Cottonseed processing waste liquid synbiotic feed additive and application thereof

Abstract

The invention belongs to the technical field of fermented feed, and relates to a cottonseed processing waste liquid synbiotic feed additive and application thereof. The invention provides free gossypol degrading bacteria in cottonseed processing waste liquid. A screening culture medium of the free gossypol degrading bacteria comprises the cottonseed processing waste liquid, yeast extract powder, ammonium sulfate, sodium chloride, magnesium sulfate, monopotassium phosphate and water. The free gossypol degrading bacteria consist of aspergillus niger, lactobacillus gasseri and saccharomyces cerevisiae. By optimizing the culture medium formula and the combination of the free gossypol degrading bacteria, the free gossypol degrading rate of the cottonseed processing waste liquid and the retention rate of oligosaccharide are improved, so that the quality and the safety of the cottonseed processing waste liquid synbiotics feed additive are ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermented feed, and relates to a symbiotic feed additive from cottonseed processing waste liquid and its application. Background Art

[0002] Cottonseed, as an important by-product of cotton, is rich in resources. Cottonseed contains high-value components such as abundant oil, protein, and raffinose, and has high commercial value. Currently, cottonseed processing enterprises mainly produce products such as cottonseed meal, cottonseed oil, and cottonseed protein. During the production process, a large amount of cottonseed processing waste liquid will be generated. These waste liquids usually contain high levels of organic matter and nutrients, but also contain some anti-nutritional factors and harmful substances (such as free gossypol). Direct discharge will not only cause environmental pollution but also waste valuable resources. To effectively utilize these waste liquids, the present invention explores methods to convert them into valuable feeds or feed additives. Liquid fermentation technology, due to its high efficiency, has become a feasible way for the resource utilization of cottonseed processing waste liquid.

[0003] Traditional cottonseed processing waste liquid is added to feed at a low dose. However, due to the high content of free gossypol, problems such as insignificant animal feeding effects, animal diarrhea, and death occur, making it difficult to meet the requirements of modern animal husbandry for high-quality feed. Although there have been some studies on using chemical methods to extract oligosaccharides from cottonseed processing waste liquid and apply them to animal feed additives, this method is efficient and safe, but the feeding cost is too high, and secondary pollution will also be brought during chemical extraction. There are many studies on using microbial fermentation methods to remove free gossypol from raw materials such as cottonseed and cottonseed meal, and the research results also prove that using microbial fermentation to remove phenol is green and has no secondary pollution, and some special functional active substances will also be produced after the raw materials are fermented. However, since most of the current relevant studies on the degradation of free gossypol in raw materials focus on the screening and functions of free gossypol highly degrading bacteria, the retention of other active components (such as oligosaccharides) in the raw materials is often ignored, and some strains cannot be used as feed additives.

[0004] Therefore, in the technical field of fermented feed, it is particularly important to screen a strain type suitable for cottonseed processing waste liquid and a composite strain with a suitable ratio. Summary of the Invention

[0005] The object of the present invention is to provide a free gossypol-degrading bacterium suitable for cottonseed processing waste liquid. By optimizing the medium formula and the combination of free gossypol-degrading bacteria, the degradation rate of free gossypol in cottonseed processing waste liquid and the retention rate of oligosaccharides are improved, thereby ensuring the quality and safety of the synbiotic feed additive of cottonseed processing waste liquid. The functions are clarified by analyzing the active ingredients in the synbiotic feed additive product of cottonseed processing waste liquid. The feeding effect and application effect of the synbiotic feed additive of cottonseed processing waste liquid are evaluated by conducting animal feeding experiments.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In the first aspect, the present invention provides a free gossypol-degrading bacterium in cottonseed processing waste liquid. The screening medium of the free gossypol-degrading bacterium comprises cottonseed processing waste liquid, yeast extract powder, ammonium sulfate, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate and water;

[0008] The free gossypol-degrading bacterium is composed of Aspergillus niger, Lactobacillus gasseri and Saccharomyces cerevisiae.

[0009] Preferably, the screening method of the free gossypol-degrading bacterium is as follows:

[0010] Per liter of the screening medium, add: 60 - 300 g of cottonseed processing waste liquid, 2.5 - 3.5 g of yeast extract powder, 4 - 6 g of ammonium sulfate, 0.5 - 1 g of sodium chloride, 0.5 - 1 g of magnesium sulfate, 1 - 2 g of potassium dihydrogen phosphate, and make up to a total volume of 1000 mL with water (preferably distilled water) to obtain the screening medium;

[0011] Under sterile conditions, inoculate the gossypol-degrading bacterium into the screening medium and carry out fermentation culture.

[0012] Preferably, the volume ratio of Aspergillus niger, Lactobacillus gasseri and Saccharomyces cerevisiae in the free gossypol-degrading bacterium is 1:2:1.

[0013] Preferably, the pH of the screening medium is 6.0 - 7.0, the inoculation amount of the free gossypol-degrading bacterium is 5%, the temperature of the fermentation culture is 20 - 30 °C, and the time of the fermentation culture is 7 - 9 d.

[0014] In the second aspect, a synbiotic feed additive of cottonseed processing waste liquid is provided. The synbiotic feed additive of cottonseed processing waste liquid comprises a cottonseed processing waste liquid medium and the free gossypol-degrading bacterium of the present invention. The cottonseed processing waste liquid medium comprises: cottonseed processing waste liquid, peptone, beef extract powder, yeast extract powder, sodium acetate, trisodium citrate, magnesium sulfate, potassium dihydrogen phosphate and water.

[0015] Preferably, the preparation method of the synbiotic feed additive of cottonseed processing waste liquid is as follows:

[0016] Add per liter of cottonseed processing waste liquid medium: 200 - 500 g of cottonseed processing waste liquid, 3 - 10 g of peptone, 0.5 - 5 g of beef extract powder, 1 - 4 g of yeast extract powder, 1 - 5 g of sodium acetate, 0.1 - 2 g of ammonium citrate, 0.1 - 1 g of magnesium sulfate, 0.1 - 2 g of potassium dihydrogen phosphate, and make up to a total volume of 1000 mL with water (preferably distilled water); after the salts are dissolved, adjust the pH of the cottonseed processing waste liquid medium to 6.0 - 7.0 with sodium bicarbonate to obtain the cottonseed processing waste liquid medium;

[0017] Under aseptic conditions, inoculate the free gossypol - degrading bacteria into the cottonseed processing waste liquid medium according to the volume ratio of Aspergillus niger:Lactobacillus gasseri:Saccharomyces cerevisiae = 1:2:1, and shake - ferment to obtain the cottonseed processing waste liquid synbiotic feed additive.

[0018] Preferably, the inoculation amount of the free gossypol - degrading bacteria is 5%, the temperature of the shake - fermentation is 20 - 30 °C, the time of the shake - fermentation is 7 - 9 d, and the rotation speed of the shake - fermentation is 90 - 200 r / min.

[0019] In the third aspect, a liquid synbiotic product is provided, and the liquid synbiotic product includes the cottonseed processing waste liquid synbiotic feed additive of the present invention.

[0020] In the fourth aspect, the application of the cottonseed processing waste liquid synbiotic feed additive of the present invention in high - density cultivation of probiotics and / or high - concentration retention of oligosaccharides is provided.

[0021] In the fifth aspect, the application of the cottonseed processing waste liquid synbiotic feed additive of the present invention in the preparation of a liquid synbiotic product is provided.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] By adding the cottonseed processing waste liquid synbiotic feed additive of the present invention to the basal diet, it can provide more comprehensive and balanced nutrition for animals.

[0024] (1) It can significantly promote the growth performance of yellow - feather broilers. In terms of average body weight, the average body weight of yellow - feather broilers in the experimental group (cottonseed processing waste liquid synbiotic feed additive of the present invention + basal diet) at 23 days of age and 65 days of age is significantly higher than that of the control group (basal diet). In terms of average daily feed intake, the average daily feed intake of yellow - feather broilers in the experimental group (cottonseed processing waste liquid synbiotic feed additive of the present invention + basal diet) from 24 to 44 days of age is significantly lower than that of the control group (basal diet). In terms of feed - to - weight ratio, the feed - to - weight ratio of yellow - feather broilers in the experimental group (cottonseed processing waste liquid synbiotic feed additive of the present invention + basal diet) during the period from 1 to 65 days of age is significantly lower than that of the control group (basal diet).

[0025] (2) It can significantly improve the slaughter performance of yellow - feather broilers. The experimental group (the cottonseed processing waste symbiotic bacteria feed additive of the present invention + basal diet) significantly increased the semi - eviscerated rate and eviscerated rate of broilers. Specifically, the semi - eviscerated rate of the experimental group was (82.68 ± 0.48)-(84.15 ± 0.33), and that of the control group was (82.31 ± 0.46). The eviscerated rate of the experimental group was (79.51 ± 0.55)-(80.81 ± 0.32), and that of the control group was (78.46 ± 0.49).

[0026] (3) It can improve the immune ability of poultry. Compared with the control group (basal diet), the experimental group (the cottonseed processing waste symbiotic bacteria feed additive of the present invention + basal diet) significantly increased the thymus index. Specifically, the thymus index of the experimental group was (4.03 ± 0.34)-(4.45 ± 0.49), and that of the control group was 2.86 ± 0.37.

[0027] (4) It can significantly increase the content of serum immunoglobulins in yellow - feather broilers. Compared with the control group (basal diet), the contents of IgM, IgA, and IgG in the serum of broilers in the experimental group (the cottonseed processing waste symbiotic bacteria feed additive of the present invention + basal diet) were all significantly increased. Specifically, the content of IgM in the serum of broilers in the experimental group was (2.37 ± 0.09)mg / mL-(2.41 ± 0.13)mg / mL, and that in the control group was (2.01 ± 0.06)mg / mL. The content of IgA in the serum of broilers in the experimental group was (2.25 ± 0.2)mg / mL-(2.74 ± 0.21)mg / mL, and that in the control group was (1.82 ± 0.11)mg / mL. The content of IgG in the serum of broilers in the experimental group was (6.54 ± 0.42)mg / mL-(8.03 ± 0.49)mg / mL, and that in the control group was (5.58 ± 0.22)mg / mL.

[0028] (5) It can improve the serum antioxidant indexes of yellow - feather broilers. Specifically, the glutathione peroxidase in the serum of broilers in the experimental group (the cottonseed processing waste symbiotic bacteria feed additive of the present invention + basal diet) was (3902.44 ± 158.28)U / L-(4443.9 ± 216.06)U / L, and that in the control group (basal diet) was (3087.8 ± 170.95)U / L.

[0029] (6) In terms of villus height, compared with the control group (basal diet), the experimental group (the cottonseed processing waste symbiotic bacteria feed additive of the present invention + basal diet) significantly increased the villus heights of the duodenum and jejunum of yellow - feather broilers. Specifically, the villus height of the duodenum of yellow - feather broilers in the experimental group was

[0030] (1889.42 ± 105.95) μm - (1943.24 ± 126.35) μm. The duodenal villus height of the yellow - feather broilers in the control group was (1587.94 ± 56.81) μm. The villus height of the jejunum of the yellow - feather broilers in the experimental group

[0031] (1682.89 ± 123.62) μm - (1841.95 ± 68.94) μm. The villus height of the jejunum of the yellow - feather broilers in the control group was (1321.24 ± 55.22) μm. In terms of the crypt depth, compared with the control group (basal diet), the experimental group (the cottonseed processing waste liquid synbiotic feed additive of the present invention + basal diet) significantly reduced the crypt depth of the duodenum and ileum of the yellow - feather broilers. Specifically, the crypt depth of the duodenum of the yellow - feather broilers in the experimental group was

[0032] (183.21 ± 10.22) μm - (217.68 ± 11.72) μm. The crypt depth of the duodenum of the yellow - feather broilers in the control group was (251.71 ± 17.09) μm. The crypt depth of the ileum of the yellow - feather broilers in the experimental group was

[0033] (149.52 ± 11.92) μm - (177.37 ± 6.68) μm. The crypt depth of the ileum of the yellow - feather broilers in the control group was (243.29 ± 9.15) μm. In terms of the villus - to - crypt ratio, compared with the control group (basal diet), the experimental group (the cottonseed processing waste liquid synbiotic feed additive of the present invention + basal diet) significantly increased the villus - to - crypt ratio of the duodenum and ileum of the broilers. Specifically, the villus - to - crypt ratio of the duodenum of the broilers in the experimental group was (8.84 ± 0.59) - (11.02 ± 1.03), and the villus - to - crypt ratio of the duodenum of the broilers in the control group was (6.61 ± 0.54). The villus - to - crypt ratio of the ileum of the broilers in the experimental group was (7.42 ± 0.43) - (8.71 ± 0.63), and the villus - to - crypt ratio of the ileum of the control group was (5.22 ± 0.2). Description of the Drawings

[0034] Figure 1 is the degradation rate of free gossypol in cottonseed processing waste liquid by free gossypol - degrading bacteria.

[0035] Figure 2 is the effect of the inoculation ratio of free gossypol - degrading bacteria on the degradation of free gossypol.

[0036] Figure 3 is the effect of the inoculation amount of free gossypol - degrading bacteria on the degradation of free gossypol.

[0037] Figure 4 is the effect of the fermentation temperature of free gossypol - degrading bacteria on the degradation of free gossypol.

[0038] Figure 5 is the effect of the fermentation time of free gossypol - degrading bacteria on the degradation of free gossypol.

[0039] Figure 6 is a multi-factor interaction; A is the interaction between fermentation temperature and inoculation ratio, B is the interaction between fermentation time and inoculation ratio, and C is the interaction between fermentation time and fermentation temperature.

[0040] Figure 7 This is a free gossypol standard curve provided by an embodiment of the present invention. Detailed implementation manners

[0041] The present invention will be specifically described below in combination with specific implementation manners and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific implementation manners and examples are used to illustrate the present invention, rather than limiting the present invention.

[0042] Next, the technical solutions of the present invention will be described in combination with examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0043] Example 1

[0044] This example provides free gossypol-degrading bacteria in cottonseed processing waste liquid. The screening medium for the free gossypol-degrading bacteria includes cottonseed processing waste liquid, yeast extract powder, ammonium sulfate, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate, and distilled water;

[0045] The free gossypol-degrading bacteria are composed of Aspergillus niger, Lactobacillus gasseri, and Saccharomyces cerevisiae;

[0046] The screening method for the free gossypol-degrading bacteria is as follows:

[0047] Per liter of the screening medium, add: 60 - 300 g of cottonseed processing waste liquid, 2.5 - 3.5 g of yeast extract powder, 4 - 6 g of ammonium sulfate, 0.5 - 1 g of sodium chloride, 0.5 - 1 g of magnesium sulfate, 1 - 2 g of potassium dihydrogen phosphate, and make up to a total volume of 1000 mL with water (preferably distilled water) to obtain the screening medium;

[0048] Under sterile conditions, inoculate the gossypol-degrading bacteria into the screening medium and perform fermentation culture;

[0049] The volume ratio of Aspergillus niger, Lactobacillus gasseri, and Saccharomyces cerevisiae in the free gossypol-degrading bacteria is 1:2:1;

[0050] The pH of the screening medium is 6.0 - 7.0; the inoculation amount of the free gossypol-degrading bacteria is 5%; the temperature of the fermentation culture is 20 - 30 °C; the time of the fermentation culture is 7 - 9 d.

[0051] Effect verification 1:

[0052] Effect verification of Example 1 (phenol removal effect of fermenting cottonseed processing waste liquid by free gossypol-degrading bacteria)

[0053] I. Strains

[0054] 1. Strains used in the test

[0055] Aspergillus niger (HQ), Phanerochaete chrysosporium (HB), Trichoderma koningii (KN), Trichoderma reesei (LS), Lactobacillus gasseri (GR), Saccharomyces cerevisiae (LJ).

[0056] 2. Isolation and identification process

[0057] Aspergillus niger (HQ), Phanerochaete chrysosporium (HB), Trichoderma koningii (KN), Trichoderma reesei (LS), Lactobacillus gasseri (GR) and Saccharomyces cerevisiae (LJ) were all purchased from the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. Among them, the strain preservation number of Aspergillus niger (HQ) is CGMCC No. 3.17612, the strain preservation number of Phanerochaete chrysosporium (HB) is CGMCC No. 5.903, the strain preservation number of Trichoderma koningii (KN) is CGMCC No. 3.17875, the strain preservation number of Trichoderma reesei (LS) is CGMCC No. 3.13241, the strain preservation number of Lactobacillus gasseri (GR) (Lactobacillus gasseri) is CGMCC No. 1.3224, and the strain preservation number of Saccharomyces cerevisiae (LJ) is CGMCC No. 2.3973.

[0058] 3. Culture media

[0059] (1) Activation medium

[0060] Potato dextrose broth PDB (g / L): potato powder 6.0 g, glucose 20.0 g, pH 5.6 ± 0.2, water 1000 mL;

[0061] MRS broth (g / L): peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, dipotassium hydrogen phosphate 2.0 g, ammonium citrate dibasic 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 g, pH 5.7 ± 0.2, water 1000 mL;

[0062] YPD medium (g / L): peptone 20.0 g, glucose 20.0 g, yeast extract powder 10.0 g, pH 6.5 ± 0.2, water 1000 mL.

[0063] (2) Screening medium (fermentation medium)

[0064] Screening medium (g / L): 300 g of cottonseed processing waste liquid, 3 g of yeast extract powder, 5 g of ammonium sulfate, 1 g of sodium chloride, 0.5 g of magnesium sulfate, 1 g of potassium dihydrogen phosphate, pH 6.5 ± 0.2, and distilled water was added to make the total volume 1000 mL.

[0065] 4. Free gossypol degradation test

[0066] The above 6 strains of bacteria were used for single-strain fermentation of cottonseed processing waste liquid, and the degradation rates of free gossypol in cottonseed processing waste liquid were 53.43%, 60.66%, 69.26%, 58.67%, 66.75%, and 60.96% respectively. Among them, the degradation rates of HQ, GR, and LJ strains on free gossypol in cottonseed processing waste liquid were relatively high, with the highest being 69.26%. As Figure 1 shown.

[0067] II. Optimization of fermentation conditions

[0068] 1. Specific formula: As shown in Example 1.

[0069] 2. Preparation method (screening method): As shown in Example 1. The cottonseed processing waste liquid (sucrose 31.2%, raffinose 22.8%, stachyose 6.77%, crude protein 12.79%, crude ash 3.12%) was purchased from Xinjiang Taikun Group Changji Feed Co., Ltd.; the water content of the cottonseed processing waste liquid was calculated after drying, and each 1 L of the medium contained 60 - 300 g of cottonseed processing waste liquid (dry matter). Under sterile conditions, the strains were inoculated into the medium at a volume ratio of Aspergillus niger: Lactobacillus gasseri: Saccharomyces cerevisiae = 1:2:1 at 5%, and fermentation culture was carried out.

[0070] 3. Single-factor optimization of fermentation conditions

[0071] 3.1 Effect of inoculation ratio of free gossypol-degrading bacteria on the degradation rate of free gossypol in cottonseed processing waste liquid

[0072] By Figure 2It can be seen that when the inoculation ratio of Aspergillus niger:Lactobacillus gasseri:Saccharomyces cerevisiae is 1:1:2, 1:2:1, 2:1:1, 1:2:2, 2:1:2, 2:2:1, the degradation rates of free gossypol in cottonseed processing waste liquid are higher than those of other groups, being 68.67%, 69.13%, 68.43%, 69.70%, 69.96%, 68.91% respectively; when the inoculation ratios are 1:1:1, 1:1:2, 1:2:1, 2:1:1, the total acid contents in cottonseed processing waste liquid are higher than those of other groups, being 124.8 g / kg, 126 g / kg, 123.6 g / kg, 125.4 g / kg respectively; when the inoculation ratios are 1:1:1, 1:2:1, 2:1:1, the reducing sugar contents in cottonseed processing waste liquid are higher than those of other groups, being 8.73 mg / g, 8.47 mg / g, 8.64 mg / g respectively. Therefore, when the inoculation ratio is 1:2:1, it can be used as the optimal inoculation ratio.

[0073] 3.2 Effect of inoculation amount of free gossypol degrading bacteria on degradation rate of free gossypol in cottonseed processing waste liquid

[0074] It can be seen from Figure 3 that the degradation rate of free gossypol in cottonseed processing waste liquid increases with the increase of inoculation amount, but there is no significant difference. When the inoculation amount is 20%, the degradation rate is the highest, being 70.02%; the total acid content in cottonseed processing waste liquid shows a trend of first decreasing and then increasing with the increase of inoculation amount. When the inoculation amount is 20%, the total acid content is the highest, being 133.2 g / kg; the reducing sugar content in cottonseed processing waste liquid shows a decreasing trend with the increase of inoculation amount. When the inoculation amount is 5%, the reducing sugar content is the highest, being 6.95 mg / g. Therefore, the inoculation amount of 5% can be used as the optimal inoculation amount.

[0075] 3.3 Effect of fermentation temperature of complex bacteria on degradation rate of free gossypol in cottonseed processing waste liquid

[0076] It can be seen from Figure 4 that when the fermentation temperatures are 28 °C and 30 °C, the degradation rates of free gossypol in cottonseed processing waste liquid are higher than those of other groups, being 68.67%, 67.75% respectively; when the fermentation temperature is 37 °C, the total acid content in cottonseed processing waste liquid is higher than those of other groups, being 134.4 g / kg; when the fermentation temperature is 34 °C, the reducing sugar content in cottonseed processing waste liquid reaches 6.64 mg / g. Therefore, when the fermentation temperature is 28 °C, it is the optimal fermentation temperature.

[0077] 3.4 Effect of fermentation time on degradation rate of free gossypol in cottonseed processing waste liquid

[0078] It can be seen from Figure 5It can be seen that the degradation rate of free gossypol in cottonseed processing waste liquid increases with the increase of fermentation time. At the 7th day, the degradation rate reaches the highest, which is 69.78%. The total acid content in cottonseed processing waste liquid increases with the increase of fermentation time. At the 7th day, the total acid content reaches the highest, which is 121.2 g / kg. The reducing sugar content in cottonseed processing waste liquid decreases with the increase of fermentation time. At the 3rd day, the reducing sugar content is the highest, which is 8.22 mg / g. Therefore, when the fermentation time is 7 days, it is the optimal fermentation time.

[0079] 4. Optimization of Free Gossypol Degradation in Cottonseed Processing Waste Liquid by Multiple Factors Based on Response Surface Methodology

[0080] 4.1 Response Surface Experimental Design

[0081] On the basis of single-factor experiments, a response surface experiment with three factors and three levels was designed. The experimental results are shown in Table 1. And regression fitting was carried out. The quadratic multiple regression equation is shown in formula (I):

[0082] Y Degradation Rate =

[0083] 71.46 + 0.2091*A - 0.6825*B + 0.7233*C + 0.242*AB -

[0084] 0.44*AC - 0.2728*BC - 1.116*A2 - 1.09*B2 - 0.5405*C2

[0085] (I)

[0086] Table 1 Response Surface Optimization of Degradation Rate Experimental Design and Results

[0087]

[0088]

[0089] 4.2 Variance Analysis

[0090] As can be seen from Table 2, further regression analysis was carried out on the degradation rate model and regression coefficients. The results show that the P value of this regression model is <0.0001, indicating that the difference is extremely significant. The P value of the lack-of-fit term is 0.8198 ≥ 0.05, indicating that the difference is not significant, indicating that the model fitting degree is good, and the corresponding regression values of the regression equation can be predicted. At the same time, the regression coefficient R 2 = 0.9826, and the adjusted R 2 = 0.9602 (greater than 0.8000), indicating that 96.02% of the data can be explained by this model, indicating that the equation has high reliability.

[0091] The magnitude of the F value is an important indicator for evaluating the influence degree of each variable on the response value. The larger the F value, the higher the contribution degree of the relevant model component to the response. By analyzing the relevant data, it can be seen that the linear terms B and C in the model have a highly significant influence on the degradation rate (P≤0.01), and their quadratic terms A 2 , B 2 , C 2 also have a highly significant influence on the degradation rate (P≤0.01). The influence of different factors on the degradation rate of free gossypol in cottonseed processing wastewater is: C > B > A. The comprehensive data results show that this model can better reflect the relationship among the inoculation ratio, fermentation temperature, and fermentation time.

[0092] Table 2 Regression analysis results of the degradation rate model and regression coefficients

[0093]

[0094]

[0095] Note: P < 0.01 is highly significant, indicated by **; P < 0.05 is significant, indicated by *; P > 0.05 is not significant, indicated by ns.

[0096] 4.3 Response surface results and analysis

[0097] The influence of the interaction between fermentation temperature and inoculation ratio on the degradation rate is as shown in Figure 6 A. The slope of the response surface is relatively steep, and the degradation rate is greatly affected by the inoculation ratio and fermentation temperature. Moreover, the degradation rate first increases and then decreases with the increase of the inoculation ratio and fermentation temperature. When the inoculation ratio is 1.5 - 2.5 and the fermentation temperature is 25 - 30 °C, the degradation rate is close to the highest, and the surface plot is consistent with the results of the regression analysis in Table 2. The influence of the interaction between fermentation time and inoculation ratio on the degradation rate is as shown in Figure 6 B. The slope of the response surface is relatively steep, and the degradation rate is greatly affected by the inoculation ratio and fermentation time. Moreover, the degradation rate first increases and then decreases with the increase of the inoculation ratio and fermentation time. When the inoculation ratio is 1.5 - 2.5 and the fermentation time is 7 - 9 d, the degradation rate is close to the highest, and the surface plot is also consistent with the results of the regression analysis in Table 2. The influence of the interaction between fermentation time and fermentation temperature on the degradation rate is as shown in Figure 6 C. The slope of the response surface is relatively steep, and the degradation rate is greatly affected by the inoculation ratio and fermentation time. Moreover, the degradation rate first increases and then levels off with the increase of the fermentation temperature and fermentation time. When the fermentation temperature is 25 - 30 °C and the fermentation time is 7 - 9 d, the degradation rate is close to the highest, and the surface plot is consistent with the results of the regression analysis in Table 2.

[0098] 4.4 Verification test results

[0099] Optimized by software Design Expert 13, the optimal process conditions for degrading free gossypol in cottonseed processing waste liquid were obtained through analysis as follows: under the conditions of inoculation ratio of Aspergillus niger:Lactobacillus gasseri:Saccharomyces cerevisiae = 1:2:1, inoculum size of 5%, fermentation temperature of 27.813 °C, and fermentation time of 8.36 d, the highest degradation rate was 72.2228%.

[0100] To verify the consistency between the predicted results and the actual situation, a verification experiment was conducted under the above optimized conditions. However, considering the actual operation situation, the optimal process conditions were revised to: Aspergillus niger:Lactobacillus gasseri:Saccharomyces cerevisiae = 1:2:1, inoculum size of 5%, fermentation temperature of 27.8 °C, and fermentation time of 8.4 d. The theoretical degradation rate was 72.22%. Three parallel experiments were carried out under these conditions, and the actual degradation rate was 72.28%. The actual value was close to the predicted value. Therefore, the optimal fermentation process parameters obtained by the response surface method are accurate and reliable, and can be used for the experimental study on degrading free gossypol in cottonseed processing waste liquid.

[0101] III. Method (Phloroglucinol method)

[0102] 1. Reagents

[0103] ① Preparation of chromogenic reagent: Dissolve 1 g of phloroglucinol in 16.6 mL of concentrated hydrochloric acid, and make up the volume to 100 mL with 95% ethanol, then mix well; transfer it to a brown bottle and store it in a refrigerator at 4 °C for later use.

[0104] ② 70% (v / v) acetone solution: A mixed solution obtained by uniformly mixing 70 mL of acetone solution (analytical pure) and 30 mL of deionized water is the 70% acetone solution.

[0105] ③ Preparation of gossypol standard solution: Accurately weigh 0.025 g of gossypol acetate, dissolve it with 70% (v / v) acetone solution, and make up the volume to 25 mL to prepare a 1 mg / mL free gossypol standard solution as the stock solution; accurately pipette 1 mL of the stock solution, dilute it with 70% (v / v) acetone solution, and make up the volume to 50 mL to prepare a 0.02 mg / mL free gossypol working solution.

[0106] 2. Specific method steps

[0107] ① Drawing of gossypol standard curve: Accurately pipette 0, 0.20, 0.40, 0.60, 0.80, 1.00 mL of free gossypol working solution, and make up the volume to 1.00 mL with 70% acetone solution in turn. Add 2 mL of chromogenic reagent, and shake well; place it in a constant temperature water bath at 55 °C for 5 min. Transfer the reaction solution to a 1 cm cuvette, and measure the absorbance at 550 nm with the sample without adding free gossypol as the blank.

[0108] ② Gossypol standard curve: As Figure 7As shown, the free gossypol content is in the range R of 0 - 100 mg / L 2 = 0.9965, indicating good linearity and enabling this method to be used as a detection method.

[0109] ③ Detection method for free gossypol in cottonseed processing waste liquid: Take 1 mL of the culture medium fermentation broth and place it in a centrifuge tube, centrifuge for 10 min (10000 rpm), take 0.5 mL of the supernatant and add it to the centrifuge tube, add 1 mL of the color reagent, shake well, keep it in a constant temperature water bath at 55 °C for 5 min, measure the absorbance value at a wavelength of 550 nm, and substitute it into the gossypol standard curve to obtain the gossypol content.

[0110] Example 2

[0111] This example provides a cottonseed processing waste liquid symbiotic feed additive. The cottonseed processing waste liquid symbiotic feed additive includes a cottonseed processing waste liquid culture medium and free gossypol - degrading bacteria. The raw materials of the cottonseed processing waste liquid culture medium include: cottonseed processing waste liquid, peptone, beef extract powder, yeast extract powder, sodium acetate, ammonium citrate, magnesium sulfate, potassium dihydrogen phosphate, and water;

[0112] The free gossypol - degrading bacteria are composed of Aspergillus niger HQ, Lactobacillus gasseri GR, and Saccharomyces cerevisiae LJ;

[0113] Per liter of the cottonseed processing waste liquid culture medium, add: 200 - 500 g of cottonseed processing waste liquid, 3 - 10 g of peptone, 0.5 - 5 g of beef extract powder, 1 - 4 g of yeast extract powder, 1 - 5 g of sodium acetate, 0.1 - 2 g of ammonium citrate, 0.1 - 1 g of magnesium sulfate, 0.1 - 2 g of potassium dihydrogen phosphate, and make up to a total volume of 1000 mL with distilled water; after the salts are dissolved, adjust the pH of the cottonseed processing waste liquid culture medium to 6.0 - 7.0 with sodium bicarbonate to obtain the cottonseed processing waste liquid culture medium;

[0114] Under sterile conditions, inoculate the free gossypol - degrading bacteria into the cottonseed processing waste liquid culture medium according to the volume ratio of Aspergillus niger HQ: Lactobacillus gasseri GR: Saccharomyces cerevisiae LJ = 1:2:1, and shake and ferment to obtain the cottonseed processing waste liquid symbiotic feed additive;

[0115] The inoculation amount of the free gossypol - degrading bacteria is 5%, the temperature of the shaking fermentation is 20 - 30 °C, the time of the shaking fermentation is 7 - 9 d, and the rotation speed of the shaking fermentation is 90 - 200 r / min.

[0116] Effect verification two:

[0117] Effect verification of Example 2

[0118] I. Preparation of cottonseed processing waste liquid symbiotic feed additive by fermenting cottonseed processing waste liquid with a composite bacterium (free gossypol - degrading bacteria)

[0119] 1. Detection of viable cell count in the waste liquid of cottonseed processed by compound bacteria fermentation

[0120] After the compound bacteria were fermented in the waste liquid of cottonseed processing for 8 days, the detection results of the viable cell counts of each strain were as follows: the viable cell count of Aspergillus niger HQ reached 3.23 ± 0.21×10 7 cfu / L, the viable cell count of Lactobacillus gasseri GR reached 8.97 ± 0.77×10 9 cfu / L, and the viable cell count of Saccharomyces cerevisiae LJ reached 6.64 ± 0.13×10 9 cfu / L.

[0121] 2. Analysis of active ingredients after the waste liquid of cottonseed processed by compound bacteria fermentation

[0122] A total of 1,969 metabolites were identified in the fermentation broth of the waste liquid of fermented cottonseed processed by liquid chromatography - mass spectrometry (LC - MS) technology. Among them, organic heterocyclic compounds (479 kinds) accounted for 19.98% of the total content, mainly including indole and its derivatives, diazines, and quinoline and its derivatives; lipids and lipid - like substances (421 kinds) accounted for 35.41% of the total content, mainly including fatty acyls, prenol lipids, and glycerides; benzenes (199 kinds) accounted for 11.24% of the total content, mainly including benzene and its substituted derivatives; organic nitrogen compounds (49 kinds) accounted for 8.33% of the total content; organic acids and their derivatives (382 kinds) accounted for 8.19% of the total content, mainly including carboxylic acids and their derivatives; organic oxides (125 kinds) accounted for 4.68% of the total content, alkaloids and their derivatives (106 kinds) accounted for 4.06% of the total content, mainly including harmine alkaloids; phenylpropanoids and polyketides (134 kinds) accounted for 3.93% of the total content, mainly including flavonoids, indolizidine alkaloids, and cinnamic acid; nucleosides, nucleolides, and analogs (31 kinds) accounted for 0.81% of the total content; organic sulfides (9 kinds) accounted for 0.03% of the total content; hydrocarbons (8 kinds) accounted for 0.72% of the total content; lignans, neolignans, and related compounds (8 kinds) accounted for 0.17% of the total content; organic halogen compounds (4 kinds) accounted for 0.03% of the total content; hydrocarbon derivatives (3 kinds) accounted for 0.72% of the total content; acetyl derivatives (1 kind) accounted for 0.04% of the total content, and other compounds (10 kinds) accounted for 0.12% of the total content. The specific results are shown in Table 3.

[0123] Table 3 Main compounds in the waste liquid of cottonseed processed by compound bacteria fermentation

[0124]

[0125]

[0126]

[0127]

[0128] 3. Raffinose retention rate

[0129] The changes in the contents of raffinose and stachyose before and after the fermentation of cottonseed processing waste liquid medium were measured using a raffinose (RH) ELISA kit [Keqiao (Jiangsu) Biotechnology Co., Ltd., KQ141913] and a stachyose (ST) ELISA kit [Keqiao (Jiangsu) Biotechnology Co., Ltd., KQ141902] respectively. From the results in Table 4, it can be seen that after 8 days, the retention rate of raffinose was 77.63%, and the retention rate of stachyose was 95.74%.

[0130] Table 4 Raffinose retention ability of the tested strains

[0131] Oligosaccharide Fermentation 0 h Fermentation 8 d Retention rate (%) Raffinose 440.43±67.49 341.92±6.97 77.63 Stachyose 315.27±32.38 301.83±50.33 95.74

[0132] 4. Results of complex enzyme activity determination

[0133] After 8 days of fermentation of cottonseed processing waste liquid by the complex bacteria, the total activity of the complex enzyme detected was 498.58 U / mL, including 135.32 U / mL of amylase, 131.71 U / mL of xylanase, and 221.45 U / mL of cellulase.

[0134] II. Methods

[0135] 2.1 Method for determining viable cell count

[0136] Viable cell count medium:

[0137] ① Lactobacillus count medium: Use MRS medium to count Lactobacillus. The medium components are: 10 g of peptone, 5 g of beef powder, 4 g of yeast powder, 2 g of glucose, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 2 g of ammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15 g of agar, and 1000 mL of distilled water.

[0138] ② Aspergillus niger count medium: Use Czapek medium to count the viable cells of Aspergillus niger. The medium components are: 3 g of sodium nitrate, 1 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate (MgSO4·7H2O), 0.5 g of potassium chloride, 0.01 g of ferrous sulfate, 30 g of sucrose, 20 g of agar, and 1000 mL of distilled water.

[0139] ③ Yeast count medium: Use PDA medium containing chloramphenicol to count Saccharomyces cerevisiae. The medium components are: 200 g of potato pulp (potato), 20 g of glucose, 15 g of agar, 0.1 g of chloramphenicol, and 1000 mL of distilled water.

[0140] Experimental operation steps:

[0141] 1) Preparation of culture medium: Weigh the components in each culture medium, then sterilize at 105°C for 15 min. When the temperature of the culture medium cools to 70 - 80°C, add it to a sterile petri dish in a sterile operation bench and let it solidify for 24 h. The petri dishes without contamination are used for strain counting.

[0142] 2) Colony counting operation: Use the dilution gradient method to coat each colony. For example, take 1 mL of the fermented culture solution and add it to 9 mL of sterile physiological saline in an oscillator and shake for 20 s, and then continue to dilute by analogy to 10 -8 Then select 10 -6 ,10 -7 ,10 -8 for coating. Pipette 100 μL of the dilution solution and coat it on these three petri dishes, and then use a sterile spreading rod to spread it in a sterile operation bench. The coated petri dishes are placed upside down and flat in an incubator. The lactic acid bacteria counting petri dishes are placed in an incubator at 37°C for 24 h, and the Aspergillus niger and yeast counting petri dishes are placed in an incubator at 30°C for 48 h. After the incubation, use a colony counter to count the colonies on the petri dishes and calculate the viable cell numbers of each colony.

[0143] 2.2 Determination of metabolites of fermented cottonseed processing waste liquid by compound bacteria

[0144] (1) Pretreatment of fermentation broth samples

[0145] Take 100 μL of the sample and place it in an EP tube, add 400 μL of 80% methanol aqueous solution; vortex and shake, let it stand in an ice bath for 5 min, centrifuge at 15000 g and 4°C for 20 min; take a certain amount of the supernatant and dilute it with mass spectrometry-grade water until the methanol content is 53%; centrifuge at 15000 g and 4°C for 20 min, collect the supernatant, and inject it into LC-MS for analysis.

[0146] (2) Chromatography and mass spectrometry conditions

[0147] Liquid phase conditions: The chromatographic column is Hypersil Gold column (C18). Mobile phase A is 0.1% formic acid, and mobile phase B is methanol. The injection volume is 2 μL, and the flow rate is 0.2 mL / min. The gradient elution program is: 0 - 1.5 min, 98% A; 1.5 - 3 min, 98% - 15% A; 3 - 10 min, 15% - 0% A; 10 - 10.1 min, 0% - 98% A; 10.1 - 11 min, 98% A; 11 - 12 min, 98% A.

[0148] (3) Mass spectrometry conditions

[0149] The scanning range is selected as m / z 100 - 1500; the settings of the ESI source are as follows: Spray Voltage: 3.5 kV; Sheath gas flowrate: 35 psi; Aux Gas flowrate: 10 L / min; Capillary Temp: 320 °C; S-lens RF level: 60; Aux gas heater temp: 350 °C; Polarity: positive, negative; The MS / MS second-level scan is a data-dependent scan.

[0150] (4) Data analysis

[0151] The original mass spectrometry data is converted into the mzXML format by ProteoWizard software. First, XCMS is used for peak extraction and peak quantification. Peak alignment is performed for different samples based on parameters such as retention time and mass-to-charge ratio. Then, according to the set 10 ppm mass deviation and adduct ion information, etc., it is compared with the high-quality second-level spectrum database for metabolite identification. Subsequently, the blank sample is used to remove background ions, and finally, the identification and relative quantification results of metabolites are obtained. The data processing part is based on the Linux operating system (CentOS version 6.6) and software R, Python. The KEGG database (https: / / www.genome.jp / kegg / pathway.html) and HMDB database (https: / / hmdb.ca / metabolites) are used to annotate the identified metabolites.

[0152] 2.3 Method for measuring the activity of complex enzyme

[0153] Preparation of crude enzyme solution: After fermentation, 1 g of sample is taken from 3 evenly distributed points in the bottle, diluted with 10 times distilled water, centrifuged at 8000 r / min for 5 min at 4 °C, and the supernatant is the crude enzyme solution.

[0154] Measurement of cellulase activity: Take 0.5 mL of the crude enzyme solution at an appropriate dilution factor, inactivate the control group enzyme solution by boiling, add 2 mL of 1% CMC-Na acetate buffer solution, mix well, and place it in a 50 °C constant temperature water bath for 30 min at the same time; add 2.5 mL of DNS solution to terminate the reaction, accurately react in a boiling water bath for 5 min and then cool immediately, make up the volume to 25.0 mL with distilled water, use the boiled and inactivated enzyme solution as the blank control, measure the OD value at 540 nm, each group of experiments is repeated 3 times, and the results are averaged as shown in formula (II).

[0155] The cellulase activity (IU / g) is defined as: under the above conditions, the amount of substrate consumed by 1 mL of enzyme solution per minute to produce reducing sugar equivalent to 1 μmol of glucose is one enzyme activity unit (IU).

[0156]

[0157] In formula (II):

[0158] G is the glucose content in the diluted sample solution, mg;

[0159] n is the dilution factor of the crude enzyme solution;

[0160] m is the volume of buffer used for leaching per gram of enzyme koji, mL / g dry koji;

[0161] V is the volume of the diluted crude enzyme solution participating in the reaction, mL;

[0162] 30 is the reaction time, min;

[0163] The method for determining xylanase activity is as follows: Take 9 μL of the crude enzyme solution into a centrifuge tube, inactivate the enzyme solution in the control group by boiling, add 200 μL of 1% xylan solution, mix well, and place it in a constant temperature water bath at 50 °C for 30 min; add 400 μL of DNS solution, accurately react in a boiling water bath for 5 min, then immediately cool, and make up the volume to 10.0 mL with distilled water. Use the boiled and inactivated enzyme solution as the blank control, measure the OD value at 540 nm, repeat the measurement 3 times for each group of experiments, and take the average value as shown in formula (III).

[0164] The definition of the enzyme activity unit for xylanase activity determination is: under the conditions of pH 4.8 and temperature 50 °C, the amount of enzyme required to hydrolyze the xylan substrate solution to produce 1 μmol of xylose per minute is one enzyme activity unit (IU).

[0165]

[0166] In formula (III):

[0167] c is the xylose content calculated from the xylose standard curve, mg / mL;

[0168] N is the total dilution factor of the enzyme solution;

[0169] V is the volume of the enzyme solution added to the reaction system, mL;

[0170] m is the volume of the liquid fermentation raw material, mL.

[0171] Example 3

[0172] This example provides an evaluation of the feeding effect and safety of a liquid synbiotic product on yellow - feather broilers. The liquid synbiotic product includes a cottonseed processing waste synbiotic feed additive, and the feeding method of the cottonseed processing waste synbiotic feed additive is as follows:

[0173] (1) Experimental animals and experimental design

[0174] Select 1600 one - day - old yellow - feather broilers that are strong and have similar weights [initial weight is (38.45 ± 0.2) g]. Randomly divide them into 4 groups, with 8 replicates in each group and 50 broilers in each replicate. The control group (CK - A group) is fed a basal diet, and the experimental groups are respectively added with 0.1% (A1 group), 1% (A2 group), and 5% (A3 group) g / kg of cottonseed processing waste synbiotic feed additive to the basal diet. The experimental period is 65 days. The basal diet used during the feeding of yellow - feather broilers is formulated with reference to the "Chicken Nutrition Standard" (NY / T33 - 2004).

[0175] (2) Feeding management

[0176] This experiment was carried out in the Xiaotuguli Farm in Hutubi County, Changji Prefecture. The feeding experiment used three - layer cage - raising. The light was provided for 16 hours and darkness for 8 hours every day. During the feeding period, the broilers had free access to food and water. The temperature, humidity, and immunity in the chicken house were controlled according to the requirements of conventional feeding management, and the health status of the chickens was observed every day.

[0177] (3) Growth performance

[0178] During the experimental period, observe the health status of the chickens every day, record the number of deaths and culls. At 23, 44, and 65 days of age of the broilers, the broilers were weighed on an empty stomach in each replicate, and the feed consumption of each replicate group of broilers during the experimental period was counted. Calculate the average body weight (ABW), average daily gain (ADG), average daily feed intake (ADFI), and feed - to - gain ratio (F / G).

[0179] (4) Slaughter performance

[0180] At 65 days of age, select one chicken close to the average weight in each replicate for slaughter, and measure the dressed weight, semi - eviscerated weight, eviscerated weight, breast muscle weight, leg muscle weight, and abdominal fat weight. Calculate the dressing percentage, semi - eviscerated percentage, breast muscle percentage, leg muscle percentage, lean meat percentage, and abdominal fat percentage.

[0181] Dressed weight: The weight after bleeding and defeathering. Use the wet - plucking method and weigh it after draining.

[0182] Semi - eviscerated weight: Based on the dressed weight, remove the trachea, esophagus, crop, intestine, spleen, gallbladder, and reproductive organs, and retain the heart, liver, kidneys, pancreas, lungs, gizzard (remove the cuticle and contents), and the surrounding fat and abdominal leaf fat.

[0183] Full eviscerated weight: Based on the semi-eviscerated weight, the weight of the heart, liver, pancreas, gizzard, fat, head, and feet is removed.

[0184] Abdominal fat: Abdominal fat is obtained by peeling off the abdominal leaf fat, fat around the gizzard and cloaca.

[0185] Dressing percentage (%) = (dressed weight / live weight) × 100%

[0186] Semi-eviscerated percentage (%) = (full eviscerated weight / dressed weight) × 100%

[0187] Full eviscerated percentage (%) = (full eviscerated weight / dressed weight) × 100%

[0188] Breast muscle percentage (%) = (leg muscle weight / full eviscerated weight) × 100%

[0189] Leg muscle percentage (%) = (leg muscle weight / full eviscerated weight) × 100%

[0190] Lean meat percentage (%) = breast muscle percentage (%) + leg muscle percentage (%)

[0191] Abdominal fat percentage = (abdominal fat weight / full eviscerated weight) × 100%

[0192] (5) Immune organ index

[0193] At 65 days of age, one chicken close to the average weight was selected from each replicate for slaughter. The thymus, pancreas, spleen, and bursa of Fabricius were dissected and weighed to calculate the immune organ index: Immune organ index (g / kg) = fresh weight of immune organ (g) / pre-slaughter live weight (kg).

[0194] (6) Serum indicators

[0195] At 65 days of age, one chicken close to the average weight was selected from each replicate. Blood was collected from the wing vein, placed at room temperature for 30 min, centrifuged at 4°C and 3000 r / min for 10 min, and the serum was collected. The serum immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were determined by enzyme-linked immunosorbent assay (ELISA); the activities of serum glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and the content of malondialdehyde (MAD) were determined by microplate method. The kits used for the above index determinations were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0196] (7) Intestinal morphological structure

[0197] At 65 days of age, take intestinal segments about 1 cm long from the duodenum, jejunum, and the terminal ileum, and place them in 10% formalin for fixation. Take the intestinal segment samples fixed for 24 h, and after flushing with water, dehydration with gradient ethanol, clearing with xylene, and paraffin embedding, make sections with a thickness of 6 μm for routine hematoxylin-eosin (HE) staining, and finally seal the sections with neutral resin. Observe the changes in the intestinal morphological structure under an electron microscope, select 10 intact and straight villi, measure the villus height and crypt depth, and calculate the villus height / crypt depth (V / C) value.

[0198] Effect verification three:

[0199] Effect verification of Example 3

[0200] 1. Effects of the compound bacteria-fermented cottonseed processing waste liquid synbiotic (cottonseed processing waste liquid synbiotic feed additive) on the growth performance of yellow-feathered broilers

[0201] As can be seen from Table 5, in terms of ABW, the ABW of yellow-feathered broilers in groups A1, A2, and A3 at 23 days of age was significantly higher than that in the CK-A group (P < 0.05), and at 65 days of age, the ABW of groups A1 and A2 was significantly higher than that in CK-A (P < 0.05); in terms of ADFI, during the period from 24 to 44 days of age, groups A1, A2, and A3 were significantly lower than the CK-A group (P < 0.05); in terms of F / G, during the period from 1 to 65 days of age, group A1 was significantly lower than the CK-A group (P < 0.05).

[0202] Table 5 Effects of the cottonseed processing waste liquid synbiotic feed additive on the growth performance of yellow-feathered broilers

[0203]

[0204] 2. Effects of adding the cottonseed processing waste liquid synbiotic feed additive on the slaughter performance of yellow-feathered broilers

[0205] As can be seen from Table 6, compared with A3, the slaughter rates of groups A1 and A2 were significantly increased (P < 0.05); compared with CK-A (control group), the semi-eviscerated rate (P < 0.05) and eviscerated rate (P < 0.05) of broilers in group A1 were significantly increased. There were no significant differences in the breast muscle rate, leg muscle rate, lean meat rate, and abdominal fat rate among groups (P > 0.05).

[0206] Table 6 Effects of the cottonseed processing waste liquid synbiotic feed additive on the slaughter performance of yellow-feathered broilers

[0207]

[0208] 3. Effects of adding the cottonseed processing waste liquid synbiotic feed additive on the immune organ indices of yellow-feathered broilers

[0209] As can be seen from Table 7, compared with the (CK-A) control group, the thymus indices of the A1, A2, and A3 groups were significantly increased (P<0.05). There were no significant differences in the pancreatic indices, spleen indices, and bursa of Fabricius indices among the groups (P>0.05).

[0210] Table 7 Effects of cottonseed processing waste liquid synbiotic feed additive on immune organ indices of yellow - feather broilers

[0211]

[0212] 4. Effects of adding cottonseed processing waste liquid synbiotic feed additive on serum immunoglobulin content of yellow - feather broilers

[0213] As can be seen from Table 8, compared with the (CK-A) control group, the serum IgM content of broilers in the A1 group was significantly increased (P<0.05); the IgA content of broilers in the A2 group was significantly increased (P<0.05); the serum IgA, IgM, and IgG contents of broilers in the A3 group were significantly increased (P<0.05).

[0214] Table 8 Effects of cottonseed processing waste liquid synbiotic feed additive on serum immunoglobulin content of yellow - feather broilers

[0215]

[0216] 5. Effects of adding cottonseed processing waste liquid synbiotic feed additive on serum antioxidant indices of yellow - feather broilers

[0217] As can be seen from Table 9, compared with the (CK-A) control group, the GSH-Px activities in the sera of broilers in the A1, A2, and A3 groups were significantly increased (P<0.001). There were no significant differences in SOD activities and MAD contents among the groups (P>0.05).

[0218] Table 9 Effects of cottonseed processing waste liquid synbiotic feed additive on serum antioxidant indices of yellow - feather broilers

[0219]

[0220] 6. Effects of adding cottonseed processing waste liquid synbiotic feed additive on intestinal morphological structure of yellow - feather broilers

[0221] The intestinal tissue sections of each group of broilers were analyzed, and the results of the villus height, crypt depth, and villus-crypt ratio of each intestinal segment are shown in Table 10. In terms of villus height, compared with CK-A (control group), the villus heights of the duodenum and jejunum of broilers in groups A1, A2, and A3 were significantly increased (P<0.05). There was no significant difference in the jejunal villus height among the groups (P>0.05); in terms of crypt depth, compared with CK-A (control group), the crypt depths of the duodenum and ileum in groups A1, A2, and A3 were significantly decreased (P<0.05); the crypt depths of the jejunum in groups A1 and A3 were significantly increased (P<0.01); in terms of the villus-crypt ratio, compared with CK-A (control group), the villus-crypt ratios of the duodenum and ileum of broilers in groups A1, A2, and A3 were significantly increased.

[0222] Table 10 Effects of cottonseed processing waste liquid synbiotic feed additive on the intestinal morphological structure of yellow-feathered broilers

[0223]

[0224] On the basis of having developed and provided the cottonseed processing waste liquid synbiotic feed additive, it is very easy for those skilled in the art to further associate that this feed additive is incorporated into a wider range of health-promoting products, such as synbiotic products. Synbiotic products usually combine probiotics, prebiotics, or other beneficial components, aiming to act synergistically on the intestinal health of animals and improve the overall health status. Therefore, providing a synbiotic product containing the above-mentioned cottonseed processing waste liquid synbiotic feed additive is a natural extension and innovation by those skilled in the art based on the existing technology.

[0225] In addition, applying the feed additive of the present invention in the process of preparing synbiotic products is also an application method that is easy for those skilled in the art to think of. This application not only expands the use range of the feed additive but also further enhances the value of the feed additive through the comprehensive benefits of synbiotic products.

[0226] Therefore, in the case where there already exists the cottonseed processing waste liquid synbiotic feed additive, it is very easy for those skilled in the art to think of and develop synbiotic products containing this feed additive and to apply this feed additive in the process of preparing synbiotic products. These innovations not only enrich the types of feed additives and synbiotic products but also provide more choices and possibilities for the health care of animals.

[0227] It can be seen from the effect verification experiment that the present invention provides a free gossypol-degrading bacterium suitable for cottonseed processing waste liquid. By optimizing the medium formula and the combination of free gossypol-degrading bacteria, the degradation rate of free gossypol in cottonseed processing waste liquid and the retention rate of oligosaccharides are improved, thereby ensuring the quality and safety of the synbiotic feed additive of cottonseed processing waste liquid. The functions are clarified by analyzing the active ingredients in the synbiotic feed additive product of cottonseed processing waste liquid. The feeding effect and application effect of the synbiotic feed additive of cottonseed processing waste liquid are evaluated by conducting animal feeding experiments.

[0228] It should be understood that the present invention disclosed is not limited to the specific methods, schemes and substances described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the present invention, the scope of which is limited only by the appended claims.

Claims

1. Free gossypol-degrading bacteria in cottonseed processing waste liquid, characterized in that, The screening medium for the free gossypol-degrading bacteria comprises cottonseed processing waste liquid, yeast extract powder, ammonium sulfate, sodium chloride, magnesium sulfate, potassium dihydrogen phosphate and water; The free gossypol-degrading bacteria are composed of Aspergillus niger, Lactobacillus gasseri and Saccharomyces cerevisiae.

2. The free gossypol-degrading bacterium according to claim 1, characterized in that, The screening method for the free gossypol-degrading bacteria is as follows: Per liter of the screening medium is added with: 60 - 300 g of cottonseed processing waste liquid, 2.5 - 3.5 g of yeast extract powder, 4 - 6 g of ammonium sulfate, 0.5 - 1 g of sodium chloride, 0.5 - 1 g of magnesium sulfate, 1 - 2 g of potassium dihydrogen phosphate, and water is added to make up the total volume to 1000 mL to obtain the screening medium; Under aseptic conditions, the gossypol-degrading bacteria are inoculated into the screening medium for fermentation culture.

3. The free gossypol-degrading bacterium according to claim 2, wherein The volume ratio of Aspergillus niger, Lactobacillus gasseri and Saccharomyces cerevisiae in the free gossypol-degrading bacteria is 1:2:

1.

4. The free gossypol-degrading bacterium according to claim 3, wherein The pH of the screening medium is 6.0 - 7.0, the inoculation amount of the free gossypol-degrading bacteria is 5%, the temperature of the fermentation culture is 20 - 30 °C, and the time of the fermentation culture is 7 - 9 d.

5. Cottonseed processing waste liquid symbiotic feed additive, characterized in that, The cottonseed processing waste liquid synbiotic feed additive comprises a cottonseed processing waste liquid culture medium and the free gossypol-degrading bacteria according to any one of claims 1 - 4. The cottonseed processing waste liquid culture medium comprises: cottonseed processing waste liquid, peptone, beef extract powder, yeast extract powder, sodium acetate, trisodium citrate, magnesium sulfate, potassium dihydrogen phosphate and water.

6. The cottonseed processing waste liquid symbiotic feed additive according to claim 5, wherein The preparation method of the cottonseed processing waste liquid synbiotic feed additive is as follows: Per liter of the cottonseed processing waste liquid culture medium is added with: 200 - 500 g of cottonseed processing waste liquid, 3 - 10 g of peptone, 0.5 - 5 g of beef extract powder, 1 - 4 g of yeast extract powder, 1 - 5 g of sodium acetate, 0.1 - 2 g of trisodium citrate, 0.1 - 1 g of magnesium sulfate, 0.1 - 2 g of potassium dihydrogen phosphate, and water is added to make up the total volume to 1000 mL; after the salts are dissolved, the pH of the cottonseed processing waste liquid culture medium is adjusted to 6.0 - 7.0 with sodium bicarbonate to obtain the cottonseed processing waste liquid culture medium; Under aseptic conditions, the free gossypol-degrading bacteria are inoculated into the cottonseed processing waste liquid culture medium according to the volume ratio of Aspergillus niger:Lactobacillus gasseri:Saccharomyces cerevisiae = 1:2:1, and shaken for fermentation to obtain the cottonseed processing waste liquid synbiotic feed additive.

7. The cottonseed processing waste liquid synbiotic feed additive according to claim 6, wherein the inoculation amount of the free gossypol-degrading bacteria is 5%, the temperature of the shaking fermentation is 20 - 30 °C, the time of the shaking fermentation is 7 - 9 d, and the rotation speed of the shaking fermentation is 90 - 200 r / min.

8. A liquid synbiotic product, characterized in that, The liquid synbiotic product comprises the cottonseed processing waste liquid synbiotic feed additive according to any one of claims 5 - 7.

9. Use of the cottonseed processing waste liquid synbiotic feed additive according to any one of claims 5 - 7 in high-density culture of probiotics and / or high-concentration retention of oligosaccharides.

10. Use of the cottonseed processing waste liquid synbiotic feed additive according to any one of claims 5 - 7 in the preparation of a liquid synbiotic product.

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