Compound bacteria for degrading free gossypol in cottonseed molasses and fermentation process thereof

Abstract

The invention belongs to the technical field of high-value utilization of agricultural product processing byproducts, and discloses compound bacteria for degrading free gossypol in cottonseed molasses and a fermentation process of the compound bacteria. The compound bacteria are prepared from aspergillus niger, saccharomyces cerevisiae and lactobacillus gadsenii according to a bacterial liquid volume ratio of 1: 2: 1; the fermentation process parameters are as follows: the inoculum size is 5%, the fermentation temperature is 27.8 DEG C, and the fermentation time is 8.4 days. According to the scheme, the degradation rate of free gossypol in the cottonseed molasses reaches 72% and is increased by 6% compared with the degradation rate of single bacteria; meanwhile, metabolic pathways of ABC transport protein, vitamin absorption and the like can be regulated and controlled, the metabolite composition of the product is changed, the nutritional value and safety of the product are improved, and the method can be used for preparing feed additives or food raw materials.

Description

Technical Field

[0001] This invention belongs to the field of high-value utilization technology of agricultural product processing by-products, and relates to a compound bacteria for degrading free gossypol in cottonseed molasses and its fermentation process. Background Technology

[0002] Cottonseed molasses is a byproduct of cottonseed processing, including oil extraction and dephenolization. It is rich in various nutrients such as crude protein, sucrose, raffinose, and stachyose, and its composition and active ingredient content are superior to similar products like soybean molasses and beet molasses, making it a promising food or feed ingredient. However, cottonseed molasses contains high concentrations of free gossypol. Excessive intake of this substance can cause toxicological effects such as gastroenteritis, liver damage, and reproductive dysfunction. This problem severely limits the direct development and utilization of cottonseed molasses. Efficiently removing free gossypol is a crucial prerequisite for its safe resource utilization.

[0003] Currently, among methods for removing free gossypol, microbial degradation has become a research hotspot due to its mild conditions, high safety, and the fact that the metabolites produced by microorganisms during fermentation can improve the nutritional quality of the material. However, existing research mainly focuses on solid materials such as cottonseed meal and cottonseed hulls, and there are no systematic reports on microbial degradation technology for cottonseed molasses, a liquid byproduct. Furthermore, existing microbial degradation studies mostly employ single-strain fermentation or simple mixed-strain combinations, which suffer from limited degradation efficiency and unclear process parameters, making it difficult to meet the requirements of stable and efficient degradation effects for industrial applications. In addition, existing technologies mostly focus only on the removal of free gossypol, without delving into the regulation of metabolic pathways during fermentation and the simultaneous improvement of the product's nutritional value, thus limiting the high-value utilization of cottonseed molasses.

[0004] With the development of the agricultural product processing industry, the output of cottonseed molasses has continued to increase. Improper disposal of this molasses not only wastes resources but may also cause environmental problems. Therefore, developing efficient free gossypol degradation technology for cottonseed molasses, optimizing fermentation process parameters, and simultaneously improving the nutritional value and safety of the product by regulating metabolic processes can not only fill existing technological gaps but also realize the resource-based and high-value utilization of cottonseed molasses, meeting the needs of green agricultural development and possessing significant economic and environmental value. Summary of the Invention

[0005] In existing technologies, research on the microbial degradation of free gossypol mainly focuses on solid materials such as cottonseed meal and cottonseed hulls. No systematic reports have been found on technologies related to cottonseed molasses, a liquid byproduct of cottonseed processing. This makes it difficult to safely utilize cottonseed molasses due to its high free gossypol content. Furthermore, existing microbial degradation schemes often employ single-strain fermentation or simple mixed-strain combinations, resulting in limited degradation efficiency (below 66%) and poor stability of the degradation effect. Moreover, a clear and industrially applicable fermentation process parameter system has not been established. In addition, existing technologies only focus on the removal of free gossypol, neglecting the regulation of metabolic pathways and the simultaneous improvement of product nutritional value during fermentation, thus failing to meet the demand for high-value utilization of cottonseed molasses. To address these technological gaps and shortcomings, this invention provides a technical solution that can efficiently degrade free gossypol in cottonseed molasses, with a clearly defined process and the ability to simultaneously improve product quality.

[0006] First, the present invention provides a compound bacteria for degrading free gossypol in cottonseed molasses. The compound bacteria are composed of Aspergillus niger, Saccharomyces cerevisiae and Lactobacillus gasseri, and the volume ratio of the bacterial culture of Aspergillus niger, Saccharomyces cerevisiae and Lactobacillus gasseri is 1:1~3:1~2.

[0007] Furthermore, for the aforementioned compound microorganism, the volume ratio of the Aspergillus niger, Saccharomyces cerevisiae, and Lactobacillus gasseri in the bacterial culture is 1:2:1; wherein the concentration of the Aspergillus niger and Saccharomyces cerevisiae in the bacterial culture is 1×10⁻⁶. 8 The bacterial concentration of Lactobacillus gasseri was 10⁶ cells / mL. 8 CFU / mL.

[0008] Secondly, the present invention provides a fermentation process for degrading free gossypol in cottonseed molasses, comprising the following steps: The above-mentioned compound bacteria were inoculated into the fermentation medium and fermented under the conditions of inoculation amount of 3-7%, fermentation temperature of 25-30℃, and fermentation time of 7-9 days. The fermentation medium uses cottonseed molasses as a base material and also contains nitrogen sources, sodium salts, magnesium salts, and phosphate salts.

[0009] Furthermore, regarding the above fermentation process, in the fermentation medium, the nitrogen source is ammonium sulfate, the sodium salt is sodium chloride, the magnesium salt is magnesium sulfate heptahydrate, and the phosphate salt is dipotassium hydrogen phosphate. The addition amounts of each component are: 1.5~2.5g of ammonium sulfate, 0.8~1.2g of sodium chloride, 0.4~0.6g of magnesium sulfate heptahydrate, and 0.4~0.6g of dipotassium hydrogen phosphate per 1000mL of medium.

[0010] Furthermore, for the above fermentation process, the inoculum amount is 4-6%.

[0011] Furthermore, for the above fermentation process, the fermentation temperature is 27~28℃, and the fermentation time is 8~8.5 days.

[0012] Thirdly, the present invention seeks protection for the use of the aforementioned compound bacteria in degrading free gossypol in cottonseed molasses.

[0013] Fourthly, the present invention seeks protection for the use of the above-mentioned compound bacteria in the preparation of feed additives.

[0014] Furthermore, the feed additive contains fermentation metabolites of the aforementioned compound bacteria.

[0015] This invention uses three harmless and safe bacterial strains: Aspergillus niger, Saccharomyces cerevisiae, and Lactobacillus gasseri. These strains can be purchased through other public channels such as the China Medical Culture Collection Center (CMCC), the China Industrial Culture Collection Center (CICC), the China General Microbiological Culture Collection Center (CGMCC), and the German Microbiological Culture Collection Center (DSMZ). Publicly available culture media and cultivation methods can be used. This invention prioritizes the cultivation of the three strains using a specially designed culture medium. After mixing the strains in the specified volume ratio, the invention exhibits significant characteristics such as no antagonism between the strains, stable effects, strong enzyme activity, high viable cell count, and nitrogen retention.

[0016] Compared with existing technologies, the composite bacteria and fermentation process for degrading free gossypol in cottonseed molasses provided by this invention have the following beneficial effects: (1) This invention is the first to develop microbial degradation technology for cottonseed molasses, a liquid byproduct, which solves the limitation of existing technology that only covers solid cottonseed processing materials and provides technical support for the resource utilization of cottonseed molasses.

[0017] (2) The compound bacteria of the present invention, through the functional synergy of Aspergillus niger, Saccharomyces cerevisiae and Lactobacillus gasseri, increase the degradation rate of free gossypol in cottonseed molasses to 72%, which is 6% higher than the degradation rate of single bacteria (maximum 66%). At the same time, the fermentation process parameters are optimized and determined by single-factor experiments and response surface methodology to ensure that the degradation effect is stable and controllable and meets the consistency requirements of industrial production.

[0018] (3) During the fermentation process of the compound bacteria, not only can free gossypol be efficiently removed (down to below the safety threshold), but also key metabolic pathways such as ABC transporter protein, vitamin digestion and absorption, arginine and proline metabolism, and phenylpropanoid biosynthesis can be regulated. This results in a significant upregulation of beneficial metabolites (such as lipids and lipid molecules, organic heterocyclic compounds) and a downregulation of harmful metabolites in the product, greatly improving the nutritional value and safety of cottonseed molasses and laying the foundation for its use as a feed additive or food ingredient.

[0019] (4) The composite bacteria screening, fermentation process and application scheme provided by the present invention have high repeatability. The required equipment is conventional microbial fermentation equipment, without the need for special expensive equipment. Moreover, the composite bacteria can be applied to different batches of cottonseed molasses raw materials. The fermented products can be directly used for feed production or further processing, providing an efficient and sustainable technical path for the resource utilization of cottonseed processing by-products and the green development of agriculture in saline-alkali areas. Attached Figure Description

[0020] Figure 1 The graph shows the degradation rate of free gossypol in cottonseed molasses by different single microbial strains. "KN" refers to *Trichoderma cornigrantrum* XQ0815; "LS" refers to *Trichoderma reesei* CICC2626; "HQ" refers to *Aspergillus niger* CMCC(F)98003; "HB" refers to *Procambarus chrysospora* Bio-116032; "LJ" refers to *Saccharomyces cerevisiae* (LJ); and "GR" refers to *Lactobacillus gasseri* (GR).

[0021] Figure 2 The diagram shows the antagonistic test results for HQ, LJ, and GR, with cross-stretched lines in each pair. "HQ+LJ" refers to the binary combination of *Aspergillus niger* (HQ) and *Saccharomyces cerevisiae* (LJ); "HQ+GR" refers to the binary combination of *Aspergillus niger* (HQ) and *Lactobacillus gasseri* (GR); and "LJ+GR" refers to the binary combination of *Saccharomyces cerevisiae* (LJ) and *Lactobacillus gasseri* (GR).

[0022] Figure 3 The graph shows the degradation rate of free gossypol in cottonseed molasses by different mixed bacterial combinations. "HQ+LJ", "HQ+GR", and "LJ+GR" have the same meaning as above; "HQ+LJ+GR" refers to the ternary combination of Aspergillus niger (HQ), Saccharomyces cerevisiae (LJ), and Lactobacillus gasseri (GR); the letters "a" and "b" indicate significant differences between groups (P<0.05), with the same letter representing no significant difference between groups and different letters representing significant differences between groups.

[0023] Figure 4 The figure shows the effect of inoculation ratio on the degradation of cottonseed molasses by the compound bacteria (single-factor experiment). It shows the effect of HQ-LJ-GR compound bacteria with seven different bacterial liquid volume ratios (1:1:1, 1:1:2, 1:2:1, etc.) on the degradation rate of free gossypol, total acid content and reducing sugar content; the letters "a", "b", and "c" in the figure are significance markers.

[0024] Figure 5 The figure shows the effect of inoculum size on the degradation of cottonseed molasses by the compound bacteria (single-factor experiment). It illustrates the effects of five different inoculum sizes (1%, 5%, 10%, 15%, and 20%) on the degradation efficiency of free gossypol, total acid content, and reducing sugar content by the compound bacteria; the letters "a" and "b" in the figure are significance markers.

[0025] Figure 6 The figure shows the effect of fermentation temperature on the degradation of cottonseed molasses by a compound microbial culture (single-factor experiment). It illustrates the effects of five different fermentation temperatures (25℃, 28℃, 30℃, 34℃, and 37℃) on the degradation efficiency of free gossypol, total acid content, and reducing sugar content by the compound microbial culture; the letters "a", "b", and "c" in the figure are significance markers.

[0026] Figure 7 The figure shows the effect of fermentation time on the degradation of cottonseed molasses by the compound bacteria (single-factor experiment). It shows the effect of five different fermentation times (3d, 4d, 5d, 6d, 7d) on the degradation efficiency of free gossypol, total acid content and reducing sugar content by the compound bacteria; the letters "a", "b", and "c" in the figure are significance markers.

[0027] Figure 8 The response surface plot (left) and contour plot (right) show the interaction between inoculation ratio (A) and fermentation temperature (B). The inoculation ratio markers "1", "2", and "3" correspond to the -1, 0, and 1 levels, respectively.

[0028] Figure 9 The response surface plot (left) and contour plot (right) show the interaction between inoculation ratio (A) and fermentation time (C). The inoculation ratio markers "1", "2", and "3" correspond to the -1, 0, and 1 levels, respectively.

[0029] Figure 10 The response surface plot (left) and contour plot (right) show the interaction between fermentation temperature (B) and fermentation time (C).

[0030] Figure 11 Principal component analysis (PCA) plot of metabolites. A represents positive ion mode, and B represents negative ion mode, showing the overall differences in metabolites between the F2 and CK groups. The horizontal axis represents PC1 (first principal component), and the vertical axis represents PC2 (second principal component). The scatter plots represent different samples. "CK": Unfermented cottonseed molasses control sample; "F2": Cottonseed molasses sample fermented with compound bacteria; "PC1 (46.35%)" / "PC1 (52.88%)": First principal component and its explained proportion of sample metabolic difference variance; "PC2 (16.41%)" / "PC2 (20.76%)": Second principal component and its explained proportion of sample metabolic difference variance.

[0031] Figure 12 The scatter plot and ordinal validation plots for PLS-DA are shown. A and B represent positive ion modes, and C and D represent negative ion modes. In the scatter plot, the horizontal axis is PC1 and the vertical axis is PC2, with different colored dots representing the control group (CK) and the experimental group (F2). In the ordinal validation plot, the horizontal axis is the correlation coefficient (Cor) and the vertical axis is R. 2 and Q 2Value, indicating the R value corresponding to the intercept. 2 and Q 2 scope.

[0032] Figure 13 This is a volcano plot of differential metabolites. It shows the distribution of differential metabolites in cottonseed molasses after fermentation (F2 group) and before fermentation (CK group) under positive ion (A) and negative ion (B) detection modes, respectively. The horizontal axis is log2(FoldChange) (logarithm of fold change), where log2(FC)>1.2 represents upregulation and log2(FC)<0.833 represents downregulation; the vertical axis is -log10(P-value) (logarithm of statistical significance), with larger values ​​indicating higher statistical significance of metabolite differences; the color / size of the scatter points represents the type of metabolite difference; "F2" refers to the cottonseed molasses sample after fermentation with the compound bacteria, and "CK" refers to the unfermented cottonseed molasses control sample; "UP": indicates significantly upregulated differential metabolites; "DW": indicates significantly downregulated differential metabolites; "NoDiff": indicates metabolites with no significant difference.

[0033] Figure 14 This is a bubble graph of KEGG enrichment in positive ion mode. The horizontal axis represents the enrichment ratio, the vertical axis represents the pathway name, the bubble size represents the number of differentially metabolites, and the color intensity represents the P-value.

[0034] Figure 15 This is a bubble diagram of KEGG enrichment in negative ion mode. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0036] The experimental supplies involved in this embodiment of the invention are as follows: I. Materials and Reagents Cottonseed molasses: sourced from Changji Feed Co., Ltd. of Xinjiang Taikun Group; basic physicochemical properties: moisture 14.56%, crude protein 12.79%, crude ash 3.12%, crude fat 0.7%, free gossypol 3242.5mg / kg.

[0037] Reagents: (NH4)2SO4, NaCl, MgSO4 7H2O, K2HPO4, 0.9% physiological saline, phloroglucinol colorimetric reagent, gossypol acetate, 80% methanol aqueous solution, mass spectrometry grade water, 0.1 mol / L NaOH standard titration solution, DNS reagent, formic acid, methanol; blank control is 53% methanol aqueous solution.

[0038] The Aspergillus niger, Saccharomyces cerevisiae, and Lactobacillus gasseri used in this invention are common bacterial strains. All three strains used in this invention are harmless and safe strains, which can be purchased through other public channels such as the China Medical Culture Collection Center (CMCC), the China Industrial Culture Collection Center (CICC), the China General Microbiological Culture Collection Center (CGMCC), and the German Microbiological Culture Collection Center (DSMZ). Publicly available culture media and cultivation methods can be used. This invention prioritizes the cultivation of the three strains using a specially designed culture medium. After mixing according to the volume ratio, the strains exhibit significant characteristics such as no antagonism between them, stable effects, strong enzyme activity, high viable cell count, and nitrogen retention.

[0039] II. Instruments and Equipment BL-75A vertical pressure steam sterilizer, SW-CJ-2D double-person clean bench, DHP-9162 electric thermostatic incubator, 3H24RI benchtop high-speed low-temperature centrifuge, DU-6GW magnetic stirring water bath, Epoch microplate spectrophotometer, pHS-25 laboratory pH meter, GeneQuant pro Biochrom UV / Vis spectrophotometer, microscope, hemocytometer, 250mL Erlenmeyer flask, EP tubes, pipettes, test tubes, thermostatic shaker.

[0040] III. Test Strains The strain purchased from the China Center for Medical Bacteriological Culture Collection (CMCC): Aspergillus niger CMCC (F) 98003 ( Aspergillus niger HQ), Proteobacterium chrysosporum Bio-116032 ( Phanerochaete chrysosporium HB), Corning Trichoderma XQ0815 ( Trichoderma koningii (KN), all purchased on October 12, 2019.

[0041] The strain purchased from the China Industrial Microbial Culture Collection Center (CICC): Trichoderma reesei CICC2626 ( Trichoderma reesei (LS), purchased on October 12, 2019.

[0042] The strain purchased from the China General Microbiological Culture Collection Center (CGMCC): Lactobacillus gasseri ( Lactobacillus gasseri (GR), with accession number CGMCC 1.3396; Saccharomyces cerevisiae ( Saccharomyces cerevisiae(LJ), with accession number CGMCC 2.1030, all purchased on October 12, 2019.

[0043] IV. Culture Medium Potato Dextrose Agar (PDA): Used for the activation and culture of molds and Phanerochaete chrysosporium; MRS Liquid Medium: Used for the activation and seed culture of Lactobacillus gasseri and Saccharomyces cerevisiae; MRS Broth Medium: Used for the preparation of seed culture of microorganisms; YPD Liquid Medium: Used for the preparation of seed culture of microorganisms.

[0044] Cottonseed molasses culture medium: using cottonseed molasses as the fermentation substrate, the specific components are: 300mL cottonseed molasses, with the addition of 2g (NH4)2SO4, 1g NaCl, and 0.5g MgSO4. It is prepared by adding 7H2O and 0.5g K2HPO4 to water to a final volume of 1000mL.

[0045] Example 1 This embodiment describes the screening of compound bacterial strains and the determination of the optimal combination.

[0046] 1. Experimental Materials and Methods 1.1 Test strains Tested strains: Aspergillus niger (HQ), Proteobacterium chrysosporum (HB), Trichoderma cornutum (KN), Trichoderma reesei (LS), Lactobacillus gassei (GR), and Saccharomyces cerevisiae (LJ).

[0047] 1.2 Activation of microbial strains and preparation of seed culture Mold activation: Glycerol-preserved HQ, HB, KN, and LS strains were spread onto sterile potato dextrose agar (PDA) medium and incubated at 28°C for 7 days, then transferred to a 4°C refrigerator for later use. Spores were collected from the 7-day PDA plates by washing with sterile 0.9% physiological saline, transferring them to Erlenmeyer flasks containing sterile glass beads, and shaking thoroughly. Spores were counted under a microscope using a hemocytometer, and the spore concentration was adjusted to 10-1. 8 The fractions per mL were used as seed solutions for HQ, HB, KN, and LS.

[0048] Bacterial and yeast activation: GR and LJ cells preserved with magnetic beads were inoculated into sterile MRS liquid medium and YPD liquid medium, respectively, and incubated in constant temperature shakers at 37℃ and 28℃ for 24 hours, respectively, to achieve a viable count of 10⁻⁶. 8 CFU / mL was transferred to a 4°C freezer for later use as seed culture for GR and LJ.

[0049] 1.3 Test of single-strain ability to degrade free gossypol Cottonseed molasses was used as the fermentation substrate, and 250 mL Erlenmeyer flasks were filled with seed culture of the above six strains at a 5% inoculation rate. The flasks were then incubated at 30℃ in a shaker (150 rpm) for 7 days. After incubation, the free gossypol content was determined according to Gong Lingfeng's method with modifications, as follows: 1 mL of fermentation broth was added to a centrifuge tube and centrifuged at 10,000 rpm for 10 min. 0.5 mL of the supernatant was added to the centrifuge tube, followed by 1 mL of colorimetric reagent. After shaking evenly, the mixture was incubated in a 55℃ water bath for 5 min, and the absorbance was measured at 550 nm. The absorbance was then measured according to the standard curve of gossypol acetate (regression equation: Y = 0.01x - 0.0158, R0). 2 =0.9992, where Y is the absorbance value and x is the free gossypol content (unit: mg / L) to calculate the free gossypol degradation rate. Each group of experiments was repeated 3 times.

[0050] 1.4 Strain Antagonism Test Strains with high single-strain degradation rates were selected (single-strain degradation test results showed that HQ, LJ, and GR had strong degradation abilities). Antagonistic tests were conducted using the streak cross method: On sterilized PDA plates, the three test strains were streaked along the cross direction of the plate, with three parallel streaks for each combination. The parallel combinations were placed in a constant temperature incubator at 28℃ (HQ+LJ, HQ+GR, HQ+LJ+GR combination) or 37℃ (LJ+GR combination). Observations were made every 12 hours, and the presence of inhibition zones at the intersection of the streaks was recorded to determine whether there was antagonistic effect between the strains.

[0051] 1.5 Test for degradation of free gossypol by mixed bacterial combinations Four mixed bacterial combinations were set up: HQ+LJ, HQ+GR, LJ+GR, and HQ+LJ+GR. For each group, 5% of the corresponding strain's seed culture was inoculated into sterilized cottonseed molasses medium (100 mL medium in a 250 mL Erlenmeyer flask) and cultured at 30℃ in a shaker (150 rpm) for 7 days. The degradation rate of free gossypol in each group was determined according to the method in 1.3. Each experiment was repeated 3 times, and the average value was taken. SPSS 26 software was used for analysis of variance to compare the differences in degradation rates among the groups.

[0052] 2. Test Results 2.1 Degradation rate of free gossypol by single bacteria As attached Figure 1 As shown (attached) Figure 1(Bar chart showing the degradation rate of free gossypol in cottonseed molasses by different strains). All six strains were able to degrade free gossypol in cottonseed molasses, but the degradation abilities differed significantly. Among them, HQ (Aspergillus niger), LJ (Saccharomyces cerevisiae), and GR (Lactobacillus gasseri) had significantly higher degradation rates than HB (Phanerochaete chrysosporium), KN (Trichoderma corniglanum), and LS (Trichoderma reesei), at 69.26%, 66.75%, and 66.96%, respectively. The other three strains had lower degradation rates. Therefore, HQ, LJ, and GR were selected for subsequent mixed strain experiments.

[0053] 2.2 Results of strain antagonism test As attached Figure 2 As shown (attached) Figure 2 The diagram shows the antagonistic test results of HQ, LJ, and GR by streaking in pairs. No inhibition zones appeared at the intersections of the streaks between HQ and LJ, HQ and GR, and LJ and GR, indicating that there is no antagonistic effect among the three strains. The strains can coexist and grow, which meets the basic conditions for mixed fermentation and can be used for subsequent mixed degradation experiments.

[0054] 2.3 Degradation effect of different mixed bacterial combinations As attached Figure 3 As shown (attached) Figure 3 The bar chart shows the degradation rates of free gossypol in cottonseed molasses by different mixed bacterial combinations. The degradation rates of free gossypol in the HQ+GR, LJ+GR, and HQ+LJ+GR combinations were significantly higher than those in the HQ+LJ combination (p<0.05). Among them, the HQ+LJ+GR combination had the highest degradation rate, reaching 68.63±0.05657%. Based on the physiological characteristics of each strain (HQ secretes hydrolytic enzymes such as cellulase and xylanase, LJ grows rapidly and has strong fermentation ability, and GR produces organic acids that can regulate the environmental pH), Aspergillus niger (HQ) + Saccharomyces cerevisiae (LJ) + Lactobacillus gasseri (GR) was determined to be the optimal mixed bacterial combination for degrading free gossypol in cottonseed molasses.

[0055] Example 2 This embodiment describes the optimization of key process parameters for compound bacterial fermentation using single-factor experiments.

[0056] 1. Experimental Materials and Methods 1.1 Test strains and seed culture preparation The compound bacteria used in the experiment were the optimal combination of strains determined in Example 1: Aspergillus niger (HQ), Saccharomyces cerevisiae (LJ), and Lactobacillus gasseri (GR); the methods for strain activation and seed culture preparation were the same as in Example 1.

[0057] 1.2 Fermentation medium Cottonseed molasses culture medium was used, dispensed into 250mL Erlenmeyer flasks (100mL each), and sterilized (121℃, 30min) for later use.

[0058] 1.3 Single-factor experimental design The degradation rate of free gossypol was used as the core evaluation index, while total acid content and reducing sugar content were measured as auxiliary indicators. The effects of four factors—inoculation ratio, inoculation amount, fermentation temperature, and fermentation time—on the fermentation effect of the compound bacteria were investigated. During the experiment, only one factor was changed, while the other factors were kept constant at the basic conditions of the previous single-strain fermentation: total inoculation amount 5%, shaker speed 150 rpm, fermentation temperature 30℃, and fermentation time 7 days. Each group of experiments was repeated 3 times. The levels of each factor were set as follows: Inoculation ratio (HQ:LJ:GR, based on bacterial volume ratio): Seven levels were set up, namely 1:1:1, 1:1:2, 1:2:1, 2:1:1, 1:2:2, 2:1:2, and 2:2:1; the bacterial concentration of Aspergillus niger and Saccharomyces cerevisiae was 1×10⁻⁶. 8 The bacterial concentration of Lactobacillus gasseri was 10⁶ cells / mL. 8 CFU / mL.

[0059] Inoculum size (calculated as seed liquid volume / culture medium volume): Five levels were set: 1%, 5%, 10%, 15%, and 20%. Fermentation temperature: Set 5 levels, namely 25℃, 28℃, 30℃, 35℃, and 37℃; Fermentation time: Set 5 levels, namely 3d, 4d, 5d, 6d, and 7d.

[0060] 1.4 Measurement Indicators and Methods Degradation rate of free gossypol: The degradation rate was determined according to the method in Example 1 and calculated based on the regression equation of the standard curve of acetic acid gossypol.

[0061] Total acid content: determined by potentiometric titration with a pH meter (refer to GB 12456-2021). The calculation formula is: Total acid content (g / kg) = [c×(V1-V2)×0.09×1000×F] / m, where c is the concentration of NaOH standard titration solution (mol / L), V1 is the volume of NaOH consumed in the titration of the sample (mL), V2 is the volume of NaOH consumed in the blank test (mL), 0.09 is the lactic acid conversion factor, F is the sample dilution factor, and m is the sample weight (mg).

[0062] Reducing sugar content: determined using the DNS method: 0.1 mL of the sample was placed in a test tube, water was added to a final volume of 0.2 mL, 0.4 mL of DNS reagent was added, the tube was boiled in a water bath for 5 min, immediately cooled, and the volume was adjusted to 1 mL. The absorbance was measured at a wavelength of 540 nm, a standard curve was plotted, and the regression equation was obtained (Y = 10.165x - 0.1148, R0). 2 =0.9997, where Y is the absorbance value and x is the reducing sugar content (in mg / g). The content is calculated based on the regression equation.

[0063] 1.5 Data Processing Data were processed using Excel 2021, ANOVA was performed using SPSS 26, and charts were generated using Origin 2024. Results are expressed as mean values, and p < 0.05 was used as the criterion for determining significance between groups (different letters in the charts indicate significant differences between groups).

[0064] 2. Test Results 2.1 Effect of inoculum ratio on fermentation efficiency As attached Figure 4 As shown (attached) Figure 4 (Combined graph showing the effect of inoculation ratio on degradation rate, total acid content, and reducing sugar content) Degradation rate: The degradation rates of the 1:1:2, 1:2:1, 2:1:1, 1:2:2, 2:1:2, and 2:2:1 groups were relatively high, at 68.67%, 69.13%, 68.43%, 69.70%, 69.96%, and 68.91%, respectively, with no significant differences between the groups; Total acid content: The total acid content of the 1:1:1, 1:1:2, 1:2:1, and 2:1:1 groups was relatively high, at 124.8 g / kg, 126 g / kg, 123.6 g / kg, and 125.4 g / kg, respectively. Reducing sugar content: The reducing sugar content of the 1:1:1, 1:2:1, and 2:1:1 groups was relatively high, at 8.73 mg / g, 8.47 mg / g, and 8.64 mg / g, respectively.

[0065] Based on the three indicators, the appropriate inoculation ratio was determined to be HQ:LJ:GR=1:2:1 (based on bacterial volume ratio).

[0066] 2.2 Effect of inoculum size on fermentation efficiency As attached Figure 5 As shown (attached) Figure 5 (Combined graph showing the effect of inoculum size on degradation rate, total acid content, and reducing sugar content) Degradation rate: The degradation rate increased with increasing inoculum amount, but there was no significant difference between groups. The degradation rate was highest at 20% inoculum amount (70.02%). Total acid content: It first decreased and then increased with the increase of inoculum amount, and the total acid content was the highest at 20% inoculum amount (133.2g / kg). Reducing sugar content: It decreased with increasing inoculum amount, and the reducing sugar content was the highest at 5% inoculum amount (6.95 mg / g).

[0067] Considering that a high inoculation rate would increase costs, and that a 5% inoculation rate resulted in the optimal reducing sugar content and a degradation rate close to that of a high inoculation rate, 5% was determined to be the appropriate inoculation rate.

[0068] 2.3 Effect of fermentation temperature on fermentation efficiency As attached Figure 6 As shown (attached) Figure 6 (Combined graph showing the effect of fermentation temperature on degradation rate, total acid content, and reducing sugar content) Degradation rate: The degradation rates of the 28℃ and 30℃ groups were relatively high, at 68.67% and 67.75% respectively, which were significantly higher than those of the 25℃, 35℃ and 37℃ groups. Total acid content: The total acid content was highest in the 37℃ group (134.4 g / kg), but the degradation rate was low at this temperature; Reducing sugar content: The 34℃ group had the highest reducing sugar content (6.64 mg / g), but the degradation rate was not optimal.

[0069] Based on the principle of prioritizing overall degradation rate, 28℃ was determined to be the suitable fermentation temperature.

[0070] 2.4 Effect of fermentation time on fermentation effect As attached Figure 7 As shown (attached) Figure 7 (Combined graph showing the effect of fermentation time on degradation rate, total acid content, and reducing sugar content) Degradation rate: The degradation rate increased with the extension of fermentation time, and the highest degradation rate (69.78%) was observed at 7 days, which was significantly higher than that of the 3-6 day group. Total acid content: It showed an increasing trend with the extension of fermentation time, and the total acid content was the highest at 7 days (121.2 g / kg). Reducing sugar content: It decreased with the extension of fermentation time, and the reducing sugar content was the highest at 3 days (8.22 mg / g), but the degradation rate was low.

[0071] Based on the combined degradation rate and total acid content, 7 days was determined to be the appropriate fermentation time.

[0072] 2.5 Summary of Single-Factor Optimization Results Through single-factor experiments, the optimal range of key process parameters for the degradation of free gossypol in cottonseed molasses by compound bacteria fermentation was determined to be: inoculation ratio HQ:LJ:GR=1:2:1 (based on bacterial volume ratio), inoculation amount 5%, fermentation temperature 28℃, and fermentation time 7d. These parameters provide the basis for subsequent multi-factor synergistic optimization (response surface methodology).

[0073] Example 3 This embodiment describes the optimization of optimal process parameters for co-culture fermentation using response surface methodology.

[0074] 1. Experimental Materials and Methods 1.1 Test strains and seed culture preparation The test strains were the optimal combination of bacteria determined in Example 1: Aspergillus niger (HQ), Saccharomyces cerevisiae (LJ), and Lactobacillus gasseri (GR).

[0075] The methods for strain activation and seed liquid preparation are the same as in Example 1.

[0076] 1.2 Fermentation medium The same fermentation medium formulation as in Example 2.

[0077] 1.3 Response Surface Design Based on the results of the single-factor experiment in Example 2, three factors that significantly affected the degradation rate of free gossypol were selected as independent variables: inoculation ratio (A), fermentation temperature (B), and fermentation time (C). The degradation rate of free gossypol (Y) was used as the response value. Using the Box-Behnken central composite design principle, a total of 17 groups of experiments with 3 factors and 3 levels were designed. The specific factor level settings are shown in Table 1.

[0078] Table 1.3 Experimental Design Table for 3-Factor Level Response Surface Analysis

[0079] 1.4 Fermentation and Determination Methods According to the response surface methodology, the compound bacterial seed culture was mixed at the set inoculation ratio and inoculated into the fermentation medium in a 250mL Erlenmeyer flask (containing 100mL of liquid) at 5% of the total inoculation amount. The flask was then placed in a constant temperature shaker (150rpm) at the set temperature and cultured for the set time. The free gossypol degradation rate was determined using the same method as in Example 1, calculated based on the regression equation of the acetate-gossypol standard curve. Each experiment was repeated three times, and the average value was taken as the response value.

[0080] 1.5 Data Processing The experimental data were fitted using Design-Expert 13 software to establish a regression model and test its effectiveness using analysis of variance. The interaction between factors was analyzed using response surface plots and contour plots. The optimal process parameters were solved using the model and then validated using experiments.

[0081] 2. Test Results The results of each group of experiments are shown in Table 2, and the analysis of variance is shown in Table 3.

[0082] Table 2. Experimental Results

[0083] Table 3. Results of Analysis of Variance

[0084] Note: P<0.01 is highly significant (denoted by **), P<0.05 is significant (denoted by *), and P>0.05 is not significant (denoted by ns).

[0085] 2.1 Regression Model Establishment and Analysis of Variance By fitting 17 sets of experimental data, a quadratic multiple regression equation was obtained for the degradation rate of free gossypol (Y) with respect to the inoculation ratio (A), fermentation temperature (B), and fermentation time (C): Y = 71.46 + 0.2091A - 0.6825B + 0.7233C + 0.242AB - 0.44AC - 0.2728BC - 1.116A 2 -1.09B 2 -0.5405C 2 ; The analysis of variance results (Table 3) show that the regression model has P < 0.0001 (highly significant) and the lack-of-fit term has P = 0.8198 (not significant), indicating that the model fits well and there are no factors affecting the model's fit; the model's coefficient of determination R0 2 =0.9826, adjusted R 2 =0.9602 (greater than 0.8000), indicating that 96.02% of the response value variation can be explained by this model, demonstrating high reliability. The order of influence of each factor on the degradation rate is: fermentation time (C) > fermentation temperature (B) > inoculum ratio (A); where the first-order terms are B and C, and the second-order term is A. 2 B 2 C 2 The interaction term AC had a highly significant effect on the degradation rate (P<0.01), while the other terms had no significant effect (P>0.05).

[0086] 2.2 Interaction Analysis of Various Factors 2.2.1 Interaction between inoculum ratio and fermentation temperature As attached Figure 8 As shown (attached) Figure 8 The response surface plot (left) and corresponding contour plot (right) show the interaction between inoculation ratio and fermentation temperature: the slope of the response surface is relatively steep, indicating that the interaction between the two factors is significant; the degradation rate first increases and then decreases with the increase of inoculation ratio and fermentation temperature. In the range of inoculation ratio of 1:2:1 and fermentation temperature of 25~30℃, the degradation rate is close to the peak, which is consistent with the interaction trend in the analysis of variance.

[0087] 2.2.2 Interaction between inoculum ratio and fermentation time As attached Figure 9 As shown (attached) Figure 9 The response surface plot (left) and corresponding contour plot (right) show the interaction between inoculation ratio and fermentation time: the response surface is steep and the interaction between the two factors is significant; the degradation rate first increases and then decreases with the increase of inoculation ratio and fermentation time. The degradation rate reaches a relatively high level in the range of inoculation ratio of 1:2:1 and fermentation time of 7-9 days, which verifies the rationality of the model prediction.

[0088] 2.2.3 Interaction between fermentation temperature and fermentation time As attached Figure 10 As shown (attached) Figure 10 The response surface plot (left) and corresponding contour plot (right) show the interaction between fermentation temperature and fermentation time: the response surface shows obvious extreme value regions, and the interaction between the two factors is significant; the degradation rate first increases and then tends to level off with the increase of fermentation temperature and fermentation time, and the degradation rate is in the optimal range when the fermentation temperature is 25~30℃ and the fermentation time is 7~9d.

[0089] 2.3 Determination and Verification of Optimal Process Parameters The optimal process parameters for the degradation of free gossypol by the compound bacteria were obtained by solving the regression model using Design-Expert 13 software: inoculation ratio HQ:LJ:GR=1:2:1 (based on the volume ratio of bacteria), inoculation amount 5%, fermentation temperature 27.813℃, and fermentation time 8.36d. Under these conditions, the predicted degradation rate of free gossypol was 72.2228%.

[0090] To adapt to practical operation, the process conditions were set as follows: inoculation ratio (HQ:LJ:GR) = 1:2:1 (based on bacterial volume ratio), inoculation amount 5%, fermentation temperature 27.8℃, and fermentation time 8.4 days. The theoretical degradation rate under these conditions was 72.22%. Three parallel experiments were conducted under these conditions, and the actual average degradation rate was 72.28%, which is close to the theoretical value. This indicates that the optimized process parameters are accurate and reliable and can be used for the efficient degradation of free gossypol in cottonseed molasses.

[0091] Example 4 This embodiment describes the metabolomics analysis of the fermentation products of the compound bacteria.

[0092] 1. Experimental Materials and Methods 1.1 Test Samples Control group (CK): Unfermented cottonseed molasses culture medium (from the same batch of raw materials used in Examples 2 and 3); Experimental group (F2): Cottonseed molasses fermentation broth fermented using the optimized process of Example 3 (inoculation ratio 1:2:1, inoculation amount 5%, fermentation temperature 27.8℃, fermentation time 8.4d). Three parallel samples were taken from each group, and the supernatant was collected after centrifugation at 10000rpm for 10min and frozen at -80℃ for later use.

[0093] 1.2 Metabolomics Detection Methods 1.2.1 Sample Pretreatment Take 100 μL of sample supernatant and place it in an EP tube. Add 400 μL of 80% methanol aqueous solution, vortex to mix, and incubate on ice for 5 min. Centrifuge at 15000 g and 4℃ for 20 min. Dilute the supernatant with mass spectrometry grade water to a methanol content of 53%. Centrifuge again at 15000 g and 4℃ for 20 min, and collect the supernatant for LC-MS analysis. Blank control: Use 53% methanol aqueous solution as a blank sample and follow the same pretreatment procedure as above.

[0094] 1.2.2 LC-MS detection conditions Chromatographic conditions: Hypersil Gold column (C18); mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was methanol; flow rate was 0.2 mL / min, column temperature was 40℃; gradient elution program: 0~1.5 min, maintain A at 98% and B at 2%; 1.5~3 min, A decreases from 98% to 15% and B increases from 2% to 85%; 3~10 min, A decreases from 15% to 0% and B increases from 85% to 100%; 10.1~12 min, restore A to 98% and B to 2%.

[0095] Mass spectrometry conditions: scan range m / z 100~1500; ESI source, spray voltage 3.5kV, sheath gas flow rate 35psi, auxiliary gas flow rate 10L / min, ion transfer tube temperature 320℃, auxiliary gas heater temperature 350℃, ion introduction RF level: 60; polarity includes positive ion and negative ion modes, MS / MS secondary scan is data dependent scan.

[0096] 1.2.3 Data Processing and Analysis Raw data conversion: The offline data was converted into mzXML format using ProteoWizard, and peak extraction, peak alignment, and peak quantification were performed using XCMS.

[0097] Metabolite identification: Based on retention time, mass-to-charge ratio (with a mass deviation of 10 ppm allowed), and adduct ion information, the metabolites were identified by comparison with the KEGG database (https: / / www.genome.jp / kegg / pathway.html), HMDB database (https: / / hmdb.ca / metabolites), and LIPIDMaps database (http: / / www.lipidmaps.org / ).

[0098] Data standardization: Remove background ions from blank samples, standardize by “original quantitative value of sample / (total quantitative values ​​of metabolites in sample / total quantitative values ​​of metabolites in QC1 sample)”, and delete compounds with a relative peak area CV > 30% in QC samples.

[0099] Differential metabolite screening: Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using MetaX software, with VIP>1.0, FC>1.2 or FC<0.833, and P<0.05 as screening criteria.

[0100] Pathway enrichment analysis: The enrichment influence factor and P-value were calculated by hypergeometric test to perform KEGG pathway enrichment analysis on differential metabolites.

[0101] 2. Test Results 2.1 Validation of the reliability of metabolomics data 2.1.1 Principal Component Analysis (PCA) As attached Figure 11 As shown (attached) Figure 11 (PCA score plot, A for positive ion mode, B for negative ion mode): In the positive ion mode, the cumulative variance contribution rate of PC1 and PC2 is 62.76%; in the negative ion mode, the cumulative variance contribution rate is 73.64%. The two groups of samples are clearly separated in both positive and negative ion modes, indicating that the composition of metabolites before and after fermentation is significantly different, and the detection data can effectively reflect the metabolic characteristics of the samples.

[0102] 2.1.2 Partial Least Squares Discriminant Analysis (PLS-DA) As attached Figure 12 As shown (attached) Figure 12 For the scatter plot and ordination verification plot of PLS-DA (A and B are positive ion modes, C and D are negative ion modes): In the positive ion mode, model R 2 Y=1.00, Q 2 Y=0.98; R in negative ion mode 2 Y=1.00, Q 2 Y=0.99, R 2 Y and Q 2 Y is close to 1 and R 2 Y>Q 2 Y; The sorting verification diagram shows R under positive and negative ion modes. 2 =0.80, Q 2 =-0.99 indicates that the model has good stability, no overfitting, and can effectively distinguish the differences between the two groups of samples.

[0103] 2.2 Results of differential metabolite screening As attached Figure 13 As shown (attached) Figure 13(Volcano plot of differential metabolites, A represents positive ion mode, B represents negative ion mode): A total of 2704 differential metabolites were detected before and after fermentation, of which 716 were upregulated and 339 were downregulated. Among them, 1877 differential metabolites were screened in positive ion mode (472 upregulated and 240 downregulated), and 827 were screened in negative ion mode (244 upregulated and 99 downregulated). The sufficient number of differential metabolites provides a reliable basis for subsequent pathway analysis.

[0104] 2.3 Classification and Functional Association of Differential Metabolites Hierarchical cluster analysis was performed on the two groups of differentially expressed metabolites. The color changes of the cluster heatmap reflected the upregulation or downregulation of various metabolites in the two groups. The VIP value was used as the basis for important metabolites. The top 10 differentially expressed metabolites under positive and negative ion modes were displayed in tables, as shown in Tables 4 and 5.

[0105] Table 4. Top 10 differentially expressed metabolites of F2 and CK with upregulation and downregulation of VIP values ​​in positive ion mode.

[0106] Table 5. Top 10 differentially expressed metabolites in F2 and CK under negative ion mode, showing upregulation and downregulation of VIP values.

[0107] Positive ion mode: Upregulated differential metabolites are mainly enriched in lipids and lipid molecules, organic heterocyclic compounds, organic acids and their derivatives, including palmitoleoylethanolamide with anti-inflammatory activity and 11-hydroxyjasmonic acid that regulates growth and development; downregulated differential metabolites are concentrated in lipids and lipid molecules, organic nitrogen compounds, etc.

[0108] Negative ion mode: Upregulates differential metabolites mainly lipids and lipid molecules, organic oxygen compounds, phenylpropanoids and polyketides, including the anticancer progenitor trans-o-hydroxyphenylmethylenepyruvate; downregulates differential metabolites mainly lipids and lipid molecules, organic acids and their derivatives, organic oxygen compounds, etc.

[0109] The changes in key differential metabolites indicate that the fermentation process not only reduces harmful substances but also generates a variety of bioactive metabolites, enhancing the nutritional value of the product.

[0110] 2.4 Differential metabolic pathway enrichment analysis As attached Figure 14 , 15 As shown (attached) Figure 14 This is a KEGG enrichment bubble diagram in positive ion mode, attached. Figure 15 (KEGG enriched bubble diagram negative ion mode) Positive ion mode: Differential metabolites are mainly enriched in the ABC transporter pathway, vitamin digestion and absorption pathway, and arginine and proline metabolic pathway. The ABC transporter can promote toxin excretion, and the vitamin absorption pathway improves nutrient utilization efficiency.

[0111] Negative ion mode: mainly enriched in the phenylpropanoid biosynthesis pathway, the alkaloid biosynthesis pathway derived from the shikimic acid pathway, and the ABC transporter pathway. Phenylpropanoid compounds have anti-inflammatory, antioxidant and other pharmacological effects.

[0112] The significant enrichment of the above pathways indicates that the fermentation of the complex microorganisms achieves a synergistic effect of free gossypol degradation, nutrient optimization and bioactive substance generation by regulating multiple metabolic pathways.

[0113] Example 5 This embodiment describes a quality comparison of cottonseed molasses before and after fermentation.

[0114] 1. Test Methods The test samples were the same as in Example 4, including: control group (CK), unfermented cottonseed molasses culture medium; and test group (F2), cottonseed molasses fermentation broth fermented using the optimized process of Example 3. The free gossypol content of the two was detected respectively.

[0115] 2. Test Results The test results showed that the concentration of free gossypol in the unfermented cottonseed molasses culture medium was 445.57 mg / L, while the concentration in the fermented cottonseed molasses broth was 123.78 mg / L, with a degradation rate of 72.22%. According to the current Chinese feed hygiene standard (GB 13078-2001), the permissible amount of free gossypol is as follows: cottonseed cake (meal) ≤1200 mg / kg, broiler and growing chicken compound feed ≤100 mg / kg, laying hen compound feed ≤20 mg / kg, and growing-finishing pig compound feed ≤60 mg / kg. Using the compound bacteria and fermentation process provided by this invention, the free gossypol content of the fermentation product added to feed is far below this permissible amount.

[0116] In summary, the composite bacteria and fermentation process for degrading free gossypol in cottonseed molasses provided by this invention significantly reduce the residual amount of free gossypol and increase the total acid content after fermentation. Metabolomics results confirm that the fermentation process reduces harmful metabolites and increases anti-inflammatory and antioxidant active metabolites by activating pathways such as ABC transporter proteins, vitamin absorption, and amino acid metabolism, thereby significantly improving the safety and nutritional value of cottonseed molasses.

[0117] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A complex microbial strain for degrading free gossypol in cottonseed molasses, characterized in that, The compound bacteria consist of Aspergillus niger, Saccharomyces cerevisiae, and Lactobacillus gasseri, with a bacterial volume ratio of 1:1 to 3:1 to 2.

2. The compound bacteria according to claim 1, characterized in that, The volume ratio of Aspergillus niger, Saccharomyces cerevisiae, and Lactobacillus gasseri in the bacterial suspension was 1:2:1; the concentration of Aspergillus niger and Saccharomyces cerevisiae in the bacterial suspension was 1×10⁻⁶. 8 The bacterial concentration of Lactobacillus gasseri was 10⁶ cells / mL. 8 CFU / mL.

3. A fermentation process for degrading free gossypol in cottonseed molasses, characterized in that, Includes the following steps: The compound bacteria described in claim 1 or 2 are inoculated into a fermentation medium and fermented under the conditions of inoculation amount of 3-7%, fermentation temperature of 25-30℃, and fermentation time of 7-9 days. The fermentation medium uses cottonseed molasses as a base material and also contains nitrogen sources, sodium salts, magnesium salts, and phosphate salts.

4. The fermentation process according to claim 3, characterized in that, In the fermentation medium, the nitrogen source is ammonium sulfate, the sodium salt is sodium chloride, the magnesium salt is magnesium sulfate heptahydrate, and the phosphate salt is dipotassium hydrogen phosphate. The amount of each component added is as follows: 1.5~2.5g of ammonium sulfate, 0.8~1.2g of sodium chloride, 0.4~0.6g of magnesium sulfate heptahydrate, and 0.4~0.6g of dipotassium hydrogen phosphate per 1000mL of medium.

5. The fermentation process according to claim 3, characterized in that, The inoculation amount is 4-6%.

6. The fermentation process according to claim 3, characterized in that, The fermentation temperature is 27~28℃, and the fermentation time is 8~8.5 days.

7. Use of the compound bacteria according to claim 1 or 2 in the degradation of free gossypol in cottonseed molasses.

8. Use of the compound bacteria as described in claim 1 or 2 in the preparation of feed additives.

9. The use according to claim 8, characterized in that, The feed additive contains fermentation metabolites of the compound bacteria.

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