From slaughterhouse waste to functional feed: pilot-scale bovine blood hydrolysate improves antioxidant activities and meat quality in slow-growing chickens
Saruttiwong Boonkong, Phanthipha Laosam, Pichitpon Luasiri, Phornpilat Senanok, Sukanya Tastub, Saranya Suwanangul, Wittawat Molee, Chatsirin nakharuthai, Jukkrapong Pinyo, Rayudika Aprilia Patindra Purba, Papungkorn Sangsawad
TL;DR
This study shows how bovine blood waste can be converted into a functional feed supplement that improves chicken health and meat quality.
Contribution
The novel contribution is the successful pilot-scale production of bovine blood hydrolysate with proven functional benefits in poultry feed.
Findings
Pilot-scale hydrolysis achieved 15.08% degree of hydrolysis and 50.23% protein recovery.
BBH supplementation improved antioxidant enzyme activities in chickens by 13 to 33%.
Meat quality improved with a 3.86% reduction in cooking loss.
Abstract
Bovine blood represents 4% of slaughter weight yet remains largely underutilised despite high protein content. This study scaled enzymatic hydrolysis from laboratory to pilot-scale production while evaluating bovine blood hydrolysate (BBH) as an alternative to commercial yeast hydrolysate (YH) in slow-growing chicken (Korat chickens) nutrition. Pilot-scale Neutrase hydrolysis of 400 kg batches achieved 15.08% degree of hydrolysis and 50.23% protein recovery, exceeding laboratory-scale performance. Comprehensive characterization revealed that BBH contained 59% of peptides within the bioactive 0.4 to 2 kDa range and exhibited 45.09% β-sheet secondary structure. Amino acid profiling demonstrated substantial enrichment in branched-chain amino acids, with leucine reaching 11,131 mg per 100 g representing 426% greater abundance than commercial YH. BBH exhibited superior in vitro antioxidant…
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Figure 9- —Thailand Science Research and Innovation (TSRI), and the National Science, Research, and Innovation Fund (NSRF)
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Taxonomy
TopicsProtein Hydrolysis and Bioactive Peptides · Insect Utilization and Effects · Meat and Animal Product Quality
Introduction
The global meat processing industry generates approximately 4% of live animal weight as blood, translating to an estimated 4 million tons annually from cattle alone. Despite containing 18–20% protein with complete amino acid profiles, this blood remains largely underutilised (Bah et al., 2013). Current disposal methods impose substantial economic burdens, with treatment costs ranging from USD 50–100 per metric ton (2025 estimates), while improper disposal contributes to elevated biochemical oxygen demand in water systems and greenhouse gas emissions from anaerobic decomposition (Purba, 2025). Yet this substantial waste stream presents genuine valorization opportunities. Blood proteins possess inherent bioactive potential; hemoglobin and plasma proteins contain encrypted peptide sequences that exhibit antioxidant, antimicrobial, and immunomodulatory activities upon enzymatic hydrolysis (Bah et al., 2013; Martínez-Alvarez et al., 2015). The technical challenge lies not in whether blood proteins can be hydrolysed—laboratory research has established this feasibility—but in whether enzymatic conversion can be scaled to commercially viable production volumes while overcoming substrate-specific obstacles.
Blood presents unique processing complexities absent in conventional protein sources. First, the fibrinogen-mediated coagulation pathway activates spontaneously upon collection, creating viscous gel structures that impede enzyme accessibility and uniform mixing. Second, heat treatment required for microbial safety denatures globular proteins, altering secondary structures and potentially affecting subsequent hydrolysis kinetics. Third, high iron content from heme groups catalyzes oxidative reactions that may degrade bioactive peptides during processing and storage. These substrate-specific challenges explain why laboratory-scale protocols fail to translate directly to industrial volumes, where mixing dynamics, heat transfer coefficients, and enzyme distribution patterns differ fundamentally from bench-scale reactors (Hou et al., 2022).
Commercial yeast hydrolysates are well-established feed additives with documented benefits for antioxidant status and growth performance in poultry, attributed to bioactive compounds including glutathione and β-glucans (Wang et al., 2022). However, production costs exceeding USD 3–5 per kg limit widespread adoption, particularly in cost-sensitive markets where feed represents 60–70% of total production costs. Animal by-product hydrolysates represent an underexplored alternative with favorable amino acid profiles closely matching poultry requirements, yet systematic benchmarking against commercial products remains absent from the literature.
Previous studies investigating blood-derived hydrolysates have focused predominantly on laboratory-scale production using 1–5 L reactors for bioactive peptide characterization (Fu et al., 2019; Laosam, 2024; Abou-Diab et al., 2020) or evaluated different blood sources—including porcine hemoglobin (Fu et al., 2019), chicken blood (Zheng et al., 2018; Hamzeh et al., 2019), and blood meal as cellular antioxidants—without direct dietary supplementation in poultry feeding trials. Only one study demonstrated pilot-scale production for duck blood hydrolysate at 1000 L volume (Laosam, 2024); bovine blood at equivalent commercial scale remains unexamined. Building upon our previous research (Boonkong et al., 2024), which demonstrated the antioxidant potential of bovine blood protein hydrolysates at laboratory scale, three fundamental gaps prevent industrial translation and limit mechanistic understanding. First, no prior research has systematically scaled bovine blood hydrolysate (BBH) production to pilot scale (≥ 400 kg batches) with comprehensive documentation of how degree of hydrolysis, protein recovery, and product consistency change with reactor volume, mixing regime, and thermal management strategies. Second, direct comparison between BBH and established commercial yeast products across structural characteristics (peptide size distribution, secondary protein conformations), nutritional composition, and biological efficacy in controlled feeding trials is absent, preventing objective assessment of functional equivalence required for industry adoption. Third, integrated understanding linking specific peptide characteristics to antioxidant enzyme induction pathways and physiological outcomes in poultry remains poorly characterized, limiting rational optimization of processing parameters and supplementation strategies.
Addressing these gaps holds substantial practical and scientific significance. Scale-up from laboratory to pilot-scale enzymatic hydrolysis involves complex optimization challenges beyond simple volumetric conversion. Substrate-to-water ratios that ensure adequate enzyme-substrate contact at laboratory scale create excessive viscosity in large reactors, impeding mixing and heat distribution. pH control regimes employing manual titration in bench-scale systems become impractical at commercial volumes, where pH drift during hydrolysis affects protease activity and peptide release kinetics. Enzyme costs—negligible at laboratory scale—become economically dominant at pilot scale, necessitating optimization of enzyme-to-substrate ratios that balance degree of hydrolysis with production economics (Tran et al., 2021). These technical transitions require systematic documentation yet remain scarce in the literature.
Slow-growing chicken breeds, exemplified by indigenous varieties like Korat chickens, present biologically appropriate models for evaluating sustained antioxidant interventions. These birds exhibit distinct metabolic profiles compared to fast-growing commercial broilers, including lower basal metabolic rates and stronger reliance on oxidative phosphorylation through superoxide dismutase-based antioxidant pathways rather than uric acid-mediated mechanisms (Coudert et al., 2023). Their extended rearing periods (9–12 weeks) create opportunities for demonstrating cumulative effects of dietary antioxidants on both systemic redox status and meat quality characteristics. The longer production cycles allow assessment of whether enhanced antioxidant enzyme activities translate to improved water-holding capacity and reduced lipid oxidation in meat—parameters increasingly important to both consumers and processors. Consistent with the scientific consensus, bioactive peptides can simultaneously enhance endogenous antioxidant defense systems through upregulation of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities while improving meat quality characteristics including water-holding capacity and oxidative stability during storage and processing (Sarmadi & Ismail, 2010; Nwachukwu & Aluko, 2019; Xu et al., 2024).
Therefore, this study aimed to: (1) scale up bovine blood protein hydrolysate production from laboratory (200 g) to pilot scale (400 kg batches), optimizing processing parameters for maximum efficiency; (2) comprehensively characterize BBH through structural analysis, nutritional profiling, and direct comparison with commercial yeast hydrolysate (YH); and (3) evaluate BBH efficacy as a feed additive through in vivo performance assessment in slow-growing chickens. This research addresses the identified knowledge gaps through systematic documentation of pilot-scale production compared to laboratory scale, quantifying improvements in degree of hydrolysis and protein recovery attributable to enhanced mixing dynamics and thermal control; direct benchmarking of BBH against commercial YH across structural parameters via size exclusion chromatography and Fourier transform infrared spectroscopy, nutritional composition with emphasis on essential amino acid profiles, and biological efficacy measured by antioxidant enzyme activities in serum and hepatic tissue alongside growth performance and meat quality characteristics; and integrated structure-function evaluation linking specific peptide characteristics (size distribution, secondary structure content) to antioxidant mechanisms (SOD, GPx, CAT activities) and physiological outcomes (meat water-holding capacity, cooking loss).
However, methodological constraints must be acknowledged. This study employed: (1) heat-treated bovine blood (90 °C, 30 min) to ensure microbial safety and inactivate fibrinogen; (2) single-condition enzymatic hydrolysis using Neutrase (pH 7.0, 50 °C) selected for neutral pH operation, avoiding sodium chloride contamination from acid-base neutralization; (3) hot-air drying (70 °C, 12 h) maintained below temperatures associated with Maillard reactions; and (4) a single supplementation level (0.1% BBH) based on practical commercial inclusion rates. While these parameters reflect operational constraints in commercial manufacturing and ensure product safety, they may influence peptide profiles compared to alternative processing conditions and limit generalizability to other enzyme-substrate combinations or thermal treatments. Within these constraints, we hypothesised that: (H1) pilot-scale BBH would demonstrate antioxidant capacity comparable to commercial YH as measured by ABTS radical scavenging, ferric-reducing power, and metal-chelating activities; (H2) 0.1% BBH supplementation would enhance antioxidant enzyme activities and meat water-holding capacity without compromising growth performance relative to unsupplemented controls; and (H3) these biological effects would correlate with predominance of peptides in the < 2 kDa molecular weight range and elevated β-sheet secondary structure content. This integrated approach provides essential technical parameters for commercial-scale manufacturing while advancing fundamental understanding of how blood protein valorization contributes to sustainable circular economy models in the meat processing industry.
Materials and methods
Chemicals and reagents
The chemicals and reagents utilised in the research were obtained from Sigma Chemical Co. in St. Louis, MO. The chemicals used in the study included 2,4,6-Trinitrobenzene-1-Sulfonic Acid (TNBS), ethylenediaminetetraacetic acid, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), Trolox, 2,4,6-tripyridyl-s-triazine (TPTZ), ferrozine, acetonitrile, trifluoroacetic acid (TFA), Cytochrome-C, aprotinin, and tyrosine. Neutrase, with an activity of 0.8 Anson Units per gram (AU-N/g), was obtained from Brenntag Ingredients (Thailand) Company Limited. All accessory chemicals and reagents utilised were of analytical quality.
Blood sample collection and Preparation
Fresh blood samples were collected from 100 Thai Indigenous Brahman Charolais bovines (30–36 months old) during standard slaughtering procedures at Pon-Yang-Khram Livestock Breeding Cooperative NSC. LTD., Sakon Nakhon Province, Thailand. Approximately 5 L of blood was collected per animal using sterile containers. This yielded a total pooled volume of 500 L from 100 bovines. Because the blood was immediately transferred to the processing facility within 30 min of collection and subjected to heat treatment at 90 °C for 30 min, no anticoagulants were added. For practical purposes, this method was adjusted to induce protein denaturation for subsequent hydrolysis and to eliminate microbial contaminants, following the facility’s standard processing protocol. The rapid heat treatment prevented clotting and ensured product safety. The heated blood was carefully cooled on ice until reaching an internal temperature of 4 °C, then transferred to a freezer maintained at −20 °C for storage. Before hydrolysis experiments, frozen samples were thawed and thoroughly ground using a commercial grinder. Proximate composition including crude protein by Kjeldahl method, crude fat by Soxhlet extraction, and moisture and ash content was analysed according to AOAC guidelines (Horwitz & Latimer, 2005).
BBH production
The experiment was conducted to build upon previous research on laboratory (LAB)-scale production by Boonkong et al. (Boonkong et al., 2024). The flow of LAB-scale and pilot-scale production and characterization is shown in Fig. 1. In the LAB-scale phase, a 200 g boiled bovine blood sample (32.5 g protein) was combined with 100 mL of deionised (DI) water and hydrolysed with 5% (enzyme/protein substrate) of Neutrase (pH 7.0, 50 °C). Then, the mixture was shaken for 4 h at 120 rpm. Heating at 95 °C for 30 min stopped hydrolysis. Then, centrifugation (10,000×g, 10 min, 4 °C) was performed, and the supernatant was stored at − 20 °C for additional analysis, including protein recovery, degree of hydrolysis, and antioxidant activity.
Fig. 1. Schematic diagram of bovine blood hydrolysate production from laboratory and pilot-scale enzymatic hydrolysis to characterization and comparison with commercial yeast hydrolysate
In the pilot-scale phase, 400 kg of the boiled bovine blood sample was combined with 200 L of DI water. Neutrase was employed as the hydrolyzing agent, applied at a concentration of 5% (enzyme/protein substrate). The hydrolysis followed specific conditions, maintaining a temperature of 50 °C and an agitation speed of 50 rpm in a jacketed cooker for 4 h. The enzymatic reaction was terminated by heating the mixture to 95 °C for 30 min. After hydrolysis, solid-liquid separation was carried out using a horizontal centrifugal sieve for 10 min, after which the liquid fraction was collected. This liquid was subsequently centrifuged at 5,000 g for 10 min at 4 °C. The resulting supernatant was stored at −20 °C until further analysis. The supernatant was used for additional analysis including protein recovery, degree of hydrolysis, and antioxidant activity. Then, the supernatant sample was dried in a hot air oven at 70 °C for 12 h. The dried hydrolysate was powdered using a pulverizer and stored in vacuum-sealed bags containing 1 kg of the product. This product hereafter is called BBH.
BBH characterization
Protein recovery and degree of hydrolysis
Protein recovery was determined using Kjeldahl method AOAC guidelines (Horwitz & Latimer, 2005). Samples were digested with H_2_SO_4_ and copper catalyst, distilled with NaOH, and titrated with HCl. Protein recovery was calculated as:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{Protein recovery }}\left( \% \right){\text{ }}=\frac{{{\text{Protein in hydrolysate recovered}}}}{{{\text{Total protein in blood of the original sample}}}} \times 100$$\end{document}The degree of hydrolysis (DH) was determined using TNBS method. The sample (20 µL) was mixed with 100 µL phosphate buffer (0.2125 M, pH 8.2) and 50 µL of 0.05% TNBS solution. The mixture was incubated at 45 °C for 30 min in dark. The reaction was stopped by adding 4 mL 0.1 N HCl. Absorbance was measured at 340 nm. DH was calculated as:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\text{DH }}\left( \% \right){\text{ }}=\frac{{\alpha - {\text{amino content of hydrolysate recovered}}}}{{{\text{Total }}\alpha - {\text{amino content of the original sample}}}} \times {\text{ 100}}$$\end{document}Antioxidant activity assays
All antioxidant assays were performed at standardised protein concentration (1.0 mg/mL) with sample blanks to correct for intrinsic absorbance and potential interference from residual heme or minerals in BBH. The ABTS^•+^ assay was performed according to the protocol described by Khongla et al. (Khongla et al., 2022). A 5 µL peptide sample (with a concentration of 1 mg/mL) was mixed with a 200 µL solution of ABTS in a 96-well microplate. The reaction was incubated in darkness for 5 min before being measured at a wavelength of 734 nm using a microplate reader (Varioskan LUX, Thermo Scientific, Vantaa, Finland). Sample blanks (hydrolysate without ABTS reagent) were measured simultaneously and subtracted from sample readings. A trolox standard curve, which ranged from 0 to 2.5 mg/mL, was used to calculate the results. The activity was then reported in terms µg/mL trolox equivalence.
The ferric-reducing antioxidant power (FRAP) assay was performed according to the protocol described by Suwanangul et al. (Suwanangul et al., 2021). A 10 µL peptide sample at 1 mg/mL concentration was mixed with a 100 µL FRAP reagent in a 96-well microplate. The reaction was incubated at ambient temperature for 15 min before being quantified at a wavelength of 593 nm using a microplate reader (Varioskan LUX, Thermo Scientific, Vantaa, Finland). Sample blanks were included to account for background absorbance. A trolox standard curve, which ranged from 0 to 2.5 mg/mL, was used to calculate the results. The activity was then reported in terms of µg/mL trolox equivalence.
The metal chelating activity assay was performed according to the protocol described by Sangsawad et al. (Sangsawad et al., 2016). A 10 µL peptide sample at a concentration of 1 mg/mL was combined with 100 µL of DI, 10 µL of 2 mM FeCl_2_, and 10 µL of 5 mM ferrozine in a 96-well microplate. The reaction was allowed to incubate at room temperature in a dark room for 20 min before being measured at a wavelength of 562 nm using a microplate reader (Varioskan LUX, Thermo Scientific, Vantaa, Finland). Sample blanks (hydrolysate with ferrozine but without FeCl₂) were measured to correct for potential interference from residual heme-iron in BBH. The results were calculated using a standard ethylenediaminetetraacetic acid (EDTA) standard curve ranging from 0 to 1 mg/mL. The activity was then expressed in terms of µg/mL EDTA equivalence.
Prior to all analyses, samples were centrifuged (10,000×g, 10 min, 4 °C) to remove particulate matter and minimize chromophore interference. Preliminary validation confirmed that BBH’s intrinsic color contributed < 5% to total absorbance across all assays, and residual iron content after processing was negligible (Fe < 0.02 mg/100 g). Blank-corrected values differed < 3% from uncorrected readings, confirming minimal matrix interference. Single-point measurements at 1.0 mg/mL protein concentration were selected to represent physiologically relevant feed supplementation levels and enable direct comparison with commercial yeast hydrolysate under practical application conditions.
Characterization and comparison of the bovine blood hydrolysate derived from pilot-scale production versus commercial yeast hydrolysate
Based on superior pilot-scale performance (Pilot-scale production and characterization: 15% degree of hydrolysis and 50% protein recovery), the optimised BBH was selected for comprehensive evaluation as a feed additive. BBH was systematically compared against commercial yeast hydrolysate (YH, Hankkija, Helsinki, Finland) using structural characterization, antioxidant activity assays, amino acid profiling, mineral content quantification, and microbial analysis to establish its suitability for subsequent in vivo trials with Korat chickens.
Scanning electron microscopy
The physical characteristics of hydrolysate samples in the solid state, including boiled bovine blood, insoluble hydrolysate, BBH, and YH, were measured. All bovine blood samples were dried using a hot-air oven, while the yeast hydrolysate was dehydrated using spray drying. Samples that could pass through a 1 mm mesh were examined with the naked eye and analysed using a SEM at 0.1 K and a 100 μm resolution controlled by the Center for Scientific and Technological Equipment at Suranaree University of Technology.
Molecular weight distribution
Peptide profiles can be characterized by analysing molecular weight distribution. This tool enables us to investigate molecular patterns, compare diverse molecular weight profiles, present the percentage of each peptide size, and determine the extent of hydrolysis. The methodology proposed by Sangsawad et al. (Sangsawad et al., 2022) was utilised to evaluate the dispersion of molecular weights. The peptide profile was analysed using size exclusion chromatography. The peptide sample (100 µL, concentration: 7 mg/mL) was subjected to size exclusion chromatography using a Superdex Peptide 10/300 GL column (GE Healthcare, Piscataway, New Jersey, USA). The elution process used an isocratic mode with a 0.7 mL/min flow rate. A mobile phase consisting of 30% acetonitrile and 0.1% trifluoroacetic acid in deionised water was employed to enhance peptide solubility and minimize non-specific interactions with the column matrix, following the validated protocol for protein hydrolysate analysis (Sangsawad et al., 2022). The peptide profile was elucidated through spectrophotometric analysis, measuring absorbance at 215 nm in the ultraviolet spectrum. Molecular weight determination was facilitated by employing a series of five standardised molecular markers (cytochrome c 12.4 kDa, aprotinin 6.5 kDa, synthetic peptides 1–3 kDa, and tyrosine 181 Da), which generated a calibration curve with linear regression. Peak integration was performed using area-percentage calculation, where each molecular weight fraction’s area was expressed as a percentage of the total chromatogram area. All analyses were conducted in triplicate to ensure reproducibility.
Fourier transform infrared spectroscopy
Fourier transform Infrared Spectroscopy (FTIR) was employed to analyze the secondary structure of BBH and YH. The spectra for the individual components were obtained using a Bruker tensor 27 DTGS detector. Measurements were taken with a platinum diamond ATR in reflection mode, performing 64 scans at a resolution of 4 cm^− 1^ across a spectral range of 4,000 to 400 cm^− 1^. Data acquisition was performed using OPUS 7.5 software (Bruker Optics Ltd., Ettlingen, Germany), with linear baseline correction applied to the amide I region (1600–1700 cm⁻¹) before deconvolution analysis. Secondary structure analysis employed Fourier self-deconvolution and second-derivative spectra calculated using Savitzky-Golay algorithm (9-point smoothing) to identify component peak positions. Curve fitting was performed using PeakFit software (version 4.12) with Gaussian function shapes, with constraints of peak width 10–30 cm⁻¹ and peak positions fixed at maxima identified from second derivatives. Component assignment covering criteria β-sheet (1620–1640 cm⁻¹), random coil (1640–1650 cm⁻¹), α-helix (1650–1660 cm⁻¹), and β-turn (1660–1690 cm⁻¹) was established. All deconvolutions achieved correlation coefficient (r²) > 0.95 with reduced chi-square < 0.001. Analysis was performed in technical triplicate, and values are reported as mean ± standard deviation.
Antioxidant activities
The BBH and YH samples were evaluated the antioxidant activity using the same methods described in Antioxidant activity assays, including the ABTS^•+^ radical scavenging assay, ferric-reducing antioxidant power assay, and metal chelating activity assay.
Amino acid profile
The amino acid composition of both BBH and YH was determined through comprehensive analysis. Amino acid analysis employed three complementary hydrolysis procedures to ensure complete amino acid recovery and prevent analytical artifacts: (1) standard acid hydrolysis with 6 N HCl at 110 °C for 24 h under nitrogen atmosphere for most amino acids, (2) performic acid oxidation (3 h at 0 °C) followed by acid hydrolysis for accurate determination of sulphur-containing amino acids (methionine and cysteine), and (3) alkaline hydrolysis with 4 N NaOH at 110 °C for 22 h specifically for tryptophan quantification. This multi-method approach prevents tryptophan destruction and underestimation of cysteine/methionine that occur with acid hydrolysis alone. The hydrolysates were analysed by an accredited laboratory (Central Laboratory Co., Ltd., Khon Kaen, Thailand). AOAC official methods (Horwitz & Latimer, 2005) using an amino acid analyser with post-column ninhydrin derivatization, enabling accurate quantification of all 20 amino acids including aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, hydroxylysine, lysine, arginine, hydroxyproline, proline, and tryptophan were still be utilised to process all of hydrolysing. This analysis comprehensively characterised the protein composition in both hydrolysates, enabling a detailed comparison of their nutritional and functional properties.
In vivo application of BBH versus YH in slow-growing chicken
Hydrolysate product quality assessment
Microbial analysis, in particular for Salmonella spp., total aerobic plate count, and yeasts and molds were investigated. The pilot-scale hydrolysate product was manufactured strictly to ensure that the product is safe Bacteriological Analytical Manual (BAM), as well as a standardised reference for microbiological testing methods were adapted to analyses. In addition, Central Laboratory (Thailand) Co., Ltd., an accredited facility specializing in microbiological assessments, executed the analytical procedures. This rigorous approach to microbial characterization provides valuable insights into the microbiological profile of the hydrolysate, which is essential for evaluating its suitability as a feed ingredient and ensuring compliance with regulatory standards.
The mineral content of BBH was evaluated following AOAC guidelines, samples were sent to Central Laboratory (Thailand) Co., Ltd. Only six interested minerals, including copper (Cu), iron (Fe), manganese (Mn), phosphorus (P), potassium (K), and zinc (Zn) were measured.
Experimental design and animal housing
The BHH as well as positive control YH were then subjected to animal diets and tested to the university animals. The animal study protocol was approved by the Institutional Animal Care and Use Committee of Rajamangala University of Technology Isan, Sakon Nakhon Campus (Animal ethics number: RMUTSN-IACUC-006–67). This study was conducted from March to May 2023, with temperatures ranging from 25.0 to 42.5 °C. The experiment took place on a smallholder farm in Loei province, Thailand. Before the trial, all equipment and rearing houses underwent thorough disinfection. A total of 405 one-day-old Korat chickens (mixed sex) were randomly allocated to 15 floor pens (1.5 × 2.0 m) with 27 birds per pen, representing 5 replicate pens per treatment. Those chickens received three design treatments. The chickens were raised for 9 weeks under controlled environmental conditions with continuous lighting and ad libitum access to feed and water. The three dietary treatments were (1) Control is basal diet without hydrolysate, (2) BBH 0.1 is basal diet with 0.1% bovine blood hydrolysate, and (3) YH 0.1 is basal diet with 0.1% yeast hydrolysate. The basal diet met NRC (1994) requirements for slow-growing chickens. Body weight and feed intake were recorded weekly on a pen basis. At week 9, two birds per pen (10 birds per treatment) at average pen weight were selected for blood collection and carcass evaluation. The nutrient requirements for Korat chickens (Table 1) were based on previous research (Tran et al., 2021; Maliwan et al., 2019). The proximate composition of Korat chicken diets, consisting of starter (0–3 weeks), grower (3–6 weeks), and finisher (6–9 weeks) phases, as well as BBH and YH, were randomly selected and minced for analysis. The samples were then analysed to determine the proximate analyses following AOAC as aforementioned procedures (Horwitz & Latimer, 2005).
Table 1. Composition of the basal experimental dietsFeed ingredients (%)Starter(0–3 weeks)Grower(3–6 weeks)Finisher(6–9 weeks)Corn50.5251.0453.14Soybean37.3235.8328.11Rice bran3.503.008.89Rice bran oil4.096.456.37Calcium carbonate1.431.501.18Mono-dicalcium phosphate 21%2.041.291.52L-lysine0.180.100.10DL-methionine0.250.160.10Salt0.170.130.09^a^Premix0.500.500.50Total (Kg)100.00100.00100.00 Calculated analysis ME for poultry (Kcal/Kg)2980.003151.003200.00Dry matter (%)88.7688.9188.99Protein (%)21.2620.4518.00Fat (%)7.089.3210.15Fiber (%)3.813.703.74Lysine (%)1.281.171.01Met + Cys (%)0.900.790.68Methionine (%)0.580.480.40Threonine (%)0.800.780.68Valine (%)1.010.980.87Iso-leucine (%)0.910.880.76Arginine (%)1.431.371.20Tryptophan (%)0.240.230.20Calcium (%)1.000.900.80Total Phos. (%)0.850.680.76Avail. Phos. (%)0.480.350.39Sodium (%)0.200.150.12^a^Mineral and vitamins premix provided the following per kilogram of diets; manganese, 86.28 mg; iron, 108.11 mg; copper, 62.74 mg; selenium, 32.75 mg; zinc, 136.99 mg; iodine, 1.64 mg; vitamin A, 1650 IU; cholecalciferol, 330 IU; vitamin E, 100 mg; vitamin K, 4.31 mg; vitamin B1, 2.53 mg; vitamin B2, 8.02 mg; nicotinic acid, 53.96 mg; vitamin B6, 2.53 mg; vitamin B12, 1.54 mg; pantothenic acid, 13.23 mg; folic acid, 1.38 mg; biotin, 5.50 mg; and choline, 2583.33 mg. ME, metabolizable energy
Performance and physiological analysis
Growth performance analysis
Initial body weight was recorded at day 0, followed by weekly measurements at 7-day intervals using a digital scale (precision ± 0.1 g) after a 12-h feed withdrawal period to ensure accuracy and consistency across measurements. Body weight gain (BWG) was calculated as the difference between final and initial body weights for each measurement period. Average daily gain (ADG) was determined by dividing the total weight gain by the number of days in each growth phase (starter: 0–3 weeks, grower: 3–6 weeks, finisher: 6–9 weeks). Feed consumption was measured daily by weighing the amount of feed offered and subtracting any remaining feed at the end of each 24-h period. Weekly feed intake was calculated by summing daily consumption values. Average daily feed intake (ADFI) was determined by dividing total feed consumed by the number of birds and days in each measurement period. Feed conversion ratio (FCR) was calculated as the ratio of total feed consumed to total body weight gain (kg feed/kg gain). Feed efficiency (FE) was expressed as the percentage of body weight gain relative to feed intake (BWG/Feed intake × 100). All parameters were calculated for each growth phase and cumulatively for the entire 9-week period.
Growth performance data were collected from all 405 birds (135 birds per treatment group) to ensure statistical reliability and account for individual variation within each treatment group. All growth performance measurements were conducted under standardised conditions, with consistent timing (morning, 8:00–10:00 AM) and environmental controls (temperature, humidity, and lighting) maintained throughout the experimental period to minimize external factors that could influence growth performance outcomes.
Antioxidant status (serum and liver) analysis
At the end of the experiment, 4 male Korat chickens per treatment group were randomly selected and weighed after a 12-h feed withdrawal. Blood samples were collected into heparinised tubes and centrifuged at 2000 × g for 15 min at 4 °C to obtain serum. The serum samples were then frozen at − 20 °C until analysis. Subsequently, hepatic samples were collected, immediately washed with phosphate buffer solution, and frozen in liquid nitrogen until further analysis of antioxidant activity, according to Wang 2022. The assessment of the antioxidant status in serum and liver of Korat chickens was conducted by measuring the concentrations of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in both the serum and liver using the CheKine™ test kit, acquired from Abbkine, Inc followed the manufacturer’s recommendations.
Carcass traits and meat quality characteristics
At the end of the 9-week feeding trial, 20 male Korat chickens (4 birds per treatment group) were randomly selected and weighed after 12-h feed withdrawal. Birds were slaughtered following standard procedures, and carcasses were chilled at 4 °C for 24 h. Meat color parameters were measured on breast muscle samples using a colorimeter to determine lightness (L*), redness (a*), and yellowness (b*) values. Three measurements were taken at different locations on each sample and averaged. Muscle pH was determined at 45 min (pH_45_) and 24 h (pH_24_) post-mortem using a portable pH meter with penetrating electrode inserted directly into the breast muscle.
Water-holding capacity was assessed through drip loss and cooking loss measurements. Drip loss was determined by suspending approximately 50 g of breast muscle samples in sealed containers at 4 °C for 48 h, followed by calculating the percentage of weight loss. Cooking loss was evaluated using 50 g breast muscle samples, which were cooked in a water bath at 80 °C until the internal temperature reached 70 °C; the percentage weight loss was then calculated after cooling to room temperature. Carcass traits were evaluated by recording the eviscerated carcass weight to calculate the dressing percentage relative to live weight. Commercial cuts, including breast and drumstick, were separated and weighed individually to determine their yields as percentages of the chilled carcass weight. All measurements were performed in triplicate, and mean values were used for statistical analysis.
Principal component analysis
To examine complex relationships among multiple parameters including growth performance, antioxidant enzyme activities, and meat quality attributes, Principal Component Analysis (PCA) was performed using The Unscrambler X version 10.1 (CAMO Software AS, Oslo, Norway). Data were autoscaled (mean-centered and unit variance scaled) prior to analysis to ensure comparable weighting across variables with different measurement units. The analysis included all measured parameters from in vivo results. No missing values were present in the dataset. Model diagnostics included Hotelling’s T² statistics to identify potential outliers, Q-residuals to assess model fit, and leverage values to evaluate sample influence. Eigenvalues were computed to determine variance explained by each principal component, and the first two PCs were visualised through score plots and correlation loading plots.
Statistical analysis
All hydrolysate characterization experiments were performed in triplicate, and results are reported as mean ± standard deviation. For the animal trial, pen served as the experimental unit (n = 5 replicate pens per treatment, 27 birds per pen) for all feed-related metrics including feed intake, feed conversion ratio, and feed efficiency. For longitudinal growth performance data (body weight and feed intake measured weekly from weeks 0–9), a linear mixed-effects model was employed with Treatment and Time as fixed effects, Treatment×Time interaction, and Pen nested within Treatment as a random effect to account for repeated measurements on the same experimental unit over time. An unstructured covariance matrix was used to model the within-pen correlation structure across time points. For endpoint measurements collected at week 9 (serum and hepatic antioxidant enzyme activities, meat quality parameters, and carcass characteristics), one-way ANOVA was conducted using pen means as the experimental unit (n = 5 per treatment), followed by Duncan’s multiple range test for pairwise comparisons. Individual bird was the experimental unit for body weight measurements and for samples collected at slaughter (n = 4–10 birds per treatment depending on the specific analysis). PCA was performed to visualize multivariate relationships among treatment groups and measured variables. All statistical analyses were conducted using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA), with statistical significance declared at p < 0.05.
Results and discussion
This investigation addresses critical knowledge gaps in blood protein valorisation through systematic evaluation bridging laboratory research to commercial viability. The research framework tests whether pilot-scale BBH achieves antioxidant capacity functionally equivalent to established commercial YH, operationally defined as activities within the same order of magnitude across mechanistically distinct in vitro assays including radical scavenging, metal chelation, and reducing power. Building upon this comparative foundation, the investigation examines whether dietary supplementation enhances endogenous antioxidant defence systems in slow-growing chickens while maintaining growth performance and improving meat quality characteristics, thereby validating biological efficacy without compromising animal productivity or product attributes. Integrating these empirical evaluations, the study explores structure-function relationships linking specific molecular features including peptide size distribution and secondary protein conformations to observed biological outcomes, establishing predictive frameworks for rational hydrolysate optimization. Comprehensive characterization spanning production validation, structural analysis, nutritional profiling, and controlled feeding trials therefore provides the systematic evidence base for evaluating these interconnected hypotheses while demonstrating commercial implementation feasibility.
Pilot-scale production and characterization
Our pilot-scale production achieved substantial performance improvements over laboratory-scale hydrolysis, demonstrating successful technology transfer to commercially relevant volumes. The degree of hydrolysis increased from 10.12% at laboratory scale to 15.08% at pilot scale, representing a 49% improvement attributable to enhanced process control throughout the 400 kg batch (Fig. 2A, p < 0.05). Protein recovery similarly improved from 42.15% to 50.23%. Three independent production batches demonstrated acceptable reproducibility with coefficients of variation at 5% for degree of hydrolysis and 8% for protein recovery. This consistency indicates suitability for commercial manufacturing scale-up. The achieved degree of hydrolysis of 15.08% falls within the optimal range of 12 to 18% reported by Lafarga et al. (Lafarga et al., 2018) for producing bioactive peptides while maintaining functional properties.
Fig. 2. The degree of hydrolysis and protein recovery (A) of BBH protein hydrolysate produced from LAB and pilot-scale processing, and its antioxidant activity after Neutrase hydrolysis (B). Different superscript letters (a,b) within the same assay indicate significantly different mean values (p < 0.05)
Critically, pilot-scale processing enhanced bioactivity beyond mere production efficiency gains. ABTS radical scavenging activity increased significantly from 300 to 320 µg Trolox equivalent per mL (Fig. 2B, p < 0.05). This represents 7% improvement in electron donating capacity. Metal chelating activity improved more substantially from 100 to 125 µg EDTA equivalent per mL, a 25% enhancement in iron binding capability. FRAP values remained comparable between scales at 35 to 40 µg Trolox equivalent per mL. These bioactivity improvements suggest that controlled hydrolysis conditions at pilot scale favour formation of specific peptide populations with enhanced antioxidant properties. The present findings reveal that these improvements result from enhanced engineering control at larger reactor volumes. The jacketed cooker maintained uniform 50 °C throughout the reaction volume. Mechanical agitation at 50 rpm ensured consistent enzyme-substrate contact across the entire batch. Our accelerated storage testing at 37 °C for 90 days confirmed maintained bioactivity and microbial stability. Total plate counts remained below 1 × 10⁴ CFU per gram. ABTS radical scavenging capacity retained 94% of initial activity. BBH powder was stored in vacuum-sealed bags at minus 20 °C throughout the nine-week feeding trial, maintaining consistent bioactive properties during the experimental period. Comprehensive stability profiling under commercial storage conditions including room temperature and refrigeration remains to be established in future work. The pilot-scale process additionally generated an insoluble fraction containing 50% protein, representing a valuable co-product for applications requiring lower solubility. These results thus validate technical feasibility for commercial-scale blood valorisation while establishing quality benchmarks for subsequent comparative evaluation against commercial yeast hydrolysate.
Our pilot-scale achievements compare favourably with published reports on protein hydrolysate scale-up, though direct comparisons remain limited by substrate and processing differences. Laosam et al. (Laosam, 2024) reported pilot-scale duck blood hydrolysate production at 1000 L volume but provided no laboratory-scale benchmarking, preventing assessment of scale-up efficiency gains. Hou et al. (Hou et al., 2022) documented industrial fish protein hydrolysate production achieving 12 to 15% degree of hydrolysis, comparable to our 15.08%, though employing continuous reactors rather than batch processing. Our protein recovery of 50.23% exceeds the 35 to 45% range reported by Martínez-Alvarez et al. (Martínez-Alvarez et al., 2015) for marine by-product hydrolysates, potentially reflecting bovine blood’s simpler protein matrix compared to fish tissue containing structural connective tissues. The enhanced antioxidant activity at pilot-scale versus laboratory-scale represents a novel finding not documented in hydrolysate scale-up literature. Most studies assume bioactivity remains constant or decreases during scale-up due to reduced mixing efficiency. Our observation of enhanced ABTS and metal chelating activities suggests that improved process control can actively improve product quality beyond mere efficiency gains, warranting investigation of scale-dependent peptide release kinetics in future research.
Structural and chemical characterisation
Our pilot-scale BBH exhibited distinct morphological and molecular characteristics differentiating it from both blood meal precursor and commercial YH. Visual examination revealed progressive transformation from coarse blood meal to fine, uniform brown powder (Fig. 3). Scanning electron microscopy confirmed this refinement at the microscopic scale. Blood meal particles appeared irregular and coarse at 100 μm magnification. BBH particles demonstrated uniform, fine structure reflecting complete enzymatic deconstruction. This morphological refinement correlates directly with enhanced solubility. In addition, size exclusion chromatography revealed that BBH contained 59% of peptides within the 0.4 to 2 kDa range compared to 70% in YH (Fig. 4). This molecular weight distribution proved critical. Peptides below 2 kDa demonstrate enhanced intestinal absorption and bioavailability. BBH additionally contained 25% peptides in the 2 to 10 kDa range versus only 8% in YH. These larger peptides may provide sustained antioxidant effects through gradual enzymatic release during gastrointestinal digestion.
Fig. 3. Appearance (Row 1) and morphology (Row 2) of bovine blood products by scanning electron microscopy. (A1,2) blood meal, (B1,2) insoluble hydrolysate, (C1,2) bovine blood hydrolysate, and (D1,2) yeast hydrolysate. Which, ^1^with the naked eye and ^2^with the scanning electron microscopy at 0.1 K, 100 μm
Fig. 4. Size exclusion chromatograms of (A) bovine blood hydrolysate and yeast hydrolysate, and (B) molecular weight distribution. Note: ^a−d^Means within the same row, while ^A,B^means within the same column with different superscripts indicate statistically significant differences (p < 0.05)
Equally important is secondary structure analysis through FTIR demonstrated substantial differences between hydrolysates (Fig. 5). The primary spectra (Fig. 5A) showed characteristic amide I (1620–1624 cm⁻¹) and amide II (1515–1580 cm⁻¹) bands for both hydrolysates, confirming peptide bond presence. BBH exhibited a weaker amide I band while YH showed a weaker amide II band, indicating differences in hydrolysis extent and peptide composition. A notable distinction was the polysaccharide peak at 1036 cm⁻¹ present in YH but absent in BBH, suggesting BBH may contain a purer protein composition with potentially more concentrated bioactive peptides. Secondary structure analysis through amide I region deconvolution (1600–1700 cm⁻¹) revealed significant compositional differences (Fig. 5B-D). BBH exhibited 45.09% β-sheet content compared to 42.18% in YH. α-helix content reached 23.47% in BBH versus 30.12% in YH. Random coil structures showed inverse patterns. These structural differences reflect distinct protein origins and processing histories. The elevated β-sheet content in BBH potentially enhances structural stability during storage and processing. FTIR analysis additionally confirmed purely proteinaceous composition in BBH. Substantially, YH displayed characteristic polysaccharide peaks at 1036 cm⁻¹ absent in BBH spectra. As demonstrated in previous research (Poduslo et al., 1999; Yu, 2005), this compositional distinction indicates that BBH bioactivity derives exclusively from peptide-mediated mechanisms rather than confounding contributions from fungal cell wall polysaccharides present in yeast extracts. These structural and compositional characteristics establish the molecular foundation for subsequent in vitro antioxidant activity evaluation presented in Comparative in vitro antioxidant activity.
Fig. 5. Fourier Transform Infrared (FTIR) spectra of bovine blood hydrolysate and commercial yeast hydrolysate. Spectra and functional group detection in yeast hydrolysate and bovine blood hydrolysate (A), Fourier deconvolution and curve fitted the amide I band of each sample of BBH (B) and YH (C), and the secondary structure conformation content (%) of samples (D). Note: ^a−d^Means within the same row, while ^A,B^means within the same column with different superscripts indicate statistically significant differences (p < 0.05)
The peptide size distribution observed in BBH aligns with established bioactive ranges reported across diverse protein hydrolysate sources, though subtle distinctions merit attention. Cheung et al. (Cheung et al., 2012) documented that Pacific hake hydrolysate fractions within 0.4 to 2 kDa exhibited optimal antioxidant properties, supporting our finding that 59% of BBH peptides reside in this range. However, YH demonstrates even higher concentration at 70% within this range yet exhibits lower ABTS and metal chelating activities in Comparative in vitro antioxidant activity, suggesting that molecular weight distribution alone insufficiently predicts bioactivity. This apparent contradiction resolves through consideration of amino acid composition documented in Amino acid composition and nutritional implications, where BBH’s 534% histidine enrichment and 327% cysteine elevation provide functional groups absent from molecular weight analysis. The elevated β-sheet content at 45.09% in BBH exceeds values typically reported for enzymatic hydrolysates, which commonly demonstrate 30 to 40% β-sheet structures. This elevation may reflect bovine haemoglobin’s native quaternary structure, where extensive β-sheet regions stabilize the tetrameric assembly. The retention of substantial β-sheet content post-hydrolysis suggests that Neutrase preferentially cleaves loop regions and α-helical segments while preserving core β-sheet domains, generating peptide populations enriched in structured conformations potentially enhancing stability during storage and gastrointestinal transit.
Comparative in vitro antioxidant activity
Comprehensive antioxidant evaluation across three mechanistically distinct assays revealed complementary strengths between BBH and YH. ABTS radical scavenging activity demonstrated BBH superiority at 335 µg Trolox equivalent per mL compared to YH at 271 µg Trolox equivalent per mL (Fig. 6, p < 0.05). This represents 24% enhanced electron donating capacity in BBH. The mechanism involves aromatic amino acids including tyrosine and tryptophan donating electrons to stabilize ABTS radical cations. Metal chelating activity similarly favoured BBH at 122.19 µg EDTA equivalent per mL versus YH at 89.78 µg EDTA equivalent per mL, representing 36% superior iron binding capability (p < 0.05). One possible explanation for these findings is peptides containing histidine, cysteine, and acidic amino acid residues that form coordination complexes with ferrous ions, preventing pro-oxidant Fenton chemistry. Meanwhile, ferric reducing antioxidant power showed inverse patterns. YH achieved 44 µg Trolox equivalent per mL compared to BBH at 33 µg Trolox equivalent per mL (p < 0.05). This 25% lower FRAP activity in BBH warrants careful interpretation. The FRAP assay operates at acidic pH 3.6, substantially altering peptide ionization states compared to physiological pH 7.4. YH contains non-peptide reducing compounds including glutathione and nucleotides from yeast extracts. BBH derives reducing capacity exclusively from peptide-mediated mechanisms. The FRAP assay under non-physiological conditions may possess limited predictive value for in vivo antioxidant efficacy. Despite this limitation, the present findings directly address the hypothesis regarding comparable antioxidant capacity between BBH and commercial yeast hydrolysate. All three activity ratios fall within the predefined twofold criterion defining functional equivalence. BBH demonstrates superior performance in ABTS radical scavenging and metal chelating, the two mechanisms most physiologically relevant in animal nutrition contexts where protection against lipid peroxidation and metal-catalysed oxidative damage constitute primary challenges. The structural characteristics identified in Structural and chemical characterisation, particularly the 59% peptide content within the 0.4 to 2 kDa bioactive range and elevated β-sheet content at 45.09%, provide mechanistic foundation for these antioxidant properties. The hypothesis regarding antioxidant equivalence stands supported with BBH achieving capacity equivalent to and in key respects superior to commercial yeast hydrolysate standard.
Fig. 6. The antioxidant activity of bovine blood hydrolysate and yeast hydrolysate. ABTS: ABTS radical scavenging activity, FRAP: ferric reducing antioxidant power, and Metal: metal chelating activity, eq.: equivalent. All measurements are conducted at a protein concentration of 1.0 mg/mL. Superscripts that differ from each other indicate significant variations between the average values within the same antioxidant activity (p ≤ 0.05)
The antioxidant activities demonstrated by BBH align with and in several respects exceed those reported for protein hydrolysates from alternative sources. Fu et al. (Fu et al., 2019) documented porcine haemoglobin hydrolysates exhibiting ABTS radical scavenging at approximately 280 µg Trolox equivalent per mL, lower than our BBH value of 335 µg Trolox equivalent per mL despite similar heme protein origins. This superiority may reflect differences in enzymatic specificity, with Neutrase employed here generating distinct peptide populations compared to the alcalase and pepsin combinations used in porcine studies. Fish protein hydrolysates characterised by Bah et al. (Bah et al., 2013) demonstrated comparable ABTS activities ranging from 300 to 350 µg Trolox equivalent per mL, positioning BBH within the performance range of established marine-derived alternatives. The lower FRAP activity in BBH compared to YH warrants contextualization within broader hydrolysate literature. Plant protein hydrolysates commonly exhibit elevated FRAP values due to contributions from phenolic compounds and non-protein reducing agents co-extracted during processing. YH similarly benefits from fungal metabolites including glutathione, ergothioneine, and nucleotides that contribute reducing power independent of peptide sequences. The purely proteinaceous nature of BBH, confirmed through FTIR analysis, necessarily limits FRAP activity to peptide-derived mechanisms. From a physiological perspective, the mechanisms where BBH excels, specifically radical scavenging and metal chelation, represent the primary antioxidant challenges in biological systems containing lipid membranes and metalloproteins, rendering FRAP performance at acidic pH a less critical predictor of in vivo efficacy subsequently validated through enzyme activity measurements in Antioxidant enzyme activities in serum and hepatic tissue.
Amino acid composition and nutritional implications
Comprehensive amino acid profiling revealed substantial compositional advantages in BBH compared to YH, with particular enrichment in nutrients critical for both metabolic function and antioxidant mechanisms (Table 2). BBH demonstrated markedly elevated branched-chain amino acids. Leucine reached 11,131 mg per 100 g, representing 426% greater abundance than YH at 2,117 mg per 100 g. This substantial enrichment carries significant implications for poultry nutrition. Leucine activates the mTORC1 pathway, the master regulatory mechanism coordinating protein synthesis with amino acid availability. Valine concentration in BBH similarly exceeded YH by 353%, achieving 8,314 mg per 100 g compared to 1,838 mg per 100 g. Isoleucine showed 32% elevation at 1,786 mg per 100 g versus 1,351 mg per 100 g in YH. Our findings advance the current understanding of collective branched-chain amino acid enrichment that creates a profile particularly well suited to growing poultry nutritional demands.
Table 2. Amino acids content expressed in mg/100 g of sampleAmino acidsBovine blood hydrolysateYeast hydrolysateAspartic acid99233010Threonine40691563Serine37301762Glutamic acid92273927Glycine40681485Alanine77642078Cystine973228Valine83141838Methionine1485537Isoleucine17861351Leucine11,1312117Tyrosine28171098Phenylalanine65421381Histidine4403694HydroxylysineNot detectedNot detectedLysine70322045Arginine29591362HydroxyprolineNot detectedNot detectedProline38201423Tryptophan1077396
Beyond branched-chain amino acids, BBH exhibited superior essential amino acid content. Lysine reached 7,032 mg per 100 g versus 2,045 mg per 100 g in YH, representing 244% greater abundance. Methionine concentration achieved 1,485 mg per 100 g compared to 537 mg per 100 g in YH, a 176% elevation. These enrichments directly support protein synthesis requirements in growing chickens. Critically, amino acids with established antioxidant functions demonstrated substantial elevation in BBH. Histidine content reached 2,538 mg per 100 g compared to YH at 400 mg per 100 g, representing 534% enrichment. Histidine serves as precursor for carnosine synthesis, a dipeptide that chelates metal ions and prevents iron-catalysed oxidative damage. Cysteine content achieved 973 mg per 100 g versus 228 mg per 100 g in YH, a 327% elevation. This outcome suggests that cysteine provides the limiting substrate for glutathione biosynthesis, the primary intracellular antioxidant system. Following this, aromatic amino acids similarly showed substantial BBH enrichment. Tyrosine reached 2,254 mg per 100 g compared to 773 mg per 100 g in YH. Phenylalanine achieved 3,662 mg per 100 g versus 1,177 mg per 100 g in YH. These aromatic residues contribute electron-donating groups for radical neutralization, directly explaining the 24% superior ABTS radical scavenging activity documented in Comparative in vitro antioxidant activity. The amino acid composition data provide molecular-level explanation for functional bioactivity patterns. The 534% histidine enrichment, 329% aspartic acid elevation, and 327% cysteine enhancement collectively explain the 36% superior metal chelating activity. This composition-activity correspondence partially fulfils the hypothesis regarding structure-function relationships, demonstrating that chemical composition possesses predictive capacity for functional bioactivity.
To note, the amino acid profile of BBH demonstrates clear advantages over conventional plant-based protein sources commonly employed in poultry nutrition. Soybean meal, the primary protein supplement in commercial feeds, contains approximately 3,100 mg leucine per 100 g, substantially lower than BBH’s 11,131 mg per 100 g. This 259% enrichment proves particularly valuable given that leucine serves as the limiting branched-chain amino acid in typical corn-soybean diets. YH, while accepted as functional additives, provide markedly lower essential amino acid concentrations than BBH, explaining their typical supplementation at 0.2 to 0.5% compared to our effective 0.1% inclusion rate. The histidine content of 2,538 mg per 100 g substantially exceeds requirements for poultry diets, where NRC specifications suggest approximately 0.35% of dietary protein. This apparent excess transforms into functional advantage when considering histidine’s dual role in both protein synthesis and carnosine-mediated antioxidant defence. Wang et al. (Wang et al., 2022) documented that YH supplementation enhanced antioxidant status in broilers through β-glucan and glutathione mechanisms. Our findings suggest that BBH achieves comparable or superior effects through distinct pathways, specifically histidine-carnosine and cysteine-glutathione axes, offering mechanistic diversity potentially advantageous under varied stress conditions. The compositional analysis of our study partially addresses why BBH demonstrates functional equivalence to YH despite disparate origins, revealing that multiple biochemical pathways can converge on similar physiological outcomes when amino acid provision matches or exceeds critical functional thresholds.
Product safety validation for in vivo application
Feed additive safety requires rigorous documentation of microbial quality and mineral composition before biological testing commences. Our comprehensive safety assessment of pilot-scale BBH demonstrated exceptional microbial control substantially exceeding regulatory requirements. Total aerobic plate count achieved 5.8 × 10³ colony forming units per gram, remaining 1,379-fold below the regulatory maximum of 8 × 10⁶ CFU per gram specified under Thailand’s Animal Feed Quality Control Act of 2015 (Table 3). This microbial load reflects effective thermal inactivation during blood treatment at 90 °C for 30 min combined with hot air drying at 70 °C for 12 h. Yeast and mold enumeration yielded 3 × 10¹ CFU per gram, remaining 3,333-fold below the regulatory ceiling of 1 × 10⁵ CFU per gram. The negligible fungal contamination demonstrates effective moisture control during drying and storage. Most critically, Salmonella species testing confirmed absence in 25-gram samples, meeting the zero-tolerance standard mandated for animal feeds. Salmonella contamination presents particular concern due to potential transmission through the food chain. The complete absence reflects both effective thermal inactivation and maintenance of hygienic conditions throughout processing. Comparative evaluation demonstrates BBH’s superior microbial quality relative to alternative protein sources. Vegetable protein hydrolysates characterised by Lafarga et al. (Lafarga et al., 2018) typically exhibit total plate counts ranging from 1 × 10⁴ to 1 × 10⁵ CFU per gram, representing 17 to 172-fold higher microbial loads. Fish protein hydrolysates reported by Bah et al. (Bah et al., 2013) demonstrated counts of approximately 1 × 10⁴ CFU per gram, 17-fold higher than present findings. Furthermore, mineral analysis of experimental diets supplemented with 0.1% BBH revealed trace contributions within regulatory safety limits (Table 4). Iron content reached 2.556 mg per kg, well within the 1,000 mg per kg safety threshold while potentially supporting haemoglobin synthesis in growing chickens. Copper at 0.003 mg per kg, zinc at 0.025 mg per kg, phosphorus at 1.744 mg per kg, potassium at 4.006 mg per kg, and magnesium at 0.256 mg per kg all remained within safety standards. While absolute concentrations appear modest, these minerals may contribute synergistically to overall dietary balance. The superior microbial quality combined with compliant mineral profile establishes BBH suitability for incorporation into Korat chicken diets without introducing pathogenic organisms or excessive mineral loads.
Table 3. Microbials content (CFU/g) of BBH compared to standard of animal feed quality controlMicrobialBBH powderStandard of feed ingredientsTotal plate count5.80 × 10^3^8.00 × 10^6^Yeasts and molds3.00 × 10^1^1.00 × 10^5^Salmonella spp.Not detected per 25 gNot detected per 25 gThe animal feed quality control act, 2015 (Nuangmek et al., 2020), BBH: bovine blood hydrolysate
Table 4. Minerals content (mg/kg) of BBH compared to standard of animal feed quality controlMineralsBBH powderDiet + 0.1% BBHStandard of feed ingredientsCopper (Cu)1.500.0030≤ 300Iron (Fe)12782.56≤ 1,000Magnesium (Mg)1280.256≤ 3,000Phosphorus (P)8721.74≤ 8,000Potassium (K)20034.01≤ 20,000Zinc (Zn)12.70.025≤ 1,000The Animal Feed Quality Control Act, 2015 (Nuangmek et al., 2020), BBH: bovine blood hydrolysate
The exceptional microbial quality achieved in BBH production merits analysis beyond simple regulatory compliance. Blood’s initial sterility within the circulatory system, contrasting sharply with fish tissue harbouring 10⁵ to 10⁷ CFU per gram endogenous microbiota or plant materials carrying soil-borne organisms, fundamentally explains the superior baseline for processing. The thermal treatment protocol employed here, combining 90 °C initial heating with 70 °C drying, proves more aggressive than protocols reported for many fish protein hydrolysates where lower temperatures around 55 to 60 °C preserve heat-labile bioactive peptides but compromise microbial reduction. This trade-off between bioactivity preservation and microbial safety represents a critical processing decision. Our results demonstrate that bovine blood tolerates higher thermal loads without compromising bioactive functionality, as evidenced by robust antioxidant activities in Comparative in vitro antioxidant activity and enzyme induction in Antioxidant enzyme activities in serum and hepatic tissue. The mineral profile reveals an important consideration for feed formulation. While absolute concentrations appear modest, the bioavailable iron from heme sources demonstrates 15 to 35% absorption efficiency in monogastric animals compared to 2 to 20% for non-heme plant iron. This bioavailability advantage suggests that even the 2.556 mg per kg iron contribution may provide meaningful support for haemoglobin synthesis in rapidly growing chickens, particularly those raised under tropical heat stress conditions where iron demands increase due to enhanced erythropoiesis supporting thermoregulatory cardiovascular function.
Experimental diet validation
The proximate composition of experimental diets demonstrated close correspondence with formulated values across all growth stages, validating feed preparation accuracy and confirming nutritional adequacy for the nine-week feeding trial (Table 5). Analysed protein content aligned closely with calculated values throughout starter, grower, and finisher phases. Starter diets achieved 23.01% protein compared to formulated 21.26%, a minor variation within acceptable ranges for commercial feed production. Grower diets reached 20.35% protein against calculated 20.00%. Finisher diets achieved 17.25% protein versus formulated 18.00%. These slight variations reflect normal analytical uncertainty and ingredient compositional variability inherent in practical feed manufacturing. The protein contents remained appropriate for Korat chicken (slow-growing chicken) requirements at each developmental stage.
Table 5. The proximate composition of basal diets (%)Growth stagesMoistureProteinFatFiberAshStarter9.44^a^23.01^a^6.92^b^3.83^b^7.07^b^Grower9.06^a^20.35^b^8.57^a^3.87^b^6.27^c^Finisher9.27^a^17.25^c^8.61^a^4.76^a^8.22^a^Significantly different ^a−c^mean values are denoted by distinct superscripts with the same column within each condition, and statistical significance was set at p < 0.05
Fat content similarly demonstrated acceptable alignment with formulated specifications. Starter diets achieved 6.92% fat, grower diets 8.57%, and finisher diets 8.61%. These values show appropriate progression corresponding to changing energy requirements as birds mature. The fibre content remained consistent with calculated values across growth phases. Starter diets achieved 3.83% fibre, grower diets 3.87%, and finisher diets 4.76%. This fibre provision proves crucial for intestinal health in slow-growing breeds. Korat chickens demonstrate superior intestinal development compared to fast-growing commercial broilers. Their extended rearing periods allow gradual adaptation to fibrous feedstuffs. The elevated finisher phase fibre content at 4.76% supports digestive function without compromising nutrient utilization. Ash content, indicating mineral composition, closely aligned with formulated values. Starter diets achieved 7.07% ash, grower diets 6.27%, and finisher diets 8.22%. This pattern reflects changing mineral requirements throughout development. The close correspondence between formulated and analysed nutrient profiles across all growth phases provides confidence that experimental diets accurately reflected intended nutritional composition. These results validate that prepared diets met specific requirements for Korat chickens at different growth stages as established by previous research (Tran et al., 2021; Maliwan et al., 2019). The demonstrated formulation accuracy ensures that subsequent performance outcomes in Antioxidant enzyme activities in serum and hepatic tissue through Meat quality characteristics and commercial validation can be attributed to hydrolysate supplementation effects rather than confounding nutritional inadequacies or imbalances in basal diets.
The close correspondence between formulated and analysed diet composition validates experimental design integrity and enables confident attribution of subsequent performance outcomes to hydrolysate supplementation rather than nutritional imbalances. This validation assumes particular importance given the challenges inherent in slow-growing chicken nutrition. Korat chickens demonstrate distinct nutritional requirements compared to fast-growing commercial broilers. Their extended rearing periods allow gradual nutrient deposition, reducing the extreme precision required in fast-growing genetics where minor amino acid imbalances rapidly manifest as growth depression. The fibre tolerance demonstrated here, with finisher diets achieving 4.76% without adverse effects, contrasts with commercial broiler recommendations typically limiting fibre below 4% to maximize energy density. This tolerance reflects Korat chickens’ traditional free-range production systems where birds regularly consume fibrous plant materials, developing enhanced intestinal capacity and microbial populations capable of fermenting structural carbohydrates. The protein levels employed across growth stages, ranging from 23.01% in starter to 17.25% in finisher diets, align with published requirements for indigenous breeds reported by Jaturasitha et al. (Jaturasitha et al., 2008). The validation extends beyond chemical composition to encompass functional adequacy, confirmed through the maintained growth trajectories and normal carcass characteristics documented in subsequent sections. This multilevel validation, from proximate analysis through biological performance, distinguishes well-designed feeding trials from studies where dietary inadequacies confound treatment comparisons.
Antioxidant enzyme activities in serum and hepatic tissue
In vivo antioxidant enzyme assessment provided direct evidence of BBH bioactivity at the systemic and tissue levels, demonstrating that in vitro antioxidant capacity documented in Comparative in vitro antioxidant activity translates to enhanced endogenous defence systems. Serum superoxide dismutase activity increased significantly with BBH supplementation, reaching 89.47 units per mL compared to control at 76.23 units per mL (Fig. 7A, p < 0.05). This represents 17% enhancement in the primary enzyme catalysing superoxide radical dismutation to hydrogen peroxide. YH supplementation achieved comparable enhancement at 87.89 units per mL, demonstrating functional equivalence between hydrolysates. Serum glutathione peroxidase activity similarly increased with BBH supplementation to 385.67 units per mL versus control at 342.18 units per mL, representing 13% elevation (Fig. 7B, p < 0.05). YH supplementation reached 378.45 units per mL. Catalase activity in serum showed parallel patterns, with BBH achieving 12.87 units per mL compared to control at 10.34 units per mL (Fig. 7C, p < 0.05). Hepatic tissue analysis revealed even more pronounced treatment effects, reflecting the liver’s central role in antioxidant defence and xenobiotic metabolism. Hepatic superoxide dismutase activity reached 156.78 units per mg protein with BBH supplementation compared to control at 128.45 units per mg protein, representing 22% enhancement (Fig. 7D, p < 0.05). Hepatic glutathione peroxidase activity increased to 487.23 units per mg protein versus control at 398.67 units per mg protein, a 22% elevation (Fig. 7E, p < 0.05). Hepatic catalase activity achieved 18.96 units per mg protein with BBH supplementation compared to control at 14.23 units per mg protein, representing 33% enhancement (Fig. 7F, p < 0.05). YH supplementation produced statistically equivalent responses across all hepatic enzymes, confirming functional parity between hydrolysates. Moreover, the coordinated upregulation of all three major antioxidant enzymes across both serum and hepatic compartments demonstrates that bioactive peptides in BBH successfully modulate endogenous defence systems. The magnitude of enhancement, ranging from 13% to 33% across enzymes and tissues, represents biologically meaningful improvements in oxidative stress resistance. These findings directly address the hypothesis regarding enhanced antioxidant defence systems without compromising animal health, demonstrating that 0.1% BBH supplementation enhances antioxidant enzyme activities in both systemic circulation and hepatic tissue. The amino acid composition documented in Amino acid composition and nutritional implications, particularly the 534% histidine enrichment supporting superoxide dismutase synthesis and 327% cysteine elevation enabling glutathione production, provides mechanistic foundation for these enzymatic responses.
Fig. 7. Serum (A–C) and hepatic (D–F) antioxidant parameters in Korat chickens (slow-growing chicken). SOD: superoxide dismutase activity; GPx: glutathione peroxidase activity and CAT: catalase activity. BBH: bovine blood hydrolysate; YH: yeast hydrolysate. Control (without hydrolysate), BBH 0.1 (0.1% BBH), and YH 0.1 (0.1% YH). Different superscripts indicate significant differences between mean values within the same figure (p < 0.05)
The magnitude of antioxidant enzyme enhancement achieved through BBH supplementation compares favourably with alternative dietary interventions documented in poultry nutrition literature. Selenium-enriched diets, widely employed for antioxidant enhancement, typically improve glutathione peroxidase activities by 15 to 25% in broiler chickens, comparable to our 13% serum and 22% hepatic improvements. Vitamin E supplementation at levels exceeding 200 mg per kg diet demonstrates similar effect magnitudes for superoxide dismutase and catalase activities. The critical distinction lies in BBH providing these benefits at substantially lower inclusion rates, 0.1% compared to 0.02 to 0.05% selenium or 200 to 400 mg vitamin E, potentially offering economic advantages. Wang et al. (Wang et al., 2022) reported that yeast hydrolysate supplementation at 0.2% enhanced hepatic superoxide dismutase and glutathione peroxidase activities by approximately 18 to 20% in broilers, closely matching our observations despite the twofold lower BBH inclusion rate employed here. This dose efficiency likely reflects BBH’s elevated histidine and cysteine content documented in Amino acid composition and nutritional implications, providing abundant precursors for carnosine and glutathione biosynthesis. The coordinated upregulation across all three major antioxidant enzymes rather than selective enhancement of individual components deserves emphasis. Many antioxidant interventions demonstrate targeted effects, with selenium predominantly affecting glutathione peroxidase and vitamin E primarily supporting membrane-associated defences (Purba et al., 2025). BBH’s capacity to simultaneously enhance superoxide dismutase, glutathione peroxidase, and catalase activities suggests multiple bioactive peptides activating distinct cellular signalling pathways, potentially through Nrf2-mediated transcriptional regulation documented for bioactive peptides in rodent, fish, and cell line (Shekoohi et al., 2024) and mammalian systems (Purba & Paengkoum, 2022). Our results extend prior work (Boonkong et al., 2024) by showing that BBH’s capacity remains valid in monogastric systems. This mechanistic breadth may provide superior protection under complex oxidative stress conditions where multiple reactive oxygen species challenge cellular homeostasis simultaneously, explaining the subsequent improvements in meat water-holding capacity documented in Meat quality characteristics and commercial validation.
The magnitude of antioxidant enzyme enhancement achieved through BBH supplementation compares favorably with established nutritional antioxidant interventions in poultry. Selenium-enriched diets, a well-documented approach for enhancing glutathione peroxidase activity, typically produce 15 to 25% increases in GPx activity at supplementation levels of 0.3 to 0.5 mg per kg diet. Our 13% serum GPx enhancement and 22% hepatic GPx elevation occur at 0.1% BBH inclusion without additional selenium beyond basal diet requirements, suggesting complementary rather than redundant mechanisms. Vitamin E supplementation, another conventional strategy, enhances antioxidant status primarily through direct radical scavenging rather than enzyme upregulation, contrasting with the enzymatic induction pathway demonstrated here. Wang et al. (Wang et al., 2022) elaborated that YH supplementation in broilers enhanced SOD activity by approximately 18%, comparable to our 17% serum SOD improvement but achieved in fast-growing birds under controlled environmental conditions. Our results in slow-growing Korat chickens under tropical heat stress conditions suggest that bioactive peptides maintain efficacy across diverse genetic backgrounds and environmental challenges. The coordinated elevation across all three enzyme systems, SOD, GPx, and CAT, proves particularly noteworthy. Many interventions enhance individual enzymes while leaving others unaffected or even suppressed through metabolic trade-offs. The simultaneous upregulation observed here indicates comprehensive antioxidant defence enhancement rather than compensatory responses, likely reflecting the multiple bioactive peptides identified in our previous proteomic characterization providing diverse signalling inputs for transcriptional activation across multiple gene families encoding antioxidant enzymes.
Growth performance and nutritional safety
Growth performance parameters validated that enhanced antioxidant enzyme activities documented in Antioxidant enzyme activities in serum and hepatic tissue occurred without compromising animal productivity, a critical requirement for commercial feed additive viability. Body weight measurements were recorded weekly from day zero through week nine demonstrated consistent trajectories across all treatment groups with no statistically significant differences detected (Fig. 8A, p > 0.05). At three weeks of age, birds achieved approximately 300 g across all treatments. By nine weeks, body weights reached approximately 1,200 g with minimal variation among groups. These growth patterns align with established performance expectations for slow-growing indigenous breeds under tropical conditions. Korat chickens exhibit characteristically gradual weight gain compared to commercial broilers achieving similar weights within five to seven weeks. Feed intake patterns demonstrated uniform consumption across treatments throughout the nine-week trial (Fig. 8B, p > 0.05). Consumption progressed from approximately 400 g per bird at three weeks to approximately 1,200 g at nine weeks, with parallel increases observed in all groups. The absence of treatment effects on voluntary intake indicates that hydrolysate supplementation did not alter feed palatability. Body weight gain calculations demonstrated age-dependent patterns consistent with avian growth biology (Fig. 8D). During the starter phase, birds achieved approximately 230 g gain across all treatments. Growth continued through grower and finisher phases, reaching final weights near 1,200 g. Average daily gain stabilised at approximately 25 g per day by nine weeks across all treatments (Fig. 8F, p > 0.05).
Fig. 8. Growth performance parameters in Korat chickens (slow-growing chicken). (A) Body weight, (B) Feed intake, (C) Feed conversion ratio, (D) Body weight gain, (E) Average daily feed intake, (F) Average daily gain, and (G) Feed efficiency. BBH: bovine blood hydrolysate; YH: yeast hydrolysate. Control (without hydrolysate), BBH 0.1 (0.1% BBH), and YH 0.1 (0.1% YH). ^a−c^Different letters indicate significant differences between treatments within the same time point, while ^A,B^different letters indicate significant differences between time points (3 and 9 weeks) within the same treatment (p < 0.05)
Feed conversion ratio provided the primary economic metric for production efficiency. FCR values demonstrated similar patterns across treatments at three weeks, achieving approximately 1.8 kg feed per kilogram gain (Fig. 8C). By nine weeks, both BBH and YH supplemented groups exhibited numerically lower FCR values compared to controls, suggesting improved efficiency, though differences did not achieve statistical significance at p < 0.05. The trial occurred during tropical heat stress conditions with temperatures fluctuating between 25 and 42.5 °C from March to May in Thailand. This thermal challenge likely increased variation in growth responses. The maintained growth performance under heat stress validates the natural thermal resilience of Korat chickens. The enhanced antioxidant enzyme activities combined with maintained growth demonstrate that bioactive peptide supplementation provides oxidative protection without imposing metabolic costs compromising productivity. The hypothesis regarding performance maintenance stands supported, establishing essential safety validation for BBH as a commercially viable feed additive without adverse effects on animal productivity.
The maintained growth performance under hydrolysate supplementation aligns with the established principle that bioactive feed additives must support rather than compromise animal productivity to achieve commercial viability. Coudert et al. (Coudert et al., 2023) documented that slow-growing chickens demonstrate distinct metabolic profiles compared to fast-growing commercial broilers, relying more heavily on superoxide dismutase pathways for antioxidant defence. This metabolic distinction suggests that antioxidant interventions targeting SOD, as demonstrated in Antioxidant enzyme activities in serum and hepatic tissue, might prove particularly beneficial for slow-growing genetics. The absence of growth depression despite enhanced antioxidant enzyme activities addresses a theoretical concern regarding metabolic resource allocation. Immune activation and antioxidant defence enhancement can impose energetic costs ranging from 15 to 30% of maintenance energy requirements under severe challenge conditions. Although this is the case, our results demonstrate that moderate antioxidant enhancement at 13 to 33% enzyme elevation does not trigger resource diversion sufficient to compromise growth. The numerical FCR improvement in hydrolysate groups, though statistically non-significant, merits cautious interpretation. Several studies report similar trends where antioxidant interventions improve feed efficiency by 3 to 7% through enhanced nutrient absorption and reduced oxidative damage to intestinal epithelium. The heat stress context assumes particular importance. Thermal challenge elevates reactive oxygen species production through increased mitochondrial respiration supporting thermoregulatory mechanisms. The maintained growth under temperatures reaching 42.5 °C, combined with enhanced antioxidant defences, suggests that bioactive peptides may provide specific protection under environmental stress conditions, though controlled temperature trials would be required to definitively separate heat stress mitigation from baseline effects under thermoneutral conditions.
Meat quality characteristics and commercial validation
Meat quality parameters translate biochemical mechanisms documented at cellular levels into marketable product characteristics determining consumer acceptance and economic value. Colour measurements demonstrated L* lightness values ranging from 58.47 to 59.41 across treatment groups with no statistically significant differences (Fig. 9A, p > 0.05). These values fall within normal range for poultry breast meat, indicating absence of pale soft exudative or dark firm dry conditions. Redness values quantified through a* measurements showed statistically significant differences, with control achieving 2.67 ± 0.09, BBH reaching 2.95 ± 0.11, and YH achieving 2.42 ± 0.03 (p < 0.05). However, the maximum absolute difference of approximately 0.5 a* units remains substantially below the delta E threshold of 2 to 3 units required for human visual perception. The observed differences, while achieving statistical significance, lack practical relevance for consumer purchasing decisions. Yellowness values demonstrated no treatment effects. During this period, pH measurements revealed no significant differences across treatments at both 45 min and 24 h post-mortem (Fig. 9B, p > 0.05). Values ranged from 6.2 to 6.4 at 45 min, declining to 5.8 to 6.0 at 24 h. This progression reflects normal post-mortem glycolysis and lactate accumulation. The maintained pH kinetics demonstrate that hydrolysate supplementation did not alter muscle metabolic status or glycogen reserves affecting meat quality development. Water-holding capacity assessment revealed treatment-specific advantages for hydrolysate supplementation. Cooking loss demonstrated significant differences, with control achieving 28.45 ± 1.23%, BBH reaching 24.59 ± 0.98%, and YH achieving 23.60 ± 1.05% **(**Fig. 9C, p < 0.05). BBH supplementation reduced cooking loss by 3.86% points, representing 14% improvement. This magnitude exceeds the 2 to 3% point threshold considered commercially significant for processing yields.
Fig. 9. Meat quality parameters in Korat chickens (slow-growing chicken). Color parameters (A), pH values (B), Water-holding capacity (C), and carcass traits (D). Control (without hydrolysate), BBH 0.1 (0.1% BBH), and YH 0.1 (0.1% YH). ^a−c^Different letters indicate significant differences between treatments within the same parameter (p < 0.05)
Drip loss showed numerical trends favouring hydrolysate groups without achieving statistical significance. Control reached 3.82 ± 0.45%, BBH achieved 3.02 ± 0.38%, and YH reached 2.95 ± 0.41% (p > 0.05). The limited sample size of four birds per treatment constrains statistical power for detecting moderate effects. Carcass characteristics including dressing percentage, breast yield, and drumstick yield demonstrated no treatment effects (Fig. 9D, p > 0.05), confirming that maintained growth performance translated to equivalent tissue deposition patterns. The selective enhancement of water-holding capacity without disrupting colour, pH, or carcass composition creates favourable commercial positioning. Processing yield improvements deliver economic value without requiring adjustments to established production practices. These findings validate that antioxidant enhancement documented through enzyme activities in Antioxidant enzyme activities in serum and hepatic tissue translates to commercially valuable product improvements, supporting the hypothesis regarding beneficial effects on meat quality parameters while maintaining animal productivity and carcass characteristics.
The cooking loss reduction achieved through BBH supplementation aligns with mounting evidence linking antioxidant status to water-holding capacity in poultry meat. Xu et al. (Xu et al., 2024) documented that natural antioxidant peptides protect muscle membrane integrity through multiple mechanisms including lipid peroxidation inhibition and protein carbonyl reduction. The 3.86% point cooking loss improvement observed here likely reflects protection of myofibrillar proteins and sarcolemmal membranes against oxidative damage during the post-mortem aging period. Muscle pH decline and protein denaturation create opportunities for oxidative modification of cysteine residues and methionine sulfoxidation, compromising protein water-binding capacity. Enhanced antioxidant enzyme activities documented in Antioxidant enzyme activities in serum and hepatic tissue provide tissue-level protection extending into the post-mortem period through residual enzyme activity and accumulated reduced glutathione reserves. The commercial significance warrants emphasis. Processing yields constitute major economic determinants in poultry production, with cooking loss directly affecting consumer satisfaction through juiciness perception and processor profitability through saleable product weight. The 3.86% point improvement translates to approximately 46 g additional cooked meat per 1,200-gram carcass, representing substantial value at commercial scale. Sarmadi and Ismail (Sarmadi & Ismail, 2010) reviewed antioxidative peptides from food proteins, noting that structure-activity relationships remain incompletely characterised despite extensive bioactivity documentation. Hence, our integrated approach linking peptide characteristics in Structural and chemical characterisation, amino acid composition in Amino acid composition and nutritional implications, enzyme activities in Antioxidant enzyme activities in serum and hepatic tissue, and functional outcomes here advances mechanistic understanding by demonstrating that molecular features predict not only in vitro activities but translate to commercially relevant product attributes, fulfilling the structure-function hypothesis proposed in the introduction.
Principal component analysis and multivariate integration
Multivariate pattern recognition through principal component analysis revealed underlying biological structures coordinating responses across diverse physiological systems, demonstrating how individual effects documented in previous sections integrate into coherent metabolic phenotypes. Analysis performed using The Unscrambler X software on autoscaled data extracted seven principal components from the complete dataset encompassing antioxidant enzyme activities, growth performance parameters, and meat quality characteristics (Fig. 10). PC1 accounted for 43% of total variance, representing the dominant axis distinguishing experimental groups. PC2 explained an additional 15% of variance, capturing secondary variation orthogonal to PC1. Together, the first two components captured 58% of cumulative variance, providing two-dimensional representation preserving more than half the information content in the original multidimensional dataset. The score plot also demonstrated clear treatment-specific clustering patterns. Control group observations occupied distinct spatial regions in component space, separated from BBH and YH supplemented groups. BBH and YH treatments clustered in overlapping regions, confirming functional equivalence established through individual parameter comparisons. This spatial relationship validates that hydrolysate supplementation produces coordinated multivariate phenotypes distinguishable from unsupplemented controls, neither BBH nor YH inclusion. The loading plot revealed that antioxidant enzyme activities including superoxide dismutase, glutathione peroxidase, and catalase loaded strongly along PC1 in positive directions. Meat quality parameters including reduced cooking loss similarly loaded positively on PC1. Growth performance variables distributed across both PC1 and PC2, with week 3 measurements associating with PC1 and week 9 measurements positioning differently. These multivariate patterns confirm biological coherence from molecular composition through enzymatic activity to functional outcomes. The 43% variance explained by PC1 positions antioxidant capacity enhancement as the central organizing principle for understanding protein hydrolysate effects. The spatial association between antioxidant enzymes and meat quality parameters in component space provides quantitative support that oxidative protection translates to commercially valuable product improvements. The overlapping BBH and YH clusters along PC1, despite compositional differences documented in Amino acid composition and nutritional implications, demonstrate that multiple protein sources achieve comparable functional performance provided adequate bioactive peptide delivery. This multivariate validation culminates the results presentation by demonstrating that diverse effects spanning molecular characterization through commercial validation integrate into a coherent biological narrative centred on antioxidant capacity enhancement, validating the mechanistic framework proposed in Pilot-scale production and characterization through Meat quality characteristics and commercial validation and establishing BBH as a scientifically validated, commercially viable, and environmentally sustainable feed ingredient.
Fig. 10. Principal component (PC) analysis score plot (A) for PC1 vs. PC2 of the different experimental groups and correlation loading plot (B) for PC1 vs. PC2. Experimental groups; Control (control diet), BBH (diet with 1.0% bovine blood hydrolysate supplementation), YH (diet with 0.1% yeast hydrolysate supplementation)
The application of multivariate pattern recognition to nutritional intervention studies addresses a fundamental challenge in biological research where reductionist measurement of individual parameters may obscure coordinated systemic responses. Traditional univariate statistical approaches, while essential for establishing treatment effects on specific variables, inherently fragment integrated biological phenomena into isolated comparisons. PCA overcomes this limitation by identifying latent factors explaining covariance across multiple measurement domains simultaneously. The 43% variance explained by PC1 proves substantial when contextualised against typical biological PCA analyses where first components commonly explain 20 to 35% of variance in complex datasets. This elevated explanatory power suggests that antioxidant capacity enhancement represents a genuinely dominant organizing principle rather than one factor among many equivalently important variables. The spatial overlap between BBH and YH clusters in component space provides quantitative validation of functional equivalence established through individual parameter comparisons in Comparative in vitro antioxidant activity through Meat quality characteristics and commercial validation. This multivariate confirmation assumes particular importance given compositional differences documented in Amino acid composition and nutritional implications, demonstrating that diverse biochemical pathways can converge on equivalent integrated phenotypes. Such convergent functionality has practical implications for feed additive development, suggesting that multiple protein sources might achieve comparable efficacy provided they deliver adequate concentrations of bioactive components targeting shared physiological mechanisms. The age-dependent positioning of growth parameters warrants future investigation into developmental windows where antioxidant interventions provide maximum benefit, potentially enabling phase-specific supplementation strategies optimizing economic returns while maintaining biological efficacy.
Regarding thermal processing and product stability, the drying conditions (70 °C/12 h) were maintained below 100 °C to minimize risks of Maillard reactions and oxidative degradation typically associated with high-temperature processing. While we did not measure specific degradation markers (e.g., furosine, peroxide value) or conduct formal shelf-life stability studies, the preserved bioactive functionality was confirmed through robust in vitro antioxidant activities and significant in vivo enhancement of antioxidant enzyme systems. BBH powder was stored in vacuum-sealed bags at −20 °C throughout the study period, during which consistent biological effects were observed. These conditions align with protein hydrolysate production protocols that report minimal thermal degradation below 80 °C. Future work should establish comprehensive stability profiles including water activity, oxidative markers, and microbial counts under various commercial storage conditions to develop evidence-based shelf-life recommendations.
Conclusion
This investigation successfully scaled BBH production to commercially relevant volumes while establishing its efficacy as a functional feed additive for slow-growing chickens. Pilot-scale processing achieved 15.08% degree of hydrolysis with enhanced antioxidant activities, challenging the assumption that scale-up compromises bioactivity. The technology produced consistent batches with reproducible quality suitable for commercial manufacturing. Our results validate the core hypotheses. BBH demonstrated antioxidant capacity equivalent to commercial YH, with superior performance in radical scavenging and metal chelating. Dietary supplementation enhanced antioxidant enzyme activities by 13 to 33% without compromising growth performance. Meat quality improved through reduced cooking loss exceeding commercial significance thresholds. Structure-function relationships connecting peptide characteristics to biological outcomes received empirical support, though complete mechanistic elucidation requires additional investigation. Most importantly, BBH and yeast hydrolysate showed functional equivalence across all major parameters, establishing blood-derived hydrolysates as viable alternatives. Several limitations warrant acknowledgment. We evaluated a single supplementation level in one breed over 9 weeks without sensory testing. Dose-response studies, multi-breed validation under varied environmental conditions, and consumer acceptance testing remain necessary for broader implementation. Comprehensively, these findings establish blood protein valorization as sustainable technology converting meat industry waste into functional feed ingredients supporting both animal health and product quality.
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