CN111154657B - Aspergillus oryzae strain, application thereof and aspergillus oryzae preparation - Google Patents
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Abstract
The invention discloses an aspergillus oryzae strain, application thereof and an aspergillus oryzae preparation. The strain is preserved in Guangdong province microorganism strain preservation center in 2019, 26.9.9, with the preservation number of GDMCC No.60791, the preservation address of No. 5,59 of Mieli Zhou Lu 100, Guangdong province, and the postal code of 510075. The aspergillus oryzae strain has at least one of the following properties: (1) has the capability of fermenting the red Zhejiang vinegar to generate aldehyde substances; (2) has the capability of fermenting the red Zhejiang vinegar to produce volatile esters; (3) having the ability to produce non-volatile flavors; (4) has the ability to produce proteases.
Description
Technical Field
The invention relates to the technical field of processing of red Zhejiang vinegar seasonings, and particularly provides an aspergillus oryzae strain, application of the aspergillus oryzae strain and an aspergillus oryzae preparation.
Background
The vinegar brewing history in China is long and has more than 3000 years from now. In the long-term vinegar brewing process, people create various special vinegar making processes according to regional climate differences and dietary habit differences, and make various brands of vinegar, such as: shanxi mature vinegar, Zhenjiang aromatic vinegar, Dahong Zhejiang vinegar, Sichuan Baoning drum vinegar, Fujian red rice mature vinegar, Tianjin preserved mature vinegar, Henan Zhangde mature vinegar and the like. The vinegar has unique preparation process and special flavor, and is reputed at home and abroad. The vinegar products obtained by fermenting the existing Aspergillus oryzae varieties have lower content of volatile aldehyde and ester.
Disclosure of Invention
Based on this, there is a need to provide a strain of Aspergillus oryzae.
The invention also provides the red Zhejiang vinegar which is prepared from the aspergillus oryzae strain and has high content of volatile aldehyde and ester and high content of non-volatile substances, such as sugar, acid, ester and alcohol.
An Aspergillus oryzae strain, which has been deposited at 26.9.2019 with the accession number GDMCC No.60791 at the Guangdong province culture Collection.
In one embodiment, the Aspergillus oryzae strain has at least one of the following properties:
(1) has the capability of fermenting the red Zhejiang vinegar to generate aldehyde substances;
(2) has the capability of fermenting the red Zhejiang vinegar to produce volatile esters;
(3) having the ability to produce non-volatile flavors;
(4) has the ability to produce proteases.
An Aspergillus oryzae preparation, wherein the Aspergillus oryzae preparation is processed by the Aspergillus oryzae strain.
The aspergillus oryzae strain can be applied to preparation of red vinegar.
A red vinegar prepared by fermenting the Aspergillus oryzae strain is provided.
In one embodiment, the number of aldehydes detected in the finished product of the red Zhejiang vinegar is not less than 12; the total content of aldehydes in the red Zhejiang vinegar is not less than 1500 mug/L.
In one embodiment, the number of the detected esters in the finished product of the red Zhejiang vinegar is not less than 16; the total content of esters in the red Zhejiang vinegar is not less than 700 mug/L.
In one embodiment, the red vinegar finished product has not less than 26 kinds of detected carbohydrate; the total content of saccharide detected in the red Zhejiang vinegar is not less than 2000 mg/L.
In one embodiment, the protease activity of the aspergillus oryzae strain is not less than 750U/g when the red vinegar is fermented for half a month.
The invention provides an aspergillus oryzae strain with high content of volatile aldehyde and ester and high content of non-volatile substances such as sugar, acid, ester and alcohol and application thereof, and researches the difference of the aspergillus oryzae strain and a standard aspergillus oryzae strain on the flavor of fermented red Zhejiang vinegar so as to select better aspergillus oryzae to prepare the fermented strain of the red Zhejiang vinegar. Comparing the volatile substance detection results, the Aspergillus oryzae strains are compared with the standard strains, and the volatile aldehydes and esters of the finished product of the red Zhejiang vinegar obtained by fermenting the Aspergillus oryzae strains are respectively 4 more than the volatile aldehydes and esters of the red Zhejiang vinegar obtained by fermenting the standard strains and 3 more than the volatile aldehydes and esters obtained by fermenting the standard strains. In addition, the content of volatile aldehyde and ester in the finished product of the bright red Zhejiang vinegar obtained by fermenting the aspergillus oryzae strain is about 1 time higher than that obtained by fermenting the standard strain. In addition, the finished product of the scarlet and Zhejiang vinegar aldehyde obtained by fermenting the aspergillus oryzae strain is more than 4 types obtained by fermenting the standard strain. Compared with the detection result of non-volatile substances, the types of sugars, acids, esters and alcohols in the non-volatile substances detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain are respectively 8, 5, 2 and 1 more than those of the standard strain, the content of the sugars and the esters is greatly different from those of the standard strain, and the content of the sugars and the esters in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain is respectively 1.88 times and 2.87 times of that in the finished product fermented by the standard strain.
In conclusion, compared with the finished product prepared by using the Aspergillus oryzae strain as a fermentation strain, the detection of volatile flavor substances and the detection of non-volatile substances have obvious advantages compared with the standard Aspergillus oryzae strain. The aspergillus oryzae strain can endow the red Zhejiang vinegar with better flavor and taste.
Drawings
FIG. 1 is a colony morphology of Aspergillus oryzae strains;
FIG. 2 is a DNA sequencing alignment chart of Aspergillus oryzae strains;
FIG. 3 is a mass spectrum diagram of the different volatile flavor substances of the Aspergillus oryzae strain and the standard strain fermented scarlet and Zhejiang vinegar.
Detailed Description
Example 1
This example provides an Aspergillus oryzae strain (Aspergillus oryzae) that was deposited at 26.9.2019 in the microbial cultures Collection of Guangdong province under the accession number GDMCC No.60791 at the accession number of 5 th Hakkaido No. 59, Calif. Miehu 100, Guangzhou, Guangdong province, and at the accession number of 510075.
The aspergillus oryzae strain is obtained by screening through the following steps:
1. material preparation
(1) Starting strains: shanghai Niang 3.042, collected in the center of culture Collection of Meizhai food Co., Ltd., Guangzhou, and derived from a culture separated and purified in the production environment of Da hong Zhe Vinegar.
(2) Expanding culture medium: cooking 1000g soybean with 5000mL water for 5 hr, filtering to obtain 1000mL soybean juice, adding ammonium sulfate 1g, magnesium sulfate 1g, potassium dihydrogen phosphate 5g, glucose 5g, soluble starch 20g, agar 20g, pH 6.40-6.80, and sterilizing at 121 deg.C for 20 min.
(3) Casein screening culture medium: 4g of casein, 0.3g of monopotassium phosphate, 3g of sodium nitrate, 1g of dipotassium phosphate, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate, 30g of glucose, 20-25 g of agar, 1000mL of distilled water and sterilization at 121 ℃ for 20 min.
2. Aspergillus oryzae strain mutagenesis and screening
(1) Preparation of spore suspension: taking the inclined plane cultured for 3 days in Shanghai Niang 3.042, washing off the spores with normal saline, scattering the spores, filtering to obtain a spore suspension, counting by using a blood cell counting plate, adjusting the number of the spores to be about 106/mL, and diluting with 10-fold gradient of sterile normal saline for later use.
(2) Ultraviolet mutagenesis: placing 10 μ L spore suspension under ultraviolet lamp radiation for mutagenesis for 10s, 20s, 60s, 80s, 120s, 150s, 180s, and 200s respectively, treating for 0s as control, diluting, spreading 100 μ L spore suspension on plate, and culturing in 30 deg.C incubator for 3 days.
(3) Calculating the lethality rate: lethality ═ control plate colony number-mutagenic plate colony number)/control plate colony number × 100%
(4) Strain screening: selecting colonies with the fatality rate of 70-90% on a flat plate after ultraviolet mutagenesis, dibbling the colonies on a casein screening culture medium, culturing for 3 days at 30 ℃, observing and recording the sizes of transparent rings and the colonies, and calculating a K value. And selecting the colony with good colony growth, large colony diameter and large K value.
3. Aspergillus oryzae strain deposit
Transferring the fine bacterial colony obtained by screening to an inclined plane of an amplification culture medium, culturing at a constant temperature of 30 ℃ for 3 days, and maturing to obtain the aspergillus oryzae bacterial colony, wherein the bacterial colony morphology of the aspergillus oryzae bacterial strain cultured on the amplification culture medium is shown in figure 1 (in figure 1, the left side is the bacterial colony morphology of a plate GDMCC No.60791 of the aspergillus oryzae bacterial strain of the invention, and the right side is the bacterial colony morphology of a plate of an aspergillus oryzae standard strain). Storing in 0-4 deg.C freezer. The Aspergillus oryzae strain is preserved in Guangdong province microorganism culture collection center in 2019, 9 and 26, and the preservation number is GDMCC No. 60791.
The Aspergillus oryzae strain is inoculated on an amplification culture medium for amplification culture, DNA is extracted, and the comparison result of sequencing NCBI is shown in figure 2.
The Aspergillus oryzae strain has the following properties:
(1) has the capability of fermenting the red Zhejiang vinegar to produce aldehyde substances with high yield;
(2) has the capability of fermenting the red Zhejiang vinegar to produce volatile ester substances with high yield;
(3) having the ability to produce non-volatile flavors;
(4) has the capability of producing protease with high yield.
Example 2
This example provides a red vinegar prepared by fermenting the Aspergillus oryzae strain described in example 1.
The aldehydes substances detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain are not less than 12; the total content of aldehydes in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain is not less than 1500 mug/L.
Not less than 16 esters are detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain; the total content of esters in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain is not less than 700 mug/L.
Not less than 26 kinds of saccharides are detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain; the total content of saccharide detected in the finished product of red Zhejiang vinegar fermented by Aspergillus oryzae is not less than 2000 mg/L; the esters in the non-volatile substances detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain are not less than 5; the total content of ester in non-volatile substances detected in the finished product of red Zhejiang vinegar fermented by Aspergillus oryzae is not less than 200 mg/L.
The protease activity of Aspergillus oryzae strain fermented glutinous rice for half a month is not less than 750U/g.
Detecting volatile flavor substances in the finished product of the red Zhejiang vinegar:
the detection method of the volatile flavor substances comprises the following steps: the volatile flavor substances are researched by adopting a headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method. Diluting a sample by 10 times with distilled water, sucking 6mL into a 15mL headspace bottle, adding 1.5g of sodium chloride and a dioctyl internal standard, adding a rotor, sealing with polytetrafluoroethylene, balancing for 10min in a magnetic stirrer water bath at 50 ℃ and 600r/m, inserting a 75 mu mCAR/PDMS extraction head (aged according to instructions before use) into the headspace for adsorption for 40min, pulling out the extraction head after adsorption is finished, quickly inserting the extraction head into a GC-MS sample inlet, analyzing for 5min at 250 ℃, and starting an instrument to collect data.
The peak spectrograms of the volatile flavor substances of the finished product of the red Zhejiang vinegar obtained by fermenting the standard strain Aspergillus oryzae 3.042 and the Aspergillus oryzae strain of the invention are shown in figure 1, wherein the curve positioned at the upper part in the figure 1 is the spectrogram of the volatile flavor substances of the finished product of the red Zhejiang vinegar obtained by fermenting the standard strain Aspergillus oryzae 3.042, and the curve positioned at the lower part is the spectrogram of the volatile flavor substances of the finished product of the red Zhejiang vinegar obtained by fermenting the Aspergillus oryzae strain.
The detection results of volatile flavor substances of the finished product of the bright red Zhejiang vinegar obtained by fermenting the standard strain Aspergillus oryzae 3.042 and the Aspergillus oryzae strain are shown in Table 1.
TABLE 1 Peak spectra of volatile flavor components of two Aspergillus oryzae fermented Da hong Zhe Vinegar
As can be seen from Table 1, the finished products of the vinegar prepared by fermenting Aspergillus oryzae of the present invention are 51 types, the finished products of the vinegar prepared by fermenting Aspergillus oryzae of the standard strain 3.042 are 44 types, and the finished products of the vinegar prepared by fermenting Aspergillus oryzae of the present invention are 7 types more than the finished products prepared by fermenting Aspergillus oryzae of the standard strain 3.042. In addition, the total content of aldehydes in the finished product obtained by fermenting the aspergillus oryzae strain is 1504.420 mug/L, the total content of esters is 756.756 mug/L, the total content of aldehydes in the finished product of the bright red Zhejiang vinegar fermented by the standard strain aspergillus oryzae 3.042 is 813.985 mug/L, and the total content of esters is 489.929 mug/L. The content difference is about 1 time. The finished product of the scarlet and Zhejiang vinegar aldehyde obtained by fermenting the aspergillus oryzae strain is more than 4 types obtained by fermenting the standard strain. Meanwhile, 16 esters are detected in a finished product obtained by fermenting in the aspergillus oryzae strain, 13 esters are obtained by fermenting the aspergillus oryzae 3.042 serving as a standard strain, and more than 3 types of reddish Zhejiang acetaldehyde are obtained by fermenting the aspergillus oryzae strain than 3.042 serving as the standard strain.
The acid substances in the finished product of the red Zhejiang vinegar obtained by fermenting the aspergillus oryzae strain are 5 in types and the content is 5897.489 mu g/L, and the acid substances obtained by fermenting the standard strain aspergillus oryzae 3.042 are 5 in types and the content is 5569.866 mu g/L.
Detecting non-volatile substances in the finished product of the red Zhejiang vinegar:
detection of non-volatile flavor substances: 50ul of vinegar sample is taken into a 2mL centrifuge tube, and after being frozen at minus 80 ℃ for 12h, vacuum freeze drying is carried out immediately at minus 80 ℃ under the condition of 0.37Pa for 12 h. Adding 100ul of derivative solvent N, N-dimethylformamide into dried edible vinegar sample, vortex and shaking for 3min to mix uniformly, ultrasonically dissolving for 15min, and centrifuging at 12000rmp for 3 min. Taking 50 mu L of supernatant, adding 5 mu L of heptadecanoic acid (10ppm), adding 100ml of derivatization reagent BSTFA decaTMCS (99; I), vortexing and shaking to fully mix the mixture, reacting for 2 hours under the condition of water bath at 80 ℃, and finally sampling 1ul of sample.
The results of the detection of the flavor substances of the red Zhejiang vinegar prepared by fermenting different aspergillus oryzae are shown in table 2.
TABLE 2 Peak spectra results of non-volatile flavor of two Aspergillus oryzae fermented Da hong Zhe Vinegar
As can be seen from Table 2, 1, there were 26 kinds of sugars, 2298.558mg/L, among the comparative sugars, and the nonvolatile substances detected in the finished product of red vinegar fermented with Aspergillus oryzae strain. The product obtained by fermenting Aspergillus oryzae 3.042 as standard strain contains 18 kinds of saccharides, and has a content of 1219.921 mg/L. The content of sugar in the finished product of the red vinegar prepared by fermenting the aspergillus oryzae strain is 1.88 times that in the finished product prepared by fermenting the standard strain aspergillus oryzae 3.042. 2. In comparison with acids, 31 non-volatile substances with the content of 2509.711mg/L are detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain, and 26 acids with the content of 2227.575mg/L are obtained by fermenting the standard strain aspergillus oryzae 3.042. 3. Compared with esters, the total content of esters in non-volatile substances detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain is 209.517mg/L, and the total content of esters in the non-volatile substances of the standard fermentation strain is 72.859 mg/L. 4. Compared with alcohols, the types of the alcohols in the non-volatile substances detected in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain are 21, and the content is 3882.359 mg/L. The standard strain Aspergillus oryzae 3.042 was used as a fermentation strain to measure 20 kinds of alcohol in the material, and the content was 3049.155 mg/L. In conclusion, the Aspergillus oryzae strain has obvious advantages compared with a finished product prepared by taking the standard strain Aspergillus oryzae 3.042 as a fermentation strain.
And (3) protease activity determination:
the determination of protease activity was performed according to the national standard SB/T10317-1999. After inoculating the Aspergillus oryzae strain, measuring the activity of the protease in half a month, and finding that the activity of the fermented glutinous rice enzyme of the Aspergillus oryzae strain is 783.5U/g, the activity of the Aspergillus oryzae Huniang 3.042 enzyme is 520.4U/g, which is 55 percent higher than the activity of the Aspergillus oryzae 3.042 enzyme of a standard strain.
The invention provides an aspergillus oryzae strain with high content of volatile aldehyde and ester and high content of non-volatile substances such as sugar, acid, ester and alcohol and application thereof, and researches the difference of the aspergillus oryzae strain and a standard aspergillus oryzae strain on the flavor of fermented red Zhejiang vinegar so as to select better aspergillus oryzae to prepare the fermented strain of the red Zhejiang vinegar.
Comparing volatile substance detection results, the Aspergillus oryzae strains are compared with a standard strain Aspergillus oryzae 3.042, and volatile aldehydes and esters of the finished product scarlet Zhejiang vinegar obtained by fermenting the Aspergillus oryzae strains are respectively 4 types and 3 types more than those obtained by fermenting the standard strain Aspergillus oryzae 3.042. In addition, the content of volatile aldehyde and ester in the finished product of the bright red Zhejiang vinegar obtained by fermenting the aspergillus oryzae strain is about 1 time higher than that obtained by fermenting the standard strain aspergillus oryzae 3.042. In addition, the finished product of the scarlet and Zhejiang vinegar aldehyde obtained by fermenting the aspergillus oryzae strain is more than 4 types obtained by fermenting the standard strain aspergillus oryzae 3.042. Compared with the detection result of non-volatile substances, the detection results of the non-volatile substances in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain are 8, 5, 2 and 1 more in types than the standard strain aspergillus oryzae 3.042, the content of the non-volatile substances is sugar and esters, and the content of the sugar and the esters in the finished product of the red Zhejiang vinegar fermented by the aspergillus oryzae strain is 1.88 times and 2.87 times of the sugar in the finished product fermented by the standard strain aspergillus oryzae 3.042.
In conclusion, compared with the finished product prepared by using the Aspergillus oryzae strain as a fermentation strain, the detection of volatile flavor substances and the detection of non-volatile substances have obvious advantages compared with the standard Aspergillus oryzae strain. The aspergillus oryzae strain can endow the red Zhejiang vinegar with better flavor and taste.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. An Aspergillus oryzae strain, wherein the Aspergillus oryzae strain (Asperqi 1lus oryzae) has been deposited at 26.9.2019 with the accession number GDMCC No.60791 in Guangdong province.
2. An Aspergillus oryzae strain according to claim 1, characterized in that it has at least one of the following properties:
(1) has the capability of fermenting the red Zhejiang vinegar to generate aldehyde substances;
(2) has the capability of fermenting the red Zhejiang vinegar to produce volatile esters;
(3) having the ability to produce non-volatile flavors;
(4) has the ability to produce proteases.
3. An Aspergillus oryzae preparation, wherein the Aspergillus oryzae preparation is processed from the Aspergillus oryzae strain of claim 1 or 2.
4. Use of an Aspergillus oryzae strain according to any one of claims 1-2 for the preparation of red vinegar.
5. A red vinegar prepared by fermentation using the Aspergillus oryzae strain of any one of claims 1-2.
6. The red vinegar according to claim 5, wherein the aldehydes detected in the finished product are not less than 12; the total content of aldehydes in the red Zhejiang vinegar is not less than 1500 mug/L.
7. The red vinegar according to claim 5, wherein not less than 16 esters are detected in the finished product; the total content of esters in the red Zhejiang vinegar is not less than 700 mug/L.
8. The red vinegar according to claim 5, wherein not less than 26 kinds of sugar are detected in the finished product.
9. The red vinegar according to claim 8, wherein the total sugar content detected in the red vinegar is not less than 2000 mg/L.
10. The red vinegar according to claim 5, wherein the protease activity of Aspergillus oryzae strain in half a month of red vinegar is not less than 750U/g.
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| CN101130741A (en) * | 2007-09-19 | 2008-02-27 | 范英祝 | Vinegar koji and biological edible vinegar, and method of producing the same |
| CN105733959A (en) * | 2016-03-25 | 2016-07-06 | 浙江五味和食品有限公司 | Aspergillus oryzae strain and application thereof in Zhejiang rose vinegar fermentation |
| CN107699499A (en) * | 2017-11-16 | 2018-02-16 | 佛山市海天(高明)调味食品有限公司 | One Aspergillus oryzae ZA127 and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101130741A (en) * | 2007-09-19 | 2008-02-27 | 范英祝 | Vinegar koji and biological edible vinegar, and method of producing the same |
| CN105733959A (en) * | 2016-03-25 | 2016-07-06 | 浙江五味和食品有限公司 | Aspergillus oryzae strain and application thereof in Zhejiang rose vinegar fermentation |
| CN107699499A (en) * | 2017-11-16 | 2018-02-16 | 佛山市海天(高明)调味食品有限公司 | One Aspergillus oryzae ZA127 and its application |
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