Zymophyte enzyme preparation containing phaffia rhodozyma and lactobacillus acidophilus and application of zymophyte enzyme preparation

Abstract

The invention discloses a zymophyte enzyme preparation containing phaffia rhodozyma and lactobacillus acidophilus and application of the zymophyte enzyme preparation. The zymophyte enzyme preparation contains phaffia rhodozyma, lactobacillus acidophilus, alkaline protease and cellulase. The zymophyte enzyme preparation is added into the soybean meal, the fermented soybean meal is obtained through fermentation, the content of glutamic acid in the fermented soybean meal can be remarkably increased, the content of antigen protein in the soybean meal can be reduced, the fermented soybean meal replaces 30% by mass of soybean meal and is evenly mixed with corn flour and bran to be directly fed to fattening pigs, the total ammonia content in pork can be remarkably reduced, and the pig fattening efficiency is improved. The fishy smell of the pigs is reduced, the meat quality is improved, and the feed has good application value in fattening pig breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a fermentation bacteria enzyme preparation containing Phaffia rhodozyma and Lactobacillus acidophilus and application thereof. Background Art

[0002] Soybean meal is a byproduct obtained after soybean oil is extracted from soybeans. It is widely used in livestock and poultry feeds due to its high protein content, multiple amino acids such as methionine, good palatability, high metabolic energy, and suitability for the needs of fast-growing animals. About 85% of soybean meal is used to feed poultry and pigs. However, soybean meal also faces many problems during use, such as the presence of anti-nutritional factors (antigenic proteins, trypsin inhibitors, urease, prothrombin, goitrogens, lipoxygenase, antigenic proteins, oligosaccharides, and phytic acid), which lead to diarrhea and indigestion in animals after consumption. Indigestion in animals can cause excessive ammonia content in the intestines, and excessive ammonia content can lead to poor flavor of pork. There are two main solutions to reduce the problem of excessive ammonia content: one is to reduce the content of anti-nutritional factor antigen protein in soybean meal; the other is to use probiotics to ferment to produce flavor amino acids.

[0003] Currently, there are many methods to improve pork quality, but there are few reports on improving pork taint. Therefore, it is very important to design and produce a functional fermented soybean meal microbial preparation product to solve the pork taint. Summary of the invention

[0004] The present invention provides a fermentation bacteria enzyme preparation containing rhodozyma and Lactobacillus acidophilus and its application. After fermenting soybean meal, the fermentation bacteria enzyme preparation can significantly reduce the antigen protein content in the soybean meal and increase the glutamic acid content in the soybean meal. After pigs eat it, it can significantly reduce the crude ammonia content in pork and reduce the pig odor.

[0005] In order to achieve the above-mentioned object of the invention, the present invention is implemented by the following technical solutions: The invention provides a fermentation bacteria enzyme preparation containing Phaffia rhodozyma and Lactobacillus acidophilus, which comprises Phaffia rhodozyma powder, Lactobacillus acidophilus powder, alkaline protease and cellulase.

[0006] Furthermore, the amount of live bacteria in the red Phaffia yeast powder is 2×10 7 CFU / g~2×10 9 CFU / g, the live bacteria amount of the Lactobacillus acidophilus powder is 1×10 7 CFU / g~1×10 9 CFU / g.

[0007] Furthermore, the addition amount of the alkaline protease is 10000U / g~100000U / g; the addition amount of the cellulase is 100U / g~1000U / g.

[0008] Furthermore, the red Phaffia yeast powder is prepared from red Phaffia yeast GOY1 with a preservation number of CGMCC NO.22504; and the Lactobacillus acidophilus powder is prepared from Lactobacillus acidophilus L100 with a preservation number of CGMCC No.10701.

[0009] Furthermore, the preparation steps of the red Phaffia yeast powder are: inoculating the red Phaffia yeast into a liquid culture medium, culturing to a logarithmic growth phase, transferring the inoculation amount of 8% to 10% to a secondary production tank for further culturing, and freeze-drying after the culturing to obtain the red Phaffia yeast powder.

[0010] The present invention also provides a fermented soybean meal, which is obtained by fermenting the fermentation bacteria enzyme preparation.

[0011] Furthermore, the preparation step of the fermented soybean meal is: adding the fermentation bacteria enzyme preparation to the soybean meal in an amount of 8g / kg-10g / kg, the moisture content of the fermented soybean meal is 38%, the fermentation temperature is 37°C, the fermentation is 5d-7d, and after the fermentation is completed, the fermented soybean meal is sieved to obtain the fermented soybean meal.

[0012] The invention also provides the use of the fermentation bacteria enzyme preparation in reducing the ammonia content in the intestinal tract of animals.

[0013] The present invention also provides the use of the fermented bacteria enzyme preparation or the fermented soybean meal in improving the mutton smell of fattened pork.

[0014] Furthermore, the fermented soybean meal is used to replace 10% to 30% of soybean meal by mass, and is mixed with corn flour and bran to feed fattening pigs.

[0015] Compared with the prior art, the advantages and beneficial technical effects of the present invention are: The fermented bacteria enzyme preparation prepared by the present invention can reduce the content of soybean meal antigen protein and increase the content of flavor amino acid glutamic acid, thereby improving the quality of meat. The present invention utilizes fermentation bacteria enzyme preparations to obtain fermented soybean meal, wherein Phaffia rhodozyma and Lactobacillus acidophilus can utilize excess ammonia in the intestine, reduce crude ammonia content in pork, and improve the occurrence of pork odor and other problems.

[0016] 3. The fermented soybean meal obtained by the fermentation bacteria enzyme preparation of the present invention is easy to operate, has a low technical threshold, is easy to achieve on-site fermentation, and does not use high-temperature treatment to retain the nutrients in the fermented soybean meal to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The colonies and microscopic morphology of Phaffia rhodozyma on the plate; Figure 2 It is the colony and microscopic morphology of Lactobacillus acidophilus on the plate; Figure 3 Comparison of antigen protein content before and after fermentation of soybean meal with different yeasts; Figure 4 Comparison of antigen protein content before and after fermentation of soybean meal by different lactic acid bacteria; Figure 5 The results of qualitative and quantitative detection of antigen protein content in soybean meal before and after fermentation with fermentation bacteria enzyme preparations; Figure 6 These are the test results of amino acid content before and after fermentation of soybean meal with fermentation bacteria enzyme preparation. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail with reference to the following specific examples.

[0019] Example 1: Screening of Phaffia rhodozyma GOY1 and preparation of bacterial powder 1. Screening, isolation and identification of Phaffia rhodotorula GOY1 The aquaculture water of Qingdao Jiaozhou sea cucumber was taken, and the water was spread on YPD medium after gradient dilution. After multiple separation and purification, a single colony was obtained, named GOY1, and preserved.

[0020] The colony morphology of strain GOY1 is as follows Figure 1 As shown in a, the colonies are large, moist, raised, orange to red, and smooth without wrinkles; Figure 1 b are all irregular ovals and are independently scattered.

[0021] The DNA of Phaffia rhodozyma GOY1 was used as a template and 18S rRNA universal primers were used for amplification. The amplified fragments were sequenced and the 18S rDNA sequencing results of strain GOY1 were compared with the sequences in GenBank. The results showed that strain GOY1 had a similar Phaffia rhodozyma The homology was the highest, so the strain GOY1 was determined to be Phaffia rhodozyma.

[0022] The screened strain GOY1 was deposited, and the depository of the red yeast GOY1 is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: May 11, 2021; red yeast Phaffia rhodozyma The deposit number is CGMCC No.22504.

[0023] 2. Preparation of Phaffia rhodozyma GOY1 powder The red Phaffia yeast GOY1 was inoculated into YPD liquid culture medium and cultured to OD=1.0 in the logarithmic growth phase. The inoculation amount was 8%~10% and transferred to the secondary production tank for further culture for 24 hours. The bacterial sludge was collected by centrifugation at 4500rpm for 15 minutes. 7.4% trehalose, 7.2% skim milk powder, 4.5% gum arabic and 6.0% sodium L-glutamate freeze-drying protective agent were added to the bacterial sludge, and the sludge was added to the emulsification tank for emulsification for 1 hour. After the emulsification was completed, the sludge was added to the vacuum freeze dryer and freeze-dried for 60 hours. After the drying was completed, it was crushed with a pulverizer to obtain the red Phaffia yeast powder.

[0024] Example 2: Screening of Lactobacillus acidophilus L100 and preparation of bacterial powder 1. Screening, isolation and identification of Lactobacillus acidophilus L100 Take the intestinal chyme from healthy broiler chickens, dilute it with sterile saline, spread it in MRS medium, culture it at 37℃ for 48h, pick a single colony, streak it on the surface of MRS medium for purification, and culture it upside down in a 37℃ constant temperature incubator. Figure 2 ) to the inclined surface and save.

[0025] The DNA of strain L100 was used as a template, 16S rDNA universal primers were used for amplification, and the amplified fragments were sequenced. The 16S rDNA sequencing results were compared and analyzed, and the results showed that strain L100 and Lactobacillus acidophilus Therefore, the strain was identified as Lactobacillus acidophilus.

[0026] The strain L100 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; deposit date: April 9, 2015; Lactobacillus acidophilus Lactobacillus acidophilus The deposit number is CGMCC No.10701.

[0027] 2. Preparation of Lactobacillus acidophilus L100 powder Lactobacillus acidophilus L100 was inoculated into MRS liquid culture medium and cultured to OD=1.0 in the logarithmic growth phase. The culture was transferred to a secondary production tank at an inoculum size of 2% to 4% and continued to be cultured for 24 hours. The bacterial sludge was collected by centrifugation at 4500rpm for 15 minutes. 6% inulin, 5% skim milk powder, 9% mannitol and 9% sodium L-glutamate freeze-drying protective agent were added to the bacterial sludge, and the sludge was added to an emulsification tank for emulsification for 1 hour. After the emulsification was completed, the sludge was added to a vacuum freeze dryer and freeze-dried for 60 hours. After the drying was completed, the sludge was crushed with a pulverizer to obtain Lactobacillus acidophilus powder.

[0028] Example 3: Comparison of soybean meal antigen proteins fermented by different yeasts Streak single colonies of Saccharomyces cerevisiae and Phaffia rhodozyma on the culture medium, pick a single colony and inoculate it into YPD medium, ferment and culture until OD=1.0, inoculate it into soybean meal at a 2% inoculum, control the moisture content of the fermented soybean meal at 38%, ferment for 5 days, and determine the antigen protein content in the fermented soybean meal.

[0029] according to Figure 3 The results showed that different types of yeast had different effects on the degradation of soybean meal antigen protein content. Among them, red yeast had the best effect on degrading soybean meal antigen protein, and the antigen protein content was 38% lower than that of the group treated with brewer's yeast.

[0030] Example 4: Experiment on fermentation of soybean meal by different lactic acid bacteria Single colonies of Lactobacillus plantarum, Pediococcus acidilactici, Lactobacillus acidophilus, Enterococcus faecalis, Lactobacillus reuteri, and Bifidobacterium lactis were streaked, and single colonies on the plate were picked and inoculated into MRS medium. The culture was cultured at 37°C and fermented until OD = 1.0. The inoculation amount was 2% and the fermentation moisture was 38%. The fermentation was carried out for 5 days, and the antigen protein content in the fermented soybean meal was determined.

[0031] according to Figure 4 The results showed that the content of antigenic protein in soybean meal fermented by different lactic acid bacteria was different, among which Lactobacillus acidophilus had the best effect in degrading soybean meal antigenic protein.

[0032] Example 5: Orthogonal combination test The invention discloses a fermentation bacterial enzyme preparation, which is composed of Phaffia rhodozyma, Lactobacillus acidophilus, alkaline protease and cellulase. Phaffia rhodozyma, Lactobacillus acidophilus, alkaline protease and cellulase are subjected to a 4-factor 3-level orthogonal test according to three addition amounts to form 9 formulations of bacterial agents, 500 g of soybean meal is weighed, the addition amount of the fermentation bacterial enzyme preparation is 8 g / kg, the moisture content is 38%, and the fermentation is carried out at 37°C for 5 days. After the fermentation is completed, the antigen protein content in the fermented soybean meal is determined.

[0033] According to Table 1, the content of antigenic protein in fermented soybean meal of test group 9 is the lowest, so the final addition amount of the fermentation bacteria enzyme preparation is based on test group 9.

[0034] Table 1 Orthogonal combination of four added ingredients

[0035] Example 6: Detection of indicators of fermented soybean meal before and after fermentation The fermentation bacteria enzyme preparation was compounded according to the addition amount in Experiment 9, and the amount of live bacteria was 2×10 9 CFU / g of Phaffia rhodozyma powder, 1×10 9CFU / g of Lactobacillus acidophilus powder, 50000U / g of alkaline protease, and 100U / g of cellulase were mixed evenly to prepare a fermented bacterial enzyme preparation. Weigh 500g of soybean meal, add 8‰ of the fermented bacterial enzyme preparation, and ferment at 37℃ for 5 days. After the fermentation, crush the soybean meal through a 60-mesh sieve for later use. The fermented soybean meal was qualitatively detected by SDS-PAGE protein gel, quantitatively detected by Longke Xinyu antigen detection kit, and the amino acid results were determined.

[0036] According to Table 2, the antigen protein content of fermented soybean meal is at the level of 28.52 mg / g, and both antigen protein bands are degraded on the antigen protein gel ( Figure 5 ), the glutamic acid content increased significantly, 1.08% higher than that of unfermented soybean meal ( Figure 6 ).

[0037] Table 2 Comparison of fermented soybean meal and soybean meal antigen protein

[0038] Example 7: Effect of fermented soybean meal on ammonia treatment Take 100g of soybean meal, add 8‰ of the fermentation bacteria enzyme preparation prepared in Example 6, add 5mL of 0.3mol / L ammonia water, and the water addition amount is 38%. The raw materials, bacterial agent and ammonia water are mixed and placed in a sealed tank, and a blank control group is set. The control group is not added with the fermentation bacteria enzyme preparation. The sealed tank is placed at 37°C for 2d. After storage, the ammonia content in the tank is measured using a pump suction gas detector.

[0039] According to the results in Table 3, fermented soybean meal can significantly reduce the ammonia content. The ammonia content in the experimental group was 5.10 ppm lower than that in the control group. This result shows that fermented soybean meal can indeed reduce the ammonia content in the animal intestine.

[0040] Table 3 Ammonia content detection

[0041] Example 8: Application test of fermented soybean meal on fattening pigs 360 healthy fattening pigs weighing about 60 kg were selected and divided into 4 treatment groups, each with 3 replicates, 30 fattening pigs in each replicate, the control group was added with 30% soybean meal + 60% corn flour + 10% bran, the experimental group 1 was added with 10% fermented soybean meal (same as Example 6) + 20% soybean meal + 60% corn flour + 10% bran, the experimental group 2 was added with 20% fermented soybean meal + 10% soybean meal + 60% corn flour + 10% bran, and the experimental group 3 was added with 30% fermented soybean meal + 60% corn flour + 10% bran. After feeding for 40 days, 10 fattening pigs were randomly selected from each replicate group, the total ammonia content in the pork was detected, and the probability of occurrence of pig taint was counted.

[0042] According to the results in Table 4, in the breeding process of fattening pigs, as the proportion of fermented soybean meal replacing soybean meal increases, the total ammonia content in meat and the incidence of meat odor decrease, so the best replacement option is 30% replacement.

[0043] Table 4 Comparison of quality of fattening pork before and after feeding fermented soybean meal

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A fermentation bacterial enzyme preparation containing Phaffia rhodozyma and Lactobacillus acidophilus, characterized in that: The fermentation bacteria enzyme preparation comprises rhodozyma powder, lactobacillus acidophilus powder, alkaline protease and cellulase.

2. The fermentation bacteria enzyme preparation according to claim 1, characterized in that The amount of live bacteria in the red Phaffia yeast powder is 2×10 7 CFU / g~2×10 9 CFU / g, the live bacteria amount of the Lactobacillus acidophilus powder is 1×10 7 CFU / g~1×10 9 CFU / g.

3. The fermentation bacteria enzyme preparation according to claim 1, characterized in that: The added amount of the alkaline protease is 10000U / g to 100000U / g; the added amount of the cellulase is 100U / g to 1000U / g.

4. The fermentation bacteria enzyme preparation according to claim 1, characterized in that: The red Phaffia yeast powder is prepared from red Phaffia yeast GOY1 with a preservation number of CGMCC NO.22504; and the lactobacillus acidophilus powder is prepared from lactobacillus acidophilus L100 with a preservation number of CGMCC No.10701.

5. The fermentation bacteria enzyme preparation according to claim 1, characterized in that: The preparation steps of the red Phaffia yeast powder are as follows: inoculating the red Phaffia yeast into a liquid culture medium, culturing to a logarithmic growth phase, transferring the inoculation amount of 8% to 10% to a secondary production tank for further culturing, and freeze-drying after the culturing to obtain the red Phaffia yeast powder.

6. A fermented soybean meal, characterized in that: The fermented soybean meal is obtained by fermenting the fermentation bacteria enzyme preparation according to any one of claims 1 to 5.

7. The fermented soybean meal according to claim 6, characterized in that The preparation steps of the fermented soybean meal are as follows: adding the fermentation bacteria enzyme preparation to the soybean meal in an amount of 8g / kg-10g / kg, the moisture content of the fermented soybean meal is 38%, the fermentation temperature is 37°C, the fermentation is performed for 5d-7d, and after the fermentation is completed, the fermented soybean meal is sieved to obtain the fermented soybean meal.

8. Use of the fermentation bacteria enzyme preparation according to claim 1 in reducing the ammonia content in the intestinal tract of animals.

9. Use of the fermented bacterial enzyme preparation according to claim 1 or the fermented soybean meal according to claim 6 in improving the mutton smell of fattening pork.

10. The use according to claim 9, characterized in that: The fermented soybean meal is used to replace 10% to 30% of soybean meal by mass, and is mixed with corn flour and bran and then fed to fattening pigs.

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