WO2022033010A1 - 糖多孢菌组合物及其在食品中的应用 - Google Patents

糖多孢菌组合物及其在食品中的应用 Download PDF

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WO2022033010A1
WO2022033010A1 PCT/CN2021/077377 CN2021077377W WO2022033010A1 WO 2022033010 A1 WO2022033010 A1 WO 2022033010A1 CN 2021077377 W CN2021077377 W CN 2021077377W WO 2022033010 A1 WO2022033010 A1 WO 2022033010A1
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saccharopolyspora
koji
content
fermented
fermentation
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French (fr)
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毛健
刘双平
张晶
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Jiangnan University
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Jiangnan University
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Priority claimed from CN202010812161.6A external-priority patent/CN111979148B/zh
Priority claimed from CN202010812299.6A external-priority patent/CN111961615B/zh
Application filed by Jiangnan University filed Critical Jiangnan University
Priority to KR1020227007359A priority Critical patent/KR102920357B1/ko
Priority to JP2021576248A priority patent/JP7329221B2/ja
Publication of WO2022033010A1 publication Critical patent/WO2022033010A1/zh
Priority to US17/720,375 priority patent/US12089619B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
    • C12J1/00—Vinegar; Preparation or purification thereof
    • C12J1/04—Vinegar; Preparation or purification thereof from alcohol
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/10—Fish meal or powder; Granules, agglomerates or flakes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/38—Other non-alcoholic beverages
    • A23L2/382—Other non-alcoholic beverages fermented
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/24—Synthetic spices, flavouring agents or condiments prepared by fermentation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
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    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/005—Solid or pasty alcoholic beverage-forming compositions
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
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    • C12N1/00—Microorganisms; 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
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    • C12N1/00—Microorganisms; 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
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/04—Actinomyces

Definitions

  • the invention relates to a saccharopolyspora composition and its application in food, and belongs to the technical field of food fermentation.
  • Yellow rice wine is a kind of brewed wine, generally using glutinous rice, corn and millet as raw materials, adding wheat koji and yeast as saccharifying agent and leavening agent. become.
  • rice wine also contains 18 kinds of amino acids, including 8 kinds of essential amino acids. These 8 kinds of amino acids are many times more than the same amount of wine and beer.
  • Regular consumption of rice wine is good for health; rice wine is rich in The antioxidant substances, such as polyphenols, polysaccharides, polypeptides, etc., have antioxidant activity.
  • the brewing of rice wine is different from beer and wine. It adopts an open fermentation process.
  • the fermentation system is rich in amino acids, complex and numerous types of microorganisms, and complex bacterial community structure.
  • the bacteria involved in fermentation are mainly acetic acid bacteria, lactic acid bacteria, Bacillus, Saccharopolyspora. Wait.
  • the metabolites of microorganisms not only bring unique flavor to rice wine, but also make rice wine contain some harmful substances, such as biogenic amines.
  • Biogenic amine is a nitrogen-containing organic basic small molecule compound formed by decarboxylation of amino acids, which is commonly found in animals, plants and microorganisms.
  • a moderate amount of biogenic amine can promote growth, scavenge free radicals, enhance metabolic activity, and enhance immunity. It has important physiological functions, but excessive intake of biogenic amines will cause the expansion of arteries, blood vessels and capillaries, resulting in adverse physiological reactions such as diarrhea, headache, abdominal cramps, vomiting, and even death.
  • Biogenic amines are widely found in a variety of foods, especially in fermented foods such as yogurt, rice wine, liquor, cooking wine, soy sauce, vinegar and wine.
  • Biogenic amines in fermented foods are mainly formed by amino acid decarboxylase produced by microbial metabolism acting on free amino acids.
  • microbial metabolism produces protease and carboxypeptidase, which act on the protein in the grain, decompose to produce low molecular weight peptides and amino acids, and provide abundant precursors for the production of biogenic amines.
  • amino acid decarboxylase it will cause biogenic amines. Generated in bulk.
  • the object of the present invention is to solve the problem that the content of biogenic amines is higher in the existing traditional brewed food, provide the saccharopolyspora with excellent performance and be applied to the fermentation process of wine (white wine and rice wine), sausage and soy sauce to carry out biological enhancement, to reduce
  • the content of biogenic amines in fermented food can improve the taste and flavor of food, and better exert the application of actinomycetes in traditional fermented food.
  • the first object of the present invention is to provide a saccharopolyspora composition comprising Saccharopolyspora hirsuta J2 and Saccharopolyspora jiangxiensis J3;
  • the Saccharopolyspora hirsuta J2 has been deposited in the China Center for Type Culture Collection on April 30, 2020, and the deposit number is CCTCC NO: M 2020103;
  • Saccharopolyspora jiangxiensis J3 has been deposited in the China Center for Type Culture Collection on April 30, 2020, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2020104.
  • the saccharopolyspora japonicum J2 and Jiangxi saccharopolyspora bacteria J3 of the present invention all have the following excellent properties:
  • the pure-bred wheat koji produced by this strain is suitable for the fermentation of rice wine, and can improve the amino acid content in the rice wine;
  • the amount of biogenic amines produced is less than 2.5 mg/L, and the detected amount of biogenic amines is very small;
  • the second object of the present invention is to provide a microbial preparation containing the saccharopolyspora composition.
  • the amount of Saccharopolyspora per gram or per milliliter of starter is ⁇ 1 ⁇ 10 6 CFU.
  • the microbial preparation is to inoculate the saccharopolyspora into an actinomycete liquid medium, and culture at 28-30° C. to obtain a starter containing saccharopolyspora cells.
  • the third object of the present invention is to provide a koji prepared by using the saccharopolyspora composition, and the preparation method is as follows: after crushing the raw materials, adding water, stirring, and sterilizing, inoculating the bacteria in a sterile environment, the inoculation amount is It is 5 ⁇ -15%, the concentration of bacterial liquid is 105 ⁇ 108cfu /mL, and it is cultivated at 25 ⁇ 55°C for 72 ⁇ 96h.
  • the saccharopolyspora koji raw material is boiled and cooled under aseptic conditions; the mixing process of adding koji and raw materials, and the later raking process are all carried out in a sterile environment; the sugar Polyspora koji was inoculated and cultured in a sterile environment.
  • the koji is a mixed wheat koji prepared by using the Saccharopolyspora polyspora polyspora japonicum J2 or the Saccharopolyspora jiangxis J3.
  • the preparation method of described wheat koji comprises the steps:
  • step (3) after the material in step (3) is cooled to below 40° C., inoculate the activated Saccharopolyspora polyspora J2 at a concentration of 10 5 to 10 6 CFU/mL;
  • the fermentation of the step (5) comprises the following steps:
  • the product temperature should be kept between 36-37°C, and the ventilation and spraying should be kept smooth. After about 20 hours, the koji material will agglomerate again, and the koji material will turn white, and the temperature should be controlled below 37°C. Carry out the second turning, and after the second turning, the product temperature should be controlled at 35 ⁇ 2°C.
  • the fourth object of the present invention is to provide the application of the mixed wheat koji in fermented rice wine.
  • the fifth object of the present invention is to provide the application of the mixed wheat koji in the preparation of fermented food, drink or seasoning.
  • the food product includes, but is not limited to, a fermented or semi-fermented food product of sausage.
  • the beverage includes, but is not limited to, rice wine or liquor fermentation.
  • the seasoning includes, but is not limited to, soy sauce or sausage.
  • the application is to ferment after mixing the mixed malt koji with the brewing raw materials.
  • the method is to ferment pure wheat koji by mixing it with raw materials such as rice and wine mother in a fermenter according to the inoculation amount of 12-16%, and the fermentation adopts a traditional fermentation process.
  • the sixth object of the present invention is to provide the application of the saccharopolyspora composition or mixed wheat koji in sausage, rice wine, white wine, soy sauce and cheese to reduce biogenic amines.
  • the biogenic amines include, but are not limited to, tyramine, histamine, putrescine, cadaverine.
  • the brewing of yellow rice wine and white wine is made by first using the Saccharopolyspora to make pure koji, and then adding it to the fermentation of the wine.
  • (1) bacterial strain of the present invention is applied to food fermentation system, and will not affect the normal fermentation of food;
  • the pure-bred wheat koji made with Saccharopolyspora sp. J2 can be used for rice wine fermentation, which can not only promote the alcohol yield but also increase the amino acid content in the rice wine; mg/L, which was increased by 40.37% compared with the control group; and the addition of this strain had no obvious effect on the flavor of traditional rice wine; the content of biogenic amines in the sample group added with S.hirsuta J2 decreased by 21.71% compared with the control group, and at the same time increased
  • the content and nutritional value of amino acids in rice wine were analyzed, and the purpose of increasing the content of amino acids and volatile substances in rice wine and improving the quality of rice wine was achieved.
  • S.hirsuta J2 The amount of biogenic amine produced by S.hirsuta J2 was all less than 2.5mg/L, the amount of biogenic amine detected was very small, and basically no biogenic amine was produced.
  • S.hirsuta J2 has a degradation rate of 77.41% for tyramine, 100% for histamine, 58.1% for putrescine, 47.71% for cadaverine, and 47.71% for total biogenic amines. The degradation rate reaches 72.98%, and it has a good ability to reduce biogenic amines.
  • the saccharopolyspora has the effect of reducing biogenic amines in fermented food at the same time. It is applied to sausage fermentation. After adding S.hirsuta J2 fermentation, the average content of biogenic amines in sausages is 129.60mg/kg, and the reduction of biogenic amines is 129.60 mg/kg.
  • the white wine added with S.hirsuta J2 reduced the biogenic amine content by 24.32%; the soy sauce added with S.hirsuta J2 reduced the biogenic amine content by 34.28% compared with the control group.
  • the cheese added with S.hirsuta J2 reduced the content of biogenic amines by 16.37%.
  • Saccharopolyspora S.hirsuta J2 has the effect of improving quality and reducing damage. It was applied to cigarette fermentation, and the harmful components of fermented tobacco leaves of S.hirsuta J2-tar content, HCN, phenol, NH3 and nitrite were added. Compared with the control group, the content of , respectively, decreased by 23.51%, 16.25%, 19.75%, 27.09% and 37.62%.
  • Saccharopolyspora S.hirsuta J2 has the effect of improving the nutrient conversion rate.
  • the content of organic acids in the fermented feed added with S.hirsuta J2 increased by 26.77% compared with the control group.
  • the amino acid content increased by 21.98%, and the crude protein content increased by 18.53%.
  • the present invention also provides a composite inoculum containing Saccharopolyspora polyspora japonicum J2 and Jiangxi Saccharopolyspora J3, and the composite inoculum has the following purposes:
  • the saccharopolyspora compound bacterial agent has the effect of reducing biogenic amines.
  • the content of biogenic amines in the stinky mandarin fish added with the compound bacterial agent decreased by 20.87% compared with the control group.
  • the content of biogenic amines in the cooking wine can be compared with the control group.
  • the content of biogenic amines in the production of vinegar decreased by 23.16%
  • the content of biogenic amines in the production of cheese was reduced by 13.33% compared with the control group.
  • the saccharopolyspora compound bacterial agent has the effect of improving quality and reducing damage. It is applied in the fermentation of cigarettes, and the harmful components of fermented tobacco leaves-tar content, HCN, phenol, NH 3 and nitrite are added to the composite bacterial agent. Compared with the control group, the content decreased by 30.12%, 17.14%, 22.01%, 16.73% and 27.15% respectively.
  • the saccharopolyspora compound inoculum has the effect of improving the nutrient conversion rate.
  • the content of organic acids in the fermented feed with the compound inoculant was increased by 29.18% compared with the control group, and the content of amino acids was increased by 29.18%. It increased by 10.22%, and the crude protein content increased by 14.48%.
  • Saccharopolyspora hirsuta J2 classified as Saccharopolyspora hirsuta J2 has been deposited in the China Center for Type Culture Collection on April 30, 2020, and the deposit address is Wuhan, China , Wuhan University, the deposit number is CCTCC NO: M 2020103.
  • Figure 1 is a phylogenetic tree of Saccharopolyspora J2.
  • Figure 2 is a phylogenetic tree of Saccharopolyspora J3.
  • Figure 3 shows the changes of physical and chemical indicators during the fermentation of rice wine; (A) alcohol content; (B) reducing sugar; (C) titratable acid; (D) amino acid nitrogen.
  • Figure 4 shows the principal component analysis of flavor compounds in Saccharopolyspora J2 rice wine fermentation samples.
  • Figure 5 shows the principal component analysis of flavor compounds in Saccharopolyspora J3 rice wine fermentation samples.
  • Detection of physical and chemical indicators of rice wine Determination of alcohol content, amino acid nitrogen and total acid were determined in accordance with GB/T 13662-2018. Biogenic amine content and flavor compounds were detected by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The reducing sugar content was determined by DNS method.
  • HPLC high performance liquid chromatography
  • GC-MS gas chromatography-mass spectrometry
  • Detection of sausage physical and chemical indicators crude protein content is determined according to GB5009.3-2010 semi-micro Kjeldahl method; moisture content is determined according to GB/T 9695.15-2008 method.
  • pH measurement Take 10 g of sample and add 90 mL of distilled water to homogenize and let stand for 2 min. Use a pH meter to measure the pH value of the supernatant.
  • Determination method of biogenic amine content accurately measure 1mL of the solution to be tested in a 15mL centrifuge tube, add 1mL saturated NaHCO 3 solution, mix well, add 2mL dansyl chloride (5mg/mL acetone) reagent, mix well and place at a constant temperature of 65°C Derivatize in the dark for 30 min in a water bath, after standing at room temperature, add 0.5 mL of saturated NaCl solution, add 5 mL of ether after mixing, vortex for 20 s, let stand for stratification, transfer the upper organic phase to a 15 mL centrifuge tube, and the lower aqueous phase Extraction was performed again, and the two extracts were combined and dried under nitrogen in a water bath at 50°C. Add 1 mL of acetonitrile, shake and mix well, dissolve the residue, pass through a 0.22 ⁇ m filter membrane, and determine by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the wheat koji samples were collected from a rice wine factory in Shaoxing City, Zhejiang province, and the collected wheat koji samples were stored in a sealed sterile plastic bag at 4°C. Weigh 5 g of wheat koji into a 50 mL centrifuge tube, add 30 mL of distilled water, and place it in a 30°C shaker incubator for 30 min.
  • Actinomycetes screening medium potassium nitrate 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.01g/L, sodium chloride 0.5g/L, soluble starch 20.0g /L, agar 15.0g/L, pH 7.2-7.4 (25°C).
  • a sterile pipette to draw 1 mL of the sample into a 15 mL sterile centrifuge tube, add sterile water to 10 mL, and mix thoroughly to prepare a 10-1 sample homogenate.
  • a sterile pipette to draw 1 mL of the 10 -1 sample homogenate into a 15mL sterile centrifuge tube, add sterile water to 10 mL, and mix thoroughly to prepare a 10 -2 sample homogenate solution.
  • a 10-1 to 10-6 ten-fold increase series of wheat koji, rice milk water, and fermented mash diluted homogenous solution are prepared.
  • the genomes of the screened strains were extracted, and the screened strains were subjected to 16S rDNA amplification and sequencing.
  • PCR amplification primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3').
  • the PCR amplification system (50 ⁇ L) is: 2 ⁇ Taq PCR Master Mix 25 ⁇ L, 1 ⁇ L of upper and lower primers, 1 ⁇ L of template, and 22 ⁇ L of sterile water to make up to 50 ⁇ L.
  • PCR amplification program pre-denaturation at 94°C for 3 min, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 2 min, a total of 35 cycles, and a final extension at 72°C for 8 min.
  • PCR products were detected by 1% agarose gel electrophoresis, and sent to a gene sequencing company for sequencing. According to the returned sequencing results, BLAST sequences were compared through the NCBI official website, and the obtained 16S rDNA sequences were used for BLAST comparison.
  • Phylogenetic analysis the results are shown in Figures 1-2, the nucleotide sequence (SEQ ID NO.1) of strain J2 is identical to that of Saccharopolyspora S.hirsuta (GenBank accession numbers: MN515057.1 and NR_118870.1). The source similarity was over 98.30%, and the strain was named Saccharopolyspora polyspora polyspora japonicum J2.
  • the nucleotide sequence (SEQ ID NO.2) of the strain J3 has a homology similarity of more than 98.84% with Saccharopolyspora S.jiangxiensis (GenBank similar sequence number: MG255179.1), and the strain is named Jiangxi sugar Polyspora J3.
  • Bacterial activation inoculate the preserved Saccharopolyspora polyspora J2 into the actinomycete liquid medium with an inoculum of 10%, incubate at 30° C. for 48 hours in a shaker, and obtain a first-class seed liquid.
  • the activated strains were inoculated into the actinomycete liquid medium, the inoculum amount was 10%, and the culture was shaken for 48 hours, the rotation speed was 150r/min, and the temperature was 30°C.
  • Sample pretreatment The strains were inoculated into the detection medium for biogenic amine production and the detection medium for biogenic amine degradation, cultured in a shaker at 28°C for 5 d, and centrifuged at 12000 r/min for 5 min to collect the supernatant.
  • Actinomycetes liquid culture medium potassium nitrate 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.01g/L, sodium chloride 0.5g/L, soluble starch 20.0g /L, pH 7.2-7.4 (25°C).
  • Detection of biogenic amine production medium add L-tyrosine 0.4g/L, L-histidine 1g/L, L-lysine 1g/L, L-ornithine 1g/L to actinomycete liquid medium L,5'-pyridoxal phosphate 0.05g/L.
  • biogenic amine degradation medium add 50 mg/L biogenic amines (including histamine, tyramine, cadaverine, putrescine, spermine, spermidine, tryptamine, ⁇ -phenethylamine) to the actinomycete liquid medium , adjust the pH to 6.0-6.2.
  • biogenic amines including histamine, tyramine, cadaverine, putrescine, spermine, spermidine, tryptamine, ⁇ -phenethylamine
  • Jiangxi Saccharopolyspora J3 had a degradation rate of 81.55% for tyramine, 100% for histamine, 51.8% for putrescine, and 40.01% for cadaverine, respectively. The degradation rate of total biogenic amines reached 69.09%. It shows that the strain has a good ability to reduce biogenic amines.
  • Actinomycetes liquid culture medium potassium nitrate 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.01g/L, sodium chloride 0.5g/L, soluble starch 20.0g /L, pH 7.2-7.4 (measured at 25°C).
  • PDA medium potato flour 6.0g/L; glucose 20.0g/L, agar 20.0g/L, pH 5.4-5.8, autoclaved at 121°C for 15min; solid medium was added on this basis.
  • MRS medium beef extract 10g/L, peptone 10g/L, yeast extract 0.5g/L, glucose 20g/L, Tween 80 0.10g/L, sodium acetate 5g/L, dipotassium hydrogen phosphate 2g/L, lemon Diammonium hydrogen acid 2g/L, magnesium sulfate 0.58g/L, manganese sulfate 0.28g/L.
  • Saccharopolyspora polyspora japonicum J2 screened in Example 1 was inoculated into the actinomycete liquid medium at an inoculation amount of 10%, and incubated at 30° C. for 48 hours in a shaker to obtain a first-class seed liquid.
  • the activated strain was inoculated into the actinomycete liquid medium, the inoculum amount was 10%, and the culture was shaken for 48 hours, the rotation speed was 150 r/min, and the temperature was 25 to 45 °C to obtain a bacterial solution with a bacterial concentration of 10 5 to 10 6 cfu/mL. , to be used for the preparation of pure-bred wheat koji after being cultivated and matured.
  • the stored Lactobacillus plantarum (Lactobacillus plantarum) was inoculated into the MRS medium, the inoculum amount was 10%, and the anaerobic culture at a constant temperature of 37°C was carried out for 24 hours to obtain a first-class seed culture liquid, and the activated seed liquid was inoculated into the MRS liquid culture again.
  • the inoculum amount was 10%, and the anaerobic culture was carried out at a constant temperature of 37°C for 24 hours to obtain a bacterial liquid with a bacterial concentration of 10 5 to 10 6 cfu/mL.
  • Jiangxi Saccharopolyspora J3 purebred wheat koji was prepared according to the same method as above, except that Saccharopolyspora japonicum J2 was replaced with Jiangxi Saccharopolyspora J3.
  • step (3) after the material in step (3) is cooled to 36° C., inoculate the bacterial strain activated according to the method of Example 2, the concentration of the inoculated bacterial liquid is 10 5 to 10 6 cfu/mL, and the inoculation amount is 5 ⁇ - 15%.
  • the product temperature should be kept between 36-37°C, and the ventilation and spraying should be kept smooth. After about 20 hours, the koji material will agglomerate again, and the koji material will turn white. It is difficult to control the temperature below 37°C. , perform the second turning, after the second turning, the product temperature should be controlled at about 35 °C.
  • Koji production culture for 72-96 hours; after the incubation, place the wheat koji in a 4-7°C freezer for storage for later use;
  • the pure wheat koji of Saccharopolyspora polyspora japonicum J2 and the pure strain of Saccharopolyspora jiangxii J3 with a cell magnitude of 10 15 CFU/g were prepared and obtained respectively.
  • the mixed wheat koji used for fermentation of the brewing alcoholic beverage is to mix the pure wheat koji of Saccharopolyspora polyspora japonicum J2 and Jiangxi Saccharopolyspora japonicus J3 in a mass ratio of 1:(0.8-1.5).
  • Example 5 Application of Saccharopolyspora pure wheat koji in rice wine fermentation
  • the raw material ratio (in every liter of fermentation volume) of the traditional rice wine fermentation selected by the present embodiment is:
  • Steamed rice 500g; water 417L; wine mother: 38g;
  • Yeast activation culture transfer the yeast in the glycerol storage tube to the YPD medium on a sterile operating table, and cultivate at 30 ° C and 150 r/min for 24 hours; The transferred yeast was cultured for 18-24 h at 30°C and 150 r/min for use.
  • the first four days are the pre-fermentation stage, the temperature is controlled at 28-30 °C, the fermentation is carried out for 4 days, the first 4 days are opened no less than once a day, and the first raking time is 8-10 hours; the post-fermentation stage, the temperature is 13-15 °C, stirring every day Open the rake once and continue to ferment for 10 to 15 days.
  • the control group (TF Control) is to adjust the pure wheat koji in c) in this example to 39.3 g/L of raw wheat koji and 6.0 g/L of cooked wheat koji sampled from the factory.
  • Example 6 Application of Saccharopolyspora mixed wheat koji in rice wine fermentation
  • the mixed wheat koji fermentation group used the compound wheat koji of the pure strain of Saccharopolyspora polyspora japonicum J2 and the pure strain of Saccharopolyspora japonica J3 and fermented with Saccharomyces cerevisiae using traditional brewing methods.
  • the compound bacterial agent group (Mix) was added with Saccharopolyspora polyspora japonicum J2 pure wheat koji and Jiangxi Saccharopolyspora J3 pure wheat koji in a ratio of 1:1, with a total amount of 45.3 g.
  • Amino acid content in rice wine fermentation samples The amino acid content in fermented rice wine was analyzed by HPLC.
  • the amino acid content in the experimental group with the addition of S.hirsuta J2 reached 6434.81 ⁇ 123.3 mg/L, an increase of 40.37% compared to the control group.
  • the amino acid contents of Mix, A.flavus and A.oryzae experimental groups were not significantly different, but they were all significantly higher than those of the control group (TF Control). The content of some amino acids was significantly higher than that of the control group.
  • gaga is ⁇ -aminobutyric acid.
  • Flavor analysis of purebred and conventionally fermented fermentations Variations and similarities of flavor components in purebred and conventionally fermented samples were analyzed using principal component analysis. Biplot analysis of all samples showed that the cumulative contribution rate of variance of the first two principal components in rice wine fermented by Saccharopolyspora polyspora J2 and Jiangxi Saccharopolyspora J3 were both 83.6%, which could explain the flavor differences in most of the fermented samples .
  • the traditional fermentation group was clustered with the S.jiangxiensis J3 group and was clearly separated from the Aspergillus (A.flavus and A.oryzae) group and the L.plantarum group. This indicated that Saccharopolyspora was involved in the synthesis of most flavor substances and played a leading role in the fermentation of rice wine.
  • Embodiment 7 the application of saccharopolyspora inoculum in rice wine fermentation
  • the rice wine fermentation was carried out with the traditional rice wine blanking formula in Example 5, and the experimental group was respectively set with Mix group and Jiangxi Saccharopolyspora J3 group, the difference was that the inoculation ratio of wheat koji was 10%; Jiangxi Saccharopolyspora J3 group was inoculated Jiangxi Saccharopolyspora J3 group pure wheat koji; Mix group was inoculated with the compound strain koji prepared according to the method of Example 3 using the mixed bacterial solution of Jiangxi Saccharopolyspora J3 and Saccharopolyspora polyspora japonicum J2. Yellow rice wine brewing process and index determination method were carried out with reference to Example 4.
  • the biogenic amine contents of the samples inoculated with compound bacterial agent Mix group and Jiangxi Saccharopolyspora J3 group were 15.57 ⁇ 0.44mg/L and 16.88 ⁇ 1.41mg/L respectively, which were lower than 26.75 ⁇ 2.39mg/L of the control group.
  • Example 8 Application of Saccharopolyspora sp. J2 in fermented sausage to reduce biogenic amine content
  • Actinomycetes liquid culture medium potassium nitrate 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.01g/L, sodium chloride 0.5g/L, soluble starch 20.0g /L, pH 7.2-7.4 (measured at 25°C).
  • the preserved saccharopolyspora S.hirsuta J2 was inoculated into the actinomycete liquid medium at 10% of the inoculum, and cultured in a shaking table at 30°C for 59 hours to obtain a first-class seed liquid.
  • the activated strain was inoculated into the actinomycete liquid medium, the inoculum amount was 10 %, the shaking table was cultivated for 48h, the rotation speed was 150r /min, and the temperature was 30°C. for fermented sausages.
  • Example 1 The method in Example 1 was used to carry out bacterial activation and determination of biogenic amine content.
  • Sample pretreatment method Weigh 5.0 g of the minced sample into a 50 mL centrifuge tube, add 20 mL of 5% trichloroacetic acid and sonicate for 30 min, transfer to a 50 mL stoppered centrifuge tube, centrifuge at 6 000 r/min for 10 min, and transfer the supernatant The solution was added to a 50 mL volumetric flask, the residue was extracted once more with 20 mL of the above solution, and the supernatant was combined and diluted to the mark.
  • Embodiment 9 Saccharopolyspora J2 is applied to reduce biogenic amine content in liquor
  • Saccharopolyspora pure wheat koji used in liquor brewing refers to the production method of wheat koji in Example 3.
  • the determination of biogenic amine content was carried out by the method in Example 1.
  • the liquor brewing method adopts two rounds of fermentation.
  • the first round of fermentation after the sorghum is steamed, it is cooled to 25°C by air cooling, 4% Aspergillus oryzae seed liquid is added, and cultured at 28°C for 24 hours.
  • 10 % of medium-temperature Daqu was added, and the seed liquid of Saccharomyces cerevisiae was added in the proportion of 1%.
  • Example 10 Application of Saccharopolyspora sp. J2 in soy sauce to reduce biogenic amine content
  • Example 1 The method in Example 1 was used to carry out bacterial activation and determination of biogenic amine content. Soy sauce is brewed by high-salt dilution method:
  • the fermented glutinous rice is subjected to plate and frame pressing to remove the fermented glutinous rice. After the pressing, diatomaceous earth filtration and membrane filtration were performed to remove the precipitate. Filter-clarified soy sauce is pasteurized and then filled.
  • Example 11 Saccharopolyspora sp. J2 is used to reduce biogenic amine content in cooking wine
  • Example 5 pure-bred fermented yellow rice wine was obtained, 10% by mass of table salt was added to the fermented yellow rice wine, and it was sterilized by a sterilizer at 85° C. for 30 minutes for hot filling.
  • the effect of bacterial strains on reducing the content of biogenic amines in cooking wine was analyzed: the content of biogenic amines in cooking wine was detected by high performance liquid phase method. Compared with the control group, the content of biogenic amines in the sample group added with Saccharopolyspora japonicum J2 decreased.
  • Example 12 Saccharopolyspora polyspora sp. J2 is used to reduce biogenic amine content in vinegar
  • Example 5 pure-bred fermented rice wine was obtained as the raw material for acetic fermentation; the method in Example 1 was used to measure the content of biogenic amines.
  • Acetic acid fermentation adopts solid-state fermentation process: mix bran, bran and rice wine in a mass ratio of 1:4:10, add 5% vinegar fermented grains, and turn fermented grains from the surface of the material every day for the first 2 days after inoculation. 35-42°C. Turn to the bottom of the material by 6-8 days. From the 8th to the 12th day, the fermented grains were turned from the bottom every day, and the temperature dropped naturally. Raw vinegar is obtained after separation from vinegar grains, which is aged for 12 months after being sterilized at 85°C for 30 minutes. Hot filling after autoclaving before filling.
  • the quality of the prepared vinegar was measured, and the acetic acid content in the obtained solid-state fermentation brewed vinegar was all 55 g/L.
  • the content of biogenic amines in vinegar was analyzed: the content of biogenic amines in cooking wine was detected by high performance liquid phase method. Compared with the control group, the content of biogenic amines in the sample group added with Saccharopolyspora sp. J2 decreased.
  • Example 13 Saccharopolyspora polyspora polyspora japonicum J2 is used to reduce biogenic amine content in cheese fermentation
  • Example 1 The method in Example 1 was used to carry out bacterial activation and determination of biogenic amine content.
  • the fermented cheese products were measured, and the content of biogenic amines in the cheese products added with Saccharopolyspora polyspora J2 decreased by 16.37% compared with the control group
  • Example 14 Saccharopolyspora sp. J2 is used to improve quality and reduce damage in cigarette fermentation
  • the quality of fermented tobacco leaves was measured, and the aroma components of the obtained fermented tobacco leaves were significantly increased, the miscellaneous gases were reduced, and the irritation was weakened; the harmful components in the fermented tobacco leaves-tar, HCN, phenol, NH 3 and nitrite content were decreased compared with the control group. up 23.51%, 16.25%, 19.75%, 27.09% and 37.62%.
  • Example 15 Saccharopolyspora polysporum sp. J2 is used to improve nutrient conversion rate in feed fermentation
  • Strain activation was performed in the same manner as in Example 1.
  • the rice bran, straw and soybean meal are uniformly mixed according to the ratio of (1-5):(1-5):2, and pulverized to prepare a fermented product.
  • the fermentation temperature is 30-40 DEG C
  • the fermentation time is 4-9 d
  • the obtained fermented feed has a special aroma, rich nutrition and balanced amino acids. Compared with the control group, the content of organic acids is increased by 26.77%, the content of amino acids is increased by 21.98%, and the content of crude protein is increased by 18.53% .
  • Example 16 Saccharopolyspora in reducing biogenic amine content in fermented fish
  • Fermentation liquid preparation take quality drinking water such as mandarin fish, take this as 100%, add 6% salt, 1% green onion, 0.6% ginger, 0.1% star anise, 0.05% fennel, 0.05% cumin, 0.01% Capsicum, 0.01% Chinese prickly ash, 300000U neutral protease, mix well to get fermentation broth;
  • Determination method of biogenic amines Weigh 5.0 g of minced fish meat sample into a 50 mL centrifuge tube, add 20 mL of 5% trichloroacetic acid and sonicate for 30 min, transfer to a 50 mL centrifuge tube with a stopper, centrifuge at 6 000 r/min for 10 min, transfer The supernatant was transferred to a 50 mL volumetric flask, the residue was extracted once more with 20 mL of the above solution, the supernatant was combined and diluted to the mark.
  • the biogenic amines of the stinky mandarin fish enhanced with the compound bacterial agent Mix decreased by 20.87% compared with the control group.
  • Example 17 Saccharopolyspora is used to reduce biogenic amine content in cooking wine
  • Example 5 pure-bred fermented yellow rice wine was obtained, 10% by mass of table salt was added to the fermented yellow rice wine, and it was sterilized by a sterilizer at 85° C. for 30 minutes for hot filling.
  • the effect of bacterial strains on reducing biogenic amines in cooking wine was analyzed: the content of biogenic amines in cooking wine was detected by high performance liquid phase method. Compared with the control group, the sample group added with compound bacterial agent Mix and Jiangxi Saccharopolyspora J3 decreased by 23.16% respectively. % and 18.91%.
  • Example 18 Saccharopolyspora is used to reduce biogenic amine content in vinegar
  • Example 5 pure-bred fermented rice wine was obtained as the raw material for acetic fermentation; the method in Example 1 was used to measure the content of biogenic amines.
  • Acetic acid fermentation adopts solid-state fermentation process: mix bran, bran and rice wine in a mass ratio of 1:4:10, add 5% vinegar fermented grains, and turn fermented grains from the surface of the material every day for the first 2 days after inoculation. 35-42°C. Turn to the bottom of the material by 6-8 days. On the 8th to 12th day, the rice grains were turned from the bottom every day, and the temperature dropped naturally. Raw vinegar is obtained after separation from vinegar grains, which is aged for 12 months after being sterilized at 85°C for 30 minutes. Hot filling after autoclaving before filling.
  • Example 19 Saccharopolyspora is used to reduce biogenic amine content in cheese fermentation
  • Example 1 The method in Example 1 was used to carry out bacterial activation and determination of biogenic amine content. Preparation of Saccharopolyspora polyspora polysporidia J2 bacterial liquid and Jiangxi Saccharopolyspora japonicum J3 bacterial liquid respectively, and the two were mixed at a ratio of 1:0.8 to 1.4 to obtain a composite bacterial liquid mix.
  • the fresh milk is subjected to homogenization and pasteurization, and after it is cooled to room temperature, a mixed bacterial liquid of Lactobacillus bulgaricus and Lactobacillus plantarum with a bacterial cell number ratio of 0.1 mL/L of 1:1 is added as a starter (bacteria liquid).
  • the fermented cheese products were measured, and the content of biogenic amines in the cheese products added with Jiangxi Saccharopolyspora J3 and the compound bacterial agent Mix decreased by 13.33% and 21.66% compared with the control group.
  • Example 20 Saccharopolyspora is used to improve quality and reduce damage in cigarette fermentation
  • the activated bacterial liquid was centrifuged at 4°C and 10 000g for 15min, the collected bacterial cells were prepared into a bacterial liquid of 10 5 -10 6 cfu/mL with sterile water, and the bacterial liquid was evenly sprayed on the surface of the tobacco leaves and mixed well.
  • Treated with an equal amount of sterile water as a control placed in a constant temperature and humidity box at 30 to 37 ° C and a humidity of 70 to 80% for 15 days of fermentation and culture, and ventilated every day. After the culture, the tobacco leaves were dried to a moisture content of less than 15% .
  • the quality of the fermented tobacco leaves was measured, and the aroma components of the obtained fermented tobacco leaves were significantly increased, the miscellaneous gas was reduced, and the irritation was weakened; the harmful components in the fermented tobacco leaves of Jiangxi Saccharopolyspora J3-tar content, HCN, phenol, NH 3 and nitrite were added.
  • the content decreased by 32.65%, 17.55%, 17.69%, 25.36% and 29.17% respectively.
  • the content of harmful components-tar, HCN, phenol, NH 3 and nitrite in the fermented tobacco leaves added with the compound bacterial agent Mix decreased by 30.12%, 17.14%, 22.01%, 16.73% and 27.15%, respectively, compared with the control group.
  • Example 21 Saccharopolyspora is used to improve nutrient conversion rate in feed fermentation
  • the rice bran, straw and soybean meal are uniformly mixed according to the ratio of (1-5):(1-5):2, and pulverized to prepare a fermented product.
  • the quality of the fermented feed was analyzed: the obtained fermented feed had a special aroma, rich nutrition and balanced amino acids.
  • the content of organic acids in the fermented feed with Jiangxi Saccharopolyspora J3 increased by 37.26%, and the content of amino acids increased by 18.57%. %, the crude protein content increased by 23.41%.
  • the content of organic acids in the fermented tobacco leaves added with the compound bacterial agent Mix increased by 29.18%, the content of amino acids increased by 10.22%, and the content of crude protein increased by 14.48%.

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Abstract

糖多孢菌组合物及其在食品中的应用，该组合物包括从麦曲中筛选获得了具有降低生物胺含量作用的披发糖多孢菌J2，和江西糖多孢菌J3。将两个菌株制备混合糖多孢菌制剂用于发酵酒精饮品、发酵食品或发酵调味品制备过程中，能够在降低生物胺含量的同时提高氨基酸含量、提高发酵产品的营养价值，进而达到增强发酵食品品质、提高发酵食品安全性的作用。

Description

糖多孢菌组合物及其在食品中的应用 技术领域
本发明涉及糖多孢菌组合物及其在食品中的应用，属于食品发酵技术领域。
背景技术
黄酒是一种酿造酒，一般以糯米、玉米和黍米为原料，添加麦曲、酵母作为糖化剂、发酵剂，经蒸煮、加曲、糖化发酵、压榨、过滤、煎酒、贮存、勾兑而成。黄酒中除含有水、乙醇主要成分外，还含有18种氨基酸，其中包括8种必需氨基酸，这8种氨基酸比同量葡萄酒、啤酒中多数倍，经常饮用黄酒有益于身体健康；黄酒中含有丰富的抗氧化物质，如多酚、多糖、多肽等，具有抗氧化活性。黄酒酿造不同于啤酒和葡萄酒，采用开放型发酵工艺，发酵体系中氨基酸含量丰富，微生物种类复杂数量繁多，细菌群落结构复杂，参与发酵的细菌主要有醋酸菌、乳酸菌、芽孢杆菌、糖多孢菌等。但是微生物的代谢产物在给黄酒带来独特风味的同时也会使黄酒中含有一些有害物质，如生物胺等。
生物胺是一种含氮有机碱性小分子化合物，由氨基酸脱羧形成，普遍存在于动植物和微生物体内，适量生物胺能够促进生长、清除自由基、增强代谢活性、增强免疫力，在人体内具有重要的生理功能，但过量生物胺的摄入则会引起动脉、血管和微血管的扩大，导致腹泻、头痛、腹部痉挛、呕吐等不良生理反应，甚至会导致死亡。生物胺广泛存在于多种食品中，在酸奶、黄酒、白酒、料酒、酱油、食醋和葡萄酒等发酵食品中含量尤为丰富。
发酵食品中的生物胺主要是由微生物代谢产生的氨基酸脱羧酶作用于游离氨基酸形成的。发酵过程中微生物代谢产生蛋白酶和羧肽酶作用于谷物中的蛋白质，分解产生低分子肽和氨基酸，为生物胺的生成提供了丰富的前体物质，在氨基酸脱羧酶，将会造成生物胺的大量生成。
目前，国内外研究中尚未有关于将糖多孢菌应用于食品发酵及降生物胺方面的报道。因此，利用现代生物技术，筛选优良性能的微生物对于生产高质量、高产量、风味独特优质发酵食品，以及提高发酵食品的安全性具有重要意义。
发明内容
本发明的目的在于解决现有传统酿造食品中生物胺含量较高的问题，提供性能优良的糖多孢菌应用于酒类(白酒和黄酒)、香肠和酱油的发酵过程进行生物强化，以降低发酵食品中的生物胺含量，提高食品的口感和风味，更好的发挥放线菌在传统发酵食品中的应用。
本发明的第一个目的是提供糖多孢菌组合物，含有披发糖多孢菌(Saccharopolyspora hirsuta)J2和江西糖多孢菌(Saccharopolyspora jiangxiensis)J3；
所述披发糖多孢菌(Saccharopolyspora hirsuta)J2，已于2020年4月30日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020103；
所述江西糖多孢菌(Saccharopolyspora jiangxiensis)J3，已于2020年4月30日保藏于中国典型培养物保藏中心，保藏地址为中国，武汉，武汉大学，保藏编号为CCTCC NO：M 2020104。
本发明的披发糖多孢菌J2和江西糖多孢菌J3，均具有如下优良的性能：
(1)应用于食品发酵体系，不会影响到食品的正常发酵；
(2)该菌株制作的纯种麦曲适用于黄酒的发酵，可以提高黄酒中氨基酸含量；
(3)生物胺产生量均小于2.5mg/L，生物胺检出量极少；
(4)对酪胺、组胺、腐胺、尸胺均有降解作用；
(5)可以适用于香肠、黄酒、白酒、奶酪和酱油发酵，并且具有降生物胺的能力。
本发明的第二个目的是提供含有所述糖多孢菌组合物的微生物制剂。
在一种实施方式中，所述每克或每毫升发酵剂中的糖多孢菌的数量≥1×10 6CFU。
在一种实施方式中，所述微生物制剂是将所述糖多孢菌接种至放线菌液体培养基中，28～30℃培养获得含有糖多孢菌细胞的发酵剂。
本发明的第三个目的是提供应用所述糖多孢菌组合物制备的曲，其制备方法为：将原料破碎经加水搅拌、灭菌后，在无菌环境下接入菌种，接种量为5‰-15％，菌液浓度为10 5～10 8cfu/mL，25～55℃培养72～96h，培养结束后，保藏备用。
在一种实施方式中，所述糖多孢菌曲原料蒸煮后在无菌条件下摊凉；接入曲和原料混匀过程，以及后期开耙过程均在无菌环境中进行；所述糖多孢菌曲在无菌环境中接种和培养得到。
在一种实施方式中，所述曲是应用所述披发糖多孢菌J2或所述江西糖多孢菌J3制备的纯种麦曲混合后的混合麦曲。
在一种实施方式中，所述麦曲的制备方法包括如下步骤：
(1)碾麦：小麦破碎程度为每粒3-5片，带少量粉末，使麦粒组织破碎，淀粉外露；
(2)润麦：向步骤(1)处理后的物料中物料质量加入30-40％的清水，搅拌15-25min，使其充分均匀的吸收水分；
(3)蒸煮灭菌：将步骤(2)处理后的物料蒸煮灭菌；
(4)接种：待步骤(3)的物料降温至低于40℃后，接种活化后的披发糖多孢菌J2，接种浓度为10 5～10 6CFU/mL；
(5)发酵。
在一种实施方式中，所述步骤(5)的发酵包括如下步骤：
a)孢子发芽期：曲料进盘6小时后，品温缓慢上升至34-35℃左右，自控模式启动小风量间接通风，每次5-10分钟，间隔2小时，使品温降到32℃，要求均匀吹透；
b)菌丝生长期：间歇通风3-5次后，菌丝开始生长，品温升至35℃以上，曲料开始结块，此时应连续通风，维持品温35±2℃；
c)菌丝繁殖期：接种后12小时，品温上升较快，此时应视第一次结块情况进行翻曲，翻曲前，先升起测温探头，开启翻曲机，后摊平，放下测温头，开启通风及喷雾系统；
d)第一次翻曲后，品温保持在36-37℃之间，保持通风喷雾顺畅，约20小时后，曲料再次结块，眼观曲料发白，温度控制在37℃以下，进行第二次翻曲，二次翻曲过后，品温应控制在35±2℃。
本发明的第四个目的是提供所述混合麦曲在发酵黄酒中的应用。
本发明的第五个目的是提供所述混合麦曲在制备发酵食品、饮品或调味品中的应用。
在一种实施方式中，所述食品包括但不限于香肠的发酵或半发酵食品。
在一种实施方式中，所述饮品包括但不限于黄酒或白酒发酵。
在一种实施方式中，所述调味品包括但不限于酱油或香肠。
在一种实施方式中，所述应用是采用所述混合麦曲与酿酒原料混合后进行发酵。
在一种实施方式中，所述方法是将纯种麦曲按照12～16％的接种量在发酵罐中与米饭、酒母等原料混匀后进行发酵，发酵采用传统发酵工艺。
本发明的第六个目的是提供所述糖多孢菌组合物或混合麦曲在香肠、黄酒、白酒、酱油、奶酪中降生物胺的应用。
在一种实施方式中，所述生物胺包括但不限于酪胺、组胺、腐胺、尸胺。
在一种实施方式中，所述酿造黄酒和白酒是先利用所述糖多孢菌制成纯种曲，添加到酒类的发酵中。
本发明的有益效果：
(1)本发明的菌株应用于食品发酵体系，不会影响到食品的正常发酵；
(2)用披发糖多孢菌J2制作的纯种麦曲可用于黄酒发酵，不仅可以促进酒精产率而且可以提高黄酒中氨基酸含量；纯种发酵黄酒中氨基酸的含量最高达到了6434.81±123.3mg/L，相比于对照组样品提高了40.37％；且该菌株的添加对传统黄酒风味影响不明显；添加S.hirsuta J2的样品组相比较对照组生物胺含量降低了21.71％，同时提高了黄酒中氨基酸态含量和营养 价值，达到了提高黄酒中氨基酸和挥发性物质含量和提升黄酒品质目的。
(3)S.hirsuta J2的生物胺产生量均小于2.5mg/L，生物胺检出量极少，基本不产生生物胺。S.hirsuta J2对酪胺的降解率达到77.41％，对组胺的降解率达到100％，对腐胺的降解率分别达到58.1％，对尸胺的降解率达到47.71％，对总生物胺的降解率达到72.98％，具有良好的降生物胺的能力。
(4)该糖多孢菌同时具有降发酵食品中生物胺的效果，将其应用于香肠发酵中，添加S.hirsuta J2发酵后香肠中生物胺平均含量为129.60mg/kg，生物胺的降低效果显著，比对照组降低了36.19％；添加S.hirsuta J2的白酒相比较对照组生物胺含量降低了24.32％；添加S.hirsuta J2的酱油相比较对照组生物胺含量降低了34.28％。添加S.hirsuta J2的奶酪相较于对照组生物胺含量降低了16.37％，
(5)糖多孢菌S.hirsuta J2具有提质降害的作用，将其应用于香烟发酵中，添加S.hirsuta J2的发酵烟叶有害成分-焦油量、HCN、苯酚、NH3和亚硝酸盐的含量相较对照组分别下降了23.51％，16.25％，19.75％，27.09％和37.62％。
(6)糖多孢菌S.hirsuta J2具有提高营养转化率的作用，将其应用于饲料发酵中，添加S.hirsuta J2的发酵饲料中有机酸的含量相较于对照组提高了26.77％，氨基酸含量提高了21.98％，粗蛋白含量提高了18.53％。
本发明还提供了含有披发糖多孢菌J2和江西糖多孢菌J3的复合菌剂，该复合菌剂具有如下用途：
(1)添加糖多孢菌复合菌剂发酵黄酒中氨基酸的含量最高；菌株的添加对传统黄酒风味影响均不明显；对生物胺含量的降低降低效果相比于对照组降低了42.17％，提高了黄酒中氨基酸态含量和营养价值，达到了提高黄酒中氨基酸和挥发性物质含量和提升黄酒品质目的；
(2)糖多孢菌复合菌剂具有降生物胺效果，添加复合菌剂的臭鳜鱼生物胺含量比对照组下降了20.87％，用于生产料酒可使料酒中的生物胺含量相比较对照组降低23.16％，用于生产食醋可使生物胺含量相比较对照组降低了25.08％，用于生产奶酪可使奶酪中生物胺含量相较于对照组降低了13.33％。
(3)糖多孢菌复合菌剂具有提质降害的作用，将其应用于香烟发酵中，添加复合菌剂的发酵烟叶有害成分-焦油量、HCN、苯酚、NH 3和亚硝酸盐的含量相较对照组分别下降了30.12％，17.14％，22.01％，16.73％和27.15％。
(4)糖多孢菌复合菌剂具有提高营养转化率的作用，将其应用于饲料发酵中，添加复合菌剂的发酵饲料中有机酸的含量相较于对照组提高了29.18％，氨基酸含量提高了10.22％，粗蛋白含量提高了14.48％。
生物材料保藏
江西糖多孢菌(Saccharopolyspora jiangxiensis)J3，分类命名为江西糖多孢菌(Saccharopolyspora jiangxiensis)J3，已于2020年4月30日保藏于中国典型培养物保藏中心，保藏地址为中国，武汉，武汉大学，保藏编号为CCTCC NO：M 2020104。
披发糖多孢菌(Saccharopolyspora hirsuta)J2，分类命名为披发糖多孢菌(Saccharopolyspora hirsuta)J2，已于2020年4月30日保藏于中国典型培养物保藏中心，保藏地址为中国，武汉，武汉大学，保藏编号为CCTCC NO：M 2020103。
附图说明
图1为糖多孢菌J2的系统发育树。
图2为糖多孢菌J3的系统发育树。
图3为黄酒发酵过程中理化指标的变化；(A)酒精度；(B)还原糖；(C)可滴定酸；(D)氨基酸态氮。
图4为糖多孢菌J2黄酒发酵样品风味物质的主成分分析。
图5为糖多孢菌J3黄酒发酵样品风味物质的主成分分析。
具体实施方式
黄酒理化指标的检测：酒精度、氨基酸态氮和总酸的测定按照GB/T 13662-2018进行测定。生物胺含量风味物质采用高效液相(HPLC)和气质联用仪器(GC-MS)进行检测。还原糖含量测定采用DNS方法。
香肠理化指标的检测：粗蛋白含量按照GB5009.3-2010半微量凯氏定氮法进行测定；水分含量按照GB/T 9695.15-2008方法进行测定。pH测定：取10g样品加入90mL蒸馏水匀浆后静置2min，用pH计测定上清液pH值。
生物胺含量测定方法：准确量取1mL待测液于15mL离心管中，加入1mL饱和NaHCO 3溶液，混匀，加入2mL丹磺酰氯(5mg/mL丙酮)试剂，混匀后置于65℃恒温水浴锅中黑暗衍生30min，室温静置后，加入0.5mL饱和NaCl溶液，混匀后加入5mL乙醚，涡旋振荡20s，静置分层后，转移上层有机相于15mL离心管中，下层水相再萃取一次，合并两次萃取液，50℃水浴下氮气吹干。加入1mL乙腈振荡混匀，溶解残留物，过0.22μm滤膜，通过高效液相色谱法(HPLC)测定。
实施例1：糖多孢菌的筛选及鉴定
(1)样品的采集及预处理
麦曲样品采自浙江省绍兴市某黄酒厂，采集的麦曲置于密封的无菌塑料袋中4℃保存。称取5g麦曲于50mL离心管中，添加30mL蒸馏水放入30℃摇床培养箱中培养30min。
(2)菌株的平板筛选
放线菌筛选培养基：硝酸钾1.0g/L、磷酸二氢钾0.5g/L，硫酸镁0.5g/L，硫酸亚铁0.01g/L，氯化钠0.5g/L，可溶性淀粉20.0g/L，琼脂15.0g/L，pH值7.2-7.4(25℃)。
在无菌操作环境下，用无菌移液枪吸取1mL样品置于15mL无菌离心管中，加无菌水至10mL，充分混匀，制成10 -1的样品匀液。用无菌移液枪吸取10 -1样品匀液1mL于15mL无菌离心管中，加无菌水至10mL，充分混匀，制成10 -2的样品匀液。按上述操作，以此制成10 -1～10 -6十倍递增系列麦曲、米浆水、发酵醪稀释匀液。
分别吸取麦曲、发酵醪、米浆水各稀释度菌液100μL涂布于放线菌筛选培养基，28℃培养1～7d。在菌落密度适中的平板上挑取单个乳白色、菌落薄、有隆起或凸面、略皱的菌落，分别划线接种到放线菌筛选培养基上，反复划线确定纯菌落，并保存筛选菌株。
(3)菌株鉴定
提取筛选菌株的基因组，并对筛选菌株进行16S rDNA扩增测序。
PCR扩增引物27F(5′-AGAGTTTGATCCTGGCTCAG-3′)和1492R(5′-GGTTACCTTGTTACGACTT-3′)。
PCR扩增体系(50μL)为：2×Taq PCR Master Mix 25μL，上下引物各1μL，模板1μL，加无菌水22μL补充至50μL。
PCR扩增程序：94℃预变性3min，95℃变性30s，58℃退火30s，72℃延伸2min，共35个循环，最后72℃延伸8min。
PCR产物用1％的琼脂糖凝胶电泳检测，并送交基因测序公司测序，根据返回的测序结果，通过NCBI官网进行BLAST序列比对，用所得的16S rDNA序列进行BLAST比对，并进行了系统发育分析，结果如图1～2所示，菌株J2的核苷酸序列(SEQ ID NO.1)与糖多孢菌S.hirsuta(GenBank登录号：MN515057.1和NR_118870.1)的同源相似性达在98.30％以上，将菌株命名为披发糖多孢菌J2。菌株J3的核苷酸序列(SEQ ID NO.2)与糖多孢菌属S.jiangxiensis(GenBank相似序列号：MG255179.1)的同源相似性在98.84％以上，将该菌株命名为江西糖多孢菌J3。
(4)披发糖多孢菌J2菌株生物胺代谢能力的分析
菌种活化：将保藏的披发糖多孢菌J2接种到放线菌液体培养基中，接种量10％，30℃摇床培养48h，得一级种子液。将活化的菌株接种到放线菌液体培养基中，接种量10％，摇床培养48h，转速150r/min，温度30℃。
样品预处理：将菌种分别接种于检测生物胺产生培养基和检测生物胺降解培养基中，28℃摇床培养5d，12000r/min离心5min收集上清液。
放线菌液体培养基：硝酸钾1.0g/L、磷酸二氢钾0.5g/L，硫酸镁0.5g/L，硫酸亚铁0.01g/L，氯化钠0.5g/L，可溶性淀粉20.0g/L，pH值7.2-7.4(25℃)。
检测生物胺产生培养基：放线菌液体培养基中添加L-酪氨酸0.4g/L，L-组氨酸1g/L，L-赖氨酸1g/L，L-鸟氨酸1g/L，5′-磷酸吡哆醛0.05g/L。
检测生物胺降解培养基：放线菌液体培养基中添加50mg/L生物胺(包括组胺、酪胺、尸胺、腐胺、精胺、亚精胺、色胺、β-苯乙胺)，调节pH至6.0-6.2。
披发糖多孢菌J2降生物胺效果分析：在检测生物胺产生培养基和检测生物胺降解培养基中培养后的披发糖多孢菌J2的生物胺产生量小于2.5mg/L，生物胺检出量极少，表明基本上没有检测到生物胺的含量，因此认为不产生生物胺。披发糖多孢菌J2对酪胺的降解率为77.41％，对组胺的降解率达到100％，对腐胺的降解率达到58.1％，对尸胺的降解率达到47.71％，对总生物胺的降解率达到72.98％，说明菌株均有良好的降生物胺的能力。
(5)糖多孢菌J3生物胺代谢能力的分析
对江西糖多孢菌J3降生物胺效果进行分析：在生物胺前体存在的培养基中培养后的江西糖多孢菌J3的各类生物胺产生量均小于2.5mg/L，生物胺检出量极少，表明基本上没有检测到生物胺的含量，因此认为不产生生物胺。江西糖多孢菌J3对酪胺的降解率分别81.55％，对组胺的降解率分别达到100％，对腐胺的降解率分别达到51.8％，对尸胺的降解率分别达到40.01％，对总生物胺的降解率达到69.09％。说明该菌株具有良好的降生物胺的能力。
实施例2：糖多孢菌的活化培养
放线菌液体培养基：硝酸钾1.0g/L、磷酸二氢钾0.5g/L，硫酸镁0.5g/L，硫酸亚铁0.01g/L，氯化钠0.5g/L，可溶性淀粉20.0g/L，pH值7.2-7.4(25℃下测定)。
PDA培养基：马铃薯粉6.0g/L；葡萄糖20.0g/L，琼脂20.0g/L，pH 5.4-5.8，121℃高压灭菌15min；固体培养基在此基础上添加。
MRS培养基：牛肉膏10g/L，蛋白胨10g/L，酵母膏0.5g/L，葡萄糖20g/L，吐温80 0.10g/L，醋酸钠5g/L，磷酸氢二钾2g/L，柠檬酸氢二铵2g/L，硫酸镁0.58g/L，硫酸锰0.28g/L。
将实施例1筛选的披发糖多孢菌J2接种到放线菌液体培养基中，接种量10％，30℃摇床培养48h，得一级种子液。将活化的菌株接种到放线菌液体培养基中，接种量10％，摇床培养48h，转速150r/min，温度25～45℃，获得菌浓为10 5～10 6cfu/mL的菌液，待培养成熟后用于纯种麦曲的制备。
将保藏的黄曲霉(Aspergillus flavus)和米曲霉(Aspergillus oryzae)接种到PDA平板上，28℃培养3～5天；然后用无菌水清洗孢子液，再次转接到PDA茄形瓶中，温度28℃培养3～5天，获得菌浓为10 5～10 6cfu/mL的菌液，待孢子成熟后用于纯种麦曲的制作。
将保藏的乳杆菌L.plantarum(Lactobacillus plantarum)接种到MRS培养基中，接种量10％，37℃恒温厌氧培养24h，得到一级种子培养液，将活化的种子液再次接种到MRS液体培养基中，接种量为10％，37℃恒温厌氧培养24h，获得菌浓为10 5～10 6cfu/mL的菌液，菌种培养成熟后用于纯种麦曲制作。
江西糖多孢菌J3纯种麦曲按照上述相同方法制备，区别在于，将披发糖多孢菌J2替换为江西糖多孢菌J3。
实施例3：糖多孢菌纯种麦曲的制备
(1)碾麦：小麦破碎程度为每粒3-5片，带少量粉末，使麦粒组织破碎，淀粉外露；
(2)润麦：向步骤(1)处理后的物料中加入约35～40％的清水，搅拌20～25min，使其充分均匀的吸收水分；
(3)蒸煮灭菌：将步骤(2)处理后的物料于121℃灭菌30min；
(4)接种：待步骤(3)的物料降温至36℃后，接种按照实施例2的方法活化的菌种，接种菌液浓度为10 5～10 6cfu/mL，接种量为5‰-15％。
(5)曲料进盘后保持适当品温及室温，静置培养六小时左右。
a)孢子发芽期：曲料进盘六小时后，品温缓慢上升至34-35℃左右，自控模式启动小风量间接通风，每次5-10分钟，间隔2小时，使品温降到32℃，要求均匀吹透。
b)菌丝生长期：间歇通风3-5次后，菌丝开始生长，品温升至35℃以上，曲料开始结块，此时应连续通风，维持品温35℃左右。
c)菌丝繁殖期：接种后12小时，品温上升较快，此时应视第一次结块情况进行翻曲，翻曲前，先升起测温探头，开启翻曲机，后摊平，放下测温头。开启通风及喷雾系统。
d)第一次翻曲后，品温保持在36-37℃之间，保持通风喷雾顺畅，约20小时后，曲料再次结块，眼观曲料发白，温度难以控制在37℃以下，进行第二次翻曲，二次翻曲过后，品温应控制在35℃左右。
(6)出曲：培养72～96h；培养结束后，将麦曲置于4-7℃冰柜中储藏备用；
按照以上方法分别制备获得菌体数量级为10 15CFU/g的披发糖多孢菌J2纯种麦曲、江西糖多孢菌J3纯种麦曲。
实施例4：糖多孢菌混合麦曲的制备
将实施例3制备的披发糖多孢菌J2纯种麦曲和江西糖多孢菌J3纯种麦曲以任意比例混合，制备获得混合麦曲。
可选地，用于酿酒酒精饮品发酵的混合麦曲是将披发糖多孢菌J2纯种麦曲和江西糖多孢菌J3以1:(0.8～1.5)的质量比混合。
实施例5：糖多孢菌纯种麦曲在黄酒发酵中的应用
(1)本实施例所选取的传统黄酒发酵的原料配比(以每升发酵体积计)为：
蒸熟的米饭：500g；清水417L；酒母：38g；
(2)传统黄酒酿造过程
a)酵母活化培养：将甘油保藏管的酵母菌，在无菌操作台进行转接到YPD培养基中，30℃，150r/min条件下培养24h；然后转接到制备好的酒母中，将转接好的酵母在30℃，150r/min条件下，进行培养18-24h，备用。
b)酒母的制作：取600g蒸熟的米饭，加入1600mL清水，60g生麦曲，800U/g米饭的糖化酶进行糖化，糖化的温度控制在55-65℃，时间3-4小时，糖化结束后外观糖度不低于12°Bx后，115℃灭菌15min，灭菌后冷却至24～31℃，接入成熟的酵母种子培养液5％，培养温度不超过30℃，培养时间24h，培养成熟后即得酒母。
c)按照步骤(1)所述的传统黄酒发酵的原料配比进行落料和发酵。实验组：纯种麦曲：45.3g对照组：生麦曲：39.3g；熟麦曲：6.0g。
前四天为前酵阶段，温度控制在28～30℃，发酵4d，前4d每天开耙不少于1次，头耙时间8-10h；后酵阶段，温度为13～15℃，每天搅拌开耙一次，持续发酵10～15d。
对照组(TF Control)是将本实施例中c)中的纯种麦曲调整为工厂取样的生麦曲39.3g/L和熟麦曲6.0g/L。
实施例6：糖多孢菌混合麦曲在黄酒发酵中的应用
混合麦曲发酵组采用披发糖多孢菌J2纯种麦曲和江西糖多孢菌J3纯种麦曲的复配麦曲与酿酒酵母采用传统酿造方法共同发酵。
复合菌剂组(Mix)按照菌体数量1:1的比例添加披发糖多孢菌J2纯种麦曲和江西糖多孢菌J3纯种麦曲，添加量共45.3g。
黄酒发酵过程中理化指标的变化：为了进一步验证糖多孢菌在黄酒发酵中的作用，对比了传统麦曲和纯种麦曲发酵过程中理化指标(酒精度，还原糖，可滴定酸和氨基酸态氮)的变化。纯种发酵共分为6组，分别为A.flavus(黄酒发酵中的常用菌),A.oryzae(日本清酒酿造中的常用菌,S.jiangxiensis J3，Mix和L.plantarum，这5种纯种麦曲分别与酿酒酵母采用传统酿造方法共同发酵。至发酵结束，除L.plantarum组外，其他各组酒精度、酸度和氨基酸态氮含量均达到黄酒国家标准(图3)。L.plantarum组可滴定酸含量迅速增加到17.50g/L，样品出现明显的酸败。说明糖多孢菌对黄酒发酵过程中重要理化指标影响不大，发酵正常。
黄酒发酵样品中氨基酸含量：采用HPLC方法对发酵黄酒中的氨基酸含量进行了分析，添加S.hirsuta J2实验组中的氨基酸含量达到6434.81±123.3mg/L，相比于对照组提高了40.37％。Mix，A.flavus和A.oryzae实验组的氨基酸含量相差不大，但是均显著高于对照组(TF Control)；而添加S.jiangxiensis J3实验组的总氨基酸含量则与对照组相差不大，部分氨基酸含量显著超出对照组。
表1 黄酒发酵样品中的氨基酸含量分析
Figure PCTCN2021077377-appb-000001
注：gaga为γ-氨基丁酸。
糖多孢菌降生物胺效果分析：所得产品用高效液相法检测黄酒中生物胺含量，添加披发糖多孢菌J2的样品组相比较对照组降低了21.71％；添加江西糖多孢菌J3的样品组相比较对照组降低了35.09％。添加披发糖多孢菌J2组和江西糖多孢菌J3的复合菌剂(Mix组)相比对照组降低了42.17％。
纯种发酵与传统发酵风味分析：采用主成分分析法对纯种和传统发酵样品中风味成分的变化和相似性进行了分析。所有样本的biplot分析表明，披发糖多孢菌J2和江西糖多孢菌J3发酵的黄酒中前两个主成分的方差累积贡献率均为83.6％，这可以解释大多数发酵样本的风 味差异。从图4可以看出，传统发酵组与S.jiangxiensis J3组聚在一起，与曲霉(A.flavus和A.oryzae)组和L.plantarum组明显分离。这说明糖多孢菌参与了大多数风味物质的合成，并在黄酒发酵中起主导作用。
实施例7：糖多孢菌菌剂在黄酒发酵中的应用
以实施例5中的传统黄酒落料配方进行黄酒发酵，实验组分别设置Mix组和江西糖多孢菌J3组，区别在于，麦曲接种比例均为10％；江西糖多孢菌J3组接种江西糖多孢菌J3组纯种麦曲；Mix组接种应用江西糖多孢菌J3和披发糖多孢菌J2混合菌液按照实施例3的方法制备的复合菌种麦曲。黄酒酿造工艺和指标测定方法参照实施例4进行。
(1)对黄酒基本理化指标的影响
根据表2可以看出发酵结束后各组酒精度达到14％v/v左右，所有样品的还原糖、总酸和氨基酸态氮含量均在4.52-5.03g/L，均符合黄酒的理化要求。显著性分析表明Mix组与江西糖多孢菌J3组的酒精度、总酸含量和氨基酸态氮含量与对照组差异均不明显(P＞0.05)，说明接种糖多孢菌披发糖多孢菌J2和江西糖多孢菌J3对黄酒发酵过程中重要理化指标影响不大，不会对黄酒发酵过程带来不利影响。
表2 发酵结束时黄酒理化指标
Figure PCTCN2021077377-appb-000002
(2)糖多孢菌对黄酒生物胺含量的影响
发酵结束后，接种复合菌剂Mix组、江西糖多孢菌J3组的样品生物胺含量分别为15.57±0.44mg/L和16.88±1.41mg/L，均低于对照组的26.75±2.39mg/L。披发糖多孢菌J2总胺含量的平均值与对照组相比降低了41.79％；江西糖多孢菌J3组与对照组相比降低了36.90％。说明复合菌剂Mix、江西糖多孢菌J3均有降低生物胺含量的作用。
综上所述，在黄酒发酵体系中接种复合菌剂Mix、江西糖多孢菌J3均不影响黄酒的正常品质，且对总胺的降解率分别达到了对照组的41.79％和36.90％。说明直接添加复合菌剂Mix、江西糖多孢菌J3均有应用于黄酒生产、调控黄酒中生物胺含量的潜力，复合菌剂Mix对黄酒中生物胺的降解效果更佳。
实施例8：披发糖多孢菌J2应用于发酵香肠中降低生物胺含量
(1)菌种的活化培养
放线菌液体培养基：硝酸钾1.0g/L、磷酸二氢钾0.5g/L，硫酸镁0.5g/L，硫酸亚铁0.01g/L，氯化钠0.5g/L，可溶性淀粉20.0g/L，pH值7.2-7.4(25℃下测定)。
将保藏的糖多孢菌S.hirsuta J2接种到放线菌液体培养基中，接种量10％，30℃摇床培养59h，得一级种子液。将活化的菌株接种到放线菌液体培养基中，接种量10％，摇床培养48h，转速150r/min，温度30℃，待培养至菌浓数量级为10 5～10 7cfu/mL后用于发酵香肠。
(2)发酵香肠的制备
按质量计，取瘦肉65～80％、肥肉20～35％。清洗，去除骨、筋腱、肌膜、淋巴、血管、病变及损伤部位。肥瘦分开，切成4～5cm肉块。将瘦肉和5～8％左右冰屑放入斩拌机内，斩拌1～3min。以猪肉质量为基数，添加亚硝酸钠0.01～0.15％、食盐2～3％、复合磷酸盐0.2～0.3％、抗坏血酸钠0.05～0.06％。香辛料、胡椒、大蒜、辣椒、肉蔻为原料肉的0.2％～0.3％。其中两组分别接种8～12％活化的糖多胞菌，一组不接种作为对照，斩拌1～2min，再加入肥肉和5～8％左右冰屑，斩拌4～6min。腌制后灌入肠衣中。香肠的发酵工艺参数如表3所示。
表3 香肠发酵工艺参数
Figure PCTCN2021077377-appb-000003
(3)披发糖多孢菌J2降生物胺效果分析
以实施例1中的方法进行菌种活化和生物胺含量的测定。
样品前处理方法：称取绞碎后样品5.0g于50mL离心管中，加入20mL 5％的三氯乙酸并超声30min，转移至50mL具塞离心管中，6 000r/min离心10min，转移上清液至50mL容量瓶中，残渣用20mL上述溶液再提取1次，合并上清液并稀释至刻度。
由表4可知，添加披发糖多孢菌J2发酵后的香肠在发酵成熟后pH分别为6.16±0.16，而对照组则为6.51±0.17，水分含量和粗蛋白含量差异均不明显；添加S.hirsuta J2发酵的香肠成熟后生物胺平均含量为129.60mg/kg，比对照组降低了36.19％。
表4 发酵成熟香肠指标
Figure PCTCN2021077377-appb-000004
实施例9：糖多孢菌J2应用于白酒中降低生物胺含量
白酒酿造使用的糖多孢菌纯种麦曲参照按照实施案例3中麦曲制作方法。以实施案例1中的方法进行生物胺含量的测定。
白酒酿造方法采用两轮发酵法，第一轮发酵时高粱蒸熟后，风冷降温至25℃，添加4％米曲霉种子液，在28℃温度下培养24h。添加稻壳10％、大曲15％、麸皮8％、纯种麦曲5～9％、按1％的比例接入酿酒酵母种子液，然后密封，发酵30天后进行蒸酒。二轮发酵时添加中温大曲10％、按1％的比例接入酿酒酵母种子液，酵母菌种子液浓度为10 10～10 12cfu/mL，继续发酵12～15天后进行蒸酒。
对糖多孢菌S.hirsuta J2降生物胺效果分析：将蒸馏后的白酒勾兑成酒精度60％(V/V)，检测勾兑后样品中生物胺含量，添加S.hirsuta J2的样品组相比较对照组降低了24.32％。
实施例10：披发糖多孢菌J2应用于酱油中降低生物胺含量
以实施例1中的方法进行菌种活化和生物胺含量的测定。酱油采用高盐稀态法进行酿造：
(1)首先将豆粕和小麦按1:1比例混匀，然后蒸熟；
(2)按照5‰～10％的比例加入菌液浓度为10 5～10 6cfu/mL的披发糖多孢菌J2种子液，然后加入约1.5～2倍物料质量的盐水，酱醪最终含盐量约为18％、含水量为65％，然后混匀。
(3)酱醅发酵：起始发酵温度控制在14～16℃，随着发酵进行温度逐渐升高到约35℃。持续发酵约5个月。
(4)发酵结束后的将酱醪进行板框压榨，去除酱醅。压榨结束后进行硅藻土过滤和膜过滤，去除沉淀。过滤澄清的酱油经过巴氏灭菌后进行灌装。
披发糖多孢菌J2降生物胺效果分析，添加披发糖多孢菌J2的酱油产品中的生物胺含量相较于对照组降低了34.28％。
实施例11：披发糖多孢菌J2用于料酒中降低生物胺含量
按照实施例5中酿造方法获得纯种发酵黄酒，向发酵黄酒中加入按质量计10％食盐，过灭菌机85℃灭菌30min热灌装。
对菌株降低料酒中生物胺含量的效果进行分析：用高效液相法检测料酒中生物胺含量，添加披发糖多孢菌J2的样品组与对照组相比，生物胺含量降低。
实施例12：披发糖多孢菌J2用于食醋中降低生物胺含量
按照实施例5中酿造方法获得纯种发酵黄酒，作为醋酸发酵原料；以实施例1中的方法进行生物胺含量的测定。
醋酸发酵采用固态发酵工艺：将大糠、麸皮、黄酒按照1:4:10的质量比拌匀，接入5％的醋醅，接种后前2天内每天从物料表面翻醅，保持温度为35-42℃。到6-8天时翻到物料底部。第8-12天，每天从底部翻醅，温度自然下降。从醋醅中分离后得生醋，在经过85℃灭菌30min后陈酿12个月。在灌装前经过高温灭菌后热灌装。
对制备获得的食醋品质进行测定，所得固态发酵酿造食醋中醋酸含量均为55g/L。对食醋中生物胺含量进行分析：用高效液相法检测料酒中生物胺含量，添加披发糖多孢菌J2的样品组与对照组相比，生物胺含量降低。
实施例13：披发糖多孢菌J2用于奶酪发酵中降低生物胺含量
以实施例1中的方法进行菌种活化和生物胺含量的测定。
将新鲜牛乳进行均质和巴氏杀菌处理后待其冷却至室温后添加0.1g/L的发酵剂，搅拌均匀，在32～35℃酸化30min后添加0.05g/L的凝乳酶，混匀待凝乳形成后切割、排乳清得到干酪凝块；在干酪凝块的表面喷洒10 5～10 6cfu/mL的披发糖多孢菌J2，置于30～37℃培养3～5d，待其生长成熟，加入3.0g/L的食盐，然后压榨成型即得干酪成品。
对发酵奶酪成品进行测定，添加披发糖多孢菌J2的干酪成品的生物胺含量相较于对照组降低了16.37％
实施例14：披发糖多孢菌J2用于香烟发酵中提质降害
以实施例1中的方法进行菌种活化。将活化的菌液于4℃、10 000g离心15min，将收集的菌体用无菌水配制成10 5～10 6cfu/mL的披发糖多孢菌J2菌液，将菌液均匀喷洒在烟叶表面，充分混匀，以等量无菌水处理做对照，置于30～37℃、湿度70～80％的恒温恒湿箱中发酵培养15d，每天进行通风，培养结束后将烟叶烘干至水分为低于15％。
对发酵烟叶品质进行测定，所得发酵烟叶香气成分明显增加、杂气减轻，刺激性减弱；发酵烟叶中有害成分-焦油量、HCN、苯酚、NH 3和亚硝酸盐的含量相较对照组分别下降了23.51％，16.25％，19.75％，27.09％和37.62％。
实施例15：披发糖多孢菌J2用于饲料发酵中提高营养转化率
以实施例1中的方法进行菌种活化。将米糠、秸秆和豆粕按照(1～5)：(1～5)：2的比例混合均匀，粉碎制成发酵物。按照料水比为1:0.5～0.9的比例加水，按照10‰～10％的比例接种菌液浓度为10 5～10 6cfu/mL的披发糖多孢菌J2，搅拌均匀，自然发酵，发酵温度为30～40℃，发酵时间为4～9d，发酵结束后干燥至含水量低于15％，即得生物发酵饲料。
对发酵饲料品质进行分析：所得发酵饲料具有特殊香气，且营养丰富、氨基酸均衡，其中有机酸的含量相较于对照组提高了26.77％，氨基酸含量提高了21.98％，粗蛋白含量提高了18.53％。
实施例16：糖多孢菌于降低发酵鱼中的生物胺含量
以实施例1中的方法进行菌种活化。分别制备披发糖多孢菌J2菌液、江西糖多孢菌J3菌液，将二者按1:0.7～1.5混合，获得复合菌液Mix。
利用中性蛋白酶发酵臭鳜鱼，具体工艺为：
(1)样品预处理：鳜鱼去内脏，称重3kg；
(2)发酵液制备：取鳜鱼等质量饮用水，以此为100％计，加入6％盐、1％大葱、0.6％姜、0.1％八角、0.05％茴香、0.05％孜然、0.01％辣椒、0.01％花椒、300000U中性蛋白酶，混匀，得发酵液；
(3)接种：发酵液分成三份，一份以10％接种量向发酵液中接种菌液浓度为10 7cfu/mL 的复合菌液Mix，另一份以10％接种量向发酵液中接种菌液浓度为10 7cfu/mL的的江西糖多孢菌J3，另一份不接种；
(4)发酵：将鳜鱼浸于步骤(3)接种后的发酵液中，最上层用石头压实，20℃下发酵6天，得臭鳜鱼。
生物胺的测定方法：称取绞碎后鱼肉样品5.0g于50mL离心管中，加入20mL 5％的三氯乙酸并超声30min，转移至50mL具塞离心管中，6 000r/min离心10min，转移上清液至50mL容量瓶中，残渣用20mL上述溶液再提取1次，合并上清液并稀释至刻度。然后准确量取1mL上清液于15mL离心管中，加入1mL饱和NaHCO 3溶液，混匀，加入2mL丹磺酰氯(5mg/mL丙酮)试剂，混匀后置于65℃恒温水浴锅中黑暗衍生30min，室温静置后，加入0.5mL饱和NaCl溶液，混匀后加入5mL乙醚，涡旋振荡20s，静置分层后，转移上层有机相于15mL离心管中，下层水相再萃取一次，合并两次萃取液，50℃水浴下氮气吹干。
加入1mL乙腈振荡混匀，溶解残留物，过0.22μm滤膜，通过高效液相色谱法(HPLC)测定。
发酵结束后，采取复合菌剂Mix强化的臭鳜鱼生物胺比对照组下降了20.87％。
实施例17：糖多孢菌用于料酒中降低生物胺含量
以实施例1中的方法进行生物胺含量的测定。
按照实施例5中酿造方法获得纯种发酵黄酒，向发酵黄酒中加入按质量计10％食盐，过灭菌机85℃灭菌30min热灌装。
对菌株在料酒中降生物胺的效果进行分析：用高效液相法检测料酒中生物胺含量，添加复合菌剂Mix和江西糖多孢菌J3的样品组与对照组相比，分别降低了23.16％和18.91％。
实施例18：糖多孢菌用于食醋中降低生物胺含量
按照实施例5中酿造方法获得纯种发酵黄酒，作为醋酸发酵原料；以实施例1中的方法进行生物胺含量的测定。
醋酸发酵采用固态发酵工艺：将大糠、麸皮、黄酒按照1:4:10的质量比拌匀，接入5％的醋醅，接种后前2天内每天从物料表面翻醅，保持温度为35-42℃。到6-8天时翻到物料底部。第8-12天，每天从底部翻醅，温度自然下降。从醋醅中分离后得生醋，在经过85℃灭菌30min后陈酿12个月。在灌装前经过高温灭菌后热灌装。
复合菌剂Mix和江西糖多孢菌J3降生物胺效果分析：所得固态发酵酿造食醋中醋酸含量均为55g/L。检测样品中生物胺含量，添加复合菌剂Mix和江西糖多孢菌J3的样品组与对照组相比，分别降低了25.08％和27.61％。
实施例19：糖多孢菌用于奶酪发酵中降低生物胺含量
以实施例1中的方法进行菌种活化和生物胺含量的测定。分别制备披发糖多孢菌J2菌液、江西糖多孢菌J3菌液，将二者按1:0.8～1.4混合，获得复合菌液mix。
将新鲜牛乳进行均质和巴氏杀菌处理后待其冷却至室温后添加0.1mL/L的菌体数量比为1:1的保加利亚乳杆菌和植物乳杆菌的混合菌液作为发酵剂(菌液中的菌体浓度为1×10 8～10 9cfu/mL)，搅拌均匀，在32～35℃酸化30min后添加0.05g/L的凝乳酶，混匀待凝乳形成后切割、排乳清得到干酪凝块；在干酪凝块的表面喷洒10 5～10 6cfu/mL的糖多孢菌菌剂，置于30～37℃培养3～5d，待其生长成熟，加入3.0g/L的食盐，然后压榨成型即得干酪成品。
对发酵干酪成品进行测定，添加江西糖多孢菌J3和复合菌剂Mix的干酪成品的生物胺含量相较于对照组降低了13.33％和21.66％。
实施例20：糖多孢菌用于香烟发酵中提质降害
以实施例1中的方法进行菌种活化。分别制备披发糖多孢菌J2菌液、江西糖多孢菌J3菌液，将二者按1:0.7～1.5混合，获得复合菌液Mix。
将活化的菌液于4℃、10 000g离心15min，将收集的菌体用无菌水配制成10 5～10 6cfu/mL的菌液，将菌液均匀喷洒在烟叶表面，充分混匀，以等量无菌水处理做对照，置于30～37℃、 湿度70～80％的恒温恒湿箱中发酵培养15d，每天进行通风，培养结束后将烟叶烘干至水分为低于15％。
对发酵烟叶品质进行测定，所得发酵烟叶香气成分明显增加、杂气减轻，刺激性减弱；添加江西糖多孢菌J3发酵烟叶中有害成分-焦油量、HCN、苯酚、NH 3和亚硝酸盐的含量相较对照组分别下降了32.65％，17.55％，17.69％，25.36％和29.17％。添加复合菌剂Mix发酵烟叶中有害成分-焦油量、HCN、苯酚、NH 3和亚硝酸盐的含量相较对照组分别下降了30.12％，17.14％，22.01％，16.73％和27.15％。
实施例21：糖多孢菌用于饲料发酵中提高营养转化率
以实施例1中的方法进行菌种活化。分别制备披发糖多孢菌J2菌液、江西糖多孢菌J3菌液，将二者按1:0.5～1.2混合，获得复合菌液Mix。
将米糠、秸秆和豆粕按照(1～5)：(1～5)：2的比例混合均匀，粉碎制成发酵物。按照料水比为1:0.5～0.9的比例加水，按照10‰～10％的比例接种菌液浓度为10 5～10 6cfu/mL的菌液，搅拌均匀，自然发酵，发酵温度为30～40℃，发酵时间为4～9d，发酵结束后干燥至含水量低于15％，即得生物发酵饲料。
对发酵饲料品质进行分析：所得发酵饲料具有特殊香气，且营养丰富、氨基酸均衡，添加江西糖多孢菌J3发酵饲料中有机酸的含量相较于对照组提高了37.26％，氨基酸含量提高了18.57％，粗蛋白含量提高了23.41％。添加复合菌剂Mix发酵烟叶中有机酸的含量相较于对照组提高了29.18％，氨基酸含量提高了10.22％，粗蛋白含量提高了14.48％。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (14)

  1. 披发糖多孢菌(Saccharopolyspora hirsuta)J2，所述糖多孢菌已于2020年4月30日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020103。
  2. 含有权利要求1所述的披发糖多孢菌J2的微生物制剂。
  3. 根据权利要求2所述的微生物制剂，其特征在于，每克或每毫升发酵剂中的披发糖多孢菌J2的数量≥1×10 6CFU。
  4. 应用权利要求1所述披发糖多孢菌J2制备的麦曲、麸曲、酒药或其他类型的曲。
  5. 根据权利要求4所述的麦曲、麸曲、酒药或其他类型的曲，其特征在于，是将制曲的原料破碎、加水搅拌、灭菌后，在无菌环境下加入所述的披发糖多孢菌J2，于25～55℃培养72～96h。
  6. 一种糖多孢菌组合物，其特征在于，含有权利要求1所述的披发糖多孢菌J2和江西糖多孢菌J3；
    所述江西糖多孢菌J3，已于2020年4月30日保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO：M 2020104。
  7. 根据权利要求6所述的糖多孢菌组合物，其特征在于，所述披发糖多孢菌J2和江西糖多孢菌J3的菌体数量之比为1：(0.8～1.2)。
  8. 含有权利要求6或7所述糖多孢菌组合物的微生物制剂，其特征在于，每克或每毫升微生物制剂中的糖多孢菌数量≥1×10 6CFU。
  9. 根据权利要求8所述的微生物制剂，其特征在于，含有所述披发糖多孢菌J2和江西糖多孢菌J3的活细胞、冷冻干燥得到的干菌体、固定化的细胞，或者以其他任何形式存在的微生物细胞。
  10. 应用权利要求6或7所述糖多孢菌组合物制备的麦曲、麸曲、酒药或其他类型的曲。
  11. 根据权利要求10所述的麦曲、麸曲、酒药或其他类型的曲，其特征在于，是将制曲的原料破碎、加水搅拌、灭菌后，在无菌环境下加入所述的糖多孢菌组合物，于25～55℃培养72～96h。
  12. 权利要求6或7所述的糖多孢菌组合物，或权利要求8或9所述的微生物制剂，或权利要求10或11所述的曲在生产发酵食品、调味品、饮品、香烟和饲料中的应用。
  13. 根据权利要求12所述的应用，其特征在于，所述食品包括发酵或半发酵食品；饮品包括黄酒或白酒；所述调味品包括食醋、酱油、调味料、奶酪或香肠。
  14. 权利要求6或7所述的糖多孢菌组合物，或权利要求8或9所述的微生物制剂，或权利要求10或11所述的曲在降低发酵食品或饮品中生物胺含量方面的应用。
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