CN116004418A

CN116004418A - Bacillus bailii Z-1 and application thereof in preparation of hydrolysate

Info

Publication number: CN116004418A
Application number: CN202210845790.8A
Authority: CN
Inventors: 卢静; 赵宇; 王淑军; 吕明生; 房耀维; 杨杰; 秦俊桓
Original assignee: Jiangsu Ocean University
Current assignee: Jiangsu Ocean University
Priority date: 2022-07-19
Filing date: 2022-07-19
Publication date: 2023-04-25

Abstract

The invention relates to the technical field of biology, in particular to bacillus beijerinus (Bacillus velezensis) Z-1 and application thereof. The invention provides a screening and identification method, growth and fermentation enzyme production conditions and protease characteristic research method of bacillus belicus (Bacillus velezensis) Z-1, and application of protease thereof in preparation of mussel antioxidant substances. The mussel is subjected to enzymolysis by using the protease of the strain Z-1, and the hydrolysate obtained by the enzymolysis has a good antioxidation function, so that the technology has potential application prospect in mussel meat high-value application.

Description

Bacillus bailii Z-1 and application thereof in preparation of hydrolysate

Technical Field

The invention relates to the technical field of biology, in particular to bacillus beijerinus (Bacillus velezensis) Z-1 and application research on oxidation resistance of mussel hydrolysate prepared by the bacillus beijerinus.

Background

Mussel belongs to the phylum mollusca, is a bivalve mollusca, is called mussel and green mouth, is widely cultivated in eastern coastal areas such as Fujian, shandong, jiangsu and the like in China, and is an important species in shellfish cultivation industry in China. Mussels are loved by consumers because of delicious meat, low price and rich nutrition, and enjoy the American names of 'Donghai ladies' and 'eggs in sea'. The mussel has protein content up to 59%, various fatty acid types, and eight essential amino acids and essential fatty acids required by human body, and is a shellfish with research value.

Mussels are shellfish with high nutritive value, but have low economic value, and the surfaces of the mussels are difficult to process because of a plurality of attachments which are difficult to remove, and are generally regarded as low-value shellfish. In 2019, the export sales of mussels in China mainly take a frozen product way, accounting for 96.7% of the total export amount, most of the mussels are sold in the market in fresh and directly sold form, and the small mussels are sold in the form of simple processing such as can and cured food, even processed in the form of feed, and compared with the existing processed seafood, the mussel has low added value of the product and unreasonable utilization of mussel resources. In order to solve the situation, the added value of mussel meat can be improved by utilizing a microbial enzymolysis method. Compared with the traditional acid-base hydrolysis method, the microbial enzymolysis method has mild protein hydrolysis conditions, does not destroy amino acid, is cheaper and easy to obtain compared with commercial enzymes, and can be used for enzymolysis to obtain richer peptide fragments, amino acids and polysaccharides, and a plurality of researches and reports at present show that mussel hydrolysis peptides have various biological activity functions of deferring aging, resisting oxidation, resisting tumor, resisting bacteria, resisting coagulation, reducing blood pressure and the like.

Bacillus beleiensis was first discovered by Ruiz in 2004 as a class of spore-forming gram-positive bacteria, and few reports on them were early on, and only after 2019 was paid attention to research. Bacillus bailii is widely applied in the fields of agriculture, forestry, environmental protection, industry and the like due to the characteristics of easy separation and culture, broad antibacterial spectrum, stable antibacterial effect and high biological safety. In addition, bacillus belicus has various protease and carbohydrase activities, such as cellulase, filter paper enzyme, amylase, proteinase, etc., and thus has a wide application prospect in the food industry.

The application research on protease produced by bacillus beliensis is less at present, and in order to fully develop ocean resources and promote reasonable utilization of mussel resources, bacillus beliensis Z-1 with high protease yield is obtained by screening ocean sludge. The protease produced by the strain Z-1 is utilized to carry out enzymolysis on mussel meat, so that the biological activity function of hydrolysate is researched, and the protease is a functional factor which is the hottest research subject and has a great development prospect in the current International food community, and meanwhile, the comprehensive application value of the mussel is improved, so that the protease has feasibility and practical significance.

Disclosure of Invention

Bacillus beleiensis (Bacillus velezensis) Z-1, which bacillus beleiensis (Bacillus velezensis) Z-1 has been stored in the China general microbiological culture collection center (CGMCC), address: beijing, chaoyang area, north Chenxi Lu No. 1, 3; preservation date: 2022, 06, 10; preservation number: CGMCC No.25059; classification naming: bacillus bailii Bacillus velezensis.

In some embodiments, the invention also provides a method for preparing the bacillus belicus Z-1 and/or the fermentation enzyme liquid thereof for enzymolysis of mussels to generate hydrolysate with antioxidant function.

In some embodiments, the present invention provides a method for culturing the bacillus belgium Z-1, comprising the steps of: bacillus bailii Z-1 was inoculated into the primary seed culture medium for cultivation, and then inoculated from the primary seed culture medium to the secondary seed culture medium at 1%.

In some embodiments, the primary and secondary seed culture medium components are 10g/L bran, 5g/L tapioca starch, 2% NaCl aged seawater preparation, pH7.0.

In some embodiments, the culture conditions in the seed medium are: culturing at 35 deg.C for 18 hr at 160-200 r/min.

In some embodiments, the invention provides a fermentation enzyme production method of bacillus belicus Z-1, which comprises the steps of culturing bacterial liquid to secondary seed liquid, inoculating the secondary seed liquid into a fermentation medium for fermentation, and centrifuging to obtain fermentation enzyme liquid.

In some embodiments, the fermentation medium comprises 10g/L maltose or tapioca starch, 10g/L casein or peanut meal, configured with aged seawater having a NaCl concentration of 0-2%, pH 7-8.

In some typical embodiments, the fermentation medium composition is 10g/L maltose, 10g/L peanut meal, and is configured with aged seawater having a NaCl concentration of 0%, pH 8.0.

In some embodiments, the fermentation culture conditions are: fermenting and culturing at 35-37deg.C for 24-32 hr at 180 r/min.

In some typical embodiments, the fermentation medium is cultured at 37 ℃ for 24 hours.

The invention also provides a mussel hydrolysate which is obtained by hydrolyzing mussels by using bacillus belicus Z-1 and/or fermentation enzyme liquid thereof.

The invention also provides a method for hydrolyzing mussels by utilizing bacillus belicus Z-1 and/or fermentation enzyme liquid thereof, which comprises the following steps: weighing a certain amount of mussel dry powder according to a solid-to-liquid ratio of 1:20 are mixed with the fermentation enzyme liquid and are subjected to enzymolysis for 24 hours in a shaking table at 37 ℃ and 180 r/min. Centrifuging the zymolyte to obtain supernatant, filtering the supernatant by suction to further remove suspended matters, and ultrafiltering the supernatant by using molecular filter membranes of 8 kkDa and 5 kkDa to obtain products with three stages of different molecular sizes. The fractions with a molecular weight of less than 5 kkDa were isolated and lyophilized and stored at-80 ℃.

The invention has the beneficial effects that: the invention provides a novel bacillus belicus (Bacillus velezensis) Z-1, which is verified by various experiments that the strain can ferment to produce protease under the conditions of 25-45 ℃, pH 5-9, 0-10% and common carbon and nitrogen sources, and has the advantages of simple culture and fermentation conditions, short fermentation period, high enzyme production, stable enzyme activity, easy operation of the whole process, low cost and high benefit, and is suitable for industrial production.

Drawings

FIG. 1 strain Z-1 colony morphology;

FIG. 2 gram of strain Z-1 (. Times.1000);

FIG. 3 phylogenetic tree diagram of strain Z-1;

FIG. 4 is a graph showing the effect of temperature on the growth of strain Z-1;

FIG. 5 is a graph showing the effect of pH on the growth of strain Z-1;

FIG. 6 shows the effect of NaCl concentration on the growth of strain Z-1;

FIG. 7 is a graph showing the effect of initial carbon source on the growth of strain Z-1;

FIG. 8 is a graph of the effect of initial nitrogen source on the growth of strain Z-1;

FIG. 9 is a graph showing the effect of fermentation temperature on protease production by strain Z-1;

FIG. 10 is a graph showing the effect of fermentation pH on protease production by strain Z-1;

FIG. 11 is a graph showing the effect of fermentation NaCl concentration on the protease production of strain Z-1;

FIG. 12 graph of the effect of fermentation carbon source on protease production by strain Z-1;

FIG. 13 shows the effect of fermentation nitrogen source on protease production by strain Z-1;

FIG. 14 is a graph of temperature versus protease activity;

FIG. 15 is a graph of temperature stability of a protease;

FIG. 16 is a graph of pH versus protease activity;

FIG. 17 shows a pH stability profile of a protease;

FIG. 18 is a graph of the effect of substrate species on protease activity;

FIG. 19 DPPH clearance plot of hydrolysates versus 10mg/mL Vc;

FIG. 20 shows a graph of hydroxyl radical scavenging of hydrolysates versus 10mg/mL Vc;

FIG. 21 is a graph of superoxide anion clearance of hydrolysate versus 10mg/mL Vc;

FIG. 22 is a graph of the reducing power of the hydrolysate versus 10mg/mL Vc;

detailed description of the preferred embodiments

The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.

Example 1 screening of strains

And (3) primary screening: weighing 1g of activated sludge A2 sample (island sludge), and carrying out gradient dilution to 10 by using sterile water ⁰ ，10 ^-1 ，10 ^-2 ，10 ^-3 ，10 ^-4 ，10 ^-5 And 10 ^-6 200. Mu.L of the culture medium was pipetted onto the screening medium by a pipette, and after being uniformly coated by a coating rod, the culture medium was placed in a constant temperature incubator at 37℃for 24-48 hours, and then observation was performed. According to the size of transparent rings produced by the strain, selecting several strains with larger transparent rings, separating, purifying and preserving the strains.

And (3) re-screening: culturing several strains obtained by primary screening to a secondary seed solution, inoculating to a fermentation enzyme production culture medium with an inoculum size of 10%, culturing in a shaking table at 37 ℃ and 180rpm/min for 24 hours, centrifuging to obtain a supernatant, measuring protease activity by using Fu Lin Fenfa, namely the strain with the highest enzyme activity, namely the strain Z-1, and carrying out the next study on the strain Z-1.

EXAMPLE 2 physiological Biochemical Studies of Strain Z-1

1.1 colony morphology characterization

Inoculating the strain to LB culture, culturing for 18h at 37deg.C, inoculating to casein culture medium with inoculating loop, culturing for 36h at 37deg.C, observing morphological characteristics of strain, and as shown in figure 1, Z-1 colony is white, slightly yellow, large in colony, irregular in edge, rough and uneven in surface, and has wrinkles, and obvious transparent ring is formed around the colony.

1.2 Strain Z-1 gram staining

The gram staining conditions of the strain were observed under an electron microscope oil microscope according to the procedure of the gram staining kit, including the steps of smear, primary staining, mordant staining, decoloration, counterstain, etc., according to the instruction manual of the gram staining kit, as shown in fig. 2. The strain Z-1 is observed to be in a rod-shaped or bar-shaped form under a microscope oil microscope, and is dyed to be purple, and the strain Z-1 is a gram-positive bacillus according to the specification of a gram-staining kit.

1.3 Strain Z-1 physiological Biochemical experiments

A physiological and biochemical reagent tube (Hangzhou Binhe microbial reagent Co., ltd.) of a proper type was selected, 100. Mu.L of seed solution was sucked by a pipette and added to each reagent tube, and the mixture was cultured in an incubator at 37℃for 24 hours, and the color change of the reagent tubes was observed, and positive and negative were judged according to the instructions of use, and the results are shown in Table 1. Ornithine, xylose, urea, glucose, maltose, lactose, lysine, nitrate, sucrose, oxidative fermentation O-F and arginine are positive, and mannitol, cellobiose, rhamnose, mushroom sugar, arabitol broth, raffinose, erythrose, sodium pyruvate, ribose and phosphate glucose peptone water are negative.

TABLE 1 physiological and biochemical experiment results of Strain Z-1

The "+" indicates positive; "-" indicates negative

1.4 identification of Strain Z-1 molecular biology

2mL of bacterial liquid cultured for 18h is sucked and added into a 2mL EP tube, the centrifugation is carried out at 8000rpm/min for 2min, the gene of the bacterial strain Z-1 is extracted according to the operation of a bacterial DNA extraction kit, and the PCR technology is utilized to amplify the gene.

(1) Genome: the genome of strain Z-1 was extracted by a bacterial DNA extraction kit (Tiangen Biochemical technologies Co., ltd.).

(2) Primer: 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3')

(3) PCR reaction System (50. Mu.L): 2×Phanta Flash Master Mix (Nanjinouzan Biotechnology Co., ltd.) 25. Mu.L, upstream primer (27F) 2. Mu.L, downstream primer (1492R) 2. Mu.L, template DNA 1. Mu.L, ddH ₂ O 20μL。

(4) The reaction procedure: denaturation at 98℃for 30S; denaturation at 98℃for 10S, annealing at 55℃for 30S, extension at 72℃for 30s,30 cycles, extension at 72℃for 2 min; preserving at 4 ℃.

(5) Agarose gel electrophoresis: the PCR amplified product was subjected to 1% agarose gel electrophoresis, and after running out of a single bright band, it was sent to Shanghai Biotechnology Co., ltd for sequencing.

(6) Phylogenetic tree: and (3) comparing the bacterial 16S rRNA sequence obtained by the sequencing result with the homology of a GenBank database, selecting 15 strains with the closest homology, and drawing a strain Z-1 phylogenetic tree by using MEGA software, wherein the strain Z-1 phylogenetic tree is shown in figure 3.

EXAMPLE 3 study of growth Properties of Strain Z-1

The growth characteristics of the strain Z-1 are studied, and the basic formula of a growth medium is 10g/L peptone, 5g/L yeast powder and aged seawater, and the pH value is 7.0.

1.1 Effect of temperature on growth of Strain Z-1

The strain was cultured in growth medium at different temperatures of 25, 30, 35, 37, 40 and 45℃for 24 hours by the controlled variable method, and the absorbance OD at 600nm was measured ₆₀₀ OD under each culture temperature condition was compared ₆₀₀ As shown in FIG. 4, the results indicate that the strain Z-1 is most suitable for growth at 35 ℃.

1.2 influence of pH on the growth of Strain Z-1

The strain was assayed by the controlled variable method for 24h in growth media at

different pH

5, 6, 7, 8, 9 and 10, and OD was assayed _600， Comparison of OD under culture conditions at each pH ₆₀₀ As shown in FIG. 5, the results indicate that the most suitable strain Z-1 grows at pH7.

Effect of 1.3NaCl concentration on growth of Strain Z-1

The strain was cultured in growth media with different NaCl concentrations of 0, 2, 4, 6, 8, 10, 12, 14 and 16g/100mL for 24h by using the controlled variable method, and OD was measured _600， Comparing eachOD under NaCl concentration condition ₆₀₀ As shown in FIG. 6, the results showed that 2% NaCl concentration was optimal for growth of strain Z-1.

1.4 Effect of initial carbon Source on growth of Strain Z-1

The strain is cultured in growth medium with different initial carbon sources such as sucrose, glucose, yeast powder, maltose, tapioca starch, lactose for 24 hr by controlled variable method, and OD is measured _600， Comparison of OD under the culture conditions of each initial carbon source ₆₀₀ As shown in FIG. 7, the results indicate that the most suitable strain Z-1 grows using tapioca starch as a carbon source.

1.5 Effect of initial Nitrogen Source on growth of Strain Z-1

The OD of the strain cultured for 24h under different initial nitrogen sources such as fish meal peptone, casein, tryptone, bran, soybean meal and peanut meal is measured by using a controlled variable method ₆₀₀ Comparison of OD under each initial Nitrogen Source culture condition ₆₀₀ As shown in FIG. 8, the results indicate that the most suitable strain Z-1 grows with bran as nitrogen source.

EXAMPLE 4 investigation of the enzyme production characteristics of Strain Z-1 fermentation

The fermentation and enzyme production characteristics of the strain Z-1 are studied, the protease activity determination method refers to the protease activity determination method of a protease preparation in national standard GB/T23527-2009, and the protease activity is determined by using the Fulin method.

1.1 Medium

Seed culture medium: peptone 10g/L, yeast powder 5g/L, and aged sea water, pH7.0

Fermentation medium: yeast powder 10g/L, casein 10g/L, aged sea water, pH7.0

1.2 Effect of fermentation temperature on enzyme production by Strain Z-1 fermentation

Culturing the bacterial liquid to a secondary seed liquid, inoculating the secondary seed liquid to a fermentation enzyme production culture medium with an inoculum size of 10%, culturing the fermentation enzyme liquid at different temperatures (25, 30, 35, 37, 40 and 45 ℃) through the culture medium, centrifuging the fermentation enzyme liquid to obtain supernatant, and measuring the relative enzyme activity under different temperature fermentation conditions, wherein the result shows that the temperature is 37 ℃ and the optimum strain Z-1 is used for fermenting enzyme production.

1.3 Effect of fermentation pH on enzyme production by Strain Z-1 fermentation

Culturing the bacterial liquid to a secondary seed liquid, inoculating the secondary seed liquid to a fermentation enzyme production culture medium with an inoculum size of 10%, culturing the fermentation enzyme liquid under different pH values (5, 6, 7, 8, 9 and 10) by the culture medium, centrifuging the fermentation enzyme liquid to obtain a supernatant, and measuring the relative enzyme activity under different pH fermentation conditions, wherein the results show that the pH is optimal for the strain Z-1 fermentation enzyme production.

1.4 Effect of fermentation NaCl concentration on the enzyme production by fermentation of Strain Z-1

The bacterial liquid is cultivated to a secondary seed liquid, 10% of inoculation amount is inoculated to a fermentation enzyme production culture medium, the fermentation enzyme liquid is centrifugated to obtain supernatant liquid through the culture medium under different NaCl concentrations (0, 2, 4, 6, 8, 10, 12, 14 and 16g/100 mL), and the relative enzyme activity under different NaCl concentration fermentation conditions is measured, as shown in figure 11, the result shows that the 0% NaCl concentration is most suitable for the strain Z-1 fermentation enzyme production.

1.5 Effect of fermentation carbon Source on the enzyme production by Strain Z-1 fermentation

Culturing the bacterial liquid to a secondary seed liquid, inoculating the secondary seed liquid to a fermentation enzyme production culture medium with an inoculum size of 10%, culturing the fermentation enzyme liquid under the culture mediums of different carbon sources (sucrose, glucose, yeast powder, maltose, tapioca starch and lactose), centrifuging the fermentation enzyme liquid to obtain supernatant, and measuring the relative enzyme activity under the fermentation conditions of different carbon sources, wherein the results show that the fermentation enzyme production of the strain Z-1 takes maltose as the most suitable fermentation carbon source.

1.6 Effect of fermentation Nitrogen Source on the enzyme production by Strain Z-1 fermentation

Culturing the bacterial liquid to a secondary seed liquid, inoculating the secondary seed liquid to a fermentation enzyme production culture medium with an inoculum size of 10%, culturing the fermentation enzyme liquid under the culture medium of different nitrogen sources (fish meal peptone, casein, tryptone, bran, bean pulp and peanut pulp), centrifuging the fermentation enzyme liquid to obtain a supernatant, and measuring the relative enzyme activity under the fermentation conditions of different nitrogen sources, wherein the results show that the fermentation enzyme production is carried out by using the peanut pulp as the most suitable strain Z-1 for fermenting the nitrogen sources.

EXAMPLE 5 investigation of the influence of temperature, pH, substrate species on protease Activity

1.1 Effect of temperature on protease Activity

The enzyme solution obtained by fermentation is placed in water baths with the temperature of 45, 50, 55, 60 and 65 ℃, protease activity is measured by using the Furlin method, and the influence of the temperature on the protease activity is compared, as shown in figure 14, and the result shows that the optimal reaction temperature of the protease is 60 ℃.

1.2 Effect of temperature on stability of protease Activity

The enzyme liquid is placed in water baths with different temperatures (45, 50, 55, 60 and 65 ℃) for 20, 40, 60, 80 and 100 minutes, relative enzyme activity under each condition is measured, and compared with the temperature stability, as shown in figure 15, the result shows that the enzyme is more stable when placed at 45-55 ℃ for less than 100 minutes, and when the placing temperature is higher than 65 ℃, the enzyme activity is lost more quickly, thus being unfavorable for the use of the enzyme.

1.3 influence of pH on protease Activity

The enzyme solution obtained by fermentation was placed under different pH (5, 6, 7, 8, 9, 10) conditions, protease activity was measured by the Furling method, and the effect of pH on protease activity was compared, as shown in FIG. 16, and the results showed that the protease had an optimum reaction pH of 9.

1.4 influence of pH on stability of protease activity

The enzyme liquid is placed for 20, 40, 60, 80 and 100min under different pH conditions (5, 6, 7, 8, 9 and 10), the relative enzyme activities under each condition are measured, the pH stability is compared, as shown in figure 17, the result shows that the relative enzyme activities are higher in the pH range of 7-9, the relative enzyme activities under the conditions of pH > 10 and pH <7 are lower, the enzyme is reacted for a long time under the environment with higher acidity and alkalinity, the loss of the enzyme activity is quick, and the enzyme is suitable for reacting for a long time under the weak alkaline condition.

1.5 influence of substrate on protease Activity

Weighing casein, gelatin, beef extract powder, skimmed milk and bovine serum albumin (1 g) respectively, dissolving in 10mL of pure water, mixing well, measuring relative enzyme activities of protease solution under five different substrates, and comparing substrate specificities, wherein the results show that casein is the optimal reaction substrate of the enzyme.

EXAMPLE 6 mussel hydrolysate study

Research on method for preparing mussel hydrolysate and measuring oxidation resistance

1.1 preparation and isolation of mussel hydrolysate

Weighing a certain amount of mussel dry powder according to a solid-to-liquid ratio of 1:20 are mixed with the fermentation enzyme liquid and are subjected to enzymolysis for 24 hours in a shaking table at 37 ℃ and 180 r/min. Taking out and centrifuging the zymolyte to obtain supernatant, filtering the supernatant, carrying out suction filtration to further remove suspended matters, and carrying out ultrafiltration on the supernatant by using molecular filter membranes of 8 kkDa and 5 kkDa to obtain products with three stages of different molecular sizes. The fractions with a molecular weight of less than 5 kkDa were isolated and lyophilized and stored at-80 ℃.

1.2 DPPH radical scavenging Activity of the hydrolysate

The mussel hydrolysate is diluted to different gradient concentrations according to the protein concentration, 0.2mL of mussel hydrolysate and 0.2mL of DPPH (prepared by ethanol solution, 0.1 mM) are sequentially added, the mixture is vibrated and mixed at room temperature, the mixture is placed in a dark place for reaction for 30min, and the absorbance is measured at 517 nm. Water as a blank, ascorbic acid (Vc) as a reference, clearance is expressed as follows:

a is the absorbance of the mixture of DPPH solution and enzyme hydrolysate, B is the absorbance of the enzymatic hydrolysate,

c is the absorbance of a mixture of DPPH solution and water.

The measurement result is shown in figure 19, the clearance of 10mg/mL Vc to DPPH free radical is 95.6%, the clearance of DPPH is taken as an index, the protein concentration of hydrolysate is taken as a variable, the clearance of Z-1 mussel hydrolysate to DPPH free radical is drawn as shown in figure 19, the clearance of DPPH free radical is increased along with the gradual increase of the protein concentration, and when the protein concentration reaches 0.5mg/mL, the clearance of DPPH is equivalent to 64% of 10mg/mL Vc, which indicates that the quantity of amino acid and active polypeptide in hydrolysate and the clearance efficiency of DPPH are positively correlated, and the hydrolysate has antioxidant activity.

1.3 hydroxy radical scavenging Activity of hydrolysates

Diluting the freeze-dried powder of the Z-1 hydrolysate to different gradient concentrations according to the protein concentration, and adding 0.1mL FeSO4.7H2O (9 mM), 0.1mL salicylic acid (anhydrous ethanol solution, 9 mM), 0.1mLZ-1 enzyme-digested product and 0.1mL 0.03% H ₂ O ₂ The mixture was incubated at 37℃for 15min and absorbance at 510nm was measured. Ultrapure water and Vc were used as blank and positive control, respectively, and repeated three times. The formula is as follows:

a is the absorbance of the control without the sample, B is the absorbance of the sample, and C is the absorbance of the blank reagent.

The measurement result is shown in FIG. 20, and the result shows that the higher the protein concentration of the hydrolysate is, the higher the clearance rate of the hydroxyl radical is, and when the protein concentration reaches 0.5mg/mL, the clearance rate of the hydroxyl radical is equivalent to 68.8% of the clearance rate of Vc of 10mg/mL, thus indicating that the Z-1 hydrolysate has better clearance effect on the hydroxyl radical.

1.4 superoxide anion radical scavenging Activity of hydrolysate

The lyophilized powder of the hydrolysate was diluted to different gradient concentrations depending on the protein concentration, 0.1mL of the Z-1 enzymatic hydrolysate was mixed with 1mL of Tris-HCl (50 mM), incubated at 25℃for 10min, 30. Mu.L of pyrogallol (6 mM) was immediately added, left at room temperature for 30min, and OD320nm was measured. Ultrapure water and Vc are respectively used as a blank control and a positive control, and the formulas are as follows:

wherein A is 1mL hydrolysate+5 mL Tris-HCl buffer+0.15 mL phloroglucinol solution; b is 1mL hydrolysate+5 mL Tris-HCl buffer+0.15mLHCl solution; c is a solution of 1mL water+5 mL Tris-HCl buffer+0.15 mL phloroglucinol.

The measurement result is shown in figure 21, the protein concentration of the hydrolysate is taken as an independent variable, the relative 10mg/mL Vc superoxide anion free radical clearance rate is taken as a dependent variable, and the curve is drawn as shown in the figure, so that the result shows that the superoxide anion free radical clearance activity of the hydrolysate is positively correlated with the protein concentration, and when the protein concentration is 0.5mg/mL, the superoxide anion clearance rate is equivalent to 74.2% of the 10mg/mL Vc clearance rate, which indicates that the Z-1 mussel hydrolysate has higher superoxide anion clearance rate.

1.5 determination of the reducing power of the hydrolysate

The freeze-dried powder of the Z-1 hydrolysate is diluted to different gradient concentrations according to the protein concentration, 0.20mL of the Z-1 hydrolysate, 0.20mL of 0.2M PBS solution (pH 6.6) and 0.20mL of 1% (w/v) K3Fe (CN) 6 are absorbed and mixed evenly, and incubated for 20min at 50 ℃. Then 0.20mL of 10% trichloroacetic acid was added and centrifuged at 5000r/min for 10min. Taking 0.5mL of supernatant, 2.5mL of ultrapure water and 0.1mL of FeCl ₃ (0.2%) were mixed well. OD700nm was measured after 10min at room temperature. Distilled water and VC were used as blank and positive controls, respectively, and repeated three times.

As shown in FIG. 22, the total reducing power increased with increasing protein concentration, and when the protein concentration reached 0.5mg/mL, the total reducing power corresponded to 27.5% of Vc at 10 mg/mL.

Bacillus belicus (Bacillus velezensis) Z-1 has the sequence as determined by 16S rRNA:

CCGATTCGGGTGCCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGTGACAGAAGG。

Claims

1. the bacillus beleiensis (Bacillus velezensis) Z-1 is characterized in that the bacillus beleiensis (Bacillus velezensis) Z-1 is stored in the China general microbiological culture collection center (CGMCC) with the storage number of 25059.

2. The bacillus beleiensis Z-1 and/or the fermentation enzyme liquid thereof according to claim 1 is used for carrying out enzymolysis on mussels to generate hydrolysate with antioxidant function.

3. The method for culturing bacillus belgium Z-1 according to claim 1, comprising the steps of: bacillus bailii Z-1 was inoculated into the primary seed culture medium for cultivation, and then inoculated from the primary seed culture medium to the secondary seed culture medium at 1%.

4. The culture method of claim 3, wherein the primary and secondary seed culture medium comprises 10g/L of bran, 5g/L of tapioca starch and 2% NaCl aged seawater, and has pH of 7.0.

5. The culture method according to claim 3, wherein the culture conditions in the seed medium are: culturing at 35 deg.C for 18 hr at 160-200 r/min.

6. The method for producing the enzyme by fermentation of bacillus bailii Z-1 according to claim 1, which comprises the steps of culturing a bacterial liquid to a secondary seed liquid, inoculating the secondary seed liquid into a fermentation medium for fermentation, and centrifuging to obtain a fermentation enzyme liquid.

7. The fermentation enzyme production method of claim 6, wherein the fermentation medium comprises 10g/L of maltose or tapioca starch, 10g/L of casein or peanut meal, and is prepared by using aged seawater with NaCl concentration of 0-2%, and the pH is 7-8; preferably, the components of the fermentation medium are 10g/L of maltose and 10g/L of peanut meal, and the fermentation medium is prepared by using aged seawater with NaCl concentration of 0 percent and has pH of 8.0.

8. The fermentation enzyme production method of claim 6, wherein 5-10% of the inoculum size of the bacterial liquid is inoculated into a fermentation culture medium, and the fermentation culture conditions are as follows: fermenting and culturing at 35-37 ℃ for 24-32h at 180 r/min: preferably, the fermentation medium is cultured at 37℃for 24 hours.

9. The method for hydrolyzing mussels by bacillus belicus Z-1 and/or its fermentation broth according to claim 1, comprising the steps of: a certain amount of mussel dry powder is weighed and mixed with the fermentation enzyme liquid according to the solid-to-liquid ratio of 1:20, and the mixture is subjected to enzymolysis for 24 hours in a shaking table at 37 ℃ and 180 r/min. Centrifuging the zymolyte to obtain supernatant, filtering the supernatant by suction to further remove suspended matters, and ultrafiltering the supernatant by using molecular filter membranes of 8 kkDa and 5 kkDa to obtain products with three stages of different molecular sizes. The fractions with a molecular weight of less than 5 kkDa were isolated and lyophilized and stored at-80 ℃.

10. A mussel hydrolysate obtained by hydrolyzing mussels using bacillus beljalis Z-1 and/or a fermentation broth thereof according to claim 1.