CN104430853A - Method for preparing fermented goat milk rich in pyrroloquinoline quinine and nattokinase by virtue of breeding and co-fermentation of bacillus natto and lactobacillus casei - Google Patents

Abstract

The invention discloses a method for preparing fermented goat milk rich in pyrroloquinoline quinine and nattokinase by virtue of breeding and co-fermentation of bacillus natto and lactobacillus casei. The method comprises the following steps: carrying out nitrosoguanidine mutagenesis respectively on bacillus natto 10261 and 10263 for producing nattokinase and pyrroloquinoline quinine, then mixing the two mutagenesis progenies, implementing cell fusion and subculture, and conducting high-throughput screening by further adopting a substrate-induced adaptive strategy on the basis of the genome shuffling technology so as to obtain bacillus natto for the high production of nattokinase and pyrroloquinoline quinine; carrying out nitrosoguanidine mutagenesis on lactobacillus casei and screening out methionine auxotrophic lactobacillus casei; and co-fermenting fresh goat milk by virtue of the screened bacillus natto and lactobacillus casei so as to obtain the fermented goat milk rich in nattokinase and pyrroloquinoline quinine. In the fermented goat milk rich in nattokinase and pyrroloquinoline quinine prepared by the method disclosed by invention, the content of the pyrroloquinoline quinine reaches 100-134ng/mL and the activity of the nattokinase reaches 1725-1900U/mL.

Description

Seed selection bafillus natto and Lactobacillus casei be the standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether

Technical field

The present invention relates to microbial project and fermentation arts, be specifically related to a kind of seed selection Lactobacillus casei and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase (NK) and PQQ (PQQ) of fermentation altogether.

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.

Sheep breast is the high nutritious milk product closest to human milk.Milk protein is casein and lactalbumin mainly, compared with cow's milk, feta protein content is low and protein content of whey is high, close with human milk, and casein can form larger coagulum under hydrochloric acid in gastric juice effect, more high protein digestion is lower, so milk protein digestibility is higher than cow's milk for its content; Goat' s milk fat content is 3.6%-4.5%, fat globule diameter 2 microns, and cow's milk fat bulb diameter is 3-4 micron, and goat' s milk fat bulb diameter is little makes it more easily digest and assimilate; Sheep breast is also rich in SCFA, and rudimentary volatile fatty acid accounts for about 25% of all content of fatty acid, then less than 10% in cow's milk; Sheep Ruzhong is also containing enriching minerals and vitamins, calcium, phosphorus, potassium, magnesium, chlorine and manganese equal size are all high than cow's milk, contained by every 100 grams of sheep breasts, 10 kinds of main vitamins total amounts are 780 micrograms, and wherein vitamin A, vitamin B1, vitamin B2, vitamin C, pantothenic acid and niacin equal size can fully meet baby's needs; Sheep Ruzhong cholesterol level is lower, and every 100 grams of sheep breast cholesterol level is 10-13 milligram, has the certain significance to reduction human artery sclerosis and hypertension.

Yogurt take sweet milk as raw material, through pasteurize again by the dairy products of cooling packing after lactobacillus-fermented.Lactic acid fermented process makes Ruzhong sugar, protein has about 20% to be hydrolyzed into as Small molecular (as galactolipin and lactic acid, peptide and amino acid etc.), the comparable sweet milk of aliphatic acid increases by 2 times, these change make yogurt more easy to digest and absorb, the utilization rate of various nutrient is improved, and during the fermentation except the whole nutritional labelings remaining sweet milk, also produce the necessary all Multivitamins of human nutrition.Sheep breast is high-quality sweet milk, 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 sheep 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, decompose the scarce capacity that milk protein obtains 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 the fresh sheep breast of certain proportion, then add certain proportion bean sprout juice when sheep milk fermentation.

Summary of the invention

The invention provides a kind of seed selection Lactobacillus casei and bafillus natto altogether fermentation prepare the standby method being rich in the fermentation sheep breast of Nattokinase (NK) and PQQ (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-101 of PQQ by high yield NK in sheep milk fermentation.Meanwhile, methionine auxotrophicity (Met is obtained by random mutagenesis ^-) Lactobacillus casei (Lactobacilluscasei Shirota-Met ^-, LcS-Met ^-).LcS-Met ^-higher than BN-NKPQQ-RWX-101 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 fermentation sheep breast being rich in NK and PQQ.

A kind of seed selection Lactobacillus casei and bafillus natto altogether fermentation prepare the standby method being rich in the fermentation sheep breast of Nattokinase (NK) and PQQ (PQQ), comprise 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-101;

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-101 and Lactobacillus casei LcS-Met ^-ferment fresh sheep breast altogether, obtains the fermentation sheep 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-101 of PQQ by high yield NK in sheep 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-101, the methionine (Met) required for it is supplied by bafillus natto, namely forms strict commensalism relation.Although LcS-Met ^-higher than BN-NKPQQ-RWX-101 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 fermentation sheep breast being rich in NK and PQQ.

Bafillus natto used and Lactobacillus casei all belong to grade-safe microorganism, to ferment altogether the fermentation sheep Ruzhong of being rich in NK and PQQ obtained, PQQ content reaches 100-134ng/mL, the natto (55-63ng/mL) the highest higher than content in wholefood and bean curd (25-30ng/mL) are suitable with (140-180ng/mL) in human milk.NK vigor reaches 1725-1900U/mL.The fermentation sheep breast obtained is except being rich in Nattokinase and PQQ, and in homogeneous milky, tissue is evenly fine and smooth, and slightly whey is separated out, and has the intrinsic flavour of yogurt and the distinctive local flavor of sheep breast.

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: (fresh sheep breast accounts for the 10%-15% of dull and stereotyped total amount 1. to prepare 2 dull and stereotyped bean sprout juice fresh sheep milk solids culture 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-101 of PQQ of the high yield NK obtained is preserved in inclined-plane bean sprout juice fresh sheep milk solids culture medium.

Described dull and stereotyped bean sprout juice fresh sheep milk solids culture medium and inclined-plane bean sprout juice fresh sheep milk solids culture medium, in 1L, are made up of the component of following amount:

Further preferably, described dull and stereotyped bean sprout juice fresh sheep milk solids culture medium and inclined-plane bean sprout juice fresh sheep milk solids culture 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 fresh sheep milk solids culture medium is conducive to bafillus natto bacterium colony and is formed and high flux screening.The formula of this inclined-plane bean sprout juice fresh sheep milk solids culture medium is conducive to bafillus natto preservation.

In the present invention, when adopting genome rearrangement technology seed selection bafillus natto BN-NKPQQ-RWX-101, a certain proportion of fermentation medium component (fresh sheep breast) 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:

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

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-101 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 fermentation sheep breast being rich in NK and PQQ.

Step 3) in, before common fermentation, by BN-NKPQQ-RWX-101 and LcS-Met ^-carry out activation culture respectively, BN-NKPQQ-RWX-101 carries out activation culture in bean sprout juice fresh sheep breast 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 fresh sheep breast fluid nutrient medium, in 1L, is made up of the component of following amount:

Fresh sheep breast 100 ~ 150mL;

Sucrose 4.0 ~ 6.0g;

Bean sprout juice surplus.

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

Fresh sheep breast 100 ~ 150mL;

Sucrose 5.0g;

Bean sprout juice surplus.

The formula of this bean sprout juice fresh sheep breast 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:

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

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

By BN-NKPQQ-RWX-101 and LcS-Met ^-ferment fresh sheep breast altogether, specifically comprises: by BN-NKPQQ-RWX-101 and LcS-Met after activation ^-mixing, forms mixed bacteria liquid, mixed bacteria liquid is seeded to the fermentation medium containing fresh sheep breast, and gauze sealing obtains being rich in the fermentation sheep breast of NK and PQQ after cultivating.

The described fermentation medium containing fresh sheep breast, in 1L, is made up of the component of following amount:

In mixed bacteria liquid, described BN-NKPQQ-RWX-101 and LcS-Met ^-hybrid density than for 1:2 ~ 1:5.

The inoculum concentration that described mixed bacteria liquid is seeded to containing the fermentation medium of fresh sheep breast 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 sheep milk fermentation culture medium (namely containing the fermentation medium of fresh sheep breast), interpolation tyrosine and glutamic acid carry out metabolic regulation to BN-NKPQQ-RWX-101, promote PQQ synthesis, add maltose and metabolic regulation is carried out to BN-NKPQQ-RWX-101, promote that NK generates, add bean sprout juice and promote the propagation of BN-NKPQQ-RWX-101 in sheep milk fermentation process.In the present invention, bafillus natto and Lactobacillus casei seed selection and the fermentation sheep breast that fermentation is standby altogether thereof, be rich in Nattokinase and PQQ, in homogeneous milky, tissue is evenly fine and smooth, and has the intrinsic flavour of yogurt and the distinctive local flavor of sheep breast.

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-101 of PQQ by high yield NK in sheep 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-101 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 fermentation sheep 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-101 in containing the fermentation medium of fresh sheep breast, promotes PQQ synthesis.By adding maltose in the fermentation medium containing fresh sheep breast, metabolic regulation being carried out to BN-NKPQQ-RWX-101, promoting that NK generates.By adding the propagation of bean sprout juice promotion BN-NKPQQ-RWX-101 in sheep milk fermentation process in containing the fermentation medium of fresh sheep breast, thus obtain the fermentation sheep breast being rich in NK and PQQ, PQQ content reaches 100-134ng/mL, the natto (55-63ng/mL) the highest far above content in wholefood and bean curd (25-30ng/mL) are suitable with (140-180ng/mL) in human milk.NK vigor reaches 1725-1900U/mL.The fermentation sheep breast obtained is except being rich in Nattokinase and PQQ, and in homogeneous milky, tissue is evenly fine and smooth, and has the intrinsic flavour of yogurt and the distinctive local flavor of sheep breast.

Detailed description of the invention

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

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

Fresh sheep breast 130mL;

Sucrose 5.0g;

Bean sprout juice surplus;

The formula of this bean sprout juice fresh sheep breast 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.

B the fresh sheep milk solids of the bean sprout juice described in () culture medium (flat board) 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 fresh sheep milk solids culture medium (flat board) is conducive to bafillus natto bacterium colony and is formed and high flux screening.

C the fresh sheep milk solids of the bean sprout juice described in () culture medium (inclined-plane) 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 fresh sheep milk solids culture 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:

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

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 fermentation medium containing fresh sheep breast described in (), in 1L, is made up of the component of following amount:

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 fermentation sheep of 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-101 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-101 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: (fresh sheep breast accounts for the 10-15% of dull and stereotyped total amount 1. to prepare 2 bean sprout juice fresh sheep milk solids culture medium flat plates, 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-101 of PQQ, is preserved in bean sprout juice fresh sheep milk solids culture 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-101

The BN-NKPQQ-RWX-101 be stored in bean sprout juice fresh sheep milk solids culture medium (inclined-plane) is got a ring, and be seeded to the activation of bean sprout juice fresh sheep breast fluid nutrient medium, cultivation temperature 35 DEG C, cultivates end inoculum density and reaches 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 fermentation sheep breast of NK and PQQ is standby

By BN-NKPQQ-RWX-101 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 the fermentation medium of 1% inoculum concentration inoculation containing fresh sheep breast, and 24h is cultivated in 42 DEG C of six layers of gauze sealings, obtains the fermentation sheep breast being rich in NK and PQQ.In fermentation sheep breast culture medium (namely containing the fermentation medium of fresh sheep breast), contained bean sprout juice, tyrosine, glutamic acid and maltose carry out metabolic regulation to BN-NKPQQ-RWX-101 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, fermentation sheep Ruzhong NK vigor 1725U/mL, PQQ content 100ng/mL.The fermentation sheep breast obtained is except being rich in Nattokinase and PQQ, and in homogeneous milky, tissue is evenly fine and smooth, and has the intrinsic flavour of yogurt and the distinctive local flavor of sheep breast.

Embodiment 2

The present invention is by bafillus natto and Lactobacillus casei seed selection and fermentation is newborn for the fermentation sheep of 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-101 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-101 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.

Mutation library high flux screening: (fresh sheep breast accounts for the 10-15% of dull and stereotyped total amount 1. to prepare 2 bean sprout juice fresh sheep milk solids culture medium flat plates, 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-101 of PQQ, is preserved in bean sprout juice fresh sheep milk solids culture 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 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-101

The BN-NKPQQ-RWX-101 be stored in bean sprout juice fresh sheep milk solids culture medium (inclined-plane) is got a ring, and be seeded to the activation of bean sprout juice fresh sheep breast fluid nutrient medium, cultivation temperature 35 DEG C, cultivates end inoculum density and reaches 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 sheep breast of NK and PQQ is standby

By BN-NKPQQ-RWX-101 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 the fermentation medium of 5% inoculum concentration inoculation containing fresh sheep breast, and 36h is cultivated in 37 DEG C of six layers of gauze sealings, obtains the fermentation sheep breast being rich in NK and PQQ.In fermentation sheep breast culture medium, contained bean sprout juice, tyrosine, glutamic acid and maltose carry out metabolic regulation to BN-NKPQQ-RWX-101 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, fermentation sheep Ruzhong NK vigor 1900U/mL, PQQ content 134ng/mL.The fermentation sheep breast obtained is except being rich in Nattokinase and PQQ, and in homogeneous milky, tissue is evenly fine and smooth, and has the intrinsic flavour of yogurt and the distinctive local flavor of sheep breast.

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 (10)

1. seed selection Lactobacillus casei and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, is characterized in that, comprise 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-101;

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-101 and Lactobacillus casei LcS-Met ^-ferment fresh sheep breast altogether, obtains the fermentation sheep breast being rich in Nattokinase and PQQ.

2. seed selection Lactobacillus casei according to claim 1 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, it is characterized in that, step 1) in, described high flux screening specifically comprises: 1. prepare 2 dull and stereotyped bean sprout juice fresh sheep milk solids culture 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. seed selection Lactobacillus casei according to claim 2 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, it is characterized in that, step 1) in, described dull and stereotyped bean sprout juice fresh sheep milk solids culture medium, in 1L, is made up of the component of following amount:

4. seed selection Lactobacillus casei according to claim 1 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, 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. seed selection Lactobacillus casei according to claim 4 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, 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. seed selection Lactobacillus casei according to claim 1 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, it is characterized in that, step 3) in, before common fermentation, by bafillus natto BN-NKPQQ-RWX-101 and Lactobacillus casei LcS-Met ^-carry out activation culture respectively, bafillus natto BN-NKPQQ-RWX-101 carries out activation culture in bean sprout juice fresh sheep breast 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. seed selection Lactobacillus casei according to claim 6 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, it is characterized in that, step 3) in, described bean sprout juice fresh sheep breast fluid nutrient medium, in 1L, is made up of the component of following amount:

Fresh sheep breast 100 ~ 150mL;

Sucrose 4.0 ~ 6.0g;

Bean sprout juice surplus;

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

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

8. seed selection Lactobacillus casei according to claim 1 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, it is characterized in that, step 3) in, by bafillus natto BN-NKPQQ-RWX-101 and Lactobacillus casei LcS-Met ^-ferment fresh sheep breast altogether, specifically comprises: by the bafillus natto BN-NKPQQ-RWX-101 after activation and Lactobacillus casei LcS-Met ^-mixing, forms mixed bacteria liquid, mixed bacteria liquid is seeded to the fermentation medium containing fresh sheep breast, and gauze sealing obtains being rich in the fermentation sheep breast of Nattokinase and PQQ after cultivating.

9. seed selection Lactobacillus casei according to claim 8 and the bafillus natto standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, it is characterized in that, step 3) in, the described fermentation medium containing fresh sheep breast, in 1L, is made up of the component of following amount:

10. seed selection bafillus natto according to claim 8 and the Lactobacillus casei standby method being rich in the fermentation sheep breast of Nattokinase and PQQ of fermentation altogether, it is characterized in that, step 3) in, the bafillus natto BN-NKPQQ-RWX-101 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 fermentation medium of fresh sheep breast 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.