Solid fermentation feed additive based on cottonseed molasses and application thereof

Abstract

The invention belongs to the technical field of fermented feed, and relates to a solid fermented feed additive based on cottonseed molasses and application thereof. The invention provides a solid fermentation feed additive based on cottonseed molasses, the solid fermentation feed additive comprises a solid fermentation medium and a compound strain, the solid fermentation medium comprises cottonseed molasses and bran; the compound strains comprise lactic acid bacteria and saccharomycetes. According to the invention, the cottonseed molasses and the bran are mixed through a solid fermentation technology, and fermentation is carried out by using a compound strain, so that detoxification of the cottonseed molasses and the trophism of the fermented feed are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermented feed, and relates to a solid fermented feed additive based on raffinose molasses and its application. Background Art

[0002] In animal husbandry, the quality and nutritional components of feed are crucial for the growth and health of animals. Traditional feeds often rely on grains, soybean meal, etc. as the main raw materials, but these resources are becoming increasingly scarce, and the nutritional value of traditional feeds is limited, making it difficult to meet the requirements of modern animal husbandry for efficient, environmentally friendly, and healthy breeding. Therefore, the development of new, efficient, and environmentally friendly feeds has become a research hotspot in current animal husbandry.

[0003] As a by-product in the process of cottonseed processing, raffinose molasses contains rich nutritional components such as sugars, amino acids, vitamins, oligosaccharides, etc., and its nutritional value is much higher than that of beet molasses and soybean molasses, having potential feed application value. However, there are some problems in directly using raffinose molasses as feed, such as raffinose molasses containing some anti-nutritional factors and harmful substances (for example, free gossypol). Solid fermentation technology is a technology that uses microorganisms to ferment on solid substrates, having the advantages of simple operation, low cost, and easy separation of products.

[0004] Traditional methods for treating raffinose molasses often have problems such as low conversion efficiency and unstable product quality, making it difficult to meet the requirements of modern animal husbandry for high-quality feeds. Due to reasons such as improper selection of strains, unreasonable selection of auxiliaries, improper proportioning, and inappropriate process temperature, the nutritional components in the waste liquid cannot be fully utilized, so the prepared feed cannot meet the growth requirements of animals.

[0005] Therefore, in the technical field of fermented feed, it is particularly important to screen a composite strain with a suitable strain type and proper proportion for raffinose molasses. Summary of the Invention

[0006] The purpose of the present invention is to mix raffinose molasses with bran through solid fermentation technology and ferment with a composite strain, realizing the detoxification of raffinose molasses and the nutrition of fermented feed.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows:

[0008] In the first aspect, the present invention provides a solid fermented feed additive based on raffinose molasses, and the solid fermented feed additive includes a solid fermentation medium and a composite strain, wherein,

[0009] the solid fermentation medium includes raffinose molasses and bran;

[0010] the composite strain includes lactic acid bacteria and yeast.

[0011] Preferably, the mass ratio of the raffinose molasses to the bran is 2:3.

[0012] Preferably, the viable count ratio of the lactic acid bacteria to the yeast is 1:10.

[0013] Preferably, the lactic acid bacteria is Pediococcus acidilactici B2, and the yeast is Saccharomyces cerevisiae A15.

[0014] Preferably, the preparation method of the solid fermentation feed additive is as follows:

[0015] (1) Dry the raffinose molasses and the bran, mix the dried raffinose molasses and the bran according to the mass ratio of 2:3, and adjust the water content to 40 - 60% to obtain a solid fermentation medium;

[0016] (2) Mix the pre-fermented Pediococcus acidilactici B2 and Saccharomyces cerevisiae A15 according to the viable count ratio of 1:10 and dissolve them in deionized water to obtain a composite strain;

[0017] (3) Stir and mix the solid fermentation medium obtained in step (1) and the composite strain obtained in step (2) evenly, and perform fermentation culture to obtain a solid fermentation feed additive.

[0018] Preferably, the inoculation amounts of Pediococcus acidilactici B2 and Saccharomyces cerevisiae A15 in the composite strain are 5 - 20%; the temperature of the fermentation culture is 20 - 35°C; the time of the fermentation culture is at least 24 - 96 h.

[0019] In a second aspect, a synbiotic product is provided, and the synbiotic product includes the solid fermentation feed additive of the present invention.

[0020] In a third aspect, the application of the solid fermentation feed additive of the present invention in the preparation of a synbiotic product is provided.

[0021] In a fourth aspect, the application of the solid fermentation feed additive of the present invention in promoting the growth performance of yellow - feather broilers and / or increasing the relative intestinal length of yellow - feather broilers is provided.

[0022] In a fifth aspect, the application of the solid fermentation feed additive of the present invention in enhancing the immunity of poultry and / or promoting the intestinal digestion and absorption of poultry is provided.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] By adding the solid fermentation feed additive obtained by solid fermentation of raffinose molasses and bran using lactic acid bacteria and yeast to the basal diet, more comprehensive and balanced nutrition can be provided for animals.

[0025] (1) It can significantly promote the growth performance of yellow - feather broilers. As the feeding time increases, from 42 days of feeding to 63 days of feeding, the average daily gain and feed - to - gain ratio both show significant differences between the experimental group (the solid fermentation feed additive of the present invention + basal diet) and the control group (basal diet). Specifically, the average daily gain of the experimental group is (58.00 ± 9.28) g - (62.03 ± 8.04) g, and the average daily gain of the control group is (56.81 ± 4.53) g; the feed - to - gain ratio of the experimental group is (2.60 ± 0.09) - (2.74 ± 0.08), and the feed - to - gain ratio of the control group is (2.85 ± 0.04); indicating that the solid - state fermentation feed additive based on raffinose molasses can significantly promote the growth performance of yellow - feather broilers.

[0026] (2) It can improve the immune ability of poultry. Compared with the yellow - feather broilers fed only with the basal diet, there are significant differences in the liver index and gizzard index between the yellow - feather broilers fed with the basal diet supplemented with the solid fermentation feed additive of the present invention. Specifically, the liver index of the yellow - feather broilers fed with the basal diet supplemented with the solid fermentation feed additive of the present invention is (16.067 ± 1.404) - (19.54 ± 2.943), and the liver index of the yellow - feather broilers fed only with the basal diet is (18.071 ± 1.708); the gizzard index of the yellow - feather broilers fed with the basal diet supplemented with the solid fermentation feed additive of the present invention is (6.676 ± 0.953) - (8.132 ± 0.738), and the gizzard index of the yellow - feather broilers fed only with the basal diet is (6.696 ± 0.648); as the feeding dose increases, the immune organs show an increasing trend, indicating that the raffinose molasses fermentation culture can delay the decline of the immune organs of yellow - feather broilers, thereby improving the immune ability of poultry.

[0027] (3) It can increase the relative intestinal length of yellow - feather broilers. Feeding the basal diet supplemented with the solid - state fermented feed additive of the present invention can increase the relative jejunum length and relative ileum length of yellow - feather broilers. Specifically, the relative jejunum length of yellow - feather broilers fed the basal diet supplemented with the solid - state fermented feed additive of the present invention is (17.934 ± 2.43) cm / kg - (20.375 ± 2.564) cm / kg, while the relative jejunum length of yellow - feather broilers fed only the basal diet is (17.602 ± 1.818) cm / kg; the relative ileum length of yellow - feather broilers fed the basal diet supplemented with the solid - state fermented feed additive of the present invention is (20.742 ± 1.554) cm / kg - (23.048 ± 2.643) cm / kg, while the relative ileum length of yellow - feather broilers fed only the basal diet is (20.32 ± 2.434) cm / kg.

[0028] (4) It can increase the serum antioxidant indexes of yellow - feather broilers. Compared with those fed only the basal diet, the superoxide dismutase, malondialdehyde and glutathione peroxidase of broilers fed the basal diet supplemented with the solid - state fermented feed additive of the present invention are all significantly increased. Specifically, the superoxide dismutase of broilers fed the basal diet supplemented with the solid - state fermented feed additive of the present invention is (360.671 ± 44.342) U / mL - (398.504 ± 51.661) U / mL, malondialdehyde is (9.146 ± 0.554) nmol / mL, and glutathione peroxidase is (2967.073 ± 631.997) U / mL - (3633.449 ± 546.105) U / mL; while the superoxide dismutase of broilers fed only the basal diet is (333.289 ± 50.897) U / mL, malondialdehyde is (7.759 ± 0.884) nmol / mL, and glutathione peroxidase is (2674.39 ± 1007.337) U / mL.

[0029] (5) It can promote the intestinal digestion and absorption rate of poultry. Compared with those fed only the basal diet, feeding the basal diet supplemented with the solid - state fermented feed additive of the present invention significantly increases the villus length of different intestinal segments of broilers, indicating that the solid - state fermented feed additive based on raffinose molasses can promote the intestinal digestion and absorption rate of poultry; in addition, the crypt depth CD of each intestinal segment of broilers fed the basal diet supplemented with the solid - state fermented feed additive of the present invention decreases. The shallower the crypt, the better, and the deeper the crypt, the worse the absorption ability. The larger the villus - to - crypt ratio, the better, indicating that feeding the solid - state fermented feed additive based on raffinose molasses can improve digestion and absorption and increase the digestion and absorption area. Specifically, the jejunum crypt depth of broilers fed the basal diet supplemented with the solid - state fermented feed additive of the present invention is (227.75 ± 12.32) μm - (244.03 ± 27.60) μm, while the jejunum crypt depth of broilers fed only the basal diet is (269.89 ± 95.91) μm.

[0030] (6) It can improve the safety of broilers. By analyzing the intestinal tissue sections of broilers, there is a certain degree of shedding or breakage of the intestinal wall villi fed with the solid-state fermented feed additive based on raffinose molasses. This is particularly obvious in the duodenum, but not significant in the jejunum and ileum, indicating that the solid-state fermented feed additive based on raffinose molasses is relatively safe for broilers. Description of the Drawings

[0031] Figure 1 It is the growth curve of lactic acid bacteria.

[0032] Figure 2 It is the growth curve of yeast.

[0033] Figure 3 It is the total acid content produced by lactic acid bacteria fermenting raffinose molasses at different time periods.

[0034] Figure 4 It is the total acid content produced by yeast fermenting raffinose molasses at different time periods.

[0035] Figure 5 It is the plate antagonism experiment.

[0036] Figure 6 It is the influence of the material ratio on the total acid production and pH value.

[0037] Figure 7 It is the influence of the strain ratio on the pH value.

[0038] Figure 8 It is the influence of the strain inoculation amount on the pH value.

[0039] Figure 9 It is the influence of the fermentation temperature on the pH value.

[0040] Figure 10 It is the influence of the fermentation time on the pH value.

[0041] Figure 11 It is the sectioning result of the solid-state fermented feed additive fed to different intestinal segments of poultry. Detailed Implementation Modes

[0042] The following will specifically elaborate on the present invention in combination with the detailed implementation modes 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 detailed implementation modes and examples are used to illustrate the present invention, rather than to limit the present invention.

[0043] 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.

[0044] Example 1 Strain Screening

[0045] 1. Fermentation Strains

[0046] The strains used for fermenting raffinose molasses include: lactic acid bacteria: Lactobacillus helveticus DLX, Lactobacillus gasseri HX, Lactobacillus fermentum A3, Pediococcus acidilactici B2, Lactobacillus panis B6; yeasts: Saccharomyces cerevisiae A1, Saccharomyces cerevisiae A2, Saccharomyces cerevisiae A10, Saccharomyces cerevisiae A11, Saccharomyces cerevisiae A12, Saccharomyces cerevisiae A13, Saccharomyces cerevisiae A15, Saccharomyces cerevisiae YM, Candida utilis 1314, Candida utilis CR, Pichia kudriavzevii KDL;

[0047] The above strains were all purchased from the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. Among them, Lactobacillus helveticus DLX refers to the lactic acid bacterium with the strain preservation number of CGMCC No. 1.9090, Lactobacillus gasseri HX refers to the lactic acid bacterium with the strain preservation number of CGMCC No. 1.3224, Lactobacillus fermentum A3 refers to the lactic acid bacterium with the strain preservation number of CGMCC No. 1.15608, Pediococcus acidilactici B2 refers to the lactic acid bacterium with the strain preservation number of CGMCC No. 1.12332, Lactobacillus panis B6 refers to the lactic acid bacterium with the strain preservation number of CGMCC No. 1.3924, Saccharomyces cerevisiae A1 refers to the yeast with the strain preservation number of CGMCC No. 2.3973, Saccharomyces cerevisiae A2 refers to the yeast with the strain preservation number of CGMCC No. 2.3889, Saccharomyces cerevisiae A10 refers to the yeast with the strain preservation number of CGMCC No. 2.3888, Saccharomyces cerevisiae A11 refers to the yeast with the strain preservation number of CGMCC No. 2.3886, Saccharomyces cerevisiae A12 refers to the yeast with the strain preservation number of CGMCC No. 2.3880, Saccharomyces cerevisiae A13 refers to the yeast with the strain preservation number of CGMCC No. 2.3875, Saccharomyces cerevisiae A15 refers to the yeast with the strain preservation number of CGMCC No. 2.3871, Saccharomyces cerevisiae YM refers to the yeast with the strain preservation number of CGMCC No. 2.3868, Candida utilis 1314 refers to the yeast with the strain preservation number of CGMCC No. 2.2876, Candida utilis CR refers to the yeast with the strain preservation number of CGMCC No. 2.3047, and Pichia kudriavzevii KDL refers to the yeast with the strain preservation number of CGMCC No. 2.5428.

[0048] 2. Culture Medium

[0049] 2.1 Activation Medium for Lactic Acid Bacteria Strains

[0050] MRS agar medium (g / L): Peptone 10.0 g, Beef extract powder 8.0 g, Yeast extract powder 4.0 g, Glucose 20.0 g, Disodium hydrogen phosphate 2.0 g, Diammonium hydrogen citrate 2.0 g, Sodium acetate 5.0 g, Magnesium sulfate 0.2 g, Manganese sulfate 0.04 g, Agar 14.0 g, Tween 80 1.0 g, pH 6.5 ± 0.2, Distilled water 1000 mL, Autoclaved at 121 °C for 15 min.

[0051] MRS broth medium (g / L): Peptone 10.0 g, Beef extract powder 8.0 g, Yeast extract powder 4.0 g, Glucose 20.0 g, Disodium hydrogen phosphate 2.0 g, Diammonium hydrogen citrate 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, Distilled water 1000 mL, Autoclaved at 118 °C for 15 min.

[0052] 2.2 Yeast strain activation medium

[0053] Yeast extract peptone dextrose agar medium YPD (g / L): Peptone 10.0 g, Glucose 20.0 g, Yeast extract powder 5.0 g, Agar 14.0 g, Distilled water 1000 mL, Autoclaved at 121 °C for 15 min.

[0054] YPD liquid medium (g / L): Peptone 20.0 g, Glucose 20.0 g, Yeast extract powder 10.0 g, pH 6.5 ± 0.2, Distilled water 1000 mL, Autoclaved at 121 °C for 15 min.

[0055] 2.3 Free gossypol degradation screening medium

[0056] Raffinose molasses medium (g / L): Raffinose molasses 60.0 g, Ammonium sulfate 5.0 g, Sodium chloride 1 g, Magnesium sulfate 0.5 g, Potassium dihydrogen phosphate 1 g, pH 6.2 ± 0.2, Distilled water 1000 mL, Autoclaved at 105 °C for 15 min.

[0057] 3. High performance liquid chromatography

[0058] 1) Instrument and equipment:

[0059] Shimadzu high performance liquid chromatography (Made in Japan), Equipment model: LC-40, Equipped with a diode array detector, Equipped with an Agilent TC-C18(2) chromatographic column, Specification: 250 mm × 4.6 mm, 5 μm.

[0060] 2) Standard reagent:

[0061] Formic acid, Acetic acid, Propionic acid, Butyric acid, Lactic acid and Citric acid (Purity ≥ 99%).

[0062] Mobile phase reagents:

[0063] Methanol (chromatographic grade), potassium dihydrogen phosphate (analytical grade), deionized water.

[0064] 3) Preparation of sample solution

[0065] Seven probiotic strains to be screened (Lactobacillus helveticus DLX, Lactobacillus gasseri HX, Lactobacillus fermentum A3, Lactobacillus parafarraginis B6, Pediococcus acidilactici B2, Saccharomyces cerevisiae A15, Pichia kudriavzevii KDL) were separately inoculated into the raffinose molasses medium. The control group CK was not inoculated with strains. The fatty acid components and contents at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h of fermentation were measured respectively.

[0066] 4) Sample treatment method

[0067] After dilution, the sample solution was filtered through a 0.22 μm aqueous filter membrane and analyzed by high performance liquid chromatography.

[0068] 5) Chromatographic conditions

[0069] Chromatographic column: Agilent TC-C18(2) chromatographic column, specification: 250 mm × 4.6 mm, 5 μm; Mobile phase: potassium dihydrogen phosphate solution, 0.05 M (pH 2.7) / methanol: 97 / 3 (V / V) (gradient elution); Flow rate: 0.6 mL / min; Injection volume: 20 μL; Column temperature: 25 °C; Detection wavelength: 214 nm.

[0070] 4. Effect verification test

[0071] 1) Screening of high-efficiency free gossypol-degrading bacteria

[0072] The results of the comparison of free gossypol degradation of these 16 strains of bacteria are shown in Table 1. Each strain was set with 5 replicates. Pediococcus acidilactici B2, Lactobacillus helveticus DLX, and Lactobacillus fermentum A3 had the highest degradation rates (degradation rate ≥ 70%) for free gossypol in raffinose molasses. Yeast strains generally had lower degradation rates for free gossypol in raffinose molasses. Candida utilis and Pichia kudriavzevii were generally higher than Saccharomyces cerevisiae. However, these three yeast strains superior to them were non-feed yeast strains. Therefore, only Saccharomyces cerevisiae A15 was considered as an alternative strain.

[0073] Table 1 Free gossypol degradation rate

[0074] Number Strain Free gossypol degradation rate (%) 1 Lactobacillus helveticus DLX 67.02±0.57 2 Lactobacillus gasseri HX 71.27±0.64 3 Lactobacillus fermentum A3 70.31±0.40 4 Lactobacillus paralimentarius B6 65.82±0.69 5 Pediococcus acidilactici B2 72.71±0.83 6 Saccharomyces cerevisiae A1 19.80±3.38 7 Saccharomyces cerevisiae A2 17.65±2.64 8 Saccharomyces cerevisiae A10 7.99±11.30 9 Saccharomyces cerevisiae A11 12.79±2.85 10 Saccharomyces cerevisiae A12 19.38±1.60 11 Saccharomyces cerevisiae A13 16.28±1.28 12 Saccharomyces cerevisiae A15 19.82±1.40 13 Saccharomyces cerevisiae YM 18.26±0.21 14 Candida utilis 1314 38.11±0.04 15 Candida utilis CR 34.42±0.71 16 Pichia kudriavzevii KDL 36.86±0.98

[0075] 2) Strain growth performance

[0076] From Figure 1It can be seen that the growth rate of lactic acid bacteria is faster under the culture conditions of MRS broth medium than that of raffinose molasses medium. For example, when lactic acid bacteria are cultured in MRS broth medium, the fermentation time of 0-4h is the lag phase, the fermentation time of 4-10h is the logarithmic growth phase of the strain, during which the growth rate of the strain is the fastest, and the stable phase is entered after 12h of fermentation (see Figure 1 the DLX, HX, A3, B2, B6 curves in Figure 1 ). When lactic acid bacteria are cultured with raffinose molasses as the medium, the fermentation time of 0-24h is the lag phase, the fermentation time of 24-36h is the logarithmic growth phase of the strain, during which the growth rate of the strain is the fastest, and the stable phase is entered after 36h of fermentation (see

[0077] the TDLX, THX, TA3, TB2, TB6 curves in Figure 2 ). Among them, lactic acid bacteria B2 has the fastest growth rate and the highest biomass both in MRS medium and raffinose molasses medium. Figure 2 It can be seen from Figure 2 that for yeast, there is little difference in the growth rate of yeast under the culture conditions of YPD liquid medium (see the growth curves of YA1, YA2, YA11, YA12, YA13, YA15, YYM, Y1314 in

[0078] ) and raffinose molasses medium (see the growth curves of TA1, TA2, TA11, TA12, TA13, TA15, TYM, T1314 in

[0079] ). Generally speaking, YPD liquid medium is slightly better than raffinose molasses medium, indicating that using raffinose molasses medium as the culture condition is very suitable for the growth of yeast, and raffinose molasses medium can be used as the growth medium for yeast. The fermentation time of yeast of 0-4h is the lag phase, the fermentation time of 4-12h is the logarithmic growth phase of the strain, during which the growth rate of the strain is the fastest, and the stable phase is entered after 18h of fermentation. Saccharomyces cerevisiae A15 has the fastest growth rate and the highest biomass both in YPD medium and raffinose molasses medium.

[0080] Furthermore, the acid production capabilities of the 5 lactic acid bacteria strains with the highest free gossypol degradation rate were compared using raffinose molasses fermentation medium. It can be seen from Figure 3It can be seen that when the fermentation time of Pediococcus acidilactici B2 and Lactobacillus fermentum A3 is 120 h, the total acid production content is the highest. At the fermentation time of 120 h, the total acid production of Pediococcus acidilactici B2 and Lactobacillus fermentum A3 reaches 97.2 ± 0.0 g / L, which is significantly higher than that of other strains (P < 0.05). With the increase of fermentation time, the total acid production content of lactic acid bacteria generally shows an upward trend, but the total acid production content of Lactobacillus parafarraginis B6, Lactobacillus gasseri HX and the uninoculated control group CK shows a decreasing trend with the increase of fermentation time.

[0081] The dynamic changes of total acid production by yeast fermenting raffinose molasses are shown in Figure 4 , the acid production of Saccharomyces cerevisiae YM is the highest at 48 h, reaching 54.00 ± 3.6 g / L, but there is no difference compared with strains such as Saccharomyces cerevisiae A1, A2, A11, A15, etc. (P > 0.05); the total acid production of Saccharomyces cerevisiae A15 is the highest at 72 h of fermentation, reaching 57.6 ± 7.2 g / L, which is significantly higher than that of other strains (P < 0.05). With the increase of fermentation time, when Saccharomyces cerevisiae A2 ferments for 120 h, the total acid production content is the highest, reaching 57.6 ± 3.6 g / L.

[0082] 4) Dynamic changes of organic acid types and contents produced by strains fermenting raffinose molasses at different time periods

[0083] By using high performance liquid chromatography (HPLC), the changes of organic acid production by strains fermenting raffinose molasses at different times were analyzed, and the results are shown in Table 2. A total of five organic acids were detected, namely formic acid, acetic acid, propionic acid, lactic acid and citric acid. Butyric acid was not detected during the whole fermentation process of all strains. The highest formic acid content was detected in Lactobacillus fermentum A3. At 72 h and 96 h of fermenting raffinose molasses, the detected amount was 1540 mg / kg; the highest acetic acid content was detected in Candida utilis CR. At 96 h of fermenting raffinose molasses, the detected amount was 3880 mg / kg; the highest propionic acid content was detected in Pediococcus acidilactici B2. At 120 h of fermenting raffinose molasses, the detected amount was 1390 mg / kg; the highest lactic acid content was detected in Lactobacillus parafarraginis B6. At 120 h of fermenting raffinose molasses, the detected amount was 15500 mg / kg; the highest citric acid content was detected in the uninoculated control group CK. At 24 h of fermenting raffinose molasses, the detected amount was 1530 mg / kg.

[0084] Table 2 Dynamic changes of organic acid contents produced by strains fermenting raffinose molasses at different times

[0085]

[0086]

[0087]

[0088] Note: "——" in the table indicates not detected.

[0089] 5) Effect of raffinose molasses on viable cell count of strains

[0090] The viable cell counts of lactic acid bacteria and yeast were evaluated using a fermentation medium containing raffinose molasses. As shown in Table 3, the viable cell count of Pediococcus acidilactici B2 was the highest among lactic acid bacteria, reaching 3.60 ± 0.44×10 9 CFU / mL, significantly higher than that of other strains (P<0.05). The viable cell count of Saccharomyces cerevisiae A15 reached the highest 1.91 ± 0.14×10 9 CFU / mL, significantly higher than that of other Saccharomyces cerevisiae strains (P<0.05).

[0091] Table 3 Determination of viable cell count of lactic acid bacteria fermenting raffinose molasses

[0092] Number Strain Viable cell count (CFU) / mL 1 Lactobacillus helveticus DLX <![CDATA[1.12±0.064×10 9 > 2 Lactobacillus gasseri HX <![CDATA[1.43±0.21×10 9 > 3 Lactobacillus fermentum A3 <![CDATA[1.97±0.31×10 9 > 4 Lactobacillus paralimentarius B6 <![CDATA[1.28±0.044×10 9 > 5 Pediococcus acidilactici B2 <![CDATA[3.60±0.44×10 9 > 6 Saccharomyces cerevisiae A1 1.05±0.057×10 7 Saccharomyces cerevisiae A2 <![CDATA[8.43±1.20×10 8 > 8 Saccharomyces cerevisiae A10 <![CDATA[1.48±0.15×10 8 > 9 Saccharomyces cerevisiae A11 <![CDATA[1.08±0.18×10 8 > 10 Saccharomyces cerevisiae A12 <![CDATA[1.19±0.024×10 8 > 11 Saccharomyces cerevisiae A13 <![CDATA[1.03±0.12×10 8 > 12 Saccharomyces cerevisiae A15 <![CDATA[1.91±0.14×10 9 >

[0093] 6) Effect of strain combination

[0094] Based on the above results, Pediococcus acidilactici B2 and Saccharomyces cerevisiae A15 have the best free gossypol degradation ability, high acid production ability, growth performance and biomass. Therefore, these two strains were used as subsequent solid fermentation strains. Further, an antagonism experiment was conducted on these two strains, and the results showed that there was no antagonism between the two strains (see Figure 5 ). In addition, the viable cell counts of the two strains after combination were compared. The results are shown in Table 4. The total viable cell count of the two strains did not change significantly. Moreover, the degradation rate of free gossypol in the raffinose molasses fermentation broth after strain combination increased from the highest 72.71% of single strain fermentation to 75.24 ± 0.49%, significantly higher than that of single strain fermentation (P<0.05). And the total acid content also increased to 112.8 ± 2.1 g / L, significantly higher than that of single strain fermentation (P<0.05). The viable cell count increased to 4.20 ± 0.27×10 9 CFU / mL, significantly higher than that of single strain fermentation (P<0.05). It can be seen that after the combination of Pediococcus acidilactici B2 and Saccharomyces cerevisiae A15, all indexes of fermented raffinose molasses were improved (Table 4).

[0095] Table 4 Comparison before and after strain combination

[0096] Item Free gossypol degradation rate (%) Total acid production content (g / L) Viable cell count (CFU) / mL B2 <![CDATA[72.71±0.83 b > <![CDATA[97.2±3.6 b > <![CDATA[3.60±0.44×10 9a > A15 <![CDATA[19.82±1.40 c > <![CDATA[57.6±3.6 c > <![CDATA[1.91±0.14×10 9b <!-- 9 -->]]> Compound of B2 and A15 <![CDATA[75.24±0.49 a > <![CDATA[112.8±2.1 a > <![CDATA[4.20±0.27×10 9a >

[0097] Example 2 Solid fermentation feed additive based on raffinose molasses

[0098] In this example, raffinose molasses was used as an energy source and wheat bran was added, and a solid fermentation was carried out using a composite bacteria of lactic acid bacteria B2 and yeast A15 to prepare a feed additive.

[0099] 1. Specific formula (fermentation conditions)

[0100] Specific formula (fermentation conditions): ① Optimal material ratio for solid fermentation of raffinose molasses: raffinose molasses: wheat bran = 2:3 (dry matter content); ② Optimal water content for solid fermentation of raffinose molasses: 40%; ③ Optimal ratio for strain compounding: lactic acid bacteria: yeast = 10:1 (order of magnitude); ④ Strain inoculation amount: 15%; ⑤ Temperature: 30 °C.

[0101] 2. Preparation method

[0102] Preparation method: Raffinose molasses comes from Xinjiang Taikun Group Changji Feed Co., Ltd., and wheat bran is commercially available. Dry the raffinose molasses and wheat bran, mix the raffinose molasses and wheat bran in a ratio of 2:3 (mass ratio), calculate the amount of liquid required at a water content of 40%, pre-ferment lactic acid bacteria B2 and yeast A15 and mix them in a ratio of 10:1 (order of magnitude) and dissolve them in deionized water (inoculation amount 15%), stir and mix evenly, and place them at 30 °C for fermentation for more than 72 h (the longer the fermentation time, the higher the gossypol degradation rate).

[0103] 3. Effect verification

[0104] 1) Optimal ratio of raffinose molasses to wheat bran

[0105] The total acid production content of raffinose molasses: wheat bran (mass ratio) was measured under the conditions that the viable cell counts of Pediococcus acidilactici B2 and Saccharomyces cerevisiae A15 strains were 1:1, the inoculation amount was 10%, the initial water content was 50%, and fermentation was carried out at 30 °C for 72 h. The results are shown in Figure 6 . When raffinose molasses: wheat bran = 40:60, the total acid production content is the highest, reaching 370.8 ± 7.2 g / kg, which is significantly higher than other groups (P < 0.05). As the proportion of raffinose molasses increases, the total acid production content shows a trend of first increasing and then decreasing. The pH value results also show that when raffinose molasses: wheat bran = 40:60, the fermentation pH value is the lowest, reaching 4.283 ± 0.031, which is significantly lower than other groups (P < 0.05). In addition, as the ratio of raffinose molasses increases, the fermentation pH value shows a trend of first decreasing, then increasing, and then decreasing again.

[0106] As can be seen from Table 5, when raffinose molasses: wheat bran = 40:60, the highest free gossypol degradation rate reaches 79.17 ± 0.22%, which is significantly higher than other groups (P < 0.05). As the raffinose molasses ratio increases, the free gossypol degradation rate shows a trend of first increasing and then decreasing.

[0107] Table 5 Effect of material ratio on free gossypol degradation rate (50% initial water content)

[0108] Number Material ratio Free gossypol degradation rate (%) 1 Raffinose molasses: Bran = 50:50 <![CDATA[25.75±0.27 c > 2 Raffinose molasses: Bran = 60:40 <![CDATA[19.51±1.05 c > 3 Raffinose molasses: Bran = 80:20 <![CDATA[0.00±0.00 d > 4 Raffinose molasses: Bran = 90:10 <![CDATA[0.00±0.00 d > 5 Raffinose molasses: Bran = 40:60 <![CDATA[79.17±0.22 a > 6 Raffinose molasses: Bran = 30:70 <![CDATA[54.24±0.50 b > 7 Raffinose molasses: Bran = 20:80 <![CDATA[51.32±0.65 b > 8 Raffinose molasses: Bran = 10:90 <![CDATA[18.14±1.25 c >

[0109] As can be seen from Table 6, when raffinose molasses: wheat bran = 40:60, the highest viable cell count reaches 6.85 ± 0.21×109 CFU / g, significantly higher than other groups (P < 0.05), the material ratio is raffinose molasses: wheat bran = 50:50. Secondly, the viable count reaches 3.70 ± 0.57×10 9 CFU / g, significantly higher than other groups (P < 0.05). As the ratio of raffinose molasses increases, the viable count first increases and then decreases. Therefore, it is determined that raffinose molasses: wheat bran (mass ratio) = 40:60.

[0110] Table 6 Effect of material ratio on viable count (50% initial water content)

[0111]

[0112]

[0113] 2) Optimal initial water content for material fermentation

[0114] The initial fermentation moisture content of the material was compared at 30%, 40%, and 50%. The fermentation conditions were: raffinose molasses: wheat bran = 40:60, Pediococcus acidilactici B2 and Saccharomyces cerevisiae A15 strains were mixed at a viable count ratio of 1:1, the inoculation amount was 10%, and fermentation was carried out at 30°C in a constant temperature incubator for 72 h. As shown in Table 7, as the initial water content increases, the degradation rate of free gossypol first increases and then decreases, and the degradation rate of free gossypol is the highest at 40% water content.

[0115] Table 7 Effect of initial water content of material on degradation rate of free gossypol

[0116] Number Initial water content of material Free gossypol degradation rate % 1 30％ <![CDATA[40.70±0.18 b > 2 40％ <![CDATA[56.69±0.73 a > 3 50％ <![CDATA[19.17±0.22 c >

[0117] The pH value changes of the fermentation products after 72 h of fermentation under different initial moisture content conditions of the material are shown in Table 8. As the water content increases, the pH value of the fermentation product also shows a gradually increasing trend, and the lower the water content, the lower the fermentation pH value. Therefore, considering comprehensively, 40% water content is selected as the best.

[0118] Table 8 Effect of initial water content of material on pH after fermentation

[0119] Number Initial water content of material pH 1 30％ 4.04 2 40％ 4.37 3 50％ 4.43

[0120] 3) Strain ratio

[0121] The different ratios of Pediococcus acidilactici B2 and Saccharomyces cerevisiae A15 were compared. The fermentation conditions were: the initial water content was 40%, the mass ratio of raffinose molasses to wheat bran = 40:60, the strain inoculation amount was 10%, the constant temperature was 30°C, and the fermentation was carried out for 72 h. Pediococcus acidilactici B2: Saccharomyces cerevisiae A15 = 1:1, 1:10, 1:100, 1:1000, 10:1, 100:1, 1000:1. By Figure 7It can be seen that when the ratio of Pediococcus acidilactici B2 to Saccharomyces cerevisiae A15 is 1000:1, the fermentation pH value is the lowest, reaching 4.4 ± 0.095, which is significantly lower than that of other groups (P < 0.05). As the ratio of Pediococcus acidilactici B2 increases, the fermentation pH value generally shows a downward trend, and when the ratio of Pediococcus acidilactici B2 to Saccharomyces cerevisiae A15 is 1:10, the fermentation pH value increases significantly. As can be seen from Table 9, when the ratio of Pediococcus acidilactici B2 to Saccharomyces cerevisiae A15 is 1:10, the highest degradation rate of free gossypol reaches 57.02 ± 1.52%, and as the ratio of Pediococcus acidilactici B2 increases, the degradation rate of free gossypol shows a trend of first increasing, then decreasing, then increasing again, and then decreasing again. Therefore, the ratio of Pediococcus acidilactici B2 to Saccharomyces cerevisiae A15 = 1:10 is selected as the optimal ratio.

[0122] Table 9 Effects of strain ratios on the degradation rate of free gossypol

[0123] Number Strain ratio Free gossypol degradation rate (%) 1 B2:A15 = 1:1 <![CDATA[48.96±1.37 d > 2 B2:A15 = 1:10 <![CDATA[57.02±1.52 a > 3 B2:A15 = 1:100 <![CDATA[55.84±0.85 ab > 4 B2:A15 = 1:1000 <![CDATA[51.65±0.46 cd > 5 B2:A15 = 10:1 <![CDATA[56.88±0.80 a > 6 B2:A15 = 100:1 <![CDATA[53.55±1.17 bc > 7 B2:A15 = 1000:1 <![CDATA[52.28±0.69 c >

[0124] 4) Optimal inoculation amount of the strain

[0125] The different inoculation amounts of the strain were compared under the fermentation conditions: the initial water content was 40%, the mass ratio of raffinose molasses to wheat bran was 40:60, the ratio of viable cell numbers of Pediococcus acidilactici B2 to Saccharomyces cerevisiae A15 was 1:10, the constant temperature was 30 °C, the fermentation time was 72 h, and the inoculation amounts of the compound bacteria were 1%, 5%, 10%, 15%, and 20%. As Figure 8 can be seen, when the inoculation amount of the strain is 10%, the fermentation pH value is the lowest, reaching 4.29 ± 0.125. As the inoculation amount of the strain increases, the fermentation pH value shows a trend of first increasing, then decreasing, and then increasing again.

[0126] As can be seen from Table 10, when the inoculation amount of the strain is 15%, the highest degradation rate of free gossypol reaches 74.23 ± 0.43%, which is significantly higher than that of other groups (P < 0.05). Therefore, the inoculation amount of 15% is selected as the optimal inoculation amount.

[0127] Table 10 Effects of strain inoculation amounts on the degradation rate of free gossypol

[0128] Number Strain inoculation amount (%) Free gossypol degradation rate (%) 1 1 <![CDATA[52.80±1.55 c > 2 5 <![CDATA[50.33±1.18 c > 3 10 <![CDATA[62.32±0.52 b > 4 15 <![CDATA[74.23±0.43 a > 5 20 <![CDATA[69.34±0.38 a >

[0129] 5) Determination of the optimal fermentation temperature

[0130] The effects of different temperatures on fermentation were compared under the fermentation conditions: the initial water content was 40%, the ratio of raffinose molasses to wheat bran (mass ratio) = 40:60, Pediococcus acidilactici B2:Saccharomyces cerevisiae A15 = 1:10, the fermentation time was 72 h, and the cultures were incubated at constant temperatures of 20 °C, 25 °C, 30 °C, 35 °C, and 40 °C. As Figure 9It can be seen that with the increase of the culture temperature, the fermentation pH shows a downward trend. The pH decreases relatively slowly at 25°C - 35°C, while when the fermentation temperature is 40°C, the pH value reaches the lowest at 4.723 ± 0.023, which is significantly lower than that of other groups (P < 0.05).

[0131] As can be seen from Table 11, when the fermentation temperature is 30°C, the highest degradation rate of free gossypol reaches (83.09 ± 1.23)%, which is significantly higher than that of other groups (P < 0.05). The second is when the fermentation temperature is 35°C, and the degradation rate of free gossypol reaches (80.75 ± 1.83)%, which is significantly higher than that of other groups (P < 0.05). When the fermentation temperature is lower than 30°C, the degradation rate of free gossypol increases with the increase of the fermentation temperature. When the fermentation temperature is higher than 30°C, the degradation rate of free gossypol decreases with the increase of the fermentation temperature. Therefore, the fermentation temperature of 30°C is selected as the best.

[0132] Table 11 Effects of fermentation temperature on the degradation rate of free gossypol

[0133] Number Fermentation temperature (°C) Free gossypol degradation rate (%) 1 20 <![CDATA[53.99±0.36 c > 2 25 <![CDATA[56.78±4.18 c > 3 30 <![CDATA[83.09±1.23 a > 4 35 <![CDATA[80.75±1.83 ab > 5 40 <![CDATA[79.45±1.19 b >

[0134] 6) Determination of fermentation time

[0135] The effects of fermentation time on fermentation were compared. Fermentation conditions: initial water content was 40%, the ratio of raffinose molasses to bran (mass ratio) = 40:60, Pediococcus acidilactici B2: Saccharomyces cerevisiae A15 = 1:10, cultured at a constant temperature of 30°C for 3d, 5d, 7d, 9d, 11d, 13d and 15d. From Figure 10 It can be seen that the fermentation pH value decreases with the increase of fermentation time. When the fermentation time is 11d, the fermentation pH value is the lowest, reaching 3.81 ± 0.00, which is significantly lower than that of other groups (P < 0.05).

[0136] As can be seen from Table 12, when the fermentation time is 15d, the highest degradation rate of free gossypol reaches (92.34 ± 0.10)%, which is significantly higher than that of other groups (P < 0.05). The second is when the fermentation time is 13d, and the degradation rate of free gossypol reaches (90.53 ± 0.52)%, which is significantly higher than that of other groups (P < 0.05). The degradation rate of free gossypol increases with the increase of fermentation time. Therefore, considering fermentation comprehensively, 7d is the best.

[0137] Table 12 Effects of fermentation time on the degradation rate of free gossypol

[0138] Number Fermentation time (d) Free gossypol degradation rate (%) 1 3d <![CDATA[40.85±0.35 f > 2 5d <![CDATA[47.71±0.79 e > 3 7d <![CDATA[74.75±0.68 d > 4 9d <![CDATA[79.80±0.69 c > 5 11d <![CDATA[86.39±0.84 b > 6 13d <![CDATA[90.53±0.52 a > 7 15d <![CDATA[92.34±0.10 a >

[0139] Example 3 Animal feeding effect and safety evaluation

[0140] This example provides an evaluation of the feeding effect and safety of a solid-fermented feed additive based on raffinose molasses for yellow-feathered broilers. The feeding method of the solid-fermented feed additive based on raffinose molasses is as follows:

[0141] 1. Experimental animals and experimental design

[0142] Select 1600 one-day-old yellow-feathered broilers that are strong and have similar weights (initial weight is 38.45 ± 0.2 g), and 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 (corn-soybean meal type diet, specific components are shown in Table 13), and the experimental groups are respectively added with 0.2% (K1 group), 0.4% (K2 group), and 0.6% (D group) g / kg of the solid-fermented feed additive of raffinose molasses in the basal diet. The experimental period is 65 days. The basal diet used in the feeding process of yellow-feathered broilers is formulated with reference to the "Chicken Nutrition Standard" (NY / T 33-2004).

[0143] Table 13 Corn-soybean meal type diet formula

[0144]

[0145]

[0146] 2. Feeding management

[0147] This experiment was carried out in the Xiaotuguli Farm in Hutubi County, Changji Prefecture, Xinjiang Uygur Autonomous Region. The feeding experiment was carried out in three-layer cages, with 16 hours of light and 8 hours of darkness 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.

[0148] 3. Growth performance

[0149] During the experimental period, the health status of the chickens was observed every day, the number of dead and culled chickens was recorded. At 22, 42, and 63 days of age of the broilers, the broilers were weighed on an empty stomach in each replicate, and the feed consumption of the broilers in each replicate group during the experimental period was counted. The average body weight (ABW), average daily gain (ADG), average daily feed intake (ADFI), and feed-to-gain ratio (F / G) were calculated.

[0150] 4. Slaughter performance

[0151] At 65 days of age, one chicken close to the average weight was selected from each replicate for slaughter, and the carcass weight, semi-eviscerated weight, fully eviscerated weight, breast muscle weight, leg muscle weight, and abdominal fat weight were measured. The slaughter rate, semi-eviscerated rate, breast muscle rate, leg muscle rate, lean meat rate, and abdominal fat rate were calculated.

[0152] Carcass weight: The weight after bleeding and defeathering, using the wet plucking method, and weighing after draining.

[0153] Semi-eviscerated weight: The weight obtained by removing the trachea, esophagus, crop, intestine, spleen, gallbladder, and reproductive organs from the dressed weight, while retaining the heart, liver, kidneys, pancreas, lungs, gizzard (after removing the cuticle and contents), and the surrounding fat and abdominal fat.

[0154] Full-eviscerated weight: The weight obtained by further removing the heart, liver, pancreas, gizzard, fat, head, and feet from the semi-eviscerated weight.

[0155] Abdominal fat: Abdominal fat is obtained by peeling off the abdominal fat and the fat around the gizzard and cloaca.

[0156] Dressing percentage (%) = (Dressed weight / Live weight) × 100%

[0157] Semi-eviscerated percentage (%) = (Full-eviscerated weight / Dressed weight) × 100%

[0158] Full-eviscerated percentage (%) = (Full-eviscerated weight / Dressed weight) × 100%

[0159] Breast muscle percentage (%) = (Breast muscle weight / Full-eviscerated weight) × 100%

[0160] Leg muscle percentage (%) = (Leg muscle weight / Full-eviscerated weight) × 100%

[0161] Lean meat percentage (%) = Breast muscle percentage (%) + Leg muscle percentage (%)

[0162] Abdominal fat percentage = (Abdominal fat weight / Full-eviscerated weight) × 100%

[0163] 5. Immune organ index

[0164] 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) / Live weight before slaughter (kg).

[0165] 6. Serum indicators

[0166] 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.

[0167] 7. Intestinal morphological structure

[0168] At 65 days of age, take about 1 cm of the duodenum, jejunum, and the terminal ileum segments, and place them in 10% formalin for fixation. Take the intestinal segment samples fixed for 24 h, after flushing with water, dehydration with gradient alcohol, clearing with xylene, and paraffin embedding, perform routine hematoxylin-eosin (HE) staining with 6-μm thick sections, and finally seal 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.

[0169] 8. Effect verification

[0170] 1) Effects of adding cottonseed molasses-based solid-state fermented feed additive to the diet on the growth performance of yellow-feathered broilers

[0171] As can be seen from Table 14, adding the cottonseed molasses-based solid-state fermented feed additive to the diet had no significant effect on the average feed intake, daily weight gain, and feed-to-weight ratio of yellow-feathered broilers from 2 to 21 days. As the feeding time increased, from 42 days of feeding to 63 days of feeding, the average daily weight gain and feed-to-weight ratio in all three experimental groups showed significant differences compared with the control group, and with the increase in the feeding dose, the average daily weight gain and feed-to-weight ratio both showed significant increases or decreases, indicating that the cottonseed molasses-based solid-state fermented feed additive could significantly promote the growth performance of yellow-feathered broilers.

[0172] Table 14 Effects of cottonseed molasses-based solid-state fermented feed additive on the growth performance of yellow-feathered broilers

[0173]

[0174]

[0175] 2) Effects of adding cottonseed molasses-based solid-state fermented feed additive to the diet on the organ indices of yellow-feathered broilers

[0176] As can be seen from Table 15, compared with the control group, only the liver index and gizzard index in the three experimental groups were significantly different, and the differences in the other indices such as spleen index, thymus index, and pancreas index in the experimental groups were not significant compared with the control group. However, with the increase in the feeding dose, the immune organs showed an increasing trend, indicating that the cottonseed molasses fermentation culture could delay the decline of the immune organs of yellow-feathered broilers, thereby improving the immune ability of poultry.

[0177] Table 15 Effects of cottonseed molasses-based solid-state fermented feed additive on the organ indices of yellow-feathered broilers

[0178]

[0179] 3) Effects of adding cottonseed molasses-based solid-state fermented feed additive to the diet on the slaughter performance of yellow-feathered broilers

[0180] As can be seen from Table 16, there were no significant differences in the slaughter rate, semi-eviscerated rate, eviscerated rate, pectoral muscle rate, leg muscle rate, lean meat rate, and abdominal fat rate of broilers in all experimental groups compared with the control group (P>0.05).

[0181] Table 16 Effects of solid-state fermentation feed additive based on raffinose molasses on slaughter performance of yellow-feathered broilers

[0182]

[0183]

[0184] 4) Effects of dietary addition of solid-state fermentation feed additive based on raffinose molasses on relative intestinal length of yellow-feathered broilers

[0185] As can be seen from Table 17, compared with the control group CK-A, the relative jejunum length and relative ileum length of broilers in experimental group K2 were significantly increased (P<0.05); compared with the control group CK-A, there were no significant differences in the relative duodenum length of broilers in all experimental groups (P>0.05). Thus, it can be seen that the soybean meal-based diet supplemented with 0.4% solid-state fermentation feed of raffinose molasses can increase the relative jejunum length and relative ileum length of yellow-feathered broilers to varying degrees, and dietary addition of solid-state fermentation feed additive based on raffinose molasses has no effect on the relative duodenum length and relative cecum length of yellow-feathered broilers.

[0186] Table 17 Effects of solid-state fermentation feed additive based on raffinose molasses on relative intestinal length of yellow-feathered broilers

[0187]

[0188] 5) Effects of dietary addition of solid-state fermentation feed additive based on raffinose molasses on serum immunoglobulin content of yellow-feathered broilers

[0189] As can be seen from Table 18, compared with the control group CK-A, immunoglobulin A, immunoglobulin G, and immunoglobulin M of broilers in all experimental groups were not increased. Thus, it can be seen that dietary addition of solid-state fermentation feed additive based on raffinose molasses does not increase the serum immunoglobulin content of yellow-feathered broilers.

[0190] Table 18 Effects of solid-state fermentation feed additive based on raffinose molasses on serum immunoglobulin content of yellow-feathered broilers

[0191]

[0192] 6) Effects of dietary addition of solid-state fermentation feed additive based on raffinose molasses on serum antioxidant indexes of yellow-feathered broilers

[0193] As can be seen from Table 19, compared with the control group CK-A, the superoxide dismutase (SOD) of broilers in experimental group D increased significantly (P<0.05); the SOD, malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) of broilers in experimental group D all increased significantly (P<0.05). Thus, it can be seen that the soybean meal-based diet supplemented with 0.6% raffinose molasses solid-state fermented feed can improve the serum antioxidant indexes of yellow-feathered broilers to varying degrees.

[0194] Table 19 Effects of solid-state fermented feed additive based on raffinose molasses on serum antioxidant indexes of yellow-feathered broilers

[0195]

[0196]

[0197] 7) Effects of dietary supplementation with solid-state fermented feed additive based on raffinose molasses on liver function indexes of yellow-feathered broilers

[0198] As can be seen from Table 20, compared with the control group CK-A, the aspartate aminotransferase (AST) of broilers in experimental groups K1 and D decreased, but there was no significant difference (P>0.05); compared with the control group CK-A, the alanine aminotransferase (ALT) of broilers in all experimental groups decreased, but there was no significant difference (P>0.05). Thus, it can be seen that dietary supplementation with solid-state fermented feed additive based on raffinose molasses has no significant effect on the liver function indexes of yellow-feathered broilers.

[0199] Table 20 Effects of solid-state fermented feed additive based on raffinose molasses on liver function indexes of yellow-feathered broilers

[0200]

[0201] 8) Effects of dietary supplementation with solid-state fermented feed additive based on raffinose molasses on intestinal morphological structure of yellow-feathered broilers

[0202] As can be seen from Table 21, compared with the control group CK-A, the villus lengths of different intestinal segments of broilers in the three experimental groups increased significantly, indicating that the solid-state fermented feed additive based on raffinose molasses can promote the intestinal digestion and absorption rate of poultry; in addition, the crypt depths (CD) of each intestinal segment of broilers in all experimental groups decreased. The shallower the crypt, the better, and the deeper the crypt, the worse the absorption ability. The larger the villus-crypt ratio, the better, indicating that feeding the solid-state fermented feed additive based on raffinose molasses can improve digestion and absorption and increase the digestion and absorption area.

[0203] Table 21 Effects of solid-state fermented feed additive based on raffinose molasses on intestinal morphological structure of yellow-feathered broilers

[0204]

[0205]

[0206] As shown by Figure 11 Figure 11 , when analyzing the intestinal tissue sections of each group of broilers, the intestinal wall villi fed with the solid-state fermented feed additive based on raffinose molasses showed a certain degree of shedding or breakage, especially in the duodenum, which was more obvious, while it was not significant in the jejunum and ileum, indicating that the solid-state fermented feed additive based on raffinose molasses is relatively safe for broilers.

[0207] It can be seen from the effect verification experiment that through the optimization of the solid-state fermentation culture conditions of raffinose molasses, the invention realizes the detoxification and feed conversion of raffinose molasses. The feeding effect and application effect of the solid-state fermented feed based on raffinose molasses are evaluated through animal feeding experiments with the solid-state fermented feed additive based on raffinose molasses.

[0208] On the basis of having developed and provided the solid-state fermented feed additive based on raffinose molasses, those skilled in the art can easily further associate that integrating this feed additive 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 feed additive based on raffinose molasses is a natural extension and innovation by those skilled in the art on the basis of the prior art.

[0209] In addition, applying the feed additive of the present invention in the process of preparing synbiotic products is also an application method that those skilled in the art can easily think of. This application not only expands the use scope of the feed additive, but also further enhances the value of the feed additive through the comprehensive benefits of synbiotic products.

[0210] Therefore, in the case of the existing solid-state fermented feed additive based on raffinose molasses, those skilled in the art can easily think of and develop a synbiotic product containing this feed additive, as well as applying 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 maintenance of animals.

[0211] It should be understood that the disclosed invention of the present invention is not limited to the specific methods, schemes and substances described, because these can all be changed. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and schemes, and are not intended to limit the scope of the present invention. The scope of the present invention is only limited by the appended claims.

Claims

1. A solid fermented feed additive based on cottonseed molasses, characterized in that: The solid fermentation feed additive comprises a solid fermentation medium and a composite strain, wherein: The solid fermentation medium includes cottonseed molasses and bran; The composite bacterial strain comprises lactic acid bacteria and yeast.

2. The solid fermented feed additive according to claim 1, characterized in that: The mass ratio of the cottonseed molasses to the bran is 2:

3.

3. The solid fermented feed additive according to claim 1, characterized in that: The ratio of the number of live bacteria of the lactic acid bacteria to that of the yeast is 1:

10.

4. The solid fermented feed additive according to claim 1, characterized in that: The lactic acid bacteria is Pediococcus cidilactici B2, and the yeast is Saccharomyces cerevisiae A15.

5. The solid fermented feed additive according to claim 4, characterized in that: The preparation method of the solid fermentation feed additive is as follows: (1) drying cottonseed molasses and bran, mixing the dried cottonseed molasses and bran in a mass ratio of 2:3, and adjusting the water content to 40-60%, to obtain a solid fermentation medium; (2) mixing the pre-fermented Pediococcus cidilactici B2 and Saccharomyces cerevisiae A15 in deionized water at a live cell count ratio of 1:10 to obtain a composite strain; (3) The solid fermentation medium obtained in step (1) and the composite strain obtained in step (2) are stirred and mixed evenly, and fermented to obtain a solid fermentation feed additive.

6. The solid fermented feed additive according to claim 5, characterized in that: The inoculation amount of Pediococcus cidilactici B2 and Saccharomyces cerevisiae A15 is 5-20%; the fermentation temperature is 20-35° C.; and the fermentation time is at least 24-96 hours.

7. A synbiotic product, characterized in that: The synbiotic product comprises the solid fermentation feed additive according to any one of claims 1 to 6.

8. Use of the solid fermentation feed additive according to any one of claims 1 to 6 in the preparation of synbiotic products.

9. Use of the solid fermented feed additive according to any one of claims 1 to 6 in promoting the growth performance of yellow-feathered broilers and / or increasing the relative length of the intestine of yellow-feathered broilers.

10. Use of the solid fermented feed additive according to any one of claims 1 to 6 in improving the immunity of poultry and / or promoting intestinal digestion and absorption of poultry.

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