A method for preparing high small peptide acidified rice residue protein by time sequence inoculation solid state fermentation

Abstract

This invention provides a method for preparing high-small-peptide acidified rice residue protein through sequential inoculation solid-state fermentation, relating to the fields of bio-fermentation engineering and deep processing of agricultural products. The method includes: preparing a mixed fermentation substrate of rice residue and wheat bran; firstly, inoculating with Bacillus subtilis for a first-stage fermentation to efficiently degrade large-molecule proteins; then, inoculating with Lactobacillus plantarum for a second-stage fermentation, utilizing the degradation products generated in the first stage to rapidly produce acid and lower the system pH to a strongly acidic range. This invention decouples and sequentially connects the two core processes of "deep protein degradation" and "rapid system acidification" on a time scale, avoiding metabolic conflicts between the two microorganisms and achieving a breakthrough increase in total acid content (≥1.95g / 100g) and small peptide content (≥6.5g / 100g). The product does not develop mold during fermentation and storage and can be used as a highly digestible protein feed, particularly suitable for eel farming.

Description

Technical Field

[0001] This invention relates to the fields of bio-fermentation engineering and deep processing of agricultural products, and in particular to a method for preparing high-small-peptide acidified rice residue protein by time-sequential inoculation solid-state fermentation. Background Technology

[0002] Rice residue is a major byproduct of rice processing for producing starch sugars, monosodium glutamate, and other products. It contains 40%–60% crude protein with a relatively balanced amino acid composition, making it a potentially valuable unconventional plant protein resource. my country generates a large amount of rice residue annually. If it could be utilized at a high value, it would not only alleviate the shortage of high-quality protein feed but also reduce environmental pollution, yielding significant economic and social benefits. However, due to its poor water solubility, large molecular weight, presence of anti-nutritional factors, and low in vitro digestibility, rice residue protein has long been primarily used for low-value purposes as fertilizer or ordinary feed filler, resulting in a significant waste of this high-quality protein resource.

[0003] Microbial solid-state fermentation is an effective technique for improving the nutritional properties of rice residue protein. Its core lies in utilizing the abundant extracellular enzyme system (mainly proteases) secreted during microbial fermentation to directionally break down large protein molecules into smaller peptides and free amino acids, thereby significantly improving their digestibility and absorption. Current technologies often employ a mixed fermentation process using Bacillus subtilis and Lactiplantibacillus plantarum. Bacillus subtilis, a typical aerobic enzyme-producing bacterium, can efficiently secrete neutral or alkaline proteases, achieving deep protein degradation; Lactiplantibacillus plantarum, on the other hand, rapidly lowers the pH of the system through metabolic acid production, creating an acidic environment to inhibit the growth and reproduction of putrefactive bacteria (such as molds).

[0004] It is particularly important to note that rice residue protein differs fundamentally from other starch-protein agricultural byproducts such as soy milk and potato residue in its protein molecular structure and physicochemical properties, and cannot be simply substituted for them. Specifically, rice residue protein is mainly composed of gluten (accounting for more than 80% of the total protein). Its molecules contain a large number of intra- and inter-chain disulfide bonds and hydrophobic interactions, forming a highly dense globular tertiary structure, which is far more difficult for proteases to approach and degrade than soy milk protein (mainly composed of glycinin and β-conglycinin, with a relatively loose structure). At the same time, rice residue protein has extremely low water solubility (nitrogen solubility index NSI < 10%), far lower than soy milk protein (NSI > 80%), and contains anti-nutritional factors unique to rice residue, such as phytic acid and rice bran lectins. The aforementioned structural characteristics result in rice residue protein exhibiting significantly higher resistance to protease degradation than soy milk protein. Simply transplanting fermentation processes suitable for substrates like soy milk to rice residue systems presents unforeseen technical obstacles, including extremely low protein degradation efficiency, difficulty in controlling pH during fermentation, and insufficient yield of target peptides. Therefore, there is an urgent need in this field to develop dedicated fermentation processes tailored to the specific structural characteristics of rice residue protein, rather than simply applying existing methods for other substrates.

[0005] Furthermore, while existing technologies employ a two-stage fermentation process using Bacillus subtilis followed by Lactobacillus plantarum to treat other substrates (such as soy milk), the fermentation mechanism upon which this approach relies is closely related to the substrate characteristics and cannot be directly transferred to the rice residue system. Firstly, soy milk proteins are primarily globulins, with a loose structure and high water solubility, allowing for rapid and effective degradation by Bacillus subtilis proteases. In contrast, rice residue proteins are primarily glutenin, highly dense, and have extremely low water solubility, resulting in significant differences in protein degradation efficiency under the same strains and processing conditions. Secondly, soy milk contains abundant fermentable sugars, directly supporting rapid acid production by Lactobacillus plantarum. Rice residue, however, has a lower content of fermentable sugars, requiring Bacillus subtilis to degrade starch and protein into reducing sugars and small peptides before providing sufficient carbon sources for Lactobacillus plantarum. This results in an extremely narrow time window for the two fermentation stages, demanding significantly higher precision in timing control compared to the soy milk system. Therefore, directly transplanting the two-stage fermentation process applicable to soy milk to rice residue substrate not only fails to achieve similar results, but also faces a series of unforeseen technical obstacles such as insufficient protein degradation, delayed acid production, and pH loss of control.

[0006] However, in the rice residue system, when the two adopt the traditional strategy of simultaneous inoculation (co-fermentation), the fermentation process often goes out of control due to the conflict of metabolic characteristics between the two microorganisms. Specifically, it manifests as the following three major technical defects: (1) pH control of the fermentation system fails, and the product has a high risk of mold growth: During the vigorous metabolism of proteins, Bacillus subtilis releases ammonia through deamination, which causes the pH of the fermentation system to not only not decrease, but also to show a "ammoniation rebound". At this time, due to the competitive consumption of carbon source, the acid production capacity of Lactobacillus plantarum is significantly inhibited, making it difficult to effectively reduce the pH at the end of fermentation to the safe anti-mold threshold (usually requiring pH < 4.80). The high pH at the end (usually > 5.0) makes the product very susceptible to secondary contamination and mold growth during subsequent drying and storage, which seriously restricts the commercial value of the product; (2) Insufficient protein degradation and extremely low yield of target small peptides: There is intense ecological niche competition between the two strains under the condition of simultaneous inoculation, which leads to an imbalance in the microbial community structure, inhibits the expression of protease in Bacillus subtilis, and the protein degradation is incomplete. At the same time, the acidic metabolic environment of *Lactobacillus plantarum* may also inhibit the enzyme activity of *Bacillus subtilis* in advance. Ultimately, this results in extremely low accumulation of acid-soluble small peptides (molecular weight <10 kDa) with high digestibility and absorption activity (usually less than 3.5 g / 100g), making it difficult to reach an effective dose to improve animal intestinal health; (3) Poor process stability, making it difficult to achieve industrial promotion: Traditional mixed fermentation process is extremely sensitive to fluctuations in inoculation ratio, environmental conditions, etc. The metabolism of the two bacteria is mutually restrictive, the process window is narrow, and there are large differences between batches. The uncontrollability of the fermentation endpoint pH and small peptide content makes it difficult to standardize product quality and cannot meet the basic requirements of modern feed industry for the stability of raw material quality.

[0007] Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing high-small-peptide acidified rice residue protein by sequential inoculation solid-state fermentation, aiming to solve a series of problems existing in the current solid-state fermentation of rice residue protein, such as difficulty in pH control leading to high risk of mold growth, low protein degradation efficiency resulting in low yield of small peptides, and poor product stability.

[0009] In a first aspect, the present invention provides a method for preparing high-peptide acidified rice residue protein by sequential inoculation solid-state fermentation, comprising the following steps:

[0010] (1) Preparation of fermentation substrate: Mix rice residue protein with carbon source auxiliary materials to prepare fermentation substrate;

[0011] (2) First-stage solid-state fermentation: Bacillus subtilis was first inoculated into the fermentation substrate obtained in step (1) for first-stage solid-state fermentation to degrade the macromolecular proteins therein; the Bacillus subtilis was Bacillus subtilis NCUTY 001, which was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number: CCTCC NO: M 2025647, and the deposit address was Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0012] (3) Second stage solid-state fermentation: After the first stage solid-state fermentation in step (2) is completed, Lactiplantibacillus plantarum is introduced into the system for the second stage solid-state fermentation to produce organic acids and reduce the pH of the system; the Lactiplantibacillus plantarum is Lactiplantibacillus plantarum NCUTUAS4, which was deposited at the China Center for Type Culture Collection on April 30, 2024, with accession number: CCTCC NO: M 2024858, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0013] (4) Post-processing: After the second stage of solid-state fermentation in step (3) is completed, the fermentation product of high small peptide acidified rice residue protein is obtained through post-processing.

[0014] Optionally, the carbon source is wheat bran, and the mass ratio of rice residue protein to wheat bran is (5-9):1.

[0015] Optionally, the initial moisture content of the fermentation substrate is 50%-60%.

[0016] Optionally, the inoculation amounts of Bacillus subtilis and Lactobacillus plantarum are independently 8%-12%.

[0017] Optionally, the temperatures of the first stage solid-state fermentation and the second stage solid-state fermentation are independently set at 28-32°C.

[0018] Optionally, the first stage of solid-state fermentation takes 35-60 hours; the second stage of solid-state fermentation takes 60-85 hours.

[0019] Optionally, the post-processing includes drying the fermentation product obtained after the second stage of solid-state fermentation at 40-60°C until the moisture content is ≤12%.

[0020] Optionally, during the first stage of solid-state fermentation and the second stage of solid-state fermentation, the material is turned over once every 12-24 hours.

[0021] Secondly, the present invention provides a fermentation product of high-peptide acidified rice residue protein prepared by any of the above-mentioned optional methods.

[0022] Optionally, the product has an acid-soluble peptide content ≥6.5g / 100g and a total acid content ≥1.95g / 100g.

[0023] Thirdly, the present invention provides the application of a fermentation product of high-small-peptide acidified rice residue protein obtained by any of the above-mentioned optional methods in the preparation of highly digestible protein feed or functional food ingredients.

[0024] Optionally, the highly digestible protein feed is used for eel farming.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The sequential inoculation solid-state fermentation method provided by the present invention decouples and connects the two core processes of "deep protein degradation" and "rapid acidification of the system" on a time scale. First, Bacillus subtilis is used to carry out aerobic fermentation independently to efficiently degrade macromolecular proteins, and then Bacillus plantarum is inoculated to carry out facultative anaerobic fermentation to rapidly produce acid. This completely avoids the metabolic conflict between the two bacteria and achieves the total acid content at the end of fermentation (≥1.95g / 100g). No signs of mold growth were observed during the entire fermentation and storage process.

[0027] (2) The sequential inoculation solid-state fermentation method provided by the present invention, by giving Bacillus subtilis sufficient independent action time, allows its protease expression to be free from competitive inhibition, and the protein degradation to be more thorough, providing a sufficient "raw material library" for the accumulation of small peptides; at the same time, the acidic environment created by the subsequently inoculated Lactobacillus plantarum effectively inhibits the extracellular protease of Bacillus subtilis from further degrading small peptides into free amino acids, realizing the "generation-protection" synergy of small peptides, so that the content of acid-soluble small peptides in the product reaches more than 6.5 g / 100g, and can reach up to 7.0 g / 100g, which is a leap of more than 100% compared with the traditional mixed co-fermentation process (<3.5 g / 100g);

[0028] It is particularly important to emphasize that the aforementioned "generation-protection" synergistic mechanism has special significance and unpredictable technical effects for rice residue protein. Because rice residue protein is primarily composed of highly dense glutenin, it possesses natural resistance to protease hydrolysis, making it difficult to achieve deep degradation using conventional fermentation processes. This invention, through a sequential inoculation strategy, provides Bacillus subtilis with sufficient independent action time to break the dense disulfide bonds and hydrophobic structure of rice residue glutenin, achieving a deep protein degradation effect that is difficult to achieve with conventional processes. Simultaneously, the strongly acidic environment created by *Lactobacillus plantarum* effectively prevents the already generated small peptides from being further degraded into free amino acids. This "generation-protection" balance is especially crucial for the rice residue protein system: the high glutenin content of rice residue protein means that once the protein backbone is opened, if the acidity of the environment is not controlled in time, the highly active proteases of *Bacillus subtilis* will rapidly over-degrade the small peptides into free amino acids, leading to the loss of the target product. This synergistic effect is unpredictable in existing fermentation schemes for loosely structured protein substrates such as soy milk, and cannot be obtained by those skilled in the art simply by replacing the substrate.

[0029] (3) The sequential inoculation solid-state fermentation method provided by the present invention enables plant lactobacillus to quickly initiate acid metabolism in the "nutrient-rich and low-competition" environment constructed by Bacillus subtilis through a sequential decoupling strategy. The total acid content of the product is as high as 1.95~2.05 g / 100g. The strong acid environment effectively inhibits the growth and reproduction of putrefactive bacteria and molds. The product can remain stable for a long time without the addition of any chemical preservatives during subsequent drying and storage, which significantly improves the biosafety and shelf life of the product.

[0030] (4) The sequential inoculation solid-state fermentation method provided by this invention uses Bacillus subtilis NCUTY001 (CCTCC NO: M 2025647) and Lactobacillus plantarum NCUTUAS4 (CCTCC NO: M 2024858) specifically preserved. The former has high protease activity and excellent temperature resistance, while the latter has a rapid acid production rate and strong acid tolerance. The synergistic effect of the strains and the process further amplifies the technical advantages of the sequential inoculation strategy, achieving a synergistic effect of "1+1>2". Moreover, the process of this invention is simple and easy to scale up industrially, providing a practical and feasible technical path for the high-value utilization of grain processing by-products such as rice residue protein. It is worth noting that the Bacillus subtilis NCUTY 001 was selected from four candidate thermostable Bacillus strains through systematic enzyme production capacity evaluation. Its protease production activity (0.38 U / mL) and α-amylase production activity (0.10 U / mL) are relatively high. The dual-enzyme advantage of this strain (U / mL, the highest among candidate strains) enables it to simultaneously and efficiently degrade protein and starch components in rice residue, providing sufficient carbon and nitrogen substrates for the second-stage acid-producing fermentation of Lactobacillus plantarum. The enzyme system characteristics of this strain are highly matched with the characteristics of rice residue substrate.

[0031] (5) The rice residue protein fermentation product prepared by the present invention is rich in small peptides and organic acids, and has good palatability and digestibility. It can be used as a high-digestibility protein feed, and is especially suitable for the breeding of high-protein aquatic animals such as eels. It can significantly promote their growth and enhance their immunity, and provides an ideal candidate to replace expensive fishmeal protein. Attached Figure Description

[0032] Figure 1 The graph shows the effect of different inoculation methods (sequential inoculation of this invention vs. traditional simultaneous inoculation and single inoculation) on the dynamic changes of pH value during solid-state fermentation of rice residue protein.

[0033] Figure 2 A comparison of the content of acid-soluble small peptides in rice residue protein products at the fermentation endpoint due to different inoculation methods;

[0034] Figure 3 The effect of different rice residue protein to bran mass ratios on pH (single-factor experimental results).

[0035] Figure 4 The effect of different moisture contents on pH (single-factor experimental results);

[0036] Figure 5 The effect of different fermentation temperatures on pH (single-factor experimental results);

[0037] Figure 6 The effect curves of various factors on the content of acid-soluble small peptides in the orthogonal experiment are shown.

[0038] Figure 7 The effect curves of each factor on the total acid content in the orthogonal experiment are shown.

[0039] Figure 8 This is a graph showing the effect of various factors on protein content in an orthogonal experiment.

[0040] Figure 9 A comparison chart showing the content of small peptides in rice residue protein fermentation products before and after process optimization. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0042] This invention provides a method for preparing high-peptide acidified rice residue protein by sequential inoculation solid-state fermentation, comprising the following steps:

[0043] (1) Preparation of fermentation substrate: Mix rice residue protein with carbon source auxiliary materials to prepare fermentation substrate;

[0044] (2) First-stage solid-state fermentation: Bacillus subtilis was first inoculated into the fermentation substrate obtained in step (1) for first-stage solid-state fermentation to degrade the macromolecular proteins therein; the Bacillus subtilis used was Bacillus subtilis NCUTY001, Latin name Bacillus subtilis NCUTY 001, which was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number: CCTCC NO: M 2025647, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0045] (3) Second stage solid-state fermentation: After the first stage solid-state fermentation in step (2) is completed, Lactiplantibacillus plantarum is introduced into the system for the second stage solid-state fermentation to produce organic acids and reduce the pH of the system. The Lactiplantibacillus plantarum used is Lactiplantibacillus plantarum NCUTUAS4, which was deposited at the China Center for Type Culture Collection on April 30, 2024, with accession number CCTCC NO: M 2024858 and deposit address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0046] (4) Post-processing: After the second stage of solid-state fermentation in step (3) is completed, the fermentation product of high small peptide acidified rice residue protein is obtained through post-processing.

[0047] Specifically, the plant lactobacillus NCUTUUAS4 used has been disclosed in Chinese patent literature (publication number: CN118813492A, title: A composite mutagenized plant lactobacillus and its mutagenization method and application).

[0048] Specifically, the Bacillus subtilis NCUTY 001 (CCTCC NO: M 2025647) selected in this invention has undergone specific screening for rice residue protein substrates. Its secreted extracellular protease has a significant preferential degradation ability for rice residue glutenin, which can effectively break the disulfide bonds and hydrophobic interactions within the glutenin molecule, achieving deep enzymatic hydrolysis. Meanwhile, the selected Lactobacillus plantarum NCUTUAS4 (CCTCC NO: M 2024858) has the ability to rapidly initiate acid production metabolism in the high-protein, low-sugar environment unique to the rice residue fermentation system. It can quickly establish an acidic environment under the limited reducing sugar conditions produced by the degradation of Bacillus subtilis in the early stage. The synergistic effect of the two strains is the result of specific screening and optimization for rice residue protein substrates.

[0049] In fact, this invention completely avoids the metabolic conflict between the two microorganisms by decoupling and orderly connecting the two core processes of "deep protein degradation" and "rapid system acidification" on a time scale, achieving a breakthrough increase in total acid content (≥1.95g / 100g) and small peptide content (≥6.5g / 100g). The product is rich in small peptides and organic acids, and no mold growth occurs during fermentation and storage. This product can be used as a highly digestible protein feed, especially suitable for eel farming, promoting the high-value utilization of grain processing by-products.

[0050] It is particularly important to emphasize that this invention employs a sequential inoculation solid-state fermentation strategy tailored to the unique physicochemical properties of rice residue protein, including its high gluten content, dense molecular structure, and low water solubility. The gluten molecules in rice residue protein contain numerous disulfide bonds and hydrophobic interactions, forming a highly dense spherical structure that exhibits natural resistance to protease degradation—a structural feature not found in other starch-protein substrates such as soy milk and potato residue. This invention first inoculates *Bacillus subtilis* for fully independent aerobic fermentation, allowing its extracellular proteases sufficient time to break down the dense structure of rice residue gluten, achieving deep degradation of the large protein molecules. Subsequently, *Lactobacillus plantarum* is inoculated for facultative anaerobic fermentation. The degradation products generated in the initial stage rapidly produce acid, creating an acidic environment that not only inhibits the growth of putrefactive bacteria but also effectively prevents the already generated small peptides from being further degraded into free amino acids by the extracellular proteases of *Bacillus subtilis*. This "generation-protection" synergistic mechanism, designed specifically for the structure of rice residue protein, is the core innovation of this invention, distinguishing it from existing technologies and cannot be achieved simply by replacing the substrate.

[0051] In some embodiments, the carbon source excipient used in step (1) is wheat bran, and the mass ratio of rice residue protein to wheat bran is (5-9):1.

[0052] Specifically, the preferred mass ratio of rice residue protein to wheat bran is 7:1. This ratio ensures sufficient protein substrate while providing adequate porosity and buffering capacity.

[0053] In some embodiments, the initial water content of the fermentation substrate is 50%-60%.

[0054] Specifically, the rice residue protein used can be defatted or uncrusted rice residue protein raw material. The rice residue protein raw material is dried, pulverized, and passed through a 60-mesh sieve. It is then mixed evenly with wheat bran, which serves as a porous medium and auxiliary carbon source, in a certain proportion. The moisture content of the system is adjusted to 50%~60% (w / w) to obtain the fermentation substrate. After being sterilized by high-pressure steam (115~121℃, 15~30min), it is cooled for later use.

[0055] Specifically, the initial water content of the fermentation substrate is preferably 52.4% to facilitate the growth of microbial cells and the diffusion of enzyme molecules.

[0056] In some embodiments, the inoculum amounts of Bacillus subtilis and Lactobacillus plantarum used are independently 8%-12%.

[0057] Specifically, activated Bacillus subtilis high-density seed liquid is preferably inoculated into the fermentation substrate obtained in step (1) at an inoculation amount of 10% relative to the dry basis mass of the substrate; specifically, after fermentation in step (2), activated Lactobacillus plantarum high-density seed liquid is directly inoculated into the fermentation system without sterilization treatment, and the inoculation amount is preferably 10% of the dry basis mass of the substrate.

[0058] In some embodiments, the temperatures of the first-stage solid-state fermentation and the second-stage solid-state fermentation are independently set at 28-32°C.

[0059] In some embodiments, the first stage of solid-state fermentation takes 35-60 hours; the second stage of solid-state fermentation takes 60-85 hours.

[0060] In fact, in the first stage of solid-state fermentation in step (2), Bacillus subtilis rapidly multiplies and secretes a large amount of extracellular protease, which efficiently cuts and degrades the poorly soluble macromolecular storage proteins in rice residue into soluble polypeptides, oligopeptides and a small amount of free amino acids.

[0061] In fact, in the second stage of solid-state fermentation in step (3), Lactobacillus plantarum makes full use of the degradation products (such as reducing sugars, small molecule peptides, amino acids, etc.) produced in the first stage of solid-state fermentation as the preferred carbon and nitrogen sources to rapidly proliferate and dominate homo- or hetero-lactic acid fermentation, accumulating a large amount of organic acids (mainly lactic acid), causing the pH of the system to drop rapidly and continuously.

[0062] Specifically, in step (2), activated Bacillus subtilis high-density seed liquid is introduced, and after being thoroughly mixed, it is preferably placed in a 28°C constant temperature incubator for shallow tray or stacked solid fermentation for 48 hours.

[0063] Specifically, in step (3), activated high-density seed liquid of *Lactobacillus plantarum* is introduced, stirred evenly, and preferably maintained at 28°C for a second solid-state fermentation for 72 hours.

[0064] In fact, the optimal timing control point is to first ferment with Bacillus subtilis for 48 hours, followed by inoculation with Lactobacillus plantarum for another 72 hours. This timing ensures that the protein backbone is fully opened, while the remaining nutrient substrate just supports the secondary growth peak of the lactic acid bacteria. The entire fermentation process is optimally controlled at a constant temperature of 28°C. This temperature balances the stability of the mesophilic protease in Bacillus subtilis with the metabolic activity of Lactobacillus plantarum.

[0065] In some embodiments, post-processing includes drying the fermentation product obtained after the second-stage solid-state fermentation at 40-60°C until the moisture content is ≤12%.

[0066] Specifically, after the fermentation of the system in step (3) is completed, the material is transferred to a blower drying oven or vacuum drying oven at 40-60℃ and dried at low temperature until constant weight. After crushing and sieving, rice residue protein fermentation product rich in acid-soluble small peptides and with pH stable in the acidic range is obtained.

[0067] In fact, the selected Bacillus subtilis NCUTY 001 with accession number CCTCC NO: M 2025647 is characterized by high protease activity and good temperature resistance; the selected Lactobacillus plantarum NCUTUAS4 with accession number CCTCC NO: M 2024858 is characterized by fast acid production rate and strong acid tolerance.

[0068] Specifically, the screening process for Bacillus subtilis NCUTY 001 was as follows: Heat-resistant Bacillus strains were screened from healthy poultry manure treated at 85℃ for 15 min, resulting in four candidate strains (numbered Z1, Z2, Z3, and Z4). The enzyme production capacity of each strain was systematically evaluated. The results showed that strain Z1 (NCUTY001) exhibited a protease activity of 0.38 U / mL, an α-amylase activity of 0.10 U / mL (the highest among the four candidate strains), and a filter paper cellulase activity of 1.25 U / mL. The α-amylase activity was the highest among all candidate strains, enabling this strain to efficiently hydrolyze the starch components in rice residue, providing sufficient reducing sugar substrate for subsequent acid-producing fermentation by *Lactobacillus plantarum*. Simultaneously, the high protease activity ensured the effective degradation of gluten in the rice residue. This synergistic dual-enzyme characteristic makes NCUTY001 particularly suitable as a starting strain for solid-state fermentation of rice residue (rich in starch and protein). Based on morphological observation, physiological and biochemical tests and 16S rRNA molecular identification, Z1 was confirmed to be Bacillus subtilis and was preserved and named Bacillus subtilis NCUTY001.

[0069] In some embodiments, the material is turned over every 12-24 hours during the first-stage solid-state fermentation and the second-stage solid-state fermentation.

[0070] The present invention also provides a fermentation product of high small peptide acidified rice residue protein prepared by any of the above embodiments.

[0071] In some embodiments, the obtained product has an acid-soluble peptide content ≥6.5g / 100g and a total acid content ≥1.95g / 100g.

[0072] The present invention also provides an application of the high-small-peptide acidified rice residue protein fermentation product prepared by any of the above embodiments in the preparation of highly digestible protein feed or functional food ingredients.

[0073] In some embodiments, the highly digestible protein feed is used for eel farming.

[0074] Seed liquid preparation method:

[0075] Activated Bacillus subtilis or Lactobacillus plantarum were inoculated into MRS liquid medium (LB medium can also be used for Bacillus subtilis), incubated statically at 37°C for 24 h, centrifuged to collect the bacterial cells, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to ≥1×10⁻⁶. 9 CFU / mL, for later use.

[0076] Detection method:

[0077] (1) pH value determination: Add the fermentation product to deionized water at a ratio of 1:10 (w / v), shake thoroughly and let stand, and use a pH meter to measure the pH value of the supernatant.

[0078] (2) Determination of total acid content: The acid-base titration method was adopted, with phenolphthalein as an indicator, and 0.1 mol / L NaOH standard solution was used to titrate until a faint red color was obtained. The result was expressed as lactic acid (g / 100g).

[0079] (3) Determination of acid-soluble small peptide content: The trichloroacetic acid precipitation-BCA method was used. After the sample was precipitated with 15% trichloroacetic acid, the supernatant was taken to determine the OD. 562 The value is used to calculate the content of acid-soluble small peptides based on the standard curve.

[0080] (4) Protein content determination: Kjeldahl method was used, in accordance with GB 5009.5-2016.

[0081] Example 1 (Sequential Inoculation - Optimal Process)

[0082] Example 1 of this invention provides a method for preparing acidified rice residue protein with high small peptide content by sequential inoculation solid-state fermentation, comprising the following steps:

[0083] (1) Preparation of fermentation substrate: Take dried rice residue protein powder (protein content of about 70%), mix it with wheat bran at a mass ratio of 7:1, put it into a 250 mL Erlenmeyer flask (each flask contains 50 g rice residue protein powder / wheat bran mixture), add 55 g deionized water (water content 52.4%: 55 g water / (55 g water + 50 g rice residue protein powder and wheat bran mixture)), stir thoroughly and mix well, sterilize at 121℃ for 20 min, cool to room temperature, and obtain fermentation substrate.

[0084] (2) First stage solid-state fermentation: The activated Bacillus subtilis NCUTY 001 seed culture (bacterial count ≥ 1×10⁻⁶) was fermented. 9 CFU / mL) at 10% (v / w) 干基 The inoculum obtained in step (1) is added to the fermentation substrate, and the mixture is thoroughly mixed under aseptic conditions. The substrate is then placed in a 28℃ constant temperature incubator and allowed to ferment for 48 h. The substrate is turned over once every 24 h during the fermentation process to obtain the first stage of fermentation.

[0085] (3) Second stage solid-state fermentation: The activated Lactobacillus plantarum seed culture NCUTUAS4 (bacterial count ≥1×10⁻⁶) was fermented. 9 CFU / mL) at 10% (v / w) 干基 The inoculum obtained in step (2) is added to the first stage fermentation product, thoroughly mixed, and then placed in a 28℃ constant temperature incubator for 72 h of fermentation. During this period, the product is turned over once every 24 h to obtain the co-fermentation product.

[0086] (4) Post-processing: Spread the co-fermented product obtained in step (3) on a tray and dry it in a 60°C forced-air drying oven until constant weight. Then crush it with a pulverizer to obtain the acidified rice residue protein fermentation product with high small peptide content.

[0087] Test results: The final pH of the rice residue protein fermentation product prepared in Example 1 was 4.31, the total acid content was 1.98 g / 100 g, the acid-soluble small peptide content was 6.99 g / 100 g, and the protein content increased by about 13.27%.

[0088] Comparative Example 1 (Traditional Simultaneous Vaccination)

[0089] Comparative Example 1 provides a traditional method for preparing rice residue protein through simultaneous inoculation and solid-state fermentation.

[0090] The difference between Comparative Example 1 and Example 1 is that Bacillus subtilis and Lactobacillus plantarum were inoculated simultaneously in step (2) (each inoculated at 10%), step (3) was not performed, and there was no stepwise fermentation; other conditions and steps were the same as in Example 1.

[0091] Test results: The final pH of the rice residue protein fermentation product prepared in Comparative Example 1 was 5.24, the total acid content was 0.70 g / 100 g, and the protein content increased by about 2.50%.

[0092] Comparative Example 2 (inoculated only with Bacillus subtilis)

[0093] Comparative Example 2 provides a method for preparing rice residue protein by solid-state fermentation using only Bacillus subtilis inoculation.

[0094] The difference between Comparative Example 2 and Example 1 is that only the first stage of solid-state fermentation (Bacillus subtilis fermentation alone for 120 h) in step (2) is performed, and step (3) is not performed; other conditions and steps are the same as in Example 1.

[0095] Test results: The final pH of the rice residue protein fermentation product prepared in Comparative Example 2 was 5.10, the total acid content was 0.80 g / 100 g, and the protein content decreased by 2.30%.

[0096] Comparative Example 3 (inoculated only with Lactobacillus plantarum)

[0097] Comparative Example 3 provides a method for preparing rice residue protein by solid-state fermentation using only Lactobacillus plantarum inoculation.

[0098] The difference between Comparative Example 3 and Example 1 is that step (2) is omitted, and *Lactobacillus plantarum* is directly inoculated into the fermentation substrate obtained in step (1) for 120 h of fermentation; other conditions and steps are the same as in Example 1.

[0099] Test results: The final pH of the rice residue protein fermentation product prepared in Comparative Example 3 was 4.80, the total acid content was 1.03 g / 100 g, and the protein content decreased by 3.50%.

[0100] Fermentation was carried out according to the methods of Example 1 and Comparative Examples 1-3. The pH value of the fermentation system was measured every 12 hours, and a pH dynamic change curve was plotted. The results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the pH of Comparative Example 1 (simultaneous inoculation) rose significantly in the later stage of fermentation, eventually reaching pH > 5.0; the pH of Comparative Example 2 (Bacillus subtilis only) continued to rise to 5.10; the pH of Comparative Example 3 (Lactobacillus plantarum only) dropped to 4.80 and then stabilized; while the pH of Example 1 (sequential inoculation) continued to decrease steadily after the inoculation of Lactobacillus plantarum in the second stage, eventually stabilizing at 4.31, indicating that the sequential inoculation strategy successfully achieved precise pH control.

[0101] The content of acid-soluble small peptides in the products obtained in Example 1 and Comparative Example 1 was determined, and the results are as follows: Figure 2 As shown. By Figure 2It can be seen that the content of small peptides prepared by the sequential seeding strategy (Example 1) (6.99 g / 100g) is significantly higher than that of traditional simultaneous seeding (Comparative Example 1, 3.50 g / 100g), indicating that the sequential seeding strategy is the key to achieving deep protein degradation and efficient small peptide generation.

[0102] Examples 2-9

[0103] Embodiments 2 to 9 of the present invention provide a method for optimizing process parameters based on single-factor experiments.

[0104] Based on verifying the feasibility of the sequential inoculation strategy, and in order to further determine the impact of each key factor on the fermentation effect, this invention conducted a single-factor optimization experiment using pH as a rapid screening index.

[0105] Example 2 (Different substrate ratios)

[0106] The difference from Example 1 is that in step (1), the mass ratio of rice residue protein to wheat bran is 5:1; 30 g of deionized water (with a water content of 37.5%) is added; the temperature is 37°C; and other conditions and steps are the same as in Example 1.

[0107] Example 3 (Different substrate ratios)

[0108] The difference from Example 1 is that in step (1), the mass ratio of rice residue protein to wheat bran is 9:1; 30 g of deionized water (with a water content of 37.5%) is added; the temperature is 37°C; and other conditions and steps are the same as in Example 1.

[0109] Example 4 (Different Moisture Contents)

[0110] The difference from Example 1 is that in step (1), 30 g of deionized water (with a water content of 37.5%) is added and the temperature is 37°C; other conditions and steps are the same as in Example 1.

[0111] Example 5 (Different Moisture Contents)

[0112] The difference from Example 1 is that in step (1), 40 g of deionized water (with a water content of 44.4%) is added and the temperature is 37°C; other conditions and steps are the same as in Example 1.

[0113] Example 6 (Different Moisture Contents)

[0114] The difference from Example 1 is that in step (1), 50 g of deionized water (with a water content of 50.0%) is added and the temperature is 37°C; other conditions and steps are the same as in Example 1.

[0115] Example 7 (Different fermentation temperatures)

[0116] The difference from Example 1 is that the fermentation temperature in steps (2) and (3) is 31°C; other conditions and steps are the same as in Example 1.

[0117] Example 8 (Different fermentation temperatures)

[0118] The difference from Example 1 is that the fermentation temperature in steps (2) and (3) is 34°C; other conditions and steps are the same as in Example 1.

[0119] Example 9 (Different fermentation temperatures)

[0120] The difference from Example 1 is that the fermentation temperature in steps (2) and (3) is 37°C; other conditions and steps are the same as in Example 1.

[0121] The following also provides a method for optimizing process parameters based on orthogonal experiments.

[0122] Based on the single-factor experiments in Examples 2 to 9, three factors that significantly affected the fermentation effect were selected: A. fermentation temperature (°C), B. moisture content (g), and C. substrate ratio (rice residue: wheat bran). Three levels were selected for each factor, according to L9(3) 4 An orthogonal array was used to conduct experiments, with the content of acid-soluble small peptides, total acid content, and protein content as evaluation indicators, to further optimize the fermentation process parameters.

[0123] Table 1: Factor Level Table for Orthogonal Experiment

[0124]

[0125] Table 2: Orthogonal experimental design and results

[0126]

[0127] Based on the data in Table 2, effect curves were plotted to show the influence of each factor on the content of acid-soluble small peptides, total acid content, and protein content. The results are as follows: Figure 6 , Figure 7 , Figure 8 As shown.

[0128]

[0129]

[0130] As shown in Table 2, the order of influence of each factor on the fermentation effect of rice residue protein is: A (fermentation temperature) > C (substrate ratio) > B (moisture content). The optimal combination is A1B2C2, namely, fermentation temperature 28℃, moisture content 55 g water, and rice residue:wheat bran = 7:1. This combination is completely consistent with the conditions in Example 1.

[0131] Verification of optimization effect:

[0132] To further verify the reliability of the orthogonal optimization results, the optimal combination obtained from the orthogonal experiment (Example 1) was compared with other combinations in the orthogonal experiment. The comparison of small peptide content in rice residue protein fermentation products before and after process optimization is shown below. Figure 9 As shown.

[0133] Depend on Figure 9 It can be seen that the content of small peptides in the rice residue protein fermentation product prepared by Example 1 (optimal combination) (6.99 g / 100g) is significantly higher than that of other combinations in the orthogonal experiment (5.68~6.82 g / 100g), indicating that the optimized process parameters provided by the present invention are reliable.

[0134] Performance verification:

[0135] (1) Effects of different inoculation methods on pH dynamics during fermentation

[0136] The pH dynamic change detection results of Comparative Examples 1-3 and Example 1 are as follows: Figure 1 As shown.

[0137] See Figure 1 In Comparative Example 1 (simultaneous inoculation), the pH significantly increased during the later stages of fermentation, eventually reaching >5.0; in Comparative Example 2 (inoculated only with Bacillus subtilis), the pH continued to rise to 5.10; in Comparative Example 3 (inoculated only with Lactobacillus plantarum), the pH decreased to 4.80 and then stabilized; while in Example 1 (sequential inoculation), the pH steadily decreased after the second stage of inoculation with Lactobacillus plantarum, eventually stabilizing at 4.31. These results demonstrate that, compared to simultaneous and single inoculation, the sequential inoculation strategy employed in this invention effectively avoids metabolic conflicts between the two bacteria and achieves precise pH control.

[0138] (2) Effect of different inoculation methods on the content of acid-soluble small peptides

[0139] The results of the acid-soluble small peptide content detection in Comparative Examples 1-3 and Example 1 are as follows: Figure 2 As shown.

[0140] See Figure 2 The peptide content of Comparative Example 1 (simultaneous inoculation) was 3.50 g / 100g, while the peptide content of Example 1 (sequential inoculation) was as high as 6.99 g / 100g. The results indicate that the peptide content of the product prepared by the sequential inoculation strategy is approximately 100% higher than that prepared by the traditional simultaneous inoculation strategy, demonstrating that the sequential inoculation strategy is key to achieving deep protein degradation and efficient peptide generation.

[0141] (3) Effect of different substrate ratios on the final pH of fermentation

[0142] The pH test results of the fermentation endpoint in Examples 1-3 are as follows: Figure 3 As shown.

[0143] See Figure 3 When the ratio of rice residue to wheat bran was 5:1, the endpoint pH was 4.78; when the ratio was 7:1, the endpoint pH was 4.80; and when the ratio was 9:1, the endpoint pH was 4.87. The results indicate that the substrate ratio has a certain impact on the endpoint pH of fermentation. The lowest pH was observed at a ratio of 5:1, which was not significantly different from the 7:1 group but significantly lower than the 9:1 group (P < 0.05).

[0144] (4) Effect of different moisture contents on the pH of the fermentation endpoint

[0145] The pH test results of the fermentation endpoint in Examples 4-6 are as follows: Figure 3 As shown.

[0146] See Figure 4 When the initial water addition was 30 g, the final pH was 4.75; when the initial water addition was 40 g, the final pH was 4.72; and when the initial water addition was 50 g, the final pH was 4.70. The results show that the initial water addition significantly affects the final pH of fermentation, with the lowest pH observed at an initial water addition of 50 g, and the difference from other water content groups is significant (P < 0.05). Therefore, an initial water content of 50.0% is preferred.

[0147] (5) Effect of different fermentation temperatures on the final pH of fermentation

[0148] The pH test results of the fermentation endpoint in Examples 1 and 7-9 are as follows: Figure 5 As shown.

[0149] See Figure 5 When the fermentation temperature was 28℃, the final pH was 4.61; when the temperature was 31℃, the final pH was 4.72; when the temperature was 34℃, the final pH was 4.86; and when the temperature was 37℃, the final pH was 4.70. The results show that fermentation temperature has a highly significant impact on the final pH, with the lowest pH at 28℃, and the difference from other temperature groups is significant (P < 0.05). Therefore, the optimal fermentation temperature is 28℃. Although the single-factor experiments showed the optimal levels for each factor using pH as an indicator, in order to comprehensively improve the small peptide content, total acidity, and protein quality of the product, subsequent orthogonal experiments will be used to synergistically optimize these parameters across multiple indicators.

[0150] (6) Effects of various factors on the content of acid-soluble small peptides in orthogonal experiments

[0151] Based on the data in Table 2, effect curves were plotted to show the influence of each factor on the content of acid-soluble small peptides. The results are as follows: Figure 6 As shown. See also Figure 6The effects of various factors on the content of acid-soluble peptides were as follows: the highest peptide content was observed at fermentation temperature of 28℃ (level 1); the highest peptide content was observed at a moisture content of 52.4% (55 g water) (level 2); and the highest peptide content was observed at a substrate ratio of 7:1 (level 2). The results indicate that the optimal levels for all three factors were the combination of A1B2C2.

[0152] (7) Effects of various factors on total acid content in orthogonal experiments

[0153] Based on the data in Table 2, effect curves were plotted to show the influence of each factor on the total acid content. The results are as follows: Figure 7 As shown. See also Figure 7 The effects of various factors on total acid content did not correlate with those on small peptide content: the highest total acid content was observed at a fermentation temperature of 34℃, a moisture content of 60%, and a substrate-to-peptide ratio of 7:1. These results indicate that the optimal combination of process parameters A3B3C3 simultaneously promotes the accumulation of total acid.

[0154] (8) Effects of various factors on protein content in orthogonal experiments

[0155] Based on the data in Table 2, effect curves were plotted to show the influence of each factor on protein content. The results are as follows: Figure 8 As shown. See also Figure 8 The effects of various factors on protein content showed a trend consistent with that of small peptides: the highest protein content was achieved at a fermentation temperature of 28℃, a moisture content of 52.4%, and a substrate ratio of 7:1. The results indicate that the optimal combination of process parameters A1B2C2 can comprehensively improve the nutritional quality of rice residue protein fermentation products.

[0156] (9) Comparison of small peptide content before and after process optimization

[0157] Comparison of small peptide content in Example 1 (corresponding to Experiment No. 2 in the orthogonal experiment) with other combinations in the orthogonal experiment (Experiment No. 1, Experiment Nos. 3-9) Figure 9 As shown.

[0158] See Figure 9 The small peptide content of the rice residue protein fermentation product prepared in Example 1 (optimal combination A1B2C2) was 6.99 g / 100g, significantly higher than that of other combinations in the orthogonal experiment (5.68~6.82 g / 100g) (P<0.05). The results indicate that the process parameters determined by the present invention through single-factor experiments and orthogonal optimization (moisture content 52.4%, fermentation temperature 28℃, rice residue:wheat bran = 7:1, Bacillus subtilis fermentation for 48 h followed by inoculation with Lactobacillus plantarum for 72 h) are reliable optimal process parameters.

[0159] In summary, the sequential inoculation solid-state fermentation method provided by this invention can effectively solve the problems of difficult pH control and low yield of small peptides during rice residue protein fermentation. The rice residue protein fermentation product prepared by the optimal process parameters determined through single-factor experiments and orthogonal optimization has an acid-soluble small peptide content ≥6.5 g / 100g, a total acid content ≥1.95 g / 100g, and an increase in protein content ≥13%, and has good storage stability, thus possessing extremely high industrial application value.

[0160] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A method for preparing high-peptide acidified rice residue protein by sequential inoculation solid-state fermentation, characterized in that, Includes the following steps: (1) Preparation of fermentation substrate: Mix rice residue protein with carbon source auxiliary materials to prepare fermentation substrate; (2) First-stage solid-state fermentation: Bacillus subtilis is first inoculated into the fermentation substrate obtained in step (1) for first-stage solid-state fermentation to degrade the macromolecular proteins therein; the Bacillus subtilis is Bacillus subtilis NCUTY 001 ( Bacillus subtilis NCUTY 001 was deposited at the China Center for Type Culture Collection on March 31, 2025, with accession number CCTCC NO: M 2025647, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. (3) Second-stage solid-state fermentation: After the first-stage solid-state fermentation in step (2) is completed, *Lactobacillus plantarum* is introduced into the system for the second-stage solid-state fermentation to produce organic acids and lower the pH of the system; the *Lactobacillus plantarum* is *NCUTUUAS4* ( Lactiplantibacillus plantarum NCUTUAS4 was deposited at the China Center for Type Culture Collection on April 30, 2024, with accession number CCTCC NO: M 2024858, and the deposit address is Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. (4) Post-processing: After the second stage of solid-state fermentation in step (3) is completed, the fermentation product of high small peptide acidified rice residue protein is obtained through post-processing.

2. The method according to claim 1, characterized in that, The carbon source is wheat bran, and the mass ratio of rice residue protein to wheat bran is (5-9):1; and / or the initial moisture content of the fermentation substrate is 50%-60%.

3. The method according to claim 1, characterized in that, The inoculum amounts of Bacillus subtilis and Lactobacillus plantarum are independently 8%-12%; and / or the temperatures of the first-stage solid-state fermentation and the second-stage solid-state fermentation are independently 28-32℃; and / or the time of the first-stage solid-state fermentation is 35-60h; and the time of the second-stage solid-state fermentation is 60-85h.

4. The method according to claim 1, characterized in that, The post-processing includes drying the fermentation product obtained after the second stage of solid-state fermentation at 40-60℃ until the moisture content is ≤12%.

5. The method according to claim 1, characterized in that, During the first and second stages of solid-state fermentation, the material is turned over every 12-24 hours.

6. A fermentation product of high-small-peptide acidified rice residue protein prepared by the method according to any one of claims 1 to 5.

7. The fermentation product of high-peptide acidified rice residue protein according to claim 6, characterized in that, The product has an acid-soluble small peptide content ≥6.5g / 100g and a total acid content ≥1.95g / 100g.

8. The use of a high-peptide acidified rice residue protein fermentation product prepared by any one of claims 1 to 5, or the high-peptide acidified rice residue protein fermentation product as described in claim 6, in the preparation of highly digestible protein feed or functional food ingredients.

9. The application according to claim 8, characterized in that, The highly digestible protein feed is used for eel farming.

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