CN112746005B - Brown mandarin orange vinegar rich in riboflavin and its production method - Google Patents

Brown mandarin orange vinegar rich in riboflavin and its production method Download PDF

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CN112746005B
CN112746005B CN202011585037.7A CN202011585037A CN112746005B CN 112746005 B CN112746005 B CN 112746005B CN 202011585037 A CN202011585037 A CN 202011585037A CN 112746005 B CN112746005 B CN 112746005B
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付彩霞
陈世贵
余红波
邹涛
陈海山
王应喜
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Hubei Tulaohan Flavouring Food Co ltd
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Abstract

The invention relates to a production method for preparing brown mandarin orange vinegar by using brown acetic acid producing bacteria, which comprises the following steps: selecting intact mandarin orange, cleaning, peeling, squeezing to obtain juice, transferring the juice to sterile fermentation tank, inoculating activated FBFS82 bacterial solution, and culturing; sterilizing after fermentation, storing the cooled FBFS fermentation liquor in a container, and sealing and storing; and (3) measuring the total acid of the orange original vinegar by adopting an acid-base titration method, preparing the orange original vinegar with FBFS fermentation liquor water, adjusting the acidity to be 5 degrees, and preparing the mandarin orange vinegar. The brown mandarin orange vinegar is rich in riboflavin, and can improve the defects of light color and strong sour irritation existing in liquid fermented vinegar.

Description

Brown orange vinegar rich in riboflavin and production method thereof
Technical Field
The invention belongs to the technical field of citrus fermentation, and relates to orange vinegar which is brown and rich in riboflavin and a production method thereof.
Background
The mandarin orange (peel) has rich nutrition, and contains carbohydrate 12.8%, protein and fat 1%, crude fiber 0.4%, and nutrients such as calcium, phosphorus, ferrum, carotene, vitamin B1, vitamin B2, vitamin C, hesperidin, citric acid, malic acid, and volatile oil. The mandarin orange is used as a main or supplementary raw material for brewing the condiment, the mandarin orange resource can be fully utilized to increase the income of farmers, the grain production cost is greatly saved, and the functional components, the aromatic components and the color in the mandarin orange endow the condiment with special nutrition, taste and flavor, and can meet the higher-level requirements of people. However, in the process of preparing vinegar by fermenting mandarin oranges, the mandarin orange vinegar has the unique mandarin orange flavor of mandarin oranges and has good taste, but the orange vinegar is light in color and luster and does not accord with the consumption habit of China.
In addition, hubei province is the main producing area of citrus in China, the planting area of citrus in 2019 is 360 ten thousand mu, and the total yield is 450 ten thousand tons, which accounts for about 10 percent of the whole country. The problem that the wenzhou mandarin oranges are used as fresh eating fruits planted mainly in Hubei province, the storage period of the fruits is short, the market period of the fresh fruits is relatively centralized, and the fruits are difficult to sell by citrus farmers is not effectively solved all the time. Meanwhile, the non-standard mandarin orange fruits accounting for 20-30% of the total yield cannot be sold, and except for part of mandarin orange can processed, no other processing, transformation and value-added channels exist, so that the increase of the income of mandarin oranges is directly influenced. Therefore, intensive studies have been required for further processing and utilization of mandarin orange.
Disclosure of Invention
In order to solve the technical problems, the invention provides orange vinegar which is brown and rich in riboflavin and a production method thereof, so as to overcome the defects of light color and strong sour stimulation existing in liquid fermented vinegar.
The scheme of the invention is as follows:
a brown and riboflavin-rich mandarin orange vinegar comprises pigment fermentation broth prepared by fermenting Gluconobacter ancient tomb FBFS82 with preservation number of CCTCCM2016387 with mandarin orange juice as substrate.
A process for the production of brown and riboflavin-rich mandarin orange vinegar, said process comprising the steps of:
(1) Preparation of FBFS82 pigment fermentation liquor: selecting intact mandarin orange, cleaning, peeling, squeezing to obtain juice, transferring to a sterile fermentation tank, inoculating activated FBFS82 bacterial liquid, fermenting and culturing to obtain FBFS82 pigment fermentation liquid;
(2) Sterilizing after fermentation is finished, storing the cooled FBFS82 pigment fermentation liquor in a container, and sealing and storing;
(3) Blending the brown mandarin orange vinegar: measuring total acid of the orange original vinegar by an acid-base titration method, mixing with FBFS82 pigment fermentation liquor according to a certain proportion, adjusting acidity, and preparing mandarin orange vinegar;
the preparation of the orange vinegar which produces brown color and is rich in riboflavin is completed.
Preferably, the mandarin orange juice in the step (1) is inoculated with activated FBFS82 bacterial liquid according to the proportion of 10-15% (v/v).
Preferably, the culture temperature in the step (1) is 20-30 ℃, and the culture lasts for 2-8 days.
Preferably, in the fermentation culture in the step (1), the citrus juice is also supplemented with 18-22g/L of carbon source and 10-15g/L of nitrogen source, and K 2 HPO 4 7-11 g/L and CaCl 2 0.04-0.08g/L。
Further preferably, the carbon source is glucose, fructose, mannose, maltose or a combination thereof, and the nitrogen source is tryptone, yeast extract, peptone or malt extract or a combination thereof.
Further preferably, the fermentation broth of FBFS82 pigment in the step (2) is cooled to 15-38 ℃.
Preferably, in the step (3), mandarin orange vinegar containing 10-25% (v/v) of FBFS82 pigment fermentation liquor is prepared, and the mandarin orange vinegar meets the quality requirement of brewed vinegar (GB 18187-2000) in China on liquid fermented vinegar.
The gluconobacter archaeofaciens FBFS82 is used for producing riboflavin by mixed fermentation, and has a preservation number of CCTCCM2016387.
The invention has the beneficial effects that:
1. the mandarin orange fermentation liquid of gluconacetobacter FBFS82 can generate brown pigment, and the mandarin orange vinegar prepared by blending the mandarin orange fermentation liquid with original mandarin orange vinegar meets the quality requirement of brewed edible vinegar (GB 18187-2000) in China on liquid fermented edible vinegar; in addition, in the fermentation process, proper carbon sources, nitrogen sources and inorganic salt components are matched, and a large amount of riboflavin can be generated, so that the color and the nutrition of the orange vinegar can be further improved, and the generated riboflavin can be purified, and the additional value of the orange juice can be further improved.
2. The fermentation liquor of the FBFS82 mandarin orange selects fructose and tryptone as a carbon source and a nitrogen source, and the fructose is 25g/L, the tryptone is 12.5g/L and the K are 2 HPO 4 9g/L and CaCl 2 0.06g/L, under which the maximum yield of riboflavin is 23.24mg/L.
3. The bacterial strain can produce brown pigment, and the generated riboflavin can be in a stable state by combining the acidic environment of the orange vinegar with the brown light-shading characteristic, so that the decomposition is avoided.
Drawings
FIG. 1 comparison of color of mandarin orange vinegar with several vinegar products;
FIG. 2 mass spectrum of riboflavin standard;
FIG. 3 is a mass spectrum of the fermentation broth;
FIG. 4 effect of fermentation time on riboflavin production.
Detailed Description
The invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the scope of the examples.
Example 1
A brown and riboflavin-rich mandarin orange vinegar comprises pigment fermentation broth obtained by fermenting Gluconobacter archaeocharis FBFS82 with preservation number of CCTCCM2016387 with mandarin orange juice as substrate.
A process for the production of brown and riboflavin-rich mandarin orange vinegar, said process comprising the steps of:
(1) Preparation of FBFS82 pigment fermentation liquor: selecting intact mandarin oranges, cleaning, peeling, juicing, transferring the mandarin orange juice to an aseptic fermentation tank, inoculating activated FBFS82 bacterial liquid, and fermenting and culturing to obtain FBFS82 pigment fermentation liquid;
(2) Sterilizing after fermentation is finished, storing the cooled FBFS82 fermentation liquor in a container, and sealing and storing;
(3) Blending of mandarin orange vinegar: measuring total acid of mandarin orange original vinegar by acid-base titration, preparing with sterile water, adjusting acidity to 5 °, and preparing mandarin orange vinegar;
the preparation of orange vinegar which is brown and rich in riboflavin is completed.
Preferably, the mandarin orange juice in the step (1) is inoculated with activated FBFS82 bacterial liquid according to 10% (v/v).
Preferably, the culture temperature in the step (1) is 28 ℃.
Preferably, in the fermentation culture in the step (1), the citrus juice is also supplemented with 18-22g/L of carbon source and 10-15g/L of nitrogen source, and K 2 HPO 4 7-11 g/L and CaCl 2 0.04-0.08g/L。
Further preferably, the carbon source is glucose, fructose, mannose, maltose or a combination thereof, and the nitrogen source is tryptone, yeast extract, peptone or malt extract or a combination thereof.
Materials and reagents involved in this example:
mandarin orange and original vinegar: the mandarin orange is produced by Yichang, and the original vinegar is prepared by peeling mandarin orange, squeezing to obtain juice, filtering, fermenting with ethanol and acetic acid, sterilizing, and packaging to obtain yellowish vinegar (6 deg.).
The flavor reaches delicious fragrant rice vinegar: jinan Yihe food Co., ltd; wangfushan apple vinegar: economic city Yuanxing industry Co., ltd; zilin 9 ° rice vinegar: shanxi Zilin Vinegar industry Co., ltd; constant 9 ° rice vinegar: jiangsu Hengshun Vinegar industry GmbH; zilin 9 ° rice vinegar: shanxi Zilin Vinegar industry Co.
1. Preparation of pigment fermentation liquor
Peeling mandarin orange, juicing, inoculating 10% (v/v) activated FBFS82 bacterial solution, and fermenting at 28 deg.C in sterile 10L fermenter for 6 days. During the fermentation, the sugar degree and acidity of the fermentation liquor are monitored. Filtering with multilayer gauze, sterilizing, storing the fermentation liquid in a sealed container, and storing at room temperature.
2. Blending of mandarin orange vinegar
And (4) measuring the total acid of the original vinegar by adopting an acid-base titration method. Blending mandarin orange vinegar with sterile water, raw vinegar and pigment fermentation liquid, wherein the acidity of the mixed vinegar is 5 °, the addition ratio of the fermentation liquid (v/v) is 10%, 15%, 20% and 25%, respectively, and the addition ratio is respectively represented by mandarin orange vinegar A, B, C and D. And placing the prepared mandarin orange vinegar into a transparent glass bottle, and storing at room temperature.
3. Detection method
1) Sensory characteristics of mandarin orange vinegar
The sensory characteristics of the mandarin orange vinegar are evaluated by referring to the national standard (GB 18187-2000) for brewing edible vinegar in China. The color of mandarin orange vinegar was compared with commercially available liquid fermented vinegar from 5 different production areas.
2) Color stability
The absorbance value (OD) of the pigment at 440nm was measured at intervals of 10d during the storage of mandarin orange vinegar 440 ) The stability of the dye was evaluated.
3) Qualitative and quantitative determination of riboflavin
Dionex Ultimate 3,000 high resolution ultra high performance liquid chromatography was used in conjunction with Q active quadrupole and orbital trap hybrid mass spectrometry. The operating conditions are as follows: 5mL of the fermentation broth was centrifuged and filtered through a 0.22 μm membrane, and 20 μ L of the filtrate was passed through a C18 polar-end-capped LC column (150 mm. Times.2.1 mm,2.6 μm) at a column temperature of 25 ℃ and a flow rate of 0.2mL/min, with 30% methanol aqueous solution and 0.1% formic acid aqueous solution as mobile phases, and analyzed at a wavelength of 270nm. The identification of riboflavin uses electrospray positive ion mode mass spectrometry, and the quantification of riboflavin is based on retention time and standards.
4. Results of the experiment
4.1 Physical and chemical index analysis of FBFS82 pigment fermentation liquor
The results of the detection and analysis of physical and chemical indexes such as pH, total sugar and total acid in the FBFS82 fermentation process are shown in Table 1. The physicochemical indexes in the 2 nd day are obviously changed, and the total sugar content is reduced from 10 percent to about 7 percent; total acid from 0.58g to 100mL -1 Increased to about 1.37 g.100 mL -1 . The mandarin orange juice is acidic, has pH of about 4.0, and gradually accumulates acid with fermentation, and decreases to 2.17.
TABLE 1 physical and chemical index detection of FBFS fermentation broth
Fermentation time (d) Total sugar (g 100 mL) -1 ) pH Acidity (g.100 mL) -1 )
0 10 4.01 0.58
2 8 2.44 1.47
4 7.33 2.41 1.39
6 7 2.45 1.23
7 7 2.17 1.37
4.2 organoleptic Properties of Mandarin orange Vinegar
The original mandarin orange vinegar is light yellow, has low taste, and has obvious pungent smell of acetic acid. After the orange vinegar is blended with FBFS82 fermentation liquor, the color of the orange vinegar is obviously improved, and the color is shown in figure 1. As can be seen from FIG. 1, from A to D, the color of the mandarin orange vinegar gradually darkens, transitioning from amber to reddish brown. Compared with the 5 types of liquid fermented vinegar sold in the market, the mandarin orange vinegar has bright and fresh color and luster and is more attractive. Meanwhile, the mandarin orange vinegar has richer taste and smell. From the mandarin orange vinegar B, the mandarin orange vinegar has obvious sauce flavor, soft sour taste, and no foreign odor. The improvement of the organoleptic properties is more obvious with the increase of the content of the fermentation liquor of the FBFS82 pigment.
4.3 Identification of riboflavin production by FBFS82
The method of this example 1 is adopted, UHPLC-MS/MS is adopted to analyze riboflavin in a riboflavin standard and FBFS82 pigment fermentation liquor, electrospray positive ion mode mass spectrometry is adopted for identification of riboflavin, and the results are shown in FIGS. 2 and 3.
As can be seen from fig. 2 and 3, the MS/MS analysis of the riboflavin peak identified in FBFS82 fermentation broth showed that the [ M + H ] + ion peak mass-to-charge ratio was 377.14551, and the [ M + H ] + ion peak mass-to-charge ratio of the riboflavin standard was 377.14563, indicating that riboflavin was present in FBFS82 fermentation broth.
4.4 Effect of fermentation time on Riboflavin production
In the method of this example 1, in the fermentation culture of step (1), 18g/L carbon source (glucose) and 10g/L nitrogen source (yeast extract) and K are also added to the citrus juice 2 HPO 4 7 g/L and CaCl 2 0.04g/L. The DO value and the riboflavin content of the FBFS82 pigment fermentation liquor are measured by sampling every 8h during the fermentation culture process in the step (1), and the result is shown in a graph 4. As can be seen from FIG. 4, the cell concentration was stable after 24 hours of culture, and after 40 hours of culture, the cell concentration was slightly decreased and remained stable. In the first 24h of culture, almost no riboflavin is synthesized, the riboflavin content is obviously increased after 32h of culture, the growth of the thalli tends to be stable, the riboflavin content reaches the maximum value of 0.33mg/L after 64h of culture, and then the riboflavin content is reduced.
4.5 FBFS82 riboflavin production response surface optimization
4.5.1 Effect of carbon sources on Riboflavin production by FBFS82
Glucose, fructose, mannose and maltose, 4 different carbon sources were selected based on the method of example 1, the addition amount was 12mg/L, and after culturing at 28 ℃ and 180r/min for 3d (72 h), the riboflavin content in the culture broth was examined to analyze the influence of the different carbon sources on riboflavin production by FBFS82, and the results are shown in Table 2.
TABLE 2 influence of carbon sources on the riboflavin production of FBFS82
Carbon source Riboflavin yield (mg/L)
Glucose 0.033±0.01
Fructose 5.31±0.33
Maltose 1.2±0.05
Mannose 3.96±0.01
As shown in Table 2, FBFS82 can produce riboflavin from glucose, fructose, maltose and mannose, and riboflavin is produced at the highest yield of 5.31mg/L when fructose is used as the sole carbon source, followed by mannose and maltose.
4.5.2 Effect of Nitrogen Source on Riboflavin production by FBFS82
In addition to the method of example 1, tryptone, yeast extract, peptone, malt extract, 4 different nitrogen sources were selected, the addition amount was 8mg/L, and after culturing at 28 ℃ and 180r/min for 3d, the riboflavin content in the culture broth was examined to analyze the influence of different nitrogen sources on riboflavin production by FBFS82, and the results are shown in Table 3.
TABLE 3 influence of nitrogen sources on riboflavin production by FBFS82
Nitrogen source Riboflavin yield (mg/L)
Tryptone 5.173±0.025
Yeast cream 4.867±0.076
Peptone 2.915±0.210
Malt extract 1.713±0.017
As shown in Table 3, FBFS82 can produce riboflavin from tryptone, yeast extract, peptone and malt extract, and the yield of riboflavin is highest at 5.173mg/L when tryptone is used as the only nitrogen source, and then yeast extract and peptone are used.
4.5.3 Central composite design (Central composite design) optimization of fermentation conditions for producing riboflavin by FBFS82 on the basis of single-factor and principal component analysis experiments, fructose, tryptone and K are selected 2 HPO 4 And CaCl 2 The central composite experiment was designed for four factors and the results are shown in table 4.
TABLE 4 center composite design experimental design and results
Figure BDA0002865424410000071
Figure BDA0002865424410000081
The Design-expert 8.06 software was used to perform multiple regression fitting on the table 4 data and to perform analysis of variance on the model and related parameters, the results are shown in table 5.
TABLE 5 regression model analysis of variance
Figure BDA0002865424410000082
Note: * Significantly, p <0.05, very significant, p <0.01.
As can be seen from Table 5, the model is extremely remarkable (p)<0.0001 ); coefficient of determination R of the model 2 Is 0.9461, which shows that the regression equation has better fitting degree with the reality, and the model can explain the yield change of the riboflavin of 94.61 percent; the mismatching term is 0.1286 which is more than 0.05, and is not significant, which shows that the regression model is very consistent with the actual value. In the independent item, fructose and tryptone have very significant influence on the production of riboflavin by FBFS82 liquid fermentation (p)<0.01 ); among the interactive items, fructose and K 2 HPO4, tryptone and K 2 HPO4 interaction is extremely significant (p)<0.01 ); in the second order, fructose, tryptone and K 2 HPO4 has a very significant influence on the production of riboflavin by FBFS82 liquid fermentation (p)<0.01)。
The optimal conditions and yield for producing riboflavin by FBFS82 liquid fermentation are predicted by a quadratic regression equation as follows: fructose 20g/L, tryptone 12.5g/L, K 2 HPO 4 9g/L and CaCl 2 0.06g/L, under which the predicted yield of riboflavin was 23.2mg/L. The FBFS82 is cultured under the optimal condition, the temperature is 28 ℃, the culture time is 3d, the yield of the riboflavin reaches 23.24mg/L, and the verification value is close to the predicted value, which indicates that the model established by the response surface is stable and reliable.
Example 2
A brown and riboflavin-rich mandarin orange vinegar comprises pigment fermentation broth prepared by fermenting Gluconobacter ancient tomb FBFS82 with preservation number of CCTCCM2016387 with mandarin orange juice as substrate.
A process for the production of brown and riboflavin-rich mandarin orange vinegar, said process comprising the steps of: the method comprises the following steps:
(1) Preparation of FBFS82 pigment fermentation liquor: selecting intact mandarin orange, cleaning, peeling, squeezing to obtain juice, transferring to sterile fermentation tank, inoculating activated FBFS82 bacterial solution, and fermenting;
(2) Sterilizing after fermentation, storing the cooled FBFS fermentation liquor in a container, and sealing and storing;
(3) Blending mandarin orange vinegar: measuring total acid of mandarin orange original vinegar by acid-base titration, preparing with sterile water, adjusting acidity to 5 °, and preparing mandarin orange vinegar;
the preparation of the orange vinegar which produces brown color and is rich in riboflavin is completed.
Preferably, the activated FBFS82 bacterial liquid is inoculated to the citrus juice in the step (1) according to 10% (v/v).
Preferably, the culture temperature in the step (1) is 28 ℃, and the culture is carried out for 3 days.
Preferably, in the fermentation culture in the step (1), a carbon source of fructose is supplemented by 20g/L, a nitrogen source of tryptone is supplemented by 12.5g/L, and K is added 2 HPO 4 9g/L and CaCl 2 0.06g/L。
Comparative example 1
The strain was changed, and other conditions were changed to those of example 2. The riboflavin production was measured as shown in Table 6.
TABLE 6 influence of the Strain species on Riboflavin production
Bacterial strains Riboflavin yield mg/L
FBFS82 23.24
Lactobacillus fermentum GKJFE 3.49
Lactobacillus plantarum NCDO1752 0.6
Leuconostoc lactis NZ9000 of Cryptococcus lactis 0.7
As can be seen from Table 6, the strain using FBFS82 produced riboflavin in much higher yields than the other strains.
Comparative example 2
The amounts of the carbon source and the nitrogen source were changed, and the conditions were changed in example 2. The riboflavin production was measured as in Table 7.
TABLE 7 Effect of different nutritive salts on riboflavin production
Figure BDA0002865424410000101
As can be seen from Table 7, in selecting a suitable strain of FBFS82, a suitable carbon source and nitrogen source is selected based on citrus juice to promote the mass production of riboflavin.
Example 3
In addition to the example 2, chitosan is added in an amount of 0.5% of the mass of the mandarin orange juice in the fermentation culture process in the step (1), and the method is otherwise the same as the example 2. The riboflavin production was measured as shown in Table 8.
TABLE 8 Effect of different nutritive salts on riboflavin product
Nutrient salt Riboflavin yield mg/L
Example 2 23.24
Example 3 28.91
As can be seen from table 8, the yield of riboflavin can be further increased by adding a certain amount of chitosan, which may be because, in the existing riboflavin fermentation process, the pH is generally controlled to be about 6.7-7, in the citrus tangerines juice fermentation process of the present application, the pH of the system is lower and lower, and the production of riboflavin is not facilitated under the low acid condition.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims (4)

1. A production method of orange vinegar which is brown and rich in riboflavin is characterized in that: the mandarin orange vinegar comprises brown fermentation liquid rich in riboflavin prepared by fermenting gluconobacter archaeocharitis FBFS82 with the preservation number of CCTCCM2016387 with mandarin orange juice as substrate; the method comprises the following steps:
(1) Preparation of FBFS82 pigment fermentation liquor: selecting intact mandarin orange, cleaning, peeling, squeezing to obtain juice, transferring to sterile fermentation tank, inoculating activated FBFS82 bacterial solution, and fermenting to obtain FBFS82 pigment fermentation broth;
(2) Sterilizing after fermentation is finished, storing the cooled FBFS82 pigment fermentation liquor in a container, and sealing and storing;
(3) Blending the brown mandarin orange vinegar: measuring total acid of the orange original vinegar by an acid-base titration method, mixing with FBFS82 pigment fermentation liquor according to a certain proportion, adjusting acidity, and preparing mandarin orange vinegar;
completing the preparation of the honey orange vinegar which produces brown color and is rich in riboflavin;
inoculating activated FBFS82 bacterial liquid in the mandarin orange juice of the step (1) according to 10% -15%;
the culture temperature in the step (1) is 20-30 ℃, and the culture lasts for 2-8 days;
in the fermentation culture in the step (1), the citrus juice is also supplemented with 18-22g/L of fructose, 10-15g/L of tryptone and K 2 HPO 4 7-11 g/L and CaCl 2 0.04-0.08 g/L。
2. The method of claim 1, wherein the FBFS82 pigment fermentation broth of step (2) is cooled to 15-38 ℃.
3. The method as claimed in claim 1, wherein the step (3) is to prepare mandarin orange vinegar containing 10% -25% FBFS82 pigment fermentation broth, which meets the quality requirements of brewed vinegar in China for liquid fermented vinegar.
4. Use of gluconobacter archaeofaciens FBFS82 with the accession number CCTCCM2016387 for the mixed fermentation production of riboflavin by the method of any one of claims 1 to 3.
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CN107904126B (en) * 2017-11-30 2021-03-19 湖北土老憨调味食品股份有限公司 Multi-strain brewed citrus type aromatic ester vinegar based on tangerine concentrated juice and preparation method thereof
CN110205232A (en) * 2019-06-14 2019-09-06 河南圆梦生物工程有限公司 A kind of date-wolfberry fruit health preserving vinegar and preparation method thereof
CN112322443B (en) * 2020-11-30 2022-12-02 华中农业大学 Orange vinegar with dark color and rich gamma-aminobutyric acid, and preparation method and application thereof

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