Method for constructing complex microbial flora for fermenting sunflower meal and optimizing fermentation process

Abstract

The application discloses a method for constructing a compound microbial flora for fermenting sunflower meal and optimizing a fermentation process, and screens and obtains bacillus velezensis FFRC LBC 01, bacillus subtilis FFRC BS 01 and lactococcus rhamnosus FFRC LR 01 with lignin, cellulose and phytic acid degradation capabilities respectively, the three strains have corresponding enzyme activities, do not have obvious antagonism among each other and have good safety; through further optimization of strain inoculation ratios, inoculation sequences and process conditions such as fermentation moisture, temperature and time, the neutral detergent fiber content in the sunflower meal can be effectively reduced, and the crude protein and small peptide contents can be increased, so that the nutritional composition of the sunflower meal is improved, and a technical basis is provided for further utilization of the sunflower meal as a plant feed raw material.

Description

Technical Field

[0001] This invention relates to a method for screening lignin, cellulose and phytic acid degrading bacteria, constructing a complex microbial community and optimizing the fermentation process of sunflower meal, specifically a method for constructing a complex microbial community for fermenting sunflower meal and optimizing the fermentation process, belonging to the field of feed microbiology technology. Background Technology

[0002] With the large-scale and intensive development of aquaculture, compound feed has become an important input in aquaculture production. The source and cost of protein raw materials directly affect the production cost of aquatic feed. Fishmeal, with its high protein content, relatively balanced amino acid composition, and good palatability, has long been widely used in aquatic animal feed. However, fishmeal production is constrained by factors such as marine fishery resources, catch volume, and market supply and demand. As aquaculture scale continues to expand, the demand for fishmeal continues to increase, leading to a growing supply-demand imbalance and high fishmeal prices that are easily affected by market fluctuations. Therefore, developing widely available, relatively low-cost protein raw materials that can partially replace fishmeal is of great significance for reducing aquatic feed costs, alleviating fishmeal resource shortages, and promoting the sustainable development of aquaculture.

[0003] Plant protein raw materials are widely available and produced in large quantities. Compared with some animal protein raw materials, they have advantages such as lower cost and better resource sustainability, making them an important raw material source to replace fishmeal in aquatic feed. In my country, the processing of oil crops generates a large amount of oilseed cakes and meals as byproducts, mainly including soybean meal, rapeseed meal, cottonseed meal, and sunflower meal. Among these, sunflower meal is the main byproduct of sunflower seed oil extraction or solvent extraction, containing a certain amount of protein, and has the potential to be developed and utilized as a plant protein source for aquatic feed.

[0004] However, sunflower meal and other miscellaneous plant protein feeds typically contain high levels of fiber and other anti-nutritional factors, limiting their further application in aquatic feed. Neutral detergent fiber (NDF) is an important component of miscellaneous plant protein feeds, generally ranging from 8.8% to 50%. NDF mainly includes structural substances such as cellulose, hemicellulose, and lignin. Cellulose molecules can form relatively stable crystalline structures through hydrogen bonds, while lignin can form complex plant cell wall structures with cellulose and other substances, thereby reducing the degree to which plant-based feeds are acted upon and utilized by digestive enzymes.

[0005] For aquatic animals, excessively high fiber content is detrimental to feed digestibility and utilization. On the one hand, higher fiber levels may increase the viscosity of digestive tract contents, affecting the sufficient contact between nutrients such as protein and digestive enzymes, thus impacting nutrient digestion and absorption. On the other hand, high-fiber meal feed ingredients are usually coarser in texture, which may adversely affect palatability and feed intake. In particular, some aquatic animals have limited ability to digest and utilize plant fiber; therefore, high neutral detergent fiber content is one of the important factors limiting the high proportion of sunflower meal and other meal feed ingredients in aquatic feeds.

[0006] Besides fibrous substances, plant-based feed ingredients also contain anti-nutritional factors such as phytic acid. Phytic acid can bind with minerals and other nutrients, thus affecting the effective utilization of some nutrients. Therefore, in improving the utilization value of sunflower meal as a feed protein source, it is necessary not only to reduce the content of structural substances such as cellulose and lignin, but also to further reduce the impact of anti-nutritional factors such as phytic acid, while improving the availability of protein.

[0007] Currently, the main methods for processing unconventional plant-based feed ingredients include physical treatment, chemical treatment, enzyme treatment, and microbial fermentation. Physical treatment can alter the physical structure of raw materials through methods such as crushing, but its actual degradation capacity for anti-nutritional factors such as cellulose, lignin, and phytic acid is limited. While some chemical treatments can alter the structure of plant cell walls, there are issues with controlling treatment conditions and subsequent applications. Directly adding exogenous enzyme preparations can act on specific substrates, but its treatment effect is affected by factors such as the type of enzyme, enzyme activity, and reaction conditions.

[0008] Microbial fermentation is an important technical means to improve the nutritional value of unconventional plant-based feed ingredients. Compared with some physical and chemical treatment methods, microbial fermentation has the advantages of relatively mild reaction conditions and simpler operation. Under suitable fermentation conditions, microorganisms can produce a variety of enzyme systems during their growth and reproduction, such as cellulase, phytase, and enzymes related to lignin degradation, thereby degrading substances such as cellulose, lignin, and phytic acid in the raw materials. At the same time, the fermentation process can also promote the conversion of macromolecular nutrients and increase the content of easily utilized nutrients such as small peptides, thus improving the nutritional value of the raw materials.

[0009] However, different microorganisms have different growth characteristics and produce different enzyme systems, so a single strain can usually only exhibit good degradation ability for one or some substrates. For example, a strain with high lignin degradation ability may not also have strong cellulose or phytic acid degradation ability, and a strain with strong phytic acid degradation ability may not have high cellulose degradation ability. Therefore, using only a single microorganism for sunflower meal fermentation may not be able to achieve effective degradation of multiple anti-nutritional factors such as lignin, cellulose, and phytic acid simultaneously.

[0010] Constructing a complex microbial community using microorganisms with different functional characteristics provides a feasible approach to improve the overall fermentation effect of plant-based feed ingredients. By combining microorganisms capable of degrading lignin, cellulose, and phytic acid respectively, it is hoped that the synergistic effect of enzyme systems produced by different strains can further disrupt the plant cell wall structure and simultaneously reduce the content of various anti-nutritional factors. However, the fermentation effect of the complex microbial community is not only related to the degradation capacity of the individual strains, but also affected by factors such as the presence of antagonistic interactions between strains, the inoculation ratio of different strains, the inoculation order, fermentation temperature, fermentation time, moisture content, inoculum concentration, and carbon source. If there is significant antagonism between the strains, or if the inoculation ratio and fermentation conditions are unsuitable, the functions of different strains may not be fully utilized, thus affecting the fermentation effect of the complex microbial community on sunflower meal.

[0011] Therefore, how to screen strains with good lignin, cellulose and phytic acid degradation capabilities and good safety from different sources, and on this basis, select strains without obvious antagonistic effects to construct a complex microbial community, while determining reasonable strain ratios, inoculation order and fermentation conditions to improve the enzyme activity of the complex microbial community and its ability to degrade anti-nutritional factors such as neutral detergent fiber in sunflower meal, and improve the nutritional value of fermented sunflower meal, are technical problems that need to be solved in the existing technology. Summary of the Invention

[0012] To address the above problems, this invention provides a method for constructing a complex microbial community for fermenting sunflower meal and optimizing the fermentation process, specifically including:

[0013] A composite microbial community for fermenting sunflower meal is constructed, the composite microbial community comprising Bacillus belye FFRCLBC 01, Bacillus subtilis FFRC BS 01, and Lactobacillus rhamnosus FFRC LR 01, wherein the inoculation ratio of Bacillus belye FFRCLBC 01, Bacillus subtilis FFRC BS 01, and Lactobacillus rhamnosus FFRC LR 01 is 4:1:1.

[0014] Preferably, the *Bacillus velezensis* FFRC LBC 01 was isolated from the Nanquan Pond of the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, with the accession number CGMCC No. 36770, the depository institution being the China General Microbiological Culture Collection Center, the deposit date being 2025-11-26, and the classification name being *Bacillus velezensis*; the *Bacillus subtilis* FFRC BS 01 was isolated from corn stalks; and the *Lactobacillus rhamnosus* FFRC LR 01 was screened from the strain bank of the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences.

[0015] Preferably, the Bacillus belye FFRC LBC 01 has lignin degradation ability, the Bacillus subtilis FFRC BS 01 has cellulose degradation ability, and the Lactobacillus rhamnosus FFRC LR 01 has phytic acid degradation ability.

[0016] This invention also discloses a method for optimizing the fermentation process of sunflower meal using a compound microbial community. The compound microbial community includes Bacillus belyssus FFRC LBC 01, Bacillus subtilis FFRC BS 01, and Lactobacillus rhamnosus FFRC LR 01. Sunflower meal is pulverized and passed through a 60-mesh sieve, sterilized at 121℃ for 20 min, and then packaged in 25 g portions into 250 mL fermentation bags. The fermentation conditions of the compound microbial community are gradually optimized through single-factor experiments, Plackett-Burman experiments, and response surface methodology experiments to obtain experimentally based process parameters for fermenting sunflower meal using the compound microbial community. The single-factor experiments are optimization experiments of fermentation temperature, fermentation time, fermentation material-to-water ratio, fermentation microbial concentration, and molasses addition ratio. Each experiment changes only one condition while keeping other conditions constant. The optimal factor level for fermentation is determined by using the content of neutral detergent fiber, phytic acid, and acid-soluble protein as comprehensive indicators. An inducer of 1‰ is added to ferment the sunflower meal.

[0017] The results of fermentation temperature optimization show that the suitable fermentation temperature range is 31℃-37℃;

[0018] The results of fermentation time optimization show that the suitable fermentation time range is 3-5 days;

[0019] The results of optimizing the fermentation material-to-water ratio show that the suitable range for fermentation moisture content is 40%-60%.

[0020] The results of the optimization of fermentation bacteria concentration showed that the suitable range of fermentation bacteria concentration was 1×10⁻⁶. 6 CFU / g-1×10 8 CFU / g;

[0021] The results of optimizing the molasses addition ratio show that the suitable range for adding molasses is 3%-6%.

[0022] The Plackett-Burman experiment was used to further screen the main factors affecting the fermentation effect. The results showed that fermentation time, fermentation moisture content and fermentation temperature were the most significant factors. The response surface methodology was used to optimize the above factors. After model prediction and actual verification, the optimal fermentation process parameters were determined to be 58.87% moisture content, 35.6℃ fermentation temperature and 4 days fermentation time.

[0023] Preferably, the optimization of fermentation temperature specifically includes an inoculum concentration of 1×10⁻⁶. 7 The fermentation conditions were: CFU / g, moisture content 40%, fermentation time 3 days, molasses addition ratio 3%, inoculation ratio of Bacillus vesiculosus FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 4:1:1, fermentation temperature settings: 28℃, 31℃, 34℃, 37℃, 40℃.

[0024] Preferably, the optimization of fermentation time specifically includes an inoculum concentration of 1×10⁻⁶. 7 The formula was: CFU / g, moisture content 40%, temperature 37℃, molasses addition ratio 3%, inoculation ratio of Bacillus vesiculosus FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 4:1:1, fermentation time set: 2d, 3d, 4d, 5d, 6d.

[0025] Preferably, the optimization of the fermentation material-to-water ratio specifically includes an inoculum concentration of 1×10⁻⁶. 7 CFU / g, fermentation time set at 3 days, temperature at 37℃, molasses addition ratio at 3%, inoculation ratio of Bacillus belyss FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 at 4:1:1, moisture content set at 30%, 40%, 50%, 60% and 70%.

[0026] Preferably, the optimization of the fermentation bacteria concentration specifically includes setting the fermentation time to 3 days, the temperature to 37°C, the molasses addition ratio to 3%, the inoculation ratio of Bacillus berberis FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR01 to 4:1:1, the moisture content to 40%, and the fermentation bacteria concentration settings to 1.0E+05, 1.0E+06, 1.0E+07, 1.0E+08, and 1.0E+09.

[0027] Preferably, the optimization of the molasses addition ratio specifically includes an inoculum concentration of 1×10⁻⁶. 7The fermentation time was set to 3 days, the moisture content was 40%, the temperature was set to 37℃, and the inoculation ratio of Bacillus belyssus FFRC LBC 01, Bacillus subtilis FFRC BS01 and Lactobacillus rhamnosus FFRC LR 01 was 4:1:1, with molasses ratios of 0%, 1%, 3%, 4.5% and 6%.

[0028] Preferably, the 1‰ inducer is lactose.

[0029] The beneficial effects of this invention are:

[0030] Compared with existing technologies, this invention constructs a complex microbial community composed of *Bacillus belye* FFRC LBC 01, *Bacillus subtilis* FFRC BS 01, and *Lactobacillus rhamnosus* FFRC LR01 by screening strains with lignin, cellulose, and phytic acid degradation capabilities. Furthermore, it optimizes the inoculation ratio, inoculation sequence, and fermentation process conditions for fermenting sunflower meal using this complex microbial community, thereby achieving fermentation treatment of sunflower meal. This invention has at least the following beneficial effects:

[0031] (1) The three strains obtained by screening have the ability to degrade lignin, cellulose and phytic acid respectively, and can form a complex microbial community with different degradation functions.

[0032] This invention screens and evaluates the degradation capabilities of bacterial strains using sodium carboxymethyl cellulose, aniline blue, and calcium phytate media, obtaining *Bacillus belye*, *Bacillus subtilis*, and *Lactobacillus rhamnosus*. *Bacillus belye* was able to form a hydrolysis zone on aniline blue medium, *Bacillus subtilis* on sodium carboxymethyl cellulose medium, and *Lactobacillus rhamnosus* on calcium phytate medium, indicating that the three strains possess corresponding lignin, cellulose, and phytate degradation capabilities, respectively.

[0033] Further analysis of the transparent zone to colony diameter ratio (D / d) of the strains revealed that *Bacillus belyssioides* had a D / d value of 3.31, *Bacillus subtilis* had a D / d value of 3.00, and *Lactobacillus rhamnosus* had a D / d value of 2.55. Therefore, by combining three strains with different substrate degradation capabilities, a complex microbial community can be formed that simultaneously targets different anti-nutritional components in sunflower meal.

[0034] (2) The three strains have corresponding enzyme activities, which provide a basis for the degradation of related substances during the fermentation of the compound bacteria.

[0035] Enzyme activity assays showed that *Bacillus belye* possessed peroxidase, manganese peroxidase, and laccase activities, with activities of 4 U / mL, 0.12 U / mL, and 0.42 U / mL, respectively; *Bacillus subtilis* had a cellulase activity of 30.23 U / mL; and *Lactobacillus rhamnosus* had a phytase activity of 4 U / mL.

[0036] Therefore, the three strains used in this invention have different enzyme activity characteristics and can play roles related to the degradation of lignin, cellulose and phytic acid, respectively, providing a basis for improving the nutritional quality of sunflower meal by using a compound microbial fermentation method.

[0037] (3) There is no obvious antagonistic effect among the three strains, and they can jointly construct a complex fermentation microbial community.

[0038] The results of the interstrain antagonism test showed that no antagonism occurred between Bacillus belyssus and Bacillus subtilis, between Bacillus subtilis and Lactobacillus rhamnosus, or between Lactobacillus rhamnosus and Bacillus belyssus, and the three strains were able to coexist.

[0039] Therefore, this invention does not simply select strains with different degradation functions, but constructs a composite microbial community based on verifying the compatibility between strains, avoiding the influence of obvious antagonism between strains on the role of each strain in the fermentation system, and providing experimental basis for the use of three strains together in sunflower meal fermentation.

[0040] (4) The three strains selected have good safety.

[0041] The hemolytic test results showed that the positive control Aeromonas hydrophila exhibited β-hemolysis, while the Bacillus belyceae, Bacillus subtilis and Lactobacillus rhamnosus used in this invention did not exhibit hemolytic activity.

[0042] The safety of the strain was further evaluated through in vivo injection experiments in giant freshwater prawns at a bacterial concentration of 1×10⁻⁶. 8 Under the condition of CFU / mL, the mortality rate of each experimental group of Bacillus belyceae, Bacillus subtilis, and Lactobacillus rhamnosus was 0%, and no inflammatory symptoms were observed. In the bacterial immersion safety test, no deaths or inflammatory symptoms were observed in any of the experimental groups, and the mortality rate was 0%. The above results indicate that the three strains used in this invention have good safety, providing a foundation for their further application in sunflower meal fermentation and aquatic feed.

[0043] (5) By optimizing the inoculation ratio and inoculation order of the three strains, it is beneficial to give full play to the role of the compound microbial community in fermenting sunflower meal.

[0044] This invention uses the content of neutral detergent fiber, acid-soluble protein, and phytic acid after fermentation as comprehensive evaluation indicators, and optimizes the inoculation ratio and inoculation order of the three strains through orthogonal experiments.

[0045] The results of the orthogonal experiment on the inoculation ratio showed that the order of importance of the microbial factors affecting the fermentation of sunflower meal was Lactobacillus rhamnosus > Bacillus belysus > Bacillus subtilis, and the optimal inoculation ratio was Bacillus belysus : Bacillus subtilis : Lactobacillus rhamnosus = 4 : 1 : 1.

[0046] The results of the orthogonal experiment on inoculation sequence showed that the order of influence of microbial factors on sunflower meal fermentation was Bacillus belye > Bacillus subtilis > Lactobacillus rhamnosus. The optimal inoculation sequence was to inoculate Bacillus belye at 0 h of fermentation, and then inoculate Bacillus subtilis and Lactobacillus rhamnosus at 24 h.

[0047] Therefore, this invention optimizes the proportion of different strains and the inoculation time in the compound microbial community to obtain a strain combination suitable for the co-fermentation of sunflower meal by three strains.

[0048] (6) The main process factors affecting the fermentation effect of compound bacteria on sunflower meal were identified, and suitable fermentation conditions were obtained.

[0049] This invention optimized single-factor conditions such as fermentation moisture content, fermentation time, fermentation temperature, fermentation bacteria concentration, and molasses addition amount. The results showed that the suitable range for fermentation moisture content was 40%–60%, the suitable range for fermentation time was 3–5 days, the suitable range for fermentation temperature was 31–37℃, and the suitable range for fermentation bacteria concentration was 1×10⁻⁶. 6 ~1×10 8 The appropriate range for the amount of molasses added is 3% to 6% (CFU / g).

[0050] Based on this, the Plackett-Burman experiment was used to further screen the main factors affecting the fermentation effect. The results showed that fermentation time, fermentation moisture content, and fermentation temperature were the most significant influencing factors. Response surface methodology was then used to optimize these factors. After model prediction and actual verification, the optimal fermentation parameters were determined to be: 58.87% moisture content, 35.6℃ fermentation temperature, and 4 days fermentation time. The actual comprehensive score reached 44.12, which is close to the theoretical prediction value of the model, indicating that the established optimization model can well reflect the actual fermentation situation.

[0051] Therefore, this invention gradually optimized the fermentation conditions of the compound bacteria through single-factor experiments, Plackett-Burman experiments, and response surface methodology, and obtained experimentally based process parameters for the fermentation of sunflower meal by the compound bacteria.

[0052] (7) Adding lactose as an inducer can further promote the degradation of sunflower meal neutral detergent fiber.

[0053] This invention compared the effects of various inducers, including lactose, manganese sulfate, copper sulfate, sophorolipid, guaiacol, oat glucan, and glucono-delta-lactone, on the degradation of neutral detergent fiber in fermented sunflower meal. At an inducer addition rate of 1‰, the addition of lactose resulted in a neutral detergent fiber degradation rate of 35.99%, higher than that of the other tested inducers. Therefore, lactose was selected as the inducer for fermentation of sunflower meal using a compound microbial culture.

[0054] (8) The optimized compound bacteria fermentation process can significantly reduce the content of neutral detergent fiber in sunflower meal.

[0055] After fermenting sunflower meal under the optimized conditions of this invention, the neutral detergent fiber content decreased from 37.67%±0.48% before fermentation to 24.18%±0.38%, a reduction of 35.81% compared with before fermentation.

[0056] This demonstrates that the composite microbial community constructed in this invention, combined with the optimized fermentation process, can effectively reduce the neutral detergent fiber content in sunflower meal, thereby improving the high fiber content in sunflower meal, which is a limiting factor for its feed utilization.

[0057] (9) Fermentation by compound bacteria can increase the content of crude protein and small peptides in sunflower meal and improve the nutritional composition of sunflower meal.

[0058] Under the optimized fermentation conditions of this invention, the crude protein content of sunflower meal before fermentation was 37.71%±0.26%, which increased to 39.74%±0.26% after fermentation, an increase of 5.38% compared with before fermentation; the content of small peptides increased from 118.30±3.54 before fermentation to 150.90±4.01 after fermentation, an increase of 27.56% compared with before fermentation.

[0059] Meanwhile, the crude fat content decreased from 1.93% ± 0.19% before fermentation to 1.72% ± 0.17% after fermentation, a reduction of 10.77%. Therefore, the compound microbial fermentation treatment of the present invention can not only reduce the neutral detergent fiber in sunflower meal, but also increase the crude protein and small peptide content, thereby improving the nutritional composition of fermented sunflower meal. Attached Figure Description

[0060] Figure 1 The diagram shows the hydrolysis zones of the strains of this invention; (A) is the hydrolysis zone of Bacillus belyssus FFRC LBC 01 with aniline blue; (B) is the hydrolysis zone of Bacillus subtilis FFRC BS 01 with sodium carboxymethyl cellulose; and (C) is the hydrolysis zone of Lactobacillus rhamnosus FFRC LR 01 with calcium phytate.

[0061] Figure 2 The diagram shows the hemolytic activity test results of the strains of this invention; (A) is Aeromonas hydrophila; (B) is Bacillus belyssus FFRC LBC 01; (C) is Bacillus subtilis FFRC BS 01; and (D) is Lactobacillus rhamnosus FFRC LR 01.

[0062] Figure 3 The diagrams show the antagonistic results of the strains of this invention; (A) shows the antagonistic results of Bacillus belyss FFRC LBC 01 and Bacillus subtilis FFRC BS 01; (B) shows the antagonistic results of Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01; and (C) shows the antagonistic results of Lactobacillus rhamnosus FFRC LR 01 and Bacillus belyss FFRC LBC 01.

[0063] Figure 4 This diagram illustrates the effect of fermentation moisture on acid-soluble protein, phytic acid, and neutral detergent fiber in fermented sunflower meal.

[0064] Figure 5 This is a graph showing the effect of fermentation time on acid-soluble protein, phytic acid, and neutral detergent fiber in fermented sunflower meal.

[0065] Figure 6 This is a graph showing the effect of fermentation temperature on acid-soluble protein, phytic acid, and neutral detergent fiber in fermented sunflower meal.

[0066] Figure 7 This is a graph showing the effect of the concentration of fermentation bacteria in this invention on acid-soluble protein, phytic acid, and neutral detergent fiber in fermented sunflower meal.

[0067] Figure 8 The graph shows the effect of adding different carbon sources on acid-soluble protein, phytic acid and neutral detergent fiber in fermented sunflower meal.

[0068] Figure 9 This is a graph showing the effect of the amount of molasses added on the acid-soluble protein, phytic acid, and neutral detergent fiber in fermented sunflower meal.

[0069] Figure 10 The diagram shows the half-normal probability effect of the normalized effect of this invention (α=0.05).

[0070] Figure 11 The Pareto plot for the normalization effect of this invention (α=0.05).

[0071] Figure 12 The contour plot and response surface plot show the effect of the interaction between fermentation temperature and fermentation time on the content of acid-soluble protein, phytic acid and neutral detergent fiber in fermented sunflower meal.

[0072] Figure 13The contour plot and response surface plot show the effect of the interaction between fermentation temperature and fermentation moisture on the content of acid-soluble protein, phytic acid and neutral detergent fiber in fermented sunflower meal.

[0073] Figure 14 The contour plot and response surface plot show the effect of the interaction between fermentation moisture and fermentation time on the content of acid-soluble protein, phytic acid and neutral detergent fiber in fermented sunflower meal.

[0074] Figure 15 This is a graph showing the effect of the type of inducing agent of the present invention on the neutral detergent fiber of fermented sunflower meal. Detailed Implementation

[0075] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0076] A method for constructing a complex microbial community for fermenting sunflower meal and optimizing the fermentation process.

[0077] Example 1: Materials and Methods

[0078] 1.1 Isolation, Screening and Identification of Bacteria

[0079] 1.1.1 Isolation of bacteria

[0080] Samples were randomly collected from soil and decaying wood along the shore of Taihu Lake, pond sediment from the South District of the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences, composted sunflower meal, composted corn stalks, and termites purchased online. 5 g of each of the collected soil, decaying wood, composted sunflower meal, composted corn stalks, and pond sediment samples were added to 45 mL of sterile water, shaken for 5-10 min, allowed to stand for 10 min to form a suspension, centrifuged at 400 r / min for 2 min, and 2 mL of the supernatant was collected in an EP tube for later use. Ten termites were soaked in 75% alcohol for 2 min, then placed in a 2 mL EP tube, and 1 mL of sterile water was added for homogenization. 100 μL of each of the supernatants prepared from the composted sunflower meal, composted corn stalks, pond sediment, soil, and termites were serially diluted with 900 μL of sterile water (10 μL each). -2 10 -3 10 -4 10 -5 10 -6100 μL of the diluted solution was spread onto alkali lignin nutrient agar, sodium carboxymethyl cellulose nutrient agar, and calcium phytate nutrient agar plates, with three plates for each dilution gradient. The plates were inverted and placed in a constant temperature incubator at 37°C for 48 h. Colony growth was observed for preliminary screening. Single colonies with different morphologies were picked from the alkali lignin nutrient agar, sodium carboxymethyl cellulose nutrient agar, and calcium phytate nutrient agar plates using a disposable inoculation loop and inoculated into the corresponding liquid medium for activation for 24 h. Then, a disposable inoculation loop was used to dab liquid from the liquid medium onto the nutrient agar plates for streaking purification. The plates were then sealed and stored at 4°C.

[0081] 1.2 Screening of strains

[0082] The strains, sealed and stored at 4℃, and those preserved at the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences, were activated in liquid alkali lignin, liquid sodium carboxymethyl cellulose, and liquid calcium phytate medium. They were then cultured at 37℃ and 180 r / min in a constant-temperature shaker for 24 h. Sterilized filter paper discs (4.7 mm in diameter) were placed in petri dishes containing aniline blue, sodium carboxymethyl cellulose, and calcium phytate agar, and 10 μL of bacterial suspension was added to each disc. The cultures were then incubated at 37℃ for 48 h. If the strain exhibited a certain degradation effect on lignin, a clear zone would appear around the colonies in the aniline blue medium. The diameter of each colony (d, cm) and the diameter of the clear zone (D, cm) were measured, and the optimal degrading strain was selected based on the D / d ratio. Similarly, if the strain exhibited a certain degradation effect on phytate, a clear zone would appear around the colonies in the calcium phytate medium. The diameter of each colony (d, cm) and the diameter of the clear zone (D, cm) were measured, and the optimal degrading strain was selected based on the D / d ratio. Stain the sodium carboxymethyl cellulose culture dish with Congo red dye for 30 min, then discard the liquid. Next, pour NaCl solution into the culture dish and decolorize for 30 min, then observe the clear zone. If the bacteria have a certain degradation effect on cellulose, a clear zone will appear around the colony. Measure the diameter of each colony (d, cm) and the diameter of the clear zone (D, cm), and select the optimal degrading strain based on the D / d value.

[0083] 1.3 Strain Identification

[0084] Strains that effectively degrade lignin, cellulose, and phytic acid were picked from purification culture medium plates and liquid-activated for 24 h before being sent to Tianlin Biotechnology Co., Ltd. for sequencing. The sequences were then compared with those in the NCBI database to determine the taxonomic names of the most similar strains: *Bacillus belyssii* FFRC LBC 01, *Bacillus subtilis* FFRC BS 01, and *Lactobacillus rhamnosus* FFRC LR 01.

[0085] Table 1. Ratio of clear zone to colony diameter of bacterial strains

[0086]

[0087] In the above embodiments,

[0088] The alkali lignin culture medium comprises: 5 g / L alkali lignin, 1 g / L K2HPO4, 1 g / L KH2PO4, 2 g / L (NH4)2SO4, 0.2 g / L MgSO4, 0.1 g / L CaCl2, 0.05 g / L FeSO4, 0.02 g / L MnSO4, 0.005 g / L CuSO4, 0.005 g / L ZnSO4, temperature (37℃), and pH (7.0±0.2).

[0089] The aniline blue culture medium comprises: 1 g / L aniline blue, 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, at a temperature of 37°C and a pH of 7.0 ± 0.2.

[0090] The sodium carboxymethyl cellulose culture medium comprises: sodium carboxymethyl cellulose 5 g / L, (NH4)2SO4 2 g / L, K2HPO4 1 g / L, MgSO4 0.5 g / L, NaCl 0.5 g / L, FeSO4 0.1 g / L, temperature (37℃), pH (7.0±0.2).

[0091] The phytate calcium culture medium comprises: 10 g / L peptone, 15 g / L glucose, 10 g / L phytate calcium, 0.5 g / L MgSO4, 0.03 g / L MnSO4, 0.03 g / L FeSO4, temperature (37℃), and pH (5.7±0.2).

[0092] The LB medium consisted of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and pH (7.0 ± 0.1).

[0093] The MRS medium consisted of: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L K₂HPO₄, 0.2 g / L MgSO₄, 0.04 g / L MnSO₄, 2 g / L diammonium hydrogen citrate, 5 g / L sodium acetate, 1 g / L Tween 80, and pH (6.5 ± 0.2).

[0094] Example 2: Safety Assessment

[0095] 2.1 Hemolytic activity: The strains screened in Example 1 were cultured at 37 °C for 24 h, and single colonies were picked and inoculated on sterile Columbia blood solid medium (Qingdao Hope Biotechnology Co., Ltd., Qingdao, China) with a sterile inoculation loop to set up the experimental group; Aeromonas hydrophila was used as the control group and cultured in an incubator at 28 °C for 48 h. The presence of hemolysis around the strains was observed and photographed.

[0096] The results are as follows Figure 2 As shown, the results of the hemolysis test of the strains showed that, except for the positive control Aeromonas hydrophila which showed β-hemolysis, Bacillus belysus FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 did not show hemolytic activity.

[0097] 2.2 Safety Experiment of In Vitro Injection in Giant Freshwater Prawns: The safety was assessed according to the method described by Divisekera et al. Giant freshwater prawns were acclimatized in a control environment for 7 days, with 10 prawns per group and three replicates. The injection concentration in the experimental group was 1×10⁻⁶. 8 CFU / mL bacterial suspension was injected at a dose of 100 μL, while the control group received 100 μL of sterile saline. Disease incidence and mortality were monitored and recorded continuously for 14 days. At the end of the experiment, an autopsy was performed to check for signs of inflammation.

[0098] The experimental results are shown in Table 2. Compared with the control group, no death or inflammatory symptoms occurred in the experimental group.

[0099] Table 2 Safety tests of Bacillus belyssus, Bacillus subtilis and Lactobacillus rhamnosus

[0100]

[0101] 2.3 Safety test of Macrobrachium rosenbergii bacterial solution immersion: The bacterial solution activated and cultured for 24 h was diluted to 1×10⁻⁶. 8 After reaching CFU / mL, the shrimp were placed in a bucket and completely submerged. After soaking for 3 hours, they were transferred to clean water. Shrimp that received no treatment served as the control group. Each group consisted of 10 shrimp, with three replicates. Disease incidence and mortality were continuously monitored and recorded for 14 days. At the end of the experiment, dissections were performed to check for signs of inflammation.

[0102] The results are shown in Table 3. Compared with the control group, no death or inflammatory symptoms occurred in the experimental group.

[0103] Table 3 Safety tests of Bacillus belyssus, Bacillus subtilis, and Lactobacillus rhamnosus

[0104]

[0105] 2.4 Determination of activities of laccase, peroxidase, manganese peroxidase, cellulase, and phytase

[0106] The activities of laccase, peroxidase, manganese peroxidase, cellulase, and phytase were determined using the Beijing BoxBIO assay kit (catalog number: BC5845; specification: 100T / 48S).

[0107] The results are shown in Table 4. The peroxidase activity of Bacillus belyss FFRC LBC 01 was 4 U / mL, the manganese peroxidase activity was 0.12 U / mL, and the laccase activity was 0.42 U / mL. The cellulase activity of Bacillus subtilis FFRC BS 01 was 30.23 U / mL, and the phytase activity of Lactobacillus rhamnosus FFRC LR 01 was 4 U / mL.

[0108] Table 4. Cellulase activity of Bacillus belyssus, Bacillus subtilis, and Lactobacillus rhamnosus.

[0109]

[0110] Example 3: Strain Antagonism Test

[0111] The previously screened Bacillus subtilis FFRC BS 01, Lactobacillus rhamnosus FFRC LR01, and Bacillus bereaves FFRC LBC 01 were inoculated into MRS or LB liquid medium at a 1% inoculation rate and cultured at 37 °C and 180 r / min for 24 h for activation. Then, 1 mL of the seed culture of the cultured strains was inoculated into 100 mL of MRS liquid medium and cultured at 37 °C in a shaker for 24 h for activation, which was then used for antagonistic experiments.

[0112] To investigate whether there is antagonistic interaction between the bacterial strains in mixed fermentation, an antagonistic experiment was conducted. LB and MRS media were used for the antagonistic experiment. The bacterial strains were streaked in pairs on LB solid medium, and the growth at the crossover points was observed. The results are as follows: Figure 3 As shown.

[0113] Figure 3 shows the antagonistic experiment between (A) Bacillus belyss FFRC LBC 01 and Bacillus subtilis FFRC BS 01, which shows that Bacillus belyss FFRC LBC 01 and Bacillus subtilis FFRC BS 01 do not antagonize each other; (B) shows the antagonistic experiment between Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01, which shows that Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 do not antagonize each other; and (C) shows the antagonistic experiment between Lactobacillus rhamnosus FFRC LR 01 and Bacillus belyss FFRC LBC 01, which shows that Lactobacillus rhamnosus FFRC LR 01 and Bacillus belyss FFRC LBC 01 do not antagonize each other.

[0114] In conclusion, Bacillus subtilis FFRC BS 01, Lactobacillus rhamnosus FFRC LR 01, and Bacillus belesiensis FFRC LBC 01 do not exhibit antagonism and can coexist. Therefore, Bacillus subtilis FFRC BS 01, Lactobacillus rhamnosus FFRC LR 01, and Bacillus belesiensis FFRC LBC 01 can be used as strains for subsequent fermentation experiments on sunflower meal.

[0115] Example 4: Method for Optimizing Sunflower Meal Fermentation Process with Compound Microbial Communities

[0116] 4.1 Optimal Inoculation Sequence Experiment for Optimizing Sunflower Meal Fermentation Process

[0117] The test strain was subjected to L9(3) 3 An orthogonal experiment was conducted to determine the optimal inoculation order among the microbial strains, using the content of neutral detergent fiber, phytic acid, and acid-soluble protein as comprehensive indicators. Sunflower meal was pulverized through a 60-mesh sieve, packaged into fermentation bags, and three fermentation strains were used as factors. Inoculation times were set at 0 h, 12 h, and 24 h as inoculation amounts, and the inoculation concentration was set at 10... 7 The fermentation conditions were: CFU / g, moisture content 40%, temperature 37℃, molasses addition ratio 3%, and fermentation time 3 days. The fermentation factor levels are shown in Table 5, and the orthogonal experimental setup is shown in Table 6.

[0118] Table 5. Factors affecting the order of inoculation of bacterial strains.

[0119]

[0120] Table 6. Inoculum Time L9(3) 3 Orthogonal experimental table

[0121]

[0122] The composite values ​​of neutral detergent fiber, acid-soluble protein, and phytic acid content after fermentation were calculated according to the proportions, as shown in Table 7. Range analysis showed that the main and secondary factors affecting sunflower meal fermentation were A (Bacillus belye FFRCLBC 01) > B (Bacillus subtilis FFRC BS 01) > C (Lactobacillus rhamnosus FFRC LR 01). The optimal inoculation sequence for sunflower meal fermentation was A1B3C3 (i.e., inoculate with Bacillus belye FFRC LBC 01 at 0 hours, and inoculate with Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 at 24 hours).

[0123] Table 7. Orthogonal Range Analysis Table for Vaccination Order

[0124]

[0125] 4.2 Experiment on the inoculation ratio of microbial community for optimizing the fermentation process of sunflower meal

[0126] The test strain was subjected to L9(3) 3 An orthogonal experiment was conducted to determine the optimal inoculation ratio among the microbial strains, using the content of neutral detergent fiber, phytic acid, and acid-soluble protein as comprehensive indicators. Sunflower meal was pulverized through a 60-mesh sieve, packaged into fermentation bags, and three fermentation strains were used as factors. Inoculation ratios of 1, 2, and 4 were set as the inoculation amount ratios, and the inoculation concentration was set at 1×10⁻⁶. 7 The fermentation conditions were: CFU / g, moisture content 40%, temperature 37℃, molasses addition ratio 3%, and fermentation time 3 days. The fermentation factor levels are shown in Table 8, and the orthogonal experimental setup is shown in Table 9.

[0127] Table 8. Factor Level Table of Inoculation Ratio of Microbial Strains

[0128]

[0129] Table 9. Proportion of bacterial species L9(3) 3 Orthogonal experimental table

[0130]

[0131] The composite values ​​of neutral detergent fiber, acid-soluble protein, and phytic acid content after fermentation were calculated according to the ratios, as shown in Table 10. The range analysis showed that the main and secondary factors affecting the fermentation of sunflower meal were C (Lactobacillus rhamnosus FFRCLR 01) > A (Bacillus belyss FFRC LBC 01) > B (Bacillus subtilis FFRC BS 01). The optimal inoculation ratio for sunflower meal fermentation was C1A3B1 (i.e., Bacillus belyss: Bacillus subtilis: Lactobacillus rhamnosus = 4:1:1).

[0132] Table 10 Orthogonal Range Analysis Table of Vaccination Ratio

[0133]

[0134] 4.3 Single-factor optimization experiment to optimize sunflower meal fermentation process

[0135] The experimental design is shown in Table 11. Sunflower meal was pulverized and passed through a 60-mesh sieve, sterilized at 121℃ for 20 min, and then packaged into 25 g portions in 250 mL fermentation bags. Single-factor optimization experiments were conducted on fermentation temperature, fermentation time, fermentation moisture and carbon source addition ratio, fermentation bacteria concentration, and inoculum size as single factors. Only one condition was changed in each experiment, while other conditions remained constant. The optimal factor levels for fermentation were determined using the content of neutral detergent fiber, phytic acid, and acid-soluble protein as comprehensive indicators.

[0136] Table 11 Single-factor experimental design and levels of sunflower meal

[0137]

[0138] 4.3.1 Optimization of water ratio in fermentation feed

[0139] The inoculum concentration is 1×10 7 The fermentation time was set at 3 days (CFU / g), 37℃, and 3% molasses was added. Moisture content was set at 30%, 40%, 50%, 60%, and 70%. After fermentation, the content of neutral detergent fiber, phytic acid, and acid-soluble protein was measured to determine the appropriate moisture content.

[0140] The results are as follows Figure 4 As shown, within the fermentation moisture range of 30%-70%, the content of neutral detergent fibers first decreases and then increases, reaching its lowest value at 50% moisture. Phytic acid content exhibits a distinct single-peak change, gradually decreasing with increasing fermentation moisture. Acid-soluble protein content shows a fluctuating trend of first increasing and then decreasing, reaching its peak at 50% moisture. This indicates that a fermentation moisture content of around 50% is more conducive to the degradation of neutral detergent fibers, while 50% is most favorable for the synthesis and accumulation of acid-soluble proteins. Fermentation moisture above 40% is more conducive to phytic acid degradation. The suitable range is 40%-60%.

[0141] 4.3.2 Fermentation Time Optimization

[0142] The inoculum concentration is 1×10 7 The optimal fermentation conditions were: CFU / g, moisture content 40%, temperature 37℃, molasses addition 3%, inoculum ratio 4:1:1, and fermentation times of 2, 3, 4, 5, and 6 days. After fermentation, the levels of neutral detergent fiber, phytic acid, and acid-soluble proteins were measured to determine the appropriate fermentation time.

[0143] like Figure 5As shown, within the fermentation time range of 2-6 days, the content of neutral detergent fiber first decreased and then stabilized, reaching its lowest value at 3 days of moisture content; the phytic acid content showed a clear single-peak change, gradually decreasing with increasing fermentation time; the acid-soluble protein content showed a fluctuating trend of first increasing and then decreasing, reaching its peak at 3 days of moisture content. That is, a fermentation time of around 3 days is more conducive to the degradation of neutral detergent fiber, while a fermentation time of 3 days is most conducive to the synthesis and accumulation of acid-soluble proteins, and a fermentation time of more than 3 days is more conducive to phytic acid degradation. The suitable range is 3-5 days.

[0144] 4.3.3 Fermentation Temperature Optimization

[0145] The inoculum concentration is 1×10 7 The fermentation parameters were: CFU / g, moisture content 40%, fermentation time 3 days, molasses addition ratio 3%, inoculum ratio 4:1:1, and fermentation temperatures set at 28℃, 31℃, 34℃, 37℃, and 40℃. After fermentation, the contents of neutral detergent fiber, phytic acid, and acid-soluble protein were measured to determine the optimal fermentation temperature.

[0146] like Figure 6 As shown, within the fermentation temperature range of 28℃-40℃, the content of neutral detergent fibers first decreased and then increased, reaching its lowest value at 34℃; the phytic acid content also first decreased and then increased, reaching its lowest value at 37℃; and the acid-soluble protein content showed a fluctuating trend of first increasing and then decreasing, reaching its peak at 34℃. This indicates that a fermentation temperature of around 34℃ is more conducive to the degradation of neutral detergent fibers, while a fermentation time of 34℃ is most favorable for the synthesis and accumulation of acid-soluble proteins, and a fermentation temperature of 37℃ is more conducive to the degradation of phytic acid. The suitable range is 31℃-37℃.

[0147] 4.3.4 Optimization of Fermentation Bacterial Concentration

[0148] Fermentation was set for 3 days at 37℃, with a molasses addition ratio of 3%, an inoculum ratio of 4:1:1, and a moisture content of 40%. The fermentation bacteria concentrations were set as follows: 1.0E+05, 1.0E+06, 1.0E+07, 1.0E+08, and 1.0E+09. After fermentation, the contents of neutral detergent fiber, phytic acid, and acid-soluble protein were measured to determine the appropriate feed-to-water ratio.

[0149] like Figure 7 As shown, in 10 5 CFU / g-10 9 Within the CFU / g fermentation bacteria concentration range, the phytic acid content of neutral detergent fibers showed a distinct single-peak change, gradually decreasing with increasing fermentation bacteria concentration; the phytic acid content, however, exhibited a fluctuating trend of first decreasing and then increasing, peaking at 10 CFU / g. 8 The CFU / g bacterial concentration drops to its lowest value; the acid-soluble protein content shows a fluctuating trend of first increasing and then decreasing, reaching its lowest value at 10. 7The peak value is reached at a CFU / g bacterial concentration, i.e., 10. 9 A fermentation bacteria concentration of around CFU / g is more conducive to the degradation of neutral detergent fibers, while 10 7 The optimal fermentation concentration of CFU / g is most conducive to the synthesis and accumulation of acid-soluble proteins. 8 A fermentation bacteria concentration of CFU / g or higher is more conducive to phytic acid degradation. The suitable range is 10. 6 CFU / g-10 8 CFU / g.

[0150] 4.3.5 Effects of adding different carbon sources on sunflower meal acid-soluble protein, neutral detergent fiber, and phytic acid

[0151] Fermentation was set for 3 days at 37°C, with a carbon source addition ratio of 3%, an inoculum ratio of 4:1:1, and a moisture content of 40%. The carbon source options included molasses, fructose, lactose, dextrin, and glucose. After fermentation, the levels of neutral detergent fiber, phytic acid, and acid-soluble proteins were measured to determine the optimal material-to-water ratio.

[0152] like Figure 8 As shown, among fermentations with different carbon sources, lactose showed the best degradation effect on neutral detergent fibers; fructose showed the best degradation effect on phytic acid; and lactose was most conducive to the synthesis and accumulation of acid-soluble proteins. Considering all factors, molasses was the optimal carbon source.

[0153] Optimization of molasses addition ratio

[0154] The inoculum concentration is 1×10 7 The fermentation time was set at 3 days (CFU / g), with a moisture content of 40% and a temperature of 37℃. The inoculum ratio was 4:1:1, and the molasses ratios were 0%, 1%, 3%, 4.5%, and 6%. After fermentation, the contents of neutral detergent fiber, phytic acid, and acid-soluble protein were measured to determine the appropriate molasses addition ratio.

[0155] like Figure 9 As shown, within the molasses addition range of 0%-6%, the neutral detergent fiber content exhibited a distinct unimodal change, gradually decreasing with increasing molasses addition; the phytic acid content also showed a distinct unimodal change, gradually decreasing with increasing molasses addition; the acid-soluble protein content showed a trend of first increasing and then stabilizing, reaching its peak at a molasses addition of 4.5%. This indicates that a molasses addition of around 6% is more conducive to the degradation of neutral detergent fibers, while a fermentation time of 4.5% is most favorable for the synthesis and accumulation of acid-soluble proteins, and a molasses addition of around 6% is more conducive to phytic acid degradation. The suitable range is 3%-6%.

[0156] Example 5: Plackett-Burman Experiment Optimizing Sunflower Meal Fermentation Process

[0157] Based on the single-factor experiment, the high and low values ​​of each factor were selected, and a PB experimental design was carried out. The content of neutral detergent fiber, phytic acid and acid-soluble protein were used as evaluation indicators. Five factors affecting the quality of fermented sunflower meal were tested: X1: fermentation time, X2: molasses addition ratio, X3: fermentation temperature, X4: moisture, X5: fermentation bacteria concentration. The experiment was divided into 12 groups, and the experimental design is shown in Table 12.

[0158] Table 12 Plackett-Burman Experimental Design Table

[0159]

[0160] Based on the single-factor experimental design, an experimental design with N=12 trials was selected, with the comprehensive score content as the response value. The experimental design and results are shown in Table 13.

[0161] Analysis of variance was performed on the experimental data. The Lenth method was used to identify significant effects in the Plackett-Burman experiment, and a half-normal probability effect diagram of the standardized effects of the factors was obtained (see...). Figure 10 ) and the Pareto plot of the standardized effects of factors (see Figure 11 ).Depend on Figure 10 It can be seen that the standardized effect points of factors B, D, and A are far from the fitted line, therefore they are significant influencing factors (P<0.05). That is, the significant factors affecting the standardized score are fermentation time, fermentation moisture content, and fermentation temperature; the standardized effect points of other factors are relatively small. The Pareto plot of the standardized effects (see...) Figure 11 The magnitude and importance of the effects were further determined, and factors B, D, and A all exceeded the t-value to be significant.

[0162] Table 13 Plackett-Burman Experimental Design and Results

[0163]

[0164] As shown in Table 14, the p-value of the model is 0.0110 < 0.05, indicating significance. Therefore, the model is reliable, meaning it fits well across the entire regression region under study; the multiple correlation coefficient R0 2 =0.9856, indicating a good correlation; the adjusted coefficient of determination R0.9856 2 adj =0.9472, indicating that 94.72% of the variability of the experimental data can be explained by this regression model; generally, the lower the coefficient of variation (CV), the higher the reliability and accuracy of the experiment. The CV value is equal to 8.41%, indicating that the Plackett-Burman experiment has good reliability and accuracy; precision is the ratio of effective signal to noise (Adeq Precisior) greater than 4.0 is considered reasonable, and the precision of this experiment reaches 17.5334.

[0165] By performing multiple regression fitting on the data, the regression equation is obtained:

[0166] Y=33.73+3.53A+10.48B-0.03C+4.12D-1.28E-2.38AD-3.41BE+2.84CE (1)

[0167] From regression equation (1), it can be seen that the partial regression coefficient of factor B is 10.48, indicating that factor D has a positive effect on the comprehensive score, that is, the comprehensive score increases with the increase of factor D; factors C and E have negative effects, while factors A and D have positive effects. However, considering more than 3 factors in the response surface methodology will significantly increase the number of experiments (3 factors require 20 treatments, and 4 factors require 31 treatments). Therefore, fermentation time, fermentation temperature, and fermentation moisture were selected for further optimization.

[0168] Table 14. Factors, levels, and significance analysis of the Plackett-Burman experiment.

[0169]

[0170] Example 6: Optimization of Sunflower Meal Fermentation Conditions Using Response Surface Methodology

[0171] Based on the results of single-factor and PB experiments, the three key factors affecting the content of neutral detergent fiber, phytic acid, and acid-soluble protein were ranked according to their F-values. Using the content of neutral detergent fiber, phytic acid, and acid-soluble protein as response values, a three-factor, three-level response surface methodology was designed and a model was established using the Box-Behnken Design (BBD) central composite principle. The factors and levels are shown in Table 15.

[0172] Table 15 Response Surface Experiment Factors and Level Design

[0173]

[0174] The Plackett-Burman experiment showed that fermentation time, fermentation moisture content, and fermentation temperature had a significant impact on the overall score. Based on this, a three-factor, three-level response surface methodology optimization experiment was conducted to explore the effects of fermentation time, fermentation moisture content, and fermentation temperature on the overall score. The overall score was used as the evaluation index to optimize the process parameters for fermenting sunflower meal using compound bacteria. The experimental design and results are shown in Table 16. Design-Expert 13.0.5 software was used to perform multivariate quadratic response surface regression fitting on the experimental results to obtain the regression equation.

[0175] Y=45.41+3.64A+0.8362B+8.02C+1.11AB+1.04AC+7.33BC-8.75A2 -7.98B 2 -7.29C 2

[0176] Table 16 Box-Behnken Experimental Design and Results

[0177]

[0178] As shown in Table 17, the regression models for the comprehensive scores showed highly significant differences (P<0.001), with no significant lack-of-fit term (P>0.05), and the coefficient of determination R0.05 was... 2 The value is 0.9567, R 2 adj A value of 0.9011 indicates that the regression model fits the experimental results well and can predict the relationship between each factor and the response value effectively. Among them, C, BC, and A... 2 B 2 C 2 Factor A (P < 0.01) has a highly significant effect on increasing the relative content of acid-soluble protein and decreasing the content of phytic acid and neutral detergent fibers. Factor C (P < 0.001) has a significant effect on increasing the relative content of acid-soluble protein and decreasing the content of phytic acid and neutral detergent fibers.

[0179] Table 17 Analysis of Variance for the Quadratic Model of the Box-Behnken Experimental Design

[0180]

[0181]

[0182] Response surface optimization results are as follows Figures 12-14 As shown, the density of contour lines indicates the interaction between two factors. The optimal feed fermentation process predicted by the response surface methodology is a moisture content of 58.8718%, under which the overall score is 44.84. To facilitate verification and practical operation, the fermentation process parameters were adjusted to a moisture content of 58.87%, a fermentation temperature of 35.6℃, and a fermentation time of 4 days. Verification showed an overall score of 44.12, close to the theoretical value. Therefore, the model can reflect the actual situation well, demonstrating that optimizing the fermentation process of sunflower meal using the response surface methodology with compound bacteria can increase the relative content of acid-soluble protein and reduce the content of phytic acid and neutral detergent fiber, proving the feasibility of this method.

[0183] Example 7: Effects of different inducers on enzyme production during sunflower meal fermentation and their screening

[0184] In solid-state fermentation, some inducers can regulate the enzymatic degradation of sunflower meal, affecting the production of cellulase and ligninase. This study used seven cellulase and ligninase inducers, including lactose, manganese sulfate, copper sulfate, sophorolipid, guaiacol, oat glucan, and glucono-delta-lactone. Inducers were added simultaneously with inoculation for solid-state fermentation of sunflower meal to select the optimal inducer at a concentration of 1‰. After fermentation, the neutral detergent fiber content was used as the evaluation index to screen for the best inducer.

[0185] The results are as follows Figure 15 As shown, the degradation rate of neutral detergent fiber in sunflower meal fermented with 1‰ inducer was 35.99% for lactose, 32.10% for manganese sulfate, 22.53% for copper sulfate, 33.05% for sophorolipid, 31.75% for guaiacol, 27.89% for oat beta-glucan, and 34.77% for glucono-delta-lactone. The degradation rate of sunflower meal fermented without inducer was 34.77%. Therefore, lactose was chosen as the inducer for fermenting sunflower meal.

[0186] Example 8: Effects of optimal fermentation conditions on the content of nutrients and anti-nutritional factors in sunflower meal.

[0187] As shown in Table 18, when sunflower meal was fermented under optimal conditions, compared with before fermentation, the content of small peptides increased significantly by 27.56%; crude protein increased significantly by 5.38%; crude fat decreased by 10.77%; and neutral detergent fiber decreased by 35.81%.

[0188] Table 18 Effects of optimal fermentation conditions on the nutritional composition and anti-nutritional factors of fermented sunflower meal

[0189]

[0190] In summary, this invention screened and obtained *Bacillus belye* FFRC LBC 01, *Bacillus subtilis* FFRC BS 01, and *Lactobacillus rhamnosus* FFRC LR 01, which respectively possess the ability to degrade lignin, cellulose, and phytic acid. These three strains exhibit corresponding enzyme activities, show no significant antagonistic effects among themselves, and demonstrate good safety. By further optimizing the inoculation ratio, inoculation sequence, and fermentation conditions such as moisture content, temperature, and time, the neutral detergent fiber content in sunflower meal can be effectively reduced, while the crude protein and small peptide content can be increased, thereby improving the nutritional composition of sunflower meal and providing a technical basis for the further utilization of sunflower meal as a plant-based feed ingredient.

[0191] In the above embodiments, the detection indicators are as follows:

[0192] (1) Viable bacteria detection

[0193] Viable bacteria testing items: total bacterial count, Bacillus, Lactic acid bacteria, and mold. Total bacteria – counted by pour method on nutrient agar plates, incubated at 37 ℃ for 48 h; Bacillus – counted by pour method on nutrient agar plates, incubated at 37 ℃ for 48 h after a 10 min water bath at 80 ℃; Lactic acid bacteria – counted by pour method on MRS solid medium, anaerobically incubated at 37 ℃ for 48 h. Bacteria that produce a clear zone on the plate are considered acid-producing lactobacilli, otherwise they are non-acid-producing lactobacilli; Molds are counted according to national standard GB / T13092-2006, by pour method on high-salt Czapek's medium, incubated at 29 ℃ for 96 h.

[0194] (2) Detection of small peptide content

[0195] The content of small peptides in feed is determined by the trichloroacetic acid method: trichloroacetic acid is used as a protein precipitant to precipitate proteins and peptides with longer peptide chains in the sample, while small peptides are dissolved in acid to determine the content of small peptides.

[0196] (3) Determination of crude protein

[0197] The protein content was determined according to the national standard GB / T 6432-2018. 0.5 ± 0.001 g of sample was weighed and mixed with 0.4 g copper sulfate, 6 g potassium sulfate, and 12 mL concentrated sulfuric acid. The mixture was added to a digestion tube and placed in a graphite digester. The digestion was carried out at 200℃ for 30 min, 300℃ for 30 min, and 420℃ for 1 h. The mixture was then cooled and brought to a final volume of 100 mL. A certain amount of the digest was then placed in a water vapor evaporator, and an alkaline solution was added. The mixture was distilled with acidic water for 10 min, and the vapor was collected using boric acid absorbent. A few drops of methyl red-bromocresol green ethanol indicator were added, and the amount of standard acid used was recorded, and the crude protein content was calculated.

[0198] (4) Determination of acid-soluble proteins

[0199] The determination of acid-soluble proteins shall be performed in accordance with the national standard NY / T 3801-2020. Specifically, accurately weigh 1.0 g of sample (accurate to 0.001 g), dissolve in 15% trichloroacetic acid (TCA) solution and bring the volume to 50 mL, mix well, let stand for 5 min, and filter; then, take 10.00–25.00 mL of the filtrate and proceed with the protein determination according to the method described herein.

[0200] (5) Detection of soluble protein content

[0201] The Coomassie Brilliant Blue method was used to determine the soluble protein content in feed: within a certain protein concentration range, the binding of protein and dye follows Beer's Law. After the dye binds to the protein, the color changes from red to blue, and the maximum absorbance changes from 465 nm to 595 nm. The protein content is determined by measuring the absorbance at 595 nm.

[0202] (9) Phytic acid content detection

[0203] Phytic acid, also known as creatine, is mainly found in the seeds, roots, and stems of plants. A sulfosalicylic acid-ferric chloride solution is purple-red and has a maximum absorbance at 500 nm. Phytic acid and iron ions combine in an environment of pH 6.0-6.5, causing the solution to lighten in color; the phytic acid content is detected by measuring the decrease in absorbance. The phytic acid content in cottonseed meal before and after fermentation was determined using the Solarbio phytic acid content detection kit.

[0204] (5) Crude fiber detection

[0205] The determination of crude fiber is performed according to the national standard GB / T 6434-2022. Specifically, accurately weigh 0.5 g of sample (accurate to 0.001 g) into a filter bag, treat the sample with boiling dilute sulfuric acid, filter to separate the residue, wash; treat with boiling potassium hydroxide solution, filter to separate the residue, wash, degrease, dry, weigh, and then ashing. Follow the same procedure as described in this crude fiber determination method.

[0206] (6) Neutral detergent fiber testing

[0207] The determination of neutral detergent fiber content is performed in accordance with the national standard GB / T 20806-2022. Specifically, accurately weigh 0.5 g of sample (accurate to 0.001 g) into a filter bag, digest the sample with neutral detergent, and wash it. Follow the same method as described in this article for the determination of neutral detergent fiber content.

[0208] (7) Detection of acid detergent fibers

[0209] The determination of crude fiber shall be performed in accordance with the national standard NY / T 1459-2022. Specifically, accurately weigh 0.5 g of sample (accurate to 0.001 g) into a filter bag, wash and treat the sample with acid, and then treat with water and acetone solution. Follow the same method as described in this acid-washed fiber determination procedure.

[0210] (6) Detection of lignin by acid washing

[0211] The determination of crude fiber follows the national standard GB / T 20805-2006. Specifically, accurately weigh 1 g of sample (accurate to 0.001 g) into a beaker, and treat the sample with acidic detergent and concentrated sulfuric acid. Follow the method for determining acid-washed lignin.

[0212] In the above embodiments, all data were analyzed for significance using one-way ANOVA and t-tests in SPSS Statistics 20.0. The test results are expressed as mean ± standard error (Mean ± SE). P < 0.05 was considered significant, and P < 0.01 was considered highly significant.

[0213] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0214] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for constructing a complex microbial community for fermenting sunflower meal, characterized in that, The complex microbial community includes Bacillus belyssus FFRC LBC 01, Bacillus subtilis FFRC BS 01, and Lactobacillus rhamnosus FFRC LR 01, with an inoculation ratio of 4:1:

1.

2. The construction of a compound microbial community for fermenting sunflower meal according to claim 1, characterized in that, The *Bacillus belye* FFRC LBC 01 was isolated from the Nanquan Pond of the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, and its preservation number is CGMCC No. 36770; the *Bacillus subtilis* FFRC BS 01 was isolated from corn stalks; and the *Lactobacillus rhamnosus* FFRC LR 01 was obtained from the strain bank of the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences.

3. The construction of a complex microbial community for fermenting sunflower meal according to claim 1 or claim 2, characterized in that, The Bacillus beryl FFRC LBC 01 has lignin degradation ability, the Bacillus subtilis FFRC BS01 has cellulose degradation ability, and the Lactobacillus rhamnosus FFRC LR 01 has phytic acid degradation ability.

4. A method for optimizing sunflower meal fermentation process using a composite microbial community, characterized in that, The complex microbial community includes Bacillus belyssus FFRC LBC 01, Bacillus subtilis FFRC BS 01, and Lactobacillus rhamnosus FFRC LR 01. Sunflower meal was pulverized and passed through a 60-mesh sieve, sterilized at 121℃ for 20 min, and packaged in 25 g portions into 250 mL fermentation bags. The fermentation conditions of the complex microbial community were gradually optimized through single-factor experiments, Plackett-Burman experiments, and response surface methodology experiments to obtain experimentally based process parameters for fermenting sunflower meal with the complex microbial community. The single-factor experiments were optimization experiments of fermentation temperature, fermentation time, fermentation material-to-water ratio, fermentation microbial concentration, and molasses addition ratio. Only one condition was changed in each experiment, while other conditions remained unchanged. The optimal factor level for fermentation was determined by the comprehensive index of neutral detergent fiber, phytic acid, and acid-soluble protein content. An inducer of 1‰ was added to ferment the sunflower meal. The results of fermentation temperature optimization show that the suitable fermentation temperature range is 31℃-37℃; The results of fermentation time optimization show that the suitable fermentation time range is 3-5 days; The results of optimizing the fermentation material-to-water ratio show that the suitable range for fermentation moisture content is 40%-60%. The results of the optimization of fermentation bacteria concentration showed that the suitable range of fermentation bacteria concentration was 1×10⁻⁶. 6 CFU / g-1×10 8 CFU / g; The results of optimizing the molasses addition ratio show that the suitable range for adding molasses is 3%-6%. The Plackett-Burman experiment was used to further screen the main factors affecting the fermentation effect. The results showed that fermentation time, fermentation moisture content and fermentation temperature were the most significant factors. The response surface methodology was used to optimize the above factors. After model prediction and actual verification, the optimal fermentation process parameters were determined to be 58.87% moisture content, 35.6℃ fermentation temperature and 4 days fermentation time.

5. The method for optimizing sunflower meal fermentation process using a composite microbial community according to claim 4, characterized in that, The optimization of fermentation temperature specifically includes setting the inoculum concentration to 1×10⁻⁶. 7 The fermentation conditions were: CFU / g, moisture content 40%, fermentation time 3 days, molasses addition ratio 3%, inoculation ratio of Bacillus vesiculosus FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR01 4:1:1, fermentation temperature settings: 28℃, 31℃, 34℃, 37℃, 40℃.

6. The method for optimizing sunflower meal fermentation process using a composite microbial community according to claim 4, characterized in that, The fermentation time optimization specifically includes setting the inoculum concentration to 1×10⁻⁶. 7 The formula was: CFU / g, moisture content 40%, temperature 37℃, molasses addition ratio 3%, inoculation ratio of Bacillus vesiculosus FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 4:1:1, fermentation time set: 2d, 3d, 4d, 5d, 6d.

7. The method for optimizing sunflower meal fermentation process using a composite microbial community according to claim 4, characterized in that, The optimization of the fermentation feed water ratio specifically includes setting the inoculum concentration to 1×10⁻⁶. 7 CFU / g, fermentation time set at 3 days, temperature at 37℃, molasses addition ratio at 3%, inoculation ratio of Bacillus belyss FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 at 4:1:1, moisture content set at 30%, 40%, 50%, 60% and 70%.

8. The method for optimizing sunflower meal fermentation process using a composite microbial community according to claim 4, characterized in that, The optimization of the fermentation bacteria concentration specifically includes setting the fermentation time to 3 days, the temperature to 37℃, the molasses addition ratio to 3%, the inoculation ratio of Bacillus belyssus FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 to 4:1:1, the moisture content to 40%, and the fermentation bacteria concentration settings to 1.0E+05, 1.0E+06, 1.0E+07, 1.0E+08, and 1.0E+09.

9. The method for optimizing sunflower meal fermentation process using a composite microbial community according to claim 4, characterized in that, The optimization of the molasses addition ratio specifically includes an inoculum concentration of 1×10⁻⁶. 7 The fermentation time was set to 3 days, with a moisture content of 40% and a temperature of 37℃. The inoculation ratio of Bacillus belyssus FFRC LBC 01, Bacillus subtilis FFRC BS 01 and Lactobacillus rhamnosus FFRC LR 01 was 4:1:1, and the molasses ratio was 0%, 1%, 3%, 4.5% and 6%, respectively.

10. The method for optimizing sunflower meal fermentation process using a composite microbial community according to claim 4, characterized in that, The 1‰ inducer is lactose.

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