and E50) respectively showed that WS yielded 2004.87 ugml\u003csup\u003e-1\u003c/sup\u003e which was the optimum lactic acid production with a retention time of 2.622 min and area cover of 364.3 5 mAU. U90 yielded 1457.67 ugml\u003csup\u003e-1\u003c/sup\u003e lactic acid production with a retention time of 2.64 min and area cover of 263.53 mAU. E50 yielded 239.10 ugml\u003csup\u003e-1\u003c/sup\u003e lactic acid production with a retention time of 2.53 min and area cover of 39.01 mAU which turned out to be the lowest production.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince sodium azide is a strong inhibitor of iron-porphyrin, it effectively stopped the growth of most fungi and non-LAB organisms. Because LAB are not iron-porphyrin synthetes, they can develop when sodium azide is present. The negative catalase test result is also due to this Lactobacillus trait. The most successful method for visual detection of LAB was determined to be bromocresol purple (0.012 gL\u003csup\u003e-1\u003c/sup\u003e). Bromocresol blue is a pH indicator that turns yellow when lactic acid is produced. The amount of lactic acid that a bacterial colony produces is indicated by the yellow circle surrounding it. Adnan and Tan (2007) state that mesophilic bacteria reach their highest reproductive and activity temperature at 37˚C, which is why this temperature was selected for the incubation period. According to Kotzamanidis \u003cem\u003eet al.\u003c/em\u003e (2002), bacterial cells undergo autolysis as a result of rising lactate concentrations, which lowers cell biomass. The reason the lactate content kept rising even soon after is that the released lactate dehydrogenase enzymes are still active. This study showed that mutagenesis of the isolate by ultraviolet ray and ethidium bromide did not result in the increase of lactic acid production compared to the production from parental strain while Banjo \u003cem\u003eet al\u003c/em\u003e., (2018) reported that the mutagenesis of \u003cem\u003eA. flavus\u003c/em\u003e by ultraviolet ray and ethidium bromide resulted in an increased ascorbic acid yield of 6.99 gL\u003csup\u003e-1\u003c/sup\u003e and 7.28 gL\u003csup\u003e-1\u003c/sup\u003e production respectively compared to a yield of 3.92 gL\u003csup\u003e-1\u003c/sup\u003e from the parental strain of \u003cem\u003eA. flavus.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe HPLC analysis also showed the effect of the exposure of the strain chemically and physically on their ability to produce more lactic acid. For the effect of the UV light on the strain which was the physical mutagenesis, there are two possible things that could have occurred such that the exposure led to the inhibition of production of the enzyme lactate dehydrogenase or the enzyme was produced and it inhibited the ability of the organism to hydrolyze the protein or possibly killed the organism during the process. For the effect of the ethidium bromide on the strain which was the chemical mutagenesis, there are also two possible actions that could have occurred during the production such that the chemical stopped the production of the enzyme lactate dehydrogenase from the organism which aids the lactic acid production or the enzyme was produced but was inhibited by the chemical from performing its activity. If it was said that the organism died, there will be no production of the enzyme or lactic acid.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the result of this study showed that the wild strain isolate yielded the optimum production of lactic acid than the chemical and physical mutant isolate. Thus, it does not agree with the work of Khanam and Prasuna (2014), who reported that ethidium bromide increased enzyme production. The result also revealed that cheese whey was a more favourable substrate for the production of lactic acid than corn steep liquor in fermentation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e \u003cp\u003eI am enclosing herewith a manuscript entitled \u0026ldquo;Biosynthesis of Lactic Acid by Mutagenetic Lactic Acid Bacteria Strains Isolated From Processed Milk\u0026rdquo; submitted to Folia Microbiologica. With the submission of this manuscript we would like to undertake that the above mentioned manuscript has not been published elsewhere, accepted for publication elsewhere or under editorial review for publication elsewhere. We also disclose that there is no potential sources of conflict of interest as may be relevant to the manuscript.\u003c/p\u003e "},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdnan AFM, Tan IKP (2007) Isolation of lactic acid bacteria from Malaysian foods of the isolates for industrial potential. Biores Technol 98: 1380-1385. https://doi.org/10.1016/j.biortech.2006.05.034\u003c/li\u003e\n \u003cli\u003eBanjo TT, Kareem SO, Banjo TO, Abayomi OS (2018) Strain improvement of \u003cem\u003eAspergillus flavus\u003c/em\u003e for enhanced ascorbic acid production by physical and chemical mutagenesis. Nig J Biotech 35 (2): 8\u0026ndash;15. https://dx.doi.org/10.4314/njb.v35i2.2\u003c/li\u003e\n \u003cli\u003eChen S, Lin J, Zheng J (2019) Enhanced lactic acid production in \u003cem\u003eLactobacillus casei\u003c/em\u003e by a mutant strain obtained via ethyl methanesulfonate mutagenesis. Biochem Engineer J 151: 107-330.\u003c/li\u003e\n \u003cli\u003eChen S, Tong Q, Ding J, Ren,D, Zhang L, Jiang M, Zhang C (2019) Engineering of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e with the Xylose Isomerase Pathway for Effective Utilization of Rice Straw Hydrolysates. Appl Environ Microbiol 85(6): 02-18.\u003c/li\u003e\n \u003cli\u003eDong Y, Hu J, Fan L, Wang J (2018) Optimization of fermentation conditions for lactic acid production from Jerusalem artichoke tuber hydrolysate by \u003cem\u003eLactobacillus paracasei\u003c/em\u003e LA104. J Microbiol Biotechnol 28(5): 815-821.\u003c/li\u003e\n \u003cli\u003eGawel D, Maliszewska-Tkacyk M, Jonczyk P, Schaaper RM, Fijalkowska IJ (2002) Lack of strand bias in UV-induced mutagenesis in \u003cem\u003eEscherichia coli\u003c/em\u003e. Bacteriol 184: 4449-4454. https://doi.org/10.1111/j.1365-2672.1960.tb00188.x\u003c/li\u003e\n \u003cli\u003eHofvendahl, K. and Hahn-H\u0026auml;gerdal, B. (2000). Factors affecting the fermentative lactic acid production from renewable resources. \u003cem\u003eEnzyme and Microbial Technology\u003c/em\u003e, 26: 87-107. https://doi.org/10.1016/S0141-0229(99)00155-6\u003c/li\u003e\n \u003cli\u003eHarris, D.C. (2010). Quantitative Chemical Analysis (8th ed.). New York, NY: W.H. Freeman and Company\u003c/li\u003e\n \u003cli\u003eJohansen E, N\u0026aelig;rdal I, Sk\u0026aring;ra T, Axelsson L Rudi K (2020) Strain level analysis of the natural and genetically modified \u003cem\u003eLactococcus lactis\u003c/em\u003e food fermentation microbiota. Front Microbiol 11: 73.\u003c/li\u003e\n \u003cli\u003eKhanam R, and Prasuna GR (2014) Strain improvement of white rot fungi \u003cem\u003ePycnoporus cinnabarinus\u003c/em\u003e with the influence of physical and chemical mutagens for enhancing laccases production. J Sci Ind Res 73: 331-337.\u003c/li\u003e\n \u003cli\u003eKotzamanidis C, Roukas T Skaracis G (2002) Optimization of lactic acid production from beet molasses by \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e NCIMB 8130. World J Microbiol Biotechnol 18: 441-448. https://doi.org/10.1023/A:1015523126741\u003c/li\u003e\n \u003cli\u003eMahwish S, Shazia AB, Muhammad S, Tanzila S Shazia N (2018) Strain improvement of newly isolated \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e MS1 for enhanced bacteriocin production. Turkish J Biochem 43(3): 323\u0026ndash;332. https://doi.org/10.1515/tjb-2017-0075\u003c/li\u003e\n \u003cli\u003eNelson DL, Cox MM (2017) Lehninger Principles of Biochemistry (7th ed.). New York, NY: W.H. Freeman and Company.\u003c/li\u003e\n \u003cli\u003eNguyen TTM, Vu TTT, Tran TM, Le TT (2022) Optimization of fermentation conditions for lactic acid production from sago starch by \u003cem\u003eLactobacillus plantarum\u003c/em\u003e. Biotechnol Rep, 33: 00658.\u003c/li\u003e\n \u003cli\u003eOshiro M, Shinto H, Tashiro Y, Miwa N, Sekiguchi T, Okamoto M, Ishizaki A, Sonomoto K, (2009) Kinetic modeling and sensitivity analysis of xylose metabolism in \u003cem\u003eLactococcus lactis\u003c/em\u003e IO-1. Biosci Bioengineer, 108: 376-384. https://doi.org/10.1016/j.jbiosc.2009.05.003\u003c/li\u003e\n \u003cli\u003eOsho MB, Sobande OE (2019) Microbiological assessment of tigernut milk as a potential probiotic product. Nig J Biotechnol 36 (1):186-193. https://dx.doi.org/10.4314/njb.v36i1.24\u003c/li\u003e\n \u003cli\u003eSinghvi M, Joshi D, Adsul M, Varma A, Gokhale D (2010) D-(\u0026minus;)-Lactic acid production from cello-biose and cellulose by \u003cem\u003eLactobacillus lactis\u003c/em\u003e mutant RM2-24. Green Chemi 12: 1106-1109. https://doi.org/10.1039/b925975a\u003c/li\u003e\n \u003cli\u003eSobrun Y, Bhaw-Luximon A, Jhurry D, Puchooa D (2012) Isolation of lactic acid bacteria from sugar cane juice and production of lactic acid from selected improved strains. Adv Biosci Biotechnol\u003cem\u003e\u0026nbsp;\u003c/em\u003e3: 398\u0026ndash;407.\u003c/li\u003e\n \u003cli\u003eSun J, Qiu T, Liu H, Zhang W, Yu L (2017) Microbial production of lactic acid: A potential approach to in situ valorization of lignocellulosic biomass\u003c/li\u003e\n \u003cli\u003eTashiro Y, Kaneko W, Sun Y, Shibata K, Inokuma K. Zendo T, Sonomoto K (2011) Continuous D-lactic acid production by a novel thermotolerant \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003elactis\u0026nbsp;\u003c/em\u003eQU 41. Appl Microbiol Biotechnol 89: 1741-1750. https://doi.org/10.1007/s00253-010-3011-7\u003c/li\u003e\n \u003cli\u003eWang G, Ma M, Zhang Y, Liu L, Liu X, Jiang X, Jiang C (2018) Ethyl nitrosourea (ENU) mutagenesis generates a glycerol utilization-deficient mutant of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e for vitamin C production from glycerol. Appl Microbiol Biotechnol 102(7): 3269-3281.\u003c/li\u003e\n \u003cli\u003eWang Q, Lu Z, Du R, Xu Y, Zhang C (2020) Atmospheric and room temperature plasma (ARTP) mutagenesis of Lactobacillus rhamnosus for improved acid tolerance and lactic acid production. Appl Biochem Biotechnol 190(1): 217-230\u003c/li\u003e\n \u003cli\u003eYadav V, Kundu S, Sen R (2021) Advances in lactic acid production by bacteria: \u003cem\u003eA review.\u0026nbsp;\u003c/em\u003eProc Biochem 111: 4-18.\u003c/li\u003e\n \u003cli\u003eYoganand S, Archana B, Dhanjay J, Daneshwar P (2012) Isolation of lactic acid bacteria from sugar cane juice and production of lactic acid from selected improved strains. Adv Biosci Biotechnol (3): 398-407. http://dx.doi.org/10.4236/abb.2012.34057\u003c/li\u003e\n \u003cli\u003eZhao J, Zhang B, Meng Y, Han Y, Wang Y (2020) Improvement of lactic acid production in \u003cem\u003eLactobacillus brevis\u003c/em\u003e by mutagenesis and optimization of fermentation conditions. J Biotechnol 10(5): 1-11.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Plates","content":"\u003cp\u003ePlate 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"McPherson University, Nigeria","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":"Lactic Acid, Synthesis, Mutagenetic LAB Strains, Processed Milk","lastPublishedDoi":"10.21203/rs.3.rs-3963690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3963690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLactic acid, a valuable weak natural organic acid extensively utilized in various industries has been conventionally synthesized through chemical processes. However, the growing demand for sustainable and eco-friendly production methods has stimulated interest in microbial synthesis. The study was to explore the potential of multi-strain lactic acid bacteria isolated from processed liquid milk to synthesize lactic acid. Microorganisms were isolated and screened on deMan Rogosa Sharpe (MRS) agar and were identified according to morphological and biochemical characteristics. Mutagenesis of positive isolate using chemical and physical means, quantitative and qualitative production using High-Performance Liquid Chromatography (HPLC) were investigated. The isolate was discovered to have a clear zone on MRS agar, which allowed for its identification as \u003cem\u003eLactobacillus\u003c/em\u003e sp. It was then chosen for mutagenesis using ethidium bromide (EB) and UV light. Thirteen mutants were identified, and three of those were examined to see if they could generate lactic acid using various substrates. HPLC confirmed mutants and parent strain to produce significant homo-fermentative lactic acid in cheese whey substrate. The parent strain gave a significant yield of 2004.87ugml\u003csup\u003e-1\u003c/sup\u003e as compared to UV and EB mutant strains with 1457.67ugml\u003csup\u003e-1\u003c/sup\u003e and 239.10ugml\u003csup\u003e-1\u003c/sup\u003e respectively. Optimum lactic acid yields were produced at 37\u0026deg;C, pH 4.5, and 150 rpm 16 h fermentation period. This study showed that mutagenesis did not influence optimum lactic acid production. The yield improvement that occurred via mutations might have diverted the metabolism from lactic acid production towards mixed acid fermentation, hence produce reduced levels of lactic acid.\u003c/p\u003e","manuscriptTitle":"Biosynthesis of Lactic Acid by Mutagenetic Lactic Acid Bacteria Strains Isolated From Processed Milk","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 12:33:42","doi":"10.21203/rs.3.rs-3963690/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":"29bddf40-c783-4da9-acdb-c91a7060e721","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28822514,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2024-02-20T12:33:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-20 12:33:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3963690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3963690","identity":"rs-3963690","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}