CN116376779A - Salt-tolerant bacillus for producing complex enzyme and application of salt-tolerant bacillus in feed preparation - Google Patents

Salt-tolerant bacillus for producing complex enzyme and application of salt-tolerant bacillus in feed preparation Download PDF

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CN116376779A
CN116376779A CN202310486556.5A CN202310486556A CN116376779A CN 116376779 A CN116376779 A CN 116376779A CN 202310486556 A CN202310486556 A CN 202310486556A CN 116376779 A CN116376779 A CN 116376779A
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salt
tolerant bacillus
bacillus
clostridium
microbial agent
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江凌
刘振磊
朱政明
杨智晗
朱丽英
刘瑜欣
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Nanjing Tech University
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Abstract

The invention discloses a salt-tolerant bacillus for producing complex enzyme and application thereof in feed preparation, and belongs to the technical field of biology. The invention provides salt-tolerant bacillus capable of simultaneously producing cellulase and xylanase, which can be used for producing the cellulase and the xylanase. The cellulase and xylanase preparation produced by the strain can efficiently degrade lignocellulose such as wheat straw, corn straw, rice straw and the like, and can obtain monosaccharides such as glucose and the like. The strain of the invention can also be used for preparing ruminant feed, and is beneficial to improving the utilization rate of the feed.

Description

Salt-tolerant bacillus for producing complex enzyme and application of salt-tolerant bacillus in feed preparation
Technical Field
The invention relates to a salt-tolerant bacillus for producing complex enzyme and application thereof in feed preparation, belonging to the technical field of biology.
Background
Under the current new background of carbon neutralization, how to improve the cultivation efficiency, enhance the feed utilization rate and reduce the methane emission has become an important attack point of carbon emission reduction in the animal husbandry. The fodder for ruminant is mainly coarse fodder with high lignification such as corn stalk, rice straw, wheat stalk, etc., and even in beef cattle raising, the coarse fodder with low quality such as corn stalk, rice straw stalk, etc. accounts for no less than 50%. Ruminants rely on cellulases/xylanases secreted by ruminal microorganisms to digest plant feed that is unavailable to monogastric animals. However, since fiber components such as cellulose and lignin contained in the roughage mainly exist in the cell wall, the cellulose/xylanase secreted by the rumen microorganism alone cannot effectively degrade the fiber components and release intracellular nutrients. Therefore, there is an urgent need to improve the utilization efficiency of cellulose in coarse feed for ruminants, and if strains with improved cellulose utilization can be found out for feeding ruminants, the economic and social benefits of animal husbandry in China will be improved.
Disclosure of Invention
In view of the above problems in the prior art, the present invention provides a salt tolerant bacillus (Bacillus halotolerans) B757 for producing cellulase and xylanase, which has been deposited at the collection of microorganism strains in Guangdong province at 10/14 of 2022 under the deposit number GDMCC No:62887 the preservation address is building 5 of Guangzhou Md.A. No. 100 university, first.
The salt-tolerant bacillus B757 is gram-positive bacteria, is aerobic, has a short rod shape, and is light green under microscopic observation; the colony is milky white, the edge is irregular and microcreped;
the salt-tolerant bacillus B757 can grow in a culture medium (such as LB culture medium) containing NaCl and a conventional carbon and nitrogen source, and the proper culture temperature is 27-38 ℃ and the pH is 6.5-7.5;
the salt-tolerant bacillus B757 can tolerate the growth environment of 12% NaCl and 1.0% bile salt, and incubate for 3 hours under the condition of pH 2.0, the viable count is hardly reduced, which indicates that the strain can resist the adverse effects of gastric acid and high-concentration bile salt;
the salt-tolerant bacillus B757 has stronger cellulase and xylanase producing capacity;
the activity of the cellulase and xylanase is as follows: the cellulase activity is 80-180U/mL, and the xylanase activity is 50-150U/mL;
preferably, the cellulase and xylanase have the following activity: cellulase activity was 159.4U/mL and xylanase activity was 106.4U/mL.
The invention also provides a microbial agent containing the salt-tolerant bacillus B757.
In one embodiment, the microbial agent contains the salt-tolerant bacillus B757 and clostridium tyrobutyrate.
In one embodiment, the microbial agent has a concentration ratio of salt-tolerant bacillus B757 to clostridium tyrobutyricum of 8-10:1.
In one embodiment, the microbial agent is compounded by microbial powder and auxiliary materials.
In one embodiment, the clostridium tyrobutyrate is clostridium tyrobutyrate (Clostridium tyrobutyricum) L319, accession No. GDMCC No:62289, which is disclosed in the publication No. CN 115305219A.
In one embodiment, the microbial agent has a bacterial concentration of 8×10 of salt-tolerant bacillus B757 10 ～8×10 12 CFU/g, clostridium casei concentration of 8X10 9 ～8×10 11 CFU/g。
In one embodiment, the microbial preparation is prepared from the following steps of obtaining single bacterial powder and counting viable bacteria, wherein the viable bacteria number ratio of the salt-tolerant bacillus B757: clostridium tyrobutyrate l319=10:1.
In one embodiment, the microbial agent comprises the following microorganisms: salt-tolerant bacillus B757 multiplied by 8 multiplied by 10 11 CFU/g, clostridium casei 8×10 10 CFU/g。
In one embodiment, the auxiliary materials in the microbial agent comprise one or more of trehalose and chitosan oligosaccharide, and the auxiliary materials mainly play roles of dispersing single bacterial powder and protecting microbial cells.
In one embodiment, the salt-tolerant bacillus B757 and clostridium tyrobutyricum L319 are respectively subjected to liquid fermentation, centrifugation and spray drying to obtain dry bacterial powder, and the dry bacterial powder is mixed with auxiliary materials to obtain the microbial agent.
The invention also provides application of the microbial preparation in preparation of ruminant feed.
The beneficial effects are that:
1. the salt-tolerant bacillus B757 obtained by screening has strong resistance to the external environment, can completely resist the gastric acid and intestinal canal bile salt high osmotic pressure environment in ruminants, and has strong tolerance and stable property compared with enzyme preparations. In addition, the microbial agent has long shelf life and is easy to transport and store.
2. The salt-tolerant bacillus separated by the invention has higher capability of producing composite enzymes such as cellulase, protease, xylanase and the like and stronger capability of degrading cellulose, and can improve the digestibility and the utilization rate of roughage.
3. The selected salt-tolerant bacillus and clostridium tyrobutyricum strains have strong complementarity and degrade cellulose through mutual synergistic effect; in addition, as a novel probiotic, butyric acid, a main metabolite of clostridium tyrobutyrate, is a main nutrient for regeneration and repair of intestinal epithelial tissue cells, and butyric acid has a remarkable effect in promoting animal growth. Therefore, the application of the compound microbial agent of the salt-tolerant bacillus and the clostridium tyrobutyrate in further improving the animal production capacity on the basis of improving the digestibility and the utilization rate of the roughage is the initiative of the invention.
Preservation of biological materials
Salt tolerant bacillus (Bacillus halotolerans) B757, classified under the name Bacillus halotolerans, was deposited at the collection of microorganism strains in the cantonese province on day 14 of 10 of 2022 under the accession number GDMCC No:62887 the preservation address is building 5 of Guangzhou Md.A. No. 100 university, first.
Drawings
Fig. 1: the invention separates the growth state of the screened salt-tolerant bacillus B757 strain on a flat plate.
Fig. 2: the isolated and screened salt-tolerant bacillus B757 strain has a growth curve under the conditions of high osmotic pressure and high bile salt.
Detailed Description
The microbial agent is produced autonomously by the inventor; other auxiliary materials are purchased from the feed market. Clostridium tyrobutyrate (Clostridium tyrobutyricum) L319 used in the examples, accession No. GDMCC No:62289, which is disclosed in the publication No. CN 115305219A.
Cellulase activity assay method reference (shi rhyme. Rumen origin 11 bacillus physiological property study and fermentation condition optimization [ D ]. University of company industry university, 2018.); xylanase activity determination methods are described (Zhao Longmei et al screening identification and characterization of xylanase-producing microorganisms [ J ]. Proc. Livestock ecological, 2022,43 (04): 14-20.).
Cellulase activity is defined as: the amount of enzyme required to hydrolyze the substrate to 1. Mu. MoL glucose per 1min at 25℃in an environment of pH7.0 was defined as 1U.
Xylanase activity is defined as: the amount of enzyme required to hydrolyze 0.01moL xylan per minute to produce 1. Mu. MoL reducing sugar (xylose) at 25℃and pH7.0 is one enzyme activity unit (U).
Example 1: isolation and identification of salt-tolerant bacillus B757
Sample collection: and selecting the optimal sampling time for microbial separation, and collecting samples in a region polluted by radiation in 4 months in spring by combining with the local actual climate condition. After removing 5cm of surface soil at each sampling point, taking 5-30cm of soil (uniformly mixing) and placing into a sterile sampling tube, and placing into a lead plate box for preservation in a refrigerator at-4 ℃ for later use.
Isolation of strains: taking 1g of the soil sample, adding the soil sample into 99ml of sterile physiological saline, uniformly mixing, absorbing 1ml of liquid, adding the mixture into a liquid culture medium taking CMC (sodium carboxymethylcellulose) as a sole carbon source, culturing for 24 hours at 30 ℃ and 200rpm, absorbing 1ml of liquid, adding the liquid culture medium taking CMC as the sole carbon source, continuously carrying out shaking culture for 24 hours at 30 ℃ and 200rpm, and repeating the steps for enrichment culture three times. Finally, the culture solution is taken, diluted in a gradient way, coated on a flat-plate culture medium with CMC as the only carbon source, and cultured for 48 hours at 30 ℃. According to the size of transparent circles generated by bacterial colonies on a plate culture medium, 50 strains which can better utilize CMC are screened out, and the larger the transparent circles are, the stronger the cellulase production capability of the strains is.
Next, the isolated single colonies were inoculated on LB medium supplemented with 2% AZCL-wheat arabinoxylan for selection, and cultured at 30℃for 48 hours to test whether the colonies had xylanase activity. And numbering the strains with large hydrolysis circles according to the blue hydrolysis circles generated on the LB plate, and transferring the strains to an inclined plane to serve as target bacteria for standby.
The cellulase activity and xylanase activity of the strain of Bacillus obtained by the 6-strain screening were measured. Single bacterial colony of the strain is selected, inoculated into LB culture medium, cultured for 24 hours at 30 ℃, and enzyme activity in fermentation broth is detected. The results are shown in Table 1. Strain 1, which has the highest cellulase and xylanase production capacity, was obtained and glycerol deposited under accession number B757 and identified by 16srDNA, resulting in Bacillus halodurans (Bacillus halotolerans). Therefore, the salt-tolerant bacillus B757 screened by the invention has stronger capability of producing cellulase and xylanase, and can be prepared into bacterial powder for research and development of various feed products.
TABLE 1 measurement results of enzyme Activity of target strains
Strain numbering Cellulase (U/ml) Xylanase (U/ml)
B757 159.4 106.4
B895 130.7 107.7
B914 140.1 65.7
B357 104.7 59.7
B154 98.4 98.4
B322 116.7 110.7
Example 2: tolerance performance verification of salt-tolerant bacillus B757
Seed medium (g/L): glucose 10, K 2 HPO 4 4.8，KH 2 PO 4 3.5，(NH 4 ) 2 SO 4 2，MgCl 2 0.16，CaCl 2 0.02，Na 2 MoO 4 ·2H 2 O 0.0024，FeCl 3 0.0018，MnCl 2 ·2H 2 O0.0015, naCl 10, initial pH7.0.
(1) Growth performance under 12% NaCl
Inoculating Bacillus halodurans B757 seed solution into seed culture medium containing 12% NaCl at an inoculum size of 2%, sampling at intervals, and measuring OD 600 The absorbance at the position is respectively plotted by taking the sampling time as the abscissa and the absorbance value as the ordinate, and as shown in FIG. 2, the bacterial concentration OD of the strain under the condition of 12% NaCl 600 Can reach 2.2.
(2) Growth performance under 1.0% bile salt condition
Inoculating Bacillus halodurans B757 seed solution into seed culture medium containing 1.0% bile salt at an inoculum size of 2%, sampling at intervals, and measuring OD 600 And (3) drawing a cell growth curve by taking the sampling time as an abscissa and the absorbance value as an ordinate. As shown in FIG. 2, the strain has a maximum concentration OD under 1.0% bile salt conditions 600 Reaching 1.52.
(3) Tolerance at pH 2.0
Inoculating salt-tolerant bacillus B757 to a seed culture medium, culturing to a mid-log phase, taking 1mL of fermentation liquor, centrifuging, pouring out supernatant, reserving thalli, re-suspending the thalli by using a culture medium with pH of 2.0 (HCl-conditioned), shaking uniformly, treating for 3 hours, diluting and coating on a flat plate, wherein each sample is 3 times in parallel, culturing at 30 ℃ for 24 hours, calculating the survival rate under the treatment condition (the survival rate=the number of viable bacteria after incubation/the number of viable bacteria before incubation), and incubating for 3 hours under the condition of pH of 2.0, wherein the survival rate still reaches 97.38 percent and has almost no influence.
Example 3: preparation of microbial agent containing salt-tolerant bacillus B757
(1) Preparation of clostridium tyrobutyrate microbial inoculum
The seed medium was a commercially available clostridium enrichment medium (Reinforced Clostridium Medium, RCM). (Qingdao sea Bo biotechnology Co., ltd., product number: HB 0316)
The isolated single colonies were inoculated into RCM medium and cultured at 30℃for 12 hours to obtain seed night.
The fermentation medium is a modified version of fermentation medium (Clostridium Growth Medium, CGM) (see Jiang Ling. Study of clostridium tyrobutyrate immobilized on fiber bed for fermentation of inexpensive biomass to produce butyric acid [ D)]University of south China, 2010.) is specifically as follows: collecting kitchen waste shrimp and crab shells, cleaning, drying, grinding into powder, and sieving with 100 mesh sieve. The obtained shrimp and crab shell powder is directly used as nitrogen source, and added into liquid culture medium according to 25g/L, wherein 5g NaCl and 1.5g K are added into the culture medium (1L) 2 HPO 4 ，0.6g MgSO 4 ·7H 2 O，0.03g FeSO 4 ·7H 2 O,20g glucose.
The batch fermentation of clostridium tyrobutyrate L319 was performed in a 50L full-automatic mechanical stirring fermenter, and the addition amount of the fermentation medium was 25L. Before fermentation, the fermenter was sterilized at 121℃for 20min, then the fermentation medium was added and sterilized at 115℃for 20min. After cooling, air in the tank was replaced with sterile nitrogen to achieve an anaerobic environment, then seed liquid was introduced into the fermenter according to an inoculum size of 5%, stirring speed and temperature were set to 100rpm and 30 ℃, respectively, and pH of the fermentation broth was controlled to 6.0 by adding 8M NaOH.
When the bacterial cells are cultured to the middle and late stage of logarithmic growth (about 12 h), the fermentation broth is split into centrifugal cups, sealed, centrifuged at 5000rpm and 4 ℃ for 15min, and the supernatant is discarded. Collecting the centrifuged sample, and spray drying to obtain bacterial powder. The spray drying operating conditions were: the air inlet temperature is 200 ℃, and the air outlet temperature is 80 ℃. Dry viable bacteria unit 4×10 9 CFU/g or more.
(2) Preparation of salt-tolerant bacillus B757 microbial inoculum
Fermentation medium composition (liquid, g/L): glucose 30, peptone 20, yeast powder 15, potassium dihydrogen phosphate 1.5, magnesium sulfate 1.0 and initial pH7.0.
Inoculating salt-tolerant bacillus B757 into a seed culture medium, and culturing for 18h at 30 ℃ by introducing air to obtain liquid seeds; then to the packaging with high temperature sterilizationInoculating liquid seeds according to an inoculum size of 10% of the volume fraction in a 50L fermentation tank in a fermentation medium, and culturing at 30 ℃ under the conditions of 200r/min and ventilation of 0.8vvm for 40 hours (logarithmic growth phase) to obtain a thallus culture; the cell culture was centrifuged at 5000r/min for 15min at 4℃and the supernatant was discarded. Collecting the centrifuged sample, and spray drying to obtain bacterial powder. The spray drying operating conditions were: the air inlet temperature is 200 ℃, and the air outlet temperature is 80 ℃. Dry viable bacteria unit 8×10 10 CFU/g or more.
(3) Preparation of composite microbial agent
And (3) compounding the clostridium tyrobutyrate bacterial agent prepared in the step (1) and the salt-tolerant bacillus B757 according to the viable cell count ratio of the salt-tolerant bacillus B757 to clostridium tyrobutyrate L319=10:1, and mixing the compounded microorganism with auxiliary materials (trehalose or chitosan oligosaccharide) to obtain the microorganism bacterial agent, wherein the volume ratio of the thallus to the auxiliary materials is 1:2. The microbial concentration of the microbial agent is xx CFU/g.
Example 4: influence of microbial agent containing salt-tolerant bacillus B757 on utilization rate of roughage
The microbial agent obtained in the example 3 is respectively and evenly mixed with wheat, rice and corn straws which are crushed and sieved by a 200-mesh sieve (namely, the added amount of the straws is 9g/100 g), and the water is supplemented, so that the microbial agent fully absorbs the water. The blank was subjected to storage fermentation at room temperature (25 ℃) for 60 days without adding a microbial agent (replaced with an auxiliary material in the microbial agent prepared in example 3), and then the residue was transferred to a constant weight 200 mesh nylon bag in total, and the content of the crude fibers was measured by the nylon bag crude fiber measurement method with reference to Hu Yan (Wu Qiu et al, test study for measuring crude fibers in feeds with nylon bags [ J. Feed industry, 2005 (01): 55-56.) and the crude fiber degradation rate was calculated.
As shown in Table 2, the microbial inoculum of example 3 has a crude fiber hydrolysis rate of 25.4% for rice straw and 20.3% for wheat straw.
TABLE 2 influence of microbial inoculants on feed crude fiber degradation rate
Project Wheat straw Rice straw Corn stalk
Crude fiber before treatment (%) 38.5％ 32.5％ 28.7％
Post-fermentation crude fiber (%) 30.7％ 24.2％ 23.4％
Crude fiber degradation rate (%) 20.3％ 25.4％ 18.4％
Comparative example 1:
the specific embodiment is the same as example 4, except that the microbial inoculum is replaced by the microbial inoculum prepared by the method of example 3 and only containing salt-tolerant bacillus B757, the adding amount of the microbial inoculum is 9g/100g of rice straw, and the result shows that the hydrolysis rate of crude fiber is only 21.4%.
Comparative example 2:
the specific embodiment is the same as example 4, except that the microbial inoculum is replaced by the microbial inoculum prepared by the method of example 3 and only containing clostridium tyrobutyrate L39, the adding amount of the microbial inoculum is 9g/100g of rice straw, and the result shows that the hydrolysis rate of crude fiber is only 19.8%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (10)

1. Salt tolerant bacillus (Bacillus halotolerans) B757, which produces cellulase and xylanase, was deposited at the cantonese province microorganism strain collection at 10 and 14 days 2022 under the accession number GDMCC No:62887.
2. a microbial agent comprising the salt-tolerant bacillus B757 of claim 1.
3. The microbial agent according to claim 2, characterized in that it contains the salt-tolerant bacillus B757 and clostridium tyrobutyricum according to claim 1.
4. A microbial agent according to claim 3, wherein the clostridium tyrobutyrate is clostridium tyrobutyrate (Clostridium tyrobutyricum) L319, accession No. GDMCC No:62289.
5. the microbial agent according to claim 3 or 4, wherein the ratio of the concentration of the salt-tolerant bacillus B757 to the concentration of the clostridium tyrobutyricum is (8-10): 1.
6. The microbial agent according to any one of claims 2 to 5, wherein the microbial agent is compounded by microbial powder and auxiliary materials.
7. The microbial agent according to any one of claims 2 to 5, wherein the salt-tolerant bacillus B757 has a bacterial concentration of 8×10 10 ～8×10 12 CFU/g, clostridium casei concentration of 8X10 9 ～8×10 11 CFU/g。
8. Use of the salt tolerant bacillus B757 of claim 1 for the production of cellulases and xylanases.
9. Use of a salt tolerant bacillus B757 of claim 1 or a microbial preparation of any of claims 2 to 7 in the preparation of ruminant feed.
10. The use according to claim 9, characterized in that the use includes, but is not limited to, degrading the crude fiber content of wheat straw, rice straw or corn straw.
CN202310486556.5A 2023-04-28 2023-04-28 Salt-tolerant bacillus for producing complex enzyme and application of salt-tolerant bacillus in feed preparation Pending CN116376779A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN116948915A (en) * 2023-08-07 2023-10-27 天典(广东)生物科技有限公司 Bacillus sojae and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116948915A (en) * 2023-08-07 2023-10-27 天典(广东)生物科技有限公司 Bacillus sojae and application thereof
CN116948915B (en) * 2023-08-07 2023-12-19 天典(广东)生物科技有限公司 Bacillus sojae and application thereof

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