The Effects of the Dietary Inclusion of By-Products Obtained after the Extraction of Vitamin B 2 from Fermented Soybean on the Performance and Meat Quality of Growing–Finishing Pigs

: The by-products obtained after the extraction of vitamin B 2 from fermented soybean (VBP), primarily consisting of soybean meal, soybean oil, vitamin B 2 , and Bacillus subtilis , may serve as a cost-effective and recycled resource in animal feed. This research aimed to assess the impact of VBP on the growth performance, nutrient utilization, blood parameters, and meat quality of pigs in the growing–finishing phase. In this 16-week feeding experiment, 140 pigs of mixed breed [Duroc × (Landrace × Yorkshire)] were used, with an initial average body weight of 26.05 ± 1.32 kg. The pigs were assigned randomly to one of four dietary groups, each consisting of five pigs, with two gilts and three barrows per pen, and a total of seven repetitions per treatment. The animals were fed different levels of by-products after vitamin B 2 extraction (0, 10, 20, and 30 g/kg as the CON, VBP10, VBP20, and VBP30 dietary groups, respectively) in a three-phase feeding trial (wk. 0–4, 4–8, and 8–13). When higher levels of VBP were added as supplements, the outcomes indicated that there were no notable variations ( p > 0.05) in growth performance and nutrient digestibility among the treatment groups throughout the trial. In phase II (4–8 weeks), the inclusion of VBP in the diets showed a reduction ( p = 0.011; with linear and quadratic effects, p = 0.003) in serum norepinephrine concentration. The sensory evaluation of meat color was linearly improved ( p = 0.043) in pigs fed with graded levels of VBP supplementation. The percentage of lean meat showed a significant improvement ( p = 0.016) with the gradual levels of VBP supplementation. The addition of 30 g/kg (on an as-fed basis) of vitamin B2 by-products in the diet can simultaneously mitigate the negative impacts of acute stress without compromising growth performance and enhance the percentage of lean meat. These findings suggest that VBP could be a viable and economical alternative to corn and soybean meal and partially replace it in pig feed.


Introduction
Vitamin B 2 is an important nutrient and an essential component of basic metabolism in humans, animals, and microorganisms [1].It has been extensively applied in food, pharmaceuticals, and feed additives.The fermentation process of soybean carried out by Bacillus subtilis is widely used in the large-scale commercial vitamin B 2 production industry to meet the tremendous demand for human and animal health supplements [2,3].The extraction of vitamin B 2 from Bacillus subtilis-fermented soybean results in the generation of a substantial quantity of by-products.The by-products generated from diet, feed, and bioenergy industries can enhance the sustainability of resource utilization and the economic profits of the swine industry [4][5][6][7][8].In several studies, high dosages of particular vitamins, such as vitamin C (500 mg/kg) and vitamin E (220 IU/kg), have been shown to improve immunological response in weanling pigs [8,9].However, previous research indicates that the growth performance of weaning piglets is not influenced by feeding on vitamin supplementation [9,10].In the last few decades, a number of studies have been conducted to determine the requirements of the essential B vitamins for pigs, given their vital role in growth, lean tissue development, and metabolic processes associated with energy, carbohydrates, and fat [11].The National Research Council (NRC) [12] provides guidelines on the recommended dietary intake of vitamins and trace minerals.Despite numerous studies conducted in recent decades to ascertain the vitamin requirements for pigs, these findings, utilized by the NRC, have resulted in minimal adjustments to the requirements estimated during this experiment.There have also been minimal updates to the requirements.Vitamin B 2 from fermented soybean (VBP) has the potential to be an economical and sustainable resource for the partial substitution of ingredients.It can also offer a cost-effective means of supplying additional vitamin B2 and Bacillus subtilis in pig feed.So far, VBP has been rarely used in livestock diets.Therefore, we hypothesized that the inclusion of VBP, containing different nutrients, may partially replace the protein and carbohydrate sources and supply additional vitamins, thereby reducing the feed cost.The aim of this study was to evaluate the impact of VBP on the growth performance, meat quality, blood parameters, and nutrient digestibility of finishing pigs.

Materials and Methods
The procedures followed in managing and caring for the animals in this study were authorized by Dankook University's Animal Care and Use Committee (protocol number: DK-3-1609).The by-products obtained after the extraction of vitamin B 2 from fermented soybean (VBP; BASF, Ansan, Republic of Korea) used in the current study were provided by a commercial company (SM Bio Tech, Sungnamsi, Republic of Korea).The VBP contained excipients (400 g/kg) and residue resulting from the extraction of vitamin B 2 from the Bacillus subtilis fermented soybean (600 g/kg).The mixture was dried (60-70 • C) by hot-air drying for 3 to 4 h.The by-products obtained after the extraction of vitamin B 2 from fermented soybean, hereafter called VBP, primarily consisted of soybean meal, soybean oil, vitamin B 2 , and Bacillus subtilis.The VBP contained 265 g/kg crude protein (CP), 121 g/kg ether extract, 4964 kcal/kg gross energy (GE), and 1782.20 mg/kg vitamin B 2 .The analyzed compositions and the peroxide value of vitamin B2 from fermented soybean VBP, provided on an as-fed basis, are shown in Table 1.

Animals and Housing
In this study, 140 pigs of mixed breed [Duroc × (Landrace × Yorkshire)], with an initial average body weight (BW) of 26.05 ± 1.32 kg, were distributed across four dietary groups.The allocation followed a complete randomization-within-blocks design based on sex (2 barrows and 3 gilts), with each pen containing five pigs and a total of seven replicates for each treatment.The diets used in this trial were developed to meet or surpass the nutritional needs specified by the NRC [12] for growing-finishing pigs (as detailed in Table 2).The diets used in this trial were developed to meet or surpass the nutritional needs specified by the NRC [12] for growing-finishing pigs NRC [12]. 1 Abbreviation: DDGS, distillers dried grains with solubles; VBP, by-products obtained after the extraction of vitamin B 2 from fermented soybean. 2,3Provided per kilogram of complete diet were followed by Sureshkumar et al., [13].
A fraction of dietary corn and soybean meal was replaced with VBP, with 0, 10, 20, and 30 g/kg (as-fed basis) inclusion levels for the CON, VBP10, VBP20, and VBP30 dietary groups, respectively.In the present study, the VBP dosages were initially established as part of an initial evaluation.These dietary regimens were administered during three distinct phases: 0-4 weeks (phase I), 4-8 weeks (phase II), and 8-13 weeks (phase III).The pigs were accommodated in a controlled environment within a room equipped with plastic-slatted flooring and a mechanical ventilation system.Each enclosure, measuring 1.8 × 1.8 m, was equipped with a self-feeding system and nipple drinkers, ensuring free access to both feed and water throughout the duration of the study.An automated mechanical ventilation system was employed, which was programmed to deliver 12 h of artificial lighting daily.The environmental temperature in the room was maintained at around 30 • C and gradually decreased by 1 • C per week.

Sampling and Measurements
Individual pig weights were measured at the start of the study, as well as the 4th, 8th, and 13th week of the experimental period.The study determined the average daily gain (ADG, kg).Also, average daily feed intake (ADFI, kg) and gain-to-feed ratio (G: F) were determined by the amount of feed ingests and scraps (pen basis).
To evaluate the digestibility of dry matter (DM), energy (E), and nitrogen (N), 2 g/kg of chromium oxide (Cr 2 O 3 ) was added to the diet.This marker was introduced for fecal collection for seven days prior to the 4th week, 8th week, and 13th week of the study.Samples of the feed were collected and preserved on a weekly basis.In the 5th and 10th weeks, these samples were combined to create a demonstrative composite sample.Fecal samples were randomly collected from at least two pigs in each pen, consisting of one male and one female, resulting in a total of 14 pigs per treatment by rectal massage; these samples were combined by pen.According to the respective treatment conditions, all feed and fecal samples were dried at 70 • C for 72 h and then finely ground to a texture that enabled them to pass through a 1 mm screen.Following the guidelines of the AOAC [14], the nutrient digestibility of DM, N, and GE and chromium absorption in feed and fecal samples were analyzed following the method used by Williams et al. [15].Regarding the blood parameters, two pigs were chosen at random from each pen (one male and one female, 14 pigs/treatment).They underwent blood collection through jugular venipuncture in the 4th week, 8th week, and 13th week of the study.The serum was separated through centrifugation at 3000 rpm for 20 min, at 3000× g at a temperature of 4 • C, and subsequently preserved at −20 • C until further analysis.Thereafter, an assessment was conducted for epinephrine, norepinephrine, cortisol, and IgG levels.The quantification of serum epinephrine and norepinephrine levels was accomplished by employing an ionexchange purification technique, followed by high-performance liquid chromatography (HPLC) and electrochemical detection, in accordance with the methodology outlined by Hay and Mormède [16].In brief, the samples were placed in cationic columns and a boric acid elution procedure was used to separate the catecholamines (epinephrine and norepinephrine).The eluents underwent analysis through high-performance liquid chromatography (HPLC) with electrochemical detection at an oxidizing potential of +0.65 V.The within-assay coefficient of variation (CV) for epinephrine and norepinephrine was recorded at 6.5% and 7.0%, respectively.The inter-assay CVs for epinephrine and norepinephrine were 11.6% and 7.1%, respectively.The level of cortisol in serum was determined by a commercial ELISA kit.The serum IgG levels were assessed using an automatic biochemistry blood analyzer (Hitachi 747, Hitachi, Tokyo, Japan).
Backfat thickness (BFT) and lean meat percentage were measured from each pig in the 4th, 8th and 13th week using a real-time ultrasound instrument (Pig-log 105, SFK Technology, Herlev, Denmark).
Upon completion of the study, all pigs were transported to a nearby commercial slaughterhouse and processed using standard slaughtering techniques.The carcasses were cooled at a temperature of 2 • C for a duration of 24 h.Subsequently, a 2.54 cm thick section of the longissimus muscle (LM) was obtained by making a perpendicular cut at the 10th and 11th rib.Meat samples were selected at random from each pen, involving two pigs from each pen, comprising one gilt and one barrow, with a total of 14 pigs per treatment.The sensory assessment, which included color, marbling, and firmness scores, was conducted using a five-point scale (1 = pale, soft, and lacking marbling; 5 = dark, very firm, with moderate to abundant marbling or more).This evaluation was carried out by a panel of six trained experts, all of whom adhered to the standards outlined by the National Pork Producers Council [17].At the same time, duplicate pH measurements for each sample were analyzed using a pH meter (Testo205, Testo, Titisee-Neustadt, Germany).In addition, drip loss estimation was conducted according to the plastic bag method using 2 g of breast meat.The assessment of cooking loss was carried out in accordance with the procedure outlined by Sullivan et al. [18].According to Sureshkumar et al. [13], we measured the water-holding capacity (WHC).In brief, a 0.3 g sample was pressed with a 3000 g weight for 3 min at 26 • C, on a piece of filter paper with a diameter of 125 mm.The delineated areas of the pressed sample and the expressed moisture were subsequently determined using a digitizing area-line sensor (MT-10S; M.T. Precision Co., Ltd., Tokyo, Japan).The smaller ratio of water: meat area indicates an increased WHC.Longissimus muscle area (LMA) was measured by tracing the longissimus muscle surface at the 10th rib using the aforementioned digitizing area-line sensor.

Statistical Analysis
All data were analyzed using the general linear model (GLM) procedure of SAS software (2000; SAS Inst.Inc., Cary, NC, USA).The polynomial orthogonal contrasts of increasing dietary VBP supplementation were examined by linear and quadratic effects.The replication was considered an experimental unit.Data variability was expressed as the standard error of the mean (SEM).Statistical significance was taken into consideration when the p-value remained under 0.05, and when it was less than 0.10, trends were considered.

Growth Performance and Nutrient Digestibility
In the current study, the effect of the dietary inclusion of VBP supplements on growingfinishing pigs' growth performance and nutrient digestibility is illustrated in Tables 3 and 4.There was no significant difference (p > 0.05) observed in body weight, ADG, ADFI, and G:F ratio among treatments over the entire experimental period.In addition, no significant differences were observed (p > 0.05) in dry matter and nitrogen digestibility among treatments during the phase I and phase II feeding trials.However, there was a tendency for the apparent total tract digestibility (ATTD) of dry matter to linearly increase (0.05 < p < 0.10) during phase III.

Blood Profiles
The dietary inclusion of VBP supplementation in blood analysis is presented in Table 5.Throughout the feeding trial, the serum cortisol and IgG were not influenced (p > 0.05) by the VBP treatment groups.During phase II, the serum norepinephrine decreased (p = 0.011, linear; p = 0.003, quadratic), but the serum epinephrine only showed trends in linear reduction (p = 0.064) associated with the inclusion of VBP.At weeks 8-13, there was a tendency for reduction (p = 0.056, linear) in serum norepinephrine concentration associated with the inclusion of VBP in the diets.

Meat Quality
Table 6 shows the impacts of dietary VBP supplementation on meat quality.There was a linear increase (p = 0.043) in meat color associated with the inclusion of VBP (Table 6).Other sensory evaluation scores (like firmness and marbling), longissimus muscle area, pH, cooking loss, drip loss, or WHC were not influenced (p > 0.05) by dietary VBP supplementation.During weeks 8-13, the lean meat percentage significantly increased (p = 0.016), while the backfat thickness showed a linearly decreasing trend (p = 0.054) associated with the inclusion of VBP (Table 7).

Discussion
Soybean meal is widely used in the pig industry as a significant plant-based protein source due to its high amino acid content and cost-effectiveness [19].Although it contains high protein, vitamins, and minerals, it has been rarely used directly because of its highly unacceptable characteristics such as its high satiety value, poor digestibility, the presence of anti-nutrients (e.g., trypsin inhibitors, hemagglutinins, and antivitamins), bitterness, and long cooking time [20][21][22].Microbial fermentation can be employed to remove anti-nutritional compounds, undigested elements, and certain large molecular nutrients from soybean meal.Previous studies have shown that the fermentation process involving B. subtilis ZJ12-1 can effectively degrade β-conglycinin and glycinin, the major antigenic proteins present in soybeans.This process results in the production of smaller peptides with antioxidant properties.[23].Furthermore, the fermented soybean meal in the feeds can introduce additional probiotics and their metabolic by-products, including vitamins, exoenzymes, and various short-chain fatty acids.This can lead to improved digestive, absorptive, and immune functions in pigs [24,25].Consequently, a range of processing methods, including heat treatment, enzymatic processes, and fermentation, have been employed to deactivate undesirable components in order to enhance the nutritional quality of soybeans for swine [19,26,27].Fermentation is a suggested method for improving the flavor, texture, and nutritional value of feedstuffs and/or agro-industrial by-products [23,28,29].Various Bacillus species have been substantially used as host strains for the industrial processing of vitamins, proteases, purine nucleotides, etc. [30][31][32].The VBP used in the present study is a residue collected after the extraction of vitamin B 2 from fermented soybeans.In this study, we substituted a portion of corn and soybean meal with VBP and investigated the impact of different levels of VBP supplementation on growth performance, blood parameters, nutrient utilization, and meat quality.

Growth Performance and Nutrient Digestibility
In the current study, growth performance and nutrient digestibility were not influenced by VBP supplementation throughout the experiment.Previous research demonstrated that B. subtilis-fermented soybean reduced the activity of anti-nutrients (e.g., raffinose, and stachyose), which is associated with indigestion and flatulence in monogastric animals.Additionally, this process demonstrated a reduction in the undesirable beany odor associated with soybeans, making it more palatable for animals.Moreover, it led to increased digestibility of soybeans or their components that are typically considered indigestible.[33][34][35].Bacillus subtilis was used to remove the bitter taste of soybean [36].This was accomplished by raising the concentration of volatile substances during the Bacillus subtilis fermentation, which is connected to extracellular hydrolase enzymes, such as lipases, amylase, and sucrose.ADG and ADFI rose when growing and finishing pigs' diets were gradually supplemented with more vitamin B, as shown by Mahan et al. [37].In rat [38] and neonatal pig [39] studies, the inclusion of fermented soybeans in diets improved ADG and feed efficiency.Moreover, the antioxidant activity of soybean isoflavones from fermented soybeans improved growth performance in broilers [40] and piglets [41].However, our findings showed that ADG, ADFI, and feed efficiency were unaffected for pigs who were fed VBP-containing isoflavones, at the growing and finishing phases.The addition of a combination of B vitamins in the diet has been documented to enhance the growth performance of young pigs [42,43].However, there is variation in opinions regarding the optimal levels of vitamin B required for achieving maximum growth performance.
Fermentation could bring substantial changes in the nutrient properties of soybeans.Visessanguan et al. [33] and Hu et al. [35] reported that B. subtilis-fermented soybean, during fermentation, releases proteinases that degrade soybean protein into non-protein and soluble nitrogen and regulates the enzymatic hydrolysis of soybean (e.g., fibrinolysis and caseinolysis), while increasing the synthesis of bacterial protein.They also showed that while total sugar and crude fiber were reduced, the ash content and overall energy from fermented soybeans remained unchanged.The carbohydrate concentration was reduced by microbes for energy supply.In the current study, the apparent total-tract digestibility of dry matter and nitrogen in pigs fed VBP diets, remained unaffected.This may result from insufficient VBP supplementation or the differences between fermented soybean and vitamin B 2 extracted fermented soybean.Thus, further investigation is needed to clarify the adaptable amount of VBP for increasing growth performance and nutrient digestibility in growing-finishing pigs.

Blood Profiles
Epinephrine, norepinephrine, and cortisol concentrations in the serum serve as indicators of the stress and anti-stress states of animals in current scientific research.Epinephrine and norepinephrine, belonging to catecholamines, are secreted by the adrenal glands and provide energy and strength to boost the main neurotransmitters of the sympathetic nervous system [44,45].Cortisol, an anti-stress hormone, is produced by the hypothalamicpituitary-adrenal axis during stress (environmental stressors, feeding management, trans-portation, social mixing, and maternal separation), and it can suppress the immune response; high cortisol level in serum can also lower immunity [46,47].In our study, the linear reduction in epinephrine or norepinephrine concentration evidenced lower acute stress in the VBP group than in the control group during phase II and III, while cortisol and IgG were not shown to be affected by VBP supplementation.These results indicate that the dietary inclusion of VBP could minimize acute stress responses and exert a positive effect on the sympathetic nervous system, but with no effect on the hypothalamic-pituitaryadrenal axis in comparison with the control.The significant reduction in epinephrine or norepinephrine concentration with increasing VBP supplementation indicates that VBP may have modulatory effects on acute stress responses through the anti-stress capacity of fermentation, soybean isoflavones, or vitamin B 2 [48,49].As there are not enough reports about fermented soybean or vitamin B 2 by-products reducing stress in pigs, and the mechanism is still unclear, more research is required to understand the mechanism involved in the reduction in acute stress.

Meat Quality
Over the course of the last forty years, the average weight of slaughtered pigs for commercial purposes has shown a consistent linear increase, with a growth rate of 5.8 kg every ten years [50].Increasing the slaughter weight of pigs offers several benefits, including reduced overhead costs, higher meat yield, and improved meat-to-bone ratio.However, this also introduces challenges such as increased backfat thickness [51].The color of meat is a crucial factor influencing consumer acceptance in the pork market.[52,53].Lean meat or marbling is generally preferred by customers to fatty pork with subcutaneous white fat [54].In the present research, the meat sensory color and lean meat percentage were linearly increased by VBP supplementation.Lipid peroxidation is a natural phenomenon that affects meat quality [55].Meat quality can be increased by using dietary antioxidants including arginine, vitamin B2, and soybean isoflavone, which have been demonstrated to decrease lipid peroxidation and improve antioxidative status [40,48,56].The vitamin B 2 contained in VBP is one of the neglected antioxidants that can protect the body against oxidative stress, and it plays an important role in the prevention of lipid peroxidation through the conversion of oxidized glutathione to its reduced form, or as a component of the glutathione redox cycle [48,56].Mahan et al. [42] observed that supplementing the diet with vitamin B led to an increase in the loin meat area, reaching up to 100% of the National Research Council (NRC) requirement estimates.In a study conducted by Mahan et al. [42], it was shown that the addition of vitamin B in the diet resulted in an increase in loin eye area up to 100% of the NRC requirement estimates.The findings suggest that vitamin B may not be a limiting factor for lean deposition in pigs when their fortification levels exceed 100-200% of the NRC requirement estimates.However, higher supplementation of B vitamins could still be advantageous for enhancing vitamin deposition in pork.The soybean isoflavone could also exert an antioxidant effect and prevent lipid peroxidation in the male broiler [40].Furthermore, the fermentation of soybean could increase the activity of soybean isoflavone to prevent lipid peroxidation, while increasing the synthesis of bacterial protein for muscle formation [57].Therefore, VBP could be used as a functional supplement in the diet to improve pork meat quality.The mechanisms of how dietary supplementation with fermented soybean or VBP can increase meat quality are awaiting further clarification.

Conclusions
In summary, dietary VBP supplements enhanced pigs' ability to withstand stress without negatively affecting their growth performance.Simultaneously, it improved meat the color and the percentage of lean meat, suggesting that this by-product has the potential to be a cost-effective alternative to corn and soybean meal in pigs' diets.

Table 1 .
Analyzed compositions and peroxide value of vitamin B 2 from fermented soybean VBP, as-fed basis 1 .

Table 2 .
Composition of the experimental growing-finishing pig diets (as-fed basis)1.

Table 3 .
Effect of dietary vitamin B 2 from fermented soybean VBP supplementation on growth performance in growing-finishing pigs 1 .
1Abbreviations: VBP, by-products obtained after the extraction of vitamin B 2 from fermented soybean.

Table 4 .
Effect of dietary vitamin B2 from fermented soybean VBP supplementation on nutrient digestibility in growing-finishing pigs 1 .

Table 5 .
Effect of dietary vitamin B 2 from fermented soybean VBP supplementation on blood profiles in growing-finishing pigs 1 .

Table 6 .
Effect of dietary vitamin B2 from fermented soybean VBP supplementation on meat quality in growing-finishing pigs 1 .
1 Abbreviations: SEM, standard error of means; VBP, by-products obtained after the extraction of vitamin B 2 from fermented soybean.a,b Means in the same row with different superscript differ significantly (p < 0.05).

Table 7 .
Effect of dietary vitamin B 2 from fermented soybean VBP supplementation on backfat thickness and lean meat percentage in growing-finishing pigs 1 .