Probiotic fermented feed and preparation method thereof

Abstract

The invention provides a probiotic fermented feed and a preparation method thereof, belongs to the technical field of biological fermented feeds, and particularly relates to the technical field of biological fermented feeds. According to the preparation method, bacillus subtilis, lactobacillus plantarum and saccharomyces cerevisiae are used as complex microbial inoculants to ferment the feed; by using phlorizin and honeysuckle glycoside in a fixed proportion in a fermentation substrate, the growth activity of bacillus subtilis in the initial stage of fermentation can be improved, the activity of protease and cellulase in a fermentation system can be improved, the nutritional ingredients of the feed can be improved, and the yield of the feed can be increased. And nutrient substances generated by rapidly decomposing a substrate by the bacillus subtilis can be supplied to growth and metabolism of lactic acid bacteria and saccharomycetes, so that the number of probiotics in the feed is increased, and the nutritional value of the feed is improved. The probiotic fermented feed and the preparation method thereof have the advantages of being low in cost, simple in preparation method, short in production period, environmentally friendly and high in nutritional value.

Description

Technical Field

[0001] The present invention belongs to the technical field of bio-fermented feed, and particularly relates to a probiotic fermented feed and a preparation method thereof. Background Art

[0002] Probiotic fermented feed refers to a mixture rich in feed raw materials, probiotic cells and probiotic metabolites obtained by inoculating a proportion of compound beneficial microorganisms into a feed raw material substrate under artificially controlled conditions, fully mixing and stirring evenly, and performing anaerobic fermentation at an appropriate temperature, pH value and moisture content for a certain period of time. After animals consume probiotic fermented feed, it not only improves the feed utilization rate of animals, promotes animal growth and development and improves animal production efficiency, but also is beneficial to improving the health level of the animal body.

[0003] Currently, common feed fermentation microorganism species on the market include lactic acid bacteria, yeasts, bacillus subtilis, etc. The participation of lactic acid bacteria in fermented feed is to obtain the required sensory characteristics and its microbial safety in the final product. Lactic acid bacteria can grow rapidly in most food matrices, thus rapidly reducing the pH value and preventing the growth of other competing organisms. Bacillus subtilis can secrete a variety of active enzymes, and by decomposing antigenic protein ANF, the feed utilization rate is improved. The cell protein of yeast can provide rich protein for animal growth and development and promote the growth of beneficial bacteria. Yeast also helps to improve palatability and increase the feed intake of animals. Mixed bacteria fermented feed can increase the number of beneficial bacteria in piglets. Highly active probiotics can regulate and improve the intestinal flora environment, thus inhibiting the growth and reproduction of harmful microorganisms; and can promote the digestion and absorption of crude protein in feed by pigs; feeding mixed bacteria fermented feed can significantly promote the growth of piglets and reduce the feeding cost.

[0004] The prior art, such as the Chinese invention patent with the authorization announcement number of CN 119184201 B, discloses a fermented feed for improving intestinal health and a preparation method thereof, which relates to the technical field of feed processing; the fermented feed for improving intestinal health is composed of ZIF-8@clove basil oil nanoparticles, porphyra polysaccharide active traditional Chinese medicine composite gel, corn flour, soybean meal powder, grass powder, bacillus subtilis, yeast and papain; the porphyra polysaccharide active traditional Chinese medicine composite gel is composed of porphyra polysaccharide, chitosan, compound traditional Chinese medicine ultramicro powder and citric acid; by combining ZIF-8@clove basil oil nanoparticles with porphyra polysaccharide active traditional Chinese medicine composite gel and then coating the fermented feed, it is effectively isolated from the external environment, maintaining the activity of microorganisms in the fermented feed, increasing the stability of the feed, and can also adsorb harmful substances in the feed, reducing the adverse effects on the digestive tract, significantly improving intestinal health, thereby promoting digestion and absorption and helping to improve the growth performance of animals. Summary of the Invention

[0005] The object of the present invention is to provide a probiotic fermented feed with low cost, simple preparation method, short production cycle, environmental protection and high nutritional value, and a preparation method thereof.

[0006] The technical solution adopted by the present invention to achieve the above object is as follows: The present invention discloses a preparation method of a probiotic fermented feed, comprising the following steps: S1. Mix the crushed corn, soybean meal, wheat bran, rice bran, dried distillers grains, maltose, and additive evenly according to a mass ratio of 120 - 150:100 - 130:30 - 50:30 - 40:15 - 25:8 - 15:0.01 - 0.03 to obtain a fermentation substrate; S2. Mix the compound bacterial solution evenly with the fermentation substrate, adjust the water content to 40wt% - 55wt% with water, and ferment in a sealed manner at 30 - 38°C for 3 - 7 days to obtain a product; The above additive is composed of phloridzin and loganin in a mass ratio of 15 - 37:4 - 8; the above compound bacterial solution is obtained by mixing Bacillus subtilis bacterial solution, Lactobacillus plantarum bacterial solution, and Saccharomyces cerevisiae bacterial solution. The fermented feed composed of lactic acid bacteria, bacillus, and yeast contains a large amount of lactic acid bacteria, as well as protease and cellulase produced by metabolism, which can increase the palatability of the feed, improve the digestion ability of animals, and is beneficial to improving the growth performance of animals. The compound fermentation of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae in the feed. Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae are in a symbiotic fermentation system, and the three strains promote and restrict each other, jointly exerting the biological synergistic and antagonistic effects of microorganisms. In the early stage of fermentation, yeast and bacillus start to grow and reproduce, consume oxygen, create an anaerobic environment for the growth of lactic acid bacteria. Lactic acid bacteria grow and metabolize to produce lactic acid, inhibiting the growth of bacillus and reducing the number of bacillus. Subsequently, lactic acid bacteria and yeast further grow and become dominant strains, increasing the acidity further and further inhibiting the growth of bacillus. Phloridzin and loganin with a mass ratio of 15 - 37:4 - 8 can promote the rapid reproduction of Bacillus subtilis in the early stage of fermentation, improve the enzyme activities of protease and cellulase in the fermentation system, improve the nutritional components of the feed, and the nutrients produced by the rapid decomposition of the substrate by Bacillus subtilis can supply the growth and metabolism of lactic acid bacteria and yeast, increasing the number of probiotics in the feed and improving the nutritional value of the feed.

[0007] Preferably, the mass ratio of the above compound bacterial solution to the fermentation substrate is 3 - 10:100.

[0008] Preferably, the viable bacteria number ratio of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae in the above compound bacterial solution is 2 - 3:2 - 2.5:1 - 1.5.

[0009] Preferably, the above-mentioned compound bacterial liquid is obtained by mixing Bacillus subtilis bacterial liquid, Lactobacillus plantarum bacterial liquid and Saccharomyces cerevisiae bacterial liquid in a volume ratio of 1:1:1.

[0010] Preferably, the viable count of the above-mentioned Bacillus subtilis bacterial liquid is 6.0 - 9.0×10 9 CFU / mL, the viable count of the Lactobacillus plantarum bacterial liquid is 6 - 7.5×10 9 CFU / mL, and the viable count of the Saccharomyces cerevisiae bacterial liquid is 3 - 4.5×10 9 CFU / mL.

[0011] Preferably, the temperature of the sealed fermentation in the above-mentioned step S2 is 33 - 37°C, and the time is 3 - 5 d.

[0012] Preferably, the product obtained in the above-mentioned step S2 also undergoes a drying treatment.

[0013] Preferably, the temperature of the above-mentioned drying treatment is 20 - 40°C, and the drying time is 36 - 60 h.

[0014] Preferably, the preparation steps of the above-mentioned compound bacterial liquid include: a. Strain activation: Take Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae, and inoculate them into liquid media for activation culture respectively; b. Bacterial liquid culture: Transfer the three kinds of bacterial liquids after activation culture in step a to the corresponding liquid media respectively for enlarged culture; c. Compound: Mix the bacterial liquids after enlarged culture and compound them in proportion to obtain the compound bacterial liquid.

[0015] Preferably, the liquid medium of the above-mentioned Bacillus subtilis is LB liquid medium.

[0016] Preferably, the liquid medium of the above-mentioned Lactobacillus plantarum is MRS liquid medium.

[0017] Preferably, the liquid medium of the above-mentioned Saccharomyces cerevisiae is YPD liquid medium.

[0018] Preferably, before mixing the compound bacterial liquid with the fermentation substrate evenly in the above step S2, 0.32 - 0.75 mg / kg of mulberry root bark alcohol is added to the fermentation substrate. In the symbiotic fermentation system of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae, a certain amount of mulberry root bark alcohol can reduce the adverse effects on Bacillus subtilis during the growth and metabolism of lactic acid bacteria and yeast, improve the growth vitality of Bacillus subtilis, increase the activities of protease and cellulase in the fermentation system, increase the content of crude protein and acid-soluble protein in the feed, reduce the content of neutral detergent fiber and acid detergent fiber in the feed, and the nutrients produced by the rapid decomposition of the substrate by Bacillus subtilis can supply the growth and metabolism of Lactobacillus plantarum and yeast in the later stage of fermentation, increase the number of probiotics in the feed, thereby increasing the content of feed nutrients, improving the nutritional value of the feed, and improving the feed quality.

[0019] The present invention discloses a probiotic fermented feed, which is prepared by using the above preparation method.

[0020] The present invention also discloses the use of phloridzin and lonicerin in increasing the number of probiotics in the symbiotic fermentation system during the compound fermentation of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae for feed.

[0021] Preferably, the mass ratio of the above phloridzin to lonicerin is 15 - 37:4 - 8.

[0022] Since the present invention uses specific proportions of phloridzin and lonicerin when compound fermenting feed with Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae, it has the following beneficial effects: it can promote the rapid reproduction of Bacillus subtilis in the early stage of fermentation, produce a large number of enzyme systems with high activity, can more fully digest the substrate to produce nutrients, and the nutrients produced by the rapid decomposition of the substrate by Bacillus subtilis can supply the growth and metabolism of Lactobacillus plantarum and Saccharomyces cerevisiae, thereby increasing the content of feed nutrients and improving the nutritional value of the feed.

[0023] Since the present invention uses a certain amount of mulberry root bark alcohol when compound fermenting feed with Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae, it has the following beneficial effects: the addition of a certain amount of mulberry root bark alcohol can reduce the adverse effects on Bacillus subtilis during the growth and metabolism of Lactobacillus plantarum and Saccharomyces cerevisiae, improve the growth vitality of Bacillus subtilis and its extracellular hydrolase activity, thereby better improving the nutritional components of the feed and improving the feed quality.

[0024] The purpose of the present invention is to provide a probiotic fermented feed and its preparation method, which has the advantages of low cost, simple method, short production cycle, environmental protection, and high nutritional value of the feed. Description of the Drawings

[0025] Figure 1Detection results of viable counts of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae at 24 h of fermentation in Test Example 1 of the present invention.

[0026] Figure 2 Detection results of protease and cellulase activities in Test Example 2 of the present invention.

[0027] Figure 3 Determination results of growth rates of crude protein and acid-soluble protein contents in Test Example 3 of the present invention.

[0028] Figure 4 Determination results of reduction rates of neutral detergent fiber and acid detergent fiber contents in Test Example 3 of the present invention.

[0029] Figure 5 Detection results of probiotic numbers in fermented feed in Test Example 1 of the present invention. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] First, the concepts involved in the present application will be described in conjunction with the accompanying drawings. It should be noted here that the descriptions of the following concepts are only for making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0032] In the embodiments of the present invention, unless otherwise stated, the equipment and materials used in the present invention are obtained by purchasing on the market.

[0033] The Bacillus subtilis involved in the embodiments provided by the present invention was purchased from the China Center for Industrial Culture Collection, with the number CICC24534; the Lactobacillus plantarum was purchased from the China Center for Industrial Culture Collection, with the number CICC20265; the Saccharomyces cerevisiae was purchased from the China Center for Industrial Culture Collection, with the number CICC1005. The distillers grains (fresh) were provided by Anheuser-Busch InBev (Foshan) Brewery Co., Ltd. in Foshan, Guangdong Province. The distillers grains were dried ventilated in an oven at 50 °C, and the obtained dried distillers grains were stored for standby under sealed and dry conditions at 4 °C.

[0034] Example 1: 1. A preparation method of a probiotic fermented feed, comprising the following steps: 1.1 Crush corn, soybean meal, wheat bran, and rice bran, and then sieve them through a 60-mesh sieve. Then, mix the crushed corn, soybean meal, wheat bran, rice bran, dried distillers grains, maltodextrin, and additive, and dried distillers grains evenly according to a mass ratio of 140:120:40:35:20:10:0.02. The mass ratio of phloridzin to lonicerin in the additive is 25:4 to obtain a fermentation substrate.

[0035] 1.2 Preparation of the complex bacterial solution, including: 1.2.1 Activation of the bacterial strains: YPD medium: 20 g of tryptone, 20 g of glucose, 10 g of yeast extract, made up to 1000 mL with distilled water, pH value is the natural value, and 18 g of agar is added for the solid medium.

[0036] LB liquid medium: 10 g of peptone, 10 g of sodium chloride, 5.0 g of yeast powder, made up to 1000 mL with distilled water, and the pH is adjusted to 7.4.

[0037] MRS medium: 10 g of peptone, 5 g of glucose, 10 g of beef extract, 5 g of yeast extract, 2 g of dipotassium hydrogen phosphate, 0.25 g of manganese sulfate, 2 g of ammonium citrate tribasic, 2 g of sodium acetate trihydrate, 1.0 mL of Tween-80, 0.5 g of magnesium sulfate, made up to 1000 mL with distilled water, adjust the pH to 6.5, and 18 g of agar is added for the solid medium.

[0038] Streak plate and activate the strains Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae preserved in glycerol. Select single colonies and inoculate them into 50 mL of liquid medium for scale-up culture. Saccharomyces cerevisiae is cultured in YPD liquid medium at 30 °C with shaking at 180 r / min for 36 h, Bacillus subtilis is cultured in LB liquid medium at 37 °C with shaking at 150 r / min for 24 h, and Lactobacillus plantarum is cultured in MRS liquid medium at 37 °C statically for 24 h. The above three kinds of cultured bacterial solutions are used as seed solutions.

[0039] 1.2.2 Inoculate the seed solution into 250 mL of liquid medium at an inoculation amount of 1.0% (v / v). Saccharomyces cerevisiae is inoculated into YPD liquid medium and cultured statically at 30 °C for 30 h, Bacillus subtilis is inoculated into LB liquid medium and cultured statically at 37 °C for 24 h, and Lactobacillus plantarum is inoculated into MRS liquid medium and cultured statically at 37 °C for 24 h. Measure the OD 600nm value and perform dilution coating on the plate to calculate the cell density. The viable count of the Bacillus subtilis bacterial solution is calculated to be 7.5×10 9 CFU / mL, the viable count of the Lactobacillus plantarum bacterial solution is 6.2×10 9 CFU / mL, and the viable count of the Saccharomyces cerevisiae bacterial solution is 3.5×10 9CFU / mL, mix the three bacterial solutions evenly according to a volume ratio of 1:1:1 to obtain a composite bacterial solution.

[0040] 1.3 Inoculate the above composite bacterial solution into the fermentation substrate at a ratio of 10 wt%, mix evenly, adjust the water content to 50 wt% with water, and ferment in a sealed manner at 35 °C for 4 days to obtain the product.

[0041] Example 2: Among them, the mass ratio of phloridzin to lonicerin in the additive is 25:8, and the rest is exactly the same as in Example 1.

[0042] Example 3: Among them, the mass ratio of phloridzin to lonicerin in the additive is 25:6, and the rest is exactly the same as in Example 1.

[0043] Example 4: Among them, the mass ratio of phloridzin to lonicerin in the additive is 25:2, and the rest is exactly the same as in Example 1.

[0044] Example 5: Among them, the mass ratio of phloridzin to lonicerin in the additive is 25:25, and the rest is exactly the same as in Example 1.

[0045] Example 6: Among them, the additive does not include lonicerin, and the rest is exactly the same as in Example 1.

[0046] Example 7: Among them, the additive does not include phloridzin, and the rest is exactly the same as in Example 1.

[0047] Example 8: Among them, the fermentation substrate does not include the additive, and the rest is exactly the same as in Example 1.

[0048] Example 9: Among them, before adding the composite bacterial solution to the fermentation substrate in step 1.3, first add 0.45 mg / kg of mulberry root bark alcohol to the fermentation substrate and mix evenly, and the rest is exactly the same as in Example 3.

[0049] Example 10: Among them, before adding the composite bacterial solution to the fermentation substrate in step 1.3, first add 0.6 mg / kg of mulberry root bark alcohol to the fermentation substrate and mix evenly, and the rest is exactly the same as in Example 3.

[0050] Example 11: Among them, before adding the composite bacterial solution to the fermentation substrate in step 1.3, first add 0.1 mg / kg of mulberry root bark alcohol to the fermentation substrate and mix evenly, and the rest is exactly the same as in Example 3.

[0051] Example 12: Before adding the complex bacterial liquid to the fermentation substrate in step 1.3, 1 mg / kg of mulberry root bark alcohol was first added to the fermentation substrate and mixed evenly, and the rest was exactly the same as in Example 3.

[0052] Test Example 1: Determination of viable bacteria count Samples were taken at 24 h of fermentation to detect the numbers of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae.

[0053] Samples of the fermented feed product were taken to detect the number of probiotics, which was the sum of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae.

[0054] The number of Bacillus subtilis was determined with reference to "Detection of Bacillus subtilis in Feed Microbial Agents" (GB / T 26428-2010), the number of Saccharomyces cerevisiae was determined with reference to "National Food Safety Standard Food Microbiology Examination - Enumeration of Molds and Yeasts" (GB 4789.15-2016), and the number of Lactobacillus plantarum was determined with reference to "National Food Safety Standard Food Microbiology Examination - Examination of Lactic Acid Bacteria" (GB 4789.35-2016). The detection results of the viable bacteria counts of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae at 24 h of fermentation are shown in Figure 1 . The detection results of the number of probiotics in the fermented feed are shown in Figure 5 .

[0055] Test Example 2: Detection of feed enzyme activity 1. Protease detection method Take 5 g of the feed sample and add sterile water to make up to 50 mL. Stir with a magnetic stirrer for 60 min, centrifuge. Mix 1.0 mL of the supernatant with 1.0 mL of casein (1% w / v) solution in 50 mM lactic acid buffer at pH 3.0, 50 mM phosphate buffer at pH 7.0, and 50 mM boric acid buffer solution at pH 10.5, and react at 40 °C for 20 min. Add 2.0 mL of 0.4 M trichloroacetic acid solution to terminate the reaction. Filter the mixed solution, take 1.0 mL of the filtrate and add it to 5.0 mL of sodium carbonate solution (0.4 M) and 1.0 mL of Folin-Ciocalteu reagent, and incubate in a water bath at 40 °C for 20 min. Measure the absorbance at OD 680. Use tyrosine to construct a standard curve. The amount of enzyme required to release 1.0 μg of tyrosine from casein per minute is equivalent to 1 unit of protease activity (U).

[0056] 2. Cellulase detection method Standard curve preparation: Take 0, 0.2, 0.4, 0.8, 1 mL of 1 mg / mL glucose solution and place them in test tubes, make up to 2 mL with distilled water, mix well, add 3 mL of DNS (3,5-dinitrosalicylic acid), shake evenly, water bath for 5 min, after cooling, make up the volume to 20 mL, zero with the glucose blank tube, measure the OD value at a wavelength of 540 nm, use the OD540 value as the ordinate and the glucose amount as the abscissa to draw the standard curve.

[0057] Take 5 g of feed sample, add sterile water to 50 mL, stir with a magnetic stirrer for 60 min, centrifuge, take 0.3 mL of the supernatant and add it to 2 mL of preheated 5 g / L sodium carboxymethylcellulose substrate, keep in a water bath at 39 °C for 20 min. Then immediately add 3 mL of DNS, transfer the test tube to boiling water and shake evenly for 5 min, after cooling, make up the volume to 20 mL, and measure the OD 540 value.

[0058] Enzyme activity unit: The amount of enzyme that produces 1 μmol of reducing sugar in 1 mL of enzyme solution in 1 min is taken as one enzyme activity unit. Cellulase activity (U / g) = (reducing sugar concentration × enzyme solution dilution factor) ÷ (enzyme action time × enzyme solution volume × glucose molecular weight) The detection results of protease and cellulase activities are shown in Figure 2 .

[0059] Test Example 3: Analyze the crude protein, acid-soluble protein, neutral detergent fiber, and acid detergent fiber in the fermentation substrates of each example respectively. Place the fermented feed in an oven at 40 °C to dry, then crush it for analysis. Calculate the growth rate of the crude protein content, the growth rate of the acid-soluble protein content, the reduction rate of the neutral detergent fiber content, and the reduction rate of the acid detergent fiber content after fermentation.

[0060] The crude protein content was determined with reference to the national standard GB / T 6432-2018 "Determination of Crude Protein in Feeds - Kjeldahl Method". The acid-soluble protein content was determined with reference to the agricultural industry standard "NY / T 3801-2020 Determination of Acid-Soluble Protein in Feed Raw Materials". The content of neutral detergent fiber in the sample was determined according to GB / T 20806-2022. The content of acid detergent fiber in the sample was determined according to NY / T 1459-2007.

[0061] The determination results of the growth rate of the crude protein content and the acid-soluble protein content are shown in Figure 3 . The determination results of the reduction rate of the neutral detergent fiber content and the acid detergent fiber content are shown in Figure 4 .

[0062] From Figure 1 , Figure 2 , Figure 3 , Figure 4 ,Figure 5 It can be seen that at 24 h of fermentation, the number of Bacillus subtilis in the fermentation systems of Example 1, Example 2, and Example 3 is significantly higher than that of Example 4, Example 5, Example 6, Example 7, and Example 8, and there is no significant difference among Example 4, Example 5, Example 6, Example 7, and Example 8; the protease and cellulase activities in the fermented feeds prepared in Example 1, Example 2, and Example 3 are significantly higher than those of Example 4, Example 5, Example 6, Example 7, and Example 8, and the differences among Example 4, Example 5, Example 6, Example 7, and Example 8 are not obvious; the growth rates of the crude protein content and acid-soluble protein content in the fermented feeds prepared in Example 1, Example 2, and Example 3 are significantly greater than those of Example 4, Example 5, Example 6, Example 7, and Example 8. The growth rate of the crude protein content in Example 4, Example 5, Example 6, and Example 7 is slightly higher than that in Example 8, and the growth rate of the acid-soluble protein content in Example 4, Example 5, Example 6, and Example 7 has no obvious difference from that in Example 8; the reduction rates of the neutral detergent fiber content and acid detergent fiber content in the fermented feeds prepared in Example 1, Example 2, and Example 3 are significantly greater than those of Example 4, Example 5, Example 6, Example 7, and Example 8, and there is no significant difference between Example 4, Example 5, Example 6, Example 7, and Example 8; the number of probiotics in the fermented feeds prepared in Example 1, Example 2, and Example 3 is significantly greater than that of Example 4, Example 5, Example 6, Example 7, and Example 8, and the differences among Example 4, Example 5, Example 6, Example 7, and Example 8 are not obvious. From this, it can be known that the addition of phloridzin and loganin with a mass ratio of 15-37:4-8 can significantly improve the growth vitality of Bacillus subtilis in the symbiotic fermentation system of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae in the initial stage of fermentation, improve the protease and cellulase activities in the fermentation system, can more fully digest the substrate to produce nutrients, increase the crude protein and acid-soluble protein contents in the feed, reduce the neutral detergent fiber and acid detergent fiber contents in the feed, and the nutrients produced by the rapid decomposition of the substrate by Bacillus subtilis can supply the growth and metabolism of Lactobacillus plantarum and Saccharomyces cerevisiae in the later stage, increase the number of probiotics in the feed, thereby increasing the nutrient content of the feed, improving the nutritional value of the feed, and improving the feed quality.

[0063] From Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5It can be seen that at 24 h of fermentation, the number of Bacillus subtilis in the fermentation systems of Example 9 and Example 10 was significantly higher than that in Example 3, Example 11, and Example 12, and there was no significant difference between Example 3 and Example 11 and Example 12; the protease and cellulase activities in the fermented feeds prepared in Example 9 and Example 10 were significantly higher than those in Example 3, Example 11, and Example 12, and there was no significant difference between Example 3 and Example 11 and Example 12; the growth rates of the crude protein content and acid-soluble protein content in the fermented feeds prepared in Example 9 and Example 10 were significantly greater than those in Example 3, Example 11, and Example 12, and there was no significant difference in the growth rates of the crude protein content and acid-soluble protein content in the fermented feed prepared in Example 3 and Example 11 and Example 12; the reduction rates of the neutral detergent fiber content and acid detergent fiber content in the fermented feeds prepared in Example 9 and Example 10 were significantly greater than those in Example 3, Example 11, and Example 12, and there was no significant difference in the reduction rates of the neutral detergent fiber content and acid detergent fiber content in the fermented feed prepared in Example 3 and Example 11 and Example 12; the number of probiotics in the fermented feeds prepared in Example 9 and Example 10 was significantly greater than that in Example 3, Example 11, and Example 12, and there was no significant difference between Example 3 and Example 11 and Example 12. From this, it can be known that adding 0.32 - 0.75 mg / kg of mulberry root bark alcohol to the fermentation substrate can reduce the adverse effects of the growth and metabolism of Lactobacillus and Saccharomyces cerevisiae on Bacillus subtilis in the symbiotic fermentation system of Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae, further improve the growth vitality of Bacillus subtilis, increase the protease and cellulase activities in the fermentation system, increase the crude protein and acid-soluble protein contents in the feed, reduce the neutral detergent fiber and acid detergent fiber contents in the feed, and the nutrients produced by the rapid decomposition of the substrate by Bacillus subtilis can supply the growth and metabolism of Lactobacillus plantarum and Saccharomyces cerevisiae in the later stage of fermentation, increase the number of probiotics in the feed, thereby increasing the nutrient content of the feed, improving the nutritional value of the feed, and improving the feed quality.

[0064] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for implementing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some changes or modifications to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0065] In this text, specific examples are used to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only for helping to understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limited nature of literal expression and objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principles of the present application, several improvements, embellishments or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, embellishments, changes or combinations, or directly applying the inventive concept and technical solution to other occasions without improvement, shall all be regarded as the protection scope of the present application.

Claims

1. A method for preparing probiotic fermented feed, characterized in that: The steps include: S1. Mix the crushed corn, soybean meal, bran, rice bran, dried distiller's grains, maltose, and additives in a mass ratio of 120-150:100-130:30-50:30-40:15-25:8-15:0.01-0.03 to obtain a fermentation substrate; S2, mixing the composite bacterial liquid and the fermentation substrate evenly, adjusting the water content to 40wt%-55wt% with water, and fermenting in a sealed manner at 30-38°C for 3-7 days to obtain the product; The additive consists of phlorizin and honeysuckle in a mass ratio of 15-37:4-8; the composite bacterial liquid is obtained by mixing Bacillus subtilis bacterial liquid, Lactobacillus plantarum bacterial liquid and Saccharomyces cerevisiae bacterial liquid.

2. The method for preparing a probiotic fermented feed according to claim 1, characterized in that: The mass ratio of the composite bacterial liquid to the fermentation substrate is 3-10:

100.

3. The method for preparing a probiotic fermented feed according to claim 1, characterized in that: The ratio of the number of live bacteria of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae in the composite bacterial liquid is 2-3:2-2.5:1-1.

5.

4. The method for preparing a probiotic fermented feed according to claim 3, characterized in that: The composite bacterial liquid is obtained by mixing Bacillus subtilis bacterial liquid, Lactobacillus plantarum bacterial liquid and Saccharomyces cerevisiae bacterial liquid in a volume ratio of 1:1:

1.

5. The method for preparing a probiotic fermented feed according to claim 4, characterized in that: The viable bacterial count of the Bacillus subtilis liquid is 6.0-9.0×10 9 CFU / mL, the number of viable bacteria of Lactobacillus plantarum liquid is 6-7.5×10 9 CFU / mL, the number of live bacteria in the brewer's yeast culture liquid is 3-4.5×10 9 CFU / mL.

6. The method for preparing a probiotic fermented feed according to claim 1, characterized in that: The product obtained in step S2 is further dried.

7. The method for preparing a probiotic fermented feed according to claim 6, characterized in that: The temperature of the drying treatment is 20-40° C., and the drying time is 36-60 hours.

8. Use of phlorizin and honeysuckle in increasing the number of probiotics in the symbiotic fermentation system when Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae are used to ferment feed.

9. The use according to claim 8, characterized in that: The mass ratio of phlorizin to honeysuckle is 15-37:4-8.

10. A probiotic fermented feed, characterized in that: The method is prepared by the method described in any one of claims 1 to 7.

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