CN116855414B - Bacillus belicus and application thereof in fermented bean products - Google Patents
Bacillus belicus and application thereof in fermented bean products Download PDFInfo
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- CN116855414B CN116855414B CN202310838933.7A CN202310838933A CN116855414B CN 116855414 B CN116855414 B CN 116855414B CN 202310838933 A CN202310838933 A CN 202310838933A CN 116855414 B CN116855414 B CN 116855414B
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- bacillus
- fermented
- fermentation
- belicus
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Classifications
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- 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
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of 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
-
- 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
- C12R2001/07—Bacillus
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
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Abstract
The invention belongs to the technical field of microbial fermented foods, and particularly relates to bacillus belicus and application thereof in fermented bean products. The strain is bacillus belicus (Bacillus velezensis), the strain number is GZUA, and the strain is preserved in China Center for Type Culture Collection (CCTCC) NO. M2023666. According to the invention, GZUA1 pure fermented soybeans are used, the amino acid nitrogen of the fermented soybean product reaches 0.94%, and the amino acid nitrogen is improved by 1.12 times compared with natural fermented soybeans; protease activity reaches 538.28U/g; the total biogenic amine level is obviously reduced by only 10.65mg/kg, which is reduced by 10.68 times compared with the traditional natural fermentation, wherein the phenethylamine, tyramine and spermine are respectively reduced by 2.60 times, 10.54 times and 42.61 times compared with the natural fermentation fermented soybean. The strain is applied under the optimal fermentation process parameters, so that the quality and edible safety of the fermented bean products can be comprehensively improved, and high-quality strain and technical support are provided for industrial safe production of the fermented bean products.
Description
Technical Field
The invention belongs to the technical field of microbial fermented foods, and particularly relates to bacillus belicus and application thereof in fermented bean products.
Background
Fermented soybean is a traditional fermented soybean product, and is generally prepared by soaking, steaming or boiling soybeans or black beans, and fermenting by microorganisms. It is used for seasoning and can be used as medicine, and is popular with people. At present, bacterial fermented soybeans are most common, natural fermentation is mainly used in traditional production, the process problems of complex bacterial phases, difficult bacterial source control and the like exist, and the product quality stability is poor and is greatly influenced by factors such as seasonal temperature/humidity, environmental sanitation conditions/microorganism systems and the like. Therefore, the artificial inoculation or flora reinforcement fermentation technology is adopted, so that the method becomes a scientific path and method for effectively controlling the growth of mixed bacteria, realizing standardized production and stabilizing the product quality; on the other hand, natural fermentation, complex bacterial phases and wild strains are randomly participated, so that potential safety hazards are brought, and especially the problem of biogenic amine formed by fermenting amino acid decarboxylase positive strains in the production of fermented bean products mainly containing proteins is more and more focused.
Biogenic amines are low molecular weight organic nitrogen-containing compounds, and are classified as aliphatic, aromatic, heterocyclic amines, and the like; aliphatic includes putrescine, cadaverine, spermine and spermidine, aromatic includes tyramine and phenethylamine, and histamine and tryptamine are heterocyclic amines. Biogenic amines can be formed by decarboxylation of amino acids produced by proteolytic cleavage by amino acid decarboxylases produced by microbial metabolism. Biogenic amines are produced in high protein fermented foods such as cheese, beer, fermented soybean foods, etc., and have become one of the potential safety problems of fermented foods. Research shows that excessive biogenic amine can damage human nervous system, even cause the formation of cancerogenic substance nitrous amine, produce adverse reactions such as headache, nausea, rash, diarrhea, etc., even induce hypertension, migraine, cancer, etc.
Therefore, the excellent fermentation strain of the bean products is screened, the high protease activity and the non-production or low-production amine characteristics of the bean products are utilized to construct a fermentation bean product production model, and the fermentation process is controlled from the process of the process by flora reinforcement and manual fermentation control, so that the product quality is improved; the safety risk factors are scientifically controlled from the production strain, so that the method is an effective path for realizing scientific, normalized and safe production of bean products, and can provide high-quality strain and feasibility scheme and technical parameters for industrial development.
Some screening technical researches aiming at fermented bean products and corresponding strains are carried out, for example, patent document with patent number of CN201710279437.7 discloses a bacillus beliensis strain for producing nattokinase and application thereof, and the screened bacillus beliensis SN-14 is derived from Guizhou local fermented soybean, so that the safety of the strain is high, and the activity of the nattokinase is high. Patent document CN201810063421.7 discloses bacillus belicus and its separating and screening method and application, and the bacillus belicus has high protease, amylase and fermented soya bean plasmin producing ability when fermenting. Literature screening of high-yield Nattokinase Strain and study of its enzymatic stability (Gao Zexin, shuoshi paper, 2018) 15 high-yield Nattokinase strains were isolated from samples of Guizhou indigenous feature food (fermented soybean) using the Casein plate method and the agarose-fibrinogen plate method, wherein SN-14 strain was Bacillus bailii (Bacillus velezensis). Through tests, the biogenic amine content of each natto product is 49.09-73.71 mg/kg, and the biogenic amine content of SN-14 is low.
The technical research only can show that bacillus belicus has the characteristics of high safety and low content of biogenic amine, but the biogenic amine is very complicated due to the formation reason, when the sources are different and the screening methods are different, the difference between strains is obvious, the biogenic amine content of each natto product tested in the literature is 49.09-73.71 mg/kg, and the biogenic amine content is still high.
Disclosure of Invention
The invention provides bacillus belicus and application thereof in fermented bean products for solving the problems.
The method is realized by the following technical scheme:
A special bacterium for fermenting bean products is characterized in that the strain is bacillus belicus (Bacillus velezensis), the strain number is GZUA, the strain is preserved in China center for type culture collection, the preservation number is CCTCC NO: M2023666, and the preservation date is 2023, 04 and 28.
Further, the bacillus belicus GZUA is separated from the high-quality fermented soybean.
Further, the bacillus belicus GZUA grows on a nutrient agar medium at 20-45 ℃ and is yellowish, dry in surface, opaque, irregularly shaped and irregular in edge; gram staining test shows positive (+), and observing the bacterial cells in a rod shape and sporulation under a microscope; h 2 S, indole and ammonia gas are not produced.
Further, the bacillus belicus GZUA has the characteristics of high protease activity and low biogenic amine.
Further, the bacillus belicus GZUA shows high sensitivity to sulfonamides, macrolides, ouinones and aminosugar-shake antibiotics; and has strong inhibition capability to escherichia coli and staphylococcus aureus.
The invention also provides application of the strain in fermentation of bean products.
In the application, bacillus belicus GZUA1 is applied to bean product fermentation, so that the quality of the fermented bean product can be remarkably improved, the total amount and composition of biogenic amine in the bean product can be effectively reduced, and the edible safety of the bean product is improved.
The application specifically comprises the following steps:
(1) Activating strains: activating bacillus beijerinckii GZUA1 obtained through separation, purification and identification, and performing slant culture after pure inspection is qualified for later use;
(2) Seed liquid preparation: inoculating Bacillus bailii GZUA1 into nutrient broth liquid culture medium, shake-culturing at 20-45deg.C and 100-200r/min for 12-72 hr to obtain seed liquid for use;
(3) Fermentation: inoculating the seed liquid prepared in the step (2) into the selected, cleaned and autoclaved soybean substrate according to the inoculum size of 4-12%, uniformly mixing, and placing in a constant temperature incubator at 20-45 ℃ for culturing and fermenting for 1-5d.
Further, the viable count of the seed liquid for the spread culture in the step (2) is more than or equal to 10 8 CFU/m L.
The invention has the beneficial effects that:
The bacillus belicus GZUA1 is obtained by screening from fermented bean products, has the characteristics of high protease activity and low production of biogenic amine, does not produce H 2 S, indole and ammonia, is highly sensitive to sulfonamides, macrolides, ouinones and aminosugar antibiotics, and has strong inhibition capability to common pathogenic bacteria such as escherichia coli, staphylococcus aureus and the like.
According to the invention, bacillus belicus GZUA is used for pure-planting fermented soybean, and a fermentation model is constructed by taking the starter, the inoculation amount, the fermentation time and the fermentation temperature as influencing factors, so that the optimal fermentation process and parameters are obtained. Under the process and parameter conditions, compared with the traditional natural fermentation, the fermented soybean product has full grain, dark yellow color, short and rich sticky filaments, rich fermented soybean fragrance, no ammonia odor and blackening phenomenon; the amino acid nitrogen of the fermented soybean product reaches 0.94%, which is improved by 1.12 times compared with natural fermented soybean; protease activity reaches 538.28U/g; and the total biogenic amine level is obviously reduced by only 10.65mg/kg, which is reduced by 10.68 times compared with the traditional natural fermentation, wherein the phenethylamine, tyramine and spermine are respectively reduced by 2.60 times, 10.54 times and 42.61 times compared with the natural fermentation fermented soybean. The strain is applied under the optimal fermentation process parameters, so that the quality and edible safety of the fermented bean products can be comprehensively improved, and high-quality strain and technical support are provided for industrial safe production of the fermented bean products.
Drawings
FIG. 1 shows colony morphology of Bacillus belicus GZUA 1.
FIG. 2 shows the form of Bacillus belicus GZUA.
FIG. 3 shows the PCR amplification result of the strain 16 SrDNA.
FIG. 4 shows a 16SrDNA phylogenetic tree of strains, identified as follows: a1, bacillus bailii (Bacillus velezensis); a2, glycine bacillus (Bacillus glycinifermentans); a3, bacillus subtilis (Bacillus subtilis).
FIG. 5 is a photograph of fermented soybean products produced by fermentation of Bacillus belicus GZUA.
Detailed Description
The technical scheme of the present invention is further defined below in conjunction with the specific embodiments, but the scope of the claimed invention is not limited to the description.
Example 1
Screening of Bacillus bailii GZUA1
(1) Separation and purification: 10g of high-quality fermented bean products are weighed and put into 80-110mL of sterile water, and are placed on a shaking table to be uniformly mixed, so as to obtain sample liquid. Adding the sample solution into sterile normal saline according to the ratio of 1:9, sequentially carrying out gradient dilution, then taking 80-120 mu L of the sample solution, coating the sample solution on a nutrient agar plate, culturing the sample solution for 12-72 hours at 20-45 ℃, picking single bacterial colonies, marking and purifying the bacterial strains for multiple times, and then preserving the bacterial strains in a slant culture medium for later use.
(2) Safety performance evaluation: the strain screened in the step (1) is subjected to safety performance evaluation, and the content comprises hydrogen sulfide, indole and ammonia gas tests: 1) Hydrogen sulfide test: inoculating the strain into ferrous sulfate semisolid culture medium, culturing at 35-37 ℃ for 24 hours, and if black is generated, determining that the strain is positive, and indicating that the strain produces hydrogen sulfide; 2) Indole test: inoculating a small amount of strain into a test culture medium tube, culturing for 24 hours at 35-37 ℃, taking about 2mL of culture solution, adding 2-3 drops of indole reagent, and slightly shaking the test tube to make red positive, so that the strain produces indole; 3) Ammonia gas test: and collecting fermentation gas, and detecting whether ammonia exists or not.
(3) Protease activity evaluation: and (3) measuring the enzyme activity of the strain screened in the step (2) by adopting a Fu Lin Fen method.
(4) Antibiotic susceptibility assessment: the strain initially screened in the step (2) is subjected to antibiotic susceptibility evaluation by a flat plate-paper sheet method. When in use, the antibiotic paper is attached to the surface of the culture medium coated with the bacteria to be detected in a sterile operation, and after culturing for 48 hours at 37 ℃, the diameter of the inhibition zone is measured. The antibiotics include oxadiazine penicillin, chloramphenicol, amikacin, erythromycin, fluoxastrobin, and compound neotame.
(5) And (3) antibacterial capacity test: and (3) performing antibacterial capacity test on the strain screened in the step (2). The escherichia coli and staphylococcus aureus are used as indicator bacteria for bacteriostasis experiments, and sterile physiological saline is used for dilution until the concentration of bacterial suspension is 1mL 10 8 CFU/mL. Absorbing 0.1mL of diluted indicator fungus suspension, coating the indicator fungus suspension on a nutrient agar culture medium, standing for 5min, punching by a sterile puncher with the diameter of 6mm, taking 50 mu L of fermentation liquor after separating strains and centrifuging, freezing and centrifuging for 10min at the temperature of 4 ℃ and 12000r, adding the fermentation liquor into the holes, culturing for 24-48 h, observing whether a bacteriostasis ring appears, accurately recording the diameter of the appearing bacteriostasis ring, and carrying out 2 parallel experiments on each strain.
Biogenic amine formation assay: and (3) selecting high-quality and safe strains obtained through the screening in the steps (2) to (5), inoculating the strains into a nutrient broth culture medium, and shake-flask culturing for 24 hours at 37 ℃ and 150 r/min. Weighing high-quality soybean, cleaning, adding water according to a certain proportion, steaming at 121 ℃ for 38min, filtering water after steaming, cooling with water, and inoculating with the bacterial liquid. The inoculum size was about 1mL of 10 8 CFU/mL of the bacterial liquid was inoculated per 100g of soybean, and the fermentation was carried out at 37℃for 3 days. And quantitatively analyzing biogenic amine in the fermented bean product by adopting a high performance liquid chromatography.
Example 2
Identification of Bacillus bailii GZUA1
The high-enzyme activity and low-yield amine safety and high-quality strains screened in example 1 are identified according to the sequence of morphological identification, physiological biochemical identification and molecular biological identification.
(1) Morphological identification: observing colony morphology of the selected strain after growing on a nutrient agar plate for 24 hours, and recording; and then gram staining the strain, observing the form, arrangement mode, special structure and staining reaction of the strain under a microscope, and carrying out preliminary identification to the genus and family.
(2) And (3) physiological and biochemical identification: the tests of acid and gas production, nitrate reduction, catalase, liquefied gelatin, citrate utilization, methyl red test, starch hydrolysis and the like of sugar fermentation (glucose, sucrose and lactose) are carried out, and the genus and species are to be identified.
(3) Molecular biology identification: and (3) carrying out 16S rDNA sequencing on the purified strain, carrying out sequence comparison on a ribosome database according to the sequencing result, carrying out homology comparison on the sequence of the related strain in NCBI, and constructing a phylogenetic tree by using MAGA7.0 software.
The strain GZUA1 grows rapidly on a nutrient agar plate, bacterial colonies are yellowish, dry and opaque on the surface, irregular in shape and irregular in edge (shown in figure 1), and bacterial bodies are rod-shaped and sporulated (shown in figure 2) when observed under an optical microscope, and meanwhile, the strain GZUA is positive in a contact enzyme test, an oxidase test, a V-P test and the like, can be identified by using starch, gelatin and the like through morphological and physiological biochemical identification, and is intended to be identified as gram-positive bacillus; identified as bacillus beijerinus by molecular biology (shown in fig. 3 and 4).
Example 3
Application of bacillus belicus GZUA1 in fermented soybeans
(1) Strain activation: activating bacillus behenensis GZUA1, performing slant culture after pure detection is qualified, and preserving for later use.
(2) Seed liquid preparation: inoculating Bacillus bailii GZUA1 into nutrient broth liquid culture medium, shake-culturing at 37deg.C and 150r/min for 48 hr to obtain seed liquid for expanding culture.
(3) Fermentation: inoculating the seed liquid prepared in the step (2) into the selected, cleaned and autoclaved soybean substrate according to the inoculation amount of 8%, uniformly mixing, and placing in a constant temperature incubator at 37 ℃ for culturing and fermenting for 3d.
Example 4
Application of bacillus belicus GZUA1 in fermented soybeans
(1) Strain activation: activating bacillus behenensis GZUA1, performing slant culture after pure detection is qualified, and preserving for later use.
(2) Seed liquid preparation: inoculating Bacillus bailii GZUA1 into nutrient broth liquid culture medium, shake-culturing at 37deg.C and 150r/min for 48 hr to obtain seed liquid for expanding culture.
(3) Fermentation: inoculating the seed liquid prepared in the step (2) into the selected, cleaned and autoclaved soybean substrate according to the inoculation amount of 6%, uniformly mixing, and placing the mixture in a 39 ℃ constant temperature incubator for culturing and fermenting for 3d.
Example 5
Application of bacillus bailii GZUA1 fermented soybeans
(1) Strain activation: activating bacillus behenensis GZUA1, performing slant culture after pure detection is qualified, and preserving for later use.
(2) Seed liquid preparation: inoculating Bacillus bailii GZUA1 into nutrient broth liquid culture medium, shake-culturing at 37deg.C and 150r/min for 48 hr to obtain seed liquid for expanding culture.
(3) Fermentation: inoculating the seed liquid prepared in the step (2) into the selected, cleaned and autoclaved soybean substrate according to the inoculation amount of 10%, uniformly mixing, and placing the mixture in a constant temperature incubator at 37 ℃ for culturing and fermenting for 2d.
Comparative example 1
Natural fermentation
Cleaning semen glycines, and steaming in an autoclave at 120deg.C for 40 min. The steamed soybeans are discharged from the pot and then placed on a bamboo sieve, then placed in a fermentation chamber at 35-40 ℃ and fermented for 3d by means of various microorganisms naturally existing in the air.
Comparative example 2
Screening of Strain A2
(1) Separation and purification: weighing 10g of fermented bean product, putting into 80-110mL of sterile water, and putting on a shaking table for uniform mixing to obtain sample liquid. Adding the sample solution into sterile normal saline according to the ratio of 1:9, sequentially carrying out gradient dilution, then taking 80-120 mu L of the sample solution, coating the sample solution on a nutrient agar plate, culturing the sample solution for 12-72 hours at 20-45 ℃, picking single bacterial colonies, marking and purifying the bacterial strains for multiple times, and then preserving the bacterial strains in a slant culture medium for later use.
(2) Safety performance evaluation: and (3) evaluating the safety performance of the strain screened in the step (1), wherein the content comprises the following steps: hydrogen sulfide, indole, ammonia gas test: 1) Hydrogen sulfide test: inoculating the strain into ferrous sulfate semisolid culture medium, culturing at 35-37 ℃ for 24 hours, and if black is generated, determining that the strain is positive; 2) Indole test: inoculating a small amount of strain into a test culture medium tube, culturing for 24 hours at 35-37 ℃, taking about 2mL of culture solution, adding 2-3 drops of indole reagent, and slightly shaking the test tube to make the strain positive in red; 3) Ammonia gas test: and collecting fermentation gas, and detecting whether ammonia exists or not.
(3) Protease activity evaluation: and (3) measuring the enzyme activity of the strain screened in the step (2) by adopting a Fu Lin Fen method.
(4) Antibiotic susceptibility assessment: the strain initially screened in the step (2) is subjected to antibiotic susceptibility evaluation by a flat plate-paper sheet method. When in use, the antibacterial agent is applied to the surface of a culture medium coated with the bacteria to be detected in an aseptic operation manner, and after culturing for 48 hours at 37 ℃, the diameter of the antibacterial zone is measured. The antibiotics include oxadiazine penicillin, chloramphenicol, amikacin, erythromycin, fluoxastrobin, and compound neotame.
(5) And (3) antibacterial capacity test: and (3) performing antibacterial capacity test on the strain screened in the step (2). The escherichia coli and staphylococcus aureus are used as indicator bacteria for bacteriostasis experiments, and sterile physiological saline is used for dilution until the concentration of bacterial suspension is 1mL 10 8 CFU/mL. Absorbing 0.1mL of diluted indicator fungus suspension, coating the indicator fungus suspension on a nutrient agar culture medium, standing for 5min, punching by a sterile puncher with the diameter of 6mm, taking 50 mu L of fermentation liquor after separating strains and centrifuging, freezing and centrifuging for 10min at the temperature of 4 ℃ and 12000r, adding the fermentation liquor into the holes, culturing for 24-48 h, observing whether a bacteriostasis ring appears, accurately recording the diameter of the appearing bacteriostasis ring, and carrying out 2 parallel experiments on each strain.
Biogenic amine formation assay: selecting the strains obtained by screening in the steps (2) to (5), inoculating the strains into a nutrient broth culture medium, and culturing the strains for 24 hours at 37 ℃ and 150r/min in a shaking flask. Weighing high-quality soybean, cleaning, steaming at 121deg.C under high pressure for 38min, filtering, cooling, and inoculating with the above bacterial liquid. The inoculum size was about 1mL of 10 8 CFU/mL of the bacterial liquid was inoculated per 100g of soybean, and the fermentation was carried out at 37℃for 3 days. And quantitatively analyzing biogenic amine in the fermented soybeans by adopting a high performance liquid chromatography method.
Comparative example 3
Screening of Strain A3
(1) Separation and purification: weighing 10g of fermented bean product, putting into 80-110mL of sterile water, and putting on a shaking table for uniform mixing to obtain sample liquid. Adding the sample solution into sterile normal saline according to the ratio of 1:9, sequentially carrying out gradient dilution, then taking 80-120 mu L of the sample solution, coating the sample solution on a nutrient agar plate, culturing the sample solution for 12-72 hours at 20-45 ℃, picking single bacterial colonies, marking and purifying the bacterial strains for multiple times, and then preserving the bacterial strains in a slant culture medium for later use.
(2) Safety performance evaluation: the strain screened in the step (1) is subjected to safety performance evaluation, and the content comprises hydrogen sulfide, indole and ammonia gas tests: 1) Hydrogen sulfide test: inoculating the strain into ferrous sulfate semisolid culture medium, culturing at 35-37 ℃ for 24 hours, and if black is generated, determining that the strain is positive, and proving that the strain produces hydrogen sulfide; 2) Indole test: inoculating a small amount of strain into a test culture medium tube, culturing for 24 hours at 35-37 ℃, taking about 2mL of culture solution, adding 2-3 drops of indole reagent, and slightly shaking the test tube to make the strain positive in red; 3) Ammonia gas test: and collecting fermentation gas, and detecting whether ammonia exists or not.
(3) Protease activity evaluation: and (3) measuring the enzyme activity of the strain screened in the step (2) by adopting a Fu Lin Fen method.
(4) Antibiotic susceptibility assessment: and (3) evaluating the antibiotic sensitivity of the strain screened in the step (2) by adopting a flat plate one-paper sheet method. When in use, the antibacterial agent is applied to the surface of a culture medium coated with the bacteria to be detected in an aseptic operation manner, and after culturing for 48 hours at 37 ℃, the diameter of the antibacterial zone is measured. The antibiotics include oxadiazine penicillin, chloramphenicol, amikacin, erythromycin, fluoxastrobin, and compound neotame.
(5) And (3) antibacterial capacity test: and (3) performing antibacterial capacity test on the strain screened in the step (2). The escherichia coli and staphylococcus aureus are used as indicator bacteria for bacteriostasis experiments, and sterile physiological saline is used for dilution until the concentration of bacterial suspension is 1mL 10 8 CFU/mL. Absorbing 0.1mL of diluted indicator fungus suspension, coating the indicator fungus suspension on a nutrient agar culture medium, standing for 5min, punching by a sterile puncher with the diameter of 6mm, taking 50 mu L of fermentation liquor after separating strains and centrifuging, freezing and centrifuging for 10min at the temperature of 4 ℃ and 12000r, adding the fermentation liquor into the holes, culturing for 24-48 h, observing whether a bacteriostasis ring appears, accurately recording the diameter of the appearing bacteriostasis ring, and carrying out 2 parallel experiments on each strain.
(6) Biogenic amine formation assay: selecting the strains obtained by screening in the steps (2) to (5), inoculating the strains into a nutrient broth culture medium, and culturing the strains for 24 hours at 37 ℃ and 150r/min in a shaking flask. Weighing high-quality soybean, cleaning, steaming at 121deg.C for 38min, filtering to dry, and inoculating with the bacterial liquid. The inoculum size was about 1mL of 10 8 CFU/mL of the bacterial liquid was inoculated per 100g of soybean, and the fermentation was carried out at 37℃for 3 days. And quantitatively analyzing biogenic amine in fermented soybeans by adopting a high performance liquid chromatography method.
The following will compare specific examples with comparative examples
Comparative analysis of example 1 with comparative examples 1 to 3
(1) Evaluation of fermentation safety performance of different strains
TABLE 1 safety Properties of strains
Note that: more than 90% of the strains are positive, "+" and more than 90% of the strains are negative
As can be seen from Table 1, none of the strains GZUA, A2, and A3 produced H 2 S, indole, and ammonia. In the natural fermentation process of the bean product, harmful substances such as H 2 S, indole, ammonia gas and the like are generated due to the action of microorganisms, so that the smell and the color of the product are influenced, the palatability and the safety of the product are reduced, and the product becomes a potential safety hazard of the fermented bean product, so that H 2 S, indole and ammonia gas are used as one of important indexes for evaluating the safety of the fermented bean product. Therefore, the strains GZUA, A2 and A3 can be used as the safety primary screening strains for bean product fermentation.
(2) Evaluation of protease Activity of different strains
TABLE 2 protease Activity of strains
As is clear from Table 2, GZUA strains 1 and A3 each exhibited high protease activity. The protease activity difference between the strain GZUA and the strain A3 is not obvious (p is more than 0.05), is higher than 150U/mL, and the protease activity difference between the strain A2 and the strains GZUA and A3 is very obvious and is only 98U/mL (p is less than 0.01).
(3) Sensitivity and bacteriostatic ability of different strains to antibiotics
TABLE 3 results of antibiotic susceptibility testing of strains
Note that: "M" means sensitive; "S" means highly sensitive, with a zone of inhibition diameter of mm.
Table 4 bacteriostatic ability of strains
As can be seen from Table 3, 3 strains showed different degrees of sensitivity to 7 general antibiotics, especially to sulfonamides, macrolides, ouinones and aminosugar-shake antibiotics. This shows that 3 strains do not contain drug-resistant plasmids of common antibiotics as production strains, so that the risk of transferring drug-resistant genes to intestinal flora does not exist, and the strain can be used as a safety strain for standby. As shown in Table 4, 3 strains had inhibitory effects on both Escherichia coli and Staphylococcus aureus, and GZUA was the most potent inhibitory effect on both Escherichia coli and Staphylococcus aureus. As can be seen, the antibacterial activity of Bacillus bailii GZUA1 is more prominent.
(4) Biogenic amine levels from fermented soybeans of different strains
TABLE 5 biogenic amine content (mg/kg) in fermented soybeans of different strains
Note that: ND is not detected, and the group marked with different letters in the data in the figure shows that the difference is significant (p < 0.05)
As can be seen from Table 5, 8 biogenic amines were detected in the fermented soybeans under the condition of comparative example 1,5 biogenic amines were detected in the fermented soybeans of comparative example 2 and comparative example 3, and only 4 biogenic amines were detected in the fermented soybeans of examples 3 to 5. Comparative example 3 fermented soybeans have the highest contents of tryptamine and phenethylamine of 7.06mg/kg and 8.78mg/kg respectively; putrescine was present in comparative example 1 and comparative example 3; cadaverine and spermidine were only present in comparative example 1, especially cadaverine up to 11.50mg/kg; histamine was also present in comparative examples 1 and 2 at 8.57mg/kg and 0.44mg/kg, respectively, but was not detected in examples 3-4. Examples 3-5 showed a significant decrease in tyramine compared to comparative examples 1-3, with an average value of only 3.29mg/kg (p < 0.05); whereas examples 3-5 and comparative examples 2-3, which were artificially inoculated with a microbiota-enhanced fermentation, had a relatively low spermine content of <4.00mg/kg, comparative example 1 (group of natural fermentation) was as high as 57.52mg/kg. From this, it is known that the artificial inoculation flora intensified fermentation can effectively control the biogenic amine level in the fermented bean product, and the biogenic amine content can be changed to different degrees according to the strain adopted. Therefore, the product safety can be effectively improved by adopting the high-quality safe fermentation strain to artificially inoculate flora to strengthen the fermented bean product.
The strain A2 was identified as Bacillus glycine (Bacillus glycinifermentans) and the strain A3 as Bacillus subtilis (Bacillus subtilis). They are dominant strains in the fermentation system screened under the same screening conditions as strain GZUA 1. However, the comprehensive evaluation analysis is carried out by combining the test results of the safety performance, the protease activity, the antibiotic sensitivity, the antibacterial capacity and the biogenic amine formation level, and the fermentation biogenic amine water level, the antibacterial capacity and the protease activity of the strain are obviously different from those of the strain GZUA, and no GZUA1 is excellent. Thus, strain GZUA was selected as a subsequent fermented soybean puree fermentation strain.
(5) Total biogenic amine levels of fermented soybeans from different strains
TABLE 6 total biogenic amine content of fermented soybeans of different strains
Note that: the group marked with capital letters in the data in the figure indicates that there is a very significant difference (p < 0.01).
Table 6 results show that the total biogenic amine levels of examples 3-5 are effectively controlled to be in the range <16.00 mg/Kg. The total biogenic amine level of the natural fermentation comparative example 1 is highest and reaches 113.69mg/Kg. Comparative example 2 and comparative example 3 were 27.89mg/Kg and 33.29mg/Kg, respectively, and were very different from examples 3-5 and comparative example 1 (p < 0.01). Therefore, the bacillus besseyi GZUA adopted in the embodiment 3-5 is fermented, the total biogenic amine level of the fermented bean product can be effectively controlled, is extremely lower than that in the comparative example 1-3, and is more suitable for being applied to fermented products as a beneficial fermentation type low biogenic amine strain.
Comparative document 1 screening of high yield Nattokinase Strain and study of its enzymatic stability (Gao Zexin) Bacillus belicus SN-14 fermentation total biogenic amine level was 54.31mg/Kg, whereas the example total biogenic amine level of the present patent strain GZUA1 was far lower than this data. The formation of biogenic amines is very cumbersome. But it is confirmed that it is closely related to microorganisms. The data of the present technology, in which the total amount of biogenic amine is lower than that of comparative document 1, includes three major reasons of screening method, strain distinction and technical purpose. The strain SN-14 is obtained by screening 9 different Guizhou indigenous special foods such as Zunyi fermented soya beans, guiyang fermented soya beans, square fermented bean curd, tongren fermented soya beans, pichia fermented soya beans, anshun fermented soya beans, green rock bean curd and the like by a casein plate method, an agarose-fibrinogen plate method, an ultraviolet spectrophotometry method and the like; in the technical scheme, the strain GZUA is a fermentation-benefiting safety strain obtained by taking a Guizhou characteristic high-quality traditional fermentation bacteria type fermented soybean product as a raw material and adopting the methods of dominant strain screening, safety performance detection, protease activity assessment, antibiotic sensitivity assessment, antibacterial capacity detection, biogenic amine level detection and the like. In taxonomy, bacillus belicus is the species name, and both strain GZUA1 and strain SN-14 belong to the genus Bacillus. They are the same species (strain) of different origin. However, in long-term habitat, natural selection, superior and inferior elimination, spontaneous mutation and the like can cause differences among different sources of the same species (strain), so that the strain has different production performances. Furthermore, the object of the present technical proposal is to solve the problem of producing biogenic amine as a harmful substance by fermenting fermented soybeans with natural bacteria, and the object of comparative document 1 is to screen a strain having a high nattokinase-producing ability for the production of natto products. Therefore, bacillus belicus GZUA1 obtained by screening in the patent has more advantages.
In conclusion, the beneficial fermentation safe strain bacillus bailii GZUA1 is adopted to ferment and produce bean products such as fermented soybeans, so that the quality of the products can be improved, the products are full, dark yellow in color and luster, short and rich in sticky filaments, have typical fermented soybean fragrance, and meanwhile, the composition and content of biogenic amine in the products can be effectively controlled, so that safe low biogenic amine fermented products (shown in figure 5) can be obtained. As a safe production strain for fermentation, bacillus beziacillus GZUA1 has extremely strong application prospect.
It is noted that the above examples and test examples are only limited to further explanation and understanding of the technical solutions of the present invention, and are not to be construed as further limiting the technical solutions of the present invention, and the invention without significant essential features and significant improvements made by those skilled in the art still falls within the scope of protection of the present invention.
Claims (3)
1. A special bacterium for fermenting bean products is characterized in that the strain is bacillus belicus (Bacillus velezensis), the strain number is GZUA, the strain is preserved in China center for type culture collection, the preservation number is CCTCC NO: M2023666, and the preservation date is 2023, 04 and 28.
2. Use of a specific bacterium according to claim 1 in fermented bean products, comprising the steps of:
(1) Activating strains: activating bacillus beijerinckii GZUA1 obtained through separation, purification and identification, and performing slant culture after pure inspection is qualified for later use;
(2) Seed liquid preparation: inoculating Bacillus bailii GZUA1 into nutrient broth liquid culture medium, shake-culturing at 20-45deg.C and 100-200r/min for 12-72 hr to obtain seed liquid for use;
(3) Fermentation: inoculating the seed liquid prepared in the step (2) into the selected, cleaned and autoclaved soybean substrate according to the inoculum size of 4-12%, uniformly mixing, and placing in a constant temperature incubator at 20-45 ℃ for culturing and fermenting for 1-5d.
3. The use according to claim 2, wherein the viable count of the seed liquid for the propagation in step (2) is not less than 10 8 CFU/mL.
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