Application of a strain of *Pediococcus lactis* NR and its combined enzyme preparation in the preparation of fermented wheat bran

Abstract

This invention relates to the fields of fermentation engineering and feed processing technology, and in particular to the application of a strain of *Pediococcus lactis* NR and its combined enzyme preparation in the preparation of fermented wheat bran. This invention provides a strain of *Pediococcus lactis* NR, with the preservation number CCTCC NO: M 20252979. 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 provides a method for preparing fermented wheat bran based on the synergistic fermentation of *Pediococcus lactis* and a complex enzyme system. Under anaerobic conditions, *Pediococcus lactis* NR converts the monosaccharides released by enzymatic hydrolysis into organic acids, further promoting the enzymatic hydrolysis process. This invention effectively solves the problems of high levels of anti-nutritional factors and difficult fiber degradation in traditional fermentation technologies, resulting in low digestible energy and poor palatability of wheat bran. It enables fermented wheat bran to partially replace corn as an economical energy feed, significantly reducing feed costs and possessing significant application value and promising prospects for promotion.

Description

Technical Field

[0001] This invention relates to the fields of fermentation engineering and feed processing technology, and in particular to the application of a strain of *Pediococcus lactis* NR and its combined enzyme preparation in the preparation of fermented wheat bran. Background Technology

[0002] As a traditional energy feed, the price of corn has been rising year by year, directly increasing the production costs of animal husbandry. Therefore, developing economical and efficient energy feed resources that can replace corn has become an important issue that the feed industry urgently needs to address.

[0003] Wheat bran is a byproduct of wheat processing. Due to its rich content of carbohydrates, proteins, and minerals, it has high potential for feed utilization. However, because wheat bran contains a large amount of complex cross-linked structures such as cellulose, hemicellulose, and lignin, its cellulose is tightly bound to other nutrients, resulting in low digestible energy and difficulty in effective absorption of nutrients by animals. Furthermore, wheat bran contains certain anti-nutritional factors, such as phytic acid and non-starch polysaccharides, further limiting its widespread use in animal feed. These problems not only cause indigestion and low nutrient utilization in animals but also seriously affect the palatability and taste of the feed.

[0004] In recent years, fermentation technology has received widespread attention and application in the feed processing field due to its green, environmentally friendly, and efficient characteristics. Microbial fermentation can effectively degrade cellulose and anti-nutritional factors, releasing encapsulated nutrients and significantly improving the digestibility and nutrient density of feed. However, traditional fermentation technologies have some shortcomings, such as substrate inhibition limiting enzymatic hydrolysis efficiency, limited degradation of fiber structures, and difficulty in achieving full release of carbohydrates from wheat bran. Therefore, how to further improve the nutrient utilization rate of wheat bran and enhance its ability as an energy feed substitute through innovative fermentation methods has become a current research hotspot and challenge. Summary of the Invention

[0005] The purpose of this invention is to provide the application of a strain of *Pediococcus lactis* NR and its combined enzyme preparation in the preparation of fermented wheat bran, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a strain of Pediococcus lactis ( Pediococcus acidilactici The preservation number of the lactic acid cocci NR is CCTCC NO: M 20252979.

[0007] More preferably, the *Pediococcus lactis* NR is used to disrupt the fibrous structure of wheat bran.

[0008] More preferably, the fiber structure comprises cellulose and hemicellulose.

[0009] More preferably, the wheat bran fermentation agent is used to improve the utilization of pentose sugars.

[0010] This invention provides the application of the above-mentioned *Pediococcus lactis* NR in any of the following: (1) Preparation of wheat bran starter culture; (2) It damages the fiber structure of wheat bran; (3) Prepare products that disrupt the structure of wheat bran fibers.

[0011] More preferably, the wheat bran fermentation agent is used to break down the fibrous structure of the wheat bran.

[0012] More preferably, the fiber structure comprises cellulose and hemicellulose.

[0013] More preferably, the wheat bran fermentation agent is used to improve the utilization of pentose sugars.

[0014] The present invention provides a wheat bran fermentation agent, wherein the wheat bran fermentation agent comprises the above-mentioned Pediococcus lactis NR.

[0015] Optionally, the wheat bran fermentation agent may also include a compound enzyme preparation; The compound enzyme preparation includes cellulase, xylanase, glucoamylase, phytase, and arabinofuranase; The wheat bran fermentation agent is used to break down the fibrous structure of wheat bran.

[0016] More preferably, the fiber structure comprises cellulose and hemicellulose.

[0017] More preferably, the wheat bran fermentation agent is used to improve the utilization of pentose sugars.

[0018] This invention provides the application of the above-mentioned *Pediococcus lactis* NR or the above-mentioned wheat bran starter in fermented wheat bran.

[0019] This invention provides a method for preparing fermented wheat bran, comprising the steps of mixing Pediococcus lactis NR bacterial solution and wheat bran, adding a compound enzyme preparation, and fermenting to obtain fermented wheat bran; The preservation number of the lactic acid porphyria NR is CCTCC NO: M 20252979; The compound enzyme preparation includes cellulase, xylanase, saccharifying enzyme, phytase, and arabinofuranase.

[0020] 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 100 U / g dry basis of cellulase, 100 U / g dry basis of xylanase, 50 U / g dry basis of saccharifying enzyme, 30 U / g dry basis of phytase, and 30 U / g dry basis of arabinofuranylase. The fermentation time was 96 hours and the temperature was 37°C.

[0021] The present invention provides fermented wheat bran prepared using the above-described method.

[0022] Optionally, the fermented wheat bran may have a lactic acid content of up to 120 mg / g dry basis, an acetic acid content of up to 12.4 mg / g dry basis, a xylose content of up to 68 mg / g dry basis, and an arabinose content of 14.6 mg / g dry basis. The dry matter loss rate of the fermented wheat bran may be less than 2%, and the in vitro digestibility may be increased from 37.5% to 56.69%.

[0023] This invention provides the application of the above-mentioned fermented wheat bran in the preparation of feed additives or feed.

[0024] The present invention provides a feed additive or feed comprising the above-mentioned fermented wheat bran.

[0025] The present invention discloses the following technical effects: This invention provides a strain of Pediococcus lactis ( Pediococcus acidilactici The *Pediococcus lactis* NR strain has the preservation number CCTCC NO: M 20252979. This invention utilizes *Pediococcus lactis* NR to improve the quality of wheat bran feed; it is low-cost and the strain is safe and reliable.

[0026] This invention also provides a wheat bran fermentation agent and a method for preparing fermented wheat bran based on the synergistic fermentation of *Pediococcus lactis* and a complex enzyme system. Under anaerobic conditions, *Pediococcus lactis* NR converts the monosaccharides released by enzymatic hydrolysis into organic acids, relieving substrate inhibition and further promoting the enzymatic hydrolysis process. Simultaneously, the acidic environment generated during fermentation promotes the further disintegration and collapse of the wheat bran fiber structure, thereby releasing carbohydrates in the wheat bran in a more efficient and environmentally friendly manner, ensuring the growth and development of fed animals and improving their nutrient utilization. This invention effectively solves the problems of low digestible energy and poor palatability caused by numerous anti-nutritional factors and difficult fiber degradation in traditional fermentation technologies. It enables fermented wheat bran to partially replace corn as an economical energy feed, significantly reducing feed costs and possessing significant application value and promotion prospects. Therefore, fermented wheat bran obtained using the method described in this invention can serve as an economical substitute for energy feeds such as corn, reducing costs.

[0027] Furthermore, the fermented wheat bran obtained by the present invention based on bacterial-enzyme co-fermentation exhibits good structural modification effects, high monosaccharide release rate, and significantly reduced cellulose and hemicellulose content in the final product. The wheat bran after bacterial-enzyme co-fermentation has a lactic acid content of up to 120 mg / g dry basis, an acetic acid content of up to 12.4 mg / g dry basis, a xylose content of up to 68 mg / g dry basis, and an arabinose content of 14.6 mg / g dry basis. The dry matter loss rate of the fermented wheat bran is less than 2%, and the in vitro digestibility increases from 37.5% to 56.69%. In the fermented wheat bran treated with enzyme co-fermentation, some insoluble dietary fiber is converted into soluble nutrients, the content of anti-nutritional factors is reduced, the texture is softer and more delicate than ordinary wheat bran products, and the antibacterial substances produced during fermentation can extend its shelf life. Attached Figure Description

[0028] 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.

[0029] Figure 1 The results show the fermentation effects of different bacterial strains on wheat bran; where A represents lactic acid, B represents xylose, and C represents acetic acid. Figure 2 The results represent the optimization of the complex enzyme system; where A is lactic acid; B is acetic acid; C is xylose; and D is arabinose. Figure 3 pH measurement results for different treatments; Figure 4 Results of determination of glucose (A), xylose (B), arabinose (C), lactic acid (D), and acetic acid (E) under different treatments; Figure 5 The results are for the determination of phosphorus. Figure 6 The results are for the determination of acid-hydrolyzed sugar and amylase-hydrolyzed sugar; where A is acid-hydrolyzed sugar - glucose; B is acid-hydrolyzed sugar - xylose; C is acid-hydrolyzed sugar - arabinose; and D is amylase-hydrolyzed sugar - glucose. Figure 7 To observe the changes in wheat bran before and after fermentation using scanning electron microscopy; where A represents before fermentation and B represents after fermentation; Figure 8 The results are from in vitro digestion assays; where A represents total digestibility; B represents glucose in digestive fluid; C represents xylose in digestive fluid; D represents arabinose in digestive fluid; E represents lactic acid in digestive fluid; F represents residual glucose; G represents residual xylose; and H represents residual arabinose. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 4. Identification and verification of strains DNA was extracted from the pure culture and amplified by PCR using primers specific to *Pediococcus lactis* (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*.

[0039] 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.

[0040] 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.

[0041] 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. 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 treated wheat bran (moisture content of 8%) and 5g of CaCO3, mix them well and add 200 mL of water.

[0042] 1.2 Grouping Enzymatic hydrolysis: Add a compound enzyme preparation solution to the pretreated wheat bran and mix well. The compound enzyme preparation contains 100 U / g dry basis (dry wheat bran) of cellulase, 50 U / g dry basis of saccharifying enzyme, 30 U / g dry basis of arabinose-furanosidase, 30 U / g dry basis of phytase, and 100 U / g dry basis of xylanase. Anaerobic fermentation is carried out at 37°C for 96 hours.

[0043] NR: 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 (cfu / mL), then add the compound enzyme preparation (same as the previous grouping: enzymatic hydrolysis). At this point, the wheat bran moisture content is 50%, mix well. Anaerobic fermentation at 37℃ for 96 hours.

[0044] 3A: Inoculate the pretreated 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 50%, mix well. Anaerobic fermentation at 37℃ for 96 hours.

[0045] 12B: Inoculate the pretreated 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 50%, mix well. Anaerobic fermentation at 37℃ for 96 hours.

[0046] 1.3 Results The results are as follows Figure 1As shown in the figure. The results show that, by comparing the contents of xylose, lactic acid, and acetic acid in the fermentation system, *Pediococcus lactis* NR has a stronger destructive effect on cellulose and hemicellulose, a stronger ability to utilize pentose sugars, and more easily absorbed energy in the system. Therefore, the fermentation effect of *Pediococcus lactis* NR is superior to that of *Pediococcus lactis* 3A and *Lactobacillus paracasei* 12B.

[0047] 2. Optimization of compound enzyme systems 2.1 Pretreatment of wheat bran before fermentation Weigh out wheat bran, remove impurities and wash it clean. 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 treated wheat bran (moisture content of 8%) and 5g of CaCO3, mix them well and add 200 mL of water.

[0048] 2.2 Grouping The initial enzyme system was cellulase, and other enzymes were added to it in combination. Lactococcus lysus NR was added to increase the content of monosaccharides and organic acids.

[0049] 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 has a moisture content of 50%. After anaerobic fermentation at 37℃ for 96 hours, it is recorded as enzyme-free.

[0050] 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). 9 (cfu / mL), then add cellulase at a concentration of 100 U / g dry basis. At this point, the wheat bran moisture content is 50%. Mix well. Anaerobic fermentation at 37℃ for 96 hours, denoted as CAE.

[0051] Cellulase + 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 (cellulase and xylanase) solution was added, wherein the amount of cellulase added was 100 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 50%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 hours, and the result was recorded as CAE+XAE.

[0052] Cellulase + Saccharifying enzyme: 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). 9Then, a compound enzyme preparation (cellulase and saccharifying enzyme) solution was added, wherein the amount of cellulase added was 100 U / g dry basis and the amount of saccharifying enzyme added was 50 U / g dry basis. At this time, the moisture content of the wheat bran was 50%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 hours, which was recorded as CAE+AGE.

[0053] Cellulase + Xylanase + Saccharifying enzyme: 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 (cellulase, xylanase, and saccharifying enzyme) solution was added, wherein the amount of cellulase added was 100 U / g dry basis, the amount of xylanase added was 100 U / g dry basis, and the amount of saccharifying enzyme added was 50 U / g dry basis. At this time, the moisture content of the wheat bran was 50%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 hours, and the result was recorded as CAE+XAE+AGE.

[0054] Cellulase + Xylanase + Glycosylase + Arabinofuranosides: 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 (cellulase, xylanase, saccharifying enzyme, and arabinofuranylase) solution was added, wherein the amount of cellulase added was 100 U / g dry basis, the amount of xylanase added was 100 U / g dry basis, the amount of saccharifying enzyme added was 50 U / g dry basis, and the amount of arabinofuranylase added was 30 U / g dry basis. At this time, the moisture content of wheat bran was 50%, and it was mixed well. Anaerobic fermentation was carried out at 37℃ for 96 hours, and the result was recorded as CAE+XAE+AGE+AFE.

[0055] Cellulase + xylanase + glucoamylase + arabinofuranosides + phytase: 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 (cellulase, xylanase, glucoamylase, arabinofuranylase, and phytase) solution was added. The compound enzyme preparation contained 100 U / g dry basis cellulase, 100 U / g dry basis xylanase, 50 U / g dry basis glucoamylase, 30 U / g dry basis arabinofuranylase, and 30 U / g dry basis phytase. At this point, the wheat bran moisture content was 50%, and the mixture was thoroughly mixed. Anaerobic fermentation was carried out at 37℃ for 96 hours, denoted as CAE+XAE+AGE+AFE+PE.

[0056] 2.3 Results The results are as follows Figure 2As shown in the figure. The results indicate that the mixed enzyme system of cellulase + xylanase + glucoamylase + arabinofuranosylase + phytase has the best fermentation effect and the highest content of organic acids and monosaccharides.

[0057] 3. Optimization of fermentation conditions 3.1 Pretreatment of wheat bran before fermentation Weigh out wheat bran, remove impurities and wash it clean. 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 treated wheat bran (moisture content of 8%) and 5g of CaCO3, mix them well and add 200 mL of water.

[0058] 3.2 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 (cellulase, xylanase, glucoamylase, arabinofuranylase, and phytase) solution was added. The compound enzyme preparation contained 100 U / g dry basis cellulase, 100 U / g dry basis xylanase, 50 U / g dry basis glucoamylase, 30 U / g dry basis arabinofuranylase, and 30 U / g dry basis phytase. At this point, the wheat bran moisture content was 50%. The mixture was thoroughly mixed. Anaerobic fermentation was carried out at 37℃ for 0 h, 24 h, 48 h, 72 h, and 96 h.

[0059] 3.3 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 (cellulase, xylanase, glucoamylase, arabinofuranylase, and phytase) solution. The compound enzyme preparation contains 100 U / g dry basis cellulase, 100 U / g dry basis xylanase, 50 U / g dry basis glucoamylase, 30 U / g dry basis arabinofuranylase, and 30 U / g dry basis phytase. At this point, the wheat bran moisture content is 50%. Mix well. Perform anaerobic fermentation at 35℃, 37℃, or 39℃ for 72 hours.

[0060] 3.4 Results The results of the optimization of fermentation time are shown in Table 1. The results show that fermentation for 72 hours yields the best results.

[0061] Table 1 Results of Fermentation Time Optimization The results of the optimization of fermentation temperature are shown in Table 2. The results show that the best effect is achieved when the fermentation temperature is 37℃.

[0062] Table 2 Results of Fermentation Temperature Optimization Example 3 Validation of bacterial-enzyme synergy 1. Pretreatment of wheat bran before fermentation Weigh out wheat bran, remove impurities and wash it clean. 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 treated wheat bran (moisture content of 8%) and 5g of CaCO3, mix them well and add 200 mL of water.

[0063] 2. Grouping Wheat bran group (R): Wheat bran is dried and pulverized to 30 mesh, which is pre-treated wheat bran.

[0064] Enzymatic hydrolysis group (E): A compound enzyme preparation (cellulase, xylanase, saccharifying enzyme, phytase and arabinofuranylase) solution was added to the pretreated wheat bran and mixed well. The compound enzyme preparation contained 100 U / g dry basis of cellulase, 100 U / g dry basis of xylanase, 50 U / g dry basis of saccharifying enzyme, 30 U / g dry basis of phytase and 30 U / g dry basis of arabinofuranylase. Anaerobic fermentation was carried out at 37°C for 72 h.

[0065] 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 72 hours.

[0066] JE (Joint Fermentation Group): Fresh Pediococcus lactis NR bacterial suspension (effective viable count 3 × 10⁻⁶) was inoculated into pretreated wheat bran at an inoculation rate of 1% (by weight / volume percentage). 9 Then, add a compound enzyme preparation (cellulase, xylanase, glucoamylase, phytase, and arabinofuranylase) solution. The compound enzyme preparation contains 100 U / g dry basis cellulase, 100 U / g dry basis xylanase, 50 U / g dry basis glucoamylase, 30 U / g dry basis phytase, and 30 U / g dry basis arabinofuranylase. At this point, the wheat bran moisture content is 50%. Mix well. Anaerobic fermentation at 37℃ for 72 hours.

[0067] 3. Indicator Survey 3.1 pH Measurement Take 1g of fermented wheat bran, add 9mL of sterile water, dissolve, and then measure the pH of the supernatant using a pH meter. The results are as follows. Figure 3 As shown in the figure. The results showed that after fermentation by the *Pediococcus lactis* fermentation group and the bacterial-enzyme co-fermentation group, the pH of wheat bran decreased and stabilized at around 4.3.

[0068] 3.2 Determination of glucose, xylose, arabinose, lactic acid and acetic acid Glucose, arabinose, and xylose were dried to constant weight in a drying oven at 105℃. Preparation of standard solutions for glucose, arabinose, xylose, lactic acid, and acetic acid: Weigh 1g of the chromatographic grade standard from each sample, dissolve it in ultrapure water, and dilute to 100mL to prepare a 10g / L standard stock solution. Dilute the standard stock solution to prepare the corresponding working solutions. Working solutions were prepared fresh each time. All samples were filtered through a 0.22μm filter before HPLC injection. The concentrations of the working solutions for lactic acid and acetic acid were 0.1g / L, 0.2g / L, 0.4g / L, 0.8g / L, 1.0g / L, 1.6g / L, and 2.0g / L, respectively; the concentrations of the working solutions for glucose, arabinose, and xylose were 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1.0g / L, 1.2g / L, and 1.4g / L, respectively.

[0069] Chromatographic column: Bio-Rad Aminex HPX-87H Column; Chromatographic conditions: mobile phase: 5 mmol / L H2SO4; column temperature: 40℃; differential refractive index detector: detector temperature: 35℃; flow rate: 0.6 mL / min; injection volume: 20 μL.

[0070] Standard curve plotting: Standard curves are plotted based on the peak areas of each substance and the corresponding concentrations of the standard substances. The standard curves for each substance and R0 are also included. 2 As shown below: Glucose: y = 262515x - 6874.9, R² = 0.999; Xylose: y = 256009x - 6856.2, R² = 0.9988; Arabinose: y = 262996x + 13729, R² = 0.9981; Lactic acid: y = 174887x + 18024, R² = 0.9999; Acetic acid: y = 78193x–84797, R²=0.9998.

[0071] Sample preparation: Take 1g of fermented wheat bran, add 9mL of sterile water, mix well, and filter the supernatant through a 0.22μm filter membrane. The results are as follows. Figure 4As shown in the figure. The results showed that after fermentation with mixed bacteria and enzymes, the xylose content reached 68 mg / g dry basis, and the arabinose content was 14.6 mg / g dry basis. After utilization by *Pediococcus lactis* NR, the glucose content decreased to about 5 mg / g dry basis, the lactic acid content reached as high as 120 mg / g dry basis, and the acetic acid content reached 12.4 mg / g dry basis.

[0072] 3.3 Determination of Phosphorus The test was performed according to the national standard GB / T6437-2018, and the results are as follows: Figure 5 As shown in the figure. The results showed that after fermentation with bacterial enzymes, wheat bran released phosphorus from phytic acid phosphorus, and the content of free phosphorus could reach 8.46 mg / g dry basis.

[0073] 3.4 Determination of Acid Hydrolysis and Amylase Enzymatic Hydrolysis of Sugars Lactic acid and acetic acid can be directly used for bodily functions. Animals lack the ability to break down cellulose and hemicellulose and therefore cannot utilize their energy. The acid hydrolysis and amylase enzymatic hydrolysis of sugars in fermented wheat bran were determined according to the national standard GB5009.8-2023, and the changes in energy content during fermentation were calculated. The results are as follows: Figure 6 As shown in Table 3. The results showed that the bacterial-enzyme co-fermentation group consumed the most cellulose and hemicellulose, converting them into energy forms of lactic acid and organic acid (acetic acid), which remained in the animal system.

[0074] Table 3. Changes in energy content of wheat bran during fermentation (mg / g dry basis) 3.5 Scanning electron microscopy observation of changes in wheat bran before and after fermentation Wheat bran before and after fermentation was freeze-dried under vacuum. Intact portions were fixed onto mica slides, sputtered with gold, and the structural changes were observed under an electron microscope. The results are as follows: Figure 7 As shown in the figure. The results showed that before fermentation, intact aleurone layer cells, transverse cells, and tubular cells were intertwined with gluten and starch granules. After synergistic fermentation with bacteria and enzymes, some starch and gluten were degraded and utilized, exposing a layered and striped fibrous skeleton, and the surface of the fibrous skeleton showed obvious signs of bio-erosion, with structural separation and detachment.

[0075] 3.6 In vitro digestion Simulated saliva, simulated gastric juice, and simulated intestinal juice were all purchased from Beijing Regen Biotechnology Co., Ltd.

[0076] 3g of sample was mixed with simulated saliva and incubated in a shaker at 37℃ for 2 min. 10mL of simulated gastric juice was added to the mixture, and the pH was adjusted to 3. The mixture was then incubated in a shaker at 37℃ and 150rpm for 2 h. Next, 20mL of simulated intestinal juice was added to the mixture, the pH was adjusted to 6.9, and the mixture was incubated in a shaker at 130rpm for 4 h. Finally, the mixture was placed in a boiling water bath for 10 min to inactivate the enzymes. The digestive fluid and digestive residue were separated by centrifugation. The digestive fluid was filtered through a 0.22μm membrane into a sample vial, and the reducing sugars and lactic acid in the digestive fluid were measured. The residue was lyophilized, and the acid-hydrolyzed sugars in the residue were measured. The cellulose, hemicellulose, and total digestibility were calculated. The results are shown in Table 4. Figure 8 As shown in the figure. The results showed that after in vitro digestion, the energy substances in wheat bran were further released during the co-fermentation of bacteria and enzymes. The glucose concentration in the digestate of wheat bran co-fermented with bacteria and enzymes reached 71.56 mg / g dry basis, the xylose concentration reached 48 mg / g dry basis, and the arabinose concentration reached 16 mg / g dry basis. Compared with other treatment groups, the digestion residue of the bacteria and enzyme fermentation group had the least amount of energy substances, and the total digestibility increased from 37.5% to 56.69%.

[0077] The calculation formula is as follows: Total digestibility = (Weight of fermented wheat bran before digestion - Weight of residue after digestion) / Weight of fermented wheat bran before digestion × 100%; Cellulose digestibility = [(Acid-hydrolyzed glucose content of fermented wheat bran - Glucose content of fermented wheat bran hydrolyzed by amylase) - (Acid-hydrolyzed glucose content of digestion residue - Glucose content of digestion residue hydrolyzed by amylase)] / (Acid-hydrolyzed glucose content of fermented wheat bran - Glucose content of fermented wheat bran hydrolyzed by amylase) × 100%; Hemicellulose digestibility = [content of xylose and arabinose hydrolyzed in fermented wheat bran - content of xylose and arabinose hydrolyzed in digestion residue] / content of xylose and arabinose hydrolyzed in fermented wheat bran × 100%.

[0078] Table 4 Digestibility of fermented wheat bran (fermentation time: 72 h) In summary, the fermented wheat bran obtained through synergistic fermentation of bacteria and enzymes can have a lactic acid content of up to 120 mg / g dry basis, an acetic acid content of up to 12.4 mg / g dry basis, a xylose content of up to 68 mg / g dry basis, and an arabinose content of 14.6 mg / g dry basis. The dry matter loss rate of the fermented wheat bran is less than 2%, and the in vitro digestibility has increased from 37.5% to 56.69%.

[0079] Example 4: Experiment on Fermented Wheat Bran as a Substitute for Corn in Weaned Piglets Eighty-fourty healthy weaned male piglets (weaned at 21 days of age and fed in pen for 3 days after birth) with uniform weight (7.06±0.73 kg) and similar genetic background were selected and randomly divided into a control group, a 2.5% replacement group, and a 5% replacement group, with four replicates per treatment and seventy piglets per replicate. The control group was fed a basal diet. The 2.5% replacement group used fermented wheat bran obtained from the co-fermentation of bacteria and enzymes in Example 3 to replace 2.5 wt% of corn. The 5% replacement group used fermented wheat bran obtained from the co-fermentation of bacteria and enzymes in Example 3 to replace 5 wt% of corn. The basal diet (New Hope Liuhe Changwang 4% Piglet Compound Premix CW400) was formulated according to the NRC-2012 piglet nutritional requirements standard. During the animal experiment, piglets had free access to feed and water and received routine immunizations as required by the pig farm. All piglets were housed in one room, with 68 piglets individually housed in a 20×40m² room. 2 Inside a concrete enclosure without a bedding layer, the room temperature was maintained at 25±2℃ for a total of 28 days. The number of piglet deaths and diarrhea were recorded during the animal experiment. At the end of the experiment, the piglets were weighed, and their feed consumption was measured. Average daily gain (ADG) was calculated as the difference between the initial and final body weight divided by the number of experimental days. Average daily feed intake (ADFI) was calculated as the feed consumption during the experimental period divided by the number of experimental days. The results are shown in Table 5. The results indicate that fermented wheat bran can replace corn in equal amounts as an energy source in feed without affecting animal growth performance.

[0080] Table 5. Results of Growth Performance Survey 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. A strain of Pediococcus lactis ( Pediococcus acidilactici NR, characterized in that, The preservation number of the lactic acid pediococcus NR is CCTCC NO: M 20252979.

2. The use of the *Pediococcus lactis* NR as described in claim 1 in any of the following: (1) Preparation of wheat bran starter culture; (2) It damages the fiber structure of wheat bran; (3) Prepare products that disrupt the structure of wheat bran fibers.

3. A wheat bran starter culture, characterized in that, The wheat bran starter culture includes the *Pediococcus lactis* NR as described in claim 1.

4. The wheat bran fermentation agent according to claim 3, characterized in that, The wheat bran fermentation agent also includes a compound enzyme preparation; The compound enzyme preparation includes cellulase, xylanase, glucoamylase, phytase, and arabinofuranase; The wheat bran fermentation agent is used to break down the fibrous structure of wheat bran.

5. The application of the *Pediococcus lactis* NR as described in claim 1 or the wheat bran starter as described in claim 3 or 4 in fermented wheat bran.

6. A method for preparing fermented wheat bran, characterized in that, The process includes mixing Pleurotus ostreatus NR bacterial solution with wheat bran, adding a compound enzyme preparation, and then fermenting to obtain fermented wheat bran. The preservation number of the lactic acid porphyria NR is CCTCC NO: M 20252979; The compound enzyme preparation includes cellulase, xylanase, saccharifying enzyme, phytase, and arabinofuranase.

7. The method according to claim 6, 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 100 U / g dry basis of cellulase, 100 U / g dry basis of xylanase, 50 U / g dry basis of saccharifying enzyme, 30 U / g dry basis of phytase, and 30 U / g dry basis of arabinofuranylase. The fermentation time was 96 hours and the temperature was 37°C.

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

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

10. A feed additive or feed, characterized in that, The feed additive or feed includes the fermented wheat bran as described in claim 8.

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