Fermented compound feed for preventing diarrhea of piglets and method for preparing and applying the same

Abstract

The application discloses a kind of fermented compound feed for preventing piglet diarrhea and its preparation and application method. Corn, soybean meal and bran are premixed according to the mass ratio of 4.5:4.5:1.0, then 10% of ZJUAF-2 fermentation broth and 10% of ZJUAF-4 fermentation broth are added, and 40% of water is added. The initial fermentation temperature is 25-30℃, the fermentation time is 48-72 h, and the material is turned over every 5-8 h. When the pH of the feed is reduced to 4.0, corn-soybean meal type fermented compound feed is obtained. Corn, soybean meal and bran are fermented by mixing one strain of Bacillus subtilis and one strain of Pediococcus pentosaceus. The contents of beta-conglycin and soybean protein decreased by 71.94% and 82.86%, respectively. The content of neutral detergent fiber decreased by 38.93%. The contents of gassy factors raffinose and stachyose decreased by 42.22% and 36.60%, respectively. The content of phytic acid phosphorus decreased by 37.50%. In addition to the significant decrease in the above-mentioned anti-nutritional factors, the lactic acid content increased by 3.5 times, and the acid-soluble protein increased by 6 times. Feeding the fermented feed to piglets can control diarrhea as effectively as feeding antibiotics.

Description

Technical Field

[0001] This invention relates to the field of microbiology and fermentation, specifically to a fermented compound feed for preventing diarrhea in piglets and its preparation and application methods. Background Technology

[0002] Diarrhea is a major cause of death in piglets. After weaning, piglets' digestive tract and enzyme systems are not yet fully developed, leading to reduced digestibility and utilization of nutrients in their feed. This creates favorable conditions for the proliferation of pathogenic bacteria in the intestines, resulting in diarrhea. Furthermore, antigenic substances in feed, such as antigenic proteins, can trigger an immune response, inducing diarrhea. Therefore, reducing anti-nutritional factors in feed and improving the digestibility and utilization of nutrients in feed can help prevent piglet diarrhea.

[0003] Bio-fermentation effectively reduces anti-nutritional factors in feed, improves the digestibility and utilization of feed nutrients by animals, improves animal health, and alleviates diarrhea in piglets caused by antibiotic reduction, making it a key focus of feed resource development in my country. However, currently, there are many types of bio-fermented feeds, and differences in strains and fermentation processes lead to variations in quality. Furthermore, to reduce costs, unconventional feed ingredients are used in the production of fermented feeds. However, there are currently no comprehensive product standards or appropriate recommended addition levels for unconventional ingredients, and there is a lack of evaluation on the residues of toxins and anti-nutritional factors in unconventional ingredients (Zhang Zhenlei, 2017). The safety and efficacy of fermented products still need further verification. Therefore, safe and effective bio-fermented feed products urgently need to be developed.

[0004] To address the issue of degrading anti-nutritional factors in feed, the applicant designed a fermented compound feed using conventional plant-based raw materials such as corn, soybean meal, and wheat bran as substrates in patent ZL201610728880.3, avoiding potential toxins present in unconventional raw materials. Simultaneously, a fermented compound feed low in soybean antigen proteins was prepared using a highly efficient Bacillus strain for degrading soybean antigen proteins screened in patent ZL201610728303.4, and its effectiveness in improving the digestibility of feed amino acids was demonstrated through ileal digestibility testing in piglets (C. Shi, 2017). However, as the scale of single-batch fermentation increased to over ten tons, and with the mixed use of multiple strains, the oxygen content within the fermentation system decreased rapidly, anaerobic acid-producing bacteria easily formed a dominant flora, feed acidification accelerated, the activity of digestive enzymes secreted by microorganisms decreased, the degradation level of anti-nutritional factors declined, and the quality of the fermented feed decreased.

[0005] Adding enzymes to feed can promote the breakdown of feed macromolecules and improve feed digestibility (as in CN201811644468.9 and CN201811295681.3, where the inventors used neutral proteases to degrade protein raw materials). However, this also limits the quality of fermented feed to the enzyme activity of the enzyme products, while increasing production steps and costs. Therefore, fully utilizing the enzyme production performance of strains and improving the activity of substrate enzymes during feed fermentation will effectively reduce anti-nutritional factors in feed, prevent piglet diarrhea, and reduce production costs. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention provides a fermented compound feed for preventing diarrhea in piglets and a method for its preparation and application.

[0007] A corn-soybean meal fermented compound feed for preventing diarrhea in piglets is disclosed. Corn, soybean meal, and wheat bran are fermented using a strain of Bacillus subtilis and a strain of Pediococcus pentosaceus. The contents of β-conglobulin and soybean globulin decreased by 71.94% and 82.86%, respectively; neutral detergent fiber decreased by 38.93%; the contents of raffinose and stachyose, the flatulence factors, decreased by 42.22% and 36.60%, respectively; and the contents of phytic acid phosphorus decreased by 37.50%. In addition to the significant decrease in the contents of the above anti-nutritional factors, the lactic acid content increased by 3.5 times and the acid-soluble protein increased by 6 times. Feeding piglets with the fermented feed has a control effect on diarrhea comparable to that of feeding antibiotics.

[0008] The Bacillus subtilis mentioned above belongs to the Bacillus subsp. subtilis subspecies. Bacillus subtilis subsp. subtilis The strain, named ZJUAF-2, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2016, with accession number CGMCC NO:12824. It is an existing strain and was first disclosed in CN106222114A.

[0009] The aforementioned *Pediococcus pentosaceus* belongs to the genus *Pediococcus pentosaceus*. Pediococcus pentosaceus It is named ZJUAF-4 and is deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is November 21, 2018, and the accession number is CGMCC NO:16767.

[0010] A method for preparing the corn-soybean meal fermented compound feed involves premixing corn, soybean meal, and wheat bran in a mass ratio of 4.5:4.5:1.0, then adding 10% ZJUAF-2 fermentation broth and 10% ZJUAF-4 fermentation broth by mass, and adding 40% water by mass. The fermentation starts at 25-30°C, and the fermentation time is 48-72 hours. The feed is turned over every 5-8 hours. When the pH of the feed drops to 4.0, the corn-soybean meal fermented compound feed is obtained.

[0011] The ZJUAF-2 fermentation broth: ZJUAF-2 was cultured in LB medium until OD. 600 When the concentration reaches 1.2, the culture medium is inoculated into the fermentation broth at a volume ratio of 5.0%. The composition of the culture medium by mass percentage includes 2.0% glycerol, 3.0% soybean meal, 0.4% NaH2PO4, 0.03% K2HPO4, 0.5-1.0% wheat bran, and the remainder is water. The fermentation broth is cultured at 30℃ and 200 r / min for 24 h, and the neutral protease activity reaches 600 U / mL.

[0012] The ZJUAF-4 fermentation broth was cultured in MRS medium until OD. 600 When the bacterial concentration reaches 1.4-1.7, inoculate the fermentation broth medium at a volume ratio of 0.1%. The medium composition, by weight, includes 0.5-2.0% yeast extract, 0.5-2.0% glucose, 0.5-1.0% tomato extract, 0.1-0.5% KH₂PO₄, and the remainder is water. Incubate at 37℃ for 24 h until the bacterial concentration reaches 1.0 × 10⁻⁶. 9 ~1.0×10 10 cfu / mL.

[0013] A method for applying the aforementioned corn-soybean meal fermented compound feed involves feeding the fermented feed to piglets, which can improve the digestibility of piglets and prevent diarrhea. The fermented feed is dried at 65°C to a moisture content of 12% by mass. It is then used to replace 5% of the feed weight of corn and 5% of the feed weight of soybean meal at a mass ratio of 10% by mass. When fed to 9 kg piglets, the jejunal trypsin activity of the piglets increases by 1.45 times, the apparent digestibility of crude protein increases by 2.87 percentage points, the gastric pH reaches 4.0, and the daily weight gain increases by 7.9%. The effect on controlling the diarrhea rate in piglets is comparable to that of feeding antibiotics.

[0014] Beneficial effects of this invention:

[0015] 1. It increases the activity of neutral protease in the early stage of fermentation to about 20 times that of the original technology, which helps to overcome the problem of insufficient degradation of antigen proteins in large-scale fermentation, eliminating the need to add protease preparations and reducing process flow and production costs.

[0016] 2. Feeding fermented feed can lower the pH in piglets' stomachs to 4.0, which helps reduce the invasion of pathogens into the gastrointestinal tract;

[0017] 3. Compared with adding antibiotics, adding fermented feed increased the activity of jejunal trypsin in piglets by 1.45 times, increased the apparent digestibility of crude protein by 2.87 percentage points, increased daily weight gain by 7.9%, and the effect of controlling diarrhea rate was comparable to that of feeding antibiotics. Attached Figure Description

[0018] Figure 1 Differences in enzyme activity in fermentation broth before and after process improvement;

[0019] Figure 2 Differences in feed protein degradation levels before and after process improvement. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] This invention improves the fermentation process, enhances the activity of proteases in the fermented feed substrate, and leverages the degradation effect of Bacillus subtilis neutral protease on soybean antigenic proteins, thereby improving the nutrient digestibility of the feed. Furthermore, the organic acids produced by microorganisms in the later stages of fermentation help lower the pH in piglets' stomachs, inhibiting the reproduction of bacteria and viruses and reducing pathogen infection of the digestive tract. For example, the pH tolerance range of African swine fever virus (3.95~10.5) is limited, and this also helps prevent diarrhea in piglets, demonstrating significant application potential.

[0022] Example 1: Preparation of fermented feed:

[0023] (1) Preparation of fermentation culture medium

[0024] Fermentation medium for Bacillus subtilis ZJUAF-2: glycerol 2.0%~4.0%, soybean meal 2.0%~4.0%, NaH2PO4 0.4%, K2HPO4 0.03%, and wheat bran 1.0%; Fermentation medium for Pediococcus pentosaceus ZJUAF-4: yeast powder 0.5-2.0%, glucose 0.5-2.0%, tomato powder 0.5-1.0%, and KH2PO4 0.1-0.5%.

[0025] (2) Preparation of fermentation broth

[0026] After activation and purification, Bacillus subtilis ZJUAF-2 was cultured, and single colonies were picked and cultured on LB medium at 37°C and 150 rpm for 12 h until OD was achieved. 600 When the pH value reaches approximately 1.0, inoculate 0.1% into the fermentation medium and incubate at 37℃, 200 rpm, with aeration and stirring for 24 h to obtain the Bacillus subtilis fermentation broth. The viable Bacillus subtilis count should reach 1.0 × 10⁻⁶ cells / year. 8~1.0×10 10 The concentration of CFU / mL and the neutral protease activity reached 600 U / mL. After activation and purification, *Pediococcus pentosaceus* ZJUAF-4 was cultured as a single colony on MRS medium and incubated statically for 24 h. The activated seed culture was then inoculated into the fermentation medium at 0.1% and incubated statically for another 24 h, with gentle stirring every 12 h to ensure homogeneity. The bacterial concentration reached 1.0 × 10⁻⁶ CFU / mL. 9 ~1.0×10 10 When cfu / mL, the fermentation broth of Pediococcus pentosaceus was obtained.

[0027] (3) Preparation of fermented compound feed

[0028] Premix corn, soybean meal, and wheat bran in a mass ratio of 4.5:4.5:1. Then add 10% ZJUAF-2 fermentation broth and ZJUAF-4 fermentation broth respectively by mass-volume ratio, and add 40% water. Stir well to form a fermentation system. Ferment the above system at 25~30℃ for 48~72 hours, turning the material every 5~8 hours to ensure the fermentation temperature does not exceed 55℃. Fermented feed is obtained when the pH of the feed drops to 4.0.

[0029] Example 2: Selection of Fermentation Medium for Bacillus subtilis

[0030] Different fermentation media were used, with an inoculation volume ratio of 5% ZJUAF-2, and fermentation was carried out for 24 h. The neutral protease activity of the fermentation broth was determined by the Folin-Ciocalteu method, and the results are shown in Table 2. Among them, medium 1 was LB medium, with the specific components being 1% fish peptone, 0.5% yeast extract, and 1.0% NaCl by mass percentage; medium 2 was the unmodified ZJUAF-2 medium, with the specific components being 2.0% soybean meal, 1.8% corn flour, 2.4% glucose, 0.4% NaH2PO4, 0.03% K2HPO4, and 0.02% CaCl2 by mass percentage; medium 3 was the modified ZJUAF-2 medium, with the specific components being 3.0% soybean meal, 2.0% glycerol, 0.4% NaH2PO4, 0.03% K2HPO4, and 1.0% wheat bran by mass percentage.

[0031] Table 1. Effects of different culture media on enzyme activity in fermentation broth

[0032] Fermentation medium 24-hour neutral protease activity (Unit / mL) Fermentation medium 1 38 Fermentation medium 2 155 Fermentation medium 3 611

[0033] The results are shown in Table 1 and Figure 1 As shown, the improved culture medium is beneficial for increasing the enzyme activity of the fermentation broth. The fermentation broth was used for a 20-ton-scale fermentation of compound feed, and the results are as follows... Figure 2As shown, the degradation effect of the two soybean antigen proteins is enhanced after the process improvement, wherein the soybean antigen proteins are soybean globulin and β-congglobulin.

[0034] Example 3: The Effects of Different Lactic Acid Bacteria on the Quality of Fermented Feed

[0035] Referring to the fermented feed preparation process in Example 1, the selection of *Pediococcus pentosaceus* was replaced with *Enterococcus faecalis*, *Lactobacillus plantarum*, and *Lactobacillus casei*. The results are shown in Table 2. After formulation, the acid-soluble protein content of the *Pediococcus pentosaceus* ZJUAF-4 fermented feed was higher than that of the other strains. Although the feed acidity was not as high as that of *Lactobacillus plantarum* and *Lactobacillus casei*, the difference was not significant. Therefore, *Pediococcus pentosaceus* ZJUAF-4 was selected as the strain used in this invention.

[0036] Table 2. Effects of different lactic acid bacteria on acid-soluble proteins and pH in fermentation products

[0037] Fermentation strains Acid-soluble protein pH Pediococcus pentosaceus ZJUAF-4 8.85 3.95 Enterococcus faecalis 8.82 4.21 Lactobacillus plantarum 7.63 3.83 Lactobacillus casei 7.72 3.85

[0038] Example 4: Chemical composition analysis of feed before and after fermentation

[0039] As shown in Table 3, the fermented feed prepared according to the present invention (45% corn, 45% soybean meal, and 10% wheat bran) showed a 71.94% and 82.86% decrease in β-conglobulin and soy globulin, respectively; a 38.93% decrease in neutral detergent fiber; a 42.22% and 36.60% decrease in the content of the flatulence factors raffinose and stachyose, respectively; and a 37.50% decrease in the content of phytic acid phosphorus. In addition to the significant decrease in the content of the above anti-nutritional factors, the lactic acid content increased by 3.5 times, and the acid-soluble protein increased by more than 6 times, thus improving the nutritional value of the feed.

[0040] Table 3. Analysis of feed chemical composition before and after fermentation

[0041] index Unfermented feed Fermented feed Crude protein, % 24.03 27.61 Acid-soluble protein, % 1.18 8.85 β-Consomglobulin, mg / g 31.76 8.91 Soy globulin, mg / g 63.74 10.92 Crude fat, % 3.67 3.37 Neutral detergent fiber, % 13.64 8.33 Acid detergent fibers, % 3.49 3.58 starch, % 30.35 27.48 Raffinose, % 0.36 0.19 Stachyose, % 2.65 1.68 Phytate phosphorus, % 0.32 0.20 Lactic acid, % 1.27 4.45

[0042] Example 5: Effects of Fermented Feed on Piglet Growth Performance

[0043] Two hundred and sixty healthy piglets weighing 9.25 ± 0.68 kg were randomly divided into two treatment groups, with six replicates per group and eighteen piglets per replicate. The antibiotic group received a basal diet supplemented with 100 mg / kg quinethol and 50 mg / kg tylosin; the fermented feed group received a basal diet (without antibiotics) plus 10% fermented compound feed. The experiment lasted 24 days. As shown in Table 4, the fermented feed effectively controlled the diarrhea rate in piglets, and its anti-diarrheal effect was comparable to that of antibiotics. Furthermore, the fermented feed group showed a 6.39% increase in feed intake and a 7.99% increase in daily weight gain. P < 0.05), thus improving growth performance.

[0044] Table 4. Effects of fermented feed on diarrhea prevention and growth performance in piglets.

[0045] project antibiotic group Fermented feed group Initial weight, kg 9.37 9.25 Final weight, kg 18.70 19.32 Daily weight gain, g <![CDATA[388 b ]]> <![CDATA[419 a ]]> Daily feed intake, g 595 633 Material consumption and weight gain ratio 1.54 1.51 Diarrhea rate, % 7.24 7.94

[0046] Example 6: Effect of Fermented Feed on Apparent Intestinal Digestibility of Nutrients in Piglets

[0047] Based on Example 5, from day 19 to 21 of the experiment, a total manure collection method was used, collecting 200g of uncontaminated manure samples from piglets daily. The manure samples covered all experimental pigs in the same pen. After drying and pulverizing the manure samples at 55°C, the apparent digestibility of nutrients was determined using the endogenous indicator method. The results are shown in Table 5. Adding fermented feed to the piglet diet significantly improved the digestibility and utilization of dry matter, crude protein, and phosphorus.

[0048] Table 5. Effects of fermented feed on apparent intestinal digestibility of nutrients in piglets.

[0049] project antibiotic group Fermented feed group Dry matter, % <![CDATA[84.08 b ]]> <![CDATA[86.67 a ]]> Crude protein, % <![CDATA[79.76 b ]]> <![CDATA[82.63 a ]]> Crude fat, % 76.87 80.56 calcium,% 76.19 82.00 phosphorus,% <![CDATA[64.15 b ]]> <![CDATA[74.02 a ]]>

[0050] Example 7: Effects of Fermented Feed on the Activity of Digestive Enzymes in the Jejunum of Piglets

[0051] Based on Example 6, six piglets were randomly selected from each treatment group to measure the enzyme activity in the jejunal contents of the piglets. The results are shown in Table 6. Adding 10% fermented feed to the piglet diet significantly increased the activity of jejunal amylase and trypsin, and promoted intestinal digestive function.

[0052] Table 6. Effects of fermented feed on the activity of digestive enzymes in the jejunum of piglets.

[0053] project antibiotic group Fermented feed group amylase, units / mg prot 335 <![CDATA[652 a ]]> Trypsin, units / g prot 19.2 <![CDATA[28.0 a ]]> Lipase, units / g prot 295 391

[0054] Example 8: Effect of fermented feed on the pH of the digestive tract of piglets

[0055] Based on the experiment in Example 5, six piglets were randomly selected from each treatment group, and the pH of the contents of the piglets' stomach, duodenum, jejunum, ileum, cecum, and colon was measured. The results are shown in Table 7. Adding fermented feed to the piglet diet reduced ( P (< 0.05) The pH in the piglet's stomach is reduced to 4.0, and the pH in the duodenum and jejunum is also reduced, which is conducive to inhibiting the growth and reproduction of pathogens and viruses.

[0056] Table 7. Effects of fermented feed on pH of digestate in the digestive tract of piglets.

[0057] project antibiotic group Fermented feed group Stomach <![CDATA[5.06 b ]]> <![CDATA[4.00 a ]]> Duodenum 6.06 5.25 jejunum 6.18 5.67 ileum 6.09 6.63 appendix 5.62 5.73 colon 6.10 5.70

Claims

1. A fermented compound feed for preventing diarrhea in piglets, characterized in that: Corn, soybean meal, and wheat bran were fermented using a mixture of Bacillus subtilis and Pediococcus pentosaceus. The content of β-conglobulin and soybean globulin decreased by 71.94% and 82.86%, respectively; neutral detergent fiber decreased by 38.93%; the contents of raffinose and stachyose, the flatulence factors, decreased by 42.22% and 36.60%, respectively; and phytic acid phosphorus content decreased by 37.50%. Besides the significant decrease in the contents of the above anti-nutritional factors, lactic acid content increased by 3.5 times, and acid-soluble protein increased by 6 times. Feeding this fermented compound feed to piglets showed comparable control of diarrhea to that of antibiotics. The Bacillus subtilis mentioned above belongs to the Bacillus subsp. subtilis subspecies. Bacillus subtilis subsp. subtilis The specimen, named ZJUAF-2, is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2016, with accession number CGMCC NO:12824. The described *Pediococcus pentosaceus* belongs to the genus *Pediococcus pentosaceus*. Pediococcus pentosaceus It was named ZJUAF-4 and deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 21, 2018, with accession number CGMCC NO:16767. The method for preparing the fermented compound feed involves premixing corn, soybean meal, and wheat bran in a mass ratio of 4.5:4.5:1.0, then adding 10% ZJUAF-2 fermentation liquid and 10% ZJUAF-4 fermentation liquid by mass, and adding 40% water by mass. The fermentation starts at 25-30℃, and the fermentation time is 48-72 hours. The feed is turned over every 5-8 hours. When the pH of the feed drops to 4.0, the corn-soybean meal fermented compound feed is obtained. The ZJUAF-2 fermentation broth: ZJUAF-2 was cultured in LB medium until OD. 600 When the concentration reaches 1.2, the medium is inoculated into the fermentation broth at a volume ratio of 5.0%. The composition of the medium by mass percentage includes 2.0% glycerol, 3.0% soybean meal, 0.4% NaH2PO4, 0.03% K2HPO4, 0.5-1.0% wheat bran, and the remainder is water. The fermentation broth is cultured at 30℃ and 200 r / min for 24 h, and the neutral protease activity reaches 600 U / mL. The ZJUAF-4 fermentation broth was cultured in MRS medium until OD. 600 When the bacterial concentration reaches 1.4-1.7, inoculate the fermentation broth medium at a volume ratio of 0.1%. The medium composition, by weight, includes 0.5-2.0% yeast extract, 0.5-2.0% glucose, 0.5-1.0% tomato extract, 0.1-0.5% KH₂PO₄, and the remainder is water. Incubate at 37℃ for 24 h until the bacterial concentration reaches 1.0 × 10⁻⁶. 9 ~1.0×10 10 cfu / mL.

Images