CN116656565A

CN116656565A - Bacillus licheniformis and application thereof

Info

Publication number: CN116656565A
Application number: CN202310719114.0A
Authority: CN
Inventors: 彭勃; 侯莎; 童星; 万吉林
Original assignee: Tiandian Biotechnology Co ltd; Tiandian Guangdong Biotechnology Co ltd; Guangdong Haitian Innovation Technology Co Ltd
Current assignee: Tiandian Biotechnology Co ltd; Tiandian Guangdong Biotechnology Co ltd; Guangdong Haitian Innovation Technology Co Ltd
Priority date: 2023-06-16
Filing date: 2023-06-16
Publication date: 2023-08-29
Anticipated expiration: 2043-06-16
Also published as: CN116656565B

Abstract

The application provides bacillus licheniformis and application thereof, and belongs to the technical field of microorganisms. The bacillus licheniformis of the application is named bacillus licheniformis (Bacillus licheniformis) phb01 and has been deposited in the cantonese province microorganism strain collection, address: the Guangzhou early China No. 100 university, no. 59 building No. 5 Guangdong province microbiological institute, deposit number is GDMCC No.63466. The protease produced by the fermentation of the bacillus licheniformis phb can effectively degrade residual proteins in fermentation raw materials, and the content of amino acid nitrogen and total nitrogen is improved; the bacillus licheniformis phb01 has high protease activity under the condition of 20 percent high salt; the bacillus licheniformis phb01 has good genetic stability and can be used as a standby strain for industrial production.

Description

Bacillus licheniformis and application thereof

Technical Field

The application belongs to the technical field of microorganisms, and particularly relates to bacillus licheniformis and application thereof.

Background

The traditional fermented seasonings such as soy sauce, bean paste and the like are usually prepared by fermenting main raw materials such as soybeans and the like through aspergillus oryzae, and the aspergillus oryzae generates rich protease in the growth process, so that protein substances in the raw materials can be hydrolyzed to form peptides or amino acids with high-quality flavor. However, soy sauce still contains about 20% crude protein in the waste residue after fermentation, which indicates that the raw material contains a part of nondegradable protein, and the protease produced by Aspergillus oryzae cannot be completely hydrolyzed during fermentation.

Proteases are a generic term for enzymes that hydrolyze protein peptide chains and can be classified into endopeptidases and exopeptidases according to the hydrolysis mode, and acidic proteases, neutral proteases and alkaline proteases according to their suitable conditions of action. The protease has wide sources, can be extracted from animals and plants or prepared by microbial fermentation, and most of proteases commonly used in the food industry mainly comprise microbial sources, and main source strains comprise aspergillus niger, bacillus subtilis, bacillus licheniformis and the like. Because the molecular weight of the protein is large, the structure is complex, the variety is rich, meanwhile, the protease also belongs to a large class of substances, and the action modes and the cleavage sites of the proteases from different sources can be different, so that completely different hydrolysis effects are generated. In recent years, there have been some studies on salt-tolerant proteases, in which the decrease in enzyme activity after storage under high salt conditions is mostly focused, and there are few studies on enzyme activity measured directly under high salt conditions. In the research, partial research results of halophilic archaea show that the protease produced by the halophilic archaea has higher enzyme activity retention rate under the high-salt condition, but the strain does not belong to the protease production strain for food in the range of national safety standards of food, and the protease produced by the halophilic archaea has strong degradation capability for brewing protein difficult to hydrolyze.

In order to improve the utilization rate of protein in fermentation raw materials, researchers have adopted various methods, in particular to improve the protease activity of koji making by improving aspergillus oryzae strains, and the method is simple in production operation but does not change the type or action mechanism of aspergillus oryzae protease, but protein substances which cannot be hydrolyzed by aspergillus oryzae are still not touched. Some researchers have improved the proteolytic rate by optimizing the temperature, time, etc. of fermentation, and such process optimization has been almost saturated for the traditional fermentation industry, and is difficult to improve greatly on the existing basis. In recent years, some manufacturers begin to try to supplement protease in the fermentation process to achieve the effect of enzyme and bacteria co-fermentation, however, at present, both domestic and foreign enzyme preparation manufacturers have food-grade protease products, but the properties of the protease products tend to be homogeneous, the protease products special for brewed foods are less, the degradation-resistant proteolytic capability of the protease products in fermentation raw materials is limited, the salt tolerance is poor, only less than 20% of the effect of the protease products can be exerted under the high osmotic pressure condition of more than 10%, and the use efficiency is extremely low.

Disclosure of Invention

In order to solve the technical problems, the application provides the bacillus licheniformis and the application thereof, and the protease produced by the bacillus licheniformis can be used for carrying out specific enzymolysis and fermentation on protein difficult to hydrolyze, and still has higher activity under the high-salt condition.

To achieve the above object, the present application provides a Bacillus licheniformis designated Bacillus licheniformis (Bacillus licheniformis) phb01 which has been deposited at the microorganism strain deposit center, guangdong province, 5 months 14, 2023, address: the Guangzhou early China No. 100 university, no. 59 building No. 5 Guangdong province microbiological institute, deposit number is GDMCC No.63466. The inventor of the application screens protease fermentation strains capable of effectively degrading the protein which is difficult to hydrolyze according to a large number of experiments aiming at the residual protein which is difficult to hydrolyze of bean fermentation seasonings, and then improves the action effect of protease produced by the protease fermentation strains under the high-salt condition by an ARTP mutagenesis method, finally obtains a protease fermentation strain, and the protease produced by the protease fermentation strain can carry out specific enzymolysis and fermentation on the protein which is difficult to hydrolyze and still has higher activity under the high-salt condition.

As a preferred embodiment of the bacillus licheniformis of the application, the sequence of the bacillus licheniformis phb is shown as SEQ ID NO. 1.

The application also provides an application of the bacillus licheniformis phb01 in producing protease.

The application also provides an application of the bacillus licheniformis phb01 in preparing bean condiments.

The application also provides protease which is prepared by fermenting the bacillus licheniformis. The bacillus licheniformis phb01 is obtained by a large number of experimental screening, the strain can ferment to produce phb protease, the phb protease can specifically degrade protein which is difficult to hydrolyze by aspergillus oryzae in soy sauce or fermented sauce raw materials, and the protease has good effect under high-salt conditions, and is a protease specially used for brewing food production.

The application also provides application of the protease in preparing bean condiments. The inventor uses an ARTP method to mutagenize a protease fermentation strain, improves the enzyme activity of phb01 protease under a high-salt system, remarkably improves the retention rate of the enzyme activity under the high-salt condition compared with other proteases, and is more suitable for a high-salt fermentation system.

As a preferred embodiment of the application of the present application, the bean seasoning includes soy sauce and bean paste.

The application also provides a method for improving amino acid nitrogen in the bean seasoning, which is to add the protease into soy sauce mash for fermentation.

Compared with the prior art, the application has the beneficial effects that: the application provides a bacillus licheniformis and application thereof, the application separates out the strain producing protease from natural fermented soy sauce mash, then uses the protein remained in soy sauce fermentation residue as enzymolysis substrate, screens out a protease fermentation strain (bacillus licheniformis phb 01), the protease in the strain culture solution can effectively degrade the protein remained in the fermentation raw material, the amino acid nitrogen in the soy sauce residue filtrate after enzymolysis is improved by 16.36%, the total nitrogen is improved by 20.29%; the bacillus licheniformis phb01 has high protease activity under the condition of 20 percent high salt; the bacillus licheniformis phb01 has good genetic stability and can be used as a standby strain for industrial production.

Drawings

FIG. 1 is a colony morphology of Bacillus licheniformis phb.

FIG. 2 is a diagram showing amino acid analysis of soy sauce without enzyme in example 4.

FIG. 3 is a diagram showing amino acid analysis of Bacillus licheniformis phb protease group soy sauce in example 4.

FIG. 4 is an amino acid analysis chart of a commercial alkaline protease group soy sauce in example 4.

Detailed Description

For a better illustration of the objects, technical solutions and advantages of the present application, the present application is further illustrated by the following examples. It will be apparent that the following examples are only some, but not all, of the examples of the application. It should be understood that the embodiments of the present application are only used for illustrating the technical effects of the present application, and are not used for limiting the scope of the present application. Unless otherwise indicated, all methods and test conditions used in the examples of the present application are those conventionally used in the art, and all reagents, equipment and media used in the art are those conventionally used in the art, and can be prepared by conventional methods or commercially available.

EXAMPLE 1 isolation and screening of strains

1. Treatment of raw materials:

fermenting with conventional high salt dilution process for 10 days, 30 days, 50 days, and 90 days, respectively adding 10g of soy sauce into 100mL of sterile physiological saline containing NaCl 0.85%, homogenizing with homogenizing bag, and diluting soy sauce homogenized solution with sterile physiological saline to 10 ^-3 ～10 ^-6 Respectively taking 0.1mL from each dilution, uniformly coating on a LB solid medium plate containing 10% of salt, placing the plate in a 38 ℃ incubator to cultivate for 24-48 h, selecting plates with colony numbers less than 300, respectively picking all the colonies, inoculating the colonies into LB broth culture medium test tubes, and cultivating for 12h in a shaking table with the rotating speed of 200r/min at 37 ℃.

2. Preliminary screening of strains

Centrifuging the cultured bacterial liquid under 10000r/min to remove bacterial liquid reserved in the bacterial liquid, detecting the enzyme activities of acid protease, neutral protease and alkaline protease in each bacterial liquid according to the method in GB1886.174, and respectively selecting 10 strains with highest acid protease, neutral protease and alkaline protease production.

3. Compound screening of bacterial

(1) Respectively inoculating the strains which are screened in the first time into LB culture medium, culturing for 12 hours in a shaking table with the rotating speed of 200r/min at 37 ℃, centrifuging to remove thalli, concentrating for 10 times at 50 ℃, and detecting the protease activity of each concentrated solution;

(2) Taking soy sauce fermentation residues, drying at low temperature, pulverizing into uniform and fine powder, subpackaging the powder into test tubes, adding 10mL of pure water into each group of 1g of soy sauce residues, sterilizing at 121 ℃ for 15min, cooling, and adding 500U of protease concentrate of each strain respectively, wherein the control group is not added; after being uniformly mixed, the mixture is subjected to shaking table enzymolysis for 72 hours at the temperature of 30 ℃;

(3) And filtering the sauce residue enzymolysis liquid by using filter paper, and detecting the content of amino acid nitrogen and total nitrogen.

The results are shown in Table 1, wherein the strains with the remarkably improved amino acid nitrogen and total nitrogen compared with the control group have 7 strains, the strain with the most improved amino acid nitrogen and total nitrogen content compared with the control group is selected as a target strain P-7, the protease mainly produced by the strain is alkaline protease, the amino acid nitrogen content in the sauce residue filtrate is improved by 16.36% after enzymolysis by the protease produced by the strain, and the total nitrogen content is improved by 20.29%.

TABLE 1 amino acid nitrogen and total nitrogen content in the enzymatic hydrolysate after enzymatic hydrolysis of sauce residues by the primary screening strain

4. Mutagenesis of bacterial species

Seasoning fermentation is usually a high salt system, and even though protease can act on protein difficult to hydrolyze in fermentation raw materials, good salt tolerance is required to fully play a role. The high-quality strain P-7 obtained through screening is subjected to mutagenesis, the retention rate of the protease produced by the high-quality strain P-7 is improved, and the specific mutagenesis method is as follows:

(1) Activating the P-7 strain with LB culture medium to logarithmic phase, subjecting the bacterial liquid to ARTP mutagenesis treatment, and gradient diluting to 10 with 0.85% sterile physiological saline ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Is a diluent of (a). Uniformly coating 0.1mL of the diluted solution with each concentration on the surface of LB agar medium, and culturing at 37 ℃ for 48 hours;

(2) Selecting single colony with larger colony, inoculating to LB broth medium, shake culturing at 37deg.C for 12 hr, centrifuging to remove thallus, detecting protease activity in supernatant, removing strain with enzyme activity significantly lower than that of initial strain, and detecting salt-tolerant protease activity of the rest strain culture solution under 20% salt condition. The enzyme activity detection method under the high-salt condition is adjusted on the basis of the alkaline protease detection method specified in GB1886.174, and 20% NaCl is respectively added into casein solution and corresponding buffer solution, so that the salt content in an enzymolysis reaction system reaches 20%, and the conditions such as reaction temperature, time and detection wavelength are not changed.

Among all the strains obtained by mutation breeding, the retention rate of high-salt enzyme activity is higher than that of 9 strains of the initial strain P-7, and the specific enzyme activity conditions are shown in Table 2.

TABLE 2 alkaline protease Activity of mutant Strain culture fluid and enzyme Activity Retention under high salt conditions

Strain name	Protease Activity U/mL	20% haloprotease Activity U/mL	High salt enzyme activity retention%
				P-7 (initial)	106.2	16.6	15.6
P7-2	123.6	23.3	18.8
				P7-7	133.8	29.8	22.3
P7-15	118.1	21.5	18.2
				P7-18	140.0	22.8	16.3
P7-31	128.2	32.4	25.3
				P7-36	152.4	72.2	47.4
P7-39	141.0	47.9	34.0
				P7-44	109.5	29.5	26.9
P7-50	144.9	60.6	41.8

According to the research purpose, the P7-36 strain with the highest high salt enzyme activity retention rate is selected as a preferable strain, the P7-36 strain is sent to Huada genes for sequencing and identification, the gene sequence is shown as SEQ ID NO.1, and the result is bacillus licheniformis (Bacillus licheniformis).

The SEQ ID NO.1 is:

the P7-36 strain was preserved by the above screening, isolation and identification. The inventors have deposited with the collection of microorganisms and cell cultures from Guangdong province at 5.14 of 2023 and named Bacillus licheniformis (Bacillus licheniformis) phb01, deposited under the accession number GDMCC NO 63466.

The colony morphology of Bacillus licheniformis phb01 obtained in this example is shown in FIG. 1.

EXAMPLE 2 passage stability of Bacillus licheniformis phb01

In this example, the preferred strain Bacillus licheniformis phb01 obtained in example 1 was continuously passaged 10 generations in LB broth, the strains of the 2 nd generation, the 5 th generation and the 10 th generation were taken out when they were cultured for 12 hours, the OD value thereof at 600nm was examined, the protease activity and 20% of the activity of the haloprotease in the bacterial liquid thereof were examined, and the results of the examination between the above generations were compared to judge the genetic stability thereof. The specific data are shown in Table 3.

TABLE 3 passage stability of Bacillus licheniformis phb01

Detecting items	Generation 2	Generation 5	Generation 10
				12hOD ₆₀₀	1.21	1.22	1.21
Differences in	0.00％	0.83％	0.00％
				Protease Activity U/mL	150.4	153	152.8
Differences in	-1.31％	0.39％	0.26％
				20% haloprotease Activity U/mL	70.8	82.5	71.7
Differences in	-1.94％	0.42％	-0.69％

From the OD value, the protease activity and the 20% salinase activity of each generation of strain, the difference of each generation of strain is smaller, the absolute value of the difference rate is smaller than 5%, and the absolute value of the difference rate is obviously higher than that of the original strain P-7, which shows that the genetic stability of bacillus licheniformis phb is better, and the strain can be used as a standby strain for industrial production.

EXAMPLE 3 preparation and Properties of Bacillus licheniformis phb01 protease

1. This example provides a method for producing protease by fermentation using Bacillus licheniformis phb of example 1, comprising the steps of:

(1) Activating strains: 0.1mL of bacterial liquid is sucked from a reserved Bacillus licheniformis phb glycerol pipe and inoculated into a conical flask with 100mL of LB culture medium, and the bacterial liquid is cultured for 10 to 12 hours at the temperature of 37 ℃ by a shaking table 220 r/min.

(2) Seed liquid culture: inoculating the activated strain into a seed culture medium with the inoculum size of 2-3%, and culturing for 8-10h at 37 ℃ in a shaking table of 220 r/min; the seed culture medium: 1% peptone, 1% glucose, 0.5% sodium chloride, and water as the rest, at 115 ℃ for 20min.

(3) Fermentation culture: preparing 200g of soybean meal, 150kg of corn steep liquor, 100g of glucose, 40g of monopotassium phosphate and 20g of dipotassium phosphate serving as a fermentation medium, adding water to a volume of 10L, loading into a 20L fermentation tank, adjusting the initial pH to 7.5 by ammonia water, sterilizing at 121 ℃ for 20min, cooling, and inoculating 800mL of activated phb bacterial liquid. Fermenting at 37deg.C under the control of rotation speed of initial 25HZ and ventilation of 600m ³ /h; after fermentation for 2 hours, the rotating speed is adjusted to 50HZ, and the air flow is adjusted to 900m ³ And/h, controlling the glucose content to be 1-2g/L in the fermentation process. Fermenting for 35h, and discharging fermentation liquor.

(4) Protease preparation: adding 4% diatomite into the fermentation broth, stirring for 30min, filtering, concentrating the filtrate with 10000Da molecular weight hollow fiber membrane to 1/10 of the initial volume, adding 20% glycerol and 0.1% potassium sorbate, and standing.

(5) The enzyme activities of the fermentation broth and the final protease were examined, and the protease activity under 20% salt conditions was examined according to the method in example 1.

TABLE 4 protease Activity

Enzyme activity of fermentation liquor	Enzyme activity of concentrated solution	Protease enzyme Activity	Protease 20% salt enzyme Activity
				8120U/mL	66000U/mL	52000U/mL	24600U/mL

As shown in Table 4, the final enzyme activity of the prepared phb01 protease solution is 52000U/mL, the enzyme activity under the condition of 20% salt is 24600U/mL, and the high-salt enzyme activity retention rate is 47.3% which is equivalent to that of the original strain culture solution.

2. According to the method for carrying out enzymolysis on soy sauce residues in strain re-screening in example 1, the action effect of phb01 protease is verified, and the specific experimental method is as follows: taking soy sauce fermentation residues, drying at low temperature, pulverizing into uniform and fine powder, subpackaging the powder into test tubes, adding 10mL of pure water into each group of 1g of soy sauce residues, sterilizing at 121 ℃ for 15min, cooling, adding 500U of phb protease, and taking a group without protease as a blank group and commercialized alkaline protease as a control group. After being mixed evenly, the mixture is subjected to shaking table enzymolysis for 72 hours at the temperature of 30 ℃. And detecting the amino nitrogen and total nitrogen content in the enzymolysis liquid after the enzymolysis is finished.

As shown in Table 5, phb01 protease can effectively act on protein difficult to hydrolyze in soy sauce residue, amino acid nitrogen in enzymolysis liquid is increased by 20.37%, total nitrogen content is obviously increased by 31.82%, and the proteolytic rate in soy sauce residue is obviously increased.

Table 5phb amino nitrogen and total nitrogen content in the enzymatic hydrolysate after the enzymatic hydrolysis of sauce residue by protease

	Amino acid nitrogen g/100mL	Total nitrogen g/100mL
			Blank group	0.054	0.066
phb01 protease enzymolysis	0.065	0.087
			Commercial alkaline protease enzymatic hydrolysis	0.056	0.073

Example 4 application of phb01 protease in soy sauce fermentation

In the embodiment, the soy sauce is fermented by adopting a high-salt dilute state process, phb protease accounting for 0.05% of the volume of the soy sauce mash is added within 0-1 day of fermenting the soy sauce mash, meanwhile, commercial alkaline protease accounting for 0.05% is added as a control group, then fermentation is continuously carried out according to a conventional production process, and after the fermentation is finished, the amino nitrogen content, the total nitrogen content and the amino acid type analysis of the soy sauce are detected.

The results are shown in tables 6 to 7 and FIGS. 2 to 4. As can be seen from Table 6, the amino acid nitrogen and total nitrogen content of the fermented soy sauce were significantly increased by using phb01 protease during fermentation, wherein the amino acid nitrogen was increased by 12.6% and the total nitrogen was increased by 14.4%, and the effect was better than that of commercial alkaline protease. As shown in Table 7, the content of various amino acids in soy sauce fermented with phb protease produced by phb01 strain was increased to various degrees as shown in Table 7.

TABLE 6 soy sauce index test results

	Amino acid nitrogen g/100mL	Total nitrogen g/100mL
			Enzyme-free group	0.95	1.18
phb01 protease group	1.07	1.35
			Commercial alkaline protease group	0.98	1.21

TABLE 7 amino acid analysis of soy sauce

Example 5 application of phb01 protease in fermentation of Bean paste

Mixing soybean after high-pressure cooking with flour, stirring, adding 3.042 kinds of yeast of Aspergillus oryzae Shanghai brewing 3.3% of total mass, simultaneously adding phb protease of 0.05% into an experimental group, stirring again, mixing uniformly, adding no control group, ventilating and making yeast at 30-32 ℃ for 42h, adding 12-14% of salt water until the yeast material is just last, fermenting at 30 ℃ for 80 days continuously to obtain soybean paste, and carrying out physical and chemical index detection and taste sensory evaluation on the soybean paste; scoring criteria for sensory evaluation: selecting 15 evaluation staff to score from 4 dimensions of delicate flavor, aroma, thick feel and comprehensive flavor, wherein the evaluation staff are sequentially unacceptable, acceptable, better and better from 1 to 5, and finally counting average, wherein the higher the delicate flavor score is, the more sufficient the delicate flavor is; the higher the fragrance score, the better the fragrance; the higher the thickness score, the more adequate the thickness; the higher the overall flavor score, the better the overall flavor.

The results are shown in tables 8 and 9. As shown in table 8, phb01 protease can be used for remarkably improving the amino acid nitrogen content of fermented soybean paste in the production process of the soybean paste, and the total acid is synchronously improved, so that the effect on the pH is not great; from table 9, it is clear that the flavor, aroma and richness of the soybean paste were improved to some extent by sensory evaluation using phb01 protease, and the overall flavor was improved.

TABLE 8 detection results of Bean jam index

TABLE 9 results of sensory evaluation of Bean paste

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present application.

Claims

1. A bacillus licheniformis, characterized in that the bacillus licheniformis is designated bacillus licheniformis (bacillus licheniformis) phb, deposited at the microorganism culture collection, cantonese province, at 14/5/2023, address: the Guangzhou City first middle road No. 100 college 59 building 5 Guangdong province microbiological institute, deposit number is GDMCCNo.63466.

2. The bacillus licheniformis according to claim 1, characterized in that the sequence of bacillus licheniformis phb01 is shown in SEQ ID No. 1.

3. Use of bacillus licheniformis phb01 according to any of the claims 1-2 for the production of proteases.

4. Use of bacillus licheniformis phb01 according to any of the claims 1-2 for the preparation of a legume condiment.

5. A protease prepared by fermentation of the bacillus licheniformis of any of claims 1-2.

6. Use of the protease of claim 5 for the preparation of a legume condiment.

7. The use according to claim 6, wherein the legume dressing comprises soy sauce, soy sauce.

8. A method for increasing the amino acid nitrogen content of a legume condiment, comprising adding the protease of claim 5 to a moromi mash for fermentation.