CN110846260B - Staphylococcus carnosus capable of reducing biogenic amine and application thereof - Google Patents
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Abstract
The staphylococcus carnosus is preserved in the China general microbiological culture Collection center in 25.7.2019, and has the preservation number of CGMCC No.18295, the preservation address of No. 3 Siro No.1 of the Beijing university Inward Yangxi district, and the institute of microbiology of the China academy of sciences. The strain has good capability of degrading biogenic amine, has obvious degradation effect on biogenic amine commonly seen in 4 kinds of fermented food, namely tyramine, spermidine, putrescine and cadaverine, has the degradation rates of 50.88%, 51.83%, 31.14% and 22.82% respectively, has stability in a bean paste production environment, and can be used for bean paste fermentation.
Description
Technical Field
The invention relates to a staphylococcus carnosus capable of reducing biogenic amine and application thereof, belonging to the technical field of biological fermentation.
Background
Staphylococcus carnosus (Staphylococcus carnosus) is a key species in the meat fermentation industry for developing the flavor of meat products and has been used as a starter culture for fermented meat products in Europe and the like for over 60 years. It does not produce any toxin, hemolysin, coagulase or aggregation factor, compared to other staphylococci, and is a well-established food grade gras (genetically regardized as safe) strain of the genus staphylococcus. Staphylococcus carnosus has low extracellular proteolysis capability, so the strain can be used as a gene cloning host strain for researching related metabolic pathways of the Staphylococcus in the aspect of molecular genetics and safely and normally expressing and secreting heterologous proteins.
The bean paste is a traditional bean flavoring food in China which is prepared by fermenting soybeans or broad beans, flour, hot pepper and saline water as main raw materials. Since the brewing process is an open process, various microorganisms participate in the fermentation of the soybean paste. After complex biochemical reaction, the seasoning with rich nutrition and integrated color, smell and taste is formed. However, during the fermentation process, a variety of substances including some toxic substances such as biogenic amines are often formed by the action of microorganisms. Biogenic amines are a class of nitrogen-containing organic compounds, and the following eight common main types of foods are putrescine, cadaverine, spermidine, spermine, tryptamine, phenethylamine, histamine and tyramine. Excessive intake of biogenic amine may cause many health problems such as causing headache, hypotension, nausea, hot flashes, local inflammation, palpitation, hypertension, and digestive problems, and therefore, in order to secure food safety, it is necessary to adopt a relevant method to control the content of biogenic amine. Modern plants can control biogenic amines by subjecting food to irradiation, modified atmosphere packaging, high hydrostatic pressure treatment, addition of food additives and preservatives, etc., but can be potentially harmful to humans. The biological method which is safe and environment-friendly is more and more accepted by modern people, such as adding bacterial strains which can degrade biogenic amine or dominant bacterial strains which can not produce biogenic amine in the process of food fermentation, thereby controlling the content of biogenic amine. Therefore, the strain which can adapt to the production environment of the soybean paste and can efficiently degrade the biogenic amine is screened, and the strain has important application value for preparing safe and healthy soybean paste.
In the literature, "influence of different leavening agents on flavor substance release and harmful biological amine control in fermented sausages" (7/5 in 2019), Wangdebao and the like inoculate staphylococcus carnosus and lactobacillus plantarum in the fermented sausages at the same time, and the results show that the inoculated staphylococcus carnosus and lactobacillus plantarum compound group shows advantages in degradation of histamine and tyramine compared with the inoculated lactobacillus plantarum alone: the degradation rates of the composite groups are respectively 4.8% and 66.6%, which are higher than those of the single group, namely 3.8% and 61.7%. From this, it can be seen that Staphylococcus carnosus has a certain ability to degrade biogenic amines.
Disclosure of Invention
The first purpose of the invention is to obtain a staphylococcus carnosus M43 strain through screening, which is preserved in China general microbiological culture Collection center (CGMCC) in 25.7.2019 with the preservation number of CGMCC No.18295 and the preservation address of Beijing university Hokko No. 3 of Shangyang district Beichen Xilu No.1, institute of microbiology of China academy of sciences.
The second purpose of the invention is to provide the method for culturing the staphylococcus carnosus M43, which comprises the steps of inoculating the staphylococcus carnosus M43 into a culture medium in an inoculation amount of 10-50mL/L, and standing and culturing for 20-30 h in a constant-temperature environment of 35-40 ℃.
The third purpose of the invention is to provide a method for reducing biogenic amine, which is to inoculate the staphylococcus carnosus M43 into liquid, semisolid or solid containing biogenic amine in an inoculation amount of 10-50 mL/L.
The fourth purpose of the invention is to provide an activation method of staphylococcus carnosus M43, which is to inoculate staphylococcus carnosus M43 into a culture medium in an inoculation amount of 10-50 mL/L.
In one embodiment of the invention, the activation method is carried out under the culture condition of 30-40 ℃ for 20-30 h.
In one embodiment of the invention, the medium is a modified MRS medium.
The fifth purpose of the invention is to provide a method for reducing the biogenic amine content in the soybean paste, which is to inoculate the staphylococcus carnosus M43 on the 0 th day of the fermentation of the soybean paste, wherein the inoculation amount is 10-50mL/L, and the soybean paste is uniformly stirred and then fermented for 30-40 days at 30-40 ℃.
In one embodiment of the invention, Staphylococcus carnosus M43 strain concentration in soy sauce is 103～109CFU/g。
The sixth object of the present invention is to provide a soybean paste product prepared using the staphylococcus carnosus M43.
In one embodiment of the invention, the seasoning comprises a soybean paste or a soybean flour paste.
The invention also provides application of the staphylococcus carnosus M43 in reducing biogenic amine content in the field of food.
In one embodiment of the invention, the food product comprises a food product that requires fermentation with soy beans or broad beans or meat.
In one embodiment of the invention, the food comprises soy sauce, sausage, fish sauce.
Has the advantages that: the staphylococcus carnosus M43 provided by the invention is a harmless bacterium in the fermentation process of the soybean paste, has a relatively obvious degradation effect on common biogenic amines in 4 fermented foods, namely tyramine, spermidine, putrescine and cadaverine, has the degradation rates of 50.88%, 51.83%, 31.14% and 22.82% respectively, and can be used for controlling the biogenic amine content in the production process of the fermented foods such as the soybean paste. And the amount of flavor substances in the staphylococcus carnosus M43 fermented soybean paste is 108.3% higher than that of the control, and the types of the flavor substances are more various.
Biological material preservation
The Staphylococcus carnosus (Staphylococcus carnosus) M43 provided by the invention has been preserved in the China general microbiological culture Collection center (CGMCC) in 25.7.2019, the preservation number is CGMCC No.18295, the preservation address is Beijing university Chen Xilu No.1 Hospital No. 3, China academy of sciences microbial research institute, and the Nanoclasia is the republic of China.
Detailed Description
Preparing a BAs culture medium: 2g of monopotassium phosphate, 2g of ammonium citrate, 50g of sodium chloride, 0.4g of magnesium sulfate heptahydrate, 0.03g of manganese sulfate, 0.04g of ferrous sulfate, 0.01g of thiamine, 2g of glucose, 100mg of putrescine dihydrochloride, 100mg of cadaverine dihydrochloride, 100mg of spermidine, 100mg of spermine, 100mg of tryptamine, 100mg of phenethylamine, 100mg of histamine dihydrochloride, 100mg of tyramine hydrochloride, 20g of agar and deionized water to a constant volume of 1L.
Preparing an improved MRS culture medium: 10g of soybean peptone, 20g of glucose, 50g of sodium chloride, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 100mg of putrescine dihydrochloride, 100mg of cadaverine dihydrochloride, 100mg of spermidine, 100mg of spermine, 100mg of tryptamine, 100mg of phenethylamine, 100mg of histamine dihydrochloride, 100mg of tyramine hydrochloride and the volume of deionized water is up to 1L.
And (3) measuring the content of biogenic amine: the determination is carried out by a liquid chromatography method, and specific steps are shown in biogenic amine in national sauce products of Zhu Tianao and the like.
The activation method of the strain comprises the following steps: the strain is inoculated into a culture medium in an inoculation amount of 20mL/L, and is statically cultured for 24 hours in a constant-temperature incubator at 37 ℃.
Example 1 screening and identification of Biodegrading strains
(1) Primary screening of the biological amine degradation strain:
adding 5g of soy sauce mash into 100mL conical flask, adding 50mL of normal saline, shaking on shaking table, mixing, and gradient diluting suspension to 10-5And 100 mu L of suspension is taken from each gradient, is coated on a BAs solid medium and a modified MRS solid medium, and is cultured in an incubator at 37 ℃ until the colony grows well and can be separated.
Determination of the degradation rate of the biogenic amine: single colonies isolated from the BAs solid medium and the modified MRS solid medium were activated in LB and modified MRS media, respectively, adjusted to an initial OD of 0.6 in a phosphate buffer solution containing 100mg/L of biogenic amine, and subjected to static culture in an incubator at 37 ℃ for 24 hours. The supernatant obtained by centrifugation at 4000 Xg for 5min is filtered through a 0.22 mu m filter membrane, 2mL of the supernatant is taken and put into a 10mL centrifuge tube, 1mL of 2mol/L NaOH solution and 20 mu L of benzoyl chloride solution are added, the centrifuge tube is immediately put into a water bath constant temperature oscillator, and oscillation is carried out for 20min at 30 ℃ and 200 r/min. And after the oscillation is finished, adding 2mL of saturated NaCl solution to stop the derivatization reaction, adding 3mL of anhydrous ether to extract biogenic amine, extracting for 1min by using a vortex oscillation instrument, carefully absorbing supernatant, placing the supernatant into a 5mL centrifuge tube, and drying by using a nitrogen blowing instrument. Dissolving the residue in the tube with 1mL of acetonitrile, filtering with a 0.22 μm organic filter membrane, detecting on a machine, and measuring the content of the biogenic amine by liquid chromatography, wherein the degradation rate of the biogenic amine is measured by comparing with a blank group. As shown in Table 1, the strains with higher degradation rate of the biogenic amine are primary screened strains (the numbers of the beginning of L and the beginning of M are respectively strains screened from a BAs solid culture medium and an improved MRS solid culture medium, the strains screened from the BAs solid culture medium are all bacillus, and the strains screened from the improved MRS solid culture medium are rich in varieties and comprise various microorganisms such as bacillus staphylococcus and lactobacillus).
TABLE 1 comparison of the degradation capacities of different strains on eight biogenic amines in the buffer
Note: ND means not detected
(2) Rescreening the biological amine degrading strain:
determination of the degradation rate of biogenic amine in fermentation liquor: the prescreened strain was activated using a BAs medium and a modified MRS medium, and the initial OD value was adjusted to 0.1 in the BAs medium and the modified MRS medium containing 100mg/L of biogenic amine, and was subjected to static culture in an incubator at 37 ℃ for 24 hours, followed by centrifugation at 4000 Xg for 5 min. Filtering the supernatant with a 0.22 mu m filter membrane, oscillating 2mL of filtrate and 6g/L trichloroacetic acid solution with the same volume in a water bath at 30 ℃ and 200r/min at constant temperature for 1h, taking the supernatant for derivatization reaction, finally determining the content of the biogenic amine by a liquid chromatography method, comparing with a blank group, determining the degradation rate of the biogenic amine, and screening the rescreened strain with higher degradation rate of the biogenic amine. As shown in Table 2, the degradation rates of Staphylococcus carnosus M43 on putrescine, cadaverine, tryptamine, phenethylamine, histamine and tyramine are respectively 12.34 + -0.66%, 8.17 + -0.35%, 1.51 + -0.81%, 17.88 + -1.70%, 50.69 + -4.27% and 40.77 + -0.73%.
TABLE 2 comparison of the degradation capacities of different strains of fermentation broth for eight biogenic amines
Note: ND means not detected
The genomic DNA of Staphylococcus carnosus M43 strain was extracted, using primers:
27F：5’AGAGTTTGATCCTGGCTCAG 3’
1492R:5’TACGGCTACCTTGTTACGACTT 3’
16S rDNA PCR amplification was performed. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 60s, annealing at 56 ℃ for 60s, and extension at 72 ℃ for 90s for 34 cycles; finally, extension was carried out at 72 ℃ for 10 min. The resulting PCR products were purified and sequenced, and the resulting sequences were BLAST sequence aligned in the NCBI database and identified as staphylococcus carnosus and designated M43.
Example 2 preparation of soybean paste using Staphylococcus carnosus
Firstly, soybeans, saline water and flour are taken as raw materials, and the adding amount is respectively 38g/100g, 50g/100g and 12g/100 g. Adding Aspergillus oryzae 3.042 as fermentation strain into soybean to make its final concentration be 107Culturing at 30 deg.C for 24 hr for starter propagation under CFU/g conditions, adding 20% (w/v) saline into jar after starter propagation to make the final concentration 10g/100g, inoculating 10-50mL/L Staphylococcus carnosus M43 to make the strain concentration in sauce 107And CFU/g, taking soy sauce mash without inoculated staphylococcus carnosus as a reference, respectively adding 5g/L of putrescine, cadaverine, spermidine, spermine, tryptamine, phenethylamine, histamine and tyramine mixed solution 5mL into the reference group and the experimental group, and standing and fermenting for 35 days in a constant-temperature incubator at 37 ℃. Measuring the biogenic amine content in the soybean paste sample after 35 days of fermentation, wherein the biogenic amine content is measured according to the measuring method in the secondary screening of the biogenic amine degrading strain in the example 1, and the biogenic amine degradation rate is calculated according to the following formula:
x is the degradation rate of the biogenic amine;
C1biological amine concentration of a sample of the non-inoculated strain;
C2inoculating a sample of a Staphylococcus carnosus strain with a concentration of biogenic amine.
As shown in table 3, the inoculation of staphylococcus carnosus M43 in the soybean paste fermentation process can effectively reduce the content of eight major biogenic amines in the soybean paste, the degradation rates of the eight biogenic amines in the soybean paste are 31.14%, 22.82%, 51.83%, 25.43%, 8.69%, 11.79% and 50.88% for putrescine, cadaverine, spermidine, spermine, phenethylamine, histamine and tyramine respectively, and the result shows that staphylococcus carnosus M43 has good biogenic amine degradation effect.
TABLE 3 degradation of eight biogenic amines by Staphylococcus carnosus M43
Note: ND means not detected
Example 3 salt tolerance of Staphylococcus carnosus
(1) Activating staphylococcus carnosus M43 and another staphylococcus carnosus M30 derived from the soybean paste by using the modified MRS culture medium, and adjusting the initial OD value to be 0.1 in the modified MRS culture medium;
(2) adding 0g, 3g, 6g, 9g, 12g, 15g and 18g of NaCl into 100g of culture medium respectively to make the final concentration of NaCl be 0%, 3%, 6%, 9%, 12%, 15% and 18% respectively;
(3) and statically culturing for 24 hours in an incubator at 37 ℃;
(4) the number of viable bacteria was calculated separately, and the number of viable bacteria of strains M43 and M30 is shown in Table 4.
TABLE 4 viable count of Staphylococcus carnosus in NaCl at different concentrations (log CFU/mL)
Example 4 thermotolerance of Staphylococcus carnosus
(1) Activating staphylococcus carnosus M43 and staphylococcus carnosus derived from soybean paste by using the modified MRS culture medium, and adjusting the initial OD value to be 0.1 in the modified MRS culture medium;
(2) standing at different temperatures of 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, and 45 deg.C for 24 hr;
(3) the viable count of the strains M43 and M30 is calculated respectively and shown in Table 5.
TABLE 5 viable count of Staphylococcus carnosus at different temperatures (log CFU/mL)
Example 5 Effect of Staphylococcus carnosus on Soy sauce flavor
The method for measuring the flavor substances of the soybean paste comprises the following steps: accurately weighing about 2g of soy sauce mash into a 15mL solid phase micro extraction bottle, respectively adding 2g of solid NaCl and 6mL of decarbonized water in sequence, adding 20 mu L of 25 mg/L2-octanol internal standard, putting the soy sauce mash into a rotor, screwing a cover, and putting the soy sauce mash into a constant temperature water bath kettle with a magnetic stirrer at 55 ℃, wherein the magnetic stirring speed is 400 r/min. Inserting the extraction head into the air part of the bottle through a solid phase micro-extraction bottle cap, pushing out the fiber head, starting a magnetic stirrer, adsorbing in the headspace for 40min, and then drawing back the fiber head.
Chromatographic conditions are as follows:
DB-Wax capillary chromatographic column with 30mm length, 0.32mm inner diameter and 0.25 μm liquid film thickness; carrier gas: he; flow rate: 10mL/min, no shunt; column temperature: the temperature of the injection port is kept at 250 ℃; the temperature-raising program is set as follows: the initial gas chromatographic column temperature is 40 ℃, the temperature is kept for 3.5min, the temperature is raised to 90 ℃ at the speed of 5 ℃/min, and then the temperature is raised to 230 ℃ at the speed of 12 ℃/min, and the temperature is kept for 10 min.
Mass spectrum conditions:
ionization mode: EI +; emission current (μ a): 200 of a carrier; drive voltage (V): 0.5; electron energy (eV): 70; lens 1 (V): lens 2 (V): 35; ion energy (eV): 1.8; ion energy ramp (mV amu)-1): 0.0; interface temperature (. degree. C.): 250 of (a); ion source temperature (. degree. C.): 200 of a carrier; low mass resolution: 18.0 of; high mass resolution: 12.6; probe voltage (V): 350 of (a); scanning mass range: 33 to 450 amu.
(1) Taking soybeans, saline water and flour as raw materials, adding 38g/100g, 50g/100g and 12g/100g respectively, adding Aspergillus oryzae 3.042 as fermentation strain into soybeans to make the final concentration of the soybean 107CFU/g, making starter at 30 ℃ for 24h, and adding 20% (w/v) saline water into a jar after starter making to make the final concentration 10g/100 g;
(2) experimental groups: inoculating 10-50mL/L Staphylococcus carnosus M43 when the strain is placed in a jar; control group: inoculating the same amount of Staphylococcus carnosus M30 when putting into jar, wherein the Staphylococcus carnosus is derived from soybean paste;
(3) standing and fermenting for 35 days in a constant-temperature incubator at 37 ℃;
(4) after the fermentation is finished, the volatile flavor substances of the soybean paste are analyzed, and the statistics of the flavor substances of the strains M43 and M30 are shown in tables 6 and 7.
In table 7, 50 types of the flavor substances of staphylococcus carnosus M43 were present, which were higher than 26 types of the 24 types of the flavor substances of staphylococcus carnosus M30, and thus the types of the flavor substances of staphylococcus carnosus M43 were more diverse.
TABLE 6 analysis of the content of volatile flavor substances in the soybean pastes inoculated with different strains (mg/kg dry basis)
TABLE 7 type and content of volatile flavor substances (mg/kg dry basis) in the soybean paste inoculated with different strains
Comparative example 1
See example 2 for a specific embodiment, except that another strain of Staphylococcus carnosus M30 from the same soybean paste was inoculated at the time of jar entry, and the results are shown in Table 8. It can be seen that Staphylococcus carnosus M30 has a relatively poor ability to degrade biogenic amines, but is not able to degrade cadaverine, phenethylamine, histamine and tyramine.
TABLE 8 degradation of eight biogenic amines by Staphylococcus carnosus M30
Note: ND means not detected
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.
Claims (10)
1. A strain of Staphylococcus carnosus (Staphylococcus carnosus) is preserved in 25.7.2019 by the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.18295 and the preservation address of Beijing university Hokko No. 3 of Shangyang district Beichen Xilu No.1, institute of microbiology of China academy of sciences.
2. A method for culturing the staphylococcus carnosus according to claim 1, wherein the staphylococcus carnosus is inoculated into a culture medium in an inoculation amount of 1-5% by volume, and is subjected to static culture for 20-30 hours in a constant-temperature environment at 35-40 ℃.
3. A method for reducing biogenic amine content, wherein Staphylococcus carnosus according to claim 1 is inoculated into a liquid, semi-solid or solid biogenic amine-containing product.
4. A method for reducing biogenic amine content in soybean paste, which comprises inoculating Staphylococcus carnosus of claim 1 during fermentation of soybean paste.
5. The method according to claim 4, wherein the staphylococcus carnosus is inoculated on the 0 th day of fermentation of the soybean paste, and after the uniform stirring, the fermentation is carried out for 30 to 40 days at 30 to 40 ℃.
6. The method of claim 4, wherein the concentration of Staphylococcus carnosus in the soy sauce is 103～109CFU/g。
7. A seasoning prepared using the Staphylococcus carnosus of claim 1.
8. Seasoning according to claim 7 wherein the seasoning comprises a soybean paste or a soybean flour paste.
9. Use of staphylococcus carnosus according to claim 1 for reducing biogenic amine content in the food field.
10. The use according to claim 9, wherein the food product comprises a food product to be fermented with soya or broad beans or meat.
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