CN103740695A - Method for screening lactobacilli producing pyrroloquinoline quinone and preparation method of pyrroloquinoline quinone - Google Patents
Method for screening lactobacilli producing pyrroloquinoline quinone and preparation method of pyrroloquinoline quinone Download PDFInfo
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- MMXZSJMASHPLLR-UHFFFAOYSA-N pyrroloquinoline quinone Chemical compound C12=C(C(O)=O)C=C(C(O)=O)N=C2C(=O)C(=O)C2=C1NC(C(=O)O)=C2 MMXZSJMASHPLLR-UHFFFAOYSA-N 0.000 title claims abstract description 161
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- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 16
- 235000015097 nutrients Nutrition 0.000 claims description 16
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- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
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- UMYDVEVERVKIFT-UHFFFAOYSA-N 6-(2-amino-2-carboxyethyl)-7,8-dioxo-1,2,3,4,7,8-hexahydroquinoline-2,4-dicarboxylic acid Chemical compound O=C1C(=O)C(CC(N)C(O)=O)=CC2=C1NC(C(O)=O)CC2C(O)=O UMYDVEVERVKIFT-UHFFFAOYSA-N 0.000 description 2
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for screening lactobacilli producing pyrroloquinoline quinone through immunoprecipitation. The method comprises the steps: cultivating lactobacillus plantarum PQQ-1 in an MRS liquid culture medium for 16-30 hours, and then spreading the lactobacillus plantarum PQQ-1 to an MRS solid plating medium, irradiating with ultraviolet, washing out all floras by using a phosphate buffer, pouring into an activation culture medium for standing culture for 18-30h, and then suctioning the culture solution for mixing with mouse anti-PQQ IgG1 monoclonal antibodies, and spreading the mixture to an agar subculture medium for standing culture for 48h-72h, wherein the larger the radius of the precipitation circle around the floras is, the higher the PQQ yield is. The invention also discloses a preparation method of pyrroloquinoline quinone, wherein the lactobacillus plantarum PQQ-1 is induced by ultraviolet irradiation, and the PQQ is synthesized from the lactobacillus plantarum PQQ-6 obtained through screening through fermentation, and as a result, the yield of PQQ is higher.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to a kind ofly by immunoprecipitation, screen the method for high yield pyrroloquinoline quinone milk-acid bacteria and the preparation method of pyrroloquinoline quinone.
Background technology
Pyrroloquinoline quinone (pyrroloquinoline quinone, be called for short PQQ) be the third prothetic group of finding in bacterium desaturase after flavin nucleotide and nicotinamide nucleotide, its chemical name is 4,5-dihydro-4,5-titanium dioxide-1-hydrogen pyrroles (2,3f) quinone-2,7,9-tricarboxylic acid, has another name called Methaxatin.Under natural condition, only the minority bacterium such as Methylovorus, Deinococcus radiodurans, Gluconobacter oxydans, Klebsiella pneumoniae can be synthesized.
Although only minority bacterium can be synthesized PQQ, in each kind of plant, animal and human body, all find to contain trace P QQ.PQQ is not only a kind of newfound mitochondrial respiratory chain component, participates in catalysis biological vivo oxidation reduction reaction, also has very various biological activity, can cause the many metabolic disturbances of Mammals during shortage, is considered to a kind of novel VITAMIN.
Research to the typical bacterium Klebsiella pneumoniae pathways metabolism with synthetic PQQ ability shows, synthesizes the necessary gene of PQQ and has 6, called after pqqABCDEF, but concrete ways is unclear, only know pqqC catalysis final step synthesis step, by 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicar boxylic acid (AHQQ) is converted into PQQ.
Carried out in recent years the research of the synthetic PQQ of microorganism fermentation and obtained certain progress.(the Xiong XH such as Xiong, Zhao Y, Ge X, et al.Production and Radioprotective Effects of Pyrroloquinoline Quinone.Inter J Mol Sci, 2011,12 (12): 8913-8923.) with the synthetic PQQ of Methylovorus sp.MP688 fermentation, productive rate reaches 125mg/L; (the Yang XP such as Yang, Zhong GF, Lin JP, et al.Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.J Indust Microbiol Biotechnol, 2010,37 (6): 575-580.) the pqqABCDE gene cluster of Gluconobacter oxydans is transformed into E.coli, makes PQQ concentration in its fermented liquid reach 6mM.But regrettably,, the equal nonfood grade safe microorganisms of the bacterium with PQQ synthesis capability that find or recombination to construct at present, therefore, for realizing the large-scale production of PQQ, is badly in need of the food grade safe microorganisms that seed selection PQQ productive rate is high, production performance is good.Milk-acid bacteria is not only generally regarded as safe food-grade microorganisms, and kind is various, and genetic resources is abundant, and the milk-acid bacteria of breeding high-yield PQQ not only has abundant feasibility, and is the first-selected approach that realizes PQQ large-scale production.
Summary of the invention
The invention provides a kind of method of screening high yield pyrroloquinoline quinone (pyrroloquinoline quinone is called for short PQQ) milk-acid bacteria by immunoprecipitation.
By immunoprecipitation, screen a method of producing pyrroloquinoline quinone milk-acid bacteria, comprise the following steps:
Plant lactobacillus Lactobacillus plantarum PQQ-1 was cultivated after 16～30 hours in MRS liquid nutrient medium, drawing fermented liquid is applied on MRS solid plate substratum, with after uviolizing, with all bacterium colonies of phosphoric acid buffer wash-out, pour standing cultivation 18h～30h in activation medium (A) into, then drawing nutrient solution mixes with the anti-PQQ IgG1 of mouse monoclonal antibody, coating agar subculture medium (B), standing cultivation 48h～72h, the larger person of periphery of bacterial colonies precipitation circle radius is the higher product pyrroloquinoline quinone milk-acid bacteria of PQQ productive rate.
In the present invention, by plant lactobacillus Lactobacillus plantarum PQQ-1 is formed to mutation library through ultraviolet radiation mutagenesis, the activated substratum of mutation library (A) activation is coated with agar subculture medium (B) flat board then, PQQ that each bacterium colony produces and the anti-PQQ IgG1 of mouse monoclonal antibody generation immunoprecipitation, in periphery of bacterial colonies, form precipitation circle, the larger person of precipitation circle radius, the productive rate of the PQQ that produces is higher.
In the present invention, plant lactobacillus Lactobacillus plantarum PQQ-1 system separates and obtains from snow lotus live body (Tibet kefir, a kind of symbiosis beneficial flora being comprised of milk-acid bacteria, acetic bacteria, yeast etc.), belongs to food grade safe microorganisms.Concrete sepn process is: to snow lotus live body (Tibet kefir, a kind of symbiosis beneficial flora being formed by milk-acid bacteria, acetic bacteria, yeast etc.) add appropriate sterilized water, after snow lotus live body particle is pulverized in milling, dilution, coating is containing the MRS solid plate substratum of the anti-PQQ IgG1 of mouse monoclonal antibody, standing cultivation 48h～72h, periphery of bacterial colonies forms precipitation circle person, for producing pyrroloquinoline quinone (PQQ) bacterium, through identifying, the product PQQ bacterium obtaining is plant lactobacillus Lactobacillus plantarum, called after Lactobacillus plantarum PQQ-1.Plant lactobacillus Lactobacillus plantarum PQQ-1 is food grade safe microorganisms, can adopt commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-S-003.
The anti-PQQ IgG1 of mouse monoclonal antibody can adopt commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-Ab-013.
Further, described MRS liquid nutrient medium, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 ± 0.2 with sulfuric acid.
Further, described MRS solid plate substratum, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 ± 0.2 with sulfuric acid.
MRS substratum (being MRS liquid nutrient medium and MRS solid plate substratum) is milk-acid bacteria proliferated culture medium, and its liquid nutrient medium is used for breeding milk-acid bacteria, and its solid medium is accepted mutagenesis after being used for cultivating milk-acid bacteria.
Further, the condition of described uviolizing is: after keeping 10cm～30cm apart from irradiation 10-12min with 254nm ultraviolet ray.Uviolizing is for the mutagenesis of plant lactobacillus Lactobacillus plantarum PQQ-1, the phenotype of producing high yield PQQ with seed selection.
Further, described activation medium (A), in 1L, comprises the component of following weight:
The formula of this activation medium is conducive to the activation culture of plant lactobacillus Lactobacillus plantarum PQQ-1.
Further, this activation medium regulates pH to 6.8 ± 0.2 with acid-base buffer, makes this activation medium have suitable pH, is more suitable in the activation culture of plant lactobacillus Lactobacillus plantarum PQQ-1.
Further, described agar subculture medium (B), in 1L, comprises the component of following weight:
The formula of this agar subculture medium is conducive to the succeeding transfer culture of plant lactobacillus Lactobacillus plantarum PQQ-1.
Further, this agar subculture medium regulates pH to 6.8 ± 0.2 with acid-base buffer, makes this agar subculture medium have suitable pH, is more suitable in the succeeding transfer culture of plant lactobacillus Lactobacillus plantarum PQQ-1.
The present invention also provides a kind of preparation method of pyrroloquinoline quinone, and Lactobacillus plantarum PQQ-1 is through ultraviolet radiation mutagenesis, and PQQ is synthesized in the fermentation of the Lactobacillus plantarum PQQ-6 obtaining after screening, and PQQ productive rate is very high.
A preparation method for pyrroloquinoline quinone, comprises the following steps:
(1) plant lactobacillus Lactobacillus plantarum PQQ-1 was cultivated after 16～30 hours in MRS liquid nutrient medium, drawing fermented liquid is applied on MRS solid plate substratum, with after uviolizing, with all bacterium colonies of phosphoric acid buffer wash-out, pour standing cultivation 18h～30h in activation medium (A) into, then drawing nutrient solution mixes with the anti-PQQ IgG1 of mouse monoclonal antibody, coating agar subculture medium (B), standing cultivation 48h～72h, the larger person of periphery of bacterial colonies precipitation circle radius is the higher product pyrroloquinoline quinone milk-acid bacteria of PQQ productive rate;
(2) the product pyrroloquinoline quinone milk-acid bacteria of periphery of bacterial colonies precipitation circle radius maximum in step (1), called after plant lactobacillus Lactobacillus plantarum PQQ-6, after activated substratum (A) activation, 20 ℃～40 ℃ standing cultivation 16h～30h become first order seed, first order seed continues 20 ℃～40 ℃ standing cultivation 16h～30h of expansion through first order seed substratum (C1) and becomes secondary seed, secondary seed enters fermentation state after secondary seed medium (C2) continues 20 ℃～40 ℃ standing cultivation 42h～54h of expansion, in production fermention medium (D), 20 ℃～40 ℃ anaerobism are cultivated 54h～66h, obtain pyrroloquinoline quinone (PQQ).
In step (2), further, described activation medium (A), in 1L, comprises the component of following weight:
The formula of this activation medium is conducive to the activation culture of plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, this activation medium regulates pH to 6.8 ± 0.2 with acid-base buffer, makes this activation medium have suitable pH, is more suitable in the activation culture of plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, 30 ℃ of standing cultivation 24h become first order seed.
Described first order seed substratum (C1), in 1L, comprises the component of following weight:
The formula of this first order seed substratum is conducive to the cultivation of plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, this first order seed substratum regulates pH to 6.8 ± 0.2 with acid-base buffer, makes this first order seed substratum have suitable pH, is more suitable in the cultivation of plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, first order seed continues 50 times of standing cultivation 24h of 30 ℃ of expansion through first order seed substratum (C1) and becomes secondary seed.
Further, described secondary seed medium (C2), in 1L, comprises the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus.
The formula of this secondary seed medium is conducive to the enlarged culturing of plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, this secondary seed medium regulates pH to 6.8 ± 0.2 with acid-base buffer, makes this secondary seed medium have suitable pH, is more suitable in the enlarged culturing of plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, secondary seed enters fermentation state after secondary seed medium (C2) continues 50 times of standing cultivation 48h of 30 ℃ of expansion.
Further, described production fermention medium, in 1L, comprises the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus.
The formula of this production fermention medium is conducive to the production fermentation of plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, this production fermention medium (D) regulates pH to 6.8 ± 0.2 with acid-base buffer, makes this production fermention medium have suitable pH, is more suitable for the production fermentation in plant lactobacillus Lactobacillus plantarum PQQ-6.
Further, producing 50 times of anaerobism cultivation 60h of 30 ℃ of expansion in fermention medium (D), obtain PQQ.
Compared with prior art, tool of the present invention has the following advantages:
One, the present invention selects plant lactobacillus Lactobacillus plantarum PQQ-1 as the bacterium that sets out, system is from snow lotus live body (Tibet kefir, a kind of symbiosis beneficial flora being formed by milk-acid bacteria, acetic bacteria, yeast etc.) in separate obtain, belong to food grade safe microorganisms.
Two, Lactobacillus plantarum PQQ-1 forms mutation library through ultraviolet radiation mutagenesis, the activated substratum of mutation library (A) activation is coated with agar subculture medium (B) flat board then, there is immunoprecipitation in the anti-PQQ IgG1 of PQQ that each bacterium colony produces and mouse, form macroscopic precipitation circle, the larger person of precipitation circle radius illustrates that PQQ productive rate is higher, by visual inspection, screens high yield PQQ plant lactobacillus Lactobacillus plantarum PQQ-6.
Three, the synthetic PQQ of plant lactobacillus Lactobacillus plantarum PQQ-6 fermentation that screening obtains, PQQ productive rate is very high, can be up to arriving 448ng/mL, the human milk (140-180ng/mL) the highest far above occurring in nature content, natto (55-63ng/mL) and bean curd (25-30ng/mL), also be significantly higher than the Methylovorus sp.MP688(Xiong XH of current discovery and structure, Zhao Y, Ge X, et al.Production and Radioprotective Effects of Pyrroloquinoline Quinone.Inter J Mol Sci, 2011, 12 (12): 8913-8923.) and introduced the E.coli(Yang XP of pqqABCDE gene cluster, Zhong GF, Lin JP, et al.Pyrroloquinoline quinone biosynthesis in Escherichia coli through expression of the Gluconobacter oxydans pqqABCDE gene cluster.J Indust Microbiol Biotechnol, 2010, 37 (6): 575-580.).
Embodiment
Embodiment 1
(1) by the component of following content, prepare each substratum respectively, wherein, yeast extract, yeast soak powder, peptone and beef extract powder and all adopt Sigma product, and barley meal adopts the prosperous product of Dafeng City, Jiangsu Province gold wheat edge wheat benevolence; Other reagent adopts analytical pure, after having prepared, all at 121 ℃ of sterilizing 15min;
(a): activation medium (A), in 1L, comprises the component of following weight:
And this activation medium is regulated to pH to 6.8 with acid-base buffer.
(b): agar subculture medium (B), in 1L, comprises the component of following weight:
And this agar subculture medium is regulated to pH to 6.8 with acid-base buffer.
(c): first order seed substratum (C1), in 1L, comprises the component of following weight:
And this first order seed substratum is regulated to pH to 6.8 with acid-base buffer.
(d): secondary seed medium (C2), in 1L, comprises the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus;
And this secondary seed medium is regulated to pH to 6.8 with acid-base buffer.
(e): produce fermention medium (D) in 1L, comprise the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus;
And this production fermention medium regulates pH to 6.8 with acid-base buffer.
(f): MRS liquid nutrient medium, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 with sulfuric acid.
(g): MRS solid plate substratum, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 with sulfuric acid.
(2) Lactobacillus plantarum PQQ-1 adopts commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-S-003.The anti-PQQ IgG1 of mouse monoclonal antibody adopts commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-Ab-013.
(3) plant lactobacillus Lactobacillus plantarum PQQ-1 is cultivated after 24h in MRS liquid nutrient medium, drawing 0.2mL fermented liquid is applied on MRS solid plate substratum, after keeping 20cm apart from irradiation 11min with 254nm ultraviolet ray, with all bacterium colonies of 10mL pH7.0 phosphoric acid buffer wash-out, pour 30 ℃ of standing cultivation 24h in 1000mL activation medium (A) into, then drawing 0.2mL nutrient solution mixes with the anti-PQQ IgG1 of 1mL mouse monoclonal antibody, coating agar subculture medium (B), 30 ℃ of standing cultivation 60h, periphery of bacterial colonies precipitation circle radius the maximum called after plant lactobacillus Lactobacillus plantarum PQQ-6.
(4) bacterial strain activation: 10 milliliters of activation mediums (A) are poured in 30 milliliters of test tubes, inoculate a ring Lactobacillus plantarum PQQ-6 in test tube, and 30 ℃ of standing cultivation 24h become first order seed;
(5) first order seed is cultivated: get in 10mL first order seed access 500mL first order seed substratum (C1), 30 ℃ of standing cultivation 24h become secondary seed;
(6) secondary seed is cultivated: get in 100mL secondary seed access 5000mL secondary seed medium (C2), after 30 ℃ of standing cultivation 48h, enter fermentation state;
(7) PQQ fermentation: get the secondary seed access 50L that 1000mL enters fermentation state and produce in fermention medium (D), 30 ℃ of standing anaerobism are cultivated 60h, obtain PQQ.
Adopt chromatography determination PQQ, produce PQQ concentration in fermention medium (D) and reach 448ng/mL.This chromatography determination PQQ method 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. ".
Embodiment 2
(1) by the component of following content, prepare each substratum respectively, wherein, yeast extract, yeast soak powder, peptone and beef extract powder and all adopt Sigma product, and barley meal adopts the prosperous product of Dafeng City, Jiangsu Province gold wheat edge wheat benevolence; Other reagent adopts analytical pure, after having prepared, all at 121 ℃ of sterilizing 15min;
(a): activation medium (A), in 1L, comprises the component of following weight:
And this activation medium is regulated to pH to 6.8 with acid-base buffer.
(b): agar subculture medium (B), in 1L, comprises the component of following weight:
And this agar subculture medium is regulated to pH to 6.8 with acid-base buffer.
(c): first order seed substratum (C1), in 1L, comprises the component of following weight:
And this first order seed substratum is regulated to pH to 6.8 with acid-base buffer.
(d): secondary seed medium (C2), in 1L, comprises the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus;
And this secondary seed medium is regulated to pH to 6.8 with acid-base buffer.
(e): produce fermention medium (D) in 1L, comprise the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus;
And this production fermention medium regulates pH to 6.8 with acid-base buffer.
(f): MRS liquid nutrient medium, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 with sulfuric acid.
(g): MRS solid plate substratum, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 with sulfuric acid.
(2) Lactobacillus plantarum PQQ-1 adopts commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-S-003.The anti-PQQ IgG1 of mouse monoclonal antibody adopts commercially available prod, and purchased from Yuan Ke bio tech ltd, Jiaxing, article number is YK-Ab-013.
(3) bacterial strain activation: 10 milliliters of activation mediums (A) are poured in 30 milliliters of test tubes, inoculate the freezing test tube strains plant lactobacillus of ring Lactobacillus plantarum PQQ-1 in test tube, and 30 ℃ of standing cultivation 24h become first order seed;
(4) first order seed is cultivated: get in 10mL first order seed access 500mL first order seed substratum (C1), 30 ℃ of standing cultivation 24h become secondary seed;
(5) secondary seed is cultivated: get in 100mL secondary seed access 5000mL secondary seed medium (C2), after 30 ℃ of standing cultivation 48h, enter fermentation state;
(6) PQQ fermentation: get the secondary seed access 50L that 1000mL enters fermentation state and produce in fermention medium (D), 30 ℃ of standing anaerobism are cultivated 60h, obtain PQQ.
Adopt chromatography determination PQQ, produce PQQ concentration in fermention medium (D) and reach 238ng/mL, method is with embodiment 1.
The result of comparing embodiment 1 and embodiment 2, the PQQ productive rate (448ng/mL) of the high yield PQQ milk-acid bacteria Lactobacillus plantarum PQQ-6 obtaining by immunoprecipitation high flux screening has improved 88.24% than the PQQ productive rate (238ng/mL) of the bacterium Lactobacillus plantarum PQQ-1 that sets out.
Claims (9)
1. by immunoprecipitation, screen a method of producing pyrroloquinoline quinone milk-acid bacteria, it is characterized in that, comprise the following steps:
Plant lactobacillus Lactobacillus plantarum PQQ-1 was cultivated after 16～30 hours in MRS liquid nutrient medium, drawing fermented liquid is applied on MRS solid plate substratum, with after uviolizing, with all bacterium colonies of phosphoric acid buffer wash-out, pour standing cultivation 18h～30h in activation medium into, then drawing nutrient solution mixes with the anti-PQQ IgG1 of mouse monoclonal antibody, coating agar subculture medium, standing cultivation 48h～72h, the larger person of periphery of bacterial colonies precipitation circle radius is the higher product pyrroloquinoline quinone milk-acid bacteria of PQQ productive rate.
2. the method for producing pyrroloquinoline quinone milk-acid bacteria of screening by immunoprecipitation according to claim 1, is characterized in that, described MRS liquid nutrient medium, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 ± 0.2 with sulfuric acid.
3. the method for producing pyrroloquinoline quinone milk-acid bacteria of screening by immunoprecipitation according to claim 1, is characterized in that, described MRS solid plate substratum, in 1L, comprises the component of following weight:
PH value is adjusted to 6.5 ± 0.2 with sulfuric acid.
4. the method for producing pyrroloquinoline quinone milk-acid bacteria of screening by immunoprecipitation according to claim 1, is characterized in that, the condition of described uviolizing is: after keeping 10cm～30cm apart from irradiation 10-12min with 254nm ultraviolet ray.
5. the method for producing pyrroloquinoline quinone milk-acid bacteria of screening by immunoprecipitation according to claim 1, is characterized in that, described activation medium, in 1L, comprises the component of following weight:
This activation medium regulates pH to 6.8 ± 0.2 with acid-base buffer.
6. the method for producing pyrroloquinoline quinone milk-acid bacteria of screening by immunoprecipitation according to claim 1, is characterized in that, described agar subculture medium, in 1L, comprises the component of following weight:
This agar subculture medium regulates pH to 6.8 ± 0.2 with acid-base buffer.
7. a preparation method for pyrroloquinoline quinone, is characterized in that, comprises the following steps:
(1) plant lactobacillus Lactobacillus plantarum PQQ-1 was cultivated after 16～30 hours in MRS liquid nutrient medium, drawing fermented liquid is applied on MRS solid plate substratum, with after uviolizing, with all bacterium colonies of phosphoric acid buffer wash-out, pour standing cultivation 18h～30h in activation medium into, then drawing nutrient solution mixes with the anti-PQQ IgG1 of mouse monoclonal antibody, coating agar subculture medium, standing cultivation 48h～72h, the larger person of periphery of bacterial colonies precipitation circle radius is the higher product pyrroloquinoline quinone milk-acid bacteria of PQQ productive rate;
(2) the product pyrroloquinoline quinone milk-acid bacteria of periphery of bacterial colonies precipitation circle radius maximum in step (1), called after plant lactobacillus Lactobacillus plantarum PQQ-6, after activated substratum activation, 20 ℃～40 ℃ standing cultivation 16h～30h become first order seed, first order seed continues 20 ℃～40 ℃ standing cultivation 16h～30h of expansion through first order seed substratum and becomes secondary seed, secondary seed enters fermentation state after secondary seed medium continues 20 ℃～40 ℃ standing cultivation 42h～54h of expansion, in production fermention medium, 20 ℃～40 ℃ anaerobism are cultivated 54h～66h, obtain pyrroloquinoline quinone.
8. the preparation method of pyrroloquinoline quinone according to claim 7, is characterized in that, in step (2), described first order seed substratum, in 1L, comprises the component of following weight:
This first order seed substratum regulates pH to 6.8 ± 0.2 with acid-base buffer;
Described secondary seed medium, in 1L, comprises the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus;
This secondary seed medium regulates pH to 6.8 ± 0.2 with acid-base buffer.
9. the preparation method of pyrroloquinoline quinone according to claim 7, is characterized in that, in step (2), described production fermention medium, in 1L, comprises the component of following weight:
Wholegrain barley meal 30g;
Yeast extract 7.5g;
Water surplus;
This production fermention medium regulates pH to 6.8 ± 0.2 with acid-base buffer.
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