CN114181848A - Bacillus acidocaldarius capable of generating fruity flavor of cream and application thereof - Google Patents
Bacillus acidocaldarius capable of generating fruity flavor of cream and application thereof Download PDFInfo
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- CN114181848A CN114181848A CN202111288463.9A CN202111288463A CN114181848A CN 114181848 A CN114181848 A CN 114181848A CN 202111288463 A CN202111288463 A CN 202111288463A CN 114181848 A CN114181848 A CN 114181848A
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- China
- Prior art keywords
- bacillus
- cream
- flavor
- acidocaldarius
- shq
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- 239000000796 flavoring agent Substances 0.000 title claims abstract description 68
- 235000019634 flavors Nutrition 0.000 title claims abstract description 68
- 241000640374 Alicyclobacillus acidocaldarius Species 0.000 title claims abstract description 65
- 239000006071 cream Substances 0.000 title claims abstract description 29
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- 239000000052 vinegar Substances 0.000 claims abstract description 60
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- 239000007788 liquid Substances 0.000 claims abstract description 34
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
- C12J1/00—Vinegar; Preparation or purification thereof
- C12J1/04—Vinegar; Preparation or purification thereof from alcohol
-
- 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
Abstract
The invention discloses a Bacillus acidocaldarius (Bacillus acidocola) capable of generating fruity flavor of cream, which is named as follows: SHQ-GD, class name: bacillus acidocaldarius Bacillus acidocola with the preservation number: CGMCC No.23421, preservation date: 2021, 9/15, the preservation unit is: china general microbiological culture Collection center, national institute of sciences, 3, west road, 1, north Chen, the area facing the sun, Beijing. The edible vinegar obtained by fermenting the bacillus acidocaldarius SHQ-GD can generate esters with fruity flavor, ketones with butter flavor, aldehydes with grass flavor and acids with cheese flavor, and greatly enriches the flavor of liquid fermented vinegar.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a vinegar fermentation strain, in particular to bacillus acidocaldarius capable of producing fruity flavor of cream and application thereof.
Background
Vinegar as a condiment has a history of over 3000 years in China. It is made up by using solid mixture of various microorganisms (i.e. yeast, mould and bacteria) and making natural fermentation. This process differs from the fermentation in western countries, which only uses one or several microorganisms. It also involves a complex process flow, often requiring the preparation of a starter culture, Starch Saccharification (SS), Alcoholic Fermentation (AF) and Acetic Acid Fermentation (AAF). The complex process flow endows vinegar with abundant flavor substances produced by fermentation, and the functional microorganisms and nutrient substances such as saccharides, proteins, grease and the like in the raw materials act together to generate micromolecular compounds through numerous biochemical reactions. Therefore, the functional microorganisms for producing fragrant substances with high yield have great significance for vinegar fermentation.
The aroma-producing functional microorganism is a generic term for microorganisms that play a key role in the formation of a particular flavor. They secrete enzymes such as proteases, lipases, amylases and the like during fermentation. The enzymes decompose various macromolecular substances in the raw materials into small molecular substances and can provide necessary carbon sources and nitrogen sources for the enzymes. In addition, besides the interaction of the small molecular substances to generate nutrients, the small molecular substances can also be used as precursor substances of other reactions such as Maillard reaction to generate substances such as TTMP and the like, thereby forming a full-bodied flavor substance.
Inoculating the aroma-producing functional microorganisms to Starch Saccharification (SS), Alcohol Fermentation (AF) and Acetic Acid Fermentation (AAF) stages is an effective means for further improving the flavor of vinegar. This is also known as microbial enhancement and in situ regulation. With the intensive research on microbial communities, the development of high-quality liquid fermented vinegar by using functional microbial communities is a feasible measure. At present, the research of developing high-quality liquid fermentation vinegar by using a functional microbial community is still deficient in China, but with the continuous development of a good functional microbial breeding technology and the deep research on synthesizing functional microbes, the functional microbes are believed to have wide application prospects in the production of liquid fermentation vinegar.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides bacillus acidocaldarius capable of generating fruity flavor of cream and application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a Bacillus acidocaldarius (Bacillus acidocola) capable of producing fruity flavor of cream, said Bacillus acidocaldarius being named: SHQ-GD, class name: bacillus acidocaldarius Bacillus acidocola with the preservation number: CGMCC No.23421, preservation date: 2021, 9/15, the preservation unit is: china general microbiological culture Collection center, national institute of sciences, 3, west road, 1, north Chen, the area facing the sun, Beijing.
Further, the Bacillus acidocaldarius SHQ-GD is obtained by separating from a fermentation sample of rice vinegar in Guangdong county.
Furthermore, the bacillus acidocaldarius SHQ-GD can generate strong cream fruity flavor, can grow in a 6% ethanol environment, and is a fragrance-producing functional strain with good fermentation performance.
Further, the bacillus acidothrix SHQ-GD is subjected to PCR amplification by adopting a bacterial universal primer 27F/1492R and taking the total genomic DNA as a template to obtain a target gene sequence consisting of 1188bp base pairs; the gene sequence obtained by sequencing is input into an NCBI database for comparison, and the similarity rate of the gene sequence to the standard strain Bacillus acidoola in Genebank is 96%.
Further, the bacillus acidothricus SHQ-GD grows well on a YPD agar medium, the bacterial colony is light yellow, the surface is smooth, the edge is irregular, the bacterial colony is opaque, the gram stain is purple and rod-shaped, and the strong cream fruit fragrance is emitted around a culture dish.
Further, the Bacillus acidocaldarius SHQ-GD has 6% ethanol resistance.
Further, after solid phase microextraction and gas chromatography mass spectrometry detection are carried out on the fermentation liquor of the bacillus acidoCurvulus SHQ-GD, main volatile flavor compounds are found to comprise: 7 alcohols: ethanol, propionaldehyde dimethyl acetal, n-butanol, trans-2-dodecen-1-ol, trans-2-decenol, 2-methyl-1-phenyl-3-buten-1-ol, isobutanol, 8 aldehydes: butyraldehyde, nonanal, heptanal, n-decanal, 5,9, 13-trimethyl-4, 8, 12-tetradecatrienal, 2, 4-dimethylbenzaldehyde, dodecanal, isovaleraldehyde, 19 esters: ethyl acetate, sec-butyl acetate, ethyl 2-methylbutyrate, methyl 2-methylbutyrate, butyl isobutyrate, butyl formate, ethyl isobutyrate, geranyl acetone, ethyl isovalerate, 2,4, 4-trimethylpentane-1, 3-diylbis isobutyrate, non-ethyl 2-methyl-propionate, 2-ethylhexyl salicylate, diisobutyl phthalate, homosalate, dibutyl phthalate, isopropyl myristate, isobutyl butyrate, butyl butyrate, 2 ketones: 4-heptanone, 3-methyl-4-heptanone, and 3 ethers: phenylsulfide, 3, 5-di-tert-butyl-4-hydroxybenzyl methyl ether and dibutyl ether. Wherein, the alcohol substance endows malt and sweet fragrance to the fermentation liquor; the aldehyde substances endow the fermentation liquor with fresh, citrus, grease and bitter almond flavors; the esters endow the fermentation liquor with a pleasant fruit flavor; the ketone substances give butter and cream fragrance to the fermentation liquid.
The application of the bacillus acidocaldarius capable of generating fruity flavor of cream in the aspect of vinegar fermentation production is disclosed.
The application of the bacillus acidocaldarius capable of generating fruity flavor of cream in improving the flavor of liquid fermented vinegar is disclosed.
The method for fermenting the liquid vinegar by utilizing the bacillus acidocaldarius capable of generating fruity flavor of cream comprises the following steps:
taking a strain fermentation liquor of bacillus acidothrix in a logarithmic phase of growth, centrifuging to obtain bacterial sludge, washing the bacteria twice by using normal saline with the mass concentration of 0.85% to obtain bacterial suspension, inoculating the bacterial suspension into rice wine, sealing by using gauze, placing in an incubator at 30 ℃, and stopping fermentation when the total acid is constant and does not rise any more, thereby obtaining the vinegar.
The invention has the advantages and positive effects that:
1. the fermentation flavor of the liquid vinegar is improved by in-situ regulation and control of the bacillus acidocaldarius SHQ-GD. After detection, the types of esters, acids, ketones and aldehydes of the liquid fermented vinegar are obviously increased. Meanwhile, after the vinegar is strengthened by the bacillus acidovorans, the total acid and reducing sugar of the fermented vinegar are reduced. Sensory evaluation results show that the fermented vinegar after the reinforcement of the bacillus acidovorus has the flavors of being sour, sweet, delicious, soft in taste and smooth and mellow.
2. The metabolite of the fermentation liquor of the strain of the bacillus acidocaldarius SHQ-GD comprises abundant flavor substances such as alcohols, aldehydes, esters and ketones, and is a strain with a fragrance producing function. The edible vinegar obtained by fermenting the bacillus acidocaldarius SHQ-GD can generate esters with fruity flavor, ketones with butter flavor, aldehydes with grass flavor and acids with cheese flavor, and greatly enriches the flavor of liquid fermented vinegar.
3. The bacillus acidoCurculiginis SHQ-GD is separated from a Guangdong village rice vinegar sample, can tolerate the ethanol content of 6 percent, can generate fruity esters, butter-fragrant ketones, green grass-fragrant aldehydes and cheese-fragrant acids in fermentation, and can bear the ethanol stress in vinegar fermentation. The strain has wide prospect when being used as a fermentation strain in the field of vinegar fermentation production.
Drawings
FIG. 1 is a colony morphology of Bacillus acidocaldarius on YPD agar medium and a microscopic image after gram staining; wherein, (a) is a colony morphology picture, and (b) is a microscopic picture after gram staining;
FIG. 2 is a phylogenetic tree diagram of the identification of Bacillus acidocaldarius in the present invention;
FIG. 3 is a graph showing growth curves of the strains according to the present invention at different ethanol contents;
FIG. 4 is a diagram showing the basic physicochemical indexes of the liquid fermented vinegar after the reinforcement of Bacillus acidocaldarius in the present invention; wherein, (a) is pH, (b) is reducing sugar, and (c) is total acid;
FIG. 5 is a diagram showing the volatile flavor of the vinegar after the liquid fermentation of the bacillus acidocaldarius after the reinforcement;
FIG. 6 shows sensory evaluation results of the liquid fermented vinegar after the fortification with Bacillus acidocaldarius in the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A Bacillus acidocaldarius (Bacillus acidocola) capable of producing fruity flavor of cream, said Bacillus acidocaldarius being named: SHQ-GD, class name: bacillus acidocaldarius Bacillus acidocola with the preservation number: CGMCC No.23421, preservation date: 2021, 9/15, the preservation unit is: china general microbiological culture Collection center, national institute of sciences, 3, west road, 1, north Chen, the area facing the sun, Beijing.
Preferably, the Bacillus acidocaldarius SHQ-GD is obtained by separating from a fermentation sample of rice vinegar in Guangdong village.
Preferably, the bacillus acidocaldarius SHQ-GD can generate strong cream fruity flavor, can grow in a 6% ethanol environment, and is a fragrance-producing functional strain with good fermentation performance.
Preferably, the bacillus acidothricus SHQ-GD is subjected to PCR amplification by using a bacterial universal primer 27F/1492R to take the total genomic DNA as a template to obtain a target gene sequence consisting of 1188bp base pairs; the gene sequence obtained by sequencing is input into an NCBI database for comparison, and the similarity rate of the gene sequence to the standard strain Bacillus acidoola in Genebank is 96%. The strain is preliminarily identified as Bacillus acidocaldarius (Bacillus acidocola). The similarity between the Bacillus acidocaldarius SHQ-GD and the Bacillus acidocaldarius (Bacillus acidocola) is only 96% as shown by a 16s rDNA sequencing result, so that the Bacillus acidocaldarius SHQ-GD is possibly a novel strain.
Preferably, the bacillus acidothricus SHQ-GD grows well on a YPD agar culture medium, the bacterial colony is light yellow, the surface is smooth, the edge is irregular, the bacterial colony is opaque, the gram stain is purple and rod-shaped, and the periphery of a culture dish emits heavy cream fruit fragrance.
Preferably, the bacillus acidocaldarius SHQ-GD has 6% ethanol resistance through a plate and liquid ethanol resistance test.
Preferably, after solid phase microextraction and gas chromatography mass spectrometry detection are carried out on the fermentation liquor of the bacillus acidopronus SHQ-GD, main volatile flavor compounds are found to comprise: 7 alcohols: ethanol, propionaldehyde dimethyl acetal, n-butanol, trans-2-dodecen-1-ol, trans-2-decenol, 2-methyl-1-phenyl-3-buten-1-ol, isobutanol, 8 aldehydes: butyraldehyde, nonanal, heptanal, n-decanal, 5,9, 13-trimethyl-4, 8, 12-tetradecatrienal, 2, 4-dimethylbenzaldehyde, dodecanal, isovaleraldehyde, 19 esters: ethyl acetate, sec-butyl acetate, ethyl 2-methylbutyrate, methyl 2-methylbutyrate, butyl isobutyrate, butyl formate, ethyl isobutyrate, geranyl acetone, ethyl isovalerate, 2,4, 4-trimethylpentane-1, 3-diylbis isobutyrate, non-ethyl 2-methyl-propionate, 2-ethylhexyl salicylate, diisobutyl phthalate, homosalate, dibutyl phthalate, isopropyl myristate, isobutyl butyrate, butyl butyrate, 2 ketones: 4-heptanone, 3-methyl-4-heptanone, and 3 ethers: phenylsulfide, 3, 5-di-tert-butyl-4-hydroxybenzyl methyl ether and dibutyl ether. Wherein, the alcohol substance endows malt and sweet fragrance to the fermentation liquor; the aldehyde substances endow the fermentation liquor with fresh, citrus, grease and bitter almond flavors; the esters endow the fermentation liquor with pleasant fruit flavor such as pineapple, apple, banana and grape skin; the ketone substances give butter and cream fragrance to the fermentation liquid.
The application of the bacillus acidocaldarius capable of generating fruity flavor of cream in the aspect of vinegar fermentation production is disclosed.
The method for fermenting the liquid vinegar by utilizing the bacillus acidocaldarius capable of generating fruity flavor of cream comprises the following steps:
taking a strain fermentation liquor of bacillus acidothrix in a logarithmic phase of growth, centrifuging to obtain bacterial sludge, washing the bacteria twice by using normal saline with the mass concentration of 0.85% to obtain bacterial suspension, inoculating the bacterial suspension into rice wine, sealing by using gauze, placing in an incubator at 30 ℃, and stopping fermentation when the total acid is constant and does not rise any more, thereby obtaining the vinegar.
Preferably, bacillus acidocaldarius SHQ-GD is used as a functional microorganism to carry out in-situ regulation and control on fermentation of liquid vinegar, 38 volatile flavor compounds are generated after the bacillus acidocaldarius SHQ-GD is used for reinforcement through headspace solid-phase microextraction and gas chromatography mass spectrometry detection, and only 14 volatile flavor compounds are generated in a control group which is not subjected to reinforcement.
Specifically, the preparation and detection examples are as follows:
example 1: isolation of Bacillus acidocaldarius
The edible vinegar samples are rice vinegar from Guangdong country, and are respectively sampled from fermentation pools for 15 days and 30 days, sampled by a five-point method, fully mixed, placed in a sterile EP tube and stored in a refrigerator at minus 80 ℃. Weighing vinegar sample 5.0mL under aseptic condition, mixing with aseptic normal saline 45mL, shaking in shaker for 2 hr to disperse microbial cells, and making into 10-1Dilutions of the dilutions and serial gradient dilutions were performed. Respectively taking 0.1mL of dilution as 10-5、10-6And 10-7Inoculating the diluted solution into YPD solid medium, coating, and mixingThe plates are respectively placed in a common incubator at 30 ℃ for culturing for 48h, and single colonies are observed and picked up, and are repeatedly streaked and purified to obtain pure strains. And (3) storing glycerol tubes, and mixing the bacterial liquid with 50% of glycerol in a sterile environment according to the ratio of 1: 1, mixing uniformly, and storing in a refrigerator at-80 ℃.
The colony morphology, the size, color, shape of the colony, the colony edges, bulges and transparencies were observed and recorded as shown in FIG. 1 (a). The color of the picked colony is mainly yellow green and light yellow; the appearance is mainly round, the surface is smooth and moist, the texture is uniform, and the picking is not easy. The periphery of the culture dish emits strong cream fruit fragrance, and gram-positive bacteria are shown in figure 1(b) in microscopic examination result.
Example 2: molecular biological identification of bacillus acidocaldarius
Culturing the activated strain to logarithmic phase, collecting 1mL bacterial liquid, centrifuging at 8000r/min, collecting thallus, adding 100 μ L sterile water, mixing to make cell concentration about 105mu.L/L. 10 μ L of the extract was diluted 10-fold with 90 μ L of sterile water to prepare 105、104、103、102And 10/L concentration of bacterial liquid, each dilution taking 3 u L inoculation on the plate, observed colony growth. Strains that can grow on the plate were selected for identification.
The DNA extraction kit comprises the steps of extracting a genome of a test strain, carrying out PCR amplification by using a bacterial 16S rDNA sequence universal primer (27F and 1492R), sending a PCR amplification product to Jinzhi biotechnology company for sequencing, carrying out BLAST homology search on a spliced sequence result in a GenBank database, and constructing a phylogenetic tree by using MEGA. The phylogenetic tree was constructed by the neighbor-joining method in MEGA, as shown in FIG. 2.
Example 3: determination of highest ethanol tolerance of bacillus acidocaldarius
And (3) selecting the bacillus acidocaldarius with good growth to further study the growth condition of the bacillus acidocaldarius in an ethanol environment. The activated strain was inoculated in a liquid medium containing 0%, 1%, 2%, 4%, 5%, 6% and 7% ethanol (v/v) at an inoculation amount of 3% (v/v), and the absorbance of the bacterial liquid was measured to observe the growth ability.
The growth curve of the strain under 7 ethanol contents is shown in figure 3, and the growth curve of the strain reflects the growth capacity of the strain. The growth vigor of the strain is best in the culture medium with 0% ethanol content, and the growth speed of the strain is slightly slow in the culture medium with 1%, 2% and 4% ethanol content, but the strain has no great difference with the strain cultured in the culture medium with 0% ethanol content. Although the growth of the strain is inhibited to a certain extent in the culture medium with 5% and 6% ethanol content in the first 30h, the growth rate of the strain begins to increase after 30h, and the influence of ethanol on the strain can be ignored. When the ethanol content is 7%, the growth of the strain is obviously inhibited, but after the strain is cultured for 40h, the strain reaches a stationary phase, OD600nmThe value reaches around 0.9. Therefore, it can be determined that the ethanol tolerance of Bacillus acidocaldarius SHQ-GD was 6% when ethanol stress was present.
Example 4: determination of volatile flavor substances in bacillus acidocaldarius fermentation liquor
Volatile flavor substances of a Bacillus acidolyticus (Bacillus acidocola) fermentation liquor are detected by Headspace solid phase microextraction (HS-SPME) and Gas chromatography mass spectrometry (GC-MS).
Inoculating the activated strain into YPD liquid culture medium with an inoculum size of 3% (v/v), performing shake culture at constant temperature of 30 ℃, sampling at 24h, centrifuging the fermentation liquor, taking 3mL of supernatant, placing the supernatant in a headspace bottle, and adding 30 μ L of 20ppm 2-octanol standard substance. HS-SPME: sealing the headspace bottle, and balancing in a 60 deg.C water bath for 20 min. After balancing, the extraction head is extracted in the air for 30min at 60 ℃ and the GC-MS sample injection is carried out for 15min at 250 ℃. GC conditions were as follows: the initial temperature is 40 deg.C for 3min, the temperature is raised to 150 deg.C at 4 deg.C/min for 1min, and then raised to 250 deg.C at 8 deg.C/min for 6 min. Helium is used as carrier gas, the injection port temperature is 250 ℃, and the injection time is 1 min. MS conditions: the ion source temperature is 200 ℃, the interface temperature is 220 ℃, the solvent delay time is 1.5min, the ionization mode is EI, the scanning mass range of 70eV is 33-500 m/z, and the scanning speed is 3.00 scans/s. Compounds with similarity of more than or equal to 80% are selected according to NIST11 database search and are characterized by combining ion fragments and retention indexes. 2-octanol is used as an internal standard substance, and the quantification is carried out by adopting an internal standard method.
As shown in Table 1, the fermentation broth of Bacillus acidocaldarius contains abundant alcohols, aldehydes, esters, ketones and alkanes. Their combined action causes the acidophilic bacillus to emit a strong fruity cream flavour. Wherein the esters can display the fragrance of pineapple, apple, banana, grape and other fruits. Ketones and aldehydes can exhibit the aroma of almonds, pine oil, fats, etc. The alcohol can exhibit the fresh aroma of malt. Through the comprehensive action of the substances, the special flavor of the bacillus acidocaldarius is formed.
TABLE 1 determination of volatile flavor substances in fermentation broth of Bacillus acidocaldarius
Example 5: bacillus acidocaldarius fermented liquid vinegar and determination of basic physicochemical indexes thereof
Taking strain fermentation liquor in the logarithmic phase of growth, centrifuging at low temperature to obtain bacterial sludge, washing the bacteria twice with 0.85% normal saline to obtain bacterial suspension, inoculating the bacterial suspension into rice wine by using a sterile liquid transfer gun, sealing by using 8 layers of gauze, placing in an incubator at 30 ℃, and stopping fermentation when the total acid is constant and does not rise any more. Fermentation samples were collected every 5 days to determine pH, total acid and reducing sugars.
The results of measuring the basic physicochemical indexes of the fermented vinegar are shown in fig. 4, and as the fermentation time is prolonged, the pH and the reducing sugar of all the samples are in a descending trend, and the total acid content is in an ascending trend. At the end of the fermentation, A, B and C had pH 2.9, 3.1 and 3.2, respectively. For reducing sugars, group C always exhibited lower reducing sugar concentrations due to some dilution prior to fermentation. At the end of the fermentation, the concentrations of reducing sugars in A, B and C were 3.2g/100mL, 1.4g/100mL, and 0.3g/100mL, respectively. Lower reducing sugar concentrations represent more complete degradation of the substrate while also producing a greater abundance of volatile flavors. At the end of the fermentation, the total acid content in A, B and C was 46g/L, 30g/L, and 28g/L, respectively. Due to the addition of the bacillus acidocaldarius, the total acid content of the fermented vinegar is reduced, and the lower total acid content can make the fermented vinegar soft in taste and reduce the sour and astringent taste.
TABLE 2 fermentation of liquid vinegar with Bacillus acidocaldarius
Example 6: difference of volatile flavor substances of liquid fermented vinegar
Detecting the change of volatile flavor substances in vinegar fermented for 30 days by using GC-MS, centrifuging the fermentation liquor, taking 3mL of supernatant, placing the supernatant in a headspace bottle, and adding 30 mu L of 20ppm 2-octanol standard substance. The volatile flavor substances of the vinegar fermentation liquid were measured according to the injection conditions in the above example 4.
The results of measuring the volatility of the vinegar fermentation liquid are shown in table 3 (fig. 5), and B and C (bacillus acidocaldarius enhanced group) contained more abundant volatile flavor substances than a (blank group). A. The total amount of esters in B and C is 0.0453mg/L, 0.1622mg/L and 1.844mg/L respectively. Among them, the esters in group B are most abundant in variety, including butyl acetate, butyl 2-methylpropionate, sec-butyl acetate, ethyl acetate, 2-ethyl-1-hexanol, trifluoroacetate, isobutyl propyl sebacate, 2-methylpropyl butyrate, diethyl succinate, and methyl acetate. Most of the substances contain abundant fruit fragrance, and the substances can reduce the bad flavor of the fermented vinegar, so that the vinegar has the fruit fragrance and is delicious and thick. A. The total amount of acid substances in B and C is 1.5441mg/L, 0.8466mg/L and 0.6211mg/L respectively. A contains high concentration of acetic acid, and B contains the most abundant acids including 2-methyl-propionic acid, 3-methyl-butyric acid and 2-ethyl-acetic acid. The acetic acid endows the fermented vinegar with strong and intense acidity, and the abundant organic acid species can weaken the harsh acidity to make the vinegar taste softer. A. The total amount of alcohols in B and C are 7.6752mg/L, 0.21254mg/L and 1.4648mg/L respectively. B, C contains lower concentrations of alcohols than group A, probably because B, C all used to generate esters. A. The total amount of ketones in B and C is 0.0153mg/L, 0.0513mg/L and 0.0211mg/L respectively, the types and concentrations of ketones in B are highest, and the ketones comprise 1-hydroxy-2-acetone, 2-nonanone, 3-acetyl-2-butanone, 3-methyl-2-hexanone and 2, 3-butanedione, and the ketones contribute to the fragrance of cream and nuts in the fermented vinegar. A. The total amount of aldehyde substances in the B and the C are 0.0473mg/L,
0.3302mg/L, 0.0062mg/L, high concentration of aldehyde substance in B endows fermented vinegar with green grass flavor.
TABLE 3 difference in volatile flavor substances of liquid fermented vinegar
Example 7: sensory evaluation of liquid fermentation vinegar
Referring to GB 18187-. 10 trained panelists were then selected to evaluate the color and flavor aroma of the vinegar samples. And team members received several training sessions, including definitions and descriptions, prior to formal sensory evaluation. Finally, the samples (50 g) were marked with a three-digit number and placed in a plastic cup for sensory evaluation.
Sensory evaluation results as shown in fig. 6, a and C exhibited yellowish colors and B exhibited a darker amber color from the color viewpoint. The scores were scored for sweet and sour taste, soft mouthfeel, and lubricous and mellow, and B gave the highest score. Therefore, the addition of the bacillus acidocaldarius improves the overall flavor of the liquid vinegar, and after the bacillus acidocaldarius is strengthened, the liquid fermented vinegar is soft, sour, sweet, tasty, and smooth and mellow in fragrance due to the lower total acid content and the richer volatile flavor substances.
Bacillus acidocaldarius SHQ-GD sequence:
GATGCAGGCG CGTGCTATAC ATGCAGTCGA GCGGACTTTT AAAGCTTGCT TTTAAAAGTT AGCGGCGGAC GGGTGAGTAA CACGTGGGTA ACCTGCCTGT AAGACTGGGA TAACTCCGGG AAACCGGGGC TAATACCGGA TAACTTCTTT CCTCGCATGA GGAAAGATTG AAAGATGGCT TCGGCTATCA CTTACAGATG GACCCGCGGC GCATTAGCTA GTTGGTGAGG TAACGGCTCA CCAAGGCGAC GATGCGTAGC CGACCTGAGA GGGTGATCGG CCACACTGGG ACTGAGACAC GGCCCAGACT CCTACGGGAG GCAGCAGTAG GGAATCTTCC GCAATGGACG AAAGTCTGAC GGAGCAACGC CGCGTGAGTG ATGAAGGTTT TCGGATCGTA AAACTCTGTT GTTAGGGAAG AACAAGTATC GTTCGAATAG GGCGGTACCT TGACGGTACC TAACCAGAAA GCCACGGCTA ACTACGTGCC AGCAGCCGCG GTAATACGTA GGTGGCAAGC GTTGTCCGGA ATTATTGGGC GTAAAGCGCG CGCAGGCGGT TTCTTAAGTC TGATGTGAAA GCCCACGGCT CAACCGTGGA GGGTCATTGG AAACTGGGAG ACTTGAGTGC AGAAGAGGAG AGTGGAATTC CACGTGTAGC GGTGAAATGC GTAGAGATGT GGAGGAACAC CAGTGGCGAA GGCGACTCTC TGGTCTGTAA CTGACGCTGA GGCGCGAAAG CGTGGGGAGC GAACAGGATT AGATACCCTG GTAGTCCACG CCGTAAACGA TGAGTGCTAA GTGTTAGAGG GTTTCCGCCC TTTAGTGCTG CAGCTAACGC ATTAAGCACT CCGCCTGGGG AGTACGGCCG CAAGGCTGAA ACTCAAAGGA ATAAACGGGC GCCCGCACAA GCGGTGGCAG CATGTGTTTA ATTACGGATG CACGCGACGA ACCTTACCAG GTCTTTGACA TCCTCTGAC
although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the embodiments disclosed.
Sequence listing
<110> Tianjin science and technology university
<120> bacillus acidopronus capable of generating fruity flavor of cream and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 979
<212> DNA/RNA
<213> Bacillus acidocaldarius SHQ-GD sequence (Unknown)
<400> 1
gatgcaggcg cgtgctatac atgcagtcga gcggactttt aaagcttgct tttaaaagtt 60
agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120
aaaccggggc taataccgga taacttcttt cctcgcatga ggaaagattg aaagatggct 180
tcggctatca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240
ccaaggcgac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300
ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360
ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aaactctgtt gttagggaag 420
aacaagtatc gttcgaatag ggcggtacct tgacggtacc taaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540
gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga 600
gggtcattgg aaactgggag acttgagtgc agaagaggag agtggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720
ctgacgctga ggcgcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga ataaacgggc 900
gcccgcacaa gcggtggcag catgtgttta attacggatg cacgcgacga accttaccag 960
gtctttgaca tcctctgac 979
Claims (9)
1. A Bacillus acidocaldarius (Bacillus acidocola) capable of producing fruity flavor of cream, characterized in that: the name of the bacillus acidocaldarius is as follows: SHQ-GD, class name: bacillus acidocaldarius Bacillus acidocola with the preservation number: CGMCC No.23421, preservation date: 2021, 9/15, the preservation unit is: china general microbiological culture Collection center, national institute of sciences, 3, west road, 1, north Chen, the area facing the sun, Beijing.
2. The bacillus acidoCurculus capable of producing fruity flavor of cream as claimed in claim 1, wherein: the bacillus acidopronus SHQ-GD is obtained by separating from a fermentation sample of rice vinegar in Guangdong county.
3. The bacillus acidoCurculus capable of producing fruity flavor of cream as claimed in claim 1, wherein: the bacillus acidocaldarius SHQ-GD can generate strong cream fruity flavor, can grow in a 6% ethanol environment, and is a functional strain with good fermentation performance and aroma production function.
4. The bacillus acidoCurculus capable of producing fruity flavor of cream as claimed in claim 1, wherein: the Bacillus acidovorus SHQ-GD is subjected to PCR amplification by using a bacterial universal primer 27F/1492R to take genome total DNA as a template to obtain a target gene sequence consisting of 1188bp base pairs, and the gene sequence obtained by sequencing is input into an NCBI database for comparison, so that the similarity rate of the gene sequence to a standard strain Bacillus acidoola in Genebank is 96%.
5. The bacillus acidoCurculus capable of producing fruity flavor of cream as claimed in claim 1, wherein: the bacillus acidothrix SHQ-GD grows well on the YPD agar culture medium, the bacterial colony is light yellow, the surface is smooth, the edge is irregular, the bacterial colony is opaque, the gram stain is purple and rod-shaped, and the periphery of the culture dish emits heavy cream fruit fragrance.
6. The bacillus acidoCurculus capable of producing fruity flavor of cream as claimed in claim 1, wherein: the bacillus acidocaldarius SHQ-GD has 6% ethanol resistance.
7. The Bacillus acidocaldarius capable of producing a fruity flavor of cream according to any one of claims 1 to 6, wherein: after solid-phase microextraction and gas chromatography mass spectrometry detection are carried out on the Bacillus acidocaldarius SHQ-GD fermentation liquor, main volatile flavor compounds are found to comprise: 7 alcohols: ethanol, propionaldehyde dimethyl acetal, n-butanol, trans-2-dodecen-1-ol, trans-2-decenol, 2-methyl-1-phenyl-3-buten-1-ol, isobutanol, 8 aldehydes: butyraldehyde, nonanal, heptanal, n-decanal, 5,9, 13-trimethyl-4, 8, 12-tetradecatrienal, 2, 4-dimethylbenzaldehyde, dodecanal, isovaleraldehyde, 19 esters: ethyl acetate, sec-butyl acetate, ethyl 2-methylbutyrate, methyl 2-methylbutyrate, butyl isobutyrate, butyl formate, ethyl isobutyrate, geranyl acetone, ethyl isovalerate, 2,4, 4-trimethylpentane-1, 3-diylbis isobutyrate, non-ethyl 2-methyl-propionate, 2-ethylhexyl salicylate, diisobutyl phthalate, homosalate, dibutyl phthalate, isopropyl myristate, isobutyl butyrate, butyl butyrate, 2 ketones: 4-heptanone, 3-methyl-4-heptanone, and 3 ethers: phenylsulfide, 3, 5-di-tert-butyl-4-hydroxybenzyl methyl ether and dibutyl ether. Wherein, the alcohol substance endows malt and sweet fragrance to the fermentation liquor; the aldehyde substances endow the fermentation liquor with fresh, citrus, grease and bitter almond flavors; the esters endow the fermentation liquor with a pleasant fruit flavor; the ketone substances give butter and cream fragrance to the fermentation liquid.
8. Use of the Bacillus acidocaldarius for producing fruity flavor of butter according to any one of claims 1 to 7 in the fermentative production of vinegar.
9. A method of fermenting liquid vinegar with the use of Bacillus acidocaldarius which can produce fruity flavor of cream as claimed in any one of claims 1 to 7, wherein: the method comprises the following steps:
taking a strain fermentation liquor of bacillus acidothrix in a logarithmic phase of growth, centrifuging to obtain bacterial sludge, washing the bacteria twice by using normal saline with the mass concentration of 0.85% to obtain bacterial suspension, inoculating the bacterial suspension into rice wine, sealing by using gauze, placing in an incubator at 30 ℃, and stopping fermentation when the total acid is constant and does not rise any more, thereby obtaining the vinegar.
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