CN111961615B

CN111961615B - Saccharopolyspora capable of reducing biogenic amine and application thereof

Info

Publication number: CN111961615B
Application number: CN202010812299.6A
Authority: CN
Inventors: 毛健; 刘双平; 张晶; 尹倩倩
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-08-13
Filing date: 2020-08-13
Publication date: 2021-07-27
Anticipated expiration: 2040-08-13
Also published as: CN111961615A

Abstract

The invention discloses saccharopolyspora for reducing biogenic amine and application thereof, belonging to the technical field of food fermentation. The invention screens and obtains a saccharopolyspora fuliginosa (Saccharomyces cerevisiae) J2 with the function of reducing the content of biological amine from wheat starter, and the bacterial strain can be used for reducing the content of the biological amine and simultaneously improving the content of amino acid and the nutritive value of a fermented product in the process of brewing alcoholic liquor (white wine and yellow wine), fermented sausage or soy sauce, thereby achieving the functions of enhancing the quality of the fermented food and improving the safety of the fermented food, and having wide application prospect.

Description

Saccharopolyspora capable of reducing biogenic amine and application thereof

Technical Field

The invention relates to saccharopolyspora for reducing biogenic amine and application thereof, belonging to the technical field of food fermentation.

Background

Yellow wine is a kind of brewed wine, which is made up by using glutinous rice, corn and husked millet as raw material, adding wheat yeast and yeast as saccharifying agent and fermenting agent, and making them pass through the processes of cooking, adding yeast, saccharifying and fermenting, squeezing, filtering, decocting, storing and blending. Besides the main components of water and ethanol, the yellow wine also contains 18 amino acids, wherein the amino acids comprise 8 essential amino acids, and the 8 amino acids are many times of those in the same amount of wine and beer, so that the yellow wine is beneficial to body health after being drunk frequently; yellow wine contains abundant antioxidant substances, such as polyphenol, polysaccharide, polypeptide, etc., and has antioxidant activity. The yellow wine brewing is different from beer and wine, an open type fermentation process is adopted, the amino acid content in a fermentation system is rich, the types and the number of microorganisms are complex, the bacterial community structure is complex, and the bacteria participating in the fermentation mainly comprise acetic acid bacteria, lactic acid bacteria, bacillus, saccharopolyspora and the like. However, the metabolites of the microorganisms bring unique flavor to the yellow wine and also cause the yellow wine to contain some harmful substances, such as biogenic amine and the like.

Biogenic amine is a nitrogenous organic basic micromolecule compound, is formed by decarboxylation of amino acid, is generally present in animals, plants and microorganisms, can promote growth, remove free radicals, enhance metabolic activity and enhance immunity, has important physiological functions in human bodies, but can cause the expansion of arteries, blood vessels and capillaries due to excessive biogenic amine intake, and can cause adverse physiological reactions such as diarrhea, headache, abdominal spasm, vomiting and the like, and even death.

Biogenic amine is produced in the production process of various foods, and is particularly rich in fermented foods such as yoghourt, yellow wine, white spirit, cooking wine, soy sauce, table vinegar, wine and the like. Biogenic amines in fermented foods are formed primarily by the action of amino acid decarboxylases produced by microbial metabolism on free amino acids. In the fermentation process, the microorganism metabolizes to produce protease and carboxypeptidase which act on proteins in the grains, low molecular peptides and amino acids are generated through decomposition, abundant precursor substances are provided for generation of biogenic amine, and a large amount of biogenic amine is generated in amino acid decarboxylase.

At present, no report about the application of saccharopolyspora in food fermentation and biogenic amine reduction is reported in domestic and foreign research. Therefore, the method has important significance for producing high-quality, high-yield, special-flavor and high-quality fermented food and improving the safety of the fermented food by screening excellent-performance microorganisms by using modern biotechnology.

Disclosure of Invention

The invention aims to solve the problem of high content of biogenic amine in the traditional brewed food, and provides a bacterial strain with excellent performance, namely saccharopolyspora fuliginosa J2, which is applied to the biological enhancement in the fermentation process of wines (white spirit and yellow wine), sausages and soy sauce so as to reduce the biogenic amine content in the fermented food, improve the taste and flavor of the food and better exert the application of actinomycetes in the traditional fermented food.

The first purpose of the invention is to provide saccharopolyspora fuliginosa J2, wherein the saccharopolyspora fuliginosa is preserved in China Center for Type Culture Collection (CCTCC) in 30 months and 4 months in 2020, the preservation address is Wuhan university in Wuhan, China, and the preservation number is CCTCC NO: m2020103.

The saccharopolyspora provided by 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 pure wheat starter prepared by the strain is suitable for fermentation of yellow wine, and can improve the amino acid content in the yellow wine;

(3) the biogenic amine production is less than 2.5mg/L, and the detected amount of biogenic amine is very little;

(4) has degradation effect on tyramine, histamine, putrescine and cadaverine;

(5) can be used for fermenting sausage, yellow wine, Chinese liquor and soy sauce, and has effect in reducing biogenic amine.

A second object of the present invention is to provide a starter culture containing s.hirsuta J2.

In one embodiment, the amount of S.hirsuta J2 per gram or ml of starter is ≥ 1X 10⁶CFU。

In one embodiment, the starter is prepared by inoculating the Saccharopolyspora fuliginosa into an actinomycete liquid culture medium, and culturing at 28-30 ℃ to obtain the starter containing Saccharopolyspora fuliginosa J2 cells.

The third purpose of the invention is to provide a yeast prepared by using the S.hirsuta J2, and the preparation method comprises the following steps: crushing raw materials for making yeast, adding water, stirring, sterilizing, inoculating strain in sterile environment with inoculation amount of 5 ‰ -15%, and bacterial liquid concentration of 10⁵～10⁸cfu/mL, culturing for 72-96 h at 25-55 ℃, and preserving for later use after the culture is finished.

In one embodiment, the raw saccharopolyspora koji is cooled by boiling under aseptic conditions; the process of uniformly mixing the inoculated yeast and the raw materials and the later rake opening process are carried out in a sterile environment; the saccharopolyspora koji is obtained by inoculation and culture in a sterile environment.

In one embodiment, the koji is a true wheat koji prepared using the s.hirsuta J2.

In one embodiment, the preparation method of the wheat koji comprises the following steps:

(1) grinding wheat: the crushing degree of the wheat is 3-5 tablets per grain, a small amount of powder is contained, the wheat grain tissue is crushed, and starch is exposed;

(2) moistening wheat: adding 30-40% of clear water into the materials treated in the step (1), and stirring for 15-25min to ensure that the materials fully and uniformly absorb water;

(3) and (3) cooking and sterilizing: cooking and sterilizing the materials treated in the step (2);

(4) inoculation: after the temperature of the material in the step (3) is reduced to be lower than 40 ℃, the activated saccharopolyspora fuliginosa J2 is inoculated, and the inoculation concentration is 10⁵～10⁶CFU/mL；

(5) And (5) fermenting.

In one embodiment, the fermentation of step (5) comprises the steps of:

a) and (3) spore germination period: after the yeast material is fed into the tray for 6 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 minutes at intervals of 2 hours, and requiring uniform blowing;

b) growth period of hyphae: after 3-5 times of intermittent ventilation, hypha starts to grow, the temperature of the product rises to above 35 ℃, the yeast material starts to agglomerate, and continuous ventilation is needed at the moment, and the temperature of the product is kept at 35 +/-2 ℃;

c) and (3) hypha propagation stage: after 12 hours of inoculation, the temperature of the product rises quickly, at the moment, the first agglomeration condition is considered to be turned, before turning, the temperature measuring probe is firstly raised, the turning machine is started, then the product is flattened, the temperature measuring head is put down, and the ventilation and spraying system is started;

d) after the first turning over, the product temperature is kept between 36 and 37 ℃, ventilation spraying is kept smooth, after about 20 hours, the koji material is caked again, the eye-watching koji material is whitish, the temperature is controlled below 37 ℃, the second turning over is carried out, and after the second turning over, the product temperature is controlled to be 35 +/-2 ℃.

The fourth purpose of the invention is to provide the application of the pure wheat koji prepared by the S.hirsuta J2 in fermented yellow wine.

The fifth purpose of the invention is to provide the application of the S.hirsuta J2 or the pure wheat koji containing the S.hirsuta J2 in preparing fermented food, beverage or seasoning.

In one embodiment, the food product includes, but is not limited to, fermented or semi-fermented food products of sausages.

In one embodiment, the beverage includes, but is not limited to, yellow wine or white wine fermentation.

In one embodiment, the seasoning includes, but is not limited to soy sauce or sausage.

In one embodiment, the application is fermentation after pure wheat koji containing S.hirsuta J2 is mixed with brewing raw materials.

In one embodiment, the method comprises the step of uniformly mixing the pure wheat koji with rice, yeast wine and other raw materials in a fermentation tank according to the inoculation amount of 12-16%, and then fermenting, wherein the fermentation adopts a traditional fermentation process.

The sixth purpose of the invention is to provide the application of the S.hirsuta J2 strain in reducing biogenic amine in sausages, yellow wine, white spirit and soy sauce.

In one embodiment, the biogenic amines include, but are not limited to, tyramine, histamine, putrescine, cadaverine.

In one embodiment, the brewed yellow wine and white wine are prepared into pure koji by utilizing the saccharopolyspora and added into the fermentation of wine.

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 pure wheat starter prepared by the strain can be used for yellow wine fermentation, so that the alcohol yield can be promoted, and the amino acid content in the yellow wine can be improved; the content of amino acid in the pure fermented yellow wine can reach 6434.81 +/-123.3 mg/L to the maximum extent, and is increased by 40.37 percent compared with a control group sample; the flavor of the traditional yellow wine is not obviously influenced by the addition of the strain; compared with a control group, the content of the biogenic amine in the sample group added with the S.hirsuta J2 is reduced by 21.71%, the amino acid state content and the nutritional value in the yellow wine are improved, and the purposes of improving the content of the amino acid and the volatile substance in the yellow wine and improving the quality of the yellow wine are achieved.

(3) S. hirsuta J2, the biogenic amine production is less than 2.5mg/L, the biogenic amine detection amount is very little, and biogenic amine is not produced basically. S. hirsuta J2 has tyramine degradation rate of 77.41%, histamine degradation rate of 100%, putrescine degradation rate of 58.1%, cadaverine degradation rate of 47.71% and total biogenic amine degradation rate of 72.98%. Both strains have good biogenic amine reducing capability.

(4) The saccharopolyspora has a biological amine reducing effect, the saccharopolyspora has the biological amine reducing effect, the average content of the biological amine in the sausage is 129.60mg/kg after the fermentation by adding S.hirsuta J2, the biological amine reducing effect is obvious, and the biological amine reducing effect is reduced by 36.19% compared with that of a control group; the content of biogenic amine in the white spirit added with S.hirsuta J2 is reduced by 24.32 percent compared with that in the control group; soy sauce with s.hirsuta J2 added reduced biogenic amine content by 34.28% compared to control.

Biological material preservation

Saccharopolyspora rosea (Saccharopolyspora jiangxiensis) J3, classified and named Saccharopolyspora rosea (Saccharopolyspora jiangxiensis) J3, has been preserved in China center for type culture collection in 30 months 4 in 2020, with the preservation address being China, Wuhan university with the preservation number being CCTCC NO: m2020104.

Saccharopolyspora fuliginosa (Saccharopolyspora hirsuta) J2, classified and named Saccharopolyspora fuliginosa (Saccharopolyspora hirsuta) J2, has been deposited in China Center for Type Culture Collection (CCTCC) in 30 months and 4 of 2020, with the deposition address of China, Wuhan university and the deposition number of CCTCC NO: m2020103.

Drawings

Fig. 1 is a phylogenetic tree of saccharopolyspora s.

FIG. 2 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.

FIG. 3 is the main component analysis of the flavor substance of the yellow wine fermentation sample.

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 biogenic amine content flavor substances are detected by adopting a High Performance Liquid Chromatography (HPLC) and a gas chromatography-mass spectrometer (GC-MS). The content of reducing sugar is measured by a DNS method.

Detecting the physical and chemical indexes of the sausage: the content of the crude protein is determined according to a GB5009.3-2010 semimicro Kjeldahl method; the moisture content was measured according to GB/T9695.15-2008. And (3) pH measurement: 10g of the sample was added to 90mL of distilled water and homogenized, and then allowed to stand for 2min, and the pH value of the supernatant was measured with a pH meter.

Example 1: screening and identification of saccharopolyspora

(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

Actinomycete screening culture medium: 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 deg.C).

Under the aseptic operation environment, a sterile pipette is used for sucking 1mL of sample, placing the sample into a 15mL sterile centrifuge tube, adding sterile water to 10mL, and fully and uniformly mixing to prepare 10^-1Homogenizing the sample. Suction with sterile pipette 10^-11mL of sample homogenizing solution is put into a 15mL sterile centrifuge tube, sterile water is added to 10mL, and the mixture is fully and uniformly mixed to prepare 10^-2Homogenizing the sample. By the above operation, 10 is made^-1～10^-6Ten times increasing series of wheat starter, rice milk water and fermented mash dilution homogenizing liquid.

Respectively sucking 100 mu L of each dilution bacterial liquid of the wheat starter, the fermented mash and the rice milk water, coating the dilution bacterial liquid on an actinomycete screening culture medium, and culturing for 1-7 days at 28 ℃. Selecting single milky white, thin, raised or convex and slightly wrinkled bacterial colonies on a flat plate with moderate bacterial colony density, streaking and inoculating the bacterial colonies on an actinomycete screening culture medium respectively, repeatedly streaking to determine pure bacterial colonies, and storing screened bacterial strains.

(3) Identification of strains

Extracting the genome of the screened strain, and carrying out 16S rDNA amplification sequencing on the screened strain.

Primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were PCR amplified.

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 electrophoresis on a 1% agarose gel and sent to sequencing company for sequencing, and the 16SrDNA result was as follows:

GACCACTCCCCCCACAAGGGTTGGGCCATGGGCTTCGGGTGTTACCGACTTTCATGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCGACTCCGACTTCACGGGGTCGAGTTGCAGACCCCGATCCGAACTGAGACCGGCTTTAAGGGATTCGCTCAACCTCACGATCTCGCAGCCCTCTGTACCGGCCATTGTAGCATGTGTGAAGCCCTGGGCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGAGTTGACCCCGGCAGTCCCCCACGAGTCCCCGGCATTACCCGCTGGCAACATAGGGCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCAACCACAAGGGAAACCCCCTCTCAGGGGCTGTCTAGTGCATGTCAAACCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCGCTTAATGCGTTAGCTACGGCACGGAAACAGTGGAACCCATCCCCACACCTAGCGCCCAACGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGACCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCAGTCTCCCCTACCGAACTCAAGTCTGCCCGTATCCACCGCAAGCCAGGAGTTAAGCTCCCGGTTTTCACGATAGACGCGACAAACCGCCTACGAGCTCTTTACGCCCAATAAATCCGGACAACGCTCGCACCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGTGCTTCTTCTACACCTACCGTCACCCGAAGGCTTCGTCGATGTCGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCCCACGCGGCGTTGCTGCGTCAGGCTTTCGCCCATTGCGCAAGATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCACCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTAGGCCACTACCCCACCAACAAGCTGATAGGCCGCGGGCTCATCCTGCACCGCCAGAACTTTCCACACCAGAACATGCCTCCAGGTGTCGTATCCGGTATTAGACCTCGTTTCCAAGGCTTATCCCGAAGTGCAGGGCAGATTACCCACGTGTTACTCACCCGTTCGCCACTCATCCACACCCGAAAGTGCTTCAGCGTTCGACT。

according to the returned sequencing result (shown as SEQ ID NO. 1), BLAST sequence alignment is carried out through NCBI official website, BLAST alignment is carried out by using the obtained 16S rDNA sequence, phylogenetic analysis is carried out, and as a result, shown as figure 1, the homology similarity of the nucleotide sequence of the strain J2 and saccharopolyspora S.hirsuta (GenBank accession numbers: MN515057.1 and NR _118870.1) reaches more than 98.30 percent, and the strain is named as saccharopolyspora fuliginospora S.hirsuta J2.

(4) Analysis of biogenic amine metabolizing ability of strain

Activating strains: inoculating the preserved Saccharopolyspora fuliginosa S.hirsuta J2 into actinomycete liquid culture medium, inoculating 10%, and shake culturing at 30 deg.C for 48h to obtain first-stage seed liquid. Inoculating the activated strain into an actinomycete liquid culture medium, wherein the inoculation amount is 10%, performing shake culture for 48h, and performing shaking culture at the rotation speed of 150r/min and the temperature of 30 ℃.

Sample pretreatment: respectively inoculating the strains in a culture medium for detecting biogenic amine production and a culture medium for detecting biogenic amine degradation, carrying out shake cultivation at 28 ℃ for 5d, and centrifuging at 12000r/min for 5min to collect supernatant.

Actinomycete liquid culture medium: 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, and pH of 7.2-7.4(25 deg.C).

Detection of biogenic amine production medium: 0.4g/L of L-tyrosine, 1g/L of L-histidine, 1g/L of L-lysine and 1g/L of L-ornithine, 0.05g/L of pyridoxal-5' -phosphate are added into an actinomycete liquid culture medium.

Detecting a biological amine degradation culture medium: adding 50mg/L biogenic amine (including histamine, tyramine, cadaverine, putrescine, spermine, spermidine, tryptamine, and beta-phenylethylamine) into actinomycete liquid culture medium, and adjusting pH to 6.0-6.2.

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 NaHCO₃The solution is mixed evenly, 2mL dansyl chloride (5mg/mL acetone) reagent is added, the mixture is placed in a constant temperature water bath kettle at 65 ℃ for dark derivatization for 30min after being mixed evenly, 0.5mL saturated NaCl solution is added after being stood at room temperature, 5mL diethyl ether is added after being mixed evenly, vortex oscillation is carried out for 20s, after standing and layering, the upper organic phase is transferred into a 15mL centrifuge tube, the lower aqueous phase is extracted once again, the two extraction solutions are combined, and nitrogen blow-drying is carried out under 50 ℃ water bath. 1mL of the solution was addedAcetonitrile was shaken well to dissolve the residue, filtered through a 0.22 μm filter and measured by High Performance Liquid Chromatography (HPLC).

Analysis of effect of saccharopolyspora shaeofaciens J2 on reducing biogenic amine: the biogenic amine production amount of the saccharopolyspora trichoderma J2 after culture in the detection biogenic amine production medium and the detection biogenic amine degradation medium is less than 2.5mg/L, and the biogenic amine detection amount is extremely small, which indicates that the biogenic amine content is not basically detected, and thus, the biogenic amine is not considered to be produced. The saccharopolyspora crenata J2 has the degradation rate of 77.41% for tyramine, the degradation rate of 100% for histamine, the degradation rate of 58.1% for putrescine, the degradation rate of 47.71% for cadaverine and the degradation rate of 72.98% for total biogenic amine, which indicates that the strains have good biogenic amine reducing capability.

Example 2: activated culture of Saccharopolyspora shawinii S.hirsuta J2

Actinomycete liquid culture medium: 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, and pH 7.2-7.4 (measured at 25 ℃).

PDA culture medium: 6.0g/L of potato powder; glucose 20.0g/L, agar 20.0g/L, pH 5.4-5.8, and autoclaving at 121 deg.C for 15 min; solid medium was added on this basis.

MRS culture medium: 10g/L of beef extract, 10g/L of peptone, 0.5g/L of yeast extract, 20g/L of glucose, 800.10 g/L of tween, 5g/L of sodium acetate, 2g/L of dipotassium phosphate, 2g/L of diammonium hydrogen citrate, 0.58g/L of magnesium sulfate and 0.28g/L of manganese sulfate.

The Saccharopolyspora shaggua J2 screened in example 1 was inoculated into an actinomycete liquid medium at an inoculum size of 10% and subjected to shake cultivation at 30 ℃ for 48 hours to obtain a primary seed solution. Inoculating the activated strain into an actinomycete liquid culture medium, wherein the inoculation amount is 10%, performing shake culture for 48h, the rotating speed is 150r/min, the temperature is 25-45 ℃, and the concentration of the obtained strain is 10⁵～10⁶cfu/mL bacterial liquid is used for preparing pure wheat koji after being cultured and matured.

Inoculating the preserved Aspergillus flavus and Aspergillus oryzae on a PDA plate, and culturing for 3-5 days at 28 ℃; then cleaning spore liquid with sterile water, and transferringInoculating into PDA eggplant-shaped bottle, culturing at 28 deg.C for 3-5 days to obtain bacterial concentration of 10⁵～10⁶cfu/mL bacterial liquid is used for preparing pure wheat koji after spores are mature.

Inoculating the preserved Lactobacillus L.plantarum (Lactobacillus plantarum) into an MRS culture medium, inoculating 10%, carrying out anaerobic culture at constant temperature of 37 ℃ for 24h to obtain a first-grade seed culture solution, inoculating the activated seed solution into the MRS liquid culture medium again, inoculating 10%, carrying out anaerobic culture at constant temperature of 37 ℃ for 24h to obtain a strain concentration of 10%⁵～10⁶cfu/mL bacterial liquid, and the strains are used for preparing pure wheat koji after being cultured to be mature.

Example 3: preparation of pure wheat yeast of saccharopolyspora

(2) moistening wheat: adding about 35-40% of clear water into the material treated in the step (1), and stirring for 20-25 min to ensure that the material fully and uniformly absorbs moisture;

(3) and (3) cooking and sterilizing: sterilizing the material treated in the step (2) at 121 ℃ for 30 min;

(4) inoculation: after the temperature of the material in the step (3) is reduced to 36 ℃, the activated strain in the example 2 is inoculated, and the concentration of the inoculated bacterial liquid is 10⁵～10⁶cfu/mL, and the inoculation amount is 5 per mill-15%.

(5) 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.

a) And (3) spore germination period: after the yeast material is fed into the tray for six hours, the temperature of the product slowly rises to about 34-35 ℃, the small-air-volume indirect ventilation is started in the self-control mode, 5-10 minutes are carried out each time, the interval is 2 hours, the temperature of the product is reduced to 32 ℃, and the product is required to be uniformly blown through.

b) Growth period of hyphae: and (3) after intermittent ventilation is carried out for 3-5 times, hypha begins to grow, the temperature of the product rises to above 35 ℃, the yeast material begins to agglomerate, and continuous ventilation is carried out at the moment, and the temperature of the product is kept about 35 ℃.

c) And (3) hypha propagation stage: and (3) after 12 hours of inoculation, the temperature of the product rises quickly, at the moment, the first agglomeration condition is considered to be turned, before turning, the temperature measuring probe is lifted, the turning machine is started, then the product is flattened, and the temperature measuring head is put down. The ventilation and spraying system is turned on.

d) After the first turning over, the product temperature is kept between 36 and 37 ℃, ventilation spraying is kept smooth, after about 20 hours, the koji material is caked again, the eye-looking koji material is whitish, the temperature is difficult to control below 37 ℃, the second turning over is carried out, and after the second turning over, the product temperature is controlled to be about 35 ℃.

(6) And (3) yeast discharging: culturing for 72-96 h; after the culture is finished, storing the wheat koji in a freezer at 4-7 ℃ for later use;

the order of magnitude of the obtained thallus is 10¹⁵CFU/g pure wheat starter cultured by Saccharopolyspora fuliginosa J2 and pure wheat starter cultured by Saccharopolyspora crassa J3.

Example 4: application of saccharopolyspora pure wheat starter in yellow wine fermentation

(1) The raw material ratio (in terms of fermentation volume per liter) of the traditional yellow wine fermentation selected in this example is as follows:

steamed rice: 500 g; 417L of clear water; and (3) yeast: 38g of the total weight of the mixture;

(2) traditional yellow wine brewing process

a) And (3) yeast activation culture: transferring the yeast in the glycerol storage tube into a YPD culture medium in a sterile operating platform, and culturing for 24h at 30 ℃ under the condition of 150 r/min; then transferring the yeast into the prepared yeast, and culturing the transferred yeast at 30 ℃ and 150r/min for 18-24h for later use.

b) Preparing yeast wine: adding 1600mL of clear water, 60g of raw wheat starter and 800U/g of saccharifying enzyme into 600g of steamed rice, saccharifying at 55-65 ℃ for 3-4 hours, sterilizing at 115 ℃ for 15min after the saccharification is finished and the apparent sugar degree is not lower than 12 degrees Bx, cooling to 24-31 ℃ after the sterilization, inoculating 5% of mature yeast seed culture solution, culturing at the temperature not higher than 30 ℃ for 24 hours, and culturing for maturation to obtain the yeast.

c) And (3) blanking and fermenting according to the raw material proportion of the traditional yellow wine fermentation in the step (1). Experimental groups: pure wheat koji: 45.3g control: raw wheat koji: 39.3 g; cooked wheat koji: 6.0 g.

The first four days are a pre-fermentation stage, the temperature is controlled to be 28-30 ℃, the fermentation is carried out for 4 days, harrowing is carried out for not less than 1 time every day for the first 4 days, and harrowing time is 8-10 hours; and in the post-fermentation stage, the temperature is 13-15 ℃, the rake is stirred and harrowed once a day, and the fermentation is continued for 10-15 days.

The Control group (TF Control) was prepared by adjusting the pure wheat koji in (3) in this example to 39.3g/L raw wheat koji and 6.0g/L cooked wheat koji sampled in the factory.

The composite microbial inoculum group (Mix) is added with pure wheat starter of Saccharopolyspora fuliginosa J2 and pure wheat starter of Saccharopolyspora Jiangxi J3 according to the ratio of the number of bacteria 1:1, and the adding amount is totally 45.3 g.

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 the traditional wheat koji and the pure-breed wheat koji is compared. The pure-strain fermentation is divided into 4 groups, namely A.flavus (common strains in yellow wine fermentation) and A.oryzae (common strains in Japanese sake brewing, S.hirsuta J2 and L.plantarum), wherein the 4 pure-strain wheat yeasts are respectively fermented together with the saccharomyces cerevisiae by adopting a traditional brewing method, the mixed wheat-yeast fermentation group adopts the compound wheat yeast of the pure-strain wheat yeast of saccharopolyspora fulgens J2 and the pure-strain wheat yeast of the saccharopolyspora rosea J3 in Jiangxi and the saccharomyces cerevisiae are fermented together by adopting the traditional brewing method, and after the fermentation is finished, the contents of alcoholic strength, reducing sugar, total acid and amino acid nitrogen all accord with the national standard of yellow wine (figure 2), which shows that the saccharopolyspora has little important physicochemical influence on the yellow wine fermentation process and the fermentation is normal.

The amino acid content in the yellow wine fermentation sample is as follows: the content of amino acid in the fermented yellow wine is analyzed by an HPLC method, the content of the amino acid in an experiment group added with S.hirsuta J2 reaches 6434.81 +/-123.3 mg/L, and is improved by 40.37 percent compared with a control group.

(3) Analysis of effect of saccharopolyspora shaeofaciens J2 on reducing biogenic amine: the content of biogenic amine in the yellow wine is detected by a high performance liquid chromatography method, and compared with a control group, the sample group added with the saccharopolyspora shafa J2 is reduced by 21.71%.

(4) Pure fermentation and traditional fermentation flavor analysis: the changes and similarities of flavor components in the pure and traditional fermentation samples were analyzed by principal component analysis. Biplot analysis of all samples showed that the cumulative contribution of variance of the first two principal components was 83.6%, which can explain the flavor difference of most fermentation samples. As can be seen from fig. 3, the conventional fermentation group was clustered with the s.hirsuta J2 group, and clearly separated from the aspergillus (a.flavus and a.oryzae) group and the l.plantarum group. This indicates that saccharopolyspora is involved in the synthesis of most flavor substances and plays a leading role in yellow wine fermentation.

Example 5: application of saccharopolyspora fuliginosa J2 in reducing content of biogenic amine in fermented sausage

(1) Activated culture of bacterial species

Inoculating the preserved saccharopolyspora S.hirsuta J2 into an actinomycete liquid culture medium, wherein the inoculation amount is 10%, and carrying out shake culture at 30 ℃ for 59h to obtain a first-level seed liquid. Inoculating the activated strain into actinomycete liquid culture medium, wherein the inoculum concentration is 10%, performing shake culture for 48h, the rotating speed is 150r/min, the temperature is 30 ℃, and culturing is performed until the bacteria concentration is 10 DEG⁵～10⁷The cfu/mL is used for fermenting the sausage.

(2) Preparation method of fermented sausage

According to the mass percent, 65-80% of lean meat and 20-35% of fat meat are taken. Cleaning, and removing bone, tendon, muscle membrane, lymph, blood vessel, pathological changes and injury parts. And (4) separating the fat and the lean, and cutting into meat blocks of 4-5 cm. And (3) placing the lean meat and about 5-8% of ice scraps into a chopping and mixing machine, and chopping and mixing for 1-3 min. Based on the mass of the pork, 0.01 to 0.15 percent of sodium nitrite, 2 to 3 percent of salt, 0.2 to 0.3 percent of composite phosphate and 0.05 to 0.06 percent of sodium ascorbate are added. Spice, pepper, garlic, pepper and nutmeg are 0.2-0.3% of the raw material meat. Wherein, two groups are respectively inoculated with 8-12% of activated saccharopolyspora, one group is not inoculated as a control, chopped and mixed for 1-2 min, then fat meat and about 5-8% of ice scraps are added, and chopped and mixed for 4-6 min. And pouring the pickled vegetable into sausage casings. The fermentation process parameters of the sausages are shown in table 1.

TABLE 1 fermentation Process parameters of sausages

(3) Analysis of effect of Saccharopolyspora capensis J2 on reducing biogenic amine

The activation of the bacterial species and the determination of the biogenic amine content were carried out in the manner described in example 1.

The sample pretreatment method comprises the following steps: weighing 5.0g of the minced sample in a 50mL centrifuge tube, adding 20mL of 5% trichloroacetic acid, performing ultrasonic treatment for 30min, transferring the sample to the 50mL centrifuge tube with a plug, performing centrifugation at 6000 r/min for 10min, transferring the supernatant to a 50m L volumetric flask, performing extraction on the residue with 20mL of the solution for 1 time, combining the supernatants and diluting the mixed solution to a scale mark.

As can be seen from Table 2, the pH values of the sausages fermented by adding the saccharopolyspora shafa J2 after the sausages are fermented to be mature are respectively 6.16 +/-0.16, while the pH values of the sausages fermented by adding the saccharopolyspora shafa J2 in the control group are respectively 6.51 +/-0.17, and the differences between the water content and the crude protein content are not obvious; after the sausage fermented by adding S.hirsuta J2 is matured, the average biogenic amine content is 129.60mg/kg, which is reduced by 36.19% compared with a control group.

TABLE 2 fermented mature sausage index

Example 6: application of saccharopolyspora sp S.hirsuta J2 in reducing content of biogenic amine in white spirit

The pure culture of the saccharopolyspora malt was prepared according to the method of example 3. The determination of the biogenic amine content was carried out in the manner as in example 1.

The liquor brewing method adopts two-round fermentation method, wherein during the first round of fermentation, sorghum is steamed, then air-cooled to 25 ℃, 4% of Aspergillus oryzae seed solution is added, and the mixture is cultured for 24h at 28 ℃. Adding 10% of rice hull, 15% of Daqu, 8% of bran and 5-9% of pure wheat starter into the saccharomyces cerevisiae seed solution according to the proportion of 1%, sealing, fermenting for 30 days, and then steaming the wine. Adding 10% of medium temperature yeast and inoculating to Saccharomyces cerevisiae seed solution at a ratio of 1% during two-round fermentation, wherein the concentration of the yeast seed solution is 10¹⁰～10¹²cfu/mL, continuing fermenting for 12-15 days, and then distilling the wine.

Analysis of the effect of saccharopolyspora sp.hirsuta J2 on reducing biogenic amine: blending distilled liquor to 60% (V/V) of alcohol content, detecting biogenic amine content in blended sample, and comparing the sample group added with S.hirsuta J2 with the control group, reducing by 24.32%.

Example 7: saccharopolyspora shawinii S.hirsuta J2 applied to soy sauce for reducing content of biogenic amine

The activation of the species and the determination of the biogenic amine content were carried out in the manner as in example 1. The soy sauce is brewed by adopting a high-salt dilute state method:

(1) firstly, uniformly mixing bean pulp and wheat according to the proportion of 1:1, and then steaming;

(2) adding bacterial liquid with the concentration of 10 percent according to the proportion of 5 per mill to 10 percent⁵～10⁶cfu/mL of saccharopolyspora shafa J2 seed liquid, then adding saline water with the mass about 1.5-2 times of that of the material, and finally mixing the sauce mash with the salt content of about 18% and the water content of 65%.

(3) Fermentation of soy sauce mash: the initial fermentation temperature is controlled at 14-16 ℃, and the temperature is gradually increased to about 35 ℃ along with the fermentation. 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 effect analysis of reducing biogenic amine of the saccharopolyspora fuliginosa J2 shows that the biogenic amine content of the soy sauce product added with the saccharopolyspora fuliginosa J2 is reduced by 34.28 percent compared with that of a control group.

Example 8: use of Saccharopolyspora capensis J2 in cooking wine for reducing biogenic amine content

The pure fermented yellow wine was obtained according to the brewing method in example 3, and 10% by mass of common salt was added to the fermented yellow wine, and the mixture was sterilized by a sterilizer at 85 ℃ for 30min and hot-filled.

Analyzing the effect of the strain on reducing the content of biogenic amine in the cooking wine: the content of biogenic amine in the cooking wine is detected by a high performance liquid chromatography method, and the content of biogenic amine is reduced in a sample group added with saccharopolyspora shafa J2 compared with a control group.

Example 9: use of Saccharopolyspora capensis J2 in lowering biogenic amine content in edible vinegar

Pure fermented yellow wine as acetic acid fermentation raw material was obtained according to the brewing method in example 3; the determination of the biogenic amine content was carried out in the manner described in example 1.

The acetic acid fermentation adopts a solid state fermentation process: uniformly stirring the chaff, the bran and the yellow wine according to the mass ratio of 1:4:10, inoculating 5% of vinegar grains, turning the grains from the surface of the material every day within 2 days before inoculation, and keeping the temperature at 35-42 ℃. Turning to the bottom of the material when the day is 6-8. And turning over the fermented grains from the bottom every day on 8-12 days, and naturally reducing the temperature. Separating from vinegar residue to obtain raw vinegar, sterilizing at 85 deg.C for 30min, and aging for 12 months. Hot filling after high temperature sterilization before filling.

The quality of the prepared vinegar is measured, and the content of acetic acid in the solid fermented vinegar is 55 g/L. Analyzing the content of biogenic amine in the vinegar: the content of biogenic amine in the cooking wine is detected by a high performance liquid chromatography method, and the content of biogenic amine is reduced in a sample group added with saccharopolyspora shafa J2 compared with a 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

<120> saccharopolyspora capable of reducing biogenic amine and application thereof

<130> BAA200602A

<160> 3

<170> PatentIn version 3.3

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cccgtcaatt cctttgagtt ttagccttgc ggccgtactc cccaggcggg gcgcttaatg 600

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Claims

1. Saccharopolyspora fuliginosa (Saccharopolyspora hirsuta) J2, which has been deposited in the chinese culture collection at 30 months 4 of 2020 with a deposit number of CCTCC NO: m2020103.

2. A microbial preparation comprising the Saccharopolyspora capensis J2 according to claim 1.

3. The microbial preparation of claim 2, wherein the amount of saccharopolyspora shagga J2 per gram or per ml of the fermentation product is not less than 1X 10⁶CFU。

4. The microbial preparation according to claim 2 or 3, which comprises live cells of the above Saccharopolyspora capensis J2, dried cells obtained by freeze-drying, or immobilized cells.

5. The microbial preparation of claim 4, wherein said microbial preparation is a liquid microbial preparation or a solid microbial preparation.

6. A pure wheat koji, bran koji or wine medicine prepared by using the Saccharopolyspora fugii J2 as defined in claim 1.

7. Use of Saccharopolyspora exigua J2 as defined in claim 1, or a microbial preparation as defined in any one of claims 2 to 5, or a pure wheat koji as defined in claim 6 for producing fermented yellow wine.

8. Use of the Saccharopolyspora exigua J2 as defined in claim 1, or the microbial preparation as defined in any one of claims 2 to 5, or the pure malt as defined in claim 6 for the preparation of food products.

9. Use according to claim 8, wherein the food product is a fermented or semi-fermented food product.

10. Use of Saccharopolyspora exigua J2 as defined in claim 1, or a microbial preparation as defined in any one of claims 2 to 5, or a pure malt as defined in claim 6 for preparing a beverage.

11. The use according to claim 10, wherein the beverage is yellow wine or white wine.

12. Use of the Saccharopolyspora exigua J2 as defined in claim 1, or the microbial preparation as defined in any one of claims 2 to 5, or the purebred malt as defined in claim 6 for preparing seasonings.

13. Use according to claim 12, wherein the flavouring is vinegar, soy sauce, sauce or sausage.

14. Use of saccharopolyspora shafa J2 according to claim 1 for reducing the content of biogenic amines in fermented food or beverage products.