CN110564580A - Method for producing vinegar containing pyrroloquinoline quinone through microbial co-culture fermentation - Google Patents

Method for producing vinegar containing pyrroloquinoline quinone through microbial co-culture fermentation Download PDF

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CN110564580A
CN110564580A CN201910796795.4A CN201910796795A CN110564580A CN 110564580 A CN110564580 A CN 110564580A CN 201910796795 A CN201910796795 A CN 201910796795A CN 110564580 A CN110564580 A CN 110564580A
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梁新乐
韩程程
季妍
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Zhejiang Gongshang University
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Abstract

a method for producing pyrroloquinoline quinone vinegar through microbial co-culture fermentation belongs to the technical field of vinegar brewing. The method utilizes saccharomyces cerevisiae, hyphomycetes and acetobacter pasteurianus to construct a co-culture system to produce the vinegar rich in pyrroloquinoline quinone. The invention adopts a method of co-culturing saccharomyces cerevisiae, hyphomycetes and Acetobacter pasteurianus microorganisms, and prepares the vinegar rich in pyrroloquinoline quinone through stages of alcoholic fermentation, acetic fermentation, fermentation stopping, product filtration and packaging and the like, wherein the concentration of the pyrroloquinoline quinone is 0.05-0.2 mg/L. The method is novel, and the microbial co-culture method is adopted, so that the nutritional factors and the vinegar are combined, the nutritional and health values of the vinegar are increased, and the feasibility is realized.

Description

Method for producing vinegar containing pyrroloquinoline quinone through microbial co-culture fermentation
Technical Field
The invention belongs to the technical field of vinegar brewing, and particularly relates to a method for producing pyrroloquinoline quinone vinegar through microbial co-culture fermentation.
background
As a basic seasoning, vinegar is also regarded for its nutritional and health-promoting functions. However, the current nutritional and healthy functional vinegar usually achieves the purpose only by changing the types of raw materials, has a single method, and has limitation on the types of added nutritional functional factors. The multi-strain co-culture method is a novel fermentation technology, has related reports in the fields of biology, food, chemistry and the like, but has less research reports on the improvement of the quality of the vinegar by introducing functional nutritional factors in a multi-strain co-culture mode.
Disclosure of Invention
aiming at the problems in the prior art, the invention aims to provide a technical scheme of a method for producing pyrroloquinoline quinone vinegar by microbial co-culture fermentation. The method realizes production of pyrroloquinoline quinone vinegar by co-fermenting Saccharomyces cerevisiae, Micromyceliophthora and Acetobacter pasteurianus, and improves quality of vinegar.
the specific technical scheme comprises the following steps:
(1) Adjusting reducing sugar and pH value based on the fermented glutinous rice, supplementing methanol, inoculating hyphomycetes and Saccharomyces cerevisiae, and co-culturing to obtain Saccharomyces cerevisiae-hyphomycetes co-culture seed solution;
(2) Taking rice and water as raw materials, performing amylase saccharification treatment, size mixing treatment and methanol supplementation, inoculating the saccharomyces cerevisiae-hyphomycete co-culture seed solution obtained in the step (1) for fermentation, and adjusting the concentration of ethanol and acetic acid after fermentation to obtain an ethanol wine base;
(3) Inoculating acetobacter pasteurianus in the alcohol base obtained in the step (2) for culture to obtain acetobacter pasteurianus seed liquid;
(4) And (3) inoculating the saccharomyces cerevisiae-hyphomycete co-culture seed liquid obtained in the step (1) and the acetobacter pasteurianus seed liquid obtained in the step (3) into the alcoholic liquor base obtained in the step (2) for multi-strain co-culture fermentation to obtain the vinegar rich in pyrroloquinoline quinone.
Further, in the step (1), the concentration of reducing sugar is adjusted to 1-3% w/v, the concentration of methanol is adjusted to 0.2-1.0% w/v, and the pH is adjusted to 6.0-7.0.
Further, the culture conditions in the step (1) are as follows: the temperature is 25-32 ℃ and the time is 48-96 hours.
Further, in the step (2), the ratio of the rice to the water is 1:3, 0.3% of alpha-amylase and 0.3% of saccharifying enzyme are added for amylase saccharification treatment, and 0.1% of CaCl is added2And (4) carrying out size mixing treatment, and supplementing methanol to the concentration of 0.2-1.0% w/v.
Further, the fermentation conditions in the step (2) are as follows: the temperature is 28-35 ℃, the time is 24-64 hours, and the inoculation amount is 1-5% v/v.
Further, after fermentation in step (2), the ethanol concentration is adjusted to 1-2% w/v and the acetic acid concentration is adjusted to 0.5-3.5% w/v.
Further, the culture conditions in step (3) are as follows: the temperature is 30-35 ℃, the time is 16-24 hours, and the inoculation amount is 4-7% v/v.
Further, ethanol wine base is added in the fermentation process in the step (4), the ethanol concentration is controlled to be 1-3% w/v, the methanol concentration is controlled to be 0.2-1.0% w/v, the fermentation temperature is 30-35 ℃, and the fermentation time is 24-48 hours.
And (3) further, adding table salt in the step (4) to stop the fermentation process, precipitating, filtering, clarifying, and adding water to prepare the pyrroloquinoline quinone-rich vinegar.
The invention adopts a method of co-culturing saccharomyces cerevisiae, hyphomycetes and Acetobacter pasteurianus microorganisms, and prepares the vinegar rich in pyrroloquinoline quinone through stages of alcoholic fermentation, acetic fermentation, fermentation stopping, product filtration and packaging and the like, wherein the concentration of the pyrroloquinoline quinone is 0.05-0.2 mg/L. The method is novel, and the microbial co-culture method is adopted, so that the nutritional factors and the vinegar are combined, the nutritional and health values of the vinegar are increased, and the feasibility is realized.
Drawings
FIG. 1 is a phylogenetic tree of Microbacterium serigenes LXL-PQ-409;
FIG. 2 is a diagram showing the colony morphology and gram staining of Microbacterium serigenes LXL-PQ-409.
Detailed Description
The invention is further illustrated by the following specific examples.
Example 1: the filamentous microbe used in the application is filamentous microbe LXL-PQ-409, which is obtained by the following steps:
Bacteria producing pyrroloquinoline quinone are separated from the Zhejiang yellow wine fermented grains. Taking fermented grains, properly diluting the fermented grains by using sterile biological saline water, and then coating the diluted fermented grains on a methanol solid culture medium. The solid medium contains 2% of methanol and (NH)4)3C6H8O4 0.3%、KH2PO4 0.14%、Na2HPO4 0.3%、MgSO4·7H20.1 percent of O, 0.07 percent of trace element liquid, 2 percent of agar and the balance of water, wherein the pH value of a culture medium is 7, the culture temperature is 30 ℃, and the culture time is 7 days. Then selecting a colony with better growth vigor, inoculating the colony in a liquid culture medium (the same as a solid culture medium except agar-free) for culturing for 7 days, and obtaining the pyrroloquinoline quinone strain after LC/MS analysis and detection.
Taking slant strains, adding 10 mL of normal saline to prepare cell suspension, and performing irradiation with 300 Gy dose137And (5) irradiating by using Cs gamma rays for 2 hours. After the irradiation is finished, the suspension is diluted and coated on a screening culture medium, and the screening culture medium contains 7% of methanol, 0.3% of ethanol and (NH)4)3C6H8O4 0.3%、KH2PO4 0.14%、Na2HPO4 0.3%、MgSO4.7H20.1% of O, 10 mmol/L ethionine, 0.07% of trace element liquid and 2% of agar, wherein the trace element liquid contains FeSO4·7H2O 0.75%、ZnSO4·7H2O 22.5%、MnSO4·4～5H2O 0.45%、CuSO4•5H2O 0.075%、NaCl 0.15%、(NH4)6Mo7O24·4H2O 0.03%、KI 0.03%、CoCl2·6H2O 0.03%、H3BO3 0.03%、CaCl2·2H2O3%, the pH value of a culture medium is 7.0, the culture temperature is 30 ℃, the culture time is 9 days, and large colonies and pyrroloquinoline quinone positive mutant strains are selected. The above-mentioned mutagenesis-directed breeding process is repeated several times. Finally, separating to obtainAccording to the pyrroloquinoline quinone high-producing strain LXL-PQ-409 disclosed by the invention, mass spectrum detection shows that LXL-PQ-409 can be used for efficiently fermenting and producing pyrroloquinoline quinone by taking methanol as a carbon source.
Extracting genome of strain LXL-PQ-409 and determining 16S rDNA gene sequence, wherein the 16S rDNA gene sequence is shown as SEQ ID NO.1, and performing systematic gene analysis result (figure 1) by adopting an ortho-position connection algorithm, wherein the homology of the gene sequence and the filamentous microbe is more than 97%. Morphological observation shows that the cells are oval and have no spores, one end of the cells has expanded short hyphae, and the cells have lateral single flagella, can move and are gram-negative. In the range of 28 to 35oc, good growth under the condition of neutral pH and partial acid. The surface of the bacterial colony grows for 5 days in a solid culture medium taking methanol as a unique carbon source, the bacterial colony gradually changes from milky white to light brown, the glossiness of the bacterial colony is reduced, the surface is wrinkled, and the morphology of the bacterial colony is shown in figure 2.
based on the morphology of the strain LXL-PQ-409 and its 16S rDNA sequence homology, it was initially identified as a filaggrin microorganism.
The above-mentioned filamentous microform LXL-PQ-409 is preserved by a preservation unit: china center for type culture Collection, Address: wuhan university in Wuhan, China, preservation date: 22 months 4 and 2019, and the accession number is: CCTCC NO: m2019284, suggested classification namedHyphomicrobium sp.LXL-PQ-409。
Example 2:
(1) Taking 10 parts of yellow wine mash, 1 part of methanol and 89 parts of water to prepare a saccharomyces cerevisiae-raw silk microbe culture medium, adjusting reducing sugar to 3% w/v, and adjusting pH to 7.0. Inoculating the raw filamentous microzyme and the saccharomyces cerevisiae for co-culture, wherein the inoculation amount is 2 percent, and the culture condition is that the culture is carried out for 96 hours at the temperature of 32 ℃. The budding rate of the microzyme is 98 percent, and the colony forming unit number of the filamentous microzyme is more than or equal to 7.0 multiplied by 108per milliliter.
(2) taking rice and water as raw materials, blending at a ratio of 1:3, adding 0.3% alpha-amylase and 0.3% saccharifying enzyme for amylase saccharification, adding 0.1% CaCl2And (3) carrying out size mixing treatment, supplementing methanol to 1.0% w/v, inoculating saccharomyces cerevisiae-hyphomycete seed liquid for fermentation, wherein the inoculation amount is 5% v/v, and culturing at 30 ℃ for 64 hours to obtain an ethanol wine base, and the ethanol concentration is 6% w/v. Adjusting ethanol concentration to2% w/v, acetic acid concentration to 2% w/v.
(3) Inoculating Acetobacter pasteurianus in the obtained alcohol base, and culturing to obtain an Acetobacter pasteurianus seed solution under the culture condition of 30 ℃ for 20 hours, wherein the inoculation amount is 5% v/v.
(4) Inoculating the acetobacter pasteurianus seed liquid into the alcohol wine base to carry out saccharomyces cerevisiae-raw silk micro-bacterium-acetobacter pasteurianus co-culture. And inoculating the hyphomycetes again for intensive fermentation at 24 hours, wherein the inoculation amount is 1% v/v. Adding alcohol liquor base in the fermentation process, controlling the alcohol concentration to be 1.5% w/v, the methanol concentration to be 1.0% w/v, the fermentation temperature to be 30 ℃, fermenting for 24 hours, adding salt to stop the fermentation process, wherein the alcohol concentration is less than or equal to 0.8% w/v, and the methanol concentration is less than or equal to 0.01% w/v. The vinegar liquid is settled, filtered and clarified, and is prepared into vinegar rich in pyrroloquinoline quinone after being mixed with water, the vinegar rich in pyrroloquinoline quinone is prepared, the acidity is 3.8 g/100 ml, and the concentration of the pyrroloquinoline quinone is 0.2 mg/L.
example 3:
(1) 10 parts of wine pre-fermentation wine mash, 1 part of methanol and 89 parts of water are taken to prepare a saccharomyces cerevisiae-raw silk microbe culture medium, reducing sugar is adjusted to 1% w/v, and the pH value is adjusted to 6.0. Inoculating the raw filamentous microsclerotia-saccharomyces cerevisiae for co-culture at the temperature of 25 ℃ for 48 hours. The budding rate of the microzyme is more than or equal to 85 percent, and the colony forming unit number of the hyphomycete is more than or equal to 0.2 multiplied by 108Per milliliter.
(2) Taking rice and water as raw materials, blending at a ratio of 1:3, adding 0.3% alpha-amylase and 0.3% saccharifying enzyme for amylase saccharification, adding 0.1% CaCl2And (3) carrying out size mixing treatment, supplementing methanol to the concentration of 0.2% w/v, inoculating saccharomyces cerevisiae-hyphomycete seed liquid for fermentation, wherein the inoculation amount is 1% v/v, and the temperature is 28 ℃ for 24 hours to prepare an ethanol wine base, and the ethanol concentration is 2.5% w/v. After fermentation, the ethanol concentration was adjusted to 1.5% w/v and the acetic acid concentration to 2.5% w/v.
(3) Inoculating acetobacter pasteurianus in the obtained alcohol base, and culturing to obtain acetobacter pasteurianus seed liquid, wherein the culture condition is 35 ℃, the time is 16 hours, and the inoculation amount is 4% v/v.
(4) Inoculating the acetobacter pasteurianus seed liquid into the alcohol wine base to carry out saccharomyces cerevisiae-raw silk micro-bacterium-acetobacter pasteurianus co-culture. And inoculating the hyphomycetes again for intensive fermentation at 24 hours, wherein the inoculation amount is 4% v/v. The wine base is added in the fermentation process, the concentration of the ethanol is controlled to be 1.5% w/v, the concentration of the methanol is controlled to be 0.2% w/v, and the fermentation temperature is 35 ℃. The fermentation time is 36 hours, salt is added to stop the fermentation process, and the concentration of the ethanol is less than or equal to 0.8% w/v and the concentration of the methanol is less than or equal to 0.01% w/v. The vinegar liquid is settled, filtered and clarified, and is prepared into the vinegar rich in pyrroloquinoline quinone, the acidity is 3.8 g/100 ml, and the concentration of the pyrroloquinoline quinone is 0.05 mg/L.
example 4:
(1) 10 parts of yellow wine mash, 1 part of methanol and 89 parts of water are taken to prepare a saccharomyces cerevisiae-raw silk microbe culture medium, reducing sugar is adjusted to 2% w/v, and the pH value is adjusted to 7.0. Inoculating the hyphomycete and the saccharomyces cerevisiae for co-culture under the culture condition of 30 ℃ for 96 hours. The budding rate of the microzyme is 98 percent, and the colony forming unit number of the filamentous microzyme is more than or equal to 2.0 multiplied by 108Per milliliter.
(2) Taking rice and water as raw materials, blending at a ratio of 1:3, adding 0.3% alpha-amylase and 0.3% saccharifying enzyme for amylase saccharification, adding 0.1% CaCl2And (3) carrying out size mixing treatment, supplementing methanol to the concentration of 0.5% w/v, inoculating saccharomyces cerevisiae-hyphomycete seed liquid for fermentation, wherein the inoculation amount is 3% v/v, and culturing for 48 hours at the temperature of 32 ℃ to obtain an ethanol wine base, wherein the ethanol concentration is 3.6% w/v. After fermentation, the ethanol concentration was adjusted to 1% w/v and the acetic acid concentration to 0.5% w/v.
(3) Inoculating Acetobacter pasteurianus in the obtained alcohol base, and culturing to obtain an Acetobacter pasteurianus seed solution under the culture condition of 30 ℃ for 24 hours, wherein the inoculation amount is 6% v/v.
(4) Inoculating the acetobacter pasteurianus seed liquid into the alcohol wine base to carry out saccharomyces cerevisiae-raw silk micro-bacterium-acetobacter pasteurianus co-culture. And inoculating the hyphomycetes again for intensive fermentation at 24 hours, wherein the inoculation amount is 6% v/v. Adding alcohol liquor base in the fermentation process, controlling the alcohol concentration to be 2% w/v, the methanol concentration to be 0.5% w/v, the fermentation temperature to be 30 ℃, fermenting for 48 hours, adding salt to stop the fermentation process, wherein the alcohol concentration is less than or equal to 0.8% w/v, and the methanol concentration is less than or equal to 0.01% w/v. The vinegar liquid is settled, filtered and clarified, and is added with water to prepare the vinegar rich in pyrroloquinoline quinone, the acidity is 3.8 g/100 ml, and the concentration of the pyrroloquinoline quinone is 0.07 mg/L.
the foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.

Claims (10)

1. A method for producing pyrroloquinoline quinone vinegar through microbial co-culture fermentation is characterized in that a co-culture system is constructed by using saccharomyces cerevisiae, hyphomycetes and acetobacter pasteurianus to produce the pyrroloquinoline quinone-rich vinegar.
2. The method according to claim 1, characterized in that it comprises in particular the steps of:
(1) Adjusting reducing sugar and pH value based on the fermented glutinous rice, supplementing methanol, inoculating hyphomycetes and Saccharomyces cerevisiae, and co-culturing to obtain Saccharomyces cerevisiae-hyphomycetes co-culture seed solution;
(2) Taking rice and water as raw materials, performing amylase saccharification treatment, size mixing treatment and methanol supplementation, inoculating the saccharomyces cerevisiae-hyphomycete co-culture seed solution obtained in the step (1) for fermentation, and adjusting the concentration of ethanol and acetic acid after fermentation to obtain an ethanol wine base;
(3) Inoculating acetobacter pasteurianus in the alcohol base obtained in the step (2) for culture to obtain acetobacter pasteurianus seed liquid;
(4) and (3) inoculating the saccharomyces cerevisiae-hyphomycete co-culture seed liquid obtained in the step (1) and the acetobacter pasteurianus seed liquid obtained in the step (3) into the alcoholic liquor base obtained in the step (2) for multi-strain co-culture fermentation to obtain the vinegar rich in pyrroloquinoline quinone.
3. The method according to claim 2, wherein the reducing sugar concentration in step (1) is adjusted to 1-3% w/v, the methanol concentration is adjusted to 0.2-1.0% w/v, and the pH is adjusted to 6.0-7.0.
4. The method according to claim 2, wherein the culture conditions in step (1) are: the temperature is 25-32 ℃ and the time is 48-96 hours.
5. The method of claim 2, wherein the ratio of rice to water in step (2) is 1:3, 0.3% of alpha-amylase and 0.3% of saccharifying enzyme are added for amylase saccharification, and 0.1% of CaCl is added2And (4) carrying out size mixing treatment, and supplementing methanol to the concentration of 0.2-1.0% w/v.
6. The method of claim 2, wherein the fermentation conditions in step (2) are: the temperature is 28-35 ℃, the time is 24-64 hours, and the inoculation amount is 1-5% v/v.
7. the method according to claim 2, wherein the ethanol concentration is adjusted to 1-2% w/v and the acetic acid concentration to 0.5-3.5% w/v after the fermentation in step (2).
8. The method according to claim 2, wherein the culture conditions in step (3) are: the temperature is 30-35 ℃, the time is 16-24 hours, and the inoculation amount is 4-7% v/v.
9. The method according to claim 2, wherein the alcoholic liquor base is supplemented in the fermentation process in the step (4), the ethanol concentration is controlled to be 1-3% w/v, the methanol concentration is controlled to be 0.2-1.0% w/v, the fermentation temperature is 30-35 ℃, and the fermentation time is 24-48 hours.
10. The method according to claim 2, wherein the fermentation process is stopped by adding table salt in step (4), and the vinegar rich in pyrroloquinoline quinone is obtained by precipitating, filtering, clarifying, and blending with water.
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CN112741241A (en) * 2020-12-30 2021-05-04 浙江大学 Chrysanthemum morifolium fermented beverage rich in pyrroloquinoline quinone and preparation method thereof
CN113337433A (en) * 2021-06-04 2021-09-03 华熙生物科技股份有限公司 Pseudomonas capable of producing pyrroloquinoline quinone and application thereof
CN113337432A (en) * 2021-06-04 2021-09-03 华熙生物科技股份有限公司 Methylophagus for producing pyrroloquinoline quinone and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112741241A (en) * 2020-12-30 2021-05-04 浙江大学 Chrysanthemum morifolium fermented beverage rich in pyrroloquinoline quinone and preparation method thereof
CN113337433A (en) * 2021-06-04 2021-09-03 华熙生物科技股份有限公司 Pseudomonas capable of producing pyrroloquinoline quinone and application thereof
CN113337432A (en) * 2021-06-04 2021-09-03 华熙生物科技股份有限公司 Methylophagus for producing pyrroloquinoline quinone and application thereof

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