CN114350553A - Bacillus amyloliquefaciens capable of producing protease in high yield and application thereof - Google Patents
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Abstract
The invention discloses a bacillus amyloliquefaciens capable of producing protease with high yield and application thereof. The strain LX-6 is classified and named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LX-6, depository: china center for type culture Collection, preservation date: 22 months 11 in 2021, with the preservation registration number of CCTCC No. M20211457. The bacillus amyloliquefaciens produces the high-efficiency protease, the protease activity is 1390.6 +/-12.5U/mL according to the determination of a SB/T10317-The soybean antigenic protein is not seen after 48 hours, and the soybean protein is a leading level in the field of solid state fermentation of soybean meal by a microbiological method. By usingB.amyloliquefaciens The LX-6 has high protease activity, has obvious degradation capability on macromolecular proteins in feeds such as soybean meal and the like, reduces anti-nutritional factors in the feeds, improves the digestibility and the use benefit of the feeds, reduces the breeding cost, and has great application potential.
Description
Technical Field
The invention relates to the technical field of microorganisms, and relates to a bacillus amyloliquefaciens LX-6 capable of producing protease and application thereof in reducing crude protein in feeds such as soybean meal and the like and reducing anti-nutritional factors and the like.
Background
The soybean meal is the most widely used feed industrial raw material at present, has the characteristics of sufficient source of goods, low probability of containing toxic and harmful substances, high safety coefficient and the like compared with animal-derived protein feed, and is an ideal material for feed. In recent years, the aquaculture industry is developed rapidly, the shortage of aquatic feed protein sources is severe day by day, and aquatic animals are different from monogastric animals such as livestock and poultry and ruminants and have higher requirements on the aspects of nutrient substances, such as intake, digestion and absorption. At home and abroad, various scholars find that the soybean has good nutritional quality, and soybean products such as soybean meal and the like can be used as fish meal substitutes for aquatic feeds. The soybean meal contains a large amount of protein, but the protein has a complex molecular structure and a large relative molecular weight, and has the problems of anti-nutritional factors and the like. Particularly, the harmful influence of soybean antigenic proteins (glycinin and beta-conglycinin) on the health of aquatic animals is more obvious, and the method mainly comprises the steps of delaying the growth speed, reducing the digestion of nutrient substances, greatly reducing the absorption and utilization of the nutrition of bean pulp by the animals, and influencing the use value and the application range of the bean pulp.
In order to realize the efficient utilization of the soybean meal, part of sensitization factors and anti-nutritional factors in the soybean meal are usually reduced by some methods in the using process, for example, the thermosensitive factors can be inactivated by puffing and cooking, the soybean protein is partially decomposed, the activity of trypsin inhibitor is reduced, and the like. Related researches show that the nutritional value of the soybean meal can be improved by utilizing microbial fermentation. On one hand, the microorganism can effectively degrade anti-nutritional factors; on the other hand, small peptides produced by degrading macromolecular proteins are easily combined with mineral elements into soluble chelate compounds in the intestinal tract to promote the absorption of inorganic salt ions. Since the bacillus has the advantages of short growth period, low fermentation cost, high content of crude protein in fermented soybean meal and the like, the production of fermented soybean meal by using the bacillus is a research trend. Liuxing and the like are fermented by utilizing bacillus subtilis, and the total protein of the soybean meal is increased from 45.28 percent to 48.37 percent. The high protease activity is the key of the bacillus for producing the fermented soybean meal. Zheng Li et al screened a protease producing bacillus, the protease producing level was maintained at 500-700U/mL, and some crude protein could not be degraded after 60h fermentation of soybean meal.
Liuxing, Zhao Rong, Li Bo, etc. several different strains have different indexes for fermentation of bean pulp, and animal nutrition 2020,1:20-23.
Zheng Li, Li Da, niu hong, etc. the influence of screening and fermentation of protease producing bacillus on soybean meal, 2019,40(20): 185-191.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, the first purpose of the invention is to provide a strain of bacillus amyloliquefaciens with stronger protease activity by screening (Bacillus amyloliquefaciens）LX-6。
A second object of the invention is to utilizeB. amyloliquefaciens LX-6 has high protease activity, and bean pulp is subjected to solid-state fermentation to degrade macromolecular protein therein, reduce anti-nutritional factors and improve the use value of the bean pulp.
The purpose of the invention is realized by the following technical means:
the invention firstly takes the soil sample collected in the scenic spot of Qiandao lake in Chunan county, Hangzhou, Zhejiang as a research object, and the soil sample is separated, fermented and cultured, and the protease activity is measuredDetermining, identifying strains, performing soybean meal fermentation, performing SDS-PAGE analysis, and analyzing total protein concentration and water-soluble protein to obtain a bacillus with potential advantages for soybean meal fermentation; taxonomic identification and designation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LX-6, which has been deposited at the China center for type culture Collection at 22.11 months 2021, the address of the deposition unit: the preservation numbers of the Wuhan university in Wuhan, China are respectively as follows: CCTCC No. M20211457.
The processes of screening the bacillus, detecting the protease activity and fermenting the soybean meal are as follows:
(1) collecting soil samples from the field (Chunan county Qiandao lake, Hangzhou, Zhejiang) and airing;
(2) separating from the soil sample, and using a hydrolysis ring of a skimmed milk powder flat plate as an indication to screen a strain producing protease;
(3) primarily detecting the protease activity of the grown strains by using an Oxford cup method, respectively, and selecting the strains with obvious hydrolysis loops by observing the sizes of the hydrolysis loops and then re-screening;
(4) performing fermentation culture on the selected strain, centrifuging and collecting fermentation liquor after the fermentation is finished, and determining the protease activity according to an SB/T10317-Bacillus amyloliquefaciens）LX-6，B. amyloliquefaciensThe protease activity of LX-6 reaches 1390.6 +/-12.5U/mL.
(5) B. amyloliquefaciensLX-6 single colony is selected to be in LB liquid culture medium, and cultured for 24h under the condition of 37 +/-l ℃ and the rotating speed of a shaking table of 180 r/min, and the viable count is 108 CFU/mL；
(6) The proportion of the soybean meal solid fermentation medium is 30g of soybean meal and 30 ml of water; will be culturedBacillus amyloliquefaciens LX-6 is inoculated into a fermentation medium according to 10 percent (v/v) and is fermented and cultured under the condition of 37 +/-l ℃;
(7) after fermentation, drying at 75 ℃, crushing, extracting protein by ultrasonic, detecting by SDS-PAGE, and after 24h fermentation, basically degrading insoluble macromolecular protein and increasing soluble micromolecular protein. After fermentation for 48 hours, the total protein concentration of the fermented soybean meal reaches 64.58%, the water-soluble protein reaches 44.45%, and the nitrogen solubility index reaches 68.8%.
The invention has the beneficial effects that:
the invention successfully screens a high-yield protease strain which is identified as bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) LX-6, the protease activity of which reaches 1390.6 +/-12.5U/mL, after the soybean meal is fermented for 24 hours, the crude protein is basically degraded and fermented for 48 hours, the total protein concentration reaches 64.58 percent, and the water-soluble protein content reaches 44.45 percent. The invention usesB. amyloliquefaciens LX-6 has the advantages of short fermentation period, low cost and the like, and has potential advantages of industrial development and application. The strain can be applied to solving the problems of large protein molecules, poor solubility and existence of anti-nutritional factors in the soybean meal, and has great application potential for improving the utilization value of feed and reducing the breeding cost.
Drawings
FIG. 1 is a schematic representation of the protease activity of the strains on skim milk powder plates.
FIG. 2 shows LX-6 colony morphology (A) and bacterial body electron micrograph (B8.0K).
FIG. 3 is a phylogenetic tree constructed from the 16S rDNA sequence of strain LX-6.
FIG. 4 is an SDS-PAGE gel of LX-6 solid-state fermented soybean meal;
wherein, M: a protein Marker; lanes 1-6 are samples of unfermented soybean meal, LX-6 solid-state fermented soybean meal for 12h, 24h, 48h, and 60h, respectively.
Detailed Description
The invention is further illustrated below with reference to the figures and examples.
Example 1: (Bacillus amyloliquefaciens Separation, screening and identification of LX-6
The method comprises the following steps:
(1) materials and culture media: the material for separating and dissociating the bacillus amyloliquefaciens LX-6 is a soil sample collected from the lake of Qiandao county, Chunan city, Hangzhou, Zhejiang province, and a culture medium used for separating the protease-producing strain is a skimmed milk powder plate (the formula is that the skimmed milk powder is 50 g/L, 20 g/L and 121 ℃, and the high-pressure sterilization is carried out for 15 min).
(2) Separation and purification: filling the freshly collected soil sample into a sampling bag, taking the sampling bag back to a laboratory, subpackaging and airing for about one week; weighing 5 g of the dried soil sample per part, respectively adding the weighed soil sample into a conical flask containing 45 ml of sterile water, adding 3-5 aseptic glass beads into the conical flask, and performing shake cultivation at 37 ℃ for 2 hours; the cultured samples were aspirated and diluted in 10-fold gradient, 10 samples were taken separately-2、10-3、10-4Coating 200 μ L of the diluted solution on a solid plate of skimmed milk powder, and culturing at 37 deg.C for 72 hr; and (3) respectively picking single colonies when colonies with obvious hydrolysis loops grow on the plate (as shown in figure 1), inoculating the single colonies on a fresh plate, and performing purification culture at 37 +/-l ℃ for 72 hours to obtain purified strains.
(3) Primary screening and secondary screening: respectively inoculating the separated strains into LB liquid culture medium, culturing at 37 deg.C under 180 r/min for 24 hr to obtain viable count of about 108 CFU/mL, adopting an Oxford cup method to preliminarily detect the protease activity of the strain, injecting the bacterial liquid into an Oxford cup on a skimmed milk powder flat plate, culturing for 24 hours at 37 +/-l ℃, and re-screening when the size of a hydrolysis ring is observed and the hydrolysis ring of LX-6 on the skimmed milk powder flat plate is 2.50 +/-0.3 cm.
The re-screening adopts a protease activity determination method, which comprises the following specific steps: preparing a liquid fermentation culture medium: 40 g/L of soybean meal and CaCl2 1.5 g/L, glucose 4.1 g/L, yeast extract powder 9.4 g/L, NaCl 3g/L, 121 ℃, and autoclaving for 15 min. Inoculating the strains with large hydrolysis loop obtained by primary screening into LB liquid culture medium, culturing at 37 deg.C under 180 r/min for 24 hr to obtain viable count of about 108 CFU/mL, 4% inoculum size was transferred to 50 mL fermentation medium, 37 ℃, 180 r/min for 48 h. After the culture is finished, centrifuging at 7800 r/min for 10min, and taking the supernatant to determine the enzyme activity. The assay was performed according to the SB/T10317-1999 protease activity assay. Hydrolysis of casein at 40 ℃ per minute produced l μ g tyrosine, defined as 1 protease activity unit, and the protease activity was calculated by repeating the test 3 times.
Drawing a standard curve: 1mL of tyrosine with different concentrations is sucked from 6 test tubes, 5 mL of 0.4 mo1 sodium carbonate is added into each test tube, and 1mL of diluted forlin reagent is added into each test tube. Shaking and placing in a water bath kettle. The color development is carried out at 40 ℃ for 20 min by a spectrophotometer (wavelength 660 nm). Typically three times, and the average value is taken. The Optical Density (OD) measured for tubes 1-6 minus the optical density measured for tube 1 (distilled water blank) is the net OD number. The net OD value is used as an abscissa and the concentration of tyrosine is used as an ordinate, and a standard curve (or the amount of tyrosine K corresponding to each degree of OD can be calculated) is drawn.
And (3) sample determination: taking 3 test tubes with the diameter of 15 multiplied by 100 mm, the number of the test tubes is 1, 2 and 3 (2 test tubes can be made), adding 1mL of supernatant of the fermentation culture sample into each test tube, placing the test tubes in a water bath with the temperature of 40 ℃ for preheating for 2 min, adding 1mL of casein preheated by the same, accurately preserving the heat for 10min, immediately adding 2 mL of trichloroacetic acid with the volume of 0.4 mol into each test tube to stop the reaction, continuing placing the test tubes in the water bath for preserving the heat for 20 min to precipitate residual protein, centrifuging or filtering the residual protein, then taking 3 test tubes with the diameter of 15 multiplied by 150 mm, the number of the test tubes is l, 2 and 3, adding 1mL of filtrate into each test tube, adding 5 mL of sodium carbonate with the volume of 0.4 mol into each test tube, shaking 1mL of diluted welrin reagent uniformly, preserving the heat at the temperature of 40 ℃ for 20 min, and then carrying out Optical Density (OD) determination.
The blank test was conducted in the same manner as above except that 2 mL of 0.4 mol of trichloroacetic acid was added to inactivate the enzyme before adding casein, and then casein was added thereto, using 3 test tubes (Nos. (1), (2) and (3)).
Hydrolysis of casein at 40 ℃ per minute produced l μ g tyrosine, defined as 1 protease activity unit.
Sample protease activity unit = a/10 × 4 × N × 1/(1-W)
In the formula: A-OD value measured from the sample, and microgram number of tyrosine equivalent (or OD value. multidot.K) found by examining the standard curve;
4-4 mL of the reaction solution was taken out for 1mL measurement (i.e., 4 times);
n is the dilution multiple of enzyme solution;
10-reaction for 10 min;
w is the percentage of sample moisture.
The measurement result shows that:Bacillus amyloliquefaciens the activity of the LX-6 protease reaches 1390.6 +/-12.5U/mL.
(4) And (3) identification:
when the LX-6 is cultured on an LB solid culture medium, the bacterial colony is milky white, the edge is neat, the surface is smooth, the center is convex, and the bacterial colony is sticky (part A in figure 2). Microscopic observation of a scanning electron microscope shows that LX-6 thalli spores are long and cylindrical and have smooth surfaces (part B of figure 2).
Combining the morphological characteristics, physiological and biochemical characteristics (Table 1) and the alignment result of the 16S rRNA sequence (FIG. 3) of LX-6, the strain is identified as Bacillus amyloliquefaciens (named Bacillus amyloliquefaciens) (III)Bacillus amyloliquefaciens LX-6), and storing.
Physiological and biochemical identification of strains of Table 1 LX-6
Example 2: (Bacillus amyloliquefaciens LX-6 soybean meal fermentation method)
The method comprises the following steps:
(1) activating and culturing strains: to be preservedBacillus amyloliquefaciens LX-6 bacterial suspension (1X 10)8 cfu·mL-1) Inoculating LB liquid culture medium, and culturing at 37 + -l deg.C for 24h for fermentation.
(2) Fermentation culture: the solid fermentation culture medium is 100 g of soybean meal and 100 mL of water, and 30g of the solid fermentation culture medium is bottled in each 300 mL triangle bottle; cultured by using LB liquid culture mediumBacillus amyloliquefaciens LX-6 is inoculated into a fermentation medium according to 10% (v/v) and is fermented and cultured for 12h, 24h, 36h, 48h and 60h respectively.
(3) And (3) fermented soybean meal protein determination: after fermentation, the mixture was dried, pulverized, and 2.5 g each was dissolved in 50 mL of 0.03M Tris-HCl (pH 8.0) and subjected to ultrasonic extraction for 30 min. After extraction, SDS-PAGE detects crude protein digestion, the sample is kept stand for 5 min, 40 mul of supernatant is taken and 10 mul of 5 Xprotein loading buffer solution are mixed evenly, boiled water bath is carried out for 5-8 min, centrifugation is carried out for 3 min at 11000 rpm, 30 mul of supernatant is taken and SDS-PAGE is carried out, the voltage is 100V, and 1 h. Dyeing with Coomassie brilliant blue for 30 min, decolorizing for 30 min, and decolorizing overnight with decolorizing solution.
The results are shown in FIG. 4, the crude protein is basically degraded and the small molecular protein is increased at 24h of the fermented soybean meal.
Example 3: (Bacillus amyloliquefaciens LX-6 fermented soybean meal protein determination)
(1) Sample treatment: precisely weighing 0.2 g of fermented soybean meal sample, transferring into a dry 100 mL or 500 mL nitrogen determination bottle, adding 0.2 g of copper sulfate, 6 g of potassium sulfate and 20 mL of sulfuric acid, slightly shaking uniformly, placing a small funnel at the bottle opening, obliquely supporting the bottle on an asbestos net with a small hole at an angle of 45 degrees, heating with small fire, after the content is completely carbonized and the foam is completely stopped, enhancing the firepower, keeping the liquid in the bottle slightly boiling, and continuing to heat for 0.5 hour after the liquid is clear and transparent in a blue-green color. Taking down and cooling, carefully adding 20 mL of water, cooling, transferring into a 100 mL volumetric flask, washing the volumetric flask with a small amount of water, merging the washing liquor into the volumetric flask, adding water to the scale, and uniformly mixing for later use. And taking copper sulfate, potassium sulfate and concentrated sulfuric acid which are the same in amount as the treated sample to perform a reagent blank test by the same method.
(2) A nitrogen determination device is arranged, a plurality of drops of methyl red indicator and a plurality of milliliters of sulfuric acid are added at about 2/3 of water filled in a water vapor generator to keep the water acidic, a plurality of glass beads are added to prevent bumping, and the water in a water vapor generation bottle is heated and boiled by a pressure regulator.
(3) 10 mL of 2% boric acid solution and 1 drop of the mixing indicator were added to the receiving flask, the lower end of the condenser tube was inserted below the liquid level, 10.0 mL of the sample digestion solution was aspirated and flowed into the reaction chamber from the cuvette, and the cuvette was washed with 10 mL of water and flowed into the reaction chamber, and the rod-shaped glass stopper of the cuvette was plugged. 10 mL of 40% sodium hydroxide solution was poured into a small glass and the glass plug was lifted to allow slow flow into the reaction chamber. The screw clamp is clamped, distillation is started, steam is introduced into the reaction chamber to ensure that ammonia enters the receiving bottle through the condensing tube, and distillation is carried out for 5 min. Moving the receiving bottle to make the lower end of the condensing tube leave the liquid container, distilling for 1 min, and washing the outer part of the lower end of the condensing tube with a small amount of water. The receiving flask was removed and the end point was set to grey or blue-violet with 0.05N sulfuric acid or 0.05N hydrochloric acid standard solution.
Simultaneously, 10.0 ml of reagent blank digestive juice is sucked up according to the operation (3).
(4) And calculating
X-percent protein in sample, g;
v1-volume of sample consumed sulfuric acid or hydrochloric acid standard solution, mL;
v2-volume of reagent blank spent sulfuric acid or hydrochloric acid standard solution, mL;
n-equivalent concentration of sulfuric acid or hydrochloric acid standard solution;
0.014-1N sulfuric acid or hydrochloric acid standard solution 1mL is equivalent to nitrogen gram;
m-mass (volume) of sample, g (mL);
f-nitrogen is converted to a coefficient of protein.
The results show that the total protein content of the LX-6 fermented soybean meal in the fermentation samples at different times is shown in Table 2.
(5) And water-soluble protein extraction assay
Refer to the method for determining water-soluble protein in appendix A of GB/T31785-2015 "Soy storage quality determination rules".
Bacillus amyloliquefaciens The test results of the water-soluble protein of the LX-6 solid-state fermented soybean meal samples at different times are shown in Table 2. As seen from Table 2, the total protein content of the soybean meal was high and gradually increased as the fermentation proceeded,Bacillus amyloliquefaciens after the soybean meal is fermented for 48 hours by the LX-6, the total protein and the water-soluble protein reach the highest values. The total protein content reached a maximum of 64.58% at 48h and decreased slightly after 60 h. The water-soluble protein in the unfermented soybean meal only accounts for about 5.74% of the total proteinBacillus amyloliquefaciens Under the action of protease generated by LX-6, the content of water-soluble protein in the fermented soybean meal is greatly increased and reaches a maximum value of 44.45% after 48 hours of fermentation.
TABLE 2 protein content of LX-6 Strain fermented Soybean meal at different times
Claims (4)
1. A bacillus amyloliquefaciens capable of producing protease with high yield is characterized in that: the strain LX-6 is classified and named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) LX-6, depository: china center for type culture Collection, preservation date: 22 months 11 in 2021, the preservation registration number is CCTCC No. 20211457.
2. The bacillus amyloliquefaciens according to claim 1, wherein: the high-efficiency protease is produced, the protease activity reaches 1390.6 +/-12.5U/mL according to the determination of a SB/T10317-1999 protease activity assay method, the strain is used for carrying out solid-state fermentation on the soybean meal, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection shows that the macromolecular protein in the soybean meal is significantly degraded after 24 hours, and the soybean antigen protein is not found after 48 hours.
3. A method for fermenting soybean meal by using bacillus amyloliquefaciens according to claim 1, which is characterized by comprising the following steps of: the method comprises the following steps:
(1) the bacillus amyloliquefaciens LX-6 is selected to be singly fallen in an LB liquid culture medium, the shaking table rotating speed is 180 r/min under the condition of 37 +/-l ℃, the cultivation is carried out for 24 hours, the viable count is 108 CFU/mL；
(2) The proportion of the soybean meal solid fermentation medium is 30g of soybean meal, 30 ml of water and 30g of solid fermentation medium; cultured in liquid medium of beef extract peptoneBacillus amyloliquefaciens LX-6 is inoculated into a fermentation medium according to 10 percent (v/v) and is fermented and cultured under the condition of 37 +/-l ℃.
4. The method for fermenting soybean meal by using bacillus amyloliquefaciens according to claim 3, wherein the method comprises the following steps: the fermentation culture time is 24-48 hours.
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