CN104694371A - Citrus fruit vinegar prepared by composite strain mixed fermentation and preparation method thereof - Google Patents

Abstract

The invention discloses citrus fruit vinegar prepared by mixed fermentation of compound strains, and belongs to the technical field of fruit vinegar brewing. It is characterized in that its preparation method is as follows: 1) preparation of compound yeast strain seed liquid; 2) preparation of compound acetic acid strain seed liquid; 3) preparation of glutinous rice saccharification liquid; 4) preparation of citrus juice; 5) preparation of fermentation liquid 6) Alcoholic fermentation; 7) Acetic acid fermentation; 8) Aging at room temperature for 2-3 months, filtering and clarifying with diatomaceous earth to obtain the finished product of citrus fruit vinegar. According to the invention, citrus and glutinous rice are used as raw materials, fruit grains are co-brewed, and compound strains are mixed and fermented, so that the prepared tangerine fruit vinegar is rich in nutrition, has a mellow taste, pleasant fruity aroma and no bitterness.

Description

A kind of citrus fruit vinegar prepared by mixed fermentation of compound strains and preparation method thereof

technical field

The invention belongs to the technical field of fruit vinegar brewing, and in particular relates to a citrus fruit vinegar prepared by mixed fermentation of complex strains and a preparation method thereof.

Background technique

Citrus fruit vinegar is made from fresh and ripe citrus fruits, which are produced by peeling, juicing, separation, debittering, alcoholic fermentation, acetic acid fermentation, aging, and sterilization. It is rich in organic acids and vitamins, have health effects such as relieving heat and thirst, eliminating fatigue, beautifying and beautifying, increasing appetite, relieving alcohol and protecting the liver, antiseptic and sterilization, etc. The curative and preventive effect, is a new generation of fermented food. Safe and efficient production of citrus fruit vinegar with strong fruity aroma and unique flavor is the key to the healthy development of its industrialization. Fruit vinegar needs to have unique flavor and nutritional components, which puts forward new requirements for strain selection, domestication and production technology suitable for brewing citrus fruit vinegar. At present, most of the strains used by fruit vinegar producers in my country are single Saccharomyces cerevisiae and vinegar strains. The flavor and quality of the products produced are not good, the yield rate is low, and the investment in equipment is relatively large. Regarding fruit vinegar production technology, there are two kinds of liquid state fermentation and solid state fermentation. Compared with the liquid fermentation method, the solid-state fermentation method has the advantages of good flavor and good taste, but there are disadvantages such as long fermentation period, high labor intensity, difficult control of the fermentation process, and easy contamination by bacteria. The advantage of the liquid fermentation method is that the fermentation cycle is short, the labor intensity is small, the fermentation process is easy to control, and the chance of contamination by bacteria is reduced. However, because it is a purebred culture and the fermentation time is limited, the flavor substances formed by the liquid fermentation method are few. Its flavor and mouthfeel are inferior to those of solid-state fermentation. At present, there is no very satisfactory method to produce fruit vinegar, and the production technology of perfecting fruit vinegar is still in the stage of exploration.

Contents of the invention

Aiming at the technical problems existing in the background technology, the object of the present invention is to provide a kind of citrus fruit vinegar prepared by mixed fermentation of complex strains and a preparation method thereof. Using citrus and glutinous rice as raw materials, after peeling, juicing, beating, and saccharification, they are mixed, and compound yeast strains and compound acetic acid strains are added. After one alcoholic fermentation and one acetic acid fermentation, the fruity aroma is produced safely and efficiently. , Citrus fruit vinegar with unique flavor, rich in amino acids, flavonoids, vitamin C and other nutrients.

The adopted technical scheme is as follows:

A kind of citrus fruit vinegar prepared by mixed fermentation of compound strains is characterized in that its preparation method is as follows:

1) Preparation of compound yeast seed liquid: the liquid volume of the yeast activation medium is 150mL/250mL, and two baits of yeast 31906, S. Transfer the culture solution, place it on a shaker at 28°C, and incubate at 180r/min for 16 hours to obtain the primary culture solution, adjust the bacterial cell concentration to 10 ⁷ cells/mL; add the four primary culture solutions to the malt according to the volume percentage as follows In the culture medium: 4% Hansenula anomaly variant 1431, 3% Saccharomyces cerevisiae 1425, 3% Saccharomyces cerevisiae 31906, 2% Saccharomyces cerevisiae 1306, and then placed in a shaker at 28°C and 180r/min for 16 hours, so that When the number of bacteria reaches 10 ⁷ -10 ⁸ /mL, it is the compound yeast seed liquid.

2) Preparation of compound acetic acid bacteria seed solution: the volume of acetic acid bacteria activation medium is 30mL/150mL, and two rings of apple acetobacter ZY.C4, Acetobacter pasteurianus Rowan subsp. and Huyao 1.01 thick bacteria were respectively transferred into the culture medium, placed on a shaker at 31°C, and cultured at 150r/min for 48 hours to obtain a primary culture medium, and the concentration of bacteria was adjusted to 10 ⁶ cells/mL; four kinds of primary culture The solution is added to citrus fruit wine with an alcohol content of 6-7% according to the following volume percentages: 4% Apple Acetobacter ZY.C4, 3% Acetobacter pasteurii subsp. 2% Shanghai brewed 1.01; then placed in a shaker at 30°C, 150r/min and cultured for 24 hours, so that the number of bacteria reached 10 ⁶ -10 ⁸ cells/mL.

3) Preparation of glutinous rice saccharification liquid: Rinse the rice twice, soak for 2-4 hours; refine the pulp according to the weight ratio of water: rice = 2.5:1, adjust the pulp to a ratio of 4:1, add CaCl ₂ and MgCl ₂ , the CaCl ₂ and The amount of MgCl ₂ added is 1g per 100mL slurry, adjust the pH to 6.2-6.4 with acetic acid and sodium carbonate, add liquefaction enzyme, the amount of liquefaction enzyme added is 100mL slurry, add 1g, heat to 90-95°C to liquefy for about 20- After 30 minutes, cool down to 60°C and adjust the pH to 4.5-5.0 to obtain liquid A, which is saccharified to obtain glutinous rice saccharification liquid;

The saccharification method is as follows: add glucoamylase and malt powder, the addition amount is 1g glucoamylase and 10g malt powder per 100mL of liquid A, keep warm and saccharify for 6-8h; or add black koji: red koji = 3:1 mixed koji, The amount of mixed koji added is 100mL of liquid A, and the saccharification time is 2-4h.

4) Preparation of citrus juice: select non-rotten citrus, peel the citrus pulp into a juice extractor, and add 0.01% (by weight) of _NaHSO solution, citrus pulp: _NaHSO solution=1 : 1 (weight ratio); squeeze the juice, add pectinase to the juice for enzymolysis, the amount of pectinase added is 0.04g of pectinase per 100mL of juice, the enzymolysis temperature is 60°C, and the enzymolysis time is 60min , after enzymolysis, filter through 4 layers of gauze to obtain citrus juice; as a preference, add malt powder before enzymolysis, and the amount of addition is 10g of malt powder per 100mL of juice.

5) Fermentation broth preparation: Mix the above-prepared citrus juice and glutinous rice saccharification solution at a volume ratio of 3:1, add sulfur dioxide to the mixed solution at a ratio of 30 mg of sulfur dioxide and 50 mg of ammonium sulfate per 1 L of the mixed solution Mix with ammonium sulfate, adjust the pH value to 4.48, adjust the sugar content to 15-19°Bx′, sterilize in hot water at 85°C for 15 minutes, cool down for later use, and obtain a fermented liquid.

6) Alcoholic fermentation: Add the compound yeast seed liquid into the fermentation liquid in step 5), the inoculum amount is 9.7-10% (volume percentage), stir at 28-28.5°C, and the rotation speed is 180r/min After 1 day of fermentation, airtight After statically fermenting for 4-5 days, the citrus fruit wine is obtained by sterilization. As preferably, also add naringinase before fermentation, the addition amount of naringinase is to add 0.04g in every 100mL fermented liquid.

7) Acetic acid fermentation: adjust the alcohol content of citrus fruit wine to 6-7%, insert the compound acetic acid bacteria seed solution, the inoculum amount is 15.2-16% (volume percentage), stir and ferment at the speed of 180r/min, 31°C 5-7d; stop the fermentation, and pass through steam at 80-100°C for 30 minutes to sterilize.

8) Add table salt to the liquid obtained in 7), the amount of table salt added is 20g of table salt per 1L, age at room temperature for 2-3 months, filter and clarify with diatomaceous earth, and obtain the finished product of citrus fruit vinegar.

The citrus fruit vinegar prepared according to the above-mentioned technological process and control conditions is rich in nutrition. The fruit vinegar has a moderate sour taste, a mellow taste, a pleasant fruity aroma, and no bitterness. It is especially rich in amino acids and various antioxidants needed by human health. Oxygenated ingredients, in addition, if properly blended and diluted, can also be developed into a popular drink. In addition, the use of compound bacteria to brew fruit vinegar has a fast propagation speed, and can produce acid with high acidity in the early stage of fermentation. At the same time, the acid production speed is fast, and the maximum acidity is reached on the fifth day of fermentation. The fermentation time is 3-4 days shorter than that of a single strain of bacteria. Therefore, the use of this process can reduce the contamination of bacteria, shorten the production cycle and stabilize the product quality.

Due to the adoption of apple vinegar bacterium ZY.C4 in the present invention, the total acid, amino nitrogen, total ester, VC, flavonoids and other fruit vinegar flavor substances and nutritional components of the prepared citrus fruit vinegar are higher than those using bacteria containing AS1.41 The fruit vinegar brewed by the complex strain of acetic acid bacteria can reduce the limonoid substances in the citrus fruit vinegar products by 70-75%, making the products soft and free of bitterness.

Detailed ways

Below in conjunction with embodiment the present invention is described in further detail.

The bacterial classification involved in the embodiment and substratum are as follows:

Yeast strains: Saccharomyces 31906, Saccharomyces cerevisiae 1306, Saccharomyces cerevisiae 1425, Hansenula anomaly var. 1431. All the above strains were purchased from China Industrial Microbiology Culture Collection Management Center.

Acetic acid bacteria strains: Shanghai Niang 1.01, Acetobacter pasteuriani subsp. Rowan subsp. ( Acetobacter malorum ) ZY.C4, obtained by self-isolation, has been preserved, preservation unit: General Microbiology Center of China Committee for Microbial Culture Collection (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), date of deposit: January 07, 2014, deposit number: CGMCC NO: 8692.

Yeast Activation Medium: Mix 5 °Be′ malt juice agar and treated glutinous rice citrus culture solution at a ratio of 2:1, autoclave at 121°C for 20 minutes, and cool for later use.

Acetic acid bacteria activation medium: glucose 1g, yeast extract 1g, alcohol (95%) 5mL, water 100mL, pH natural. Alcohol is added after sterilization.

Embodiment 1: the preparation technology one of citrus fruit vinegar

1. Material preparation process

(1) Preparation of compound yeast seed liquid: the volume of yeast activation medium is 150mL/250mL, and two bait yeasts 31906, Saccharomyces cerevisiae 1306, Saccharomyces cerevisiae 1425, and Hansenula anomaly var. Transfer the culture solution respectively, place it on a shaker at 28°C, and incubate at ^180r /min for 16 hours to obtain the first-level culture solution. In the malt medium: 4% Hansenula anomaly variant 1431, 3% Saccharomyces cerevisiae 1425, 3% Saccharomyces cerevisiae 31906, 2% Saccharomyces cerevisiae 1306, and then placed in a shaker at 28°C, 180r/min and cultured for 16h, Make the number of bacteria reach 10 ⁷ -10 ⁸ /mL, which is the compound yeast seed liquid.

(2) Preparation of compound acetic acid bacteria seed solution: the volume of acetic acid bacteria activation medium is 30mL/150mL, and two rings of apple acetobacter ZY.C4, Acetobacter pasteuriani subsp. 22518 and Huyao 1.01 thick bacteria were respectively transferred into the culture medium, placed on a shaker at 31°C, 150r/min and cultivated for 48 hours to obtain the first-grade culture medium, and the concentration of bacteria was adjusted to 10 ⁶ cells/mL; the four kinds of first-grade The culture solution is added to citrus fruit wine with an alcohol content of 6-7% according to the following volume percentages: 4% Apple Acetobacter ZY.C4, 3% Acetobacter pasteurii subsp. , 2% Shanghai Brewing 1.01; then placed in a shaker at 30°C, 150r/min for 24 hours, so that the number of cells reached 10 ⁶ -10 ⁸ cells/mL.

(3) Preparation of glutinous rice saccharification liquid: Rinse the rice twice, soak for 2-4 hours; grind the pulp according to the weight ratio of water: rice = 2.5:1, adjust the pulp to a ratio of 4:1, add CaCl ₂ and MgCl ₂ , the CaCl ₂ The amount of MgCl2 and _MgCl2 added is 1g per 100mL slurry, adjust the pH to 6.2-6.4 with acetic acid and sodium carbonate, add liquefaction enzyme, the amount of liquefaction enzyme added is 100mL slurry, add 1g, heat to 90-95°C to liquefy for about 20 -30min, lower the temperature to 60°C, adjust the pH to 4.5-5.0, add glucoamylase and malt powder in an amount of 1g glucoamylase and 10g malt powder per 100mL of liquid, keep saccharification for 6-8h to obtain glutinous rice saccharification liquid.

(4) Preparation of citrus juice: select non-rotten citrus, peel the citrus pulp into a juice extractor, and add 0.01% (by weight) of NaHSO ₃ solution, citrus pulp: NaHSO ₃ solution = 1:1 (weight ratio); squeeze the juice, add pectinase and malt powder to the juice, the amount of pectinase added is 0.04g of pectinase per 100mL of juice, and the amount of malt powder added is per 100mL of juice Malt powder 10g. The enzymolysis temperature is 60°C, and the enzymolysis time is 60 minutes. After enzymolysis, 4 layers of gauze are filtered to obtain citrus juice.

(5) Fermentation broth preparation: Mix the above-prepared citrus juice and glutinous rice saccharification solution at a volume ratio of 3:1, and add 30 mg of sulfur dioxide and 50 mg of ammonium sulfate to the mixed solution per 1 L of the mixed solution. Mix sulfur dioxide and ammonium sulfate, adjust the pH value to 4.48, adjust the sugar content to 15-19°Bx', sterilize in hot water at 85°C for 15 minutes, cool down for later use, and obtain a fermentation broth.

2. Alcoholic fermentation (first fermentation)

Add 9.7% compound yeast seed liquid to the above-mentioned fermentation broth (filling volume is 4/5 of the volume), stir at 28.5°C (180r/min) and ferment for 1d, then keep it in a sealed container for fermentation for 4-5d, and test the fermentation When the alcohol content in the liquid is 10%-12% V/V, the fermentation is terminated, and after filtration, it is sterilized by steam at 80-100°C for 30 minutes.

3. Acetic acid fermentation (second fermentation)

Adjust the alcohol content of the above-mentioned fruit wine to 6-7%, put it into the acetic acid fermentation tank according to the liquid content of 30%, insert the compound acetic acid bacteria seed liquid according to 15.2%, and then add 0.04% of naringinase (that is, every 100mL Add 0.04g to citrus fruit wine, stir at 31°C (180r/min) and ferment for 5-7d, when the residual alcohol is detected to be 0.3%-0.5%, stop the fermentation, and pass steam at 80-100°C for 30min .

4. Aging, filtering and clarification

Add 2% salt to the fruit vinegar that has completed acetic acid fermentation (that is, add 20g of salt per 1L), store it in a clean and airtight tank, and age it at room temperature for 2 months. The diatomaceous earth is used as a filter medium to filter and clarify the vinegar fermented grains to obtain a finished product of citrus fruit vinegar.

Embodiment 2: the preparation process two of citrus fruit vinegar

1. Material preparation process

(1) Preparation of compound yeast seed liquid: the volume of yeast activation medium is 150mL/250mL, and two bait yeasts 31906, Saccharomyces cerevisiae 1306, Saccharomyces cerevisiae 1425, and Hansenula anomaly var. Transfer the culture solution respectively, place it on a shaker at 28°C, and incubate at ^180r /min for 16 hours to obtain the first-level culture solution. In the malt medium: 4% Hansenula anomaly variant 1431, 3% Saccharomyces cerevisiae 1425, 3% Saccharomyces cerevisiae 31906, 2% Saccharomyces cerevisiae 1306, and then placed in a shaker at 28°C, 180r/min and cultured for 16h, Make the number of bacteria reach 10 ⁷ -10 ⁸ /mL, which is the compound yeast seed liquid.

(2) Preparation of compound acetic acid bacteria seed solution: the volume of acetic acid bacteria activation medium is 30mL/150mL, and two rings of apple acetobacter ZY.C4, Acetobacter pasteuriani subsp. 22518 and Huyao 1.01 thick bacteria were respectively transferred into the culture medium, placed on a shaker at 31°C, 150r/min and cultivated for 48 hours to obtain the first-grade culture medium, and the concentration of bacteria was adjusted to 10 ⁶ cells/mL; the four kinds of first-grade The culture solution is added to citrus fruit wine with an alcohol content of 6-7% according to the following volume percentages: 4% Apple Acetobacter ZY.C4, 3% Acetobacter pasteurii subsp. , 2% Shanghai Brewing 1.01; then placed in a shaker at 30°C, 150r/min for 24 hours, so that the number of cells reached 10 ⁶ -10 ⁸ cells/mL.

(3) Preparation of glutinous rice saccharification liquid: Rinse the rice twice, soak for 2-4 hours; grind the pulp according to the weight ratio of water: rice = 2.5:1, adjust the pulp to a ratio of 4:1, add CaCl ₂ and MgCl ₂ , the CaCl ₂ The amount of MgCl2 and _MgCl2 added is 1g per 100mL slurry, adjust the pH to 6.2-6.4 with acetic acid and sodium carbonate, add liquefaction enzyme, the amount of liquefaction enzyme added is 100mL slurry, add 1g, heat to 90-95°C to liquefy for about 20 -30min, cool down to 60°C, adjust pH to 4.5-5.0, add black koji: red koji = 3:1 mixed koji, add 1g of mixed koji to 100mL liquid, saccharify for 2-4h; get glutinous rice saccharification liquid .

(4) Preparation of citrus juice: select non-rotten citrus, peel the citrus pulp into a juice extractor, and add 0.01% (by weight) of NaHSO ₃ solution, citrus pulp: NaHSO ₃ solution = 1:1 (weight ratio); squeeze the juice, add pectinase and malt powder to the juice, the amount of pectinase added is 0.04g of pectinase per 100mL of juice, and the amount of malt powder added is per 100mL of juice Malt powder 10g. The enzymolysis temperature is 60°C, and the enzymolysis time is 60 minutes. After enzymolysis, 4 layers of gauze are filtered to obtain citrus juice.

(5) Fermentation broth preparation: Mix the above-prepared citrus juice and glutinous rice saccharification solution at a volume ratio of 3:1, and add 30 mg of sulfur dioxide and 50 mg of ammonium sulfate to the mixed solution per 1 L of the mixed solution. Mix sulfur dioxide and ammonium sulfate, adjust the pH value to 4.48, adjust the sugar content to 15°Bx′, sterilize in hot water at 85°C for 15 minutes to kill miscellaneous bacteria, and cool down for later use.

2. Alcoholic fermentation (first fermentation)

Add 10% compound yeast seed liquid to the above-mentioned fermentation broth (filling volume is 4/5 of the volume), stir at 28°C (180r/min) and ferment for 1d, then leave it to ferment for 6-7d in a sealed container, and test the fermentation When the alcohol content in the liquid is 10%-12% V/V, the fermentation is terminated, and after filtration, it is sterilized by steam at 80-100°C for 30 minutes.

3. Acetic acid fermentation (second fermentation)

Adjust the alcohol content of the above-mentioned fruit wine to 6-7%, put it into the acetic acid fermentation tank according to the liquid content of 30%, insert the seed liquid of compound acetic acid bacteria at 16%, and stir at 32°C (180r/min) for fermentation After 5-7 days, when the residual alcohol content is detected to be 0.3%-0.5%, the fermentation is terminated, and the steam is sterilized at 80-100°C for 30 minutes.

4. Aging, filtering and clarification

Add 2% salt to the fruit vinegar that has completed the acetic acid fermentation, store it in a clean and airtight tank, and age it at room temperature for 3 months. The diatomaceous earth is used as a filter medium to filter and clarify the vinegar fermented grains to obtain a finished product of citrus fruit vinegar.

Example 3: Isolation and Identification of Apple Acetobacter ZY.C4

(1) Bacterial source preparation Commercially purchased citrus without rot, washed, beaten, and subjected to natural alcoholic fermentation and natural acetic acid fermentation. Measure its sugar content with a sugar meter every day, when the sugar content drops to 5 ° BX, and does not change for a week, start to filter. Filter with a double layer of boiled 80 mesh gauze, and store the filtrate in a stainless steel pot after filtering, that is, citrus wine. Cover the surface of the pot with gauze, and carry out natural fermentation at 32°C to obtain naturally fermented vinegar liquid. After 5 days, the bacteria source was taken from the vinegar solution for screening and isolation.

(2) Separation and screening process

Bacteria source—proliferation culture—dilution plate separation—preliminary screening—incline culture—liquid Erlenmeyer flask culture—confirmation of primary screening strains—preservation strains on slant—re-screening—confirmation of excellent acid-producing strains—incline Preserved strains

(3) Primary screening method

Proliferation culture: Take 10mL of fermented vinegar solution of natural acetic acid, put it into 90mL of proliferation medium, and culture in a constant temperature shaker (32°C, 120r/min) for 2 days.

Calcium plate separation experiment: Gradiently dilute the proliferation culture solution for coating and separation, culture it in a 32°C incubator for 2 days, observe the results, select the one with a large calcium dissolution zone, and transfer a single colony with different colony forms into the slant medium. Repeat 3-4 times to obtain purified strains.

(4) Re-screening

Pick two rings of the strains isolated on the calcium plate, transfer them into 20mL liquid basal medium (plus 6% absolute ethanol), culture them in a constant temperature shaker (32°C, 120r/min) for 7 days, and perform quantitative determination of acetic acid. According to the results of acidity determination, the excellent strains with high acid production were screened out.

The excellent strain with high acid production was identified as Acetobacter malorum ZY.C4.

The medium formula used for above-mentioned isolation culture is as follows:

Proliferation medium: yeast powder 1%, glucose 1%, 0.02% crystal violet 0.5%, nystatin 2.5×l0 ⁶ units/L, pH 5.5, add 3% (v/v) 95% absolute ethanol .

Isolation medium: yeast powder 1%, glucose 1%, CaCO ₃ 1.5%, agar 2%, pH 5.5, add 4% (v/v) 95% absolute ethanol and dry heat sterilized CaCO ₃ .

Incline medium: 1% yeast powder, 1% glucose, 1.5% CaCO ₃ , 2% agar, pH 5.5, aliquot into test tubes, add 4% (v/v) 95% absolute ethanol.

Liquid basal medium: yeast powder 1%, glucose 1%, pH 5.5.

Embodiment 4: citrus fruit vinegar nutritional labeling and bitter substance content comparison

 Compare the citrus fruit vinegar nutritional components, debitterness rate and mouthfeel in Example 1, Example 2 and the control. In the control, the commonly used fruit vinegar brewing strain AS1.41 Acetobacter putida turbid variant is used for the apple vinegar bacteria ZY.C4 Replacement, all the other processes are the same as in Example 1. The specific nutritional components are compared as shown in Table 1. The total acid, amino nitrogen, total ester, VC, flavonoids and other fruit vinegar flavor substances and nutritional components of citrus fruit vinegar prepared by using the compound acetic acid bacteria seed solution containing apple acetobacter ZY.C4 were higher than those using the acetic acid bacteria containing AS1.41 The fruit vinegar brewed by compound bacteria can reduce the limonoids in citrus fruit vinegar products by 70-75%, making the products soft and free of bitterness.

Table 1 Comparison of nutritional components of citrus fruit vinegar

  

Example 5 Optimization of the ratio of compound yeast strains

First-level expanded culture of yeast: the volume of yeast activation medium is 150mL/250mL, two baits of each yeast species to be tested are transferred into the culture medium, placed on a shaking table (28°C, 180r/min) for about 16 hours.

Preparation of fermentation broth: Mix the citrus juice and glutinous rice saccharification solution prepared above at a ratio of 3:1, adjust the sugar content to 11 degrees, adjust the pH to 4.2 with citric acid, and sterilize the adjusted juice in hot water at 85°C for 15 minutes to kill the bacteria, then cool for later use.

Optimization of the proportion of compound yeast strains: using 4 factors and 3 levels of orthogonal experiments, the inoculation proportions of four strains of yeast 31906, Saccharomyces cerevisiae 1306, Saccharomyces cerevisiae 1425, and Hansenula anomaly var. 1431 were optimized. The factor levels are as follows: Table 2 shows. Adjust the cell concentration of the first-level expansion culture solution to 10 ⁸ cells/mL, put the four kinds of first-level culture solutions into the new malt medium according to the proportion of the orthogonal experiment scheme, and place them in a shaker (28°C, 180r/min) for about 16 hours to make the number of bacteria reach 10 ⁷ -10 ⁸ cells/mL. The prepared mixed strains were inserted into the mixture of citrus and glutinous rice with an inoculum size of 10%, stirred at 28°C (180r/min) and fermented for 1 day, then sealed and left to ferment. The fermentation broth is weighed every day, and the total sugar is determined when the weight basically does not change. After the total sugar content was stabilized at 4-5°Be′, various physical and chemical indicators such as alcohol content, amino nitrogen content, acid production were measured and sensory indicators were evaluated.

Table 2 The factor level table of the orthogonal test for the optimization of the proportion of compound yeast strains

  

Optimizing results of compound yeast strain ratio: When the inoculum amounts of Hansenula anomaly var. , amino nitrogen content and total ester content are high, and the wine has a strong aroma and fruity aroma.

Embodiment 6 : compound yeast strain alcoholic fermentation process optimization

Seed liquid preparation of compound yeast strains: the liquid volume of the yeast activation medium is 150mL/250mL, and four kinds of bait yeasts 31906, S. cerevisiae 1306, S. Bacteria were respectively transferred into the culture medium, placed on a shaker (28°C, 180r/min) for about 16 hours, and the concentration of the bacteria was adjusted to 108 cells/mL; Variation 1431, 3% Saccharomyces 1425, 3% Saccharomyces 31906, 2% Saccharomyces 1306 were inserted into the new malt medium, placed in a shaker (28°C, 180r/min) for about 16 hours, Make the number of bacterial cells reach 10 ⁷ -10 ⁸ cells/mL.

Alcohol fermentation process optimization: First, single-factor experiments are carried out on various factors such as ammonium sulfate addition amount, sulfur dioxide addition amount, composite strain inoculation amount, fermentation time, fermentation temperature, initial sugar content and initial pH, so as to select a better level for optimization . The compound yeast was inserted into the mixture of citrus and glutinous rice, and the alcohol content and amino nitrogen content were determined after alcohol fermentation, and the sensory evaluation was carried out. The test levels of each factor are as follows: sulfur dioxide addition: 0, 30, 60, 90 mg/L; ammonium sulfate addition: 0, 25, 50, 75, 100 mg/L; 8%, 10%, 12%, 14%; fermentation time is 1, 2, 3, 4, 5, 6, 7, 8d; fermentation temperature is 24°C, 26°C, 28°C, 30°C, 32°C; There are 5 sugar gradients of 13°Bx', 15°Bx', 17°Bx', 19°Bx', 21°Bx'; the initial pH is 2.8, 3.5, 4.5, 5.5, 6.5. On the basis of the above single factor test results, determine the value range of the main influencing factors of initial sugar content, initial pH value, inoculum size, time and temperature in the alcohol fermentation process, and carry out a quadratic orthogonal rotation combined test with 5 factors and 5 levels (semi-implementation, 36 combinations in total), with the alcohol content as the evaluation index, the results of the test are analyzed, and the results are verified by the comprehensive sensory evaluation scores, and the optimized alcohol fermentation process conditions are obtained. Table 3 shows the coding table of factor levels in the alcohol fermentation process test.

Table 3 Alcohol fermentation process test factor level coding value table

Optimum process conditions for alcohol fermentation of compound yeast strains: add 30 mg/L sulfur dioxide and 50 mg/L ammonium sulfate to the mixture of citrus juice and glutinous rice saccharification liquid, adjust the initial sugar content to 19°Bx', and the initial pH value to 4.48, Add 9.7% compound yeast strains to it, ferment at 28.5°C for 1 day with stirring at 28.5°C (rotation speed: 180r/min), and then leave it to ferment in a sealed container for 4-5 days. The alcohol content of the obtained citrus fruit wine can reach 11.7%. , and the bouquet is rich and fruity.

Embodiment 7: the ratio of compound acetic acid bacteria is preferred

First-level expansion culture of acetic acid bacteria: the liquid volume of the yeast activation medium is 30mL/150mL, two rings of four different thick bacteria are respectively transferred into the culture medium, placed on a shaking table (30°C, 150r/min) for about 48 Hours.

Preparation of fermentation broth: Mix the citrus juice and glutinous rice saccharification solution prepared above at a ratio of 3:1, add 30 mg/L sulfur dioxide and 50 mg/L ammonium sulfate to the mixture of citrus juice and glutinous rice saccharification solution, adjust The initial sugar content is 19°Bx', and the pH is adjusted to 4.48 with citric acid. The adjusted fruit juice is sterilized in hot water at 85°C for 15 minutes to kill the miscellaneous bacteria, and then cooled for later use.

Citrus fruit wine fermentation: Add 9.7% compound yeast seed liquid to the above fermentation liquid (the liquid content is 4/5 of the volume), stir at 28.5°C (rotation speed: 180r/min) and ferment for 1 day, then seal and let stand for 4 days -5d, when the alcohol content in the fermentation broth is detected to be 10%-12% V/V, the fermentation is terminated, and after filtration, it is sterilized by steam at 80-100°C for 30 minutes.

Optimization of the proportion of compound acetic acid bacteria: using 4 factors and 3 levels of orthogonal experiment, the inoculation ratio of the four strains of Shanghai brewing 1.01, apple vinegar ZY.C4, Acetobacter pasteurian subsp. Rowan subsp. For optimization, the factor levels are shown in Table 4. Adjust the cell concentration of the primary culture solution of the four strains to 10 ⁷ cells/mL, transfer it into citrus fruit wine with alcohol content of 6-7% according to the proportion of the orthogonal experiment plan, and place it on a shaker (30°C, 150r/min) for about 24 hours, so that the number of bacteria reaches 10 ^{6 ~} 10 ⁸ /mL, and the mixed acetic acid bacteria are obtained.

Acetic acid fermentation: Add the prepared mixed strains to alcohol with an alcohol content of 6-7%, and stir at 31°C (180r/min) for fermentation. When the residual alcohol is detected to be 0.3%-0.5%, stop the fermentation and measure Its acid production, total ester content and amino acid peptide nitrogen content were evaluated sensory evaluation of its flavor. Combined with various physical and chemical indicators and sensory evaluation results, the optimal proportion of acetic acid bacteria inoculum suitable for the fermentation of ponkan fruit vinegar was selected.

Table 4 The optimal orthogonal test factor level table of the proportion of compound acetic acid bacteria strains

Optimizing results of compound acetic acid bacteria strain ratio: When the ratio of 4 kinds of acetic acid bacteria strains is 4% apple vinegar ZY.C4, 3% Acetobacter pasteurii subsp. Rowan subsp. Bacillus 22518, the measured acidity, amino acid peptide nitrogen content and total ester content reached the maximum, and the fruit vinegar produced had a soft sour taste and pleasant aroma, which had both the aroma of fruit vinegar and grain vinegar.

Embodiment 8 Composite acetic acid bacteria strain acetic acid fermentation process optimization

Preparation of compound acetic acid bacteria seed solution: the volume of acetic acid bacteria activation medium is 30mL/150mL, and two rings of apple vinegar ZY.C4, Acetobacter pasteurii subsp. 1.01 and other four kinds of thick bacteria were respectively transferred into the culture medium, placed on a shaker (30°C, 150r/min) for about 48 hours, and after about 48 hours, the concentration of the bacteria was adjusted to ¹⁰⁷ /mL; According to the ratio of 4% Apple Acetobacter ZY.C4, 3% Acetobacter pasteurianus Rowan subsp. Place on a shaker (30°C, 150r/min) and incubate for about 24 hours, so that the number of bacteria reaches 10 ^{6 ~} 10 ⁸ cells/mL.

Optimization of acetic acid fermentation process: Firstly, a single factor experiment was carried out on various factors such as fermentation time, initial pH, liquid volume, inoculum volume, initial alcohol content and fermentation temperature, and compound acetic acid bacteria were inserted into citrus fruit wine, and then determined after acetic fermentation Acetic acid content, amino nitrogen content, total ester content, and sensory evaluation to screen out the optimum level of each factor. The test levels of each factor are as follows: the fermentation time is 1, 2, 3, 4, 5, 6, 7, 8 days; the initial pH of the fermented wine liquid is 2.8, 3.5, 4.5, 5.5, 6.5; the filling volume is 10%, 20% , 30%, 40%, 50%; the inoculation amount of compound acetic acid bacteria is 5%, 10%, 15%, 20%; the initial alcohol content is 5%, 6%, 7%, 8%, 9%; fermentation temperature 25°C, 28°C, 31°C, 34°C, 37°C. On the basis of the above single factor test, a quadratic orthogonal rotation combination test with 5 factors and 5 levels (semi-implementation, a total of 36 combinations) was used to investigate the mixing of time, initial pH, liquid content, initial alcohol content and the best combination The influence of factors such as the inoculum amount of strains on the effect of acetic acid fermentation, the total acid content of the fermentation broth, the content of amino acid peptide nitrogen and the content of total esters were used as evaluation indicators to optimize the parameters of the acetic acid fermentation process of ponkan fruit vinegar. The coding table of factor levels in the acetic acid fermentation process test is shown in Table 5.

The optimum process conditions for acetic acid fermentation of compound acetic acid bacteria strains: fermentation time 5.3 days, initial alcohol content 6.4%, initial pH 3.51, inoculum volume 15.2%, liquid volume 30%, under these conditions, the acid production is as high as 66.4g /L, the amino nitrogen content is 0.712g/L, and the total ester content is 0.962g/L. Using this process can greatly improve the acid production rate and acid production of citrus fruit vinegar, and the fruit vinegar has a moderate sour taste and a mellow taste , fruity and pleasant.

Table 5 Level coding table of acetic acid fermentation process factors

 

Claims (6)

1. the organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare, is characterized in that its preparation method is as follows:

1) preparation of mulriple yeasts kind seed liquor: yeast activation medium liquid amount is 150mL/250mL, respectively get two bait yeast 31906, yeast saccharomyces cerevisiae 1306, yeast saccharomyces cerevisiae 1425, the thick bacterium of the abnormal mutation 1431 of Hansenula anomala move into nutrient solution respectively, be placed in shaking table in 28 DEG C, 180r/min cultivates 16h, obtain one-level nutrient solution, adjustment cell concentration is 10 ⁷individual/mL; Four kinds of one-level nutrient solutions are linked in Fructus Hordei Germinatus substratum according to following volume percent: the abnormal mutation 1431 of 4% Hansenula anomala, 3% yeast saccharomyces cerevisiae 1425,3% yeast saccharomyces cerevisiae 31906,2% yeast saccharomyces cerevisiae 1306, then shaking table is placed in 28 DEG C, cultivate 16h under 180r/min, make thalline quantity reach 10 ⁷-10 ⁸individual/mL, is mulriple yeasts kind seed liquor;

2) preparation of compound acetic acid bacteria strain seed liquor: acetic bacteria activation medium liquid amount is 30mL/150mL, respectively get two ring cider vinegar bacillus ZY.C4, Acetobacter pasteurianus Luo Wang subspecies 7002, acetify acidfast bacilli 22518 and Shanghai to make 1.01 thick bacterium and move into nutrient solution respectively, be placed in shaking table in 31 DEG C, 150r/min cultivates 48 hours, obtain one-level nutrient solution, adjustment cell concentration is 10 ⁶individual/mL; It is in the Citrus Fruit Wine of 6-7% that four kinds of one-level nutrient solutions are linked into alcoholic strength according to following volume percent: 4% cider vinegar bacillus ZY.C4, and 3% Acetobacter pasteurianus Luo Wang subspecies 7002,2% acetify acidfast bacilli 22518,2% Shanghai makes 1.01; Then be placed in shaking table in 30 DEG C, 150r/min cultivates 24 hours, makes thalline quantity reach thalline quantity and reaches 10 ⁶-10 ⁸individual/mL;

3) glutinous rice saccharified liquid preparation: rice rinses 2 times, soaks 2-4h; By weight water: rice=2.5:1 defibrination, size mixing to 4:1 ratio, add CaCl ₂and MgCl ₂, described CaCl ₂and MgCl ₂add-on be every 100mL slurries and add 1g, pH to 6.2-6.4 is adjusted with acetic acid and sodium carbonate, add α-amylase, the add-on of α-amylase is that 100mL slurries add 1g, be heated to the 90-95 DEG C of about 20-30min that liquefies, be cooled to 60 DEG C, tune pH is 4.5-5.0, obtain liquid A, liquid A obtains glutinous rice saccharified liquid through saccharification;

4) preparation of citrus juice: select the citrus without rotting, peeling, puts into juice extractor by citrus pulp, and adds the NaHSO of 0.01% (weight) ₃solution, citrus pulp: NaHSO ₃solution=1:1(weight ratio); Squeeze the juice, in juice, add pectinase enzymatic hydrolysis, the add-on of described polygalacturonase is add polygalacturonase 0.04g in every 100mL juice, hydrolysis temperature 60 DEG C, enzymolysis time 60min, and after enzymolysis, 4 layers of filtered through gauze, obtain citrus juice;

5) fermented liquid preparation: citrus juice obtained above and glutinous rice saccharified liquid are mixed with the volume ratio of 3:1, sulfurous gas and ammonium sulfate is added in the ratio adding sulfurous gas 30mg and ammonium sulfate 50 mg in every 1L mixed solution respectively in mixed solution, mixing is adjust ph 4.48 also, regulate pol to 15-19 ° of Bx ', sterilization 15min in 85 DEG C of hot water, cool for subsequent use, obtain fermented liquid;

6) zymamsis: access mulriple yeasts kind seed liquor in the fermented liquid of step 5), inoculum size is 9.7-10% (volume percent), 28-28.5 DEG C of stirring, rotating speed is after 180r/min fermentation 1d, after airtight standing for fermentation 4-5d, sterilization, obtains Citrus Fruit Wine;

7) acetic fermentation: Citrus Fruit Wine alcoholic strength is adjusted to 6-7%, access compound acetic acid bacteria strain seed liquor, inoculum size is 15.2-16% (volume percent), is 180r/min at rotating speed, and 31 DEG C are stirred fermentation 5-7d; Stop fermentation, pass into 80-100 DEG C of steam sterilizing 30min;

8) 7) in add salt in the liquid that obtains, the add-on of salt is add salt 20g in every 1L, normal temperature ageing 2-3 month, with diatomite filtration clarification, obtains organge fruit vinegar finished product.

2. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, it is characterized in that: the method for saccharifying of described step 3) is: add saccharifying enzyme and malt meal, add-on is add 1g saccharifying enzyme and 10g malt meal in every 100mL liquid A, insulation saccharification 6-8h.

3. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, it is characterized in that: the method for saccharifying of described step 3) is: add black song: the mixed starters of red colouring agent for food, also used as a Chinese medicine=3:1, the add-on of mixed starters is add 1g in 100mL liquid A, and saccharification time is 2-4h.

4. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, is characterized in that: described step 3) also adds malt meal before enzymolysis, and add-on is add malt meal 10g in every 100mL juice.

5. a kind of organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to claim 1, is characterized in that: described step 6) also adds naringinase before fermentation, and the add-on of naringinase is add 0.04g in every 100mL Citrus Fruit Wine.

6. the preparation method of the organge fruit vinegar utilizing composite bacteria mixed fermentation to prepare according to any one in claim 1-5.