WO2021120597A1 - 一株降低生物胺的乳酸片球菌及其应用 - Google Patents

一株降低生物胺的乳酸片球菌及其应用 Download PDF

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WO2021120597A1
WO2021120597A1 PCT/CN2020/100370 CN2020100370W WO2021120597A1 WO 2021120597 A1 WO2021120597 A1 WO 2021120597A1 CN 2020100370 W CN2020100370 W CN 2020100370W WO 2021120597 A1 WO2021120597 A1 WO 2021120597A1
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pediococcus
soybean paste
pediococcus lactis
lactis
microbial preparation
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French (fr)
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李崎
赵佳迪
钮成拓
王金晶
刘春凤
郑飞云
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of 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
    • 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
    • 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

Definitions

  • the invention relates to a strain of Pediococcus lactis capable of reducing biogenic amines and its application, and belongs to the technical field of bioengineering fermentation.
  • the cells of Pediococcus lactis are spherical and divide alternately in two planes at right angles to form a quadruple. Generally, the cells are in pairs, but solitary cells are rare, and they are not arranged in chains. Gram-positive, no exercise, facultative anaerobic. The colonies on MRS medium are small and white. The growth along the puncture line of agar is filamentous. Exposure to enzymes is negative and does not produce cytochromes. It can produce acid, regulate the gastrointestinal flora, and maintain the intestinal microecological balance.
  • Biogenic amines are a type of nitrogen-containing organic compounds, widely present in fermented foods such as soy sauce, soy sauce, sausage, fish sauce, cheese, wine, etc., and are usually synthesized by microorganisms using precursor amino acids for decarboxylation.
  • precursor amino acids for decarboxylation For example, cadaverine, tryptamine, phenethylamine, histamine, and tyramine are produced from the corresponding precursor amino acids lysine, tryptophan, phenylalanine, histidine, and tyrosine, respectively.
  • putrescine is produced by ornithine decarboxylase and agmatine deaminase
  • spermidine and spermine are produced by putrescine through amine synthetase, and they can be converted into each other.
  • biogenic amines have some biological functions, such as providing access to energy, restoring pH in the body, and protecting damaged DNA
  • excessive intake of biogenic amines may cause many health problems, such as headaches, low blood pressure, nausea, and tides. Heat, local inflammation, palpitations, high blood pressure, and digestive problems, especially in people who have reduced amine oxidase due to medication. Therefore, it is necessary to reduce the content of biogenic amines in food and improve food safety.
  • the first purpose of the present invention is to screen and obtain a strain of Pediococcus lactis M28, which has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on July 25, 2019.
  • the deposit number is CGMCC No. 18294, and the deposit address is Beijing. No. 3, No. 1, Beichen West Road, Chaoyang District, Institute of Microbiology, Chinese Academy of Sciences.
  • the second object of the present invention is to provide the method for culturing Pediococcus lactis, which is to inoculate Pediococcus lactis M28 into a modified MRS medium at 35-40°C at an inoculum amount of 10-50 mL/L, and cultivate for 20-30 hours.
  • the third object of the present invention is to provide a microbial preparation containing the Pediococcus lactis M28.
  • the microbial preparation includes dry cells or wet cells of Pediococcus lactis.
  • the microbial preparation contains Pediococcus lactis cells with a viable cell count ⁇ 10 7 CFU/g or a viable cell count ⁇ 10 7 CFU/mL.
  • the fourth object of the present invention is to provide a method for reducing biogenic amines.
  • the method is to inoculate the Pediococcus lactis M28 into a liquid, semi-solid or solid containing biogenic amine at an inoculum of 10-50 mL/L , Or adding the microbial preparation to a liquid, semi-solid or solid containing biogenic amine.
  • the fifth object of the present invention is to provide a method for activating the Staphylococcus carnosus M43, which is to inoculate the Staphylococcus carnosus M43 into the culture medium at an inoculum amount of 10-50 mL/L.
  • the culture condition of the activation method is 30-40°C static culture for 20-30 hours.
  • the medium is a modified MRS medium.
  • the sixth object of the present invention is to provide a method for reducing the content of biogenic amines in soybean paste.
  • the method is to inoculate the Pediococcus lactis M28 on the 0th day of soybean paste production, or to add the microbial preparation.
  • the inoculation amount of the Pediococcus lactis M28 is 10-50 mL/L, and the mixture is uniformly stirred and fermented at 30-40°C for 30-40 days.
  • the strain concentration of Pediococcus lactis M28 in the soybean paste is 10 3 to 10 9 CFU/g.
  • the seventh object of the present invention is to provide a method for improving the flavor of food, the method is adding the Pediococcus lactis M28 or adding the microbial preparation during the food fermentation process.
  • the Pediococcus lactis M28 or the microbial preparation is added on the 0th day of soybean paste fermentation, and the fermentation is carried out at 30-40°C for 30-40 days.
  • the concentration of the Pediococcus lactis M28 in the soybean paste is 10 3 to 10 9 CFU/g.
  • the eighth object of the present invention is to provide the application of the Pediococcus lactis or the microbial preparation in the preparation of condiments.
  • the condiment includes soybean paste, bean paste or bean noodle paste.
  • the present invention also provides the application of the Pediococcus lactis or the microbial preparation in the field of fermented food for reducing the content of biogenic amines.
  • the food includes foods that need to be fermented with soybeans, broad beans or meat.
  • the food includes soy sauce, sausage, fish sauce, and cheese.
  • the Pediococcus lactis M28 provided by the present invention is a harmless bacterium from the fermentation process of fava bean paste samples from the factory, and has a relatively obvious degradation effect on the common biogenic amines in the two fermented foods of tyramine and spermidine.
  • the degradation rate of tyramine and spermidine in soybean paste can reach 59.33% and 58.41%, which can be used to control the bio-amine content in fermented foods such as soybean paste.
  • the umami-related aspartic acid and glutamic acid in the soybean paste brewed by Pediococcus lactis M28 provided by the present invention are 13.98% and 6.43% higher than the control, respectively, making the soybean paste of the present invention more delicious.
  • the content of lactic acid in the soybean paste is 23.2% higher than the control, making the soybean paste softer, thus forming a unique flavor.
  • the Pediococcus acidilactici M28 provided by the present invention has been deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on July 25, 2019, and the deposit number is CGMCC No. 18294, and the deposit address is Beichen, Chaoyang District, Beijing No. 3, No. 1 West Road, Institute of Microbiology, Chinese Academy of Sciences.
  • Strain activation method inoculate the strain into the culture medium at an inoculum of 20mL/L, and cultivate it anaerobicly at 37°C for 24h.
  • Example 1 Screening and identification of biogenic amine degrading strains
  • MRS medium peptone 10g, beef extract 10g, glucose 20g, sodium chloride 50g, yeast powder 5g, sodium acetate 5g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, Tween-80 1.0mL, magnesium sulfate 0.58g, 0.25g manganese sulfate, 1L deionized water, pH 5.6.
  • BAs medium 2g potassium dihydrogen phosphate, 2g ammonium citrate, 50g sodium chloride, 0.4g magnesium sulfate heptahydrate, 0.03g manganese sulfate, 0.04g ferrous sulfate, 0.01g thiamine, 2g glucose, putrescine Dihydrochloride 100mg, cadaverine dihydrochloride 100mg, spermidine 100mg, spermine 100mg, tryptamine 100mg, phenethylamine 100mg, histamine dihydrochloride 100mg, tyramine hydrochloride 100mg, agar 20g, Deionized water 1L, pH5.5.
  • soy sauce Take 5g of soy sauce in a 100mL Erlenmeyer flask, add 50mL of physiological saline, shake and mix well on a shaker, take the suspension and dilute it to 10 -5 , and take 100 ⁇ L of the suspension in BAs solid medium and modified MRS for each gradient
  • the culture medium is coated and cultured in a 37°C incubator. Only when the colonies grow well, they can be separated.
  • Determination of the degradation rate of biogenic amine in phosphate buffer First, use MRS medium to activate the isolated single colony, and adjust the initial OD value to make the OD value of 0.6 in the phosphate buffer containing 100mg/L biogenic amine , And placed in a 37°C incubator for 24h, and then centrifuged at 4000g for 5min. Take the supernatant and filter through a 0.22 ⁇ m filter membrane, take 2mL of the filtrate into a 10mL centrifuge tube, add 1mL of 2mol/L NaOH solution, then add 20 ⁇ L of benzoyl chloride solution, immediately put the centrifuge tube into the water bath constant temperature shaking Circulator, 30°C, 200r/min oscillate for 20min.
  • the degradation rates of strain M28 to eight biogenic amines in phosphate buffer were 13.78 ⁇ 0.31%, 8.44 ⁇ 0.25%, 6.81 ⁇ 0.17%, 6.41 ⁇ 0.28%, 8.93 ⁇ 0.36%, 4.32 ⁇ 0.18%, 14.10 ⁇ 0.98% and 14.04 ⁇ 0.55%, were screened out in the fermentation broth environment for further determination of degradation rate.
  • Determination of biogenic amine degradation rate in fermentation broth Use modified MRS medium to activate the primary screened strains, and adjust the initial OD value so that the OD value is 0.1 in the modified MRS medium containing 100 mg/L biogenic amine. Place in a 37°C incubator for 24 hours, and then centrifuge at 4000g for 5 minutes. Take the supernatant and filter through a 0.22 ⁇ m filter membrane, take 2mL of the filtrate into a 10mL centrifuge tube, add the same volume of the trichloroacetic acid solution with a mass fraction of 6g/L, and shake at 30°C, 200r/min in a water bath constant temperature oscillator For 1h, take the supernatant to carry out the derivatization reaction.
  • the degradation rate of the bioamine is determined, and the strains with the higher degradation rate are selected to obtain the re-screened strains.
  • the degradation rates of strain M28 to putrescine, cadaverine, spermidine, spermine, tryptamine, phenethylamine, histamine, and tyramine were 11.32 ⁇ 0.61%, 6.97 ⁇ 1.19%, 2.10 ⁇ 0.80%, respectively. 10.57 ⁇ 0.57%, 3.68 ⁇ 0.17%, 1.75 ⁇ 3.83%, 42.18 ⁇ 4.25% and 5.80 ⁇ 0.81%.
  • the reaction conditions were as follows: pre-denaturation at 94°C for 5min into the following cycle, denaturation at 94°C for 60s, annealing at 56°C for 60s, extension at 72°C for 90s, 34 cycles; extension at 72°C for 10min. It was sent to Tianlin Company for sequencing, and the obtained sequence was compared with the BLAST sequence in the NCBI database. It was identified as Pediococcus lactis and named M28.
  • Soybeans, brine, and flour are used as raw materials, and the added amounts are 38g/100g, 50g/100g, and 12g/100g respectively.
  • 10mL/L of Pediococcus lactis is inoculated on the 0th day after entering the vat to make
  • the concentration of the strain in the sauce was 10 7 CFU/g
  • the sauce without Pediococcus lactis was used as a control.
  • the control group and the experimental group were added with 5 mL, 5 g/L putrescine, cadaverine, spermidine, and sperm.
  • X is the bio-amine degradation rate
  • C 1 is the bio-amine concentration of the uninoculated strain sample
  • C 2 is the bio-amine concentration of the inoculated experimental strain sample.
  • the inoculation of Pediococcus lactis M28 during the fermentation of soybean paste can effectively reduce the content of eight major biogenic amines in soybean paste: putrescine, cadaverine, spermidine, spermine, tryptamine, and phenethyl.
  • the degradation rates of amine, histamine and tyramine are 16.92 ⁇ 1.98%, 11.02 ⁇ 0.95%, 58.41 ⁇ 3.50%, 12.02 ⁇ 3.85%, 8.95 ⁇ 2.75%, 4.54 ⁇ 0.01%, 4.49 ⁇ 0.14% and 59.33 ⁇ 3.36, respectively .
  • the specific implementation mode is the same as in Example 2, but the difference is that M28 is replaced with Pediococcus lactis M10 from the soy sauce sample from the factory.
  • the Pediococcus lactis M10 has a relatively poor degradation ability for biogenic amines, and has no degradation ability for spermidine, spermine, tryptamine, and histamine.
  • the amino acid content of the soybean paste obtained by fermentation in Example 2 and Comparative Example 1 was determined.
  • the amino acid content determination method weigh about 1.0000g of soybean paste sample with an analytical balance, place it in a mortar and grind it evenly, using 5g/L three Dissolve the chloroacetic acid solution and make the volume up to 25mL. Put it in an ultrasonic cleaner for 20 minutes at room temperature and let it stand for at least two hours after the end.
  • the amino acid content is shown in Table 3 below:
  • the soybean paste brewed by Pediococcus lactis M28 and the soybean paste brewed by Pediococcus lactis M10 have higher glutamic acid and aspartic acid content, which are 6.43% and 13.98% higher respectively.
  • Aspartic acid and glutamic acid are the main umami-flavored amino acids, which proves that soybean paste fermented by Pediococcus lactis is more umami-flavored.
  • the soybean paste brewed by Pediococcus lactis M28 is 4.59% higher than the soybean paste brewed by Pediococcus lactis M10, reflecting the higher nutritional value of the soybean paste in the experimental group.
  • the soybean paste obtained by fermentation in Example 2 and Comparative Example 1 was measured for organic acid content.
  • the method for determining organic acid content Weigh 2g-10g of soy sauce into a 50mL EP tube, add 40mL ultrapure water and soak for 2h, Shake once every 15 minutes and centrifuge at 7500 rpm for 5 minutes.
  • Organic acid standards oxalic acid, pyruvic acid, acetic acid, lactic acid, and succinic acid have concentrations of 0.05, 0.05, 0.50, 0.50, 0.50, and 1.00 mg/mL, respectively.
  • Chromatographic column Waters Atlantis T3 (5um, 4.6 ⁇ 250mm); column temperature: 40°C; mobile phase: accurately weigh 3.12g of NaH 2 PO 4 ⁇ 2H 2 O, dilute to 1000 mL with deionized water, and use H 3 PO 4 adjusts the pH value of the solution to 2.7; injection volume: 10uL; flow rate: 0.7mL/min; detection wavelength: UV210nm.
  • the organic acid content is shown in Table 4 below:
  • the organic acids in soybean paste after fermentation are mainly acetic acid and lactic acid. Because Pediococcus lactis M28 acts on fermentable carbohydrates in the sauce to produce more lactic acid, which is 23.2% higher than the control. The small molecule lactic acid makes the soybean sauce taste softer, thus forming a unique flavor.
  • the sardines After the sardines are washed, 20%-30% of the fish weight is added with edible salt, the salt is mixed with the fish, and then the Pediococcus lactis of the present invention is added, fermented under natural conditions for 1 year, and filtered to obtain fish sauce. .

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Abstract

提供了一株降低生物胺的乳酸片球菌及其应用，属于生物工程发酵技术领域。乳酸片球菌已于2019 年7 月25 日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.18294，保藏地址为北京市朝阳区北辰西路1 号院3 号，中国科学院微生物研究所。菌株具有较好的降解生物胺能力，对于豆酱中酪胺、亚精胺的降解率可达59.33％和58.41％，可用于控制豆酱等发酵食品生产过程中的生物胺含量。

Description

一株降低生物胺的乳酸片球菌及其应用 技术领域
本发明涉及一株降低生物胺的乳酸片球菌及其应用，属于生物工程发酵技术领域。
背景技术
乳酸片球菌的细胞为圆球状，在直角两个平面交替分裂形成四联状，一般细胞成对生，单生者罕见，不成链状排列。革兰氏阳性，不运动，兼性厌氧。在MRS培养基上菌落小，呈白色。沿洋菜穿刺线的生长物呈丝状。接触酶阴性，不产细胞色素。它能够产酸，调节胃肠道菌群，维持肠道微生态平衡。在动物体内对病原微生物有颉颃作用，可竞争性地抑制病原微生物，增强动物机体的免疫功能，产生有益的代谢产物，激活酸性蛋白酶的活性，参与机体的新陈代谢，防止有害物质产生。
生物胺是一类含氮有机化合物，广泛存在于酱油、豆酱、香肠、鱼露、奶酪、葡萄酒等发酵食品中，通常是微生物利用前体氨基酸进行脱羧作用合成的。例如，尸胺、色胺、苯乙胺、组胺和酪胺分别由相应的前体氨基酸赖氨酸、色氨酸、苯丙氨酸、组氨酸和酪氨酸产生。然而，腐胺是由鸟氨酸脱羧酶和胍丁胺脱氨酶产生的，亚精胺和精胺是由腐胺经过胺合成酶产生的，它们可以相互转化。尽管生物胺具有一些生物学功能，如提供获取能量的途径、恢复体内pH值和保护受损DNA等，但过量摄入生物胺可能会导致许多健康问题，如引发头痛、低血压、恶心、潮热、局部炎症、心悸、高血压和消化问题，特别是在那些由于药物治疗而导致胺氧化酶减少的人群中。因此，降低食品中生物胺含量，提高食品安全性是很有必要的。现在大都采用生物学方法去降解生物胺，如在食品发酵过程中添加降解生物胺的菌株或不产生生物胺的优势菌株。因此筛选一株既能适应豆酱生产环境又能高效降解生物胺的菌株，对于制备安全健康的豆酱具有重要的应用价值。
发明内容
本发明的第一个目的是筛选得到一株乳酸片球菌M28，已于2019年7月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.18294，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
本发明的第二个目的是提供所述的乳酸片球菌的培养方法，是将乳酸片球菌M28以10～50mL/L接种量接种至改良MRS培养基中35～40℃，培养20～30h。
本发明第三个目的是提供含有所述乳酸片球菌M28的微生物制剂。
在本发明的一种实施方式中，所述微生物制剂包括乳酸片球菌干菌体或湿菌体。
在本发明的一种实施方式中，所述微生物制剂中含有活菌数≥10 7CFU/g或活菌数≥10 7CFU/mL的乳酸片球菌细胞。
本发明的第四个目的是提供一种降低生物胺的方法，所述方法是将所述乳酸片球菌M28以10～50mL/L的接种量接种至含生物胺的液体、半固体或固体中，或将所述微生物制剂加入至含生物胺的液体、半固体或固体中。
本发明的第五个目的是提供一种所述肉葡萄球菌M43的活化方法，所述方法是将肉葡萄球菌M43以10～50mL/L的接种量接种至培养基中。
在本发明的一种实施方式中，所述活化方法的培养条件为30～40℃静置培养20～30h。
在本发明的一种实施方式中，所述培养基为改良MRS培养基。
本发明的第六个目的是提供一种降低豆酱中生物胺含量的方法，所述方法是在豆酱生产第0天接种所述乳酸片球菌M28，或加入所述微生物制剂。
在本发明的一种实施方式中，所述乳酸片球菌M28的接种量为10～50mL/L，搅拌均匀后30～40℃发酵30～40天。
在本发明的一种实施方式中，所述乳酸片球菌M28在豆酱中菌株浓度为10 3～10 9CFU/g。
本发明第七个目的是提供一种改善食品风味的方法，所述方法为在食品发酵过程中加入所述乳酸片球菌M28，或加入所述微生物制剂。
在本发明的一种实施方式中，在豆酱发酵第0天加入所述乳酸片球菌M28或所述微生物制剂，在30～40℃发酵30～40天。
在本发明的一种实施方式中，所述乳酸片球菌M28在豆酱中的浓度为10 3～10 9CFU/g。
本发明的第八个目的是提供所述乳酸片球菌或所述微生物制剂在制备调味品中的应用。
在本发明的一种实施方式中，所述调味品包括黄豆酱、豆瓣酱或豆面酱。
本发明还提供所述乳酸片球菌或所述微生物制剂在发酵食品领域降低生物胺含量方面的应用。
在本发明的一种实施方式中，所述食品包括需要用黄豆、蚕豆或肉类发酵的食品。
在本发明的一种实施方式中，所述食品包括酱油、香肠、鱼露、奶酪。
有益效果：本发明提供的乳酸片球菌M28是来自工厂蚕豆酱样品发酵过程中的无害菌，对于酪胺、亚精胺这2种发酵食品中常见的生物胺具有较明显的降解作用，对于豆酱中酪胺、亚精胺的降解率可达59.33％和58.41％，可用于控制豆酱等发酵食品生产过程中的生物胺含量。另外，利用本发明提供的乳酸片球菌M28酿造的豆酱中与鲜味相关的天冬氨酸与谷氨酸分别比对照高出了13.98％和6.43％，使得本发明的豆酱鲜味更好，同时，该豆酱中的乳酸含 量比对照高出了23.2％，使得该豆酱味道更加柔和，从而形成独特的风味。
生物材料保藏
[根据细则91更正 28.07.2020]　
本发明所提供的乳酸片球菌(Pediococcus acidilactici)M28，己于2019年7月25日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCNo.18294，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
具体实施方式
生物胺含量测定：使用液相色谱法方法测定，具体步骤参见朱天傲等《国产酱类产品中的生物胺》。
菌株的活化方法：将菌株以20mL/L的接种量接种至培养基中，37℃厌氧培养24h。
实施例1：生物胺降解菌株的筛选及鉴定
配制MRS培养基：蛋白胨10g，牛肉膏10g，葡萄糖20g，氯化钠50g，酵母粉5g，乙酸钠5g，柠檬酸氢二铵2g，磷酸氢二钾2g，吐温-80 1.0mL，硫酸镁0.58g，硫酸锰0.25g，去离子水1L，pH 5.6。
配制BAs培养基:磷酸二氢钾2g、柠檬酸铵2g、氯化钠50g、七水合硫酸镁0.4g、硫酸锰0.03g、硫酸亚铁0.04g、硫胺素0.01g、葡萄糖2g、腐胺二盐酸盐100mg、尸胺二盐酸盐100mg、亚精胺100mg、精胺100mg、色胺100mg、苯乙胺100mg、组胺二盐酸盐100mg，酪胺盐酸盐100mg、琼脂20g、去离子水1L，pH5.5。
配制改良MRS培养基：大豆蛋白胨10g，葡萄糖20g，氯化钠50g，乙酸钠5g，柠檬酸氢二铵2g，磷酸氢二钾2g，硫酸镁0.58g，硫酸锰0.25g，吐温1mL,腐胺二盐酸盐100mg，尸胺二盐酸盐100mg，亚精胺100mg，精胺100mg，色胺100mg，苯乙胺100mg，组胺二盐酸盐100mg，酪胺盐酸盐100mg，去离子水1L，pH 5.5。
取5g酱醅于100mL锥形瓶中，加入50mL生理盐水，于摇床上充分振荡混匀，取悬浮液进行梯度稀释至10 -5，每个梯度取100μL悬浮液于BAs固体培养基和改良MRS培养基进行涂布，并于37℃培养箱进行培养，待菌落生长良好，方可分离。
磷酸盐缓冲液中生物胺降解率的测定：首先使用MRS培养基对分离的单菌落进行活化，并调节初始OD值，使其在含有100mg/L生物胺的磷酸盐缓冲液中OD值为0.6，并在37℃培养箱中放置24h，随后4000g离心5min。取上清液经过0.22μm滤膜过滤，取2mL滤液放入10mL离心管中，加入1mL浓度为2mol/L的NaOH溶液后，再加入20μL苯甲酰氯溶液，立即将离心管放入水浴恒温振荡器，30℃，200r/min振荡20min。振荡完毕后加入2mL饱和NaCl溶液停止衍生化反应，再加入3mL无水乙醚进行生物胺的萃取，使用涡旋 震荡仪萃取1min，小心吸取上清液置于5mL离心管中，使用氮吹仪吹干。用1mL乙腈溶解管中残留物，0.22μm有机滤膜过滤后上机检测，通过液相方法测定生物胺含量，与空白组对比，测定生物胺的降解率。其中菌株M28在磷酸盐缓冲液中对八种生物胺的降解率分别为13.78±0.31％、8.44±0.25％、6.81±0.17％、6.41±0.28％、8.93±0.36％、4.32±0.18％、14.10±0.98％和14.04±0.55％，被筛选出来在发酵液的环境中作进一步的降解率的测定。
发酵液中生物胺降解率的测定：使用改良MRS培养基对初筛菌株进行活化，并调节初始OD值，使其在含有100mg/L生物胺的改良MRS培养基中OD值为0.1，并在37℃培养箱中放置24h，随后4000g离心5min。取上清液经过0.22μm滤膜过滤，取2mL滤液放入10mL离心管中，加入同体积质量分数为6g/L的三氯乙酸溶液，在30℃，200r/min的水浴恒温振荡器下振荡1h，取上清液进行衍生反应，步骤同上，最终通过液相方法测定生物胺含量，并与空白组对比，测定生物胺的降解率，筛选降解率较高的菌株得到复筛菌株。其中，菌株M28对腐胺、尸胺、亚精胺、精胺、色胺、苯乙胺、组胺、酪胺的降解率分别为11.32±0.61％、6.97±1.19％、2.10±0.80％、10.57±0.57％、3.68±0.17％、1.75±3.83％、42.18±4.25％和5.80±0.81％。
提取乳酸片球菌M43菌株基因组DNA，使用引物:
27F：5’GAGAGTTTGATCCTGGCTCAG 3’
1492R:5’CTACGGCTACCTTGTTACGA 3’
进行16S rDNA PCR扩增。反应条件为：94℃预变性5min进入以下循环，94℃变性60s，56℃退火60s，72℃延伸90s，34个循环；在72℃延伸10min。送往天霖公司测序，将所得序列在NCBI数据库中进行BLAST序列比对，经鉴定为乳酸片球菌，并命名为M28。
实施例2：乳酸片球菌M28在豆酱发酵中的应用
以黄豆、盐水、面粉为原料，加入量分别为38g/100g、50g/100g、12g/100g，30℃恒温24h制曲完成后，入缸第0天接种10mL/L的乳酸片球菌，使得其在酱中菌株浓度为10 7CFU/g，以没有接入乳酸片球菌的酱醅作为对照，于对照组和实验组分别加入5mL、5g/L的腐胺、尸胺、亚精胺、精胺、色胺、苯乙胺、组胺、酪胺，并于37℃恒温培养箱静置发酵35天。取样测定发酵35天后豆酱样品中的生物胺的降解率，并以如下公式计算生物胺降解率：
Figure PCTCN2020100370-appb-000001
其中X为生物胺降解率；C 1为未接种菌株样品生物胺浓度；C 2为接种实验菌株样品生物胺浓度。
如表1所示，乳酸片球菌M28在黄豆酱发酵过程中进行接种可以有效降低黄豆酱中八种 主要生物胺的含量，腐胺、尸胺、亚精胺、精胺、色胺、苯乙胺、组胺和酪胺的降解率分别为16.92±1.98％、11.02±0.95％、58.41±3.50％、12.02±3.85％、8.95±2.75％、4.54±0.01％、4.49±0.14％和59.33±3.36。
表1 乳酸片球菌M28对八种生物胺降解能力
Figure PCTCN2020100370-appb-000002
对比例1
具体实施方式同实施例2，不同点在于，将M28替换为来源于工厂豆酱样品中的乳酸片球菌M10
表2 乳酸片球菌M10对八种生物胺降解能力
Figure PCTCN2020100370-appb-000003
注：ND表示未检测到
可见，该乳酸片球菌M10对于生物胺的降解能力比较差，对于亚精胺、精胺、色胺、组胺均没有降解能力。
实施例3
将实施例2与对比例1中发酵得到的黄豆酱进行氨基酸含量的测定，氨基酸含量测定方法：用分析天平称取黄豆酱样品1.0000g左右，置于研钵中研磨均匀，使用5g/L三氯乙酸溶液溶解并定容至25mL。放入超声波清洗仪中常温超声20min，结束后至少静置两个小时。使用双层滤纸过滤容量瓶中的溶液，取1mL澄清滤液于1.5mL离心管中，12000rpm离心10min，取上清液约500μL(可通过0.22μm水膜再次过滤)于液相取样瓶。
分析条件：Agillent1100高效液相色谱仪，4.0mm×250mm ODS柱，柱温40℃；流动相A为20mmol醋酸钠，流动相B为20mmol醋酸钠：甲醇：乙腈为l：2：2(V/V)，流动相流速为1.0mL/min；检测波长338nm，脯氨酸以262nm检测。
氨基酸含量见如下表3：
表3 不同乳酸片球菌发酵的豆酱中氨基酸含量比较
Figure PCTCN2020100370-appb-000004
Figure PCTCN2020100370-appb-000005
其中，经乳酸片球菌M28酿造的豆酱与经乳酸片球菌M10酿造的豆酱相比，谷氨酸与天冬氨酸含量均比较高，分别高出了6.43％和13.98％。天冬氨酸与谷氨酸是主要的呈鲜味氨基酸，证明乳酸片球菌发酵过的黄豆酱鲜味更好。且从氨基酸总量来看，经乳酸片球菌M28酿造的豆酱也比乳酸片球菌M10酿造的豆酱高出了4.59％，反映出实验组的黄豆酱营养价值较高。
实施例4
将实施例2与对比例1中发酵得到的黄豆酱进行有机酸含量的测定，有机酸含量测定方法：称取2g-10g酱醅放入50mL EP管中，加入40mL超纯水浸泡2h，每隔15min震荡一次，7500rpm离心5min。取上清液1mL于2mL EP管中，加入106g/L亚铁氰化钾溶液和硫酸锌溶液各0.4mL去除蛋白质和色素，静止沉淀2h(涡旋振荡)后7500rpm离心3min，取上清液10000rpm离心5min，然后0.22μm滤膜(绿色水系)过膜。
有机酸标样：草酸、丙酮酸、乙酸、乳酸、琥珀酸浓度分别为0.05、0.05、0.50、0.50、0.50和1.00mg/mL。
分析条件：色谱柱：Waters Atlantis T3(5um,4.6×250mm)；柱温：40℃；流动相：精确称取NaH 2PO 4·2H 2O 3.12g，用去离子水定容至1000mL，用H 3PO 4调节溶液pH值至2.7；进样体积：10uL；流速：0.7mL/min；检测波长：UV210nm。
有机酸含量见如下表4：
表4 不同乳酸片球菌发酵的豆酱中有机酸含量比较
Figure PCTCN2020100370-appb-000006
如表4所示，发酵结束之后的黄豆酱中的有机酸主要为乙酸和乳酸。由于乳酸片球菌M28作用于酱中可发酵碳水化合物生成乳酸较多，比对照高出了23.2％，小分子的乳酸使黄豆酱味道更加柔和，从而形成独特的风味。
实施例5：乳酸片球菌在制备酱油中的应用
将豆粕加水并蒸熟；在蒸熟的原料中，与面粉充分混匀并接入扩大培养米曲霉菌种，充分拌匀；接种后的曲料进行通风培养，控制制曲温度，制得成曲；在成曲中加入盐水拌匀入发酵池，再接入本发明的乳酸片球菌，品温42～45℃发酵至酱醅基本成熟；将成熟的酱醅进行后加工得到后处理酱油，后处理酱油加热灭菌，再配制(勾兑)、澄清及质量检验，得到符合质量标准的成品。
实施例6：乳酸片球菌在制备豆瓣酱中的应用
以蚕豆、盐水、面粉为原料，蚕豆100千克、面粉4.3千克、种曲0.29～0.43千克。将大豆进行蒸煮并与面粉、种曲混匀，在30℃恒温48h制曲，得到成曲。制曲完成后，拌盐水入缸第0天接种本发明的乳酸片球菌，于37℃恒温发酵至酱醅成熟；酱醅成熟之后进行加热灭菌，即得到豆瓣酱成品。
实施例7：乳酸片球菌在制备奶酪中的应用
将鲜奶经消毒后，加入无菌乳酸菌发酵剂和本发明的乳酸片球菌，使牛奶发酵产酸，控制温度在32～34℃，搅拌并发酵45～50分钟；再加入凝乳酶，搅拌10分钟后，静置使牛奶凝乳，凝乳大约35～40min，凝乳后即得到奶酪。
实施例8：乳酸片球菌在制备鱼露中的应用
将沙丁鱼洗净后，加入鱼重量20％～30％的食用盐，盐与鱼混匀后再加入本发明的乳酸片 球菌，在自然条件下发酵1年，经过过滤等后处理即得到鱼露。
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 (18)

  1. 一株乳酸片球菌(Pediococcus acidilactici)，于2019年7月25日由中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏编号为CGMCC No.18294，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。
  2. 一种培养权利要求1所述乳酸片球菌的方法，其特征在于，将乳酸片球菌以体积比为1％～5％的接种量接种于培养基中，于35～40℃恒温培养箱静置培养20～30h。
  3. 含有权利要求1所述乳酸片球菌的微生物制剂。
  4. 根据权利要求3所述的微生物制剂，其他特征在于，所述微生物制剂包括乳酸片球菌干菌体或湿菌体。
  5. 根据权利要求3所述的微生物制剂，其特征在于，所述微生物制剂中含有活菌数≥107CFU/g或活菌数≥107CFU/mL的乳酸片球菌细胞。
  6. 一种降低生物胺的方法，其特征在于，所述方法是将权利要求1所述的乳酸片球菌加入含生物胺的液体、半固体或固体中，或将权利要求3～5任一所述微生物制剂加入含生物胺的液体、半固体或固体中。
  7. 一种降低豆酱中生物胺含量的方法，其特征在于，所述方法是在豆酱生产过程中加入权利要求1所述乳酸片球菌，或加入权利要求3～5任一所述微生物制剂。
  8. 根据权利要求7所述的方法，其特征在于，在豆酱发酵第0天加入所述乳酸片球菌，在30～40℃发酵30～40天。
  9. 根据权利要求7所述的方法，其特征在于，在豆酱发酵第0天加入所述微生物制剂，在30～40℃发酵30～40天。
  10. 根据权利要求7～9任一所述的方法，其特征在于，所述乳酸片球菌在豆酱中的浓度为103～109CFU/g。
  11. 一种改善食品风味的方法，其特征在于，在食品发酵过程中加入权利要求1所述乳酸片球菌，或加入权利要求3～5任一所述微生物制剂。
  12. 根据权利要求11所述的方法，其特征在于，在豆酱发酵第0天加入所述乳酸片球菌或所述微生物制剂，在30～40℃发酵30～40天。
  13. 根据权利要求11或12所述的方法，其特征在于，所述乳酸片球菌在豆酱中的浓度为103～109CFU/g。
  14. 权利要求1所述的乳酸片球菌或权利要求3～5任一所述微生物制剂在制备调味品中的应用。
  15. 根据权利要求14所述的调味品，其特征在于，所述调味品包括黄豆酱、豆瓣酱或甜面酱。
  16. 权利要求1所述乳酸片球菌，或3～5任一所述微生物制剂在发酵食品领域降低生物胺含量方面的应用。
  17. 根据权利要16所述的应用，其特征在于，所述食品包括需要用黄豆、蚕豆或肉类发酵的食品。
  18. 根据权利要16所述的应用，其特征在于，所述食品包括酱油、香肠、鱼露、奶酪。
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CN113308501A (zh) * 2021-07-09 2021-08-27 甘肃省科学院生物研究所 一种乳酸菌胞外多糖的制备方法
CN113969305A (zh) * 2021-09-30 2022-01-25 广东美味鲜调味食品有限公司 一种调味酱在生产过程中的菌落总数快速检测方法
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CN114395512B (zh) * 2022-02-22 2024-01-16 宁夏大学 一株可降解生物胺的乳酸菌及其应用
CN114540231A (zh) * 2022-02-25 2022-05-27 江南大学 一种在发酵食品中促进风味物质产生的乳酸片球菌及其应用

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