WO2021120618A1 - 二元复合发酵剂及其应用 - Google Patents

二元复合发酵剂及其应用 Download PDF

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WO2021120618A1
WO2021120618A1 PCT/CN2020/103450 CN2020103450W WO2021120618A1 WO 2021120618 A1 WO2021120618 A1 WO 2021120618A1 CN 2020103450 W CN2020103450 W CN 2020103450W WO 2021120618 A1 WO2021120618 A1 WO 2021120618A1
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parts
vinegar
compound
mash
acid
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French (fr)
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李国权
李信
朱胜虎
崔鹏景
张俊红
陆平
奚宽鹏
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江苏恒顺醋业股份有限公司
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Priority to JP2021570889A priority Critical patent/JP7159492B2/ja
Publication of WO2021120618A1 publication Critical patent/WO2021120618A1/zh

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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
    • C12J1/00—Vinegar; Preparation or purification thereof
    • C12J1/04—Vinegar; Preparation or purification thereof from alcohol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)

Definitions

  • the present invention relates to a binary compound starter, and the present invention also relates to the application of the binary compound starter in vinegar brewing.
  • Vinegar is a liquid acid condiment brewed by microbial fermentation using various materials containing starch, sugar, and edible alcohol alone or in combination.
  • European and American countries generally use a single strain of fermentation technology to brew vinegar, while China and other Southeast Asian countries mainly use multi-bacterial fermentation to brew vinegar.
  • the single strain fermentation is mainly based on pure acetic acid bacteria, which has a single flavor and poor taste.
  • the traditional multi-strain fermentation process mostly adopts the natural multi-strain open fermentation mode.
  • the source of the fermentation strains is mostly brought in in the form of seed ferment (or seed liquid) from the previous generation, that is, the previous generation of vinegar or acetic acid fermentation liquid is The amount of access to the next generation, and in this way, from generation to generation.
  • direct-investment starter has entered the industrial application stage in the preparation of yogurt, dough, kimchi and other fermented foods, but it is suitable for the special environment and requirements of traditional vinegar brewing. (High-acid, high-alcohol, oligo-nutrition, coexistence of eutrophic and anaerobic microorganisms, synergistic fermentation of upper and lower layers, etc.) Research on starters is very few.
  • the present invention aims to provide a binary compound starter with low dosage, fast fermentation efficiency, high product yield and stable product quality. Another object of the present invention is to provide the binary starter in vinegar. Application in brewing.
  • the binary compound starter of the present invention includes compound inoculum I and compound inoculum II, characterized in that the compound inoculum I includes Lactobacillus helveticus CGMCC12225, Lactobacillus fermentum CGMCC12226, Lactobacillus acidatus CGMCC16938, Sonora Bacillus deserticus CGMCC15824, Bacillus coagulans CGMCC17801, acid-resistant meso-temperature ⁇ -amylase, pullulanase, glucoamylase, cellulase, and acid protease; compound inoculum II contains Acetobacter pasteurella CGMCC17802, Crimson vulgaris CGMCC16345 and glucose .
  • CGMCC No. 15824, CGMCC No. 17801, CGMCC No. 17802, CGMCC No. 16345, and the dates of preservation are respectively CGMCC No. 12225, CGMCC No. 12226, CGMCC No. 16938, CGMCC No. 15824, CGMCC No. 17801, CGMCC No. 17802, and CGMCC No. 16345. It is March 18, 2016, March 18, 2016, December 13, 2018, May 30, 2018, May 13, 2019, May 13, 2019, August 27, 2018.
  • the compound bacterial agent I contains 10-25 parts by weight of Lactobacillus helveticus CGMCC12225, 5-15 parts of Lactobacillus fermentum CGMCC12226, 5-20 parts of acid-fast Lactobacillus CGMCC16938, and 3-15 parts of Sonora Desert Bacillus CGMCC15824.
  • the compound bacterial agent II contains 35 to 55 parts by weight of Acetobacter pasteurii CGMCC17802, 2 to 10 parts of European covus CGMCC16345 and 40 to 60 parts of glucose.
  • the number of viable bacteria of Lactobacillus helveticus CGMCC12225, Lactobacillus fermentum CGMCC12226, acid-fast Lactobacillus CGMCC16938, Sonora Desert Bacillus CGMCC15824 and Bacillus coagulans CGMCC17801 in the compound bacterial agent I are 1.0 ⁇ 10 8 to 1.0 ⁇ 10 10 CFU/g
  • the enzyme activities of acid-tolerant mid-temperature ⁇ -amylase, pullulanase, glucoamylase, cellulase and acid protease are all 20,000 to 100,000 U/g.
  • the number of viable bacteria of Acetobacter pasteurii CGMCC17802 and European covus CGMCC16345 in the composite bacterial agent II is 1.0 ⁇ 10 7 to 1.0 ⁇ 10 9 CFU/g.
  • the composite bacterial agent I is added before the acetic acid fermentation, and the composite bacterial agent II is added during the acetic acid fermentation stage.
  • the binary compound starter can also be used in combination with seed mash.
  • the vinegar brewing is solid vinegar fermentation or liquid vinegar fermentation.
  • the binary compound starter is added in a direct way.
  • the vinegar is Zhenjiang aromatic vinegar, Shanxi mature vinegar, rice vinegar or apple cider vinegar.
  • the binary compound starter of the present invention solves the problems of single starter producing vinegar products with single flavor, stimulating taste, not soft and full and low yield;
  • the binary composite starter of the present invention is suitable for the special environment of vinegar brewing, has outstanding effects and wide application scenarios. It can be used directly without traditional seeds, or can be used as an enhanced starter after traditional seeds are brought in. use.
  • the fermentation efficiency is fast, the yield is high, and the quality is stable, which can significantly improve the fermentation efficiency.
  • the content of volatile acid, amino acid nitrogen, esters and other flavor substances, the product has rich flavor, full and soft taste, and outstanding aroma, which is unmatched by a single or common compound starter.
  • the binary compound starter of the present invention can be used as a solid vinegar starter or a liquid vinegar starter, and can be used alone or in combination with seed ferment;
  • the binary compound starter of the present invention is applied to solid vinegar fermentation, and can significantly increase the content of non-volatile acid, amino acid nitrogen, esters and other flavor substances; when applied to liquid vinegar fermentation, it can significantly increase the content of non-volatile acid, amino acid nitrogen, and esters.
  • the content of volatile acids and ester flavor substances are not limited to
  • Figure 1 shows the colony morphology of Acetobacter pasteurii of the present invention.
  • the preparation method of the composite inoculum I and the composite inoculum II of the present invention is as follows: each bacteria is separately subjected to three-stage expansion and fermentation according to its corresponding medium, and after the fermentation is completed, the hollow fiber membrane is used to concentrate the fermentation broth to the original fermentation 1/5 of the liquid volume, then mix the sterile skimmed milk powder 20g/100ml, sodium glutamate 12g/100ml, sorbitol 4g/100ml with the concentrated fermentation broth, and put it in the -80°C ultra-low temperature refrigerator for pre-freezing 2 ⁇ 5h, finally put the sample plate on the freeze dryer for 24-48h freeze-drying, and use the plate counting method to detect that the number of viable cells meets the requirements (the number of viable cells is 1.0 ⁇ 10 8 ⁇ 1.0 ⁇ 10 10 CFU/g) before use ; Purchase relevant enzyme preparations (enzyme activity of 20,000 to 100,000 U/g) and food grade glucose that meet the requirements of commercially available activity units, mix them in a clean room according to the
  • the detection methods of various indicators in the examples are as follows: the total acid and non-volatile acid are measured according to the "GB18187-2000” method, the total acid is calculated as acetic acid, and the non-volatile acid is calculated as lactic acid; the amino acid nitrogen is determined according to the "GB18186-2000” method. Determination; the total esters are determined with reference to the "GB/T19777-2013” method.
  • the binary compound starter of the present invention can not only be applied to the brewing of the following vinegar, but also applicable to the brewing of other solid and liquid fermented vinegar.
  • Example 1 Isolation and identification of the strain Acetobacter pasteurii
  • the strains from the preliminary screening were inoculated on a re-screened solid plate (20 g glucose, 10 g yeast extract, 30 ml acetic acid, 30 ml ethanol, and 15 g/L agar powder added). After 3 days of culture at 30°C, the strain with the largest transparent circle was selected.
  • the selected strains were inoculated into a re-screened liquid medium (20g glucose, 10g yeast extract, 30ml acetic acid, 50ml ethanol and 1L distilled water), cultured at 30°C at 200rpm for 20h, and titrated the total acid with sodium hydroxide (calculated as acetic acid) ) Content, measured every 24h, and record the changes in acid production.
  • a re-screened liquid medium (20g glucose, 10g yeast extract, 30ml acetic acid, 50ml ethanol and 1L distilled water
  • the measured 16S rDNA sequence was compared and analyzed in the NCBI database, and combined with physiological and biochemical characteristics, the strain of the present invention was named Acetobacter pasteurianus HSCY1085 (Acetobacter pasteurianus), and the 16S rDNA sequence is shown in SEQ ID No. 1.
  • the strain is deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the preservation location is the Institute of Microbiology, Chinese Academy of Sciences, No. 3 Beichen West Road, Chaoyang District, Beijing.
  • the preservation date is May 13, 2019, and the preservation number is CGMCC No.17802, its classification is named Acetobacter pasteurianus.
  • This example provides an application in Zhenjiang aromatic vinegar brewing without using traditional seed mash and inoculation, and only using the binary compound starter of the present invention.
  • the addition amount is 0.1 ⁇ (w/w) of the quality of the mash
  • the 30°C mash is added Stir about 1000g in a small bucket and sprinkle it on the top of a tank of vinegar, then go down about 10cm by hand and mix well, and finally cover the top with big chaff to keep it warm.
  • the mash is turned layer by layer, and the vinegar mash is fermented until the total acid does not increase, that is, the fermentation is over. Add salt and seal the glutinous rice for 15 days, then add the fried rice to pour the vinegar. After decoction, aging, sterilization and filling, the finished product is obtained.
  • step (3) Take the three 400kg big tanks in the above step (3), add 200kg of mash, 80kg of bran, and 45kg of bran to each tank, and mix the mash and grains evenly (ie vinegar mash). Take 26kg of traditional seed mash on the upper part of vinegar mash, cover with big bran for heat preservation, and the subsequent steps are the same as the above step (5).
  • the test group using the binary compound starter of the present invention has a faster heat-raising speed (the time for the first temperature to reach above 40°C is shortened from 38h to 20h, and 18h); the fermentation time is shortened from 22d to 15d, which is shortened 7d; After the end of sealing, the total acid content in the fermented bittern increased by 14.73%, the non-volatile acid content increased by 82.84%, the amino acid nitrogen content increased by 105.56%, and the total ester content increased by 126.05%; the product yield increased by 17.01%; the obtained product aroma Obviously, the flavor is rich, the taste is soft and full, and the sensory score is high.
  • the fermentation time of the test group using the binary compound starter of the present invention was shortened from 19 days to 15 days, which was shortened by 4 days; the total acid content in the brine increased by 10.22% and the non-volatile acid content increased by 21.89 after the end of sealing. %, the amino acid nitrogen content increased by 76.19%, the total ester content increased by 84.79%; the product yield increased by 11.95%; the overall flavor and taste of the obtained product has the characteristics of typical Zhenjiang balsamic vinegar, and the aroma is more obvious, and the taste is softer and richer , Fuller.
  • Example 3 Intensified application in Zhenjiang aromatic vinegar brewing
  • This embodiment provides an enhanced application of the binary compound starter of the present invention in the brewing of Zhenjiang aromatic vinegar while maintaining the traditional seed mash and inoculation method.
  • the mash is turned layer by layer, and the vinegar mash is fermented until the total acid does not increase, that is, the fermentation is over. Add salt and seal the glutinous rice for 15 days, then add the fried rice to pour the vinegar. After decoction, aging, sterilization and filling, the finished product is obtained.
  • step (3) Take the three 400kg big tanks in the above step (3), add 200kg of mash, 80kg of bran, and 45kg of bran to each tank, and mix the mash and grains evenly (ie vinegar mash). Take 26 kg of seed mash on the top of vinegar mash, cover with big bran for heat preservation, and the subsequent steps are the same as the above step (5).
  • step (3) Take the three 400kg big tanks in the above step (3), add 200kg of mash, 80kg of bran, and 45kg of bran to each tank, and mix the mash and grains evenly (ie vinegar mash). Take 26kg of seed mash on the upper part of the vinegar mash, mix in each tank with the commercially available Guangming brand vinegar acetic acid inoculum evenly and cover with big bran for heat preservation. The addition amount is 0.3 ⁇ (w/w) of the quality of the mash, follow-up The steps are the same as the above step (5).
  • the test group enhanced with the binary compound starter of the present invention shortened the fermentation time from 19 days to 13 days, which was shortened by 6 days; the total acid content in the brine increased by 11.80% and the non-volatile acid content after the end of sealing 27.36%, the amino acid nitrogen content is increased by 85.71%, the total ester content is increased by 91.25%; the product yield is increased by 14.15%; the obtained product has a more prominent aroma, a stronger mouthfeel, and a higher sensory score.
  • the test group strengthened with the binary compound starter of the present invention has shortened the fermentation time from 18 days to 13 days, which is 5 days shorter; after the end of sealing, the total acid content in the brine is increased by 8.52%, and the non-volatile acid content is increased 18.52%, amino acid nitrogen content increased by 69.57%, total ester content increased by 84.25%; product yield increased by 10.03%; the obtained product has obvious aroma, soft taste and high sensory score.
  • This embodiment provides a way to avoid the use of traditional seed mash and inoculation, and only use the compound starter of the present invention in the brewing of old aged vinegar in Shanxi.
  • the addition amount is 0.1 ⁇ (w/w) of the quality of the mash
  • the 30°C mash is added Stir about 2000g in a small bucket and then sprinkle it on the top of a cylinder of vinegar vinegar and mix well. Perform the mashing operation according to the technique of Shanxi old aged vinegar.
  • step (2) Take the three 400kg large tanks in the above step (2), add 200kg of mash, 90kg of bran, and 100kg of bran to each tank, and mix the mash and grains evenly.
  • step (3) Take 40g of the commercially available Guangming brand vinegar acetic acid inoculum, add 0.5 ⁇ (w/w) of the quality of the mash, add about 2000g of the mash at 30°C, mix it evenly in a small barrel, and sprinkle it on the top of a cylinder of vinegar. Evenly, the mashing operation is carried out according to the process of Shanxi old aged vinegar, and the subsequent steps are the same as the above step (4).
  • the test group strengthened with the binary compound starter of the present invention shortened the fermentation time from 9d to 7d, which was shortened by 2d.
  • the total acid content in the vinegar was increased by 18.26%, and the non-volatile acid content was increased by 59.18. %, the amino acid nitrogen content is increased by 94.44%, the total ester content is increased by 45.48%; the product yield is increased by 19.35%; the obtained product has rich flavor, soft and full taste, and high sensory score.
  • Example 5 Application in rice vinegar brewing
  • This embodiment provides an application of the binary compound starter of the invention in the brewing of rice vinegar.
  • the composite bacterial agent I of the present invention (20 parts of Lactobacillus helveticus CGMCC12225, 10 parts of Lactobacillus fermentum CGMCC12226, 9 parts of acid-fast Lactobacillus CGMCC16938, 5 parts of Sonora Desert Bacillus CGMCC15824, 6 parts of Bacillus coagulans CGMCC17801, 8 parts Acid-tolerant mid-temperature alpha-amylase, 3 parts pullulanase, 5 parts glucoamylase, 6 parts cellulase and 2 parts acid protease), the addition amount is 0.2 ⁇ (w/w) of the quality of the mash, and the fermentation is continued until alcohol The degree is 8% vol.
  • the start-up speed of the test group was faster, the fermentation cycle of the test group was shortened by 23.75%, the total acid content was increased by 11.52%, the non-volatile acid content was increased by more than 12.5 times, and the total ester content was increased by more than 14 times.
  • the apple cider vinegar prepared by the test group has significantly reduced irritation, a soft taste, a significant comprehensive aroma, and a significant improvement in quality.
  • Example 6 Application in the brewing of apple cider vinegar
  • This embodiment provides an application of the compound starter of the present invention in the brewing of apple cider vinegar.
  • the start-up speed of the test group was faster, the fermentation cycle of the test group was shortened by 18.89%, the total acid content was increased by 14.82%, the non-volatile acid content was increased by more than 10.5 times, and the total ester content was increased by more than 19 times.
  • the apple cider vinegar prepared by the test group has significantly reduced irritation, a softer taste, a significant comprehensive aroma, and a significant improvement in quality.
  • This example provides a comparison of the binary compound starter formula and the addition method of the present invention, and further illustrates the importance of the composition of the binary compound starter formula and the addition method of the present invention to the application effect.
  • the addition amount is 0.1 ⁇ (w/w) of the quality of the mash
  • the 30°C mash is added Stir about 1000g in a small bucket and sprinkle it on the top of a tank of vinegar, then go down about 10cm by hand and mix well, and finally cover the top with big chaff to keep it warm.
  • the mash is turned layer by layer, and the vinegar mash is fermented until the total acid does not increase, that is, the fermentation is over. Add salt and seal the glutinous rice for 15 days, then add the fried rice to pour the vinegar. After decoction, aging, sterilization and filling, the finished product is obtained.
  • the addition amount is 0.1 ⁇ (w/w) of the quality of the mash
  • the 30°C mash is added Stir about 1000g in a small bucket and sprinkle it on the top of a tank of vinegar, then deep down about 10cm by hand and mix well. Finally, cover the top with big chaff to keep warm.
  • the subsequent steps are the same as the above step (5).
  • the addition amount is 0.1 ⁇ (w/w) of the quality of the mash
  • the 30°C mash is added Stir about 1000g in a small bucket and sprinkle it on the top of a tank of vinegar, then go down about 10cm by hand and mix well, and finally cover the top with big chaff to keep it warm.
  • the addition amount is 0.15 ⁇ (w/w) of the quality of the mash, add 80 kg of bran and 45 kg of bran after stirring evenly, and mix the mash and grains evenly (namely vinegar mash).
  • the addition amount is 0.1 ⁇ (w/w) of the quality of the mash, add about 1000g of the mash at 30°C and stir evenly in a small barrel Sprinkle it on the top of a tank of vinegar, then go down about 10cm by hand and mix well. Finally, cover the top with large chaff to keep warm.
  • the subsequent steps are the same as the above step (5).
  • the addition amount is 0.1 ⁇ (w/w) of the quality of the mash
  • the 30°C mash is added Stir about 1000g in a small bucket and sprinkle it on the top of a tank of vinegar, then deep down about 10cm by hand and mix well. Finally, cover the top with big chaff to keep warm.
  • the subsequent steps are the same as the above step (5).
  • the indexes of total acid, non-volatile acid, amino acid nitrogen and total ester in the control group A to E did not reach the effect of the control group, indicating that the binary compound starter formula of the present invention is effective in ensuring good results.
  • the effect plays an important role.
  • control group 1 Compared with the test group, the heat-raising speed of control group 1 was slow. The time for the first temperature to reach above 40°C was extended from 20h to 103h, which was extended by 83h; the fermentation time was extended from 15d to 21d, which was extended by 6d. The non-volatile acid content of the control group 2 was significantly reduced, and the total ester content was significantly reduced.

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Abstract

一种二元复合发酵剂，包含复合菌剂I和复合菌剂II，所述二元复合发酵剂在食醋酿造中的应用，其中复合菌剂I在醋酸发酵前加入，复合菌剂II在醋酸发酵阶段加入。所述二元复合发酵剂可用于酿造固态发酵食醋和液态发酵食醋，发酵剂用量少、发酵效率快、出率高、品质稳定，可显著提高不挥发酸、氨基酸态氮、酯类等风味物质的含量，产品风味丰富、口感饱满柔和、香气突出。

Description

二元复合发酵剂及其应用 技术领域
本发明涉及一种二元复合发酵剂，本发明还涉及所述二元复合发酵剂在食醋酿造中的应用。
背景技术
食醋是单独或混合使用各种含有淀粉、糖的物料、食用酒精，经微生物发酵酿制而成的液体酸性调味品。欧美国家普遍采用单一菌种的发酵技术酿制食醋，中国及其他东南亚国家主要采用多菌发酵工艺酿制食醋。单菌种发酵主要以纯种的醋酸菌为主，风味单一，口感差。传统多菌种发酵过程多采用天然多菌种开放式发酵模式，发酵菌种的来源多由上一代以种醅(或种子液)的形式带入，即将上一代醋醅或醋酸发酵液以一定的量接入下一代，并以此方式代代传递。传统接种方式易出现核心菌种衰退，微生物群落结构变化，变化后的菌群结构难以调控和恢复，对产品的质量和稳定性带来巨大的挑战。同时具有带种量大，不易储存和运输，使用条件受到多方面限制，极大限制了食醋酿造工艺的发展。
现有技术已有直投式发酵剂及其应用，如直投式发酵剂在制备酸奶、发面团、泡菜等部分发酵食品中已进入工业化应用阶段，但适用于传统食醋酿造特殊环境和要求(高酸、高醇、寡营养、好养与厌氧微生物共存、上下层协同发酵等)的发酵剂研究极少。
发明内容
发明目的：本发明旨在提供一种用量少、发酵效率快、产品出率高、产品品质稳定的二元复合发酵剂，本发明的另一目的是提供所述二元发酵剂在食醋酿造中的应用。
技术方案：本发明的二元复合发酵剂，包含复合菌剂I和复合菌剂II，其特征在于，复合菌剂I包含瑞士乳杆菌CGMCC12225、发酵乳杆菌CGMCC12226、耐酸乳杆菌CGMCC16938、索诺拉沙漠芽孢杆菌CGMCC15824、凝结芽孢杆菌CGMCC17801、耐酸中温α-淀粉酶、普鲁兰酶、糖化酶、纤维素酶和酸性蛋白酶；复合菌剂II包含巴氏醋杆菌CGMCC17802、欧洲驹形杆菌CGMCC16345和葡萄糖。
所述瑞士乳杆菌(Lactobacillus helveticus)CGMCC12225，发酵乳杆菌(Lactobacillus fermentum)CGMCC12226，耐酸乳杆菌(Lactobacillus acetotolerans)CGMCC16938，索诺拉沙漠芽孢杆菌(Bacillus sonorensis)CGMCC15824，凝结芽孢杆菌(Bacillus coagulans)CGMCC17801，巴氏醋杆菌(Acetobacter pasteurianus)CGMCC17802，欧 洲驹形杆菌(Komagataeibacter europaeus)CGMCC16345，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地点为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏登记入册的编号分别为CGMCC No.12225，CGMCC No.12226，CGMCC No.16938，CGMCC No.15824，CGMCC No.17801，CGMCC No.17802，CGMCC No.16345，保藏日期分别为2016年03月18日，2016年03月18日，2018年12月13日，2018年05月30日，2019年05月13日，2019年05月13日，2018年08月27日。
优选，所述复合菌剂I按重量份包含10～25份瑞士乳杆菌CGMCC12225、5～15份发酵乳杆菌CGMCC12226、5～20份耐酸乳杆菌CGMCC16938、3～15份索诺拉沙漠芽孢杆菌CGMCC15824、2～10份凝结芽孢杆菌CGMCC17801、5～13份耐酸中温α-淀粉酶、1～3份普鲁兰酶、2～5份糖化酶、3～9份纤维素酶和1～5份酸性蛋白酶；复合菌剂II按重量份包含35～55份巴氏醋杆菌CGMCC17802、2～10份欧洲驹形杆菌CGMCC16345和40～60份葡萄糖。
优选，所述复合菌剂I中瑞士乳杆菌CGMCC12225、发酵乳杆菌CGMCC12226、耐酸乳杆菌CGMCC16938、索诺拉沙漠芽孢杆菌CGMCC15824和凝结芽孢杆菌CGMCC17801的活菌数均为1.0×10 8～1.0×10 10CFU/g，耐酸中温α-淀粉酶、普鲁兰酶、糖化酶、纤维素酶和酸性蛋白酶的酶活力均为2～10万U/g。
优选，所述复合菌剂II中巴氏醋杆菌CGMCC17802和欧洲驹形杆菌CGMCC16345的活菌数1.0×10 7～1.0×10 9CFU/g。
本发明的二元复合发酵剂在食醋酿造中的应用。
优选，所述复合菌剂I在醋酸发酵前加入，所述复合菌剂II在醋酸发酵阶段加入。
优选，所述二元复合发酵剂还可与种子醅联合使用。
优选，所述食醋酿造为固态食醋发酵或液态食醋发酵。
优选，所述二元复合发酵剂采用直投方式加入。
优选，所述食醋为镇江香醋、山西老陈醋、米醋或苹果醋。
研究发现，微生物多样性是实现酿造食醋风味丰富，口感饱满柔和的重要基础。在保持并提高风味丰富，口感饱满柔和的前提下，本发明的二元复合发酵剂，解决了单一发酵剂生产食醋产品风味单一、口感刺激、不柔和饱满和出率低等问题；避免了传统带种过程中易出现的核心菌种衰退，微生物群落结构变化，变化后的菌群结构难以调控和恢复，对产品的质量和稳定性带来不良影响等问题；同时解决了传统带种量大，不易储存与运输的问题。
本发明的二元复合发酵剂，适宜食醋酿造的特殊环境，效果突出，应用场景广泛，既可以在不带入传统种子的条件下直接使用，也可以在带入传统种子后作为强化发酵剂 使用。
在不带入传统种子的条件下直接使用本发明的二元发酵剂或在正常带入传统种子的条件下作为强化发酵剂使用后，发酵效率快、出率高、品质稳定，可显著提高不挥发酸、氨基酸态氮、酯类等风味物质的含量，产品风味丰富、口感饱满柔和、香气突出，是单一或普通复配发酵剂所无法比拟的。
有益效果：与现有技术相比，本发明具有如下显著优点：
(1)本发明的二元复合发酵剂可作为固态食醋发酵剂或液态食醋发酵剂，可单独使用或与种子醅联合使用；
(2)本发明的二元复合发酵剂应用于固态食醋发酵中，可显著提高不挥发酸、氨基酸态氮、酯类等风味物质的含量；应用于液态食醋发酵中，可显著提高不挥发酸和酯类风味物质的含量。
附图说明
图1为本发明的巴氏醋杆菌的菌落形态。
具体实施方式
下面结合实施例对本发明的技术方案作进一步说明。
本发明的复合菌剂I和复合菌剂II制备方法如下：单独将每种菌按照其相应的培养基进行三级扩培并进行发酵，待发酵结束后采用中空纤维膜浓缩发酵液至原发酵液体积的1/5，然后将无菌脱脂奶粉20g/100ml、谷氨酸钠12g/100ml、山梨醇4g/100ml与浓缩发酵液混匀，放入-80℃超低温冰箱中进行预冻2～5h，最后将样品盘至于冷冻干燥机上进行24～48h的冻干处理，采用平板计数法检测活菌数符合要求(活菌数均为1.0×10 8～1.0×10 10CFU/g)后备用；购买市售活力单位符合要求的相关酶制剂(酶活力为2～10万U/g)和食品级葡萄糖，在洁净室按照本发明的二元复合发酵剂配比将其混匀，最后分装于真空包装袋中；分装后的产品于4℃保存。
实施例中各项指标检测方法如下：总酸和不挥发酸参照《GB18187-2000》方法进行测定，总酸以乙酸计，不挥发酸以乳酸计；氨基酸态氮参照《GB18186-2000》方法进行测定；总酯参照《GB/T19777-2013》方法进行测定。
本发明的二元复合发酵剂不仅可应用于下述食醋的酿造，也适用于其它固态和液态发酵食醋的酿造。
实施例1：菌株巴氏醋杆菌的分离与鉴定
本实施例中碳酸钙、葡萄糖、琼脂粉、无水乙醇、乙酸和氢氧化钠均购自国药集团化学试剂有限公司，酵母提取物购自英国OXOID公司。
1、菌株分离
取镇江香醋醋醅样10g，加入90ml已灭菌生理盐水中，于摇床摇匀，后取100μl样品加入到900μl生理盐水中，于漩涡振荡器混匀，之后进行梯度稀释。混匀依次涂布到添加20g碳酸钙的固体培养基(每1L含葡萄糖20g、酵母提取物10g、琼脂粉15g，121℃灭菌20min后冷却，加入3％乙醇)上，30℃培养3天。观察平板上有无透明圈，挑取相应菌株。
2、菌株复筛
将初筛出的菌株接种到复筛固体平板(葡萄糖20g、酵母提取物10g、乙酸30ml和乙醇30ml，添加琼脂粉15g/L)上，30℃培养3天后选出透明圈最大的菌株。
将筛选出的菌株接种到复筛液体培养基(葡萄糖20g、酵母提取物10g、乙酸30ml、乙醇50ml和蒸馏水1L)中，30℃转速200rpm培养20h，以氢氧化钠滴定总酸(以醋酸计)含量，每隔24h进行一次测定，记录产酸变化情况。
经过两轮复筛，最终得到一株产酸性能优良的巴氏醋酐菌HSCY1085菌株，其菌落形态见图1。
3、菌株鉴定
测得的16S rDNA序列在NCBI数据库中进行比对分析，并结合生理生化特征，将本发明菌株命名为巴氏醋杆菌HSCY1085(Acetobacter pasteurianus)，16S rDNA序列见SEQ ID No.1。
该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地点为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏日期为2019年5月13日，保藏编号为CGMCC No.17802，其分类命名为Acetobacter pasteurianus。
实施例2：在镇江香醋酿造中的应用
本实施例提供了一种免用传统种子醅和接种方式，仅使用本发明的二元复合发酵剂在镇江香醋酿造中的应用。
1、试验组
(1)取9个400kg大缸，各取糯米50kg加水浸泡过夜。利用蒸汽将糯米蒸熟，以冷水淋饭至温度约40℃加入酒药0.3kg，拌匀后入缸搭窝成喇叭状。
(2)待窝中有一定的酒液出现时，每缸加入麦曲2.5kg，然后加水150kg，搅拌均匀。
(3)酒精发酵过程中，定期搅拌，温度控制在30℃左右，约发酵5～7d结束。
(4)取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、15份索诺拉沙漠芽孢杆菌CGMCC15824、10份凝结芽孢 杆菌CGMCC17801、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、9份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温。
(5)按照镇江香醋酿造工艺进行逐层翻醅，待醋醅发酵至总酸不在增加，即发酵结束。加盐封醅15d，然后加入炒米色进行淋醋。经煎醋、陈酿、杀菌灌装，即得成品。
2、对比试验
(1)对照组1
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。每缸取市售光明牌酿醋醋酸菌剂，添加量为酒醪质量的0.5‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温，后续步骤同上述步骤(5)。
(2)对照组2
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取传统种子醅26kg于醋醅的上部，盖上大糠保温，后续步骤同上述步骤(5)。
表1 醅卤主要指标对比(g/100ml)
Figure PCTCN2020103450-appb-000001
与对照组1相比使用本发明的二元复合发酵剂的试验组，提热速度快(首次温度达到40℃以上时间由38h缩短至20h，缩短18h)；发酵时间由22d缩短至15d，缩短7d；封醅结束后醅卤中总酸含量提高14.73％，不挥发酸含量提高82.84％，氨基酸态氮含量提高105.56％，总酯含量提高126.05％；产品出率提高17.01％；获得的产品香气明显，风味丰富，口感柔和饱满，感官评分较高。
与对照组2相比使用本发明的二元复合发酵剂的试验组，发酵时间由19d缩短至15d，缩短4d；封醅结束后醅卤中总酸含量提高10.22％，不挥发酸含量提高21.89％，氨基酸态氮含量提高76.19％，总酯含量提高84.79％；产品出率提高11.95％；获得产品的整体 风味和口感具有典型的镇江香醋特点，且香气更明显，口感更柔和、更丰富、更饱满。
实施例3：在镇江香醋酿造中的强化应用
本实施例提供了一种保持采用传统种子醅和接种方式，本发明的二元复合发酵剂于镇江香醋酿造的强化应用。
1、试验组
(1)取9个400kg大缸，各取糯米50kg加水浸泡过夜。利用蒸汽将糯米蒸熟，以冷水淋饭至温度约40℃加入酒药0.3kg，拌匀后入缸搭窝成喇叭状。
(2)待窝中有一定的酒液出现时，每缸加入麦曲2.5kg，然后加水150kg，搅拌均匀。
(3)酒精发酵过程中，定期搅拌，温度控制在30℃左右，约发酵5～7d结束。
(4)取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(10份瑞士乳杆菌CGMCC12225、5份发酵乳杆菌CGMCC12226、5份耐酸乳杆菌CGMCC16938、3份索诺拉沙漠芽孢杆菌CGMCC15824、2份凝结芽孢杆菌CGMCC17801、5份耐酸中温α-淀粉酶、1份普鲁兰酶、2份糖化酶、3份纤维素酶和1份酸性蛋白酶)，添加量为酒醪质量的0.1‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取种子醅26kg于醋醅的上部，每缸取本发明的复合菌剂II(35份巴氏醋杆菌CGMCC17802、2份欧洲驹形杆菌CGMCC16345和40份葡萄糖)，添加量为酒醪质量的0.05‰(w/w)，均匀拌入种子醅后盖上大糠保温。
(5)按照镇江香醋酿造工艺进行逐层翻醅，待醋醅发酵至总酸不在增加，即发酵结束。加盐封醅15d，然后加入炒米色进行淋醋。经煎醋、陈酿、杀菌灌装，即得成品。
2、对比试验
(1)对照组1
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取种子醅26kg于醋醅的上部，盖上大糠保温，后续步骤同上述步骤(5)。
(2)对照组2
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取种子醅26kg于醋醅的上部，每缸取市售光明牌酿醋醋酸菌剂均匀拌入种子醅后盖上大糠保温，添加量为酒醪质量的0.3‰(w/w)，后续步骤同上述步骤(5)。
表2 醅卤主要指标对比(g/100ml)
Figure PCTCN2020103450-appb-000002
Figure PCTCN2020103450-appb-000003
与对照组1相比使用本发明的二元复合发酵剂强化的试验组，发酵时间由19d缩短至13d，缩短6d；封醅结束后醅卤中总酸含量提高11.80％，不挥发酸含量提高27.36％，氨基酸态氮含量提高85.71％，总酯含量提高91.25％；产品出率提高14.15％；获得的产品香气更突出，口感柔和性更强，感官评分更高。
与对照组2相比使用本发明的二元复合发酵剂强化的试验组，发酵时间由18d缩短至13d，缩短5d；封醅结束后醅卤中总酸含量提高8.52％，不挥发酸含量提高18.52％，氨基酸态氮含量提高69.57％，总酯含量提高84.25％；产品出率提高10.03％；获得的产品香气明显，口感柔和，感官评分较高。
实施例4：在山西老陈醋酿造中的应用
本实施例提供了一种免用传统种子醅和接种方式，仅使用本发明的复合发酵剂在山西老陈醋酿造中的应用。
1、试验组
(1)将高粱粉碎为5～10瓣后，加入温水，煮料1～2h后，每100kg高粱加入62.5kg大曲粉混匀。
(2)将上述原料放入6个大缸中进行酒精发酵，入缸初始温度约25℃，后期保持18～25℃。前4天敞口发酵，后8天封口发酵，共约12～15天结束。
(3)取上述步骤(2)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(20份瑞士乳杆菌CGMCC12225、8份发酵乳杆菌CGMCC12226、15份耐酸乳杆菌CGMCC16938、7份索诺拉沙漠芽孢杆菌CGMCC15824、6份凝结芽孢杆菌CGMCC17801、10份耐酸中温α-淀粉酶、1.5份普鲁兰酶、3份糖化酶、6份纤维素酶和2份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，搅拌均匀后加入麸皮90kg，谷糠100kg，将酒醪和谷物混合均匀。取本发明的复合菌剂II(40份巴氏醋杆菌CGMCC17802、8份欧洲驹形杆菌CGMCC16345和45份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约2000g于小桶内搅拌均匀后洒在1缸醋醅的上部拌匀，按照山西老陈醋的工艺进行翻醅操作。
(4)待醋醅发酵至总酸不在增加，即发酵结束。加盐后封醅10d，然后经过熏醅、淋醋，最后经煎醋、陈酿、杀菌灌装，即得成品。
2、对比试验
(1)对照组
取上述步骤(2)中的3个400kg大缸，每个缸中加入酒醪200kg，麸皮90kg，谷糠100kg，将酒醪和谷物混合均匀。取市售光明牌酿醋醋酸菌剂40g，添加量为酒醪质量的0.5‰(w/w)，加入30℃酒醪约2000g于小桶内搅拌均匀后洒在1缸醋醅的上部拌匀，按照山西老陈醋的工艺进行翻醅操作，后续步骤同上述步骤(4)。
表3 醋醅主要指标对比(g/100g)
Figure PCTCN2020103450-appb-000004
与对照组相比使用本发明的二元复合发酵剂强化的试验组，发酵时间由9d缩短至7d，缩短2d；熏醅结束后醋醅中总酸含量提高18.26％，不挥发酸含量提高59.18％，氨基酸态氮含量提高94.44％，总酯含量提高45.48％；产品出率提高19.35％；获得产品的风味丰富，口感柔和饱满，感官评分较高。
实施例5：在米醋酿造中的应用
本实施例提供了一种使用发明的二元复合发酵剂在米醋酿造中的应用。
1、试验组
(1)挑选颗粒饱满、无霉变的糯米，除尘后经过粉碎机将糯米粉碎成70～80目的细粉。将粉碎的糯米粉和水，按照糯米粉：水＝1：5(w/w)的比例加入糊化罐中。加入2.5万U/ml高温α-淀粉酶，添加量为每吨糯米加入0.4～0.5L。同时升温至90～95℃，保温30～40min。
(2)利用螺旋式冷却器将醪液温度降至55～60℃，同时转入糖化罐中。加入10万U/g糖化酶，添加量为每吨糯米加入40～50g。温度控为55～60℃，保温30～60min。保温结束后，利用螺旋式冷却器将温度降至25～35℃，同时转入酒精发酵罐。
(3)添加酿酒酵母(购自CICC，菌株保藏编号为CICC 1001)，接种量为5％(v/v)。控制温度为25～35℃，发酵至酒精度约为5％vol。然后添加本发明的复合菌剂I(20份瑞士乳杆菌CGMCC12225、10份发酵乳杆菌CGMCC12226、9份耐酸乳杆菌CGMCC16938、5份索诺拉沙漠芽孢杆菌CGMCC15824、6份凝结芽孢杆菌CGMCC17801、8份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、6份纤维素酶和2份酸性蛋白酶)，添加量为酒醪质量的0.2‰(w/w)，继续发酵至酒精度为8％vol。
(4)选取上述步骤(3)中的1个500L发酵罐，加入200L经过压滤机过滤的澄清酒液，加入本发明的复合菌剂II(35份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和55份葡萄糖)，添加量为酒醪质量的0.15‰(w/w)，调整通气量为0.3～0.4vvm，搅拌速度为250r/min进行醋酸发酵。
(5)发酵结束的醋液采用0.01μm陶瓷膜过滤后，调配酸度，灌装即得成品。
2、对比试验
(1)对照组
选取上述步骤(3)中的1个500L发酵罐，加入200L经过压滤机过滤的澄清酒液，加入市售光明牌酿醋醋酸菌剂，添加量为酒醪质量的0.7‰(w/w)，调整通气量为0.3～0.4vvm，搅拌速度为250r/min进行醋酸发酵，后续步骤通上述步骤(5)。
表4 发酵结束后醋液主要指标对比(g/100ml)
Figure PCTCN2020103450-appb-000005
与对照组相比，试验组的启动速度快，试验组发酵周期缩短23.75％，总酸含量提高11.52％，不挥发酸含量提高12.5倍以上，总酯含量提高14倍以上。试验组制得的苹果醋刺激性显著降低、口感绵柔，具有显著的综合性香气，品质得到显著提升。
实施例6：在苹果醋酿造中的应用
本实施例提供了一种使用本发明的复合发酵剂在苹果醋酿造中的应用。
1、试验组
(1)在2个500L发酵罐中分别加入200L经过调配，糖含量为16～18％(w/w)，pH值为4.5～5.0的苹果汁，接种酵母菌(购自CICC，菌株保藏编号为CICC 1001)，接种量为5％(v/v)，28～30℃发酵至酒精度约为4％vol。
(2)选取上述步骤(1)中的1个发酵罐，加入本发明的复合菌剂I(15份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、10份耐酸乳杆菌CGMCC16938、3份索诺拉沙漠芽孢杆菌CGMCC15824、2份凝结芽孢杆菌CGMCC17801、5份耐酸中温α-淀粉酶、1份普鲁兰酶、2份糖化酶、8份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，保持发酵罐温度为30℃，压力0.05Mpa，搅拌速度为60r/min，发酵至酒精度约7％vol。
(3)在发酵罐中加入本发明的复合菌剂II(50份巴氏醋杆菌CGMCC17802、8份欧洲驹形杆菌CGMCC16345和45份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)。
(4)发酵结束的醋液采用陶瓷膜过滤后，调配酸度，灌装即得成品。
2、对比试验
(1)对照组
选取上述步骤(1)中的1个发酵罐，保持发酵罐温度为30℃，压力0.05Mpa，搅拌速度为60r/min，发酵至酒精度约7％vol；然后加入市售光明牌酿醋醋酸菌剂，添加 量为酒醪质量的0.5‰(w/w)，调整通气量为0.3～0.4vvm，搅拌速度为250r/min进行醋酸发酵，后续步骤同上述步骤(4)。
表5 发酵结束后醋液主要指标对比(g/100ml)
Figure PCTCN2020103450-appb-000006
与对照组相比，试验组的启动速度快，试验组发酵周期缩短18.89％，总酸含量提高14.82％，不挥发酸含量提高10.5倍以上，总酯含量提高19倍以上。试验组制得的苹果醋刺激性显著降低、口感更加绵柔，具有显著的综合性香气，品质得到显著提升。
实施例7：二元复合发酵剂配方及添加方式的对比
本实施例提供了本发明的二元复合发酵剂配方及添加方式的对比，进一步说明本发明的二元复合发酵剂配方组成及其添加方式对应用效果的重要性。
1、试验组
(1)取24个400kg大缸，各取糯米50kg加水浸泡过夜。利用蒸汽将糯米蒸熟，以冷水淋饭至温度约40℃加入酒药0.3kg，拌匀后入缸搭窝成喇叭状。
(2)待窝中有一定的酒液出现时，每缸加入麦曲2.5kg，然后加水150kg，搅拌均匀。
(3)酒精发酵过程中，定期搅拌，温度控制在30℃左右，约发酵5～7d结束。
(4)取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、15份索诺拉沙漠芽孢杆菌CGMCC15824、10份凝结芽孢杆菌CGMCC17801、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、9份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温。
(5)按照镇江香醋酿造工艺进行逐层翻醅，待醋醅发酵至总酸不在增加，即发酵结束。加盐封醅15d，然后加入炒米色进行淋醋。经煎醋、陈酿、杀菌灌装，即得成品。
2、对比试验
(1)对照组A
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复 合菌剂I(15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、10份凝结芽孢杆菌CGMCC17801、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、9份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温，后续步骤同上述步骤(5)。
(2)对照组B
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、15份索诺拉沙漠芽孢杆菌CGMCC15824、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、9份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温。
(3)对照组C
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、15份索诺拉沙漠芽孢杆菌CGMCC15824、10份凝结芽孢杆菌CGMCC17801、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、9份纤维素酶、5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温，后续步骤同上述步骤(5)。
(4)对照组D
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、15份索诺拉沙漠芽孢杆菌CGMCC15824、10份凝结芽孢杆菌CGMCC17801、3份普鲁兰酶、9份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的 0.15‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温，后续步骤同上述步骤(5)。
(5)对照组E
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、15份索诺拉沙漠芽孢杆菌CGMCC15824、10份凝结芽孢杆菌CGMCC17801、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，搅拌均匀后加入麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温，后续步骤同上述步骤(5)。
(6)对照组1
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，加入本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、15份索诺拉沙漠芽孢杆菌CGMCC15824、10份凝结芽孢杆菌CGMCC17801、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、9份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，及本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)，麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)，最后在顶部盖上大糠保温，后续步骤同上述步骤(5)。
(7)对照组2
取上述步骤(3)中的3个400kg大缸，每个缸中加入酒醪200kg，麸皮80kg，大糠45kg，将酒醪和谷物混合均匀(即醋醅)。取本发明的复合菌剂I(25份瑞士乳杆菌CGMCC12225、15份发酵乳杆菌CGMCC12226、20份耐酸乳杆菌CGMCC16938、15份索诺拉沙漠芽孢杆菌CGMCC15824、10份凝结芽孢杆菌CGMCC17801、13份耐酸中温α-淀粉酶、3份普鲁兰酶、5份糖化酶、9份纤维素酶和5份酸性蛋白酶)，添加量为酒醪质量的0.15‰(w/w)，及本发明的复合菌剂II(55份巴氏醋杆菌CGMCC17802、10份欧洲驹形杆菌CGMCC16345和60份葡萄糖)，添加量为酒醪质量的0.1‰(w/w)， 加入30℃酒醪约1000g于小桶内搅拌均匀后洒在1缸醋醅的上部，然后用手向下深入约10cm拌匀，最后在顶部盖上大糠保温，后续步骤同上述步骤(5)。
表6 醅卤主要指标对比(g/100ml)
Figure PCTCN2020103450-appb-000007
与试验组相比，对照组A～E中的总酸、不挥发酸、氨基酸态氮和总酯指标均未达到对照组的效果，说明本发明的二元复合发酵剂配方对确保达到良好的效果具有重要作用。
与试验组相比，对照组1提热速度慢，首次温度达到40℃以上时间由20h延长至103h，延长83h；发酵时间由15d延长至21d，延长6d。对照组2不挥发酸含量显著降低，总酯含量显著降低。
本发明的二元复合发酵剂的配方组成及其添加方式对产生突出的应用效果具有重要影响。

Claims (10)

  1. 一种二元复合发酵剂，包含复合菌剂I和复合菌剂II，其特征在于，复合菌剂I包含瑞士乳杆菌CGMCC12225、发酵乳杆菌CGMCC12226、耐酸乳杆菌CGMCC16938、索诺拉沙漠芽孢杆菌CGMCC15824、凝结芽孢杆菌CGMCC17801、耐酸中温α-淀粉酶、普鲁兰酶、糖化酶、纤维素酶和酸性蛋白酶；复合菌剂II包含巴氏醋杆菌CGMCC17802、欧洲驹形杆菌CGMCC16345和葡萄糖。
  2. 根据权利要求1所述的二元复合发酵剂，其特征在于，所述复合菌剂I按重量份包含10～25份瑞士乳杆菌CGMCC12225、5～15份发酵乳杆菌CGMCC12226、5～20份耐酸乳杆菌CGMCC16938、3～15份索诺拉沙漠芽孢杆菌CGMCC15824、2～10份凝结芽孢杆菌CGMCC17801、5～13份耐酸中温α-淀粉酶、1～3份普鲁兰酶、2～5份糖化酶、3～9份纤维素酶和1～5份酸性蛋白酶；复合菌剂II按重量份包含35～55份巴氏醋杆菌CGMCC17802、2～10份欧洲驹形杆菌CGMCC16345和40～60份葡萄糖。
  3. 根据权利要求1所述的二元复合发酵剂，其特征在于，所述复合菌剂I中瑞士乳杆菌CGMCC12225、发酵乳杆菌CGMCC12226、耐酸乳杆菌CGMCC16938、索诺拉沙漠芽孢杆菌CGMCC15824和凝结芽孢杆菌CGMCC17801的活菌数均为1.0×10 8～1.0×10 10CFU/g，耐酸中温α-淀粉酶、普鲁兰酶、糖化酶、纤维素酶和酸性蛋白酶的酶活力均为2～10万U/g。
  4. 根据权利要求1所述的二元复合发酵剂，其特征在于，所述复合菌剂II中巴氏醋杆菌CGMCC17802和欧洲驹形杆菌CGMCC16345的活菌数1.0×10 7～1.0×10 9CFU/g。
  5. 权利要求1至4任一所述的二元复合发酵剂在食醋酿造中的应用。
  6. 根据权利要求5所述的应用，其特征在于，所述复合菌剂I在醋酸发酵前加入，所述复合菌剂II在醋酸发酵阶段加入。
  7. 根据权利要求5所述的应用，其特征在于，所述二元复合发酵剂与种子醅联合使用。
  8. 根据权利要求5所述的应用，其特征在于，所述食醋酿造为固态食醋发酵或液态食醋发酵。
  9. 根据权利要求5所述的应用，其特征在于，所述二元复合发酵剂采用直投方式加入。
  10. 根据权利要求5所述的应用，其特征在于，所述食醋为镇江香醋、山西老陈醋、米醋或苹果醋。
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