Application of *Pediococcus lactis* NR and its combined compound enzyme preparation in increasing the free ferulic acid content of wheat bran

Abstract

This invention relates to the fields of fermentation engineering and feed processing technology, particularly to the application of *Pediococcus lactis* NR and its combined complex enzyme preparations in increasing the free ferulic acid content of wheat bran. This invention provides a new application of *Pediococcus lactis* NR, specifically including the preparation of wheat bran fermentation agents, increasing the free ferulic acid content of wheat bran, and preparing preparations to increase the free ferulic acid content of wheat bran. This invention utilizes *Pediococcus lactis* NR to improve the quality of wheat bran feed, with low cost and a safe and reliable strain. Simultaneously, this invention also provides a method for preparing fermented wheat bran and / or increasing the free ferulic acid content of wheat bran. This method utilizes the synergistic fermentation of *Pediococcus lactis* and a complex enzyme system to fully release the free ferulic acid in wheat bran, while simultaneously endowing it with abundant beneficial metabolites, giving the fermented wheat bran excellent antioxidant and anti-inflammatory effects.

Description

Technical Field

[0001] This invention relates to the fields of fermentation engineering and feed processing technology, and in particular to the application of *Pediococcus lactis* NR and its combined complex enzyme preparation in increasing the free ferulic acid content of wheat bran. Background Technology

[0002] Wheat bran, a byproduct of wheat processing, has significant applications in the feed industry due to its rich nutritional content and low price. Wheat bran contains abundant phenolic acids, especially ferulic acid, a natural plant compound with significant antioxidant, anti-inflammatory, and immunomodulatory effects. As an important functional component, ferulic acid has broad application potential in improving animal health, enhancing antioxidant capacity, and reducing inflammatory responses. However, in natural wheat bran, ferulic acid mainly exists in a bound form, tightly bound to components such as cellulose and hemicellulose through complex cross-linking structures, making it difficult to release and utilize, thus hindering its full antioxidant activity. This characteristic greatly limits the high-value development and utilization of wheat bran.

[0003] In recent years, microbial fermentation technology has been widely used in feed processing. Through synergistic fermentation of bacteria and enzymes, not only can the cross-linked structures in wheat bran be effectively broken down, promoting the conversion of bound ferulic acid into free ferulic acid and thus significantly improving its bioavailability, but also various beneficial metabolites, such as organic acids and oligosaccharides, are produced during fermentation. These metabolites have significant probiotic functions, improving animal gut health and increasing feed utilization. Furthermore, the free ferulic acid and other phenolic acid antioxidants enriched in this fermented wheat bran endow it with excellent antioxidant and anti-inflammatory effects, significantly reducing broiler mortality and further enhancing its value as a functional feed ingredient. Currently, there are relatively few strains available for releasing free ferulic acid from wheat bran, necessitating the discovery of new strains. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Pediococcus lactis NR and its combined compound enzyme preparation in increasing the free ferulic acid content of wheat bran, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides *Pediococcus lactis* ( Pediococcus acidilactici ) NR application in any of the following (1) Preparation of wheat bran starter culture; (2) Increase the content of free ferulic acid in wheat bran; (3) Preparation of formulations to increase the content of free ferulic acid in wheat bran; The preservation number of the lactic acid pediococcus NR is CCTCC NO: M 20252979.

[0006] This invention provides a wheat bran fermentation agent, comprising Pediococcus lactis NR and a compound enzyme preparation; the preservation number of Pediococcus lactis NR is CCTCC NO: M 20252979.

[0007] Preferably, the complex enzyme preparation includes ferulic acid esterase, xylanase, cellulase, and arabinofuranase.

[0008] This invention provides the application of *Pediococcus lactis* NR or the above-mentioned wheat bran starter in fermenting wheat bran and / or increasing the free ferulic acid content of wheat bran, wherein the preservation number of *Pediococcus lactis* NR is CCTCC NO: M 20252979.

[0009] This invention provides a method for preparing fermented wheat bran and / or increasing the free ferulic acid content of wheat bran, comprising the steps of mixing Pyrococcus lactis NR bacterial solution and wheat bran, then adding a compound enzyme preparation, and carrying out fermentation. The preservation number of the lactic acid porphyria NR is CCTCC NO: M 20252979; The compound enzyme preparation includes ferulic acid esterase, xylanase, cellulase, and arabinofuranase.

[0010] Optionally, the effective viable count of *Pediococcus lactis* NR in the *Pediococcus lactis* NR bacterial solution is 3 × 10⁻⁶. 9 cfu / mL; The amount of the *Pediococcus lactis* NR bacterial solution used is 1% of the mass of the wheat bran; The compound enzyme preparation contains 30 U / g dry basis of ferulic acid esterase, 100 U / g dry basis of xylanase, 100 U / g dry basis of cellulase, and 10 U / g dry basis of arabinofuranyl glycoside enzyme. The fermentation time was 96 hours and the temperature was 37°C.

[0011] The present invention provides fermented wheat bran prepared using the above-described method.

[0012] More preferably, the fermented wheat bran contains 20 billion CFU / g dry basis of Pediococcus lactis, 1606.41 μg / g dry basis of free ferulic acid, and 2026 μg / g dry basis of ferulic acid released after in vitro digestion.

[0013] This invention provides the application of the above-mentioned fermented wheat bran in the preparation of feed additives or feed.

[0014] The present invention provides a feed additive comprising the above-mentioned fermented wheat bran.

[0015] The present invention provides a feed comprising the above-mentioned feed additives and base feed.

[0016] The present invention discloses the following technical effects: This invention provides novel applications for *Pediococcus lactis* NR, specifically including the preparation of wheat bran starter cultures, increasing the free ferulic acid content in wheat bran, and preparing formulations to increase the free ferulic acid content in wheat bran. This invention utilizes *Pediococcus lactis* NR to improve the quality of wheat bran feed, with low cost and a safe and reliable strain.

[0017] This invention also provides a wheat bran fermentation agent and a method for preparing fermented wheat bran and / or increasing the free ferulic acid content in wheat bran. This method utilizes the synergistic fermentation of *Pediococcus lactis* and a complex enzyme system to fully release the free ferulic acid in wheat bran, while simultaneously endowing it with abundant probiotic metabolites, giving the fermented wheat bran excellent antioxidant and anti-inflammatory effects. This invention not only effectively taps into the high-value resources of wheat bran but also provides a new technical path and practical foundation for its application in the field of functional feed, possessing significant economic value and promising prospects for promotion.

[0018] Furthermore, the wheat bran fermented with bacteria and enzymes exhibits a Pediococcus lactis count of 20 billion CFU / g dry basis and a free ferulic acid concentration of 1606.41 μg / g dry basis, with a ferulic acid release of 2026 μg / g dry basis after in vitro digestion. The fermented wheat bran contains high concentrations of free ferulic acid and other phenolic acid antioxidants, as well as abundant probiotic metabolites and oligosaccharides from lactic acid bacteria, demonstrating excellent antioxidant activity and significantly reducing broiler mortality. This invention fully utilizes the resources of wheat bran, a traditional feed ingredient, and endows it with probiotic functions to improve feeding efficiency and the health of raised animals. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The fermentation effects of different strains on wheat bran; Figure 2 The results represent the optimization of the complex enzyme system; Figure 3 The result is the optimized fermentation time; Figure 4 Results of fermentation temperature optimization Figure 5 This represents the result of optimizing the fermentation method; Figure 6 Results of viable cell count Figure 7 The results are for the determination of total phenolic acids; Figure 8 A statistical graph showing the concentration of ferulic acid before and after wheat bran fermentation; Figure 9 The results are from Fourier transform infrared spectroscopy analysis. Figure 10 A statistical chart showing the release of ferulic acid after in vitro digestion of fermented wheat bran; Figure 11 The results are for the antioxidant assay of phenolic acid extracts; where A represents DPPH scavenging rate; B represents ABTS scavenging rate; and C represents FRAP. Figure 12 High performance liquid chromatography peak chromatogram of phenolic acid extract from wheat bran before fermentation (R); Figure 13 High performance liquid chromatography peak chromatogram of phenolic acid extract from wheat bran after fermentation (JE); Figure 14 Data from fecal sample screening at 47 days; where A is the control group; B is the 5% replacement group; and C is the 10% replacement group. Figure 15 The results of a survey on protein metabolism-related indicators are shown; where A represents BUN; B represents TP; C represents ALB; and D represents GLB. Figure 16 The results of a survey on liver enzyme profile-related indicators are as follows: A is TC; B is TG; C is HDL-C; D is LDL-C; E is FFA. Figure 17 The results of the survey on liver enzyme profile-related indicators; where A is ALT; B is ALP; and C is AST. Figure 18 The results of the survey on antioxidant-related indicators are as follows: A represents SOD; B represents GSH-Px; and C represents MAD. In the figure, R represents the wheat bran group; E represents the enzymatic hydrolysis group; J represents the Pediococcus lactis fermentation group; and JE represents the bacterial-enzyme co-fermentation group. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Pediococcus lactis ( Pediococcus acidilactici Obtaining NR 1. Preparation of basal culture medium MRS liquid medium: glucose 20.0 g / L; peptone 10.0 g / L; yeast extract 5 g / L; beef extract powder 5.0 g / L; dipotassium hydrogen phosphate 2.0 g / L; sodium acetate 5.0 g / L; triammonium citrate 2.0 g / L; Tween-80 1.0 mL / L; MRS solid medium is made by adding 2 wt% agar powder to MRS liquid medium; All culture media were sterilized at 115°C for 20 minutes.

[0027] 2. Isolation and screening of strains Collect an appropriate amount of sample from the liquor mash. The sample size is generally determined based on experimental needs, but should be sufficient for subsequent operations. Quickly transfer the sample to a sterile container and add an appropriate amount of sterile saline or buffer solution for dilution and mixing to reduce impurities and microbial concentration variations. Perform serial dilutions of the pretreated suspension to obtain bacterial solutions of different concentrations. This helps isolate individual colonies in subsequent cultures. Select an appropriate dilution and spread the bacterial solution evenly on MRS solid medium. Multiple parallel plates can be prepared for each dilution to increase the reliability of screening. Place the spread plates in a constant temperature biochemical incubator and anaerobically culture at 37°C for 24 hours.

[0028] 3. Isolation and purification of strains After culturing, observe the colony morphology on the plates and select colonies suspected to be lactic acid bacteria. Lactic acid bacteria colonies are characterized by being small, round, with neat edges, mostly milky white or grayish white, and having a glossy surface. Inoculate the selected colonies onto new MRS medium plates using the three-zone or four-zone streak method for further purification. Repeat this step until a pure culture is obtained.

[0029] 4. Identification and verification of strains DNA was extracted from the pure culture and amplified by PCR using Pediococcus lactis-specific primers (Forward primer: 5'-GATACGCTGTTGCTGCTGAC-3', SEQ ID NO.1; Reverse primer: 5'-CTTGCGTTCATCGTTGTCAC-3', SEQ ID NO.2). The amplified products were then detected by gel electrophoresis. Simultaneously, 16S rRNA gene sequencing was performed, and the sequencing results were compared with sequences from known strains to confirm that it was Pediococcus lactis.

[0030] 5. Preservation of bacterial strains The *Pediococcus lactis* strain obtained through the above screening is named *Pediococcus lactis* (…). Pediococcus acidilactici NR, category name Pediococcus acidilactici The strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20252979 and address: Wuhan University, Wuhan, China.

[0031] 6. Activation of Pediococcus lactis NR Perform the procedure in a clean bench or sterile room. First, inoculate *Pediococcus lactis* NR into MRS liquid medium at a bacterial count of 1%, and incubate for 24 hours at 37°C to obtain a fresh *Pediococcus lactis* NR bacterial suspension (effective viable count of 3 × 10⁻⁶). 9 (cfu / mL), used for subsequent wheat bran fermentation.

[0032] Example 2 Optimization of bacterial-enzyme co-fermentation conditions 1. Fermentation effects of different strains on wheat bran 1.1 Pretreatment of wheat bran before fermentation Weigh out wheat bran, remove impurities and wash it clean. Then, perform physical crushing pretreatment on the wheat bran. Determine the moisture content of the wheat bran according to the method of national standard GB5009.3-2016. Take 250g of the treated wheat bran and add 250mL of water.

[0033] 1.2 Grouping Enzymatic hydrolysis: Add a compound enzyme preparation (ferulinase, xylanase, cellulase and arabinofuranoside) solution to the treated wheat bran and mix well. The compound enzyme preparation contains 30 U / g dry basis (dried wheat bran), 100 U / g dry basis xylanase, 100 U / g dry basis cellulase and 10 U / g dry basis arabinofuranoside. Anaerobic fermentation is carried out at 37℃ for 96 h.

[0034] NR: Inoculate the treated wheat bran with fresh Pediococcus lactis NR bacterial suspension at a rate of 1% (by weight / volume) (effective viable count of 3 × 10⁻⁶). 9 (cfu / mL), then add the compound enzyme preparation (same as the previous grouping: enzymatic hydrolysis). At this point, the wheat bran moisture content is 60%, mix well. Anaerobic fermentation at 37℃ for 96 hours.

[0035] 3A: Inoculate the treated wheat bran with fresh Pediococcus lactis S204 bacterial suspension at a rate of 1% (by weight / volume) (effective viable count of 3 × 10⁻⁶). 9 (cfu / mL), then add the compound enzyme preparation (same as the previous grouping: enzymatic hydrolysis). At this point, the wheat bran moisture content is 60%, mix well. Anaerobic fermentation at 37℃ for 96 hours.

[0036] 12B: Inoculate the treated wheat bran with fresh Lactobacillus paracasei (purchased from Zhengzhou Best Food Additives Co., Ltd.) bacterial culture at a rate of 1% (by weight / volume) (effective viable count of 3 × 10⁻⁶). 9 (cfu / mL), then add the compound enzyme preparation (same as the previous grouping: enzymatic hydrolysis). At this point, the wheat bran moisture content is 60%, mix well. Anaerobic fermentation at 37℃ for 96 hours.

[0037] 1.3 Results The results are as follows Figure 1 As shown in the figure. The results showed that the concentration of free ferulic acid in the fermented wheat bran of the NR group was significantly higher than that of other groups, proving that *Pediococcus lactis* NR can assist the enzyme system in releasing ferulic acid.

[0038] 2. Optimization of compound enzyme systems 2.1 Pretreatment of wheat bran before fermentation Weigh out wheat bran, remove impurities and wash it clean. Then, perform physical crushing pretreatment on the wheat bran. Determine the moisture content of the wheat bran according to the method of national standard GB5009.3-2016. Take 250g of the treated wheat bran and add 250mL of water.

[0039] 2.2 Grouping The initial enzyme system was ferulic acid esterase, and other enzymes were added to it to improve the enzymatic hydrolysis effect and the release of free ferulic acid.

[0040] Enzyme-free: Inoculate the pretreated wheat bran with fresh Pediococcus lactis NR bacterial suspension at a rate of 1% (w / v) (effective viable count of 3 × 10⁻⁶). 9 (cfu / mL), mix well. At this point, the wheat bran moisture content is 60%. After anaerobic fermentation at 37℃ for 96 hours, it is recorded as enzyme-free.

[0041] Ferulic acid esterase: Inoculate pretreated wheat bran with fresh Pediococcus lactis NR bacterial suspension (effective viable count 3 × 10⁻⁶) at an inoculation rate of 1% (by weight / volume). 9 Then, ferulic acid esterase (30 U / g dry basis) was added and mixed well. At this point, the wheat bran moisture content was 60%. Anaerobic fermentation was carried out at 37℃ for 96 hours, denoted as FAE.

[0042] Ferulic acid esterase + xylanase: Inoculate pretreated wheat bran with fresh Pediococcus lactis NR bacterial suspension at a rate of 1% (w / v) (effective viable count of 3 × 10⁻⁶). 9 Then, a compound enzyme preparation (feruloesterase and xylanase) solution was added, wherein the amount of feruloesterase added was 30 U / g dry basis and the amount of xylanase added was 100 U / g dry basis. At this time, the moisture content of the wheat bran was 60%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 hours, and the result was recorded as FAE+XAE.

[0043] Ferulic acid esterase + cellulase: Inoculate pretreated wheat bran with fresh Pediococcus lactis NR bacterial suspension at a rate of 1% (by weight / volume) (effective viable count of 3 × 10⁻⁶). 9 Then, a compound enzyme preparation (ferulinase and cellulase) solution was added, wherein the amount of ferulinase added was 30 U / g dry basis and the amount of cellulase added was 100 U / g dry basis. At this time, the moisture content of the wheat bran was 60%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 hours, which was recorded as FAE+CAE.

[0044] Ferulic acid esterase + xylanase + cellulase: Inoculate pretreated wheat bran with fresh Pediococcus lactis NR bacterial suspension at a rate of 1% (w / v) (effective viable count of 3 × 10⁻⁶). 9Then, a compound enzyme preparation (feruloesterase, xylanase, and cellulase) solution was added, wherein the amount of feruloesterase added was 30 U / g dry basis, the amount of xylanase added was 100 U / g dry basis, and the amount of cellulase added was 100 U / g dry basis. At this time, the moisture content of the wheat bran was 60%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 hours, and the result was recorded as FAE+XAE+CAE.

[0045] Ferulic acid esterase + xylanase + cellulase + arabinofuranoside: Inoculate pretreated wheat bran with fresh Pediococcus lactis NR bacterial suspension at a rate of 1% (w / v) (effective viable count of 3 × 10⁻⁶). 9 Then, a compound enzyme preparation (feruloesterase, xylanase, cellulase, and arabinofuranoside) solution was added, wherein the addition amount of ferulic acid esterase was 30 U / g dry basis, the addition amount of xylanase was 100 U / g dry basis, the addition amount of cellulase was 100 U / g dry basis, and the addition amount of arabinofuranoside was 10 U / g dry basis. At this time, the moisture content of wheat bran was 60%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 h, and the result was recorded as FAE+XAE+CAE+AFE.

[0046] 2.3 Results The results are as follows Figure 2 As shown in the figure. The results indicate that, considering the structural characteristics of wheat bran, as well as the distribution, binding sites, and bond characteristics of ferulic acid within it, a combination of enzyme systems was developed to help open the wheat bran structure and release ferulic acid. The results showed that the ferulic acid release effect of the multi-enzyme combination group was significantly better than that of the single-enzyme fermentation group. In particular, the FAE+XAE+CAE+AFE combination exhibited the best effect.

[0047] 3. Optimization of fermentation conditions 3.1 Optimization of fermentation time Fresh Pediococcus lactis NR bacterial suspension (effective viable count of 3 × 10⁻⁶) was inoculated into the pretreated wheat bran at an inoculation rate of 1% (by weight / volume percentage). 9 Then, a compound enzyme preparation (feruloesterase, xylanase, cellulase, and arabinofuranoside) solution was added, wherein the addition amount of ferulic acid esterase was 30 U / g dry basis, the addition amount of xylanase was 100 U / g dry basis, the addition amount of cellulase was 100 U / g dry basis, and the addition amount of arabinofuranoside was 10 U / g dry basis. At this time, the moisture content of wheat bran was 60%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 0, 24, 48, 72, 96, and 120 h.

[0048] 3.2 Optimization of fermentation temperature Fresh Pediococcus lactis NR bacterial suspension (effective viable count of 3 × 10⁻⁶) was inoculated into the pretreated wheat bran at an inoculation rate of 1% (by weight / volume percentage). 9 Then, add a compound enzyme preparation (feruloesterase, xylanase, cellulase, and arabinofuranoside) solution, wherein the compound enzyme preparation contains 30 U / g dry basis for ferulic acid esterase, 100 U / g dry basis for xylanase, 100 U / g dry basis for cellulase, and 10 U / g dry basis for arabinofuranoside. At this point, the wheat bran moisture content is 60%, and mix well. Anaerobic fermentation is then carried out at 35℃, 37℃, 39℃, or 41℃ for 96 hours.

[0049] 3.3 Optimization of Fermentation Method The effects of oxygen on the synergistic fermentation of bacteria and enzymes were compared. Fresh *Pediococcus lactis* NR culture (with an effective viable count of 3 × 10⁻⁶) was inoculated into pretreated wheat bran at a 1% (w / v) inoculation rate. 9 Then, add a compound enzyme preparation (feruloesterase, xylanase, cellulase, and arabinofuranoside) solution, wherein the compound enzyme preparation contains 30 U / g dry basis for ferulic acid esterase, 100 U / g dry basis for xylanase, 100 U / g dry basis for cellulase, and 10 U / g dry basis for arabinofuranoside. At this point, the wheat bran moisture content is 60%, and mix well. Perform anaerobic fermentation at 37℃ for 96 hours or aerobic fermentation for 96 hours.

[0050] 3.4 Results The results of optimizing fermentation time are as follows: Figure 3 As shown, the results indicate that the concentration of free ferulic acid gradually increases with the extension of fermentation time, reaching the highest level at 96 hours, thus determining the fermentation time to be 96 hours.

[0051] The results of optimizing the fermentation temperature are as follows: Figure 4 As shown, the results indicate that the concentration of ferulic acid increases with increasing fermentation temperature, but decreases after the temperature exceeds 40℃, with the optimal fermentation temperature being 37℃.

[0052] The results of optimizing the fermentation method are as follows: Figure 5 As shown in the figure, the results indicate that the synergistic effect of bacteria and enzymes is the best under anaerobic conditions, resulting in a higher concentration of free ferulic acid in the fermentation system.

[0053] Example 3: Verification of the synergistic effect of bacteria and enzymes 1. Pretreatment of wheat bran before fermentation Weigh out wheat bran, remove impurities and wash it clean. Then, perform physical crushing pretreatment on the wheat bran. Determine the moisture content of the wheat bran according to the method of national standard GB5009.3-2016. Take 250g of the treated wheat bran and add 250mL of water. 2. Grouping Wheat bran group (R): Wheat bran is ground to 30 mesh, which is a pre-treated wheat bran.

[0054] Enzymatic hydrolysis group (E): A compound enzyme preparation solution (cellulase, xylanase, ferulic acid esterase and arabinofuranylase) was added to the pretreated wheat bran. The compound enzyme preparation contained 100 U / g dry basis of cellulase, 100 U / g dry basis of xylanase, 30 U / g dry basis of ferulic acid esterase and 10 U / g dry basis of arabinofuranylase. Anaerobic fermentation was carried out at 37°C for 96 h.

[0055] Pediococcus lactis fermentation group (J): Fresh Pediococcus lactis NR bacterial suspension (effective viable count of 3 × 10⁻⁶) was inoculated into pretreated wheat bran at an inoculation rate of 1% (by weight / volume percentage). 9 Mix well (cfu / mL). Perform anaerobic fermentation at 37℃ for 96 hours.

[0056] JE (Joint Fermentation of Microorganisms and Enzymes): Fresh Pediococcus lactis NR bacterial suspension (effective viable count of 3 × 10⁻⁶) was inoculated into the treated wheat bran at an inoculation rate of 1% (by weight / volume percentage). 9 Then, add a compound enzyme preparation solution (cellulase, xylanase, ferulic acid esterase, and arabinofuranylase), wherein the amount of cellulase added is 100 U / g dry basis, the amount of xylanase added is 100 U / g dry basis, the amount of ferulic acid esterase added is 30 U / g dry basis, and the amount of arabinofuranylase added is 10 U / g dry basis. At this time, the moisture content of wheat bran is 60%. Mix well. Anaerobic fermentation at 37℃ for 96 h.

[0057] 3. Indicator Survey 3.1 Viable Bacteria Count Viable cell counts were performed using the dilution plating method. 1g of fermented wheat bran was added to 9mL of sterile water, mixed thoroughly, and then serially diluted 10-fold. 0.1mL of each dilution was evenly spread onto MRS solid agar plates, with three replicates. The plates were inverted and incubated at 37℃. The colony counts were then recorded. Results are shown below. Figure 6 As shown in the figure. The results showed that the bacterial count in the bacterial-enzyme co-fermentation group reached a maximum of 20 billion CFU / g dry basis.

[0058] 3.2 Extraction of phenolic acids and determination of total phenolic acids Take 3g of fermented wheat bran, add 20mL of 80% (v / v) methanol, and extract by ultrasonic extraction at 50℃ for 2h. Centrifuge and separate. Add another 20mL of 80% methanol and extract by ultrasonic extraction at 60℃ for 1h. Centrifuge and collect the supernatant. Add another 20mL of 80% (v / v) methanol and extract by ultrasonic extraction at 70℃ for 1h. Centrifuge and collect the supernatant. Collect the supernatant from the three extractions into a 100mL centrifuge tube. Use a rotary evaporator to concentrate the supernatant under vacuum at 45℃ until the sample volume is less than 5mL. Finally, redissolve the extract in distilled water (30mL) to prepare fermented wheat bran phenolic acid extract and store it at -40℃ for later use.

[0059] The total phenol content in wheat bran extract was determined by the Folin-Ciocalteu colorimetric method. 0.1 mL of fermented wheat bran phenolic acid extract was mixed with 0.1 mL of Folin-Ciocalteu reagent and 0.4 mL of distilled water. After reacting for 6 min, 0.8 mL of distilled water and 1.0 mL of 7% sodium carbonate were added. The mixture was then incubated at room temperature for 90 min, and the absorbance was measured at 760 nm using a microplate reader. The total phenol content was expressed as mg gallic acid equivalent (GAE) / 100 g FW. Results are shown below. Figure 7 As shown in the figure. The results showed that the synergistic fermentation of bacteria and enzymes significantly improved the release of phenolic acids compared to the other three groups, with the total phenolic acid content reaching 3937.08 ug GAE / gFW ( Figure 7 ).

[0060] 3.3 Determination of ferulic acid concentration The fermented wheat bran phenolic acid extract was filtered through a 0.22 μm membrane into a sample vial.

[0061] HPLC determination conditions: An Agilent RX-C18 rapid separation column (4.6 mm × 150 mm, 3.5 μm) was used, with an injection volume of 10 μL. The mobile phase consisted of acetonitrile (A) and 1% glacial acetic acid (B), with gradient elution: 10% A 0-5 min, 20% A 5-10 min, 30% A 10-12 min, 40% A 12-15 min, 50% A 15-17 min, 70% A 17-22 min, 85% A 22-27 min, 100% A 27-30 min, and 10% A 30-31 min. The wavelength was 280 nm, the flow rate was 0.8 mL / min, and the column temperature was 35 °C. Take 1.0 mg of ferulic acid standard and use anhydrous ethanol as solvent to prepare solutions with concentrations of 20, 40, 60, 80, and 100 μg / mL. Plot a standard curve. The standard curve is y = 18.101x - 48.473 with R² 0.9992.

[0062] The statistical results of ferulic acid content before and after wheat bran fermentation are as follows: Figure 8As shown. The results showed that fermented wheat bran (after fermentation) obtained by synergistic fermentation of bacteria and enzymes had a significant effect on the release of ferulic acid, with the final concentration of free ferulic acid reaching 1606.41 μg / g dry basis. Figure 8 The free ferulic acid concentration in group R was 26.91 μg / g dry basis, the free ferulic acid concentration in group E was 1045.82 μg / g dry basis, and the free ferulic acid concentration in group J was 122.102 μg / g dry basis.

[0063] 3.4 Fourier transform infrared spectroscopy analysis The infrared absorption spectrum of fermented wheat bran was determined using the KBr tableting method. 1-2 mg of freeze-dried fermented wheat bran sample and 200 mg of pre-dried spectrally pure KBr powder were weighed and placed together in a dry agate mortar and ground thoroughly. The mixture was then pressed into tablets using a tableting machine. Using KBr as a control, the spectrum was scanned at 450-4000 cm⁻¹ using a Fourier transform infrared spectroscopy (FTIR) spectrometer. -1 Infrared spectra of fermented wheat bran samples within the specified range, results are as follows: Figure 9 As shown. The results show: 1550-1700 cm -1 The wavelength represents carbonyl stretching vibration, which indicates ester bonds in hemicellulose or lignin. A decrease in peak intensity indicates ester bond breakage, a looser structure, and the release of ferulic acid.

[0064] 3.5 In vitro digestion Simulated saliva, simulated gastric juice, and simulated intestinal juice were all purchased from Beijing Regen Biotechnology Co., Ltd.

[0065] Prepare artificial saliva, artificial gastric juice, and artificial small intestinal juice. Each sample's simulated saliva contains 10 mL of artificial saliva, 72 U / mL of α-amylase, and 25 μL of 0.3 M CaCl2. Each sample's simulated gastric juice contains 10 mL of artificial gastric juice, which contains 2000 U / mL of pepsin and 2.5 μL of 0.3 M CaCl2. Each sample's simulated intestinal juice contains 20 mL of artificial intestinal juice, which contains 100 U / mL of pancreatic enzyme and 0.2 g of bile salts.

[0066] Mix 3g of sample with simulated saliva, place in a shaker at 37℃ and 150rpm for 2min, add 10mL of simulated gastric juice to the mixture, then adjust the pH to 3, and place in a shaker at 37℃ and 150rpm for 2h. Add 20 mL of simulated intestinal fluid to the mixture, adjust the pH to 6.9, and incubate at 130 rpm for 4 hours. Finally, remove the mixture and incubate in a boiling water bath for 10 minutes to inactivate the intestinal fluid. Centrifuge to separate the digestive fluid, filter through a 0.22 μm membrane into a sample vial, and the results are as follows: Figure 10As shown in the figure. The results showed that after in vitro digestion, ferulic acid was further released from the fermented wheat bran obtained in group JE, reaching 2026 μg / g dry basis. In contrast, only 71.86 μg / g dry basis was released from group R. The concentration of free ferulic acid in group E was 1358.86 μg / g dry basis, and the concentration of free ferulic acid in group J was 239.79 μg / g dry basis.

[0067] 3.6 Antioxidant assay of phenolic acid extract 3.6.1 DPPH Determination Method Mix 1 mL of fermented wheat bran phenolic acid extract with 3 mL of DPPH free radical solution (0.1 mM). Shake vigorously for 1 min, react in the dark for 30 min, and then quickly measure the absorbance at a wavelength of 517 nm.

[0068] 3.6.2 ABTS Determination Method Equal volumes of 7 mmol / L ABTS solution were mixed with 2.45 mmol / L potassium persulfate solution, and the mixture was allowed to react in the dark for 12–16 hours to prepare ABTS. + · ABTS with methanol + • Dilute the solution until its absorbance at 734 nm is 0.70 ± 0.02. Add 25 μL of fermented wheat bran phenolic acid extract to 2 mL of ABTS. + • In the solution, the absorbance (Ai) at a wavelength of 734 nm was measured after 6 minutes. 2 mL of ABTS was tested. + • Measure the absorbance (A0) at 517 nm after mixing the solution with 25 μL of methanol; measure the absorbance (Aj) at 517 nm for 2 mL of methanol solution and 25 μL of sample solution. Calculate the free radical scavenging rate using the following formula: Clearance rate = [1 - (Ai - Aj) / A0] × 100%.

[0069] 3.6.3 FRAP Determination Method Take 0.1 mL of fermented wheat bran phenolic acid extract and add 1.8 mL of TPTZ working solution (composed of 25 mL of 0.3 mol / L acetate buffer, 2.5 mL of 10 mmol / L TPTZ solution, and 2.5 mL of 20 mmol / L FeCl3 solution). React at 25℃ for 8 min, and measure the absorbance at 593 nm. A standard curve was prepared using different concentrations (0, 100, 200, 400, 800, 1200, and 2000 μmol / L) of FeSO4. The standard curve is y = 0.0013x + 0.3269, R² = 0.9975. The antioxidant activity (FRAP value) of the sample is the amount of Fe required per gram of dry weight to achieve the same absorbance. 2+ (μM Fe) 2+ / g dry basis).

[0070] 3.6.4 Results The results are as follows Figure 11 As shown in the figure. The results showed that the synergistic fermentation of wheat bran by bacteria and enzymes significantly improved the scavenging rate of DPPH and ABTS free radicals, and also significantly improved the reducing capacity.

[0071] Example 4 Comparison of ferulic acid concentration in fermented wheat bran Currently, most methods for extracting ferulic acid from wheat bran utilize liquid fermentation, with few studies using solid-state fermentation to release ferulic acid. Most existing solid-state fermentation processes for releasing ferulic acid employ Aspergillus fermentation, achieving a maximum free ferulic acid release of 589.62 ± 10.33 μg / g ("Mechanism Study of Active Ingredient Release During Solid-State Fermentation of Wheat Bran"). The highest release of free ferulic acid in all current studies is 3835.34 ± 119.78 μg / g ("The impact of processing on the release and antioxidant capacity of ferulic acid from wheat: A systematic review"), achieved using high-temperature and high-pressure pretreatment and liquid enzymatic fermentation. The fermented wheat bran prepared using the microbial-enzyme co-fermentation method (solid-state fermentation process) in Example 3 of this invention releases 1606.41 μg / g of free ferulic acid. Compared to liquid fermentation, the solid-state fermentation method in this invention offers simpler post-processing, generates less pollution, produces virtually no wastewater, has low energy consumption, is easy to operate, and has low equipment requirements.

[0072] The comparison chart of ferulic acid content in wheat bran before and after fermentation is shown below. Figure 8 , Figure 12 and Figure 13 As shown in the figure. The results showed that the elution time of ferulic acid was around 8.7 min. Before wheat bran fermentation, the peak height of ferulic acid was around 300, and after fermentation, the peak height of ferulic acid was around 2000. Quantitative analysis of ferulic acid in the phenolic acid extract was performed. Before wheat bran fermentation, the concentration of free ferulic acid was 45.45 μg / g dry basis, and after fermentation, the concentration of free ferulic acid reached 1606.41 μg / g dry basis.

[0073] In the "3.5 In vitro digestion" section of Example 3, it was determined whether wheat bran fermentation affected the release of free ferulic acid from wheat bran in a simulated intestine. After co-fermentation with microorganisms and enzymes, the release amount in the simulated intestinal fluid reached 2026.18 μg / g dry basis, while unfermented wheat bran only released 71.86 μg / g dry basis. Figure 10 The total ferulic acid content in wheat bran is approximately 5 mg / g, with a release rate of up to 40%.

[0074] Example 5: Experiment on Fermented Wheat Bran as a Substitute for Corn and Cottonseed Meal in Broiler Chickens This experiment selected 1920 16-day-old yellow-feathered broilers and randomly divided them into 3 treatment groups. Each treatment had 4 replicates, with 8 cages per replicate and 20 chickens per cage. During the experiment, the broilers were allowed free access to food and water and were fed for a total of 31 days.

[0075] The control group was fed a conventional diet (CP Broiler Feed 511). The 5% replacement group replaced 4 wt% of corn and 1 wt% of cottonseed meal in the conventional diet with 5 wt% fermented wheat bran obtained from the co-fermentation of bacteria and enzymes in Example 3; the 10% replacement group replaced 7.5 wt% of corn and 2.5 wt% of cottonseed meal in the conventional diet with 10 wt% fermented wheat bran obtained from the co-fermentation of bacteria and enzymes in Example 3. Body weight and fecal samples were measured every 7 days, and feed intake and mortality were measured daily. The results are shown in Table 1 and [Table data missing]. Figure 14 As shown in the figure. The results showed that there were no significant differences in average daily weight gain and average daily feed intake among the groups from 16 to 47 days of age (P > 0.05). However, the mortality rate in the experimental group was significantly lower than that in the control group, and the residual amounts of corn and cottonseed meal in the feces were significantly lower in the experimental group than in the control group.

[0076] Table 1 Survey Results After the experiment, serum markers of yellow-feathered broilers were investigated, and the results are as follows: 1. Results of the survey on protein metabolism-related indicators, such as... Figure 15 As shown.

[0077] 1.1 When fermented wheat bran replaced 1 / 3 of the soybean meal by mass (10% replacement group), there was no significant difference in the content of urea nitrogen (BUN), indicating that the amino acid balance in the animal body was high and the protein synthesis efficiency was high.

[0078] 1.2 The serum total protein (TP) and albumin (ALB) levels in the 10% replacement group were significantly higher than those in the control group, indicating enhanced protein synthesis capacity. Albumin is synthesized by the liver, indicating vigorous liver synthesis function. Globulin (GLB), an indicator of inflammation and immune stimulation, showed no significant difference, demonstrating a stable immune status.

[0079] 2. Survey results of lipid metabolism-related indicators, such as Figure 16 As shown.

[0080] The total cholesterol (TC), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), and triglycerides (TG) in the 10% replacement group (CK vs. 10% replacement group) were significantly higher than those in the control group, demonstrating vigorous lipid transport and metabolism in broilers. In particular, the significantly increased HDL content indicates vigorous cholesterol transport from peripheral tissues to the liver for metabolism. There was no significant difference in free fatty acids, indicating normal fat metabolism in broilers.

[0081] 3. Results of the investigation of liver enzyme spectrum related indicators, such as Figure 17 As shown.

[0082] 3.1 Alanine aminotransferase (ALT) is a specific indicator of hepatocellular damage. The ALT concentrations in the 5% and 10% replacement groups were significantly lower than those in the control group, demonstrating that the hepatocellular membranes were intact and the cells were in good condition.

[0083] 3.2 Elevated alkaline phosphatase (ALP) levels are usually associated with active bone development. The concentration of alkaline phosphatase in the 10% replacement group was significantly higher than that in the control group, suggesting vigorous bone growth in broilers.

[0084] 3.3 Aspartate aminotransferase (AST) is one of the important indicators for assessing liver health. When broilers are growing rapidly, the liver and muscles are metabolically active, which may lead to a slight increase in AST.

[0085] 4. Survey results of antioxidant-related indicators, such as Figure 18 As shown.

[0086] 4.1 The activity of superoxide dismutase (SOD) in the 10% replacement group was significantly increased, and showed an upward trend with the increase of replacement amount. This indicates that fermented wheat bran significantly enhances the ability of broilers to scavenge free radicals and improves antioxidant levels.

[0087] 4.2 The activity of glutathione peroxidase (GSH-Px) was significantly reduced between the 5% substitution group and the control group, which may be due to changes in the antioxidant mechanism under high doses.

[0088] 4.3 Malondialdehyde (MDA) is a product of lipid peroxidation, and its concentration shows a decreasing trend. Although it is not statistically significant, it can corroborate the reduction of oxidative damage.

[0089] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Pediococcus acidilactici ( Pediococcus acidilactici ) NR application in any of the following (1) Preparation of wheat bran starter culture; (2) Increase the content of free ferulic acid in wheat bran; (3) Preparation of formulations to increase the content of free ferulic acid in wheat bran; The preservation number of the lactic acid pediococcus NR is CCTCC NO: M 20252979.

2. A wheat bran starter culture, characterized in that, It includes Pediococcus lactis NR and a compound enzyme preparation; the preservation number of Pediococcus lactis NR is CCTCC NO: M 20252979.

3. The wheat bran fermentation agent according to claim 2, characterized in that, The compound enzyme preparation includes ferulic acid esterase, xylanase, cellulase, and arabinofuranase.

4. The application of *Pediococcus lactis* NR or the wheat bran starter according to claim 2 or 3 in fermenting wheat bran and / or increasing the free ferulic acid content of wheat bran, characterized in that, The preservation number of the lactic acid pediococcus NR is CCTCC NO: M 20252979.

5. A method for preparing fermented wheat bran and / or increasing the free ferulic acid content of wheat bran, characterized in that, This includes the steps of mixing Pediococcus lactis NR bacterial solution with wheat bran, then adding a compound enzyme preparation, and fermenting the mixture. The preservation number of the lactic acid porphyria NR is CCTCC NO: M 20252979; The compound enzyme preparation includes ferulic acid esterase, xylanase, cellulase, and arabinofuranase.

6. The method according to claim 5, characterized in that, The effective viable count of *Pediococcus lactis* NR in the *Pediococcus lactis* NR bacterial solution was 3 × 10⁻⁶. 9 cfu / mL; The amount of the *Pediococcus lactis* NR bacterial solution used is 1% of the mass of the wheat bran; The compound enzyme preparation contains 30 U / g dry basis of ferulic acid esterase, 100 U / g dry basis of xylanase, 100 U / g dry basis of cellulase, and 10 U / g dry basis of arabinofuranyl glycoside enzyme. The fermentation time was 96 hours and the temperature was 37°C.

7. Fermented wheat bran prepared using the method of claim 5 or 6.

8. The use of the fermented wheat bran according to claim 7 in the preparation of feed additives or feed.

9. A feed additive, characterized in that, The feed additive includes the fermented wheat bran as described in claim 7.

10. A feed comprising the feed additive of claim 9 and a base feed.

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