Proximate composition of fermented Gallus gallus foot keratin‒bone composites as viable high-protein concentrates intended for dilution in pisciculture feeds

Proximate composition of fermented Gallus gallus foot keratin‒bone composites as viable high-protein concentrates intended for dilution in pisciculture feeds | 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 Short Report Proximate composition of fermented Gallus gallus foot keratin‒bone composites as viable high-protein concentrates intended for dilution in pisciculture feeds Samuel Victor Viñas, Jhoechell Francisco, David Dean Cape This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6965350/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Feb, 2026 Read the published version in BMC Research Notes → Version 1 posted 13 You are reading this latest preprint version Abstract A cofermentation process involving the fermentation of five chicken foot biomass treatments with various keratin-to-bone inputs was conducted with Saccharomyces cerevisiae and Lactiplantibacillus plantarum at 30°C for 28 days. The fermented samples were analyzed for proximate composition through standardized gravimetric and colorimetric methods, with the results subjected to one-way multivariate analysis of variance, univariate analysis of variance for every parameter, and Pearson correlation analysis. Significant compositional differences were observed among the treatments, Wilks’ Λ = 4.64 × 10⁻¹² , p < < .001, with large effect sizes for all the parameters, η² ≥ .87. The highest protein content, 76.67%, on a dry weight basis, was recorded in the pure keratin variant, whereas the ash content peaked at 26.26% in the bone-dominant samples. Moisture, protein, fat, and fiber positively covaried, with r ≥ .934, contrasting significantly with ash, with | r | ≥ .905. Formulations may therefore be selectively tuned to align with dietary strategies for certain ontogenetic stages of the target species upon proportioning keratin and bone inputs to yield an ideal protein-to-ash ratio to be diluted within pisciculture feeds. To validate the in vitro proximate predictions with in vivo efficacy data, studies can quantify performance outcomes across these inclusion rates, including but not limited to, feed conversion efficiency, the somatic growth rate, the innate immune response, and gut histomorphology. protein concentrate fermented poultry waste keratin bone biomass Introduction High-protein concentrates, whether derived from fishmeal (60–72% crude protein, CP, by weight) or plant sources such as soy protein concentrate (65–70% CP, on a moisture-free basis), are processed because of their high protein content and then diluted within feed rations [1, 2]. These concentrates can precisely influence the total and digestible protein content necessary for specific species, whereas dilution precludes the excess of raw fishmeal or whole‐meal ingredients so that every gram of feed delivers essential amino acids rather than surplus nitrogenous ballast. Certain farmed species in pisciculture present significantly varied protein requirements. Carnivores such as Atlantic salmon and rainbow trout thrive on diets containing nearly 42–49% CP to support rapid muscle accretion and high metabolic rates, resulting in feed‐conversion ratios (FCRs) near 1.0–1.25 [3, 4]. Omnivores, e.g., juvenile Nile tilapia, perform optimally at approximately 267 g/kg digestible protein (298 g/kg CP), yielding an FCR of 1.81, given the optimal water quality parameters [5]. Catfish typically require 32–35% CP, whereas penaeid shrimp consume 30–57% CP [6, 7]. A high‐protein concentrate can carry through these species-specific targets without over‐ or undershooting nutritional requirements. Economically, protein is the single costliest feed component, while feed itself accounts for 30–50% of the variable operating costs in most aquaculture operations [8]. Through concentrated protein sources that can precisely dial in (neither lower nor exceeding) dietary protein requirements using nonfish alternatives, producers can trim ingredient costs by 44% according to initial trial runs [9]. For carnivorous species whose feed bills are greatest, this precision can translate to thousands of dollars in savings per metric ton of fish produced. In terms of environmental sustainability, minimizing surplus protein in feed reduces nitrogenous waste; unbalanced amino acid intake triggers deamination of excess dietary protein, resulting in increased ammonia (NH₃) excretion [10]. Studies suggest that reformulating fish feeds on an ideal protein basis typically reduces total nitrogen output, which in turn eases the load on biofilters in recirculating systems and improves pond water quality [11]. Diluting high‐protein concentrates is a cost-effective, efficient, and environmentally responsible strategy, particularly if these concentrates have originated from biomass wastes (e.g., discarded chicken foot keratin and bone residues). Annually, the global poultry industry generates approximately 45.9 million tons of processing waste, with chicken feet constituting approximately 8.5% of that total, equating to nearly 3.9 million tons of chicken feet discarded [12, 13]. Chicken foot keratin biomass, derived from epidermal scutes, reticulate scales, and claw sheaths, has biochemical and structural features that can be rendered as viable high-protein concentrates in pisciculture feeds. They are predominantly composed of β -keratin, a structural protein characterized by its β -pleated sheet conformation, which imparts remarkable mechanical strength and chemical resistance and results in a highly stable, insoluble network that withstands prolonged exposure to aquatic settings [14]. Chicken foot keratin comprises specialized isoforms that assemble into tightly packed filamentous networks for rigidity and elasticity [15], whereas such duality creates an ideal physical scaffold in fish farming systems. A durable substrate is necessary to support biofilm formation and microbial colonization, processes that improve nutrient cycling and water quality [16]. Although keratin is highly recalcitrant, controlled biochemical treatments can gradually degrade it into bioactive peptides and amino acids [17]. These released compounds may provide low-nitrogen macronutrients for aquatic organisms or stimulate beneficial microbial communities that improve water conditions. This nutritional aspect is further highlighted in the context of keratin’s potential as an alternative protein source in food and nutrition, where even its partial hydrolysis yields a compound with functional bioactivity [18]. Its minimal fat and carbohydrate content reduces the risk of oxidative deterioration and imbalances in the gut microbiome, whereas residual indigestible fibers contribute to structural reinforcement without impeding nutrient diffusion [19, 20]. Processing raw keratin biomass , through methods such as grinding, which mechanically reduces it to powder, and fermentation , can partially compromise its original scaffold, which supports biofilm formation [21]. However, in feed applications, it could primarily be intended to be a high-protein concentrate that can offer digestible nutrients and bioactive compounds to the fish rather than preserving long-term structural integrity as a scaffold. Even if the natural filamentous network is diminished, residual structural integrity (specifically when modestly treated) and the increased surface area of pellets (when binders are used) can nevertheless maintain adequate physical strength for in‐water exposure. Chicken foot bone-derived biomass from the tarsometatarsus and distal phalanx consists of a type I collagen matrix interwoven with hydroxyapatite crystals [22]. During digestion, the collagen in chicken foot biomass undergoes hydrolysis, a process facilitated by proteolytic enzymes that break down high-molecular-weight proteins into smaller peptides and amino acids [23]. This enzymatic degradation begins in the stomach and continues in the small intestine, where enzymes such as pepsin, trypsin, and chymotrypsin sequentially cleave peptide bonds [24]. The breakdown process produces collagen hydrolysates, consisting of bioactive dipeptides and tripeptides, along with unbound amino acids such as glycine, proline, and hydroxyproline, which are essential for nutritional health [25]. In addition to collagen peptides, mineral ions such as calcium and phosphorus are essential for fish nutrition, specifically for skeletal development, growth, and metabolic health [26]. During digestion, these minerals are also gradually released, becoming more bioavailable and easily incorporated into new bone structures and other cellular functions. This slow-release mechanism can prolong the postprandial increase in plasma amino acid levels [27]. Such a sustained supply of amino acids is particularly beneficial for fish, supporting continuous growth and repair processes, especially during periods of low feed intake. Fermentation relies on microorganisms such as Saccharomyces cerevisiae and Lactiplantibacillus plantarum (formerly Lactobacillus plantarum ), which are known for their ability to degrade resistant protein structures, thereby improving the nutritional quality of fermented substrates. Although these microbes do not produce the specialized keratinolytic enzymes required for complete keratin degradation, a function attributed to certain Bacillus species and fungi, they exhibit general proteolytic activity and induce acidification that can weaken the dense disulfide-bond network of keratin [28, 29]. This proteolysis and partial structural weakening could be intended to modestly preserve the keratin structure, preventing unnecessary extreme degradation that is likely to result in the rapid depletion of bioactive nutrients when submerged in fish culture water. In various bioprocesses, the synergistic interaction of multiple microbes regulates the extent of degradation. The literature suggests that Saccharomyces cerevisiae and Lactiplantibacillus plantarum , individually or in combination with more potent degraders, contribute to improving the efficiency and quality of fermentation [30, 31]. A comparable synergistic process may occur in bone residues, where instead of collagenase-producing microbes, S. cerevisiae and L. plantarum are employed for similar fundamental and strategic purposes, facilitating proteolysis (not degraded to a greater or lesser extent, with further proteolysis occurring during digestion when fed), acidification, and partial structural weakening, specifically hydroxyapatite dissolution and collagen softening of chicken foot bone biomass [32]. This mechanism promotes peptide release and optimizes calcium and phosphorus bioavailability while preserving residual structural integrity, similarly addressing the risks of nutrient leaching, eutrophication, increased biochemical oxygen demand, and water pollution caused by uneaten feed settling in sediments when bioactive nutrients are depleted too rapidly in pisciculture water [33]. Methods The process began with procuring 5 kg of raw chicken feet from a local wet market in San Pablo City, Laguna, Philippines. Quality standards were adhered to by inspecting the visual and tactile properties of the chicken feet, which exhibited a pinkish color, moist texture, and no off-odors; feet displaying discoloration, cuts, or excessive dirt were rejected. This initial mass yielded approximately 300 g of keratin and 700 g of bone residue, with the remaining materials set aside for purposes beyond the scope of this research. During pretreatment and fermentation, food-grade gloves, laboratory aprons, heat-resistant borosilicate glassware, an analytical balance, a thermometer, and a precise timing device were used for procedural accuracy and sterility. The biomass components were first separated for accurate weighing and then thoroughly washed with clean, potable water while being placed on a sterile metal strainer to remove dirt and contaminants. The biomass that was separated with a sterile knife are meticulously processed and subsequently steam sterilized and placed on borosilicate glassware in a stovetop pressure cooker (Chef's Classics Zinnia Stainless Steel Pressure Cooker, 8 L) at medium heat for 5 minutes for uniform heat distribution. Steam sterilization through high-temperature saturated steam is preferred over boiling or chemical disinfection to preserve nutrient integrity and avoid potentially toxic residues from chemical disinfectants [34]. After sterilization, the biomass was oven-dried (Oven: La Germania FS8041 30XTR) on a sterile tray in a thin, even layer (approximately 1 cm) at a controlled medium heat for 15 minutes for uniform moisture removal. Once dried, the biomass was reduced to fine particles via industrial grinding equipment (Philips HR1744 Cucina) and a mortar and pestle in preparation for fermentation. For the fermentation stage, active Saccharomyces cerevisiae and Lactiplantibacillus plantarum strains were obtained from the Philippine National Collection of Microorganisms, National Institute of Molecular Biology & Biotechnology, University of the Philippines Los Baños, Laguna 4031 Philippines, under accession numbers BIOTECH 2055 and BIOTECH 10968, respectively. These strains, with microbial loads of 1 × 10 8 CFU/mL, were inoculated at a concentration of 5% relative to the substrate mass and mixed at a 1:1 ratio. Fermentation was conducted with five substrate combinations: (T 1 ) 65 g of keratin; (T 2 ) 48.75 g of keratin and 16.25 g of bones; (T 3 ) 32.5 g of keratin and 32.5 g of bones; (T 4 ) 16.25 g of keratin and 48.75 g of bones; and (T 5 ) 65 g of bones. Cofermentation of S. cerevisiae and L. plantarum was carried out under optimal conditions at 30°C for 28 days. pH levels were regularly monitored via an EDZO PH5011A pH meter after approximately 1 g of each sample was dissolved in 2.5 mL of purified water (pH 7.00). The liquid inoculum was absorbed into the substrate without being transformed into a wet slurry, resulting in a slightly moistened biomass with uniformly distributed microbial strains. The final product was stored as a powder in airtight, food-grade containers under controlled conditions to prevent nutrient degradation. For proximate analysis, 50 g of each sample was sent to the Institute of Plant Breeding Analytical Services Laboratory, University of the Philippines Los Baños, Laguna 4031 Philippines. The moisture content was quantified via gravimetric analysis through constant-weight oven drying at 50 °C for 48 hours. Ash content was determined via incineration in a muffle furnace at 550 °C for 8 hours until a white or light gray residue was obtained, indicating the complete combustion of organic matter. Crude fat was extracted via the Soxhlet method with a nonpolar solvent under reflux, followed by evaporation and gravimetric determination of the lipid content. Crude protein was assessed through Kjeldahl digestion involving acid digestion, neutralization, and subsequent nitrogen quantification via a colorimetric assay [35]. Crude fibers were analyzed via the Weende system, which involves sequential acid and alkali digestion to isolate indigestible fibrous residues. All the determinations were conducted in triplicate. The resulting data are then analyzed further via XLSTAT version 2024.4.2 (Addinsoft, Paris, France), which was released in October 2024. Results The one-way multivariate analysis of variance indicates that there is a significant difference in the dependent vector between the different groups, F (20, 20.85) = 2723.14, p << .001, Wilk's Λ = 4.64 × 10 -12 , η p 2 = .9996. The univariate, one-way analysis of variance for every parameter further revealed significant differences among the five experimental treatments in all cases: crude protein, F (4, 10) = 17.10, p << .001, η 2 = .87; moisture, F (4, 10) = 8015.52, p << .001, η² = 1.00; crude fat, F (4, 10) = 468.98, p << .001, η² = .99; crude fiber, F (4, 10) = 100.93, p << .001, η² = .98; and ash, F (4, 10) = 274.59, p << .001, η² = .99. Pearson correlation analysis among proximate parameters revealed consistently strong linear relationships. Moisture, crude protein, crude fat, and crude fiber are highly positively correlated with one another ( r ≥ .934). In contrast, ash is very strongly negatively correlated with all other parameters (| r | ≥ .905), with the highest inverse correlation observed between ash and moisture ( r = -.993). These relationships are reported in the correlation table (Table 2). Table 1 Proximate compositions of various keratin-bone composites. Parameter Treatment (in% dry weight) T 1 T 2 T 3 T 4 T 5 Moisture 19.33 ± 0.09 a 16.20 ± 0.09 b 13.20 ± 0.08 c 11.32 ± 0.07 d 8.66 ± 0.07 e Crude protein 76.67 ± 0.73 a 71.69 ± 5.10 ab 62.00 ± 5.52 bc 59.06 ± 3.89 c 53.19 ± 2.75 c Crude fat 17.00 ± 0.29 a 14.37 ± 0.19 b 12.35 ± 0.43 c 10.56 ± 0.20 d 8.42 ± 0.09 e Crude fiber 6.46 ± 0.58 a 4.95 ± 0.59 b 1.85 ± 0.17 cd 2.38 ± 0.17 c 1.02 ± 0.18 d Ash 0.33 ± 0.05 a 7.24 ± 0.33 b 14.29 ± 0.35 c 19.77 ± 0.32 d 26.26 ± 2.31 e The values are expressed as the means ± standard deviations (SDs; n = 3). Means in the same row with different superscript letters are significantly different at the p < .05 level. Table 2 Pearson correlation coefficient ( r ) for proximate parameters across various keratin-bone composites (n = 15). Parameter Moisture Crude protein Crude fat Crude fiber Ash Moisture .934*** .946*** .997*** -.993*** Crude protein .937*** .943*** -.905*** Crude fat .937*** -.924*** Crude fiber -.990*** Ash *** p < .001. Discussion The multivariate analysis of variance denotes a highly significant separation among the five cofermented treatments (T₁–T₅), confirming that all of them significantly varied in proximate composition. One-way ANOVAs conducted on individual parameters produced very large effect sizes (η² = .87–1.00), with moisture accounting for η² ≈ 1.00. Tukey’s HSD post hoc tests indicated that every adjacent pair of treatments differed significantly: the moisture content decreased in the order T₁ » T₂ » T₃ » T₄ » T₅, whereas the protein, fat, and fiber contents displayed the same stepwise decrease; the ash content followed an inverse pattern to that of the organic fractions. This suggests that the keratin-to-bone inputs yield reproducible, treatment-defining shifts in compositional profiles. The observed compositional gradient aligns with the expected dilution‒concentration trade-off. Substituting bone (high ash, low organic matter) with keratin (high protein, moderate fat) systematically reduces the organic fraction and elevates energy-bearing nutrients. The selection of an appropriate keratin–bone ratio therefore allows a formulator to maintain moisture and ash within the thresholds required for pellet integrity while targeting a species-specific range of requirements for protein and lipid content. Pearson correlation analysis revealed that moisture, protein, fat, and fiber covary strongly (r ≥ .934), whereas ash correlates inversely with these components (r ≈ -.99 versus moisture, -.90 to -.99 versus organics). In practical terms, any adjustment intended to, for example, reduce fiber will concurrently alter protein, fat, and moisture in lockstep , and will only increase ash when more bone is incorporated. This consistent interdependence implies that simple input ratios are sufficient to predict the proximate profile for precise diets that weigh protein–energy ratios against ash levels, which could compromise pellet stability and induce mineral leaching [36]. When complete feeds are formulated, dilution of a protein-dense concentrate, e.g., T₁, yields end products that retain elevated protein content. In contrast, high-protein concentrates with relatively high ash contents, i.e., T₅, are more satisfactory for inclusion when protein requirements are generally less than 50% , and supplemental minerals, at levels of approximately 25% or less, are desired without introducing excessive organic matter. Beyond proximate shifts, keratin–bone cofermentation engenders functional modifications. High-keratin blends (T₁, T₂) contain disulfide-rich proteins that microorganisms may hydrolyze into soluble oligopeptides, which act as natural emulsifiers and pellet binders [37]. Increasing the bone content (T₃–T₅) tilts the substrate toward calcium‒phosphate enrichment and residual collagen peptides that confer pellet firmness and facilitate the slow release of minerals [38]. Moreover, cofermentation with Saccharomyces cerevisiae and Lactiplantibacillus plantarum results in the production of a complex mixture of microbial metabolites, including amino acids, short peptides, organic acids, B vitamins, and cell wall polysaccharides, that extend the functionality of the concentrate [39]. At inclusion levels in tilapia diets, these byproducts improve digestibility, modulate gut health, and provide antioxidant benefits [40]. Thus, the concentrate functions as a bioactive ingredient rather than an inert filler. Collectively, these results establish a modular nutrient matrix whose composition can be precisely tuned by selecting a treatment level (T₁–T₅). This predictive model allows the formulation of stage-specific feeds, for example, a certain percentage of T₂ to target an increase in protein, pellet integrity, and gut resilience, or the inclusion of T₃ for supplemental minerals without being far from the required protein, yielding reproducible nutritional and functional outcomes. Limitations Only the proximate composition and the statistical evaluation of the resulting nutritional data were examined in this study, without extending to bioefficacy testing, which restricts discussion on actual nutritional utilization and physiological outcomes in vivo. No in vivo or in vitro assays were performed to determine nutrient bioavailability, digestibility, or metabolisable energy (ME), nor to evaluate feed conversion efficiency, somatic growth rate, innate immune response, or intestinal histomorphology. Declarations Acknowledgements We thank the Philippine National Collection of Microorganisms, National Institute of Molecular Biology and Biotechnology (BIOTECH, University of the Philippines Los Baños) for offering the active microbial strains used in this study and the Analytical Services Laboratory at the Institute of Plant Breeding (University of the Philippines Los Baños), for the analysis of our samples. Authors' contributions SVPV led the study design, analysis, and writing; JPF performed data encoding, validation, and drafting; DDOC contributed to validation, investigation, and drafting. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. 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These concentrates can precisely influence the total and digestible protein content necessary for specific species, whereas dilution precludes the excess of raw fishmeal or whole‐meal ingredients so that every gram of feed delivers essential amino acids rather than surplus nitrogenous ballast.\u003c/p\u003e\n\u003cp\u003eCertain farmed species in pisciculture present significantly varied protein requirements. Carnivores such as Atlantic salmon and rainbow trout thrive on diets containing nearly 42–49% CP to support rapid muscle accretion and high metabolic rates, resulting in feed‐conversion ratios (FCRs) near 1.0–1.25 [3, 4]. Omnivores, e.g., juvenile Nile tilapia, perform optimally at approximately 267 g/kg digestible protein (298 g/kg CP), yielding an FCR of 1.81, given the optimal water quality parameters [5]. Catfish typically require 32–35% CP, whereas penaeid shrimp consume 30–57% CP [6, 7]. A high‐protein concentrate can carry through these species-specific targets without over‐ or undershooting nutritional requirements.\u003c/p\u003e\n\u003cp\u003eEconomically, protein is the single costliest feed component, while feed itself accounts for 30–50% of the variable operating costs in most aquaculture operations [8]. Through concentrated protein sources that can precisely dial in (neither lower nor exceeding) dietary protein requirements using nonfish alternatives, producers can trim ingredient costs by 44% according to initial trial runs [9]. For carnivorous species whose feed bills are greatest, this precision can translate to thousands of dollars in savings per metric ton of fish produced. In terms of environmental sustainability, minimizing surplus protein in feed reduces nitrogenous waste; unbalanced amino acid intake triggers deamination of excess dietary protein, resulting in increased ammonia (NH₃) excretion [10]. Studies suggest that reformulating fish feeds on an ideal protein basis typically reduces total nitrogen output, which in turn eases the load on biofilters in recirculating systems and improves pond water quality [11]. Diluting high‐protein concentrates is a cost-effective, efficient, and environmentally responsible strategy, particularly if these concentrates have originated from biomass wastes (e.g., discarded chicken foot keratin and bone residues). Annually, the global poultry industry generates approximately 45.9 million tons of processing waste, with chicken feet constituting approximately 8.5% of that total, equating to nearly 3.9 million tons of chicken feet discarded [12, 13].\u003c/p\u003e\n\u003cp\u003eChicken foot keratin biomass, derived from epidermal scutes, reticulate scales, and claw sheaths, has biochemical and structural features that can be rendered as viable high-protein concentrates in pisciculture feeds. They are predominantly composed of \u003cem\u003eβ\u003c/em\u003e-keratin, a structural protein characterized by its \u003cem\u003eβ\u003c/em\u003e-pleated sheet conformation, which imparts remarkable mechanical strength and chemical resistance and results in a highly stable, insoluble network that withstands prolonged exposure to aquatic settings [14]. Chicken foot keratin comprises specialized isoforms that assemble into tightly packed filamentous networks for rigidity and elasticity [15], whereas such duality creates an ideal physical scaffold in fish farming systems. A durable substrate is necessary to support biofilm formation and microbial colonization, processes that improve nutrient cycling and water quality [16]. Although keratin is highly recalcitrant, controlled biochemical treatments can gradually degrade it into bioactive peptides and amino acids [17]. These released compounds may provide low-nitrogen macronutrients for aquatic organisms or stimulate beneficial microbial communities that improve water conditions. This nutritional aspect is further highlighted in the context of keratin’s potential as an alternative protein source in food and nutrition, where even its partial hydrolysis yields a compound with functional bioactivity [18]. Its minimal fat and carbohydrate content reduces the risk of oxidative deterioration and imbalances in the gut microbiome, whereas residual indigestible fibers contribute to structural reinforcement without impeding nutrient diffusion [19, 20].\u003c/p\u003e\n\u003cp\u003eProcessing raw keratin biomass\u003cdel cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003e,\u003c/del\u003e through methods such as grinding, which mechanically reduces it to powder, and fermentation\u003cdel cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003e,\u003c/del\u003e can partially compromise its original scaffold, which supports biofilm formation [21]. However, in feed applications, it could primarily be intended to be a high-protein concentrate that can offer digestible nutrients and bioactive compounds to the fish rather than preserving long-term structural integrity as a scaffold. Even if the natural filamentous network is diminished, residual structural integrity (specifically when modestly treated) and the increased surface area of pellets (when binders are used) can nevertheless maintain adequate physical strength for in‐water exposure.\u003c/p\u003e\n\u003cp\u003eChicken foot bone-derived biomass from the tarsometatarsus and distal phalanx consists of a type I collagen matrix interwoven with hydroxyapatite crystals [22]. During digestion, the collagen in chicken foot biomass undergoes hydrolysis, a process facilitated by proteolytic enzymes that break down high-molecular-weight proteins into smaller peptides and amino acids [23]. This enzymatic degradation begins in the stomach and continues in the small intestine, where enzymes such as pepsin, trypsin, and chymotrypsin sequentially cleave peptide bonds [24]. The breakdown process produces collagen hydrolysates, consisting of bioactive dipeptides and tripeptides, along with unbound amino acids such as glycine, proline, and hydroxyproline, which are essential for nutritional health [25].\u003c/p\u003e\n\u003cp\u003eIn addition to collagen peptides, mineral ions such as calcium and phosphorus are essential for fish nutrition, specifically for skeletal development, growth, and metabolic health [26]. During digestion, these minerals are also gradually released, becoming more bioavailable and easily incorporated into new bone structures and other cellular functions. This slow-release mechanism can prolong the postprandial increase in plasma amino acid levels [27]. Such a sustained supply of amino acids is particularly beneficial for fish, supporting continuous growth and repair processes, especially during periods of low feed intake.\u003c/p\u003e\n\u003cp\u003eFermentation relies on microorganisms such as \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e (formerly \u003cem\u003eLactobacillus plantarum\u003c/em\u003e), which are known for their ability to degrade resistant protein structures, thereby improving the nutritional quality of fermented substrates. Although these microbes do not produce the specialized keratinolytic enzymes required for complete keratin degradation, a function attributed to certain \u003cem\u003eBacillus\u003c/em\u003e species and fungi, they exhibit general proteolytic activity and induce acidification that can weaken the dense disulfide-bond network of keratin [28, 29]. This proteolysis and partial structural weakening could be intended to modestly preserve the keratin structure, preventing unnecessary extreme degradation that is likely to result in the rapid depletion of bioactive nutrients when submerged in fish culture water.\u003c/p\u003e\n\u003cp\u003eIn various bioprocesses, the synergistic interaction of multiple microbes regulates the extent of degradation. The literature suggests that \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e, individually or in combination with more potent degraders, contribute to improving the efficiency and quality of fermentation [30, 31].\u003c/p\u003e\n\u003cp\u003eA comparable synergistic process may occur in bone residues, where instead of collagenase-producing microbes, \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e are employed for similar fundamental and strategic purposes, facilitating proteolysis (not degraded to a greater or lesser extent, with further proteolysis occurring during digestion when fed), acidification, and partial structural weakening, specifically hydroxyapatite dissolution and collagen softening of chicken foot bone biomass [32]. This mechanism promotes peptide release and optimizes calcium and phosphorus bioavailability while preserving residual structural integrity, similarly addressing the risks of nutrient leaching, eutrophication, increased biochemical oxygen demand, and water pollution caused by uneaten feed settling in sediments when bioactive nutrients are depleted too rapidly in pisciculture water [33].\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe process began with procuring 5 kg of raw chicken feet from a local wet market in San Pablo City, Laguna, Philippines. Quality standards were adhered to by inspecting the visual and tactile properties of the chicken feet, which exhibited a pinkish color, moist texture, and no off-odors; feet displaying discoloration, cuts, or excessive dirt were rejected. This initial mass yielded approximately 300 g of keratin and 700 g of bone residue, with the remaining materials set aside for purposes beyond the scope of this research.\u003c/p\u003e\n\u003cp\u003eDuring pretreatment and fermentation, food-grade gloves, laboratory aprons, heat-resistant borosilicate glassware, an analytical balance, a thermometer, and a precise timing device were used for procedural accuracy and sterility. The biomass components were first separated for accurate weighing and then thoroughly washed with clean, potable water while being placed on a sterile metal strainer to remove dirt and contaminants. The biomass that was separated with a sterile knife are meticulously processed and subsequently steam sterilized and placed on borosilicate glassware in a stovetop pressure cooker (Chef\u0026apos;s Classics Zinnia Stainless Steel Pressure Cooker, 8 L) at medium heat for 5 minutes for uniform heat distribution. Steam sterilization through high-temperature saturated steam is preferred over boiling or chemical disinfection to preserve nutrient integrity and avoid potentially toxic residues from chemical disinfectants [34].\u003c/p\u003e\n\u003cp\u003eAfter sterilization, the biomass was oven-dried (Oven: La Germania FS8041 30XTR) on a sterile tray in a thin, even layer (approximately 1 cm) at a controlled medium heat for 15 minutes for uniform moisture removal. Once dried, the biomass was reduced to fine particles via industrial grinding equipment (Philips HR1744 Cucina) and a mortar and pestle in preparation for fermentation.\u003c/p\u003e\n\u003cp\u003eFor the fermentation stage, active \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e strains were obtained from the Philippine National Collection of Microorganisms, National Institute of Molecular Biology \u0026amp; Biotechnology, University of the Philippines Los Ba\u0026ntilde;os, Laguna 4031 Philippines, under accession numbers BIOTECH 2055 and BIOTECH 10968, respectively. These strains, with microbial loads of 1 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e CFU/mL, were inoculated at a concentration of 5% relative to the substrate mass and mixed at a 1:1 ratio. Fermentation was conducted with five substrate combinations: (T\u003csub\u003e1\u003c/sub\u003e) 65 g of keratin; (T\u003csub\u003e2\u003c/sub\u003e) 48.75 g of keratin and 16.25 g of bones; (T\u003csub\u003e3\u003c/sub\u003e) 32.5 g of keratin and 32.5 g of bones; (T\u003csub\u003e4\u003c/sub\u003e) 16.25 g of keratin and 48.75 g of bones; and (T\u003csub\u003e5\u003c/sub\u003e) 65 g of bones. Cofermentation of \u003cem\u003eS. cerevisiae\u003c/em\u003e and \u003cem\u003eL. plantarum\u003c/em\u003e was carried out under optimal conditions at 30\u0026deg;C for 28 days. pH levels were regularly monitored via an EDZO PH5011A pH meter after approximately 1 g of each sample was dissolved in 2.5 mL of purified water (pH 7.00).\u003c/p\u003e\n\u003cp\u003eThe liquid inoculum was absorbed into the substrate without being transformed into a wet slurry, resulting in a slightly moistened biomass with uniformly distributed microbial strains. The final product was stored as a powder in airtight, food-grade containers under controlled conditions to prevent nutrient degradation.\u003c/p\u003e\n\u003cp\u003eFor proximate analysis, 50 g of each sample was sent to the Institute of Plant Breeding Analytical Services Laboratory, University of the Philippines Los Ba\u0026ntilde;os, Laguna 4031 Philippines. The moisture content was quantified via gravimetric analysis through constant-weight oven drying at 50 \u0026deg;C for 48 hours. Ash content was determined via incineration in a muffle furnace at 550 \u0026deg;C for 8 hours until a white or light gray residue was obtained, indicating the complete combustion of organic matter. Crude fat was extracted via the Soxhlet method with a nonpolar solvent under reflux, followed by evaporation and gravimetric determination of the lipid content. Crude protein was assessed through Kjeldahl digestion involving acid digestion, neutralization, and subsequent nitrogen quantification via a colorimetric assay [35]. Crude fibers were analyzed via the Weende system, which involves sequential acid and alkali digestion to isolate indigestible fibrous residues. All the determinations were conducted in triplicate. The resulting data are then analyzed further via XLSTAT version 2024.4.2 (Addinsoft, Paris, France), which was released in October 2024.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe one-way multivariate analysis of variance indicates that there is a significant difference in the dependent vector between the different groups, \u003cem\u003eF\u003c/em\u003e(20, 20.85) = 2723.14, \u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026lt; .001, Wilk's \u003cem\u003eΛ\u003c/em\u003e = 4.64 × 10\u003csup\u003e-12\u003c/sup\u003e, η\u003csub\u003ep\u003c/sub\u003e\u003csup\u003e2\u003c/sup\u003e = .9996.\u003c/p\u003e\n\u003cp\u003eThe univariate, one-way analysis of variance for every parameter further revealed significant differences among the five experimental treatments in all cases: crude protein, \u003cem\u003eF\u003c/em\u003e(4, 10) = 17.10, \u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026lt; .001, η\u003csup\u003e2\u003c/sup\u003e = .87; moisture, \u003cem\u003eF\u003c/em\u003e(4, 10) = 8015.52, \u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026lt; .001, η² = 1.00; crude fat, \u003cem\u003eF\u003c/em\u003e(4, 10) = 468.98, \u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026lt; .001, η² = .99; crude fiber, \u003cem\u003eF\u003c/em\u003e(4, 10) = 100.93, \u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026lt; .001, η² = .98; and ash, \u003cem\u003eF\u003c/em\u003e(4, 10) = 274.59, \u003cem\u003ep\u003c/em\u003e \u0026lt;\u0026lt; .001, η² = .99.\u003c/p\u003e\n\u003cp\u003ePearson correlation analysis among proximate parameters revealed consistently strong linear relationships. Moisture, crude protein, crude fat, and crude fiber are highly positively correlated with one another (\u003cem\u003er\u003c/em\u003e ≥ .934). In contrast, ash is very strongly negatively correlated with all other parameters (|\u003cem\u003er\u003c/em\u003e| ≥ .905), with the highest inverse correlation observed between ash and moisture (\u003cem\u003er\u003c/em\u003e = -.993). These relationships are reported in the correlation table (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProximate compositions of various keratin-bone composites.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"741\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003eTreatment (in% dry weight)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eT\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eT\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eT\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eT\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eT\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMoisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e19.33 ± 0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16.20 ± 0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e13.20 ± 0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e11.32 ± 0.07\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.66 ± 0.07\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrude protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e76.67 ± 0.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e71.69 ± 5.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e62.00 ± 5.52\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e59.06 ± 3.89\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e53.19 ± 2.75\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrude fat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e17.00 ± 0.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14.37 ± 0.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12.35 ± 0.43\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10.56 ± 0.20\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e8.42 ± 0.09\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrude fiber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e6.46 ± 0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.95 ± 0.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.85 ± 0.17\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.38 ± 0.17\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.02 ± 0.18\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAsh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e0.33 ± 0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.24 ± 0.33\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14.29 ± 0.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e19.77 ± 0.32\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e26.26 ± 2.31\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe values are expressed as the means ± standard deviations (SDs; n = 3). Means in the same row with different superscript letters are significantly different at the \u003cem\u003ep\u003c/em\u003e \u0026lt; .05 level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePearson correlation coefficient (\u003cem\u003er\u003c/em\u003e) for proximate parameters across various keratin-bone composites (n = 15).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"741\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eMoisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCrude protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCrude fat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eCrude fiber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eAsh\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMoisture\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e.934***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e.946***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e.997***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-.993***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrude protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e.937***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e.943***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-.905***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrude fat\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e.937***\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-.924***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eCrude fiber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e-.990***\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAsh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e***\u003cem\u003ep\u003c/em\u003e \u0026lt; .001.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe multivariate analysis of variance denotes a highly significant separation among the five cofermented treatments (T₁–T₅), confirming that all of them significantly varied in proximate composition. One-way ANOVAs conducted on individual parameters produced very large effect sizes (η² = .87–1.00), with moisture accounting for η² ≈ 1.00. Tukey’s HSD post hoc tests indicated that every adjacent pair of treatments differed significantly: \u003cins cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003ethe \u003c/ins\u003emoisture content decreased in the order T₁ » T₂ » T₃ » T₄ » T₅, whereas the protein, fat, and fiber contents displayed the same stepwise decrease; the ash content followed an inverse pattern to that of the organic fractions. This suggests that the keratin-to-bone inputs yield reproducible, treatment-defining shifts in compositional profiles.\u003c/p\u003e\n\u003cp\u003eThe observed compositional gradient aligns with the expected dilution‒concentration trade-off. Substituting bone (high ash, low organic matter) with keratin (high protein, moderate fat) systematically reduces \u003cins cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003ethe \u003c/ins\u003eorganic fraction and elevates energy-bearing nutrients. The selection of an appropriate keratin–bone ratio therefore allows a formulator to maintain moisture and ash within \u003cins cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003ethe \u003c/ins\u003ethresholds required for pellet integrity while targeting \u003cins cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003ea \u003c/ins\u003especies-specific range of requirements for protein and lipid content.\u003c/p\u003e\n\u003cp\u003ePearson correlation analysis revealed that moisture, protein, fat, and fiber covary strongly (r ≥ .934), whereas ash correlates inversely with these components (r ≈ -.99 versus moisture, -.90 to -.99 versus organics). In practical terms, any adjustment intended to, for example, reduce fiber will concurrently alter protein, fat, and moisture in lockstep\u003cdel cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003e,\u003c/del\u003e and will only increase ash when more bone is incorporated. This consistent interdependence implies that simple input ratios are sufficient to predict the proximate profile for precise diets that weigh protein–energy ratios against ash levels, which could compromise pellet stability and induce mineral leaching [36].\u003c/p\u003e\n\u003cp\u003eWhen complete feeds are formulated, dilution of a protein-dense concentrate, e.g., T₁, yields end products that retain elevated protein content. In contrast, high-protein concentrates with relatively high ash contents, i.e., T₅, are more satisfactory for inclusion when protein requirements are generally less than 50%\u003cins cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003e,\u003c/ins\u003e and supplemental minerals, at levels of approximately 25% or less, are desired without introducing excessive organic matter.\u003c/p\u003e\n\u003cp\u003eBeyond proximate shifts, keratin–bone cofermentation engenders functional modifications. High-keratin blends (T₁, T₂) contain disulfide-rich proteins that microorganisms may hydrolyze into soluble oligopeptides, which act as natural emulsifiers and pellet binders [37]. Increasing the bone content (T₃–T₅) tilts the substrate toward calcium‒phosphate enrichment and residual collagen peptides that confer pellet firmness and facilitate the slow release of minerals [38].\u003c/p\u003e\n\u003cp\u003eMoreover, cofermentation with \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e results in the production of a complex mixture of microbial metabolites, including amino acids, short peptides, organic acids, B vitamins, and cell wall polysaccharides, that extend the functionality of the concentrate [39]. At inclusion levels in tilapia diets, these byproducts improve digestibility, modulate gut health, and provide antioxidant benefits [40]. Thus, the concentrate functions as a bioactive ingredient rather than an inert filler.\u003c/p\u003e\n\u003cp\u003eCollectively, these results establish a modular nutrient matrix whose composition can be precisely tuned by selecting a treatment level (T₁–T₅). This predictive model allows \u003cins cite=\"mailto:Editor%202\" datetime=\"2025-06-24T01:21\"\u003ethe \u003c/ins\u003eformulation of stage-specific feeds, for example, a certain percentage of T₂ to target an increase in protein, pellet integrity, and gut resilience, or the inclusion of T₃ for supplemental minerals without being far from the required protein, yielding reproducible nutritional and functional outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eOnly the proximate composition and the statistical evaluation of the resulting nutritional data were examined in this study, without extending to bioefficacy testing, which restricts discussion on actual nutritional utilization and physiological outcomes in vivo.\u003c/li\u003e\n\u003cli\u003eNo in vivo or in vitro assays were performed to determine nutrient bioavailability, digestibility, or metabolisable energy (ME), nor to evaluate feed conversion efficiency, somatic growth rate, innate immune response, or intestinal histomorphology.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Philippine National Collection of Microorganisms, National Institute of Molecular Biology and Biotechnology (BIOTECH, University of the Philippines Los Baños) for offering the active microbial strains used in this study and the Analytical Services Laboratory at the Institute of Plant Breeding (University of the Philippines Los Baños), for the analysis of our samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSVPV led the study design, analysis, and writing; JPF performed data encoding, validation, and drafting; DDOC contributed to validation, investigation, and drafting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\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\u003eConsent for publication\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 declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no specific grant was received from public, commercial, or non-profit funding agencies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMiles RD, Chapman FA. 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Aquacult Nutr. 2015;22(5):956\u0026ndash;65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/anu.12312\u003c/span\u003e\u003cspan address=\"10.1111/anu.12312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"protein concentrate, fermented poultry waste, keratin, bone biomass","lastPublishedDoi":"10.21203/rs.3.rs-6965350/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6965350/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eA cofermentation process involving the fermentation of five chicken foot biomass treatments with various keratin-to-bone inputs was conducted with\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eSaccharomyces cerevisiae\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eand\u003c/span\u003e \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eat 30\u0026deg;C for 28 days. The fermented samples were analyzed for proximate composition through standardized gravimetric and colorimetric methods, with the results subjected to one-way multivariate analysis of variance, univariate analysis of variance for every parameter, and Pearson correlation analysis. Significant compositional differences were observed among the treatments, Wilks\u0026rsquo;\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003eΛ\u003c/span\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e= 4.64 \u0026times; 10⁻\u0026sup1;\u0026sup2;\u003c/span\u003e, \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003ep\u003c/span\u003e\u0026thinsp;\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;.001, with large effect sizes for all the parameters, η\u0026sup2; \u0026ge; .87. The highest protein content, 76.67%, on a dry weight basis, was recorded in the pure keratin variant, whereas the ash content peaked at 26.26% in the bone-dominant samples. Moisture, protein, fat, and fiber positively covaried, with\u003c/span\u003e \u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003er\u003c/span\u003e\u0026thinsp;\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e\u0026ge;\u0026thinsp;.934, contrasting significantly with ash, with |\u003c/span\u003e\u003cspan type=\"ItalicSmallCaps\" class=\"ItalicSmallCaps\" name=\"Emphasis\"\u003er\u003c/span\u003e\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003e| \u0026ge; .905. Formulations may therefore be selectively tuned to align with dietary strategies for certain ontogenetic stages of the target species upon proportioning keratin and bone inputs to yield an ideal protein-to-ash ratio to be diluted within pisciculture feeds. To validate the in vitro proximate predictions with in vivo efficacy data, studies can quantify performance outcomes across these inclusion rates, including but not limited to, feed conversion efficiency, the somatic growth rate, the innate immune response, and gut histomorphology.\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e","manuscriptTitle":"Proximate composition of fermented Gallus gallus foot keratin‒bone composites as viable high-protein concentrates intended for dilution in pisciculture feeds","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 15:02:06","doi":"10.21203/rs.3.rs-6965350/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-02T09:28:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-05T04:41:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T11:24:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T02:41:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166422347325167065398368261312982554701","date":"2025-07-29T06:44:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73086684092113280654291850136387205861","date":"2025-07-22T08:18:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309046708967161326411514345372189888830","date":"2025-07-22T05:50:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287003411864511521826119359643508259288","date":"2025-07-22T05:00:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-21T17:07:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-25T12:07:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-24T23:15:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-24T23:15:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-06-24T11:30:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2d70a47b-ca0e-48cc-9e62-66412c48603e","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:03:24+00:00","versionOfRecord":{"articleIdentity":"rs-6965350","link":"https://doi.org/10.1186/s13104-026-07717-6","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2026-02-17 15:58:22","publishedOnDateReadable":"February 17th, 2026"},"versionCreatedAt":"2025-07-25 15:02:06","video":"","vorDoi":"10.1186/s13104-026-07717-6","vorDoiUrl":"https://doi.org/10.1186/s13104-026-07717-6","workflowStages":[]},"version":"v1","identity":"rs-6965350","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6965350","identity":"rs-6965350","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}