Method for preparing microecological preparation by using waste cellulase and application thereof

Abstract

The application discloses a method for preparing microecological preparation by using waste cellulase and application thereof. The method comprises the following steps: adding yellow rice wine lees into waste (expired or damp) cellulase as a main raw material, mixing and homogenizing, crushing, adding distilled water for dilution, heating at 80 DEG C for 30 min, cooling, inoculating lactobacillus acidophilus for fermentation for 24 h, supplementing bacillus subtilis for fermentation for 24 h, and preparing the microecological preparation through freeze drying. The enzyme preparation has high live bacteria number, good safety performance, and can significantly reduce the diarrhea rate and feed-meat ratio of pigs.

Description

[0001] Description TECHNICAL FIELD

[0002] The present application belongs to the field of microbial preparation, and particularly relates to a method for preparing microecological preparation by using waste cellulase and application thereof. BACKGROUND

[0003] Cellulase can specifically degrade cellulose, and the final product is oligosaccharide or glucose. There are many types of cellulase, generally including exo-beta-glucanase, endo-beta-glucanase and beta-glucosidase, etc. Cellulase is widely used in the feed industry. It can be used for enzymatic hydrolysis of crops such as grains, beans and cereals, or their processed products, which helps to improve the palatability and digestibility of feed. According to GB 7300.403-2022, the minimum standard of enzyme activity of feed cellulase is 5000 U / g. The quality of cellulase is directly related to the enzyme activity. When stored improperly or for too long, it may be damp, and the enzyme activity may decrease significantly. At this time, the enzyme preparation has lost its value and can only be destroyed as solid waste.

[0004] Distiller's grains are the residues left after juice is taken from wine, containing rich nutrients such as crude fiber, crude protein and amino acids, and leaving the wine aroma after brewing. Small batches of distiller's grains can be used to make distiller's fish and distiller's meat as food additives, but large batches of distiller's grains from distillery plants are difficult to digest and still face disposal problems.

[0005] Lactobacillus and Bacillus subtilis are both feed probiotics, which play an important role in the intestinal health of farmed organisms. However, the preparation of their microecological preparations usually requires the purchase of a large amount of culture medium raw materials, such as carbon sources like rice flour and nitrogen sources like casein, which has a high cost of materials. SUMMARY

[0006] The present application aims to overcome the shortcomings of the prior art, address the problems of waste cellulase and distiller's grains resource waste, and provide a method for preparing microecological preparation by using waste cellulase and application thereof.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0008] A method for preparing microecological preparation by using waste cellulase, comprising the following steps:

[0009] The waste cellulase is mixed with distiller's grains uniformly, and then 10 times the weight of the mixture of distilled water is added for homogenization. The homogenized slurry is transferred to a fermenter, and then diluted with distilled water in an equal weight to the slurry. The mixture is sterilized at 80℃ for 30 minutes. After being cooled to 37℃, 1wt% of Lactobacillus acidophilus seed liquid is poured into the mixture, and then the mixture is fermented at 37℃ and 150r / min for 24 hours. The pH of the fermented liquid is adjusted to 7.0 by using 4mol / L sodium hydroxide. Then, 1wt% of Bacillus subtilis seed liquid is added to the mixture, and the mixture is continuously fermented at 37℃ and 150r / min for 24 hours. The fermented liquid is centrifuged at 10000r / min for 10 minutes to obtain residue, and the residue is freeze-dried to obtain the microecological preparation.

[0010] In the above preparation method, the waste cellulase is expired or damp cellulase, the enzyme activity of which is less than 5000U / g, or the water content of which is higher than 10%, or a large amount of agglomerates has been formed. The waste cellulase is pre-dried at 60℃ to a water content of less than 10% before being mixed with the distiller's grains.

[0011] In the above preparation method, the distiller's grains are the distiller's grains of yellow rice wine brewed with wheat koji. The alcohol content of the distiller's grains is adjusted to 18-20%vol, and the water content is adjusted to 60-70% before being mixed with the waste cellulase.

[0012] In the above preparation method, the mixing ratio of the waste cellulase and the distiller's grains is one of 5:1, 2.5:1, 1:1, 1:2.5 and 1:5. Preferably, the mixing ratio of the waste cellulase and the distiller's grains is 1:2.5.

[0013] In the above preparation method, the homogenization parameters are as follows: the homogenization pressure is 100MPa, and the homogenization times is 3.

[0014] In the above preparation method, the preparation method of the Lactobacillus acidophilus seed liquid is as follows: 10μL of Lactobacillus acidophilus is taken from a frozen tube and coated on MRS solid culture medium, and then incubated at 37℃ for 24 hours. Then, a single colony is picked and inoculated in 250mL of MRS liquid culture medium, and then incubated at 37℃ and 180r / min for 24 hours. The formula of the MRS liquid culture medium is as follows: proteose peptone 10g / L, beef extract 10g / L, yeast powder 5g / L, K2HPO42g / L, ammonium citrate 2g / L, sodium acetate 5g / L, glucose 20g / L, 1g / L Tween 80, MgSO4·7H2O 0.2g / L, MnSO4·4H2O 0.05g / L, and pH 6.2. The formula of the MRS solid culture medium is based on the formula of the MRS liquid culture medium, and 20g / L of agar is additionally added.

[0015] In the above preparation method, the preparation method of the bacillus subtilis seed liquid is: 10 muL of bacillus subtilis is taken from a frozen tube and coated on LB solid culture medium, and then incubated at 37 DEG C for 24 hours, and then a single colony is picked and inoculated in 250 mL of LB liquid culture medium, and then incubated at 37 DEG C and 180 r / min for 24 hours; the formula of the LB liquid culture medium is: 10 g / L of proteose peptone, 5 g / L of yeast powder, 5 g / L of sodium chloride, 1 g / L of glucose, and pH 7.0; the formula of the LB solid culture medium is that 20 g / L of agar is added on the basis of the formula of the LB liquid culture medium;

[0016] The microecological preparation is prepared by using the above preparation method.

[0017] The above microecological preparation is applied in pig breeding.

[0018] The beneficial effects of the present application are:

[0019] The present application turns waste cellulase and distiller's grains into a raw material for the preparation of a feed microecological preparation, realizes resource recycling, improves the energy utilization rate, and does not need to supplement additional carbon source, nitrogen source, vitamins and other nutrients in the fermentation process, thereby reducing the cost. The microecological preparation prepared by the present application has higher viable cell count and can reduce the feed conversion ratio and diarrhea rate of pigs compared with conventional fermentation component microecological preparations. In addition, the fermentation pretreatment process used in the present application can fully exert the characteristics of distiller's grains. The alcohol content and homogenization technology of distiller's grains are used to preliminarily sterilize the waste cellulase and distiller's grains, and then the pasteurization process is used for further treatment, thereby reducing the energy consumption and preserving the flavor of the distiller's grains as much as possible, and improving the pig breeding effect. The addition of the microbial preparation of the present application to pig feed can significantly reduce the diarrhea rate of pigs. DETAILED DESCRIPTION

[0020] The present application will be further described below in conjunction with specific examples, but the present application is not limited to only these examples.

[0021] In the following examples, the cellulase is purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., the product model is H type, the product number is SDG-2425, the original enzyme activity is 10000 U / g, and the shelf life is 12 months. 1000 g of cellulase near expiration is stored at room temperature in the dark for 2 months before the experiment. At this time, the experimental enzyme (waste cellulase) has been damp and clumped, and the enzyme activity has decreased to about 3000 U / g. The enzyme activity detection method refers to NY / T 912-2020 Determination of Cellulase Activity in Feed Additives by Spectrophotometry.

[0022] In the following examples, the production process of distiller's grains is as follows: mix the wheat starter (purchased from Angel Company) with steamed and cooled glutinous rice at a ratio of 1:200, then add an equal amount of distilled water and stir evenly, main fermentation at 30℃ for 5 days, turn over evenly 2 times a day during the period, then transfer to 18℃ for post-fermentation for 20 days, press to extract juice after fermentation, and the remaining residue is the distiller's grains. Adjust the moisture content of the distiller's grains to 60%-70% before the experiment. The moisture content is detected using a rapid water activity meter, with a setting temperature of 105℃.

[0023] In the following examples, the Lactobacillus acidophilus is: Lactobacillus acidophilus CCTCCM2016139; and the Bacillus subtilis is Bacillus subtilis CGMCC 16477.

[0024] Example 1 Comparison of sterilization effects of different homogenization conditions on the mixture of waste cellulase and distiller's grains

[0025] Take one part of waste cellulase and one part of distiller's grains (alcohol content 18-20%vol, moisture content 60%-70%, alcohol content detected using GMK-600 type portable alcohol content tester produced by Korean GWON Company, according to the instruction manual), mix evenly, then add 10 times the weight of the mixture of distilled water, fully stir, homogenize in an ultra-high pressure homogenization equipment (Guangzhou Juncheng Biotechnology Co., Ltd., JC-02C type, maximum processing pressure 207MPa, maximum flow rate 2L / h), the feeding temperature is room temperature 25℃, take 200μL of homogenate after homogenization and dilute and spread on PCA agar plates (purchased from Huizhou Dot Biological Technology Co., Ltd., formula containing 5.0g / L tryptone, yeast extract powder 2.5g / L, glucose 1.0g / L, agar 15g / L, pH 7.0) to calculate the total bacterial count. The results of different homogenization conditions are shown in Table 1.

[0026] Table 1 Total bacterial count of homogenate under different homogenization conditions (unit: lg(CFU / mL))

[0027]

[0028]

[0029] Note: a, b, c, d in the upper right corner of the table represent the degree of significant difference. In the same comparison data, groups marked with the same letter have no significant difference (P value≥0.05), and groups marked with different letters have significant difference (P value<0.05). The larger the mean value, the earlier the order of the letter marked.

[0030] From Table 1, with the increase of homogenization pressure, the total bacteria count in the homogenate liquid is continuously decreased. Since there is no significant difference in the total bacteria count under the conditions of 100 MPa and 150 MPa, from the perspective of energy consumption, the homogenization pressure of 100 MPa is selected. The homogenization times are optimized. Under the homogenization pressure of 100 MPa, there is no significant difference in the total bacteria count between the homogenization for 3 times and the homogenization for 4 times. From the perspective of energy consumption, the homogenization for 3 times is selected.

[0031] Example 2 Comparison of sterilization effects of different alcohol contents in distiller's grains on the mixture of discarded cellulase and distiller's grains

[0032] A portion of discarded cellulase is mixed with a portion of distiller's grains with different alcohol contents (water content 60% to 70%), and then 10 times the weight of the mixture of distilled water is added, and stirred thoroughly. The mixture is subjected to homogenization treatment in an ultrahigh pressure homogenization equipment (the same as that in Example 1). The feeding temperature is room temperature 25°C, the homogenization pressure is 100 MPa, and the homogenization times are 3. After homogenization, 200 μL of the homogenate liquid is taken and diluted and plated on PCA agar plates (the same as those in Example 1) to calculate the total bacteria count. The experimental results are shown in Table 2. The alcohol content is tested in the same manner as in Example 1, and after detection, anhydrous ethanol or distilled water is used to adjust the alcohol content to the target alcohol content.

[0033] Table 2 Total bacteria count of homogenate liquid under different alcohol contents in distiller's grains (unit: lg(CFU / mL))

[0034]

[0035] Note: In the table, a, b, c, d, e on the right top represent the degree of significant difference. In the same comparison data, the groups marked with the same letter have no significant difference (P value≥0.05), and the groups marked with different letters have significant difference (P value<0.05). The larger the average value is, the earlier the order of the letter marked is.

[0036] From Table 2, the increase of alcohol content in distiller's grains is beneficial to the sterilization of the homogenate liquid. When the alcohol content in distiller's grains reaches 18% to 20% vol, the sterilization ability stops. From the perspective of cost, the alcohol content of 18% to 20% vol is selected.

[0037] Example 3 Comparison of effects of homogenate liquid of mixture of discarded cellulase and distiller's grains under different sterilization conditions on microecological preparation and pig breeding

[0038] A portion of the discarded cellulase was mixed with a portion of the distiller's grains (moisture content 60% to 70%, alcohol content 18% to 20% vol) to obtain a mixture, and 10 times the weight of the mixture of distilled water was added to the mixture, and the mixture was stirred thoroughly. The mixture was subjected to homogenization treatment in an ultrahigh-pressure homogenizer (the same as that used in Example 1) at a feed temperature of 25°C, a homogenization pressure of 100 MPa, and 3 times of homogenization. The homogenized slurry was diluted with distilled water in an amount equal to the weight of the homogenized slurry, and sterilized. After sterilization, 200 μL of the nutrient solution was spread on a PCA agar plate (the same as that used in Example 1) to calculate the total number of bacteria. The effects of different sterilization conditions are shown in Table 3.

[0039] Table 3 Total number of bacteria in the nutrient solution under different sterilization conditions (unit: lg(CFU / mL))

[0040] Sterilization conditions 60°C, 40 min 80°C, 30 min 100°C, 20 min 121°C, 15 min Total bacterial count 1.76±0.13 0 0 0

[0041] In combination with Tables 1, 2, and 3, it can be seen that the total number of bacteria in the nutrient solution can be reduced again by homogenization sterilization combined with high-temperature sterilization. From the perspective of energy consumption, 80°C and 30 min are the optimal conditions for high-temperature sterilization.

[0042] Example 4 Optimization of the ratio of discarded cellulase to distiller's grains in the micro-ecological preparation

[0043] Lactobacillus acidophilus seed solution: 10 μL of Lactobacillus acidophilus was taken from a frozen tube and spread on MRS solid medium, and incubated at 37°C for 24 h. Then, a single colony was picked and inoculated into 250 mL of MRS liquid medium, and incubated at 37°C and 180 r / min for 24 h. The formula of the MRS liquid medium was as follows: 10 g / L of proteose peptone, 10 g / L of beef extract, 5 g / L of yeast powder, 2 g / L of ammonium citrate, 5 g / L of sodium acetate, 20 g / L of glucose, 1 g / L of Tween 80, 0.2 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4·4H2O, and pH 6.2. The formula of the MRS solid medium was the same as that of the MRS liquid medium, except that 20 g / L of agar was additionally added.

[0044] Bacillus subtilis seed solution: 10 μL of Bacillus subtilis was taken from a frozen tube and spread on LB solid medium, and incubated at 37°C for 24 h. Then, a single colony was picked and inoculated into 250 mL of LB liquid medium, and incubated at 37°C and 180 r / min for 24 h. The formula of the LB liquid medium was as follows: 10 g / L of proteose peptone, 5 g / L of yeast powder, 5 g / L of sodium chloride, and 1 g / L of glucose, and pH 7.0. The formula of the LB solid medium was the same as that of the LB liquid medium, except that 20 g / L of agar was additionally added.

[0045] The waste cellulase and distiller's grains (moisture content 60%~70%, alcohol content 18~20%vol) were mixed uniformly in a desired ratio, then 10 times the weight of the mixture of distilled water was added, stirred well, and then homogenized in an ultrahigh pressure homogenizer (same as in Example 1) at a feed temperature of 25°C, a homogenization pressure of 100 MPa, and 3 times of homogenization. The homogenized homogenate was transferred to a fermenter, diluted with distilled water in an amount equal to the weight of the homogenate, and sterilized at 80°C for 30 min; after cooling to 37°C, 1 wt% of Lactobacillus acidophilus seed liquid was poured in, and then fermented at 37°C and 150 r / min for 24 h; the pH was adjusted to 7.0 with 4 mol / L sodium hydroxide, and then 1 wt% of Bacillus subtilis seed liquid was added, and then the fermentation was continued at 37°C and 150 r / min for 24 h; the obtained fermentation broth was centrifuged at 10,000 r / min for 10 min to obtain the bacterial residue, and the bacterial residue was freeze-dried to obtain the microecological preparation.

[0046] 0.2 g of the prepared microecological preparation was added to 10 mL of distilled water, shaken and mixed, then gradient diluted, and then coated on PCA agar plates (same as in Example 1) and MRS solid medium to calculate the total bacterial count and Lactobacillus acidophilus count, respectively, and the Bacillus subtilis count = total bacterial count - Lactobacillus acidophilus count.

[0047] Different microecological preparations were mixed into pig feed at a ratio of 0.1‰ (weight ratio) to investigate the effect of the microbial preparation on the feed conversion ratio and diarrhea rate during pig breeding. The pigs weighed 40±0.5 kg, and there were 30 pigs in each group. The feeding time was 2 weeks, and the blank group did not add the microecological preparation. Among them, the feed conversion ratio = total amount of feed consumed (kg) / total weight gain (kg), and the diarrhea rate = number of pigs with diarrhea / total number of pigs x 100%.

[0048] The different ratios of waste cellulase and distiller's grains on the bacterial count and pig breeding effect are shown in Table 4.

[0049] Table 4 Comparison of the effect of different ratios of waste cellulase and distiller's grains on the number of probiotics and pig breeding

[0050]

[0051] Note: In the table, a, b, c, d on the right top represent the degree of significant difference. In the same comparison data, the groups marked with the same letter have no significant difference (P value≥0.05), and the groups marked with different letters have significant difference (P value<0.05). The larger the mean value, the earlier the order of the letter marked.

[0052] Because the content of waste cellulase protein is high, it mainly acts as a nitrogen source in the fermentation process, and the content of carbohydrates in the distiller's grains is high, which mainly acts as a carbon source in the fermentation process. When the ratio of the two is appropriate, the number of microorganisms can be increased. As can be seen from Table 4, the ratio of waste cellulose to distiller's grains is 1:2.5, which is the best, at this time the number of lactic acid bacteria, bacillus subtilis and total bacteria are the best, and can significantly reduce the feed-meat ratio and diarrhea rate during pig breeding.

[0053] Example 5 Comparison of the effects of different microecological preparations on pig breeding

[0054] In order to confirm whether the waste cellulose and distiller's grains can be replaced by ordinary medium components, and whether the relay fermentation of lactobacillus acidophilus and bacillus subtilis has significant progress, the following microecological preparations are prepared, and the method of example 4 is used for pig breeding.

[0055] Microecological preparation A (waste cellulase + distiller's grains + relay fermentation): using the preparation method of example 4, wherein the ratio of waste cellulase to distiller's grains is 1:2.5.

[0056] Microecological preparation B (casein + distiller's grains + relay fermentation): using the preparation method of example 4, wherein the waste cellulase is replaced by casein in equal amount, and the ratio of casein to distiller's grains is 1:2.5.

[0057] Microecological preparation C (waste cellulase + rice flour + relay fermentation): using the preparation method of example 4, wherein the distiller's grains are replaced by rice flour in equal amount, and the ratio of waste cellulase to rice flour is 1:2.5.

[0058] Microecological preparation D (waste cellulase + distiller's grains + separate fermentation and then mixed use): using the homogenate of example 4, wherein the ratio of waste cellulase to distiller's grains is 1:2.5. After homogenization, the homogenate is transferred to the fermentation tank, and the same weight of distilled water as the homogenate is added for dilution, 80℃ sterilization for 30min, to obtain a nutrient solution. Lactobacillus acidophilus and bacillus subtilis are inoculated into the nutrient solution respectively, and fermented at 37℃, 150r / min, wherein lactobacillus acidophilus is fermented for 48h, and bacillus subtilis is fermented for 24h. The obtained fermentation broth is centrifuged at 10000r / min for 10min to obtain the residue, and the residue is freeze-dried to obtain lactobacillus acidophilus microecological preparation and bacillus subtilis microecological preparation respectively. Before breeding pigs, the single microecological preparation is mixed into microecological preparation D according to the proportion of the number of live bacteria of lactobacillus acidophilus and bacillus subtilis in microecological preparation A.

[0059] The number of lactobacillus acidophilus, bacillus subtilis and total bacteria in each microecological preparation and the feed-meat ratio and diarrhea rate during pig breeding are detected according to the method of example 4, and the blank group does not add microecological preparation, and the results are shown in table 5.

[0060] Table 5 Comparison of effects of different microecological preparations on probiotic bacteria and pig breeding

[0061]

[0062]

[0063] Note: a, b, c, d, e in the upper right corner of the table represent the degree of significant difference. In the same comparison data, groups marked with the same letter have no significant difference (P value ≥ 0.05), and groups marked with different letters have significant difference (P value < 0.05). The larger the mean value, the earlier the order of the letter marked.

[0064] As can be seen from Table 5, it is difficult to replace the waste cellulase and vinasse with ordinary medium components, because after replacement, the number of Lactobacillus acidophilus, the number of Bacillus subtilis, and the total number of bacteria all decrease, and the feed-meat ratio and the diarrhea rate of pigs increase. On the other hand, even if the medium of the present application is used, the effect comparison of relay fermentation and mixing after separate fermentation, the microecological preparation of relay fermentation has better effect on pig breeding, and the feed-meat ratio and the diarrhea rate are both lower.

[0065] Example 6 Safety evaluation of microecological preparation

[0066] Because the waste enzyme preparation and vinasse are used, safety detection needs to be performed after the production of the microecological preparation. The detection object is the microecological preparation A in Example 5. The relevant indicators are detected according to the reference standards “NY / T 1231-2012 Bacillus subtilis as a feed additive” and “GB 7300.504-2023 Feed additives Part 5: Lactobacillus acidophilus”, and the results show that the microecological preparation A does not detect aflatoxin, zearalenone, vomitoxin, mold, Escherichia coli, Salmonella, Shigella, and Staphylococcus aureus, which meets the relevant safety specifications, and the viable count of Lactobacillus acidophilus (1.57×10 9 CFU / g) is higher than the standard (1×10 8 CFU / g), and the viable count of Bacillus subtilis (2.31×10 9 CFU / g) is higher than the standard (1×10 9 CFU / g).

[0067] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made within the scope of the patent application of the present application shall be included in the scope of the present application.

Claims

1. A method for preparing a microecological preparation using waste cellulase, characterized in that, The process includes the following steps: Waste cellulase and distiller's grains are mixed evenly, and 10 times the weight of the mixture of distilled water is added for homogenization. The homogenized slurry is then transferred to a fermentation tank, diluted with an equal weight of distilled water, sterilized, and cooled. A 1 wt% Lactobacillus acidophilus seed culture is inoculated, and after 24 hours of fermentation, the pH is adjusted to 7.

0. Another 1 wt% Bacillus subtilis seed culture is then added, and fermentation continues for another 24 hours. The fermentation broth is centrifuged to obtain the microbial residue, which is then freeze-dried to produce a microecological preparation. The Lactobacillus acidophilus is CCTCC M2016139; The Bacillus subtilis strain is Bacillus subtilis CGMCC 16477; The mixing ratio of the waste cellulase to the distiller's grains is (0.2-5):1; The fermentation conditions were 37℃, 150r / min, and 24h. The waste cellulase is expired or damp cellulase with an enzyme activity of less than 5000 U / g, or a moisture content of more than 10%, or has formed a large number of clumps. The waste cellulase is pre-dried at 60°C to a moisture content of less than 10% before being mixed with the lees. The lees are the lees from rice wine brewed with wheat koji. Before mixing with waste cellulase, the alcohol content is adjusted to 18-20% vol and the water content is adjusted to 60-70%.

2. The method according to claim 1, characterized in that, The method for preparing the Lactobacillus acidophilus seed culture is as follows: take 10 μL of Lactobacillus acidophilus from the cryopreservation tube, spread it on MRS solid medium, and incubate it at 37°C for 24 h. Then pick a single colony and inoculate it into 250 mL of MRS liquid medium, and incubate it at 37°C and 180 r / min for 24 h.

3. The method according to claim 1, characterized in that, The method for preparing the Bacillus subtilis seed culture is as follows: 10 μL of Bacillus subtilis from the cryopreservation tube is spread on LB solid medium and incubated at 37°C for 24 h. Then, a single colony is picked and inoculated into 250 mL of LB liquid medium and incubated at 37°C and 180 r / min for 24 h.

4. A microecological preparation obtained by any of the preparation methods described in claims 1-3.

5. The application of the microecological preparation as described in claim 4 in pig farming.

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