CN114350553B - Bacillus amyloliquefaciens capable of producing protease at high yield and application thereof - Google Patents
Bacillus amyloliquefaciens capable of producing protease at high yield and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus amyloliquefaciens with high protease yield and application thereof. The strain LX-6 is classified and named as bacillus amyloliquefaciens @Bacillus amyloliquefaciens) LX-6, preservation unit: china center for type culture Collection, preservation date: 2021, 11 and 22 days, the preservation registration number is CCTCC No. M20211457. The bacillus amyloliquefaciens produces high-efficiency protease, the protease activity of the bacillus amyloliquefaciens reaches 1390.6 +/-12.5U/mL according to the protease activity determination method of SB/T10317-1999, the strain is used for solid state fermentation of soybean meal, SDS-PAGE detection shows that macromolecular protein in the soybean meal is obviously degraded after 24 hours, soybean antigen protein is not seen after 48 hours, and the leading level is reached in the field of solid state fermentation of the soybean meal by using a microbiological method. By means ofB.amyloliquefaciens The LX-6 has high protease activity, has obvious degradation capability on macromolecular proteins in feeds such as soybean meal, reduces anti-nutritional factors in the feeds, improves the digestion and absorption rate of the feeds and the use benefits, reduces the cultivation cost, and has great application potential.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus amyloliquefaciens LX-6 for producing protease and application thereof in reducing crude protein in feeds such as soybean meal and the like, reducing anti-nutritional factors and the like.
Background
Compared with animal protein feed, the soybean meal is the most widely used feed industrial raw material, has the characteristics of sufficient goods sources, low probability of toxic and harmful substances, high safety coefficient and the like, and is an ideal material for the feed. In recent years, the aquaculture industry develops rapidly, the protein source of the aquatic feed is deficient and becomes serious, and the aquatic animals are different from monogastric animals and ruminants such as livestock and poultry, and have higher requirements on the aspects of nutrient substances, ingestion, digestion, absorption and the like. A plurality of scholars at home and abroad find that soybean has good nutrition 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 has the problems of complex protein molecular structure, relatively large molecular weight, anti-nutritional factors and the like. In particular, the soybean antigen proteins (glycinin and beta-conglycinin) have more remarkable harmful effects on the health of aquatic animals, mainly reflected in the aspects of delaying the growth speed, reducing the digestion of nutrient substances, greatly reducing the absorption and utilization of the animal to the nutrition of the soybean meal, and influencing the use value and the application range of the soybean meal.
In order to realize the efficient utilization of the soybean meal, in the use process, partial sensitization factors and anti-nutritional factors in the soybean meal are often required to be reduced by some methods, such as inactivation of the thermosensitive factors by puffing and stewing, partial decomposition of soybean proteins, reduction of the activity of trypsin inhibitor and the like. Related researches show that the nutrition value of the soybean meal can be improved by utilizing microbial fermentation. On the one hand, the microorganisms can effectively degrade the anti-nutritional factors; on the other hand, small peptides produced by degradation of macromolecular proteins readily combine with mineral elements in the intestine to form soluble chelates that facilitate absorption of inorganic salt ions. As the bacillus has the advantages of short growth period, low fermentation cost, high crude protein content of the fermented soybean meal and the like, the production of the fermented soybean meal by using the bacillus has become a research trend. Liu Xing and the like are fermented by using bacillus subtilis, and the total protein of the soybean meal is improved from 45.28% to 48.37%. High protease activity is the key for producing fermented soybean meal by bacillus. Zheng Li et al screened a strain of Bacillus subtilis producing protease, the protease level was maintained at 500-700U/mL, and part of crude protein remained after fermentation of soybean meal 60h was not degraded.
Liu Xing, zhao Rong, li Bo, etc. several different strains fermented soybean meal fermentation process indexes are compared, animal nutrition 2020,1:20-23.
Zheng Li, li Da, niu Gonggong, et al screening of Bacillus protease-producing and the effect of its fermentation on soybean meal, food science 2019,40 (20): 185-191.
Disclosure of Invention
In order to overcome the defects of the prior art, the first aim of the invention is to provide a bacillus with stronger protease activity, namely bacillus amyloliquefaciensBacillus amyloliquefaciens）LX-6。
A second object of the present invention is to utilizeB. amyloliquefaciensLX-6 has high protease activity, and the soybean meal is solid-state fermented to degrade macromolecular proteins in the soybean meal, reduce anti-nutritional factors and improve the use value of the soybean meal.
The invention aims at realizing the following technical means:
firstly, taking a soil sample collected in a scenic spot of Qianlia lake in Chun-an county of Hangzhou of Zhejiang province as a research object, separating, fermenting and culturing, measuring protease activity, identifying strains, fermenting bean pulp, analyzing SDS-PAGE, and analyzing total protein concentration and water-soluble protein to obtain bacillus with potential advantages for fermenting the bean pulp; taxonomic identification and designation of Bacillus amyloliquefaciensBacillus amyloliquefaciens) LX-6 was deposited in the China center for type culture Collection, accession number: the preservation numbers of the university of Wuhan in China are respectively: cctccc No. M20211457.
The screening of bacillus, the detection of protease activity and the fermentation of soybean meal are as follows:
(1) Collecting soil samples from the wild (Qiandan lake of Chun An county in Hangzhou of Zhejiang province), and airing;
(2) Separating from soil sample, and selecting protease-producing strain using hydrolysis ring of skimmed milk powder plate as indication;
(3) The grown strains are respectively subjected to preliminary detection on protease activity of the strains by an oxford cup method, and strains with obvious hydrolysis circles are selected to enter a re-screening mode by observing the sizes of the hydrolysis circles;
(4) Fermenting and culturing the selected strain, centrifuging after fermentation, collecting fermentation liquor, and determining protease activity according to SB/T10317-1999 protease activity determination method, thereby screening out a microorganism with highest protease activity, namely Bacillus amyloliquefaciensBacillus amyloliquefaciens）LX-6，B. amyloliquefaciensThe protease activity of LX-6 reaches 1390.6 +/-12.5U/mL.
(5)B. amyloliquefaciensLX-6 single colony is selected in LB liquid culture medium, and cultured at 37+/-l deg.C and shaking table rotation speed of 180 r/min for 24h and viable count of 10 8 CFU/mL；
(6) The solid state fermentation medium proportion of the bean pulp is 30g bean pulp, 30 ml water; will be culturedBacillus amyloliquefaciensLX-6 is inoculated into a fermentation culture medium according to 10% (v/v) and is fermented and cultured at 37+/-l ℃;
(7) Drying at 75 ℃ after fermentation, crushing, ultrasonically extracting protein, detecting by SDS-PAGE, basically degrading insoluble macromolecular protein after 24h fermentation, and increasing soluble micromolecular protein. Fermenting for 48h, wherein the total protein concentration of the fermented soybean meal reaches 64.58%, the water-soluble protein of the fermented soybean meal reaches 44.45%, and the nitrogen solubility index of the fermented soybean meal reaches 68.8%.
The invention has the beneficial effects that:
the invention successfully screens a high-yield protease strain, and the high-yield protease strain is identified as bacillus amyloliquefaciensBacillus amyloliquefaciens) LX-6, the protease activity reaches 1390.6 +/-12.5U/mL, the crude protein is basically degraded after the soybean meal is fermented for 24 hours, the total protein concentration reaches 64.58 percent after the fermentation for 48 hours,the water-soluble protein content reaches 44.45%. The invention usesB. amyloliquefaciensLX-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 solve the problems of larger protein molecules, poor solubility and anti-nutritional factors in soybean meal, and has great application potential in improving the utilization value of feed and reducing the cultivation cost.
Drawings
FIG. 1 is a schematic of protease activity of strains on skim milk powder plates.
FIG. 2 shows LX-6 colony morphology (A) and cell electron micrographs (B8.0K).
FIG. 3 is a phylogenetic tree constructed from the 16S rDNA sequence of strain LX-6.
FIG. 4 is a SDS-PAGE gel of LX-6 solid state fermented soybean meal;
wherein M: protein markers; lanes 1-6 are samples of unfermented soybean meal, LX-6 solid state fermented soybean meal 12h, 24h, 48h, 60h, respectively.
Detailed Description
The invention is further illustrated in the following figures and examples.
Example 1: (Bacillus amyloliquefaciensSeparation, screening and identification of LX-6
The method comprises the following steps:
(1) Materials and culture medium: the bacillus amyloliquefaciens LX-6 is prepared from soil samples of Qiandan lake of Chun An county, hangzhou, zhejiang province, and the culture medium used for separating protease-producing strains is a skim milk powder plate (formula: skim milk powder 50 g/L,20 g/L,121 ℃ C.) and is subjected to high-pressure sterilization for 15 min.
(2) And (3) separating and purifying: the freshly collected soil samples are filled in sampling bags, taken back to a laboratory, split-packed and dried for about one week; weighing 5 parts of dried soil sample g, respectively adding into conical flasks containing 45 ml sterile water, adding 3-5 sterile glass beads into the conical flasks, and shake culturing at 37deg.C for 2 h; absorbing the cultured sample, diluting with 10 times gradient, and respectively taking 10 times of the sample -2 、10 -3 、10 -4 200 mu L of diluted solution is coated on the surface of the substrateCulturing 72 h on solid milk powder plates at 37 ℃; and (3) respectively picking single colonies to be detected from colonies with obvious hydrolysis circles (shown in figure 1) growing on the flat plate, inoculating the single colonies on a fresh flat plate, and purifying and culturing the single colonies at 37+/-l ℃ for 72 h to obtain the purified strain.
(3) Primary screening and secondary screening: inoculating the separated strains into LB liquid medium, culturing at 37deg.C and 180 r/min for 24h, and culturing with viable count of about 10 8 CFU/mL, the protease activity of the strain is initially detected by adopting an oxford cup method, bacterial liquid is injected into an oxford cup on a skim milk powder plate, the culture is carried out at 37 ℃ plus or minus l ℃ for 24h, and the re-screening is carried out by observing the size of a hydrolysis ring, namely, the hydrolysis ring of LX-6 on the skim milk powder plate is 2.50 plus or minus 0.3 cm.
The re-screening adopts a protease activity assay method, and the specific method comprises the following steps: preparing a liquid fermentation medium: bean pulp 40 g/L, caCl 2 1.5 g/L, glucose 4.1 g/L, yeast extract 9.4 g/L, naCl 3g/L,121 ℃, and autoclaving for 15 min. Inoculating strain with large hydrolysis circle obtained by primary screening into LB liquid culture medium, culturing at 37deg.C and 180 r/min for 24h to obtain viable count of about 10 8 CFU/mL was transferred to 50 mL fermentation medium at 4% inoculum size, and cultured at 37℃at 180 r/min for 48 h. After the completion of the culture, the mixture was centrifuged at 7800/r/min for 10min, and the supernatant was collected and assayed for enzyme activity. The assay was performed according to the SB/T10317-1999 protease activity assay. The casein was hydrolyzed at 40℃per minute to yield l. Mu.g of tyrosine, defined as 1 protease activity unit, and the protease activity was calculated by repeating 3 times of the test.
Drawing a standard curve: 6 test tubes were taken to draw tyrosine 1mL of different concentrations, 0.4 mo1 sodium carbonate 5 mL was added to each tube, and diluted furin reagent 1mL was added to each tube. Shaking up and placing in a water bath kettle. The color development was performed at 40℃for 20 minutes with a spectrophotometer (wavelength 660, nm). Typically three measurements are taken and averaged. The optical density measured for tubes 1-6 (OD) minus the optical density measured for tube 1 (distilled water blank) was the net OD number. The net OD value is plotted on the abscissa and the tyrosine concentration is plotted on the ordinate, and a standard curve is plotted (or the amount K of tyrosine corresponding to each degree OD can be obtained).
Sample measurement: taking 3 test tubes of 15×100 mm (2 can be used) and 1,2,3 of the fermentation culture sample supernatant 1mL is added into each tube, placing the test tubes in a water bath at 40 ℃ for preheating for 2 min, then adding 1mL of casein which is preheated in the same way, precisely preserving heat for 10min, immediately adding 2 mL of trichloroacetic acid 0.4 mol respectively after the time is up, stopping the reaction, continuously placing the test tubes in the water bath for 20 min, centrifuging or filtering after precipitating residual protein, then taking 3 test tubes of 15×150 mm, 1mL of filtrate is added into each tube, adding 5 mL of sodium carbonate 0.4 mol, shaking the diluted furin reagent 1mL uniformly, preserving heat for 20 min at 40 ℃ and then measuring Optical Density (OD).
The blank test also takes 3 test tubes, with numbers (1), (2) and (3), and the measurement method is the same as that above, except that 0.4 mol of trichloroacetic acid 2 mL is added before casein is added, so that enzyme is deactivated, and then casein is added.
Casein was hydrolyzed at 40℃per minute to yield l. Mu.g of tyrosine, defined as 1 protease activity unit.
Sample protease activity unit = a/10 x 4 x n x 1/(1-W)
Wherein: a—measuring an OD value from a sample, looking up a standard curve to obtain equivalent micrograms of tyrosine (or OD value x K);
4-4 mL of reaction solution 1mL of the reaction solution was taken out for measurement (i.e., 4 times);
n-fold of enzyme dilution;
10-reacting for 10min;
w-percent moisture of sample.
The measurement result shows that:Bacillus amyloliquefaciensthe activity of LX-6 protease reaches 1390.6 +/-12.5U/mL.
(4) And (3) identification:
the LX-6 of the present invention has milky white colony, regular edge, smooth surface, raised center and thick colony when cultured on LB solid medium (part A of FIG. 2). Scanning electron microscopy revealed that LX-6 bacterial spores were oblong to cylindrical with smooth surface (FIG. 2 part B).
Combining the morphological characteristics of LX-6, the physiological and biochemical characteristics (Table 1) and the alignment of the 16S rRNA sequences (FIG. 3), the strain was identified as Bacillus amyloliquefaciensNamed as bacillus amyloliquefaciens @Bacillus amyloliquefaciensLX-6), preservation.
Physiological and biochemical identification of the LX-6 Strain of Table 1
Example 2: (Bacillus amyloliquefaciensLX-6 fermented soybean meal method
The method comprises the following steps:
(1) Activating and culturing strains: to be preservedBacillus amyloliquefaciensLX-6 bacterial suspension (1X 10) 8 cfu·mL -1 ) Inoculating LB liquid culture medium, and culturing at 37+ -l deg.C for 24h fermentation.
(2) Fermentation culture: the solid state fermentation culture medium is 100 g bean pulp, 100 ml water, 30g of solid state fermentation culture medium is bottled in 300 mL triangular flasks; will be cultivated by LB liquid mediumBacillus amyloliquefaciensLX-6 is inoculated into a fermentation culture medium according to 10% (v/v) and is fermented and cultured for 12h, 24h, 36h, 48h and 60h respectively.
(3) And (3) measuring protein of fermented soybean meal: after fermentation, the mixture was dried and crushed, and 2.5. 2.5 g portions were dissolved in 50 mL of 0.03M Tris-HCl (pH 8.0), followed by ultrasonic extraction for 30 min. After the extraction is finished, detecting the digestion condition of crude protein by SDS-PAGE, standing the sample for 5 min, taking 40 μl of supernatant and 10 μl of 5 Xprotein loading buffer solution, uniformly mixing, carrying out boiling water bath for 5-8 min and centrifuging at 11000 rpm for 3 min, taking 30 μl of supernatant, loading the sample on SDS-PAGE, and applying a voltage of 100V for 1 h. Coomassie brilliant blue is dyed for 30 min, decolorized for 30 min and then decolorized overnight by changing the decolorization solution.
As a result, as shown in FIG. 4, the crude protein was substantially degraded and the small molecular protein was increased at 24 hours in the fermented soybean meal.
Example 3: (Bacillus amyloliquefaciensLX-6 fermented soybean meal protein determination
(1) Sample treatment: precisely weighing 0.2 g fermented soybean meal sample, transferring into a dry 100 mL or 500 mL nitrogen-fixing bottle, adding 0.2 g copper sulfate, 6 g potassium sulfate and 20 mL sulfuric acid, shaking slightly, placing a small funnel on the bottle mouth, obliquely supporting the bottle on an asbestos net with small holes at an angle of 45 degrees, heating with small fire, after the whole content is carbonized, completely stopping foaming, strengthening the fire, keeping the liquid in the bottle slightly boiling until the liquid is blue-green, clear and transparent, and continuing heating for 0.5 hour. Taking down, cooling, carefully adding 20 mL water, cooling, transferring into a 100 mL volumetric flask, washing the nitrogen bottle with a small amount of water, mixing the washing liquid with the volumetric flask, adding water to the scale, and uniformly mixing for later use. Taking the same amount of copper sulfate, potassium sulfate and concentrated sulfuric acid as the treated sample, and performing a reagent blank test.
(2) The nitrogen-fixing device is installed, several drops of methyl red indicator and several milliliters of sulfuric acid are added at about 2/3 of the position of water in the water vapor generator so as to keep the water acidic, several glass beads are added to prevent bumping, and the pressure regulator is used for controlling the water vapor generator to heat and boil the water in the water vapor generating bottle.
(3) 10 mL of 2% boric acid solution and 1 drop of mixed indicator were added to the receiving flask, the lower end of the condenser tube was inserted under the liquid surface, 10.0. 10.0 mL sample digest was sucked up and flowed into the reaction chamber from the glass, and the glass stopper was closed by washing the glass with 10. 10 mL water. 10 mL of 40% sodium hydroxide solution was poured into a small glass, and the glass plug was lifted to slowly flow into the reaction chamber. The screw clamp was clamped and distillation was started, and the vapor was introduced into the reaction chamber to allow ammonia to pass through the condenser tube into the receiving flask and distilled for 5 min. The receiving bottle was moved so that the lower end of the condensing tube was separated from the liquid dish, and distilled for another 1 min, and then the outside of the lower end of the condensing tube was rinsed with a small amount of water. The receiving flask was removed and terminated with a standard solution of 0.05N sulfuric acid or 0.05N hydrochloric acid to gray or bluish purple.
Simultaneously, 10.0 ml reagent blank digestive juice is sucked up and operated according to the step (3).
(4) Calculation of
X is the percentage content of protein in the sample, g;
v1-the volume of sulfuric acid or hydrochloric acid standard solution consumed by the sample, mL;
v2-reagent blank consumes the volume of sulfuric acid or hydrochloric acid standard solution, mL;
n-equivalent concentration of sulfuric acid or hydrochloric acid standard solution;
1mL of 0.014-1N sulfuric acid or hydrochloric acid standard solution corresponds to the gram number of nitrogen;
m-mass (volume) of sample, g (mL);
f-the nitrogen is converted to a coefficient of protein.
The results show that LX-6 fermented soybean meal has total protein content of fermented samples at different times as shown in Table 2.
(5) Extraction and measurement of water-soluble protein
The method for measuring the water-soluble protein in annex A in GB/T31785-2015 'rules for judging quality of soybean storage'.
Bacillus amyloliquefaciensThe results of the water-soluble protein tests of the LX-6 solid state fermentation soybean meal samples at different times are shown in Table 2. As shown in Table 2, the total protein content of the soybean meal is higher, and gradually increases as the fermentation proceeds,Bacillus amyloliquefaciensthe total protein and the water-soluble protein reach the highest after LX-6 fermented bean pulp 48 h. The total protein content reached a maximum of 64.58% at 48h and decreased slightly after 60h. The water-soluble protein in unfermented soybean meal only accounts for about 5.74% of the total protein, inBacillus amyloliquefaciensUnder the action of protease generated by LX-6, the content of water-soluble protein in fermented soybean meal is greatly increased, and the maximum value of the water-soluble protein reaches 44.45% in fermentation 48 and h.
TABLE 2 protein content of fermented soybean meal by different time of LX-6 Strain
。
Claims (3)
1. A bacillus amyloliquefaciens with high protease yield is characterized in that: the strain LX-6 is classified and named as bacillus amyloliquefaciens @Bacillus amyloliquefaciens) LX-6, preservation unit: china center for type culture Collection, preservation date: 2021, 11 and 22 days, the preservation registration number is CCTCC No. M20211457.
2. A method for fermenting soybean meal by using the bacillus amyloliquefaciens according to claim 1, which is characterized in that: the method comprises the following steps:
(1) The bacillus amyloliquefaciens LX-6 single colony is placed in LB liquid culture medium, and is cultured at the rotation speed of 180 r/min of a shaking table at 37+/-l ℃ for 24h, and the number of viable bacteria is 10 8 CFU/mL；
(2) The solid state fermentation culture medium proportion of the bean pulp is 30g bean pulp, 30 ml water and 30g; will be cultured by using beef extract peptone liquid culture mediumBacillus amyloliquefaciens LX-6 is inoculated into a fermentation medium according to 10% (v/v) and is fermented and cultured at 37+/-l ℃.
3. The method for fermenting soybean meal by using bacillus amyloliquefaciens according to claim 2, which is characterized in that: the fermentation culture time is 24-48 hours.
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| 豆粕发酵用高产蛋白酶芽孢杆菌的筛选及鉴定;刘旭辉等;《饲料工业》;第35卷(第8期);第55页左栏第1.1.2节和右栏第1.2.2节 * |
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