CN104522181A

CN104522181A - Method for preparing pea milk through symbiotic culture of bacillus natto and lactobacillus casei

Info

Publication number: CN104522181A
Application number: CN201410751349.9A
Authority: CN
Inventors: 阮子琦; 莫凡; 张哲滔; 吴渊; 邓惟
Original assignee: HANGZHOU YUANPEITE BIOTECHNOLOGY Co Ltd
Current assignee: HANGZHOU YUANPEITE BIOTECHNOLOGY Co Ltd
Priority date: 2014-12-10
Filing date: 2014-12-10
Publication date: 2015-04-22

Abstract

The invention discloses a method for preparing pea milk through symbiotic culture of bacillus natto and lactobacillus casei. The method comprises the following steps: performing nitrosoguanidine mutagenesis on bacillus natto 10261 and 10263 for producing nattokinase and pyrroloquinoline quinine respectively, mixing mutagenesis descendants of bacillus natto 10261 and 10263, performing cell fusion subculture, and performing high-flux screening, thereby obtaining bacillus natto with high productivity of nattokinase and pyrroloquinoline quinine; performing nitrosoguanidine mutagenesis on lactobacillus casei, and screening methionine auxotrophic lactobacillus casei; co-fermenting pea milk by using the screened bacillus natto and the lactobacillus casei, thereby obtaining the pea milk which is rich in bacillus natto and lactobacillus casei. The content of pyrroloquinoline quinine in the pea milk which is rich in bacillus natto and lactobacillus casei is up to 75-125ng/mL, and the activity of nattokinase is up to 1012-1260U/mL.

Description

The method preparing pea breast is cultivated in bafillus natto and Lactobacillus casei symbiosis

Technical field

The present invention relates to microbial project and fermentation arts, be specifically related to a kind of bafillus natto and the method preparing pea breast is cultivated in Lactobacillus casei symbiosis.

Background technology

Natto is formed through bafillus natto (Bacillus natto) fermentation by soybean, has multiple physiological effect:

1. natto is to the effect of cardiovascular system: a kind of significant physiological activator Nattokinase (Nattokinase in natto, NK) be can thrombolytic protein, be made up of 275 amino acid residues, molecular weight about 27KD, nontoxic, have no side effect, be a kind of serine protease, its thrombolytic effect even exceedes Thrombolytic agent urokinase.Nattokinase, except having direct thrombolytic effect, also has and promotes that venous endothelial cell produces the ability of PLA, thus indirectly show its thrombolysis activity.Natto Linoleic acid accounts for 50% of full-cream matter, and linoleic acid can reduce cholesterol level in blood plasma, has prevention of arterial sclerosis, heart disease and hypertensive effect, has thrombolytic effect;

2. natto is to the effect of digestive system: soybean protein changes into soluble polypeptide and amino acid through the effect of Nadou enzyme, is conducive to absorption of human body.Containing more than 1,000,000,000 Bacillus nattos in 1g natto, Bacillus natto breeding is fast, can consume the oxygen in enteron aisle, can suppress to cause the spoilage organisms such as the shigella dysenteriae of abnormal fermentation in intestines, promote lactic acid bacteria breeding, regulate intestinal bacterium balance, prevention dysentery, enteritis and constipation etc.Soybean have accumulated the enzyme groups such as a large amount of protease, amylase, lipase, esterase, cellulase and urase through fermenting bacillus natto, and these biology enzymes can effectively be nursed one's health promotion and digest and assimilate in digestive system.Bafillus natto is utilizing soybean protein to carry out in the process of breeding by decomposing, and can produce the stickums such as a large amount of ferment, vitamin, polyglutamic acid and polyfructosan, it is overlayed on gastrointestinal mucosal surfaces and plays a protective role, and can alleviate drunk when drinking.Bacillus natto can also kill intestinal bleeding Escherichia coli;

3. natto is to immune effect: found by the zoopery inoculating Bacillus natto, natto can improve Abwehrkraft des Koepers, gets rid of the staphylococcus invaded, and can also bring out body interferon and generate, strengthen immunity of organisms; Active material is contained as saponin, Cobastab in natto ₂, vitamin E etc., every day is edible can remove carcinogen in body, and pre-anti-cancer occurs, and can softening blood vessel, promotes that skin is smooth;

4. skeleton development is promoted: vitamin K ₂calcium and protein bound can be promoted thus promote that bone is formed, playing an important role in skeleton-forming process, and vitamin K in natto ₂content is hundreds of times of other fermented foods, has effect of the disease of old people such as prevention of osteoporosis, pain in the back.

In recent years find that natto also has other abundanter physiological function widely, come from its another kind of significant physiological activator---PQQ (pyrroloquinoline quinone, PQQ), it is the third prothetic group found in bacterial dehydrogenase after flavin nucleotide and nicotinamide nucleotide, and chemical name is 4,5-dihydro-4,5-titanium dioxide-1-hydrogen pyrroles (2,3f) quinone-2,7,9-tricarboxylic acids, has another name called Methaxatin.Although only minority bacterium such as Methylovorus, Deinococcus radiodurans, Gluconobacter oxydans, Klebsiella pneumoniae etc. can synthesize PQQ under natural conditions, all find containing trace P QQ in each Plants, animal and human body.Japanese Scientists determines the PQQ content in 26 kinds of common food, to find in 1 gram of food PQQ content at 3.65-61 nanogram not etc., such as, parsley in vegetables, green pepper, Chinese grooseberry in fruit, pawpaw, the green tea in drink, oolong tea, and the bean curd that people often eat.It should be noted that in the middle of japanese traditional food natto, PQQ content is the highest, reaches 61 ng/g.

PQQ is as a kind of oxidoreducing enzyme prothetic group be prevalent in animal and plant tissue, although content is very low, but act on important, be not only a kind of newfound mitochondrial respiratory chain component, participate in catalysis biological vivo oxidation reduction reaction, also there is very various biologically active, the many dysbolisms of mammal during shortage, can be caused, be considered to a kind of novel vitamin.PQQ has mammiferous major physiological effect:

1. improve immune function of human body: PQQ, as the necessary factor of human developmental, can stimulate human body cell to grow, especially human activin B cell, T cell comprehensively, improve immune function of human body;

2. prevent and treat hepatic injury: PQQ can significantly reduce serum bilirubin and gpt level, conditioning hepatic injury, has fabulous curative effect to liver diseases;

3. the injury of radical pair human body is reduced: PQQ is a kind of oxidoreducing enzyme prothetic group, participate in organism internal oxidition reduction reaction, effectively can remove interior free yl, reduce the various diseases that radical pair human injury causes, as heart disease, cancer and all kinds of inflammation;

4. various the nervous system disease is nursed one's health: PQQ can promote that neural factor synthesizes, and nurses one's health various the nervous system disease in vivo;

5. promote Amino Acid Absorption: PQQ is the prothetic group of quinoprotein enzyme, participate in respiratory chain electron transmission, can Amino Acid Absorption be promoted in human body;

6. growth promoting effects factor synthesis: growth factor is the hormone of human growth, and PQQ can stimulate human body cell to grow, increase cell density, trace P QQ just can improve metabolic capability and the GF of bio-tissue;

7. senile dementia prevention and cure: PQQ has and repairs nerve fibre, activation neuron, activates the ability of dormancy nerve cell, can effectively senile dementia prevention and cure and Improving memory power;

8. promote glutathione synthesis: glutathione has important antioxidation and integrates detoxication, and PQQ can promote body glutathione synthesis, prevent cataract from occurring and the accumulation of liver bilirubin;

9. it is ANK cell that extremely strong anti-cancer function: PQQ activates NK (NK) cell, makes it have and kills function of tumor; The immunocytes such as NK cell are assembled, more effectively killing off tumor cells; Closed tumour cell carries Human Placental Ferritin Receptor, blocks its function, Tumor suppression growth, transfer; Destroy tumour cell double-layer of lipoid, make oncolysis dead; DNA of tumor cell is stoped to copy, its apoptosis short.

In wholefood, the exclusive NK of natto, and PQQ content is the abundantest.NK and PQQ in natto is produced by bafillus natto.Natto is routine health food, bafillus natto is generally regarded as safe food-grade microorganisms (GRAS microorganism), directive breeding is carried out to bafillus natto, then ferment altogether with lactic acid bacteria, the food of NK and PQQ is rich in preparation, supplements NK and PQQ, to give full play to the physiology opsonic action of NK and PQQ significant for people in diet.

Pea (Pisum sativum L.) is nutritious, every 100g pea contains: 7.4 grams, protein, 0.3 gram, fat, 21.2 grams, carbohydrate, folic acid 82.6 microgram, dietary fiber 3 grams, 3-Hydroxyretinol 7 microgram, carrotene 220 microgram, thiamine 0.43 milligram, 0.09 milligram, riboflavin, 2.3 milligrams, nicotinic acid, vitamin C 14 milligrams, vitamin e1 .21 milligram, calcium 21 milligrams, 127 milligrams, phosphorus, 332 milligrams, potassium, 1.2 milligrams, sodium, iodine 0.9 microgram, 43 milligrams, magnesium, iron 1.7 milligrams, 1.29 milligrams, zinc, selenium 1.74 microgram, copper 0.22 milligram, 0.65 milligram, manganese.Pea taste is sweet, property is put down, return spleen, stomach warp, there is gas in benefit, antidiarrheal dysentery, adjust that battalion defends, effect of diuresis, subduing inflammation, solution mammary calculus poison, beriberi, carbuncle are swollen, alactation, taste discomfort, hiccup vomiting, trusted subordinate's distending pain, the thirsty illnesss such as dysentery of letting out have certain dietotherapy effect.Pea breast is with the drink that processes of pea high temperature grinding after soaking, and remains pea nutritional labeling, after lactobacillus-fermented, will form many dietotherapy materials such as lactein, surfactant fatty acid, organic acid based on lactic acid further.

Lactobacillus-fermented decapacitation produces outside many dietotherapy compositions, can also give pleasant sweet sour mouthfeel.Bacillus natto to ferment then can produce NK and PQQ.The present invention, by seed selection bafillus natto and Lactobacillus casei and common fermentation thereof, develops the dietotherapy fermentation pea breast being rich in NK and PQQ.If but Lactobacillus casei and bafillus natto grow imbalance in same system that a kind of bacterium can be caused in competition to repel another kind, fermentation cannot be carried out smoothly altogether.About physiological property, even if under wild state, lactic acid bacteria protein in cell wall enzyme PrtS also only has minority bacterial strain to have and vigor is not high, decomposing protein obtains the scarce capacity of essential amino acid, and bafillus natto has stronger prolease activity, its decomposing protein produces amino acid and peptide can meet lactic acid bacteria to amino acid whose demand.Bafillus natto amphimicrobian, also promotes lactobacter growth by consuming oxygen and produces acid.In the present invention, except bafillus natto and Lactobacillus casei exist not strict commensalism relation between the two, more make Lactobacillus casei because forming methionine auxotrophicity (Met by seed selection means ^-) and form strict commensalism relation with bafillus natto, thus ensure that the common fermentation of Lactobacillus casei and bafillus natto is carried out smoothly.

The metabolic pathway of bafillus natto synthesis NK and PQQ is not yet completely clear so far, thus cannot be improved the productive rate of bafillus natto synthesis NK and PQQ by rationality optimization means.And due to sugar, amino acid, lipid, communicating and close association between the large organic metabolism of nucleotides four, microbial cell metabolism network is made not only to demonstrate rigidity (Rigidity) but also demonstrate flexibility (Plasticity), be in strictly Guaranteed, rigidity shows as pulls one hair and move the whole body, thus in most cases, even if understand metabolic pathway, the sudden change of fixed point rationality also can not obtain desired phenotypes, and flexibility shows as being in different node in whole metabolism network but after relevant approach carries out collaborative sudden change, the metabolism attribute made new advances can be showed.For improving the productive rate of bafillus natto synthesis NK and PQQ, do not understand that its metabolic pathway does not become impassable obstacle, as long as there is suitable irrational optimization means (Unrationaloptimization technique), to likely synthesize relevant main flow with NK and PQQ and collateral branch's approach is planned as a whole to optimize, realize the collaborative sudden change of multidigit point, and make multidigit point work in coordination with the phenotype of sudden change from mutation library saliency by high flux screening, the target improving bafillus natto synthesis NK and PQQ productive rate can be realized.

Random mutation is modal irrational optimization means, although it can produce the enough large mutation library of mutational site quantity, but in mutation library, each mutational site disperses independent of in individual cells often, each other cannot optimum organization, and scarcely produce visible phenotypic, thus most sudden change is disappeared can not be screened in mutation library.

Genome rearrangement (Genome Shuffling) is the novel mutation breeding technologies grown up on the basis of random mutation.It is being produced on the basis of the enough large mutation library of mutational site quantity by random mutation, is making the optimum organization of each mutational site further by Fusion of Cells, thus make the rising of mutation index level particularly increase a large amount of phenotype visible mutation.Genome rearrangement is that the strain improvement utilizing this flexibility of microbial metabolism network to carry out multi-point cooperative sudden change provides technical support.Zhang etc. (2002) first obtain a mutation library with classic mutagenesis method, filter out several forward mutants as strain of setting out, then by the mode of many wheel recursion protoplast fusions, numerous gene is recombinated at random, realize genome rearrangement, from mutation library, finally filter out proterties by the object bacterial strain promoted; Stephanopoulos (2002) has transformed Bacterial phenotype by genome rearrangement; Patnaik etc. (2002) utilize genome product technology to achieve the improvement of a strain industrial strain of Bacillus acidi lactici, make it be 3 times of wild strain of setting out at pH 4.0 times lactic acid productions; Gao etc. (2012), by two-wheeled genome rearrangement, make Clostridium acetobutylicumCICC 8012 produce Acetone-Butanol-Ethanol (ABE) and improve 34.53%; Ge etc. (2012) obtain Saccharomyces cerevisiae GS3-10 by genome rearrangement, it reaches 69.48% and 100% to the conversion ratio of pentose and hexose, and starting strain is 14.83% and 100%, alcohol yied reaches 47.08g/L, and starting strain is 18.12g/L; Kang etc. (2011), by genome rearrangement, make Aureobasidium pullulans produce the blue polysaccharide in general Shandong and increase substantially, and inheritance stability; Zheng etc. (2010) improve D-ALPHA-Hydroxypropionic acid productive rate and the acid resistance of Sporolactobacillus inulinus ATCC 15538 by genome rearrangement; John etc. (2008) by the genome rearrangement of protoplast fusion formula, the Lactic Acid High-yield Strains that to obtain with cassava-bagasse be matrix; Yu etc. (2008) obtain the resistance to degree of sugar by genome rearrangement and improve thus the Lactobacillus rhamnosus of lactic acid yield increase; Wang etc. (2007) obtain acid resistance by genome rearrangement and improve thus the Lactobacillus rhamnosus of lactic acid yield increase.

Genome rearrangement also provides support for carrying out microbial metabolism group Epidemiological Analysis.Through genome rearrangement, its genetic background of object bacterium of metabolism optimization derives from the bacterium that sets out, therefore by the protein expression profiles difference of comparison object bacterium with the bacterium that sets out, be aided with metabolic fluxes and metabolic control analysis technology, the key enzyme that causes metabolism to be optimized and the regulatory mechanism about approach can be resolved.Luo etc. (2012) make natamycin (natamycin) productive rate of Streptomyces gilvosporeus ATCC 13326 obviously raise by genome rearrangement, the protein expression profiles of Streptomyces gilvosporeus ATCC 13326 occurs obviously to change, 34 kinds of protein expressions rise and 20 kinds of protein expressions declines, and prompting is synthesized the relevant metabolism network widely that involves with natamycin and is optimized; Zhang etc. (2010), by genome rearrangement, make Propionibacteriumshermanii produce V _b12increase substantially, after resetting, bacterial strain 38 kinds of protein expressions change, and wherein 22 kinds of protein expressions rise and 16 kinds of protein expressions declines, expressing in the 22 kinds of albumen risen, have 6 kinds to be V _b12enzyme in route of synthesis, prompting and V _b12synthesize the relevant metabolism network widely that involves to be optimized.

But at present there is certain defect, mainly onrelevant between seed selection culture medium and fermentation medium in genome rearrangement on strategy, the bacterial strain that seed selection is arrived when fermenting target product productive rate far below expection.For this problem, the present invention is when carrying out genome rearrangement seed selection, a certain proportion of fermentation medium component is added in seed selection flat board, and in fermentation medium, add the dull and stereotyped component of a certain proportion of seed selection, make to be formed between seed selection with fermentation two links to associate, ensured that the bacterial strain that seed selection link shows high yield phenotype still shows high yield phenotype when fermenting, this is substrate for induction adaptive strategy of the present invention.Therefore, when PQQ phenotype of holding concurrently to bafillus natto high yield NK carries out genome rearrangement seed selection, in seed selection flat board, add certain proportion pea juice, then add certain proportion bean sprout juice when pea milk fermentation.

Summary of the invention

The invention provides a kind of bafillus natto and the method preparing pea breast is cultivated in Lactobacillus casei symbiosis, the present invention adopts substrate for induction adaptive strategy further in genome rearrangement technical foundation, to bafillus natto likely synthesize relevant main flow with NK and PQQ and collateral branch's approach is planned as a whole to optimize, realize the collaborative sudden change of multidigit point, and then make multidigit point work in coordination with the phenotype of sudden change from mutation library saliency by high flux screening, capacitation enough in pea milk fermentation high yield NK to hold concurrently the bafillus natto BN-NKPQQ-RWX-208 of PQQ.Meanwhile, methionine auxotrophicity (Met is obtained by random mutagenesis ^-) Lactobacillus casei (Lactobacilluscasei Shirota-Met ^-, LcS-Met ^-).LcS-Met ^-higher than BN-NKPQQ-RWX-208 in the speed of growth, but the former relies on the latter's nutrition, is formed between the two and stablizes symbiosis, ensured that common fermentation completes smoothly, thus obtains the pea breast being rich in NK and PQQ.

The method preparing pea breast is cultivated in bafillus natto and Lactobacillus casei symbiosis, comprises the following steps:

1), nitrosoguanidine mutagenesis is carried out respectively by producing the hold concurrently bafillus natto 10261 of PQQ and bafillus natto 10263 of NK; Then mutagenic progeny both mixing, carry out Fusion of Cells Secondary Culture, genome rearrangement technical foundation further adopts substrate for induction adaptive strategy, and high flux screening obtains high yield NK and to hold concurrently the bafillus natto of PQQ, called after bafillus natto BN-NKPQQ-RWX-208;

Fusion of Cells Secondary Culture realizes genome segment S9, and the predominant mutation of all mutagenic progeny of bafillus natto 10261 and 10623 is optimized integration.If necessary, second can be carried out take turns or take turns more mutagenesis and fusion fusion offspring, until it is high and grow vigorous, inheritance stability, be suitable as the high yield NK producing bacterial strain and hold concurrently PQQ bafillus natto to select NK PQQ productive rate of holding concurrently.

2), by Lactobacillus casei (Lactobacillus casei Shirota) carry out nitrosoguanidine mutagenesis, therefrom filter out the Lactobacillus casei of methionine auxotroph, called after Lactobacillus casei LcS-Met ^-(Lactobacillus casei Shirota-Met ^-);

3) by bafillus natto BN-NKPQQ-RWX-208 and Lactobacillus casei LcS-Met ^-ferment pea juice altogether, obtains the pea breast being rich in NK and PQQ.

The present invention adopts substrate for induction adaptive strategy further in genome rearrangement technical foundation, to bafillus natto likely synthesize relevant main flow with NK and PQQ and collateral branch's approach is planned as a whole to optimize, realize the collaborative sudden change of multidigit point, and then make multidigit point work in coordination with the phenotype of sudden change from mutation library saliency by high flux screening, obtain and can to hold concurrently the bafillus natto BN-NKPQQ-RWX-208 of PQQ by high yield NK in pea milk fermentation.Meanwhile, methionine auxotrophicity (Met is obtained by random mutagenesis ^-) Lactobacillus casei LcS-Met ^-.

Lactobacillus casei and bafillus natto are fermented smoothly altogether, should symbiosis be formed between the two.Strategy of the present invention makes to compete the Lactobacillus casei be dominant under normal circumstances to become methionine auxotrophicity (LcS-Met ^-), thus in metabolism, dependence being formed to bafillus natto BN-NKPQQ-RWX-208, the methionine (Met) required for it is supplied by bafillus natto, namely forms strict commensalism relation.Although LcS-Met ^-higher than BN-NKPQQ-RWX-208 in the speed of growth, but the former relies on the latter's nutrition, is formed and stablizes symbiosis, ensured that common fermentation completes smoothly between two bacterium, thus obtains the pea breast being rich in NK and PQQ.

Bafillus natto used and Lactobacillus casei all belong to grade-safe microorganism, to ferment altogether the pea Ruzhong of being rich in NK and PQQ obtained, PQQ content reaches 75-125ng/mL, the natto (55-63ng/mL) the highest higher than content in wholefood and bean curd (25-30ng/mL).NK vigor reaches 1012-1260U/mL.The pea breast obtained is rich in Nattokinase and PQQ, has the pea intrinsic flavour of breast and the rear distinctive local flavor of fermentation.Step 1) in, bafillus natto 10261 and bafillus natto 10263 adopt prior art, bafillus natto 10261 from Chinese industrial Microbiological Culture Collection administrative center (CICC), address: No. 24, Road, Jiuxianqiao, Chaoyang District, Beijing City institute No. 6 building; Preservation date is 2002, and bacterial classification keeps being numbered 10261; Bafillus natto 10263 from Chinese industrial Microbiological Culture Collection administrative center (CICC), address: No. 24, Road, Jiuxianqiao, Chaoyang District, Beijing City institute No. 6 building; Preservation date is 2002, and bacterial classification keeps being numbered 10263; Lactobacillus casei (Lactobacillus casei Shirota) is from chlorella yakult (Shanghai) Co., Ltd.; Little mouse-anti PQQ IgG monoclonal antibody PQQ-mAb adopts commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-Ab-013.

High flux screening after described substrate for induction adaptibility gene group rearrangement specifically comprises: (pea juice accounts for the 10%-15% of dull and stereotyped total amount 1. to prepare 2 dull and stereotyped bean sprout juice pea juice solid mediums, seed selection culture medium is associated with between fermentation medium, this is substrate for induction adaptive strategy of the present invention), little mouse-anti PQQ IgG monoclonal antibody PQQ-mAb allocates into by one of them, form dull and stereotyped A, agarose-fibrin is allocated into by another, forms dull and stereotyped B; 2. the mutagenesis of bafillus natto 10261 and bafillus natto 10263 merged after offspring is coated with one of them flat board, the photocopy of reprocess fibre element film is dull and stereotyped to another, make dull and stereotyped A and dull and stereotyped B photocopy each other dull and stereotyped; 3. in dull and stereotyped A, high yield PQQ mutagenesis is merged offspring's periphery of bacterial colonies and is formed some visual precipitation line, judges that producing PQQ measures size according to the large I naked eyes of precipitation loop diameter; 4. in dull and stereotyped B, high yield NK mutagenesis is merged offspring's periphery of bacterial colonies and is formed transparent circle, can judge to produce NK vigor height by naked eyes according to transparent circle diameter; 5., in dull and stereotyped A and dull and stereotyped B, high yield PQQ bacterium colony and high yield NK bacterium colony are in same position person, is high yield NK and holds concurrently the bafillus natto of PQQ.

The hold concurrently bafillus natto BN-NKPQQ-RWX-208 of PQQ of the high yield NK obtained is preserved in inclined-plane bean sprout juice pea juice solid medium.

Described dull and stereotyped bean sprout juice pea juice solid medium and inclined-plane bean sprout juice pea juice solid medium, in 1L, are made up of the component of following amount:

Further preferably, described dull and stereotyped bean sprout juice pea juice solid medium and inclined-plane bean sprout juice pea juice solid medium, in 1L, are made up of the component of following amount:

The preparation of described bean sprout juice: the soybean 500g just sprouted, add water 2500mL, boils after being concentrated into 1000mL and filter, obtain bean sprout juice.

The formula of this dull and stereotyped bean sprout juice pea juice solid medium is conducive to bafillus natto bacterium colony and is formed and high flux screening.The formula of this inclined-plane bean sprout juice pea juice solid medium is conducive to bafillus natto preservation.

In the present invention, when adopting genome rearrangement technology seed selection bafillus natto BN-NKPQQ-RWX-208, a certain proportion of fermentation medium component (pea juice) is added in seed selection flat board, and in fermentation medium, add the dull and stereotyped component (bean sprout juice) of a certain proportion of seed selection, make to be formed between seed selection with fermentation two links to associate, the bacterial strain impelling seed selection link to show high yield phenotype still shows high yield phenotype when fermenting, and this is substrate for induction adaptive strategy of the present invention.

Step 2) in, the described Lactobacillus casei therefrom filtering out methionine auxotroph, specifically comprise: 1. prepare 2 dull and stereotyped MRS solid mediums, methionine (Met) is allocated into (Met addition is 10-15mg/L) by one of them, for dull and stereotyped C, another is simple MRS solid medium flat board (dull and stereotyped D), is dull and stereotyped D; 2. after the Lactobacillus casei (Lactobacillus caseiShirota) after mutagenesis being coated with one of them flat board, the photocopy of reprocess fibre element film is dull and stereotyped to another, makes dull and stereotyped C and dull and stereotyped D photocopy each other flat board; 3. at the same position of dull and stereotyped C and dull and stereotyped D, there is bacterium colony in dull and stereotyped C and bacterium colony does not appear in dull and stereotyped D, or the bacterium colony on dull and stereotyped C is significantly greater than the bacterium colony on dull and stereotyped D, then this bacterium colony is methionine auxotroph Lactobacillus casei, called after Lactobacillus casei LcS-Met ^-, be preserved in inclined-plane MRS solid medium.

Described dull and stereotyped MRS solid medium and inclined-plane MRS solid medium, in 1L, are made up of the component of following amount:

This MRS solid medium is conducive to the screening of the formation of Lactobacillus casei bacterium colony and methionine auxotroph Lactobacillus casei.

In the present invention, random mutagenesis techniques is adopted to obtain methionine auxotrophicity (Met ^-) Lactobacillus casei (Lactobacillus casei Shirota-Met ^-, LcS-Met ^-).LcS-Met ^-higher than BN-NKPQQ-RWX-208 in the speed of growth, but the former relies on the latter's nutrition, formed between the two and stablize symbiosis, realized the common fermentation of bafillus natto and Lactobacillus casei by the strategy building symbiosis, obtain the pea breast being rich in NK and PQQ.

Step 3) in, before common fermentation, by BN-NKPQQ-RWX-208 and LcS-Met ^-carry out activation culture respectively, BN-NKPQQ-RWX-208 carries out activation culture in bean sprout juice pea juice fluid nutrient medium, and cultivation temperature is 34 ~ 36 DEG C (preferably 35 DEG C), LcS-Met ^-in MRS fluid nutrient medium, carry out activation culture, cultivation temperature is 36 ~ 38 DEG C (preferably 37 DEG C).

Described bean sprout juice pea juice fluid nutrient medium, in 1L, is made up of the component of following amount:

Pea juice 100 ~ 150mL;

Sucrose 4.0 ~ 6.0g;

Bean sprout juice surplus.

Described bean sprout juice pea juice fluid nutrient medium, in 1L, preferably, is made up of the component of following amount:

Pea juice 100 ~ 150mL;

Sucrose 5.0g;

Bean sprout juice surplus.

The formula of this bean sprout juice pea juice fluid nutrient medium is conducive to bafillus natto Multiplying culture.Prepared by bean sprout juice: the soybean 500g just sprouted, add water 2500mL, boils after being concentrated into 1000mL and filter, obtain bean sprout juice.

Described MRS fluid nutrient medium, in 1L, is made up of the component of following amount:

This MRS fluid nutrient medium is conducive to Lactobacillus casei Multiplying culture.

By BN-NKPQQ-RWX-208 and LcS-Met ^-fermentation is containing the pea milk fermentation culture medium of pea juice altogether, specifically comprises: by BN-NKPQQ-RWX-208 and LcS-Met after activation ^-mixing, forms mixed bacteria liquid, mixed bacteria liquid is seeded to the pea milk fermentation culture medium containing pea juice, and gauze sealing obtains being rich in the pea breast of NK and PQQ after cultivating.

Described pea milk fermentation culture medium, in 1L, is made up of the component of following amount:

The preparation of described pea juice comprises: with pea in water, soaks 2 ~ 12 hours, and 90 DEG C ~ 99 DEG C are milled into juice, obtain pea juice, and wherein, the volume of described water and the mass ratio of pea are 100mL:10 ~ 25g.Particularly, as added 10 ~ 25g pea in 100mL water, soak after more than 2 hours, more than 90 DEG C are milled into juice.

In mixed bacteria liquid, described BN-NKPQQ-RWX-208 and LcS-Met ^-hybrid density than for 1:2 ~ 1:5.

The inoculum concentration that described mixed bacteria liquid is seeded to containing the pea milk fermentation culture medium of pea juice is 1% ~ 5%.

The condition that described gauze sealing is cultivated is: 24h ~ 36h is cultivated in 37 DEG C ~ 42 DEG C six layers of gauze sealings.

When fermenting altogether, in pea breast culture medium, interpolation tyrosine and glutamic acid carry out metabolic regulation to BN-NKPQQ-RWX-208, promote PQQ synthesis, add maltose and metabolic regulation is carried out to BN-NKPQQ-RWX-208, promote that NK generates, add bean sprout juice and promote the propagation of BN-NKPQQ-RWX-208 in pea milk fermentation process.In the present invention, the pea breast obtained is rich in Nattokinase and PQQ, has the pea intrinsic flavour of breast and the rear distinctive local flavor of fermentation.

Compared with prior art, tool of the present invention has the following advantages:

One, the present invention use bafillus natto and Lactobacillus casei all belong to grade-safe microorganism.

Two, genome rearrangement technical optimization NK and PQQ route of synthesis is adopted first, to bafillus natto likely synthesize relevant main flow with NK and PQQ and collateral branch's approach is planned as a whole to optimize, realize the collaborative sudden change of multidigit point, and then make multidigit point work in coordination with the phenotype of sudden change from mutation library saliency by high flux screening.Simultaneously, genome rearrangement technical foundation adopts substrate for induction adaptive strategy further, ensureing that the bacterial strain that seed selection link shows high yield phenotype still shows high yield phenotype when fermenting, having obtained and can to hold concurrently the bafillus natto BN-NKPQQ-RWX-208 of PQQ by high yield NK in pea milk fermentation.

Three, the strategy building symbiosis is adopted to realize the common fermentation of bafillus natto and Lactobacillus casei first.Methionine auxotrophicity (Met is obtained by random mutagenesis ^-) Lactobacillus casei LcS-Met ^-.LcS-Met ^-higher than BN-NKPQQ-RWX-208 in the speed of growth, but the former relies on the latter's nutrition, is formed between the two and stablizes symbiosis, ensured that common fermentation completes smoothly, thus obtains the pea breast being rich in NK and PQQ.

Four, from structure, PQQ forms by forming peptide bond connection cyclisation between tyrosine and glutamic acid.The present invention, by adding tyrosine and glutamic acid carries out metabolic regulation to BN-NKPQQ-RWX-208 in pea milk fermentation culture medium, promotes PQQ synthesis.By adding maltose in pea milk fermentation culture medium, metabolic regulation being carried out to BN-NKPQQ-RWX-208, promoting that NK generates.The propagation of BN-NKPQQ-RWX-208 in pea milk fermentation process is promoted by adding bean sprout juice in pea breast culture medium, thus obtain the pea breast being rich in NK and PQQ, PQQ content reaches 75-125ng/mL, the natto (55-63ng/mL) the highest higher than content in wholefood and bean curd (25-30ng/mL).NK vigor reaches 1012-1260U/mL.The pea breast obtained is rich in Nattokinase and PQQ, has the pea intrinsic flavour of breast and the rear distinctive local flavor of fermentation.

Detailed description of the invention

In an embodiment, relevant culture medium is formulated as follows,

A the bean sprout juice pea juice fluid nutrient medium described in () has been prepared after, 121 DEG C of sterilizing 15min, in 1L, have been made up of the component of following amount:

Pea juice 130mL;

Sucrose 5.0g;

Bean sprout juice surplus;

B bean sprout juice pea juice solid medium (flat board) described in () has been prepared after, 121 DEG C of sterilizing 15min, in 1L, are made up of the component of following amount:

The formula of this bean sprout juice pea juice solid medium (flat board) is conducive to bafillus natto bacterium colony and is formed and high flux screening.

C bean sprout juice pea juice solid medium (inclined-plane) described in () has been prepared after, 121 DEG C of sterilizing 15min, in 1L, are made up of the component of following amount:

The formula of this bean sprout juice pea juice solid medium (inclined-plane) is conducive to the preservation of bafillus natto.

D the MRS fluid nutrient medium described in () has been prepared after, 121 DEG C of sterilizing 15min, in 1L, are made up of the component of following amount:

E the MRS solid medium (flat board) described in () has been prepared after, 121 DEG C of sterilizing 15min, in 1L, are made up of the component of following amount:

This MRS solid medium is conducive to Lactobacillus casei bacterium colony and is formed and screening.

F the MRS solid medium (inclined-plane) described in () has been prepared after, 121 DEG C of sterilizing 15min, in 1L, are made up of the component of following amount:

This MRS solid medium (inclined-plane) is conducive to Lactobacillus casei preservation.

G the pea milk fermentation culture medium described in (), in 1L, is made up of the component of following amount:

H the preparation of () pea juice comprises: add 20g pea in 1000mL water, and soak 7 hours, 95 DEG C are milled into juice, obtain pea juice.

In the embodiment of the present invention, bafillus natto 10261 and bafillus natto 10263 adopt prior art, bafillus natto 10261 from Chinese industrial Microbiological Culture Collection administrative center (CICC), address: No. 24, Road, Jiuxianqiao, Chaoyang District, Beijing City institute No. 6 building; Preservation date is 2002, and bacterial classification keeps being numbered 10261; Bafillus natto 10263 from Chinese industrial Microbiological Culture Collection administrative center (CICC), address: No. 24, Road, Jiuxianqiao, Chaoyang District, Beijing City institute No. 6 building; Preservation date is 2002, and bacterial classification keeps being numbered 10263; Lactobacillus casei (Lactobacillus casei Shirota) is from chlorella yakult (Shanghai) Co., Ltd.; Little mouse-anti PQQ IgG monoclonal antibody PQQ-mAb adopts commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-Ab-013.

Embodiment 1

The present invention is by bafillus natto and Lactobacillus casei seed selection and fermentation is newborn for the pea being rich in NK and PQQ altogether, comprises the following steps:

(1) high yield NK holds concurrently the seed selection of bafillus natto BN-NKPQQ-RWX-208 of PQQ

Adopt substrate for induction adaptive strategy, obtain high yield NK by genome rearrangement and High Throughput Screening Assay to hold concurrently the bafillus natto BN-NKPQQ-RWX-208 of PQQ, concrete grammar is: first build bafillus natto mutation library, and the bafillus natto 10261 and 10263 of the PQQ that held concurrently by acquired product NK carries out nitrosoguanidine mutagenesis respectively; Then mutagenic progeny both mixing, carry out Fusion of Cells (fusogen is Macrogol 4000), Secondary Culture, realize genome segment S9, the predominant mutation of all mutagenic progeny of bafillus natto 10261 and 10623 is optimized integration, selects NK PQQ productive rate of holding concurrently high and grow vigorous, inheritance stability, be suitable as the high yield NK producing bacterial strain and hold concurrently the bafillus natto of PQQ.

Mutation library high flux screening: (pea juice accounts for the 10-15% of dull and stereotyped total amount 1. to prepare 2 bean sprout juice pea juice solid medium flat boards, seed selection culture medium is associated with between fermentation medium, this is substrate for induction adaptive strategy of the present invention), little mouse-anti PQQ IgG monoclonal antibody PQQ-mAb allocates into by one of them, form dull and stereotyped A, agarose-fibrin is allocated into by another, forms dull and stereotyped B; 2. the mutagenesis of bafillus natto 10261 and bafillus natto 10263 merged after offspring is coated with one of them flat board, the photocopy of reprocess fibre element film is dull and stereotyped to another, make dull and stereotyped A and dull and stereotyped B photocopy each other dull and stereotyped; 3. in dull and stereotyped A, high yield PQQ mutagenesis is merged offspring's periphery of bacterial colonies and is formed some visual precipitation line, judges that producing PQQ measures size according to the large I naked eyes of precipitation loop diameter; 4. in dull and stereotyped B, high yield NK mutagenesis is merged offspring's periphery of bacterial colonies and is formed transparent circle, can judge to produce NK vigor height by naked eyes according to transparent circle diameter; 5., in dull and stereotyped A and dull and stereotyped B, high yield PQQ bacterium colony and high yield NK bacterium colony are in same position person, is high yield NK and holds concurrently the bafillus natto of PQQ.The high yield NK obtained holds concurrently the bafillus natto called after BN-NKPQQ-RWX-208 of PQQ, is preserved in bean sprout juice pea juice solid medium (inclined-plane).

(2) methionine auxotrophicity (Met ^-) Lactobacillus casei (LcS-Met ^-) seed selection

Lactobacillus casei (Lactobacillus casei Shirota) is carried out nitrosoguanidine mutagenesis, therefrom filters out methionine auxotroph (LcS-Met ^-).

LcS-Met ^-screening: 1. prepare 2 MRS solid mediums dull and stereotyped, methionine (Met) is allocated into (dull and stereotyped C, Met addition is 10-15mg/L) by one of them, and another is simple MRS flat board (dull and stereotyped D); 2. after the mutagenic progeny of Lactobacillus casei (Lactobacillus casei Shirota) being coated with one of them flat board, the photocopy of reprocess fibre element film is dull and stereotyped to another, makes dull and stereotyped C and dull and stereotyped D photocopy each other flat board; 3. at the same position of dull and stereotyped C and dull and stereotyped D, there is bacterium colony in dull and stereotyped C and bacterium colony does not appear in dull and stereotyped D, or the bacterium colony on dull and stereotyped C is significantly greater than the bacterium colony on dull and stereotyped D, then this bacterium colony methionine auxotroph Lactobacillus casei, called after LcS-Met ^-, be preserved in MRS solid medium (inclined-plane).

(3) activation culture of BN-NKPQQ-RWX-208

The BN-NKPQQ-RWX-208 be stored in bean sprout juice pea juice solid medium (inclined-plane) is got a ring, is seeded to the activation of bean sprout juice pea juice fluid nutrient medium, cultivation temperature 35 DEG C, cultivate end inoculum density and reach 10 ⁹about cfu/mL.

(4) LcS-Met ^-activation culture

The LcS-Met in MRS solid medium (inclined-plane) will be stored in ^-get a ring, be seeded to MRS fluid nutrient medium 37 DEG C of activation culture, cultivate end inoculum density and reach 10 ⁹about cfu/mL.

(5) fermentation being rich in the pea breast of NK and PQQ is standby

By BN-NKPQQ-RWX-208 and the LcS-Met of activation ^-mixing, makes the two density ratio in mixed bacteria liquid be 1:2 (1 × 10 ⁸cFM/mL:2 × 10 ⁸cFM/mL), mixed bacteria liquid is with 1% inoculum concentration inoculation pea milk fermentation culture medium, and 24h is cultivated in 42 DEG C of six layers of gauze sealings, obtains the pea breast being rich in NK and PQQ.In pea milk fermentation culture medium, contained bean sprout juice, tyrosine, glutamic acid and maltose carry out metabolic regulation to BN-NKPQQ-RWX-208 synthesis NK and PQQ, bean sprout juice is conducive to bafillus natto propagation, maltose is conducive to NK synthesis, and PQQ is connected cyclisation with glutamic acid by peptide bond by tyrosine and forms.After fermentation ends, pea Ruzhong NK vigor 1012U/mL, PQQ content 75ng/mL.The pea breast obtained is rich in Nattokinase and PQQ, has the pea intrinsic flavour of breast and the rear distinctive local flavor of fermentation.

Embodiment 2

Adopt substrate for induction adaptive strategy, obtain high yield NK by genome rearrangement and High Throughput Screening Assay to hold concurrently the bafillus natto BN-NKPQQ-RWX-208 of PQQ, concrete grammar is: first build bafillus natto mutation library, and the bafillus natto 10261 and 10263 of the PQQ that held concurrently by acquired product NK carries out nitrosoguanidine mutagenesis respectively; Then mutagenic progeny both mixing, carry out cytoplasmic fusion (fusogen is Macrogol 4000), Secondary Culture, realize genome segment S9, the predominant mutation of all mutagenic progeny of bafillus natto 10261 and 10623 is optimized integration, selects NK PQQ productive rate of holding concurrently high and grow vigorous, inheritance stability, be suitable as the high yield NK producing bacterial strain and hold concurrently the bafillus natto of PQQ.

LcS-Met ^-screening: 1. prepare 2 MRS solid mediums dull and stereotyped, methionine (Met) is allocated into (dull and stereotyped C, Met addition is 10-15mg/L) by one of them, and another is simple MRS solid medium flat board (dull and stereotyped D); 2. after the mutagenic progeny of Lactobacillus casei (Lactobacillus casei Shirota) being coated with one of them flat board, the photocopy of reprocess fibre element film is dull and stereotyped to another, makes dull and stereotyped C and dull and stereotyped D photocopy each other flat board; 3. at the same position of dull and stereotyped C and dull and stereotyped D, there is bacterium colony in dull and stereotyped C and bacterium colony does not appear in dull and stereotyped D, or the bacterium colony on dull and stereotyped C is significantly greater than the bacterium colony on dull and stereotyped D, then this bacterium colony methionine auxotroph Lactobacillus casei, called after LcS-Met ^-, be preserved in MRS solid medium (inclined-plane).

(3) activation culture of BN-NKPQQ-RWX-208

(4) LcS-Met ^-activation culture

(5) fermentation being rich in the pea breast of NK and PQQ is standby

By BN-NKPQQ-RWX-208 and the LcS-Met of activation ^-mixing, makes the two density ratio in mixed bacteria liquid be 1:5 (1 × 10 ⁸cFM/mL:5 × 10 ⁸cFM/mL), mixed bacteria liquid is with 5% inoculum concentration inoculation pea milk fermentation culture medium, and 36h is cultivated in 37 DEG C of six layers of gauze sealings, obtains the pea breast being rich in NK and PQQ.In pea milk fermentation culture medium, contained bean sprout juice, tyrosine, glutamic acid and maltose carry out metabolic regulation to BN-NKPQQ-RWX-208 synthesis NK and PQQ, bean sprout juice is conducive to bafillus natto propagation, maltose is conducive to NK synthesis, and PQQ is connected cyclisation with glutamic acid by peptide bond by tyrosine and forms.After fermentation ends, pea Ruzhong NK vigor 1260U/mL, PQQ content 125ng/mL.The pea breast obtained is rich in Nattokinase and PQQ, has the pea intrinsic flavour of breast and the rear distinctive local flavor of fermentation.

Chromatography determination PQQ is adopted in the present invention, concrete steps are with reference to " Noji N; Nakamura T; Kitahata N; et al.Simple and sensitive method for pyrroloquinoline quinone (PQQ) analysis in various foods using liquid chromatography/electrospray-lonization tandem mass spectrometry.J Agri Food Chem; 2007,55 (18): 7258-7263. ".

Agarose-fibrinogen plate assay is adopted to measure NK vigor in the present invention.Determination step: by dibbling after filtering fermentation liquor on agarose-fibrin plate, hatches 18h for 37 DEG C, using urokinase as standard items, is equivalent to the unit of activity (U/mL) of urokinase by measuring transparent circle diameter calculating Nattokinase.

Claims

1. the method preparing pea breast is cultivated in bafillus natto and Lactobacillus casei symbiosis, it is characterized in that, comprises the following steps:

1), nitrosoguanidine mutagenesis is carried out respectively by producing the hold concurrently bafillus natto 10261 of PQQ and bafillus natto 10263 of Nattokinase; Then mutagenic progeny both mixing, carries out Fusion of Cells Secondary Culture, and high flux screening obtains high-yield nattokinase and to hold concurrently the bafillus natto of PQQ, called after bafillus natto BN-NKPQQ-RWX-208;

2), by Lactobacillus casei carry out nitrosoguanidine mutagenesis, therefrom filter out the Lactobacillus casei of methionine auxotroph, called after Lactobacillus casei LcS-Met ^-;

3) by bafillus natto BN-NKPQQ-RWX-208 and Lactobacillus casei LcS-Met ^-ferment pea juice altogether, obtains the pea breast being rich in Nattokinase and PQQ.

2. the method preparing pea breast is cultivated in bafillus natto according to claim 1 and Lactobacillus casei symbiosis, it is characterized in that, step 1) in, described high flux screening specifically comprises: 1. prepare 2 dull and stereotyped bean sprout juice pea juice solid mediums, little mouse-anti PQQ IgG monoclonal antibody PQQ-mAb allocates into by one of them, form dull and stereotyped A, agarose-fibrin is allocated into by another, forms dull and stereotyped B; 2. the mutagenesis of bafillus natto 10261 and bafillus natto 10263 merged after offspring is coated with one of them flat board, the photocopy of reprocess fibre element film is dull and stereotyped to another, make dull and stereotyped A and dull and stereotyped B photocopy each other dull and stereotyped; 3. in dull and stereotyped A, the mutagenesis of high yield PQQ is merged offspring's periphery of bacterial colonies and is formed some visual precipitation circle, judges to produce PQQ amount size according to the large I naked eyes of precipitation loop diameter; 4. in dull and stereotyped B, the mutagenesis of high-yield nattokinase is merged offspring's periphery of bacterial colonies and is formed transparent circle, can judge to produce Nattokinase vigor height by naked eyes according to transparent circle diameter; 5., in dull and stereotyped A and dull and stereotyped B, high yield PQQ bacterium colony and high-yield nattokinase bacterium colony are in same position person, is high-yield nattokinase and holds concurrently the bafillus natto of PQQ.

3. the method preparing pea breast is cultivated in bafillus natto according to claim 2 and Lactobacillus casei symbiosis, it is characterized in that, step 1) in, described dull and stereotyped bean sprout juice pea juice solid medium, in 1L, is made up of the component of following amount:

4. the method preparing pea breast is cultivated in bafillus natto according to claim 1 and Lactobacillus casei symbiosis, it is characterized in that, step 2) in, the described Lactobacillus casei therefrom filtering out methionine auxotroph, specifically comprise: 1. prepare 2 dull and stereotyped MRS solid mediums, methionine is allocated into by one of them, is dull and stereotyped C, another is that simple MRS solid medium is dull and stereotyped, is dull and stereotyped D; 2. after the Lactobacillus casei after mutagenesis being coated with one of them flat board, the photocopy of reprocess fibre element film is dull and stereotyped to another, makes dull and stereotyped C and dull and stereotyped D photocopy each other flat board; 3. at the same position of dull and stereotyped C and dull and stereotyped D, there is bacterium colony in dull and stereotyped C and bacterium colony does not appear in dull and stereotyped D, or the bacterium colony on dull and stereotyped C is significantly greater than the bacterium colony on dull and stereotyped D, then this bacterium colony is methionine auxotroph Lactobacillus casei.

5. the method preparing pea breast is cultivated in bafillus natto according to claim 4 and Lactobacillus casei symbiosis, it is characterized in that, step 2) in, described dull and stereotyped MRS solid medium, in 1L, is made up of the component of following amount:

The pH value of this MRS solid medium remains on 6.2 ~ 6.4.

6. the method preparing pea breast is cultivated in bafillus natto according to claim 1 and Lactobacillus casei symbiosis, it is characterized in that, step 3) in, before common fermentation, by bafillus natto BN-NKPQQ-RWX-208 and Lactobacillus casei LcS-Met ^-carry out activation culture respectively, bafillus natto BN-NKPQQ-RWX-208 carries out activation culture in bean sprout juice pea juice fluid nutrient medium, and cultivation temperature is 34 ~ 36 DEG C, Lactobacillus casei LcS-Met ^-in MRS fluid nutrient medium, carry out activation culture, cultivation temperature is 36 ~ 38 DEG C.

7. the method preparing pea breast is cultivated in bafillus natto according to claim 6 and Lactobacillus casei symbiosis, it is characterized in that, step 3) in, described bean sprout juice pea juice fluid nutrient medium, in 1L, is made up of the component of following amount:

Pea juice 100 ~ 150mL;

Sucrose 4.0 ~ 6.0g;

Bean sprout juice surplus;

The pH value of this MRS fluid nutrient medium remains on 6.2 ~ 6.4.

8. the method preparing pea breast is cultivated in bafillus natto according to claim 1 and Lactobacillus casei symbiosis, it is characterized in that, step 3) in, by bafillus natto BN-NKPQQ-RWX-208 and Lactobacillus casei LcS-Met ^-ferment pea juice altogether, specifically comprises: by the bafillus natto BN-NKPQQ-RWX-208 after activation and Lactobacillus casei LcS-Met ^-mixing, forms mixed bacteria liquid, mixed bacteria liquid is seeded to the pea milk fermentation culture medium containing pea juice, and gauze sealing obtains being rich in the pea breast of Nattokinase and PQQ after cultivating;

9. the method preparing pea breast is cultivated in bafillus natto according to claim 8 and Lactobacillus casei symbiosis, it is characterized in that, step 3) in, the bafillus natto BN-NKPQQ-RWX-208 in described mixed bacteria liquid and Lactobacillus casei LcS-Met ^-bacterium number density than for 1:2 ~ 1:5;

The inoculum concentration that described mixed bacteria liquid is seeded to containing the pea milk fermentation culture medium of pea juice is 1% ~ 5%;

10. the method preparing pea breast is cultivated in bafillus natto according to claim 1 and Lactobacillus casei symbiosis, it is characterized in that, step 3) in, the preparation of described pea juice comprises: with pea in water, soak 2 ~ 12 hours, 90 DEG C ~ 99 DEG C are milled into juice, obtain pea juice, wherein, the volume of described water and the mass ratio of pea are 100mL:10 ~ 25g.