CN113249268A - Saccharopolyspora rosea for reducing biogenic amine and application thereof - Google Patents

Saccharopolyspora rosea for reducing biogenic amine and application thereof Download PDF

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CN113249268A
CN113249268A CN202110710175.1A CN202110710175A CN113249268A CN 113249268 A CN113249268 A CN 113249268A CN 202110710175 A CN202110710175 A CN 202110710175A CN 113249268 A CN113249268 A CN 113249268A
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rosea
saccharopolyspora
fermentation
biogenic amine
reducing
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CN113249268B (en
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毛健
刘双平
孙梦菲
孙红根
徐岳正
王兰
陈斌
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Industrial Technology Research Institute Of Jiangnan University Shaoxing
Zhejiang Guyue Longshan Shaoxing Wine Co ltd
Jiangnan University
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Industrial Technology Research Institute Of Jiangnan University Shaoxing
Zhejiang Guyue Longshan Shaoxing Wine Co ltd
Jiangnan University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/30—Removing undesirable substances, e.g. bitter substances
    • A23L11/37—Removing undesirable substances, e.g. bitter substances using microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
    • A23L13/45—Addition of, or treatment with, microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
    • A23L13/60—Comminuted or emulsified meat products, e.g. sausages; Reformed meat from comminuted meat product
    • A23L13/65—Sausages
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/65—Addition of, or treatment with, microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/50—Soya sauce
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
    • A—HUMAN NECESSITIES
    • A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
    • A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
    • A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
    • A24B15/18—Treatment of tobacco products or tobacco substitutes
    • A24B15/20—Biochemical treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C12G3/022—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn of botanical genus Oryza, e.g. rice
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Abstract

The invention discloses a saccharopolyspora rosea for reducing biogenic amine and application thereof, belonging to the technical field of food fermentation. The saccharopolyspora rosea F2014 capable of degrading biogenic amine is obtained by screening, and the degradation rate of the strain on tryptamine, phenethylamine, cadaverine, putrescine, histamine, tyramine, spermidine and spermine is up to more than 88%. The roseosacrasis bacteria are used for producing yellow wine, cigarettes, sausages, soy sauce, fermented fish sauce and broad bean paste, can effectively degrade the biogenic amine content of fermented products, and does not bring adverse effects on the quality of the fermented products.

Description

Saccharopolyspora rosea for reducing biogenic amine and application thereof
Technical Field
The invention relates to a saccharopolyspora rosea strain for reducing biogenic amine and application thereof, belonging to the technical field of food fermentation.
Background
Biogenic amine is a nitrogen-containing organic base with low molecular weight, is mainly generated by the decarboxylation of amino acid under the action of amino acid decarboxylase of microorganisms, and is widely present in fermented foods. Proper amount of biogenic amine can promote normal physiological activities of human bodies, and excessive biogenic amine intake can cause the expansion of arteries, blood vessels and capillaries, cause adverse physiological reactions such as diarrhea, headache, abdominal cramp, vomiting and the like, and even cause death.
Yellow wine as a traditional alcoholic beverage with national characteristics is rich in various amino acids and functional components and is deeply favored by consumers. The yellow wine brewing is carried out in an open environment, so that the content of the biogenic amine in the yellow wine is much higher than that of other brewed wines (beer, wine and the like). At present, methods for controlling biogenic amine in food mainly comprise production process optimization, selection of proper storage conditions, reduction of biogenic amine producing microorganisms in a fermentation system or control of amine producing microorganisms to produce amine, addition of biogenic amine degrading strains, biogenic amine degrading enzymes and the like. The added biogenic amine degrading strain is used as a biological means, has the advantages of high efficiency, safety and environmental protection, and is accepted by more and more yellow wine enterprises and consumers.
The strains with the effect of degrading the biogenic amine, which are obtained by screening at present, have fewer varieties and poor effect. Therefore, the screening of the bacterial strain capable of efficiently degrading the biogenic amine has important significance for enriching the microbial strain bank of the biogenic amine degrading bacterial strain and improving the quality of fermented food.
Disclosure of Invention
The invention aims to solve the problem of high content of biogenic amine in the traditional fermented food and tobacco products, and provides the strain S.rosea F2014 with excellent performance for biological enhancement in the production process of wines (white spirit and yellow wine), cigarettes, sausages, soy sauce and broad bean paste, so that the biogenic amine content in the traditional fermented food and tobacco products is reduced, and the quality of the traditional fermented food and tobacco products is improved.
The first purpose of the invention is to provide 1 Saccharopolyspora rosea (Saccharopolyspora rosea) F2014 with stronger biogenic amine degradation capability, wherein the Saccharopolyspora rosea is preserved in China center for type culture collection (CCTCC NO) within 21 months in 2020, and the preservation address is China, Wuhan and Wuhan university with the preservation number of CCTCC NO: m2020952.
The saccharopolyspora rosea of the invention has the following excellent performances:
(1) the method is applied to a food fermentation system, and the normal fermentation of food is not influenced;
(2) the production amount of biogenic amine in the fermentation product is less than 10mg/L, and the detection amount of biogenic amine is very little;
(3) has degradation effect on tryptamine, phenethylamine, cadaverine, putrescine, histamine, tyramine, spermidine and spermine;
(4) can be used for producing yellow wine, Chinese liquor, bean paste, soy sauce fermentation and cigarette, and has effect in reducing biogenic amine.
The second purpose of the invention is to provide a leaven containing the saccharopolyspora rosea F2014.
In one embodiment, the content of the saccharopolyspora rosea F2014 in the leavening agent is more than or equal to 1 × 106CFU/mL。
In one embodiment, the fermentation agent comprises live cells of the thalli of saccharopolyspora rosea F2014, dry thalli obtained by freeze-drying, immobilized cells, a liquid microbial inoculum, a solid microbial inoculum, or a strain of saccharopolyspora rosea F2014 in any other form.
In one embodiment, the starter culture further comprises other types of bacteria or fungi.
In one embodiment, the starter culture further comprises yeast or aspergillus.
The third purpose of the invention is to provide the application of the saccharopolyspora rosea F2014 or the metabolite thereof in reducing the content of biogenic amine in the field of fermentation.
In one embodiment, the metabolites include, but are not limited to, supernatants from which the somatic cells were removed after fermentation of saccharopolyspora rosea F2014.
In one embodiment, the metabolite is prepared by a method comprising: mixing Saccharopolyspora rosea F2014 at a ratio of 5 × 106Inoculating the CFU/mL initial concentration into a Gauss I liquid culture medium, fermenting for 48-72 h at 37 ℃ under the condition of 150r/min, centrifuging, and collecting the supernatant of the fermentation liquid.
In one embodiment, the field of fermentation includes the preparation of fermented food, drink or seasoning.
In one embodiment, the beverage is an alcoholic beverage, including but not limited to yellow wine or white wine.
In one embodiment, the seasoning includes, but is not limited to, soy sauce, sausage, or fish sauce.
In one embodiment, the application is that the polyspora rosea F2014 is added into the sauce mash, and after fermentation is carried out for a period of time at 14-16 ℃, the yeast is added to continue fermentation.
In one embodiment, the application is to treat saccharopolyspora rosea F2014 at 5 × 106And (3) fermenting for 48-72 hours at the initial concentration of CFU/mL and the temperature of 37 ℃ under the condition of 150r/min, collecting and fermenting, uniformly spraying the supernatant of the fermentation liquor into the tobacco shreds by using a throat sprayer according to the amount of 1% by mass, and fermenting for 42-72 hours at the temperature of 30-37 ℃.
In one embodiment, the application is to add saccharopolyspora rosea F2014 to the koji and then ferment the broad bean paste with the koji containing saccharopolyspora rosea F2014.
The invention provides application of saccharopolyspora rosea F2014 in reducing content of biogenic amine in tobacco shreds.
In one embodiment, the biogenic amines include, but are not limited to, tryptamine, phenethylamine, cadaverine, putrescine, histamine, tyramine, spermidine, spermine.
The invention also provides application of the saccharopolyspora rosea F2014 in preparing fermented food, drinks or tobacco.
The invention has the beneficial effects that:
(1) the strain is applied to a food fermentation system, and the normal fermentation of food cannot be influenced;
(2) the strain is applied to cigarette production, and the contents of carbohydrate and protein of cigarettes are not influenced;
(3) and the production amount of the biogenic amine of the rosea F2014 is less than 10mg/L, the detection amount of the biogenic amine is very little, and the biogenic amine is not produced basically. The degradation rates of rosea F2014 to tryptamine, phenethylamine, cadaverine, putrescine, histamine, tyramine, spermidine and spermine are respectively 93.5%, 88.88%, 97.12%, 94.05%, 97.83%, 99.12%, 96.27% and 99.27%, and the degradation rate to total biogenic amine reaches 96.21%
(4) The saccharopolyspora has the effect of reducing biogenic amine, and is applied to yellow wine fermentation, and the yellow wine fermented by S.rosea F2014 is added, so that the content is reduced by 22.99% compared with that in a control group; the content of the S.rosea F2014 in the fermented cigarette is reduced by 30.78% compared with that in a control group when the S.rosea F2014 is added in cigarette production; the soybean paste fermented by S.rosea F2014 is added in the soybean paste fermentation, and the soybean paste is reduced by 24.28% compared with a control group; the soy sauce is applied to soy sauce fermentation, and the soy sauce fermented by adding S.rosea F2014 is reduced by 25.37% compared with a control group; the fish gravy is applied to fish gravy fermentation, and the fish gravy fermented by S.rosea F2014 is reduced by 25.57% compared with a control group; when the strain is applied to sausage fermentation, the sausage fermented by adding S.rosea F2014 is reduced by 35.37% compared with a control group.
Biological material preservation
Saccharopolyspora rosea (saccharomyces rosea) F2014, classified and named as Saccharopolyspora rosea (saccharomyces rosea) F2014; the saccharopolyspora is preserved in China Center for Type Culture Collection (CCTCC) in 12 months and 21 days in 2020, the preservation address is China, Wuhan and Wuhan university, and the preservation number is CCTCC NO: m2020952.
Drawings
Fig. 1 colony morphology of saccharopolyspora s. rosea F2014.
Fig. 2 phylogenetic tree of saccharopolyspora s
FIG. 3 shows the variation of physicochemical index (A) of alcoholic strength during the fermentation process of yellow wine; (B) a reducing sugar; (C) titratable acid; (D) amino acid nitrogen.
Detailed Description
Detecting physical and chemical indexes of yellow wine: the alcohol content, amino acid nitrogen and total acid are measured according to GB/T13662-. The content of reducing sugar is measured by a DNS method. The biogenic amine content was determined by High Performance Liquid Chromatography (HPLC).
Detecting the physical and chemical indexes of the cut tobacco: the total sugar and reducing sugar in tobacco shreds are obtained by Somotetrary method, and the total nitrogen is obtained by Kyojeldahl method.
The method for measuring the content of the biogenic amine comprises the following steps: accurately measuring 1mL of solution to be detected in a 15mL centrifuge tube, and adding 1mL of saturated NaHCO3Mixing the solutions, adding 2mL dansyl chloride (5mg/mL acetone) reagent, mixing, placing in 65 deg.C water bath kettle, deriving in dark for 30min, standing at room temperature, adding 05mL of saturated NaCl solution, mixing uniformly, adding 5mL of diethyl ether, carrying out vortex oscillation for 20s, standing for layering, transferring an upper organic phase into a 15mL centrifuge tube, extracting a lower aqueous phase once again, combining two extraction solutions, and drying by blowing nitrogen at 50 ℃ in a water bath. 1mL of acetonitrile was added and mixed well with shaking to dissolve the residue, which was then filtered through a 0.22 μm filter and measured by High Performance Liquid Chromatography (HPLC).
Example 1: screening and identification of Saccharopolyspora rosea
(1) Sample collection and pretreatment
The wheat koji sample is collected from a yellow wine factory in Shaoxing city, Shaoxing, Zhejiang province, and the collected wheat koji is stored in a sealed sterile plastic bag at 4 ℃.5g of malt is weighed into a 50mL centrifuge tube, added with 30mL of distilled water and put into a shaking incubator at 30 ℃ for 30 min.
(2) Plate screening of strains
Solid culture medium No. kao: 1.0g/L potassium nitrate, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.5g/L sodium chloride, 20.0g/L soluble starch, 15.0g/L agar, and pH 7.2-7.4 (25 ℃).
Taking the suspension for gradient dilution 10-1To 10-6And (5) diluting the uniform solution. Respectively sucking 100 mu L of each dilution bacterial liquid of the wheat starter and the fermented mash, coating the diluted bacterial liquid on a Gao's No. one solid culture medium, and culturing for 4-7 d at 37 ℃. Single milky white, thin and raised colony or convex colony is selected from the plate with moderate colony density and streaked and inoculated to the Gao's number one solid plate.
(3) Identification of strains
Extracting the genome of the screened strain, and carrying out 16S rDNA amplification sequencing on the screened strain.
PCR amplification primers 27F (5 '-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5 '-TACGGY-TACCTTGTTACGACTT-3').
The PCR amplification system (50. mu.L) was: 2 XTaq PCR Master Mix 25. mu.L, upper and lower primers 1. mu.L each, template 1. mu.L, sterile water 22. mu.L make up to 50. mu.L.
PCR amplification procedure: pre-denaturation at 94 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min for 35 cycles, and final extension at 72 ℃ for 8 min.
The PCR product was detected by 1% agarose gel electrophoresis and submitted to the Gene sequencing company for sequencing, BLAST sequence alignment was performed through the NCBI official website based on the returned sequencing result (shown in SEQ ID NO. 1), BLAST alignment was performed using the obtained 16S rDNA sequence, and phylogenetic analysis was performed, as shown in FIG. 2, the nucleotide sequence of the strain F2014 had a homology of 99% or more with the Saccharopolyspora S.rosea (GenBank accession number: NR-042711.1) having the highest sequence homology in the database. The colony morphology of the strain F2014 is circular, the hypha in the substrate is brown yellow, the aerial hypha is yellow, pink diffusible pigment can be generated on the Gao's No. one culture medium, and the colony is convex and difficult to pick up. The physiological and biochemical characteristics of the strain F2014 are shown in Table 1, the strain F2014 can degrade adenine, casein, esculin, starch, tyrosine, urea and other substances, can grow at 25-40 ℃, is consistent with the characteristics of Saccharopolyspora rosea, and is named as Saccharopolyspora S.rosea F2014. The same batch is screened to obtain another saccharopolyspora sp.hordei F2001 with similar characteristics.
Table 1 physiological and biochemical identification of strain F2014 part of the experimental results
Figure BDA0003133308480000041
Figure BDA0003133308480000051
Note: -represents a negative result and + represents a positive result
(4) Analysis of biogenic amine metabolizing ability of strain
Activating strains: inoculating the preserved saccharopolyspora S.rosea F2014 into a Gao-shi No. one liquid culture medium, wherein the inoculation amount is 5%, and performing shake culture at 30 ℃ for 48h to obtain a first-level seed solution. Inoculating the activated strain into Gao's first liquid culture medium, wherein the inoculation amount is 5%, performing shake culture for 48h, rotating speed is 150r/min, and temperature is 30 ℃, thus obtaining secondary seed liquid.
Sample pretreatment: inoculating the second-stage seed liquid to the first Gao's liquid containing amino acid and biogenic amineThe inoculation amount of the culture medium is 5 percent, so that the initial bacterial concentration after inoculation is about 5 multiplied by 106CFU/mL, shake culture at 37 ℃ for 5d, and centrifugation at 12000r/min for 5min to collect supernatant.
The production amounts of tryptamine, phenethylamine, cadaverine, putrescine, histamine, tyramine, spermidine and spermine of the rosea F2014 are respectively 0.15mg/L, 0.00mg/L, 1.29mg/L, 0.28mg/L, 1.37mg/L, 0.00mg/L, 1.18mg/L, 0.32mg/L, 4.59mg/L and 0.10mg/L, the total biogenic amine yield is less than 10mg/L, the detected amount of biogenic amine is very small, and biogenic amine is not produced basically. Rosea F2014 has strong degradation capacity on 8 kinds of biogenic amines, the degradation rates on tryptamine, phenethylamine, cadaverine, putrescine, histamine, tyramine, spermidine and spermine are 93.5%, 88.88%, 97.12%, 94.05%, 97.83%, 99.12%, 96.27% and 99.27% respectively, and the degradation rate on total biogenic amines reaches 96.21%. Saccharopolyspora sp.hordei F2001 has degradation rates of 1.37%, 2.56%, 3.29%, 0.00%, 4.59%, 7.24%, 6.32% for tryptamine, phenethylamine, cadaverine, putrescine, histamine, tyramine, spermidine, and spermine, respectively.
Example 2: application of saccharopolyspora s.rosea F2014 in yellow wine fermentation
(1) Preparing pure saccharopolyspora cerealis malt:
crushing 3-5 pieces of wheat per grain to crush the wheat grain tissue, exposing starch, adding about 35-40% of clear water, stirring fully, standing for about 30min to fully and uniformly absorb water, and sterilizing at 121 ℃ for 30 min. After the sterilized wheat is cooled to room temperature, saccharopolyspora bacterium liquid is inoculated, and the concentration of the inoculated bacterium liquid is 105～106CFU/mL, the inoculation amount is 5% -15%. After the yeast material is fed into the tray, the proper product temperature and room temperature are kept, and the yeast material is kept standing and cultured for about six hours. After six hours, slowly raising the temperature of the product to about 34-35 ℃, starting small-air-volume indirect ventilation in a self-control mode, reducing the temperature of the product to 32 ℃ every 5-10 min at intervals of 2 hours, and requiring uniform blowing. And (3) after intermittent ventilation is carried out for 3-5 times, hypha starts to grow, the temperature of the product rises to above 35 ℃, the yeast material starts to agglomerate, and continuous ventilation is carried out at the moment to maintain the temperature of the product to be about 35 ℃. The temperature of the product rises quickly 12 hours after inoculation, the yeast is turned over according to the situation of first agglomeration, and the product is kept warm after the yeast is turned over for the first timeKeeping the temperature between 36 and 37 ℃, keeping ventilation and spraying smooth, after about 20 hours, enabling the yeast material to agglomerate again, enabling the eye yeast material to turn white, and controlling the temperature to be below 37 ℃ difficultly, carrying out secondary yeast turning, and controlling the product temperature to be about 35 ℃ after the secondary yeast turning. Continuously culturing for 72-96 h; after the culture is finished, the wheat koji is stored in a freezer at 4-7 ℃ for later use.
(2) Different wheat yeasts are used for yellow wine fermentation:
in the experimental group raw material formulation for traditional yellow wine fermentation selected in this example, as shown in table 2, the pure saccharopolyspora koji in table 2 was replaced by factory raw wheat koji (natural fermentation of wheat, added in an amount of 11.8%) and factory mature wheat koji (pure SU-16 aspergillus flavus, added in an amount of 1.8%) in the control group.
TABLE 2 raw materials proportioning Table (Experimental group)
Figure BDA0003133308480000061
(2) Traditional yellow wine brewing process
(a) And (3) yeast activation culture: transferring yeast in glycerol storage tube into YPD culture medium in sterile operation table, culturing at 30 deg.C and 150r/min for 24 hr to make the yeast number reach 107CFU/mL; then transferring the yeast to the prepared yeast with the inoculation amount of 5 percent, and culturing the transferred yeast for 18-24 hours at the temperature of 30 ℃ and at the speed of 150r/min for later use.
(b) Preparing yeast wine: adding 1600mL of clear water, 60g of raw wheat starter and 1.1 per mill of saccharifying enzyme (the final concentration is 3 multiplied by 10 according to the unit of enzyme activity) into 600g of steamed rice5U/mL) is carried out, the temperature of saccharifying is controlled to be 55-65 ℃ for 3-4 hours, the apparent sugar degree is not lower than 12 degrees Bx after saccharifying is finished, then the yeast is sterilized at 115 ℃ for 15 minutes, the yeast is cooled to 24-31 ℃ after being sterilized, 5 percent of mature yeast seed culture solution is inoculated, the culture temperature is not higher than 30 ℃, the culture time is 24 hours, and the yeast is obtained after mature culture.
(c) Blanking and fermenting according to the raw material proportion of the traditional yellow wine fermentation. The first 5d is a pre-fermentation stage, the temperature is controlled to be 28-30 ℃, the fermentation is carried out for 5d, harrowing is carried out for not less than 1 time every day for the first 5d, and harrowing time is 8-10 hours; and in the post-fermentation stage, the temperature is 13-15 ℃, the rake is stirred and harrowed for 1 time every day, and the fermentation is continued for 10-15 days.
The change of physical and chemical indexes in the fermentation process of yellow wine: in order to further verify the effect of saccharopolyspora in yellow wine fermentation, the change of physicochemical indexes (alcoholic strength, reducing sugar, titratable acid and amino acid nitrogen) in the fermentation process of yellow wine brewed by using traditional malt and pure saccharopolyspora malt is compared. The results are shown in fig. 3, and the contents of the alcoholic strength, reducing sugar, total acid and amino acid nitrogen of the yellow wine brewed by using the pure saccharopolyspora multivora wheat koji all meet the national standard of the yellow wine, which shows that the saccharopolyspora s.rosea F2014 has little influence on important physicochemical indexes in the fermentation process of the yellow wine and the fermentation is normal.
(3) Saccharopolyspora s. rosea F2014 analysis of effect on reducing biogenic amine: the content of biogenic amine in the yellow wine is detected by a high performance liquid chromatography method, and the result is shown in table 3, and the sample group added with the S.rosea F2014 is reduced by 22.99% compared with the control group.
TABLE 3 content of biogenic amine in yellow wine brewed by saccharopolyspora and saccharomyces cerevisiae
Figure BDA0003133308480000071
Note: ND means not detected
Example 3: saccharopolyspora s.rosea F2014 applied to cigarettes for reducing content of biogenic amine
Adding the saccharopolyspora s.rosea F2014 fermentation broth supernatant into cigarette production, and taking the added sterile water as a control, wherein the method comprises the following specific steps: saccharopolyspora sp.rosea F2014 at 5 × 106Fermenting in Gao's No. I liquid culture medium at the initial concentration of CFU/mL and the temperature of 37 ℃ and 150r/min for 48h, and collecting fermentation supernatant. Uniformly spraying the supernatant of the fermentation liquor into tobacco shreds by using a throat sprayer in an inoculation amount of 1% by mass, uniformly turning, filling into a clean plastic bag, placing the bag opening without tightening, placing into a constant-temperature incubator, culturing at 30 ℃ for 48h, and taking a sample every 4h to measure the number of viable bacteria in the sample. Fermenting for 48 hr, and measuring biogenic amine, total sugar and reducing sugar with a small amount of tobacco shredAnd total nitrogen content, the results are shown in table 4, and the total sugar, reducing sugar and total nitrogen content of the experimental group are not much different from those of the control group.
TABLE 4 tobacco shred chemical composition
Figure BDA0003133308480000072
Analysis of saccharopolyspora s. rosea F2014 biogenic amine effect: the sample group to which s.rosea F2014 was added was reduced by 30.78% compared to the control group.
Example 4: saccharopolyspora sp S.rosea F2014 applied to broad bean paste for reducing content of biogenic amine
Use of s. rosea F2014 for thick broad-bean sauce fermentation:
(1) preparing a seed starter: inoculating Aspergillus oryzae and S.rosea F2014 (the inoculation amount is 10%) to a wheat bran culture medium, placing the wheat bran culture medium in a constant-temperature incubator at 30 ℃ for culturing for 48-72 h, and turning over the koji at intervals by shaking a bottle.
(2) Preparing broad bean koji: and (3) adding water to soak the shelled dry broad bean petals for 1-2 hours. Draining the soaked wet broad bean segments, putting the broad bean segments into a steamer, steaming for 30min, stewing for 10 mm, and taking out of the steamer. After the steamed broad beans are taken out of the pot, the surface moisture of the steamed broad beans is dried in the air under the aseptic condition, and the steamed broad beans are uniformly mixed with the sterilized flour and the koji in a certain proportion.
(3) Mixing broad bean koji with 180g/L saline water according to the proportion of 1: 1.2, placing the mixture in a closed container for fermentation for 30 days, and turning over the sauce once a day.
The control group replaced saccharopolyspora s.rosea F2014 with s.hordei F2001, with the rest of the steps unchanged.
Analysis on the effect of saccharopolyspora sp.rosea F2014 in reducing the biogenic amine shows that the biogenic amine content in the broad bean paste product added with the saccharopolyspora F2014 is reduced by 24.28 percent compared with that in the control group.
Example 5: saccharopolyspora s.rosea F2014 applied to soy sauce for reducing content of biogenic amine
(1) Uniformly mixing the soybean meal and the wheat according to the proportion of 1:1, and steaming;
(2) preparing yeast wine: will 109～1011Inoculating soy sauce yeast seed liquid with bacterial liquid concentration to cooled soybean mealIn the mixture with wheat, the inoculation amount is 5%, and the bacterial liquid concentration is 109～1012cfu/mL, then adding 2 times of clear water volume, at 28 degrees C, 150r/min under culture for 24 h.
(3) Fermentation of soy sauce mash: adding 10 percent of bacterial liquid into the yeast wine in the step (2) according to the proportion of 5-10 percent5～106cfu/mL saccharopolyspora F2014 seed liquid, then adding saline water with the mass being 1.5-2 times that of the materials to ensure that the final salt content of the sauce mash is 18 +/-0.5% and the water content is 65 +/-1%, and then uniformly mixing. The initial fermentation temperature is controlled at 14-16 deg.C, the temperature gradually increases to about 34-36 deg.C along with the fermentation, and yeast is inoculated when the temperature increases to about 20 + -1 deg.C, the yeast amount is 2 × 106CFU/g soy mash. The fermentation was continued for about 5 months.
(4) And (4) squeezing the fermented soybean paste by a plate frame after the fermentation is finished, and removing the fermented soybean paste. After squeezing, diatomite filtration and membrane filtration are carried out to remove the precipitate. And (4) carrying out pasteurization on the filtered and clarified soy sauce and then filling.
The control group replaced saccharopolyspora s.rosea F2014 with s.hordei F2001, with the rest of the steps unchanged.
Analysis on the effect of saccharopolyspora s.rosea F2014 in reducing the biogenic amine shows that the biogenic amine content in the fermentation product added with s.rosea F2014 is reduced by 25.57% compared with that in the control group.
Example 6: saccharopolyspora s.rosea F2014 applied to fermented fish gravy for reducing content of biogenic amine
The fish sauce is produced according to the following process flow, namely: mixing raw fish and salt (mass ratio of 3:1 or 2:1) → adding 10000U/g protease, hydrolyzing at 60 deg.C for 12h → inoculating to final concentration of 105～106S. hordei F2004, cfu/mL, fermentation at 30 ℃ 240d → late stage fermentation (incubation at 60 ℃ for one week) → filtration collection of liquid → fish sauce.
The control group replaced saccharopolyspora s.rosea F2014 with s.hordei F2001, with the rest of the steps unchanged.
Analysis on the effect of saccharopolyspora s.rosea F2014 in reducing the biogenic amine shows that the biogenic amine content in the fermentation product added with s.rosea F2014 is reduced by 25.37% compared with that in the control group.
Example 7: saccharopolyspora s.rosea F2014 applied to fermented sausages for reducing content of biogenic amine
Taking pork (65-80% (w/w) of lean meat and 20-35% (w/w) of fat meat), cleaning, separating fat and lean meat, and cutting into 4-5 cm meat blocks. Putting the lean meat and 5-8% of ice scraps into a cutting and mixing machine, and cutting and mixing for 1-3 min. Taking the mass of pork as a base number, 0.01-0.15% of sodium nitrite, 2-3% of salt, 0.2-0.3% of composite phosphate, 0.05-0.06% of sodium ascorbate and 0.2-0.3% of spice, pepper, garlic, hot pepper and nutmeg which are taken as raw material meat are added (the mass ratio of the spice to the pepper to the garlic to the hot pepper to the nutmeg is 1: 1: 1: 1). Inoculating 8-12% activated saccharopolyspora bacterium liquid (the bacterium concentration in the bacterium liquid is 10)6～107CFU/mL), chopping and mixing for 1-2 min, adding fat meat and about 5-8% of ice chips, and chopping and mixing for 4-6 min. And pouring the pickled vegetable into sausage casings.
The control group replaced saccharopolyspora s.rosea F2014 with s.hordei F2001, with the rest of the steps unchanged.
Analysis on the effect of saccharopolyspora s.rosea F2014 in reducing the biogenic amine shows that the biogenic amine content in the fermentation product added with the saccharopolyspora F2014 is reduced by 35.37% compared with that in the control group.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
South of the Yangtze university (Shaoxing) industry and technology research institute
Zhejiang Guyue Longshan Shaoxing Wine Co.,Ltd.
<120> saccharopolyspora rosea for reducing biogenic amine and application thereof
<130> BAA201689A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1432
<212> DNA
<213> Saccharopolyspora rosea
<400> 1
ccaagtggcg ggcatgctta cacatgcaag tcgaacgctg aagcccgctt cggtgggtgg 60
atgagtggcg aacgggtgag taacacgtgg gtaatctgcc ctgcactctg ggataagcct 120
tggaaacggg gtctaatacc ggataggacg catggccgca tggtctgtgt gtggaaagtt 180
ccggcggtgt aggttgagcc cgcggcctat cagcttgttg gtggggtgat ggcctaccaa 240
ggcgacgacg ggtagccggc ctgagagggt gaccggccac actgggactg agacacggcc 300
cagactccta cgggaggcag cagtggggaa tcttgcgcaa tgggcgaaag cctgacgcag 360
caacgccgcg tgggggatga cggccttcgg gttgtaaacc tctttcgaca tcgacgaagc 420
cttcgggtga cggtaggtgt agaagaagca ccggctaact acgtgccagc agccgcggta 480
atacgtaggg tgcgagcgtt gtccggattt attgggcgta aagagctcgt aggcggtttg 540
tcgcgtcggc cgtgaaaacc tgcagcttaa ctgtgggctt gcggtcgata cgggcagact 600
tgagttcggt aggggagact ggaattcctg gtgtagcggt gaaatgcgca gatatcagga 660
ggaacaccgg tggcgaaggc gggtctctgg gccgatactg acgctgagga gcgaaagcgt 720
ggggagcgaa caggattaga taccctggta gtccacgccg taaacgttgg gcgctaggtg 780
tggggactgg ttccacggtg tccgtgccgt agctaacgca ttaagcgccc cgcctgggga 840
gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag cggcggagca 900
tgtggattaa ttcgatgcaa cgcgaagaac cttacctggg tttgacatgc accagattgc 960
tatggagaca tagtttccct tgtggttggt gtacaggtgg tgcatggctg tcgtcagctc 1020
gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgcccc atgttgccag 1080
cgattcggtc ggggactcgt gggggactgc cggggtcaac tcggaggaag gtggggatga 1140
cgtcaagtca tcatgcccct tatgtccagg gcttcacaca tgctacaatg gctggtacag 1200
agggtggcga taccgtgagg tggagcgaat cccttaaagc cggtctcagt tcggatcggg 1260
gtctgcaact cgaccccgtg aagtcggagt cgctagtaat cgcagatcag cagtgctgcg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcacg tcatgaaagt cggtaacacc 1380
cgaagcccat ggcccaaccg gttccggggg gagtgtcgaa ggtgaccggg gg 1432

Claims (10)

1. A strain of Saccharopolyspora rosea (Saccharopolyspora rosea) F2014, which is preserved in China center for type culture Collection (CCTCC NO) within 12 months and 21 days in 2020, and the preservation address is China, Wuhan and Wuhan university with the preservation number of CCTCC NO: m2020952.
2. A fermentation agent comprising the Saccharopolyspora rosea F2014 according to claim 1.
3. The leavening agent according to claim 2, comprising live cells of the saccharopolyspora rosea F2014 according to claim 1, dried cells obtained by freeze-drying, immobilized cells, mycelia, or a saccharopolyspora rosea F2014 strain existing in any other form.
4. A starter culture according to claim 2 or 3, further comprising other types of bacteria or fungi.
5. A fermentation starter prepared by applying the saccharopolyspora rosea F2014 of claim 1.
6. The use of saccharopolyspora rosea F2014 or metabolites thereof according to claim 1 in reducing biogenic amine content in the field of fermentation.
7. The metabolites according to claim 6 including but not limited to saccharopolyspora rosea F2014 from which the supernatant of the somatic cells is removed after fermentation.
8. Use according to claim 6 or 7, wherein the field of fermentation comprises the preparation of fermented food, drinks or condiments.
9. The use of saccharopolyspora rosea F2014 or a metabolite thereof according to claim 1 for reducing the content of biogenic amines in tobacco shreds.
10. Use of the saccharopolyspora rosea F2014 according to claim 1 for preparing fermented food, beverages or tobacco.
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