CN117721028B - Hansenula polymorpha strain YN321 in grape juice and application thereof - Google Patents
Hansenula polymorpha strain YN321 in grape juice and application thereof Download PDFInfo
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- CN117721028B CN117721028B CN202410172246.0A CN202410172246A CN117721028B CN 117721028 B CN117721028 B CN 117721028B CN 202410172246 A CN202410172246 A CN 202410172246A CN 117721028 B CN117721028 B CN 117721028B
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- CRDAMVZIKSXKFV-FBXUGWQNSA-N (2-cis,6-cis)-farnesol Chemical compound CC(C)=CCC\C(C)=C/CC\C(C)=C/CO CRDAMVZIKSXKFV-FBXUGWQNSA-N 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)
Abstract
The invention belongs to the technical field of edible vinegar fermentation, and particularly relates to Hansenula polymorpha YN321 and application thereof. The strain YN321 is a non-saccharomyces cerevisiae which can improve the content of non-volatile acid and aroma substances acetoin, isobutyric acid and ethyl acetate of the fresh medlar fruit, and the strain is used for fermenting the fruit vinegar to enrich the aroma substances in the fruit vinegar and improve the content of the non-volatile acid of the fruit vinegar, so that the flavor and the taste of the fruit vinegar are improved. The Hansenula polymorpha (Hanseniaspora uvarum) YN321 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 29509 in the year 2024, 1 and 4.
Description
Technical Field
The invention belongs to the technical field of edible vinegar fermentation, and particularly relates to Hansenula polymorpha YN321 and application thereof.
Background
The fruit vinegar is a sour flavoring which is brewed by taking fruits or processing leftovers thereof as raw materials through alcoholic fermentation and acetic fermentation. The fruit vinegar not only can provide the nutritional value of the edible vinegar, but also has the nutrition and aromatic substances of the fruit.
The microorganism plays a key role in the fermentation process, and specifically, the fermentation of sugar substances in fruits by saccharomycetes and acetic acid bacteria promotes the generation of carboxylic acid-containing substances. The content of the non-volatile acid is closely related to the fermentation metabolism of microorganisms, and the content of the non-volatile acid in the fruit vinegar is lower, but the non-volatile acid is one of important flavor substances forming the fruit vinegar, so that the non-volatile acid can play a role in buffering the irritation of the acetic acid, and the taste of the fruit vinegar is more long-lasting and soft.
Yeast plays a main role in fermentation in vinegar brewing. Saccharomyces cerevisiae has a major advantage in the alcohol fermentation stage in terms of high sugar fermentation metabolism and alcohol tolerance, and has the main effects of converting sugar substances into alcohol under anaerobic conditions and generating other secondary byproducts; some non-saccharomyces cerevisiae, such as ester-producing yeast, can secrete esterase, catalyze alcohols to react with acid substances to generate ester compounds with special aroma, and the ester compounds can influence aroma components of vinegar through metabolism alone or in combination with other microbial interactions, for example, candida, tectorial membrane yeast and pichia pastoris metabolism can generate flavor compounds such as alcohols, aldehydes and the like, so that the ester compounds can contribute to the flavor of vinegar to a certain extent. Therefore, yeast is an indispensable important functional microorganism in the fermentation process of fruit vinegar.
Hansenula polymorpha (Hanseniaspora uvarum) is considered to contribute to the accumulation of floral flavors of wine, and is often used in wine and wine fermentation. For example:
Chinese patent application 202210429351.9 (publication No. CN114989995A; publication day: 2022, 9, 2) discloses a strain of Hansenula polymorpha (Hanseniaspora uvarum) HX17, which is a grape juice strain with strong alcohol producing ability, the grape juice fermented by the strain has strong fragrance, rich ester fragrance and can produce some special fragrant substances, such as 3-methyl-2-butanol, 2-hexadecanol, 2-phenyl ethyl acetate, ethyl nonanoate, heptyl formate and the like. The Chinese patent application 201911406443.X (publication No. CN110951631A; publication No. 2020, 4/3) discloses a strain A14 of Hansenula polymorpha (Hanseniaspora uvarum) with a preservation number of CGMCC No.18666, which has high capacity of fermenting white spirit to produce geraniol, and the concentration of geraniol can reach 63.48 mug/L after 48 hours of fermentation. Chinese patent application 202010265892.3 (publication No. CN111235307A; publication day: 6/5/2020) discloses a composite strain for fermented red date wine comprising Hansenula polymorpha (Hanseniaspora uvarum) in grape juice, which solves the problems of excessive methanol, high residual sugar, high acidity, single flavor and the like, and maintains the rich nutritive value of the fermented red date wine product. Chinese patent application No. 202110090851.X (publication No. CN112746029A; publication day: no. 2021, no. 5, no. 4) provides a strain QTX22 of Hansenula polymorpha (Hanseniaspora uvarum) strain for producing fragrance substances such as farnesol, 2-heptanol, n-hexanol and nerol by fermenting grape juice as a substrate. Chinese patent application 202110308104.9 (publication No. CN113061512A; publication day: 2021, 7, 2) provides a method for multi-strain composite fermentation of honey vinegar, which can improve the non-volatile acid of honey vinegar to 12.89g/L.
The Hansenula polymorpha used for the grape juice for improving the content of the aroma substances such as the non-volatile acid, the acetoin, the isobutyric acid, the ethyl acetate and the like in the fruit vinegar is not reported in the field.
Disclosure of Invention
In order to enrich the sour feeling of the fruit vinegar and improve the taste and flavor of the fruit vinegar, the inventor screens a strain of non-saccharomyces cerevisiae which can improve the content of non-volatile acid and aroma substances acetoin, isobutyric acid and ethyl acetate in fresh fruits of the fruit vinegar, namely, hansenula polymorpha YN321 in grape juice, and the strain is used for fermenting the fruit vinegar to enrich the aroma substances in the fruit vinegar and improve the content of the non-volatile acid in the fruit vinegar, so that the flavor and taste of the fruit vinegar are improved.
The invention provides a technical scheme, which is a strain of Hansenula polymorpha, in particular to Hansenula polymorpha (Hanseniaspora uvarum) YN321, which is preserved in China general microbiological culture Collection center (CGMCC) for 1 month 4 days of 2024, and addresses: the Qingyang area North Star West way 1, 3 China academy of sciences of microbiology, post code 100101, with the preservation number of CGMCC No. 29509;
The Hansenula polymorpha YN321 has the following characteristics:
(1) The colony morphology is: round bump, neat edge, smooth surface, moist, opaque, creamy;
(2) Withstand high sugar pressures: can tolerate 500g/L glucose;
(3) Withstand high alcohol pressures: can tolerate 14% ethanol;
(4) Withstand low pH pressure: tolerance to pH2.75;
(5) Has the performance of producing volatile flavor substances such as benzyl alcohol, 2, 3-butanedione, 2, 4-di-tert-butylphenol, valeric acid, furfural, thearubigin, menthol and the like;
(6) Can increase the content of volatile flavor substances such as acetoin, isobutyric acid, ethyl acetate, phenethyl alcohol, acetoin acetate, caproic acid, 2-acetylpyrrole and the like in the fruit vinegar;
(7) Can increase the content of non-volatile acid in fruit vinegar.
The second technical scheme provided by the invention is the application of Hansenula polymorpha YN321 in grape juice;
Further, the method is applied to vinegar fermentation, particularly to fruit vinegar fermentation, and more particularly to medlar fruit vinegar fermentation;
Further, the method is applied to improving the flavor or the taste of the vinegar; in particular to the application in improving the flavor or taste of fruit vinegar; more particularly in increasing the content of non-volatile acids and volatile flavors in fruit vinegar.
The third technical scheme provided by the invention is a fruit vinegar fermentation method, comprising the following steps:
S1, preparing seed liquid: respectively activating Saccharomyces cerevisiae, hansenula polymorpha YN321 and Acetobacter pasteurella to prepare seed culture solutions;
further, the Saccharomyces cerevisiae is selected from the group consisting of Indonesia Italiana Red vintage wine (ENARTISFERM VINTAGE RED);
Further, the acetobacter pasteurii is selected from acetobacter pasteurii (Acetobacter Pasteurianus) CGMCC No.3089;
s2, preparing fermentation raw materials: squeezing the fruits, adding pectase for enzymolysis, and regulating the sugar degree to 15-20 Brix by using white granulated sugar or sucrose;
Further, the fruits include, but are not limited to, wolfberry, orange, grape, hawthorn, and the like; preferably wolfberry;
further, the enzymolysis method in step S2 is as follows: adding 0.35% -0.4% (w/w) pectase into the juice, and carrying out enzymolysis for 2-24 hours at 50-55 ℃;
further, after enzymolysis, centrifuging and/or filtering to obtain clear juice, and further regulating sugar degree;
s3, alcohol fermentation:
(1) 2% -3% (v/v) of Hansenula polymorpha YN321 seed culture solution of grape juice is inoculated into the fruit juice obtained in the step S2, and the fruit juice is cultured for 2-3 d under the anaerobic condition of 28-30 ℃;
(2) Then 3% -4% (v/v) saccharomyces cerevisiae seed culture solution is inoculated, and anaerobic condition culture is carried out for 5-6 d at 28-30 ℃; fermenting to obtain fruit wine fermentation liquor;
S4, acetic acid fermentation: adding 9.7% -10% (v/v) acetobacter pasteurism seed culture solution into the fruit wine obtained in the step S3, and fermenting to obtain fruit vinegar;
Further, rotating the speed of 180-220 r/min in a shake flask, and culturing at 30-32 ℃ for 5-6 d to obtain fruit vinegar;
Continuously introducing oxygen into the fermentation tank, wherein the oxygen introducing amount is 0.6-0.8L/min, the rotating speed is 1500-2000 rpm, and culturing for 5-6 d at 30-32 ℃ to obtain fruit vinegar;
Further, the method for activating and preparing the seed culture solution in the step S1 comprises the following steps: inoculating loop to select thallus to inoculate in slant culture medium, and culturing at 30 deg.c for 1-2 d; inoculating thalli in the slant culture medium into a test tube liquid culture medium, and standing or shaking culture at 30 ℃ for 24 hours; inoculating the first generation seed liquid into a seed liquid culture medium according to the inoculation amount of 2%, standing or shaking at 30 ℃ for 24 hours to obtain a seed culture liquid;
The preparation method of the seed liquid culture medium comprises the following steps: adding white sugar or sucrose into the juice to adjust sugar degree to 15Brix, and sterilizing at 115deg.C for 20 min.
Further, the fruit vinegar obtained by fermentation in the step S4 is subjected to centrifugal clarification for 10min at 8000r/min, and bacterial sludge is filtered out by using gauze to obtain a finished product.
Compared with the prior art, the invention has the following beneficial effects:
(1) The Hansenula polymorpha YN321 in the grape juice is derived from fresh fruits of medlar, and can tolerate 500g/L glucose, 14% ethanol and pH value of 2.75. Not only is suitable for the synergism of the alcohol fermentation stage and the saccharomyces cerevisiae, but also is suitable for acetic fermentation by synergism of acetic acid bacteria in a low-acidity environment.
(2) The total acid content of the grape juice Hansenula polymorpha YN321 fermented medlar fruit vinegar can reach 74.3g/L, and detection shows that the medlar fruit vinegar has a significant improvement of 43% of the nonvolatile acid content, and the contents of volatile flavor substances such as acetoin, isobutyric acid and ethyl acetate are respectively improved by 4 times, 11 times and 180 times, and meanwhile, the contents of phenethyl alcohol, acetoin acetate, caproic acid and 2-acetylpyrrole are also improved, and the volatile fragrance substances such as benzyl alcohol, 2, 3-butanedione, 2, 4-di-tert-butylphenol, valeric acid, furfural, tea-scented ketone and menthol are produced by fermentation. Therefore, the strain YN321 can be used as a functional bacterium for fermenting fruit vinegar to improve the flavor and the taste of the fruit vinegar, so that the fruit vinegar has soft taste, rich sour feel, rich vinegar fragrance, softness and no irritation, prominent fruit fragrance and pleasant and comfortable fragrance.
(3) The Hansenula polymorpha YN321 in the grape juice can be also applied to brewing of other liquid state fermented vinegar, so that the contents of substances such as fixed acid, acetoin, isobutyric acid, ethyl acetate and the like in the liquid state fermented vinegar can be improved, and the flavor and the taste of the liquid state fermented vinegar are improved.
Drawings
FIG. 1 is a colony chart of Hansenula polymorpha YN321 in grape juice;
FIG. 2 is an electron microscope image of Hansenula polymorpha YN321 in grape juice;
FIG. 3 is a phylogenetic tree of Hansenula polymorpha YN321 in grape juice;
FIG. 4 is a flow chart of the fermentation process of Hansenula polymorpha YN321 for fruit vinegar according to the invention;
FIG. 5 is a graph showing fermentation rates of different strains in the examples of the present invention.
The Hansenula polymorpha (Hanseniaspora uvarum) YN321 of the grape juice is preserved in China general microbiological culture Collection center (CGMCC) for 1 month and 4 days in 2024, and the address is: the collection number of the microbial institute of China, national academy of sciences, no. 3, of West Lu 1, no. North Star, beijing, is CGMCC No. 29509.
Detailed Description
The following detailed description of the embodiments of the invention is given by way of illustration only and not by way of limitation of the scope of the invention, as will be appreciated by those skilled in the art in conjunction with the accompanying drawings and examples. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.
The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.
Unless otherwise indicated, the reagents, methods and apparatus employed in the present invention are conventional food-grade reagents, methods and apparatus in the art.
The process flow of the Hansenula polymorpha YN321 used for fermenting fruit vinegar is shown in figure 4.
The strains adopted in the embodiment of the invention are respectively as follows:
grape juice has Hansenula polymorpha YN321 and a preservation number CGMCC No.29509;
Saccharomyces cerevisiae is selected from the group consisting of Indonesia, italy, red vintage wine (ENARTISFERM VINTAGE RED), official website: https:// www.enartis.com/en/products/wire/yeast/enartisferm-vintage-red/;
Acetobacter pasteurium is selected from Acetobacter pasteurium (Acetobacter Pasteurianus) CGMCC No.3089.
Unless otherwise indicated, the experimental conditions used in the examples of the present invention are conventional experimental conditions in the art. Reagents used in the examples of the present invention are all commercially available unless otherwise specified.
The invention is further illustrated by the following examples.
Example 1 isolation and screening of YN321 Strain
Strain isolation YPD medium: 2% glucose, 2% peptone, 1% yeast extract;
10 strains of yeast are obtained by co-separation of the surface and the endophytic part of the fresh medlar, 6 strains are from endophytic, and 4 strains are from appearance. The specific separation steps are as follows:
S1, separating endophytic saccharomycetes of fresh fruits of Chinese wolfberry:
(1) Pretreatment of fresh wolfberry fruits: and (3) washing out soil and scraps on the surface of the plant by using sterile water, thoroughly washing the plant by using the sterile water, and airing for later use. The plant tissue is put into 0.5 percent calcium hypochlorite solution (or 75 percent alcohol solution) for disinfection of 0.5 to 1 min, then rinsed 3 times in sterile water, dried and ground for use.
(2) Separating: taking 5g of sample and 45mL of sterile water for full grinding; 1mL of the polishing liquid was slowly poured into a test tube containing 9mL of sterile water, to obtain 10 -2 of the dilution liquid, and so on. Selecting proper dilution gradient, coating on YPD solid culture medium plate added with 0.1% ampicillin (inhibiting bacterial growth), culturing at 30deg.C for 48 hr, after colony grows out, picking single colony streak, purifying in YPD culture medium plate, and preserving at 4deg.C for use.
S2, separating the apparent saccharomycetes of the fresh fruits of the medlar:
(1) Pretreatment of fresh wolfberry fruits: and accurately weighing 25g of medlar samples under aseptic operation by adopting a surface sampling method, putting the medlar samples into an triangular flask filled with 225ml of aseptic normal saline, and slightly oscillating for 8-15 minutes. And (5) standby application.
(2) Separating: 1mL of the suspension was slowly poured into a test tube containing 9mL of sterile water for gradient dilution, and so on. The proper dilution gradient liquid is selected and coated on a YPD solid culture medium plate added with 0.1% ampicillin (for inhibiting bacterial growth), the culture is carried out for 48 hours at 30 ℃, after bacterial colonies grow out, single bacterial colonies are selected and streaked on the YPD culture medium plate for purification, and the bacterial colonies are preserved at 4 ℃ for standby.
Bacterial strain primary screening in order to screen out yeast strains suitable for fermentation, the separated yeast is primarily judged whether to have ester fragrance or mellow fragrance by a sniffing method, and bacterial strains with stronger ester fragrance or special fruit fragrance are screened out. The specific operation is as follows:
Inoculating 10 strains of saccharomycetes obtained through separation and purification into a seed liquid culture medium, performing activation culture at 30 ℃ for 24 h%, inoculating the 10 strains of saccharomycetes into a triangular flask filled with 200mL medlar juice in an inoculum size of 4.0%, performing culture at 30 ℃ for 3-5 d, and judging strains with stronger ester aroma or special fruit aroma through sniffing;
The seed liquid culture medium specifically comprises the following components: squeezing fructus Lycii, filtering, adding white sugar to adjust sugar degree to 15Brix, and sterilizing at 115deg.C for 20 min.
The pure yeast 10 strains are obtained by separation and purification. Wherein 6 plants are separated from the interior of fresh fruits of Chinese wolfberry, and 4 plants are separated from the appearance of fresh fruits of Chinese wolfberry. After the Chinese wolfberry juice is further inoculated by a sniffing method, 5 yeasts with stronger fruit fragrance and wine fragrance are screened out, namely YN321, YN127, YN123, YN121 and YW121.
The strain re-screening further performs tolerance and fermentation rate re-screening on 5 strains of yeast obtained by primary screening so as to obtain a strain with good tolerance and fast fermentation rate, and the specific steps are as follows:
s1, inoculating 5 strains obtained by primary screening into YPD culture media with different glucose concentrations (100 g/L, 200g/L, 300g/L, 400g/L and 500 g/L), culturing at 30 ℃ for 48 hours, and observing the growth condition of the strains, wherein the growth condition is shown in table 1:
TABLE 1 results of glucose tolerance experiments with different yeasts
As shown in Table 1, 5 yeasts grow well at sugar concentrations of 100g/L to 300g/L, YN121 and YW121 can tolerate to a sugar concentration of 400 g/L; YN321, YN127 can tolerate sugar concentration up to 500g/L and grow vigorously; in addition, YN123 and YN121 have weak gas production capacity when fermenting glucose, and YW121 does not produce gas when fermenting glucose.
S2, inoculating 5 strains obtained through primary screening into YPD culture media with different ethanol concentrations (6%, 8%, 10%, 12%, 14%), culturing at 30 ℃ for 48 hours, and observing the growth condition of the strains, wherein the growth condition is shown in table 2:
TABLE 2 results of ethanol tolerance experiments with different yeasts
As shown in Table 2, the ethanol tolerance of the strains YN127 and YW121 is slightly poorer than that of the other 3 strains, and the other 3 strains of yeasts can tolerate the ethanol concentration of 6% -12% to different degrees, wherein the strain YN321 can tolerate the ethanol to 14%.
S3, inoculating 5 strains obtained by primary screening into YPD culture mediums with different pH values (2.5, 2.75, 3.0, 3.5 and 4.0), culturing at 30 ℃ for 48 hours, and observing the growth condition of the strains, wherein the growth condition is shown in table 3:
TABLE 3 results of pH tolerance experiments with different yeasts
The brewing process of edible vinegar in China comprises an alcohol fermentation stage and an acetic acid fermentation stage, the total amount of organic acid is continuously increased in the whole process, wherein the concentration of the most main acetic acid and lactic acid can reach 1% -7% and 1% -6% respectively, the whole system is in an acidic environment, and therefore the synergistic effect of the main acetic acid and lactic acid can be exerted in the acetic acid fermentation stage when saccharomycetes have strong acid resistance. As is clear from Table 3, YN321, YN123 and YW121 were among the strains having a tolerance of pH3.0 or less, and the strains YN321 and YW121 had the highest acid resistance and were able to grow at pH 2.75.
S4, further inoculating 5 strains into triangular flasks filled with 200mL of medlar juice respectively, standing at 30 ℃ for 8 days, and monitoring the loss of weight of CO 2 every 1d to evaluate the fermentation rate of saccharomycetes in the fermentation process. The detection results are shown in FIG. 5.
As can be seen from FIG. 5, the fermentation rate of strain YN321 was the fastest during fermentation of the juice of Lycium barbarum, and the fermentation rate of strain YN127 was comparable to that of strain YN321 in the first three days, but YN321 was far ahead of YN127 from the fourth day later.
In summary, in combination with the experimental results of glucose, ethanol, pH tolerance and fermentation rate of the strain, the strain YN321 has the best performance of fermenting the fruit vinegar of Lycium barbarum, and therefore, the strain YN321 is taken as the target strain of the invention.
Bacterial colony of strain YN321 on YPD plate culture medium is milky white, raised, round, smooth in surface, regular in edge, opaque and creamy, and has a colony morphology as shown in FIG. 1 and a microscopic morphology as shown in FIG. 2. The strain YN321 is identified by ITS gene sequence, the similarity of the result and Hansenula polymorpha (Hanseniaspora uvarum) in grape juice is 100%, and a phylogenetic tree is constructed by adopting MEGA7.0 software and is shown in figure 3, so that the strain YN321 can be determined to be a strain of Hansenula polymorpha, the strain is named as Hansenula polymorpha (Hanseniaspora uvarum) YN321 in grape juice and is preserved in China general microbiological culture collection center with the preservation number of CGMCC No. 29509.
Example 2 grape juice Hansenula polymorpha YN321 for liquid fermentation of fruit vinegar of Lycium barbarum
S1, respectively activating Saccharomyces cerevisiae (Saccharomyces cerevisiae) ENARTISFERM VINTAGE RED, hansenula polymorpha (Hanseniaspora uvarum) YN321, acetobacter pasteurella (Acetobacter pasteurianus) CGMCC No.3089 to prepare a seed culture solution; the method comprises the following steps:
The method for activating the saccharomycetes and preparing the seed culture solution comprises the following steps: inoculating loop to select thallus to inoculate in YPD slant culture medium, and culturing at 30 deg.C for 1-2 d; inoculating thalli in the slant culture medium into a YPD test tube liquid culture medium, and performing stationary culture at 30 ℃ for 24 hours; inoculating the first generation seed liquid into a seed liquid culture medium according to the inoculation amount of 2%, and standing and culturing for 24 hours at the temperature of 30 ℃ to obtain a seed culture liquid;
The method for activating the acetic acid bacteria and preparing the seed culture solution comprises the following steps: inoculating loop to select thallus to be inoculated in GY slant culture medium, and culturing at 30 ℃ for 1-2 d; inoculating the thallus in the slant culture medium into GY test tube liquid culture medium, shake culturing at 30deg.C at 180r/min for 24 hr; inoculating the first generation seed liquid into a seed liquid culture medium according to an inoculum size of 2%, and performing shake culture at 30 ℃ for 24 hours at 180r/min to obtain a seed culture liquid;
YPD medium: 20.0g of peptone, 10.0g of yeast extract and 20.0g of glucose were added to 1L of pure water; the slant culture medium is added with 2% (w/w) agar powder.
GY medium: glucose 20g, yeast extract 20g and ethanol 3.5% (v/v) were added to 1L of pure water; the slant culture medium is added with 2% (w/w) agar powder and no ethanol.
Seed liquid culture medium: re-hydrating fructus Lycii, squeezing, filtering, adding white sugar to adjust sugar degree to 15Brix, and sterilizing at 115deg.C for 20 min;
S2, rehydrating and juicing the dried medlar fruits, adding 0.35% (w/w) pectase, and carrying out water bath at 50 ℃ for 2 hours; centrifuging at 8000r/min for 10min after enzymolysis, filtering with 4 layers of gauze to obtain clear juice, and regulating sugar degree to 15Brix with white sugar;
The rehydration conditions are as follows: adding purified water into the mixture according to the feed liquid ratio of 1:5 (w/w) for soaking for 12 hours; squeezing all the feed liquid after soaking;
s3, inoculating 3% of Hansenula polymorpha YN321 seed culture solution of grape juice into the medlar juice obtained in the step S2, and culturing for 3 days at 30 ℃ under anaerobic conditions; then inoculating 3% of saccharomyces cerevisiae ENARTISFERM VINTAGE RED seed culture solution, and culturing for 6d at 30 ℃ under anaerobic condition; fermenting to obtain fermentation liquor of the medlar wine;
s4, adding 10% of Acetobacter pasteurism CGMCC No.3089 seed culture solution into the medlar wine obtained in the step S3, culturing for 6d at the temperature of 30 ℃ at 180r/min, and fermenting to obtain medlar fruit vinegar. This was used as an experimental group.
Blank control: taking medlar fruit vinegar which is not inoculated with YN321 for fermentation as a blank control, and the rest steps are the same as the steps;
Control group 1: the strain with the number of YN123 screened in the inoculation example 1 is used as a control group 1 instead of YN 321;
After fermentation, volatile flavor substances in the medlar fruit vinegar are detected by adopting an adsorption pen and a gas chromatography-mass spectrometer, and the results are shown in table 4:
TABLE 4 detection results of Main volatile flavor substances of Lycium fruit Vinegar
As can be seen from Table 4, 1) the volatile flavor of the fruit vinegar of Lycium barbarum fermented by Hansenula polymorpha YN321 is more abundant than that of the blank group, and the non-detected substances of the blank group are newly added, for example: benzyl alcohol, 2, 3-butanedione, 2, 4-di-tert-butylphenol, valeric acid, furfural, thearubigin and menthol; there is a significant increase in the content of the same volatile flavour, for example: acetoin is increased by 4 times, isobutyric acid is increased by 11 times, and ethyl acetate is increased by 180 times. 2) Compared with the control group 1, the new volatile flavor substances are detected in the medlar fruit vinegar fermented by YN 321: caproic acid, theaketone and menthol, and the same volatile flavor content was higher than that of the control group. It can be seen that acetoin, isobutyric acid, and ethyl acetate were also produced in the medlar fruit vinegar fermented with strain YN123, but the content and variety of volatile flavor substances were far less abundant than YN 321.
Example 3 detection of non-volatile acid and sensory evaluation of Lycium Barbarum fruit Vinegar
The content of the non-volatile acid is closely related to the fermentation metabolism activity of microorganisms, and the content of the non-volatile acid is lower, but the non-volatile acid is one of important flavor substances forming the medlar vinegar, and has a buffering effect on acetic acid irritation, so that the flavor of the fruit vinegar is more durable and softer. The fermented medlar fruit vinegar in example 2 was subjected to non-volatile acid detection, and meanwhile, medlar fruit vinegar in a control group 1 without YN321 fermentation was used as a control, and the detection results are shown in Table 5:
TABLE 5 detection results of non-volatile acids in Lycium barbarum fruit vinegar
As can be seen from the results of Table 5, the use of Hansenula polymorpha YN321 in grape juice can significantly increase the content of the non-volatile acid in the fruit vinegar, effectively reduce the irritation of the volatile acid and improve the taste of the fruit vinegar.
In order to verify the quality of the medlar fruit vinegar fermented by Hansenula polymorpha YN321, sensory evaluation was performed by a sensory evaluation group. The sensory evaluation panel consisted of 10 experienced professional sensory evaluators (5 men and 5 women), the sensory scores are shown in table 6:
TABLE 6 sensory evaluation criteria for Lycium Chinense Vinegar
The sensory scoring results are shown in table 7:
TABLE 7 sensory scoring results for Lycium barbarum fruit vinegar
In summary, fermenting the medlar fruit vinegar by using the Hansenula polymorpha YN321 in grape juice can improve the content of fixed acid and the quality taste of the medlar fruit vinegar, which indicates that YN321 can be used as a functional production strain of the medlar fruit vinegar.
EXAMPLE 4 Hansenula polymorpha YN321 in grape juice was used for 5L fermentation tank cultivation in an enlarged scale
S1, rehydrating and juicing dried medlar, adding 0.4% pectase, and carrying out water bath at 50 ℃ for 2 hours; centrifuging at 8000r/min for 10min after enzymolysis, filtering with 4 layers of gauze to obtain clear juice, and regulating sugar degree to 15Brix with white sugar;
The rehydration conditions are as follows: adding purified water into the mixture according to the feed liquid ratio of 1:5 (w/w) for soaking for 12 hours; squeezing all the feed liquid after soaking;
S2, adding 3.5L of medlar juice into a fermentation tank, inoculating 3% grape juice Hansenula polymorpha YN321 seed culture solution into the fermentation tank, and culturing for 3d at 30 ℃ under anaerobic condition; then inoculating 3% of saccharomyces cerevisiae ENARTISFERM VINTAGE RED seed culture solution, and culturing for 6d at 30 ℃ under anaerobic condition; fermenting to obtain fermentation liquor of the medlar wine; the preparation method of the fermentation strain and the seed liquid is the same as in example 2;
s3, adding 10% of Acetobacter pasteurisum seed culture solution into the medlar wine obtained in the step S2, regulating the fermentation temperature to 30 ℃, continuously introducing oxygen, wherein the oxygen introducing amount is 0.6L/min, the rotating speed is 2000rpm, and fermenting for 6d to obtain medlar vinegar.
The medlar fruit vinegar prepared by the method has good flavor, total acid of 78.0g/L, nonvolatile acid of 19.01g/L, strong vinegar fragrance of Yu Rou, no irritation, prominent fruit fragrance, pleasant fragrance and comfort, and sensory score of 85.9 minutes.
The GC-MS was used to detect the volatile aroma substances in the prepared medlar fruit vinegar, and the total of 60 volatile aroma substances in the medlar fruit vinegar prepared in this example includes 2 kinds of acids, 16 kinds of alcohols, 16 kinds of esters, 10 kinds of ketones, 8 kinds of aldehydes, 1 kind of furans, 2 kinds of pyrazines, 2 kinds of phenols, 2 kinds of alkanes and 1 kind of pyrroles. See in particular table 8:
table 8 volatile flavor results for matrimony vine fruit vinegar
Example 5 grape juice Hansenula polymorpha YN321 for fermenting orange fruit Vinegar
S1, selecting ripe fruits which are free of rot and insect damage to juice, removing kernels and peeling before juice extraction.
S2, adding 0.35% pectase, carrying out enzymolysis for 2 hours at 50 ℃, and filtering by using 4 layers of gauze.
S3, determining the initial sugar content in the orange juice, and regulating the sugar content to 20Brix by using white granulated sugar.
S4, alcohol fermentation: taking clarified orange juice with adjusted sugar degree as a raw material, inoculating 3% grape juice Hansenula polymorpha YN321 seed culture solution, and fermenting for 3d at 28 ℃; then inoculating 3% of saccharomyces cerevisiae seed culture solution, and fermenting for 6d at 28 ℃; obtaining orange wine; the preparation of the fermentation strain and seed fluid was different from example 2 only in the seed fluid medium.
The preparation method of the seed liquid culture medium comprises the following steps: squeezing fructus Citri sinensis, filtering, adding white sugar to regulate sugar degree to 15Brix, and sterilizing at 115deg.C for 20 min;
s5, adding naringinase into the orange wine obtained in the step S4 to remove the bitter taste, wherein the addition amount of the naringinase is 0.6 g/L; the debittering condition is pH7, the enzymolysis temperature is 50 ℃, and the enzymolysis time is 60 min.
S6, adding 10% acetobacter pasteurism seed culture solution into the debitterized orange wine obtained in the step S5, culturing at 220r/min and 30 ℃ for 6d, and fermenting to obtain orange fruit vinegar.
The orange fruit vinegar prepared by the method has good flavor, total acid of 64.7g/L, non-volatile acid of 15.64g/L, strong vinegar fragrance Yu Rou without irritation, prominent fruit fragrance, pleasant fragrance and comfort, and sensory score of 80 minutes.
Example 6 grape juice Hansenula polymorpha YN321 for fermenting Hawthorn fruit Vinegar
S1, preparing a hawthorn liquid: cleaning hawthorns, removing cores, and performing processing on the hawthorns: the water is 1:1, adding water in proportion, crushing, squeezing juice, adding 0.35% pectase, carrying out water bath at 50 ℃ for 2 hours, and filtering; the sugar degree of the haw juice is regulated to 15Brix by white granulated sugar.
S2, inoculating 3% of Hansenula polymorpha YN321 seed culture solution of grape juice into the haw juice obtained in the step S1, and fermenting for 3d at 28 ℃; then inoculating 3% of saccharomyces cerevisiae seed culture solution, and fermenting for 6d at 28 ℃; obtaining hawthorn wine; the fermentation strain and seed fluid preparation method were the same as in example 2, except that the seed fluid medium was different.
The preparation method of the seed liquid culture medium comprises the following steps: squeezing fructus crataegi with water (1:1), filtering, adding white sugar to adjust sugar degree to 15Brix, and sterilizing at 115deg.C for 20 min;
S3, adding 9.7% of acetobacter pasteurism seed culture solution into the hawthorn wine obtained in the step S2, culturing for 5 days at the temperature of 30 ℃ at 180r/min, and fermenting to obtain the hawthorn vinegar.
The haw vinegar prepared by the method has good flavor, total acid content of 70.5g/L, non-volatile acid content of 14.63g/L, strong vinegar fragrance Yu Rou, no irritation, pleasant fragrance and comfort, and sensory score of 77.5 minutes.
EXAMPLE 7 Hansenula polymorpha YN321 in grape juice for liquid fermentation of grape Vinegar
S1, sorting grapes: and (3) taking the grapes with good maturity, removing green, malformation, diseases and insects, broken spike grains and sundries such as grape leaves.
S2, squeezing grapes: and (3) removing stems manually, squeezing the grapes by a small squeezer, and crushing seeds without tearing peel.
S3, clarifying grape juice: adding 0.4% pectase to grape juice, standing for 24 hr, separating clear juice, and regulating sugar degree to 15Brix with sucrose to obtain clarified grape juice.
S4, inoculating 2% of Hansenula polymorpha YN321 into the clarified grape juice prepared in the step S3, fermenting at 28 ℃ for 2d, inoculating 4% of saccharomyces cerevisiae seed culture solution, controlling the fermentation temperature at 28 ℃, and fermenting for 6d to obtain grape wine; the fermentation strain and seed fluid preparation method were the same as in example 2, except that the seed fluid medium was different.
The preparation method of the seed liquid culture medium comprises the following steps: squeezing grape to obtain juice, filtering, adding sucrose into grape juice to adjust sugar degree to 15Brix, and sterilizing at 115deg.C for 20 min;
s5, adding 10% acetobacter pasteurii seed culture solution into the wine obtained in the step S4, culturing at 220r/min and 30 ℃ for 5d, and fermenting to obtain the grape vinegar.
The grape vinegar prepared by the method has good flavor, total acid content of 68.9g/L, non-volatile acid content of 16.83g/L, strong vinegar fragrance Yu Rou, no irritation, prominent fruit fragrance, pleasant and comfortable fragrance, and sensory score of 78.1 minutes.
It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (9)
1. The Hansenula polymorpha strain is characterized by specifically comprising Hansenula polymorpha (Hanseniaspora uvarum) YN321 with a preservation number of CGMCC No. 29509.
2. The use of hansenula polymorpha YN321 in grape juice according to claim 1, characterized in that it is used in vinegar fermentation.
3. The use of hansenula polymorpha YN321 in grape juice according to claim 1, characterized in that it is used in fruit vinegar fermentation.
4. The use according to claim 3, in fermentation of fruit vinegar of Lycium barbarum.
5. A fruit vinegar fermentation method, which is characterized by comprising the following steps:
S1, preparing seed liquid: respectively activating Saccharomyces cerevisiae, hansenula polymorpha YN321 and Acetobacter pasteurella to prepare seed culture solutions;
s2, preparing fermentation raw materials: squeezing the fruits, adding pectase for enzymolysis, and regulating the sugar degree to 15-20 Brix by using white granulated sugar or sucrose;
s3, alcohol fermentation:
(1) Inoculating Hansenula polymorpha YN321 seed culture solution with grape juice into the fruit juice obtained in the step S2, and culturing for 2-3 d at 28-30 ℃ under anaerobic conditions;
(2) Then inoculating a saccharomyces cerevisiae seed culture solution, and culturing for 5-6 d at 28-30 ℃ under anaerobic condition; fermenting to obtain fruit wine fermentation liquor;
S4, acetic acid fermentation: adding Acetobacter pasteurism seed culture solution into the fruit wine obtained in the step S3, and fermenting to obtain fruit vinegar;
the grape juice has Hansenula polymorpha YN321 with a preservation number of CGMCC No. 29509.
6. The method according to claim 5, wherein S1 is selected from the group consisting of Indonesia, italian Red vintage ENARTISFERM VINTAGE RED; the Acetobacter pasteurium is selected from Acetobacter pasteurium (Acetobacter Pasteurianus) CGMCC No.3089.
7. The method for fermenting fruit vinegar according to claim 5, wherein the enzymolysis method in S2 is as follows: and adding 0.35-0.4% pectase into the juice, and carrying out enzymolysis for 2-24 hours at 50-55 ℃.
8. The method for fermenting fruit vinegar according to claim 5, wherein the conditions for fermenting the fruit vinegar in the step S4 are as follows: rotating the speed of 180-220 r/min in a shaking flask, and culturing at 30-32 ℃ for 5-6 d to obtain fruit vinegar; the fermentation conditions of the fermentation tank are as follows: continuously introducing oxygen into the fermentation tank, wherein the oxygen introducing amount is 0.6-0.8L/min, the rotating speed is 1500-2000 rpm, and culturing for 5-6 d at 30-32 ℃ to obtain the fruit vinegar.
9. The method for fermenting fruit vinegar according to claim 5, wherein the fruit vinegar obtained by fermenting in step S4 is obtained by centrifuging and clarifying 8000r/min for 10min, and filtering out bacterial sludge to obtain the fruit vinegar finished product.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694371A (en) * | 2015-02-06 | 2015-06-10 | 丽水学院 | Citrus fruit vinegar prepared by composite strain mixed fermentation and preparation method thereof |
| CN106434264A (en) * | 2016-11-14 | 2017-02-22 | 天津科技大学 | Method for strengthening traditional solid fermentation of vinegar by mixed agent and application of mixed agent |
| CN111607621A (en) * | 2020-05-08 | 2020-09-01 | 晶叶(银川)生物科技有限公司 | Yeast capable of producing rose fragrance and application of yeast in Lingwu jujube enzyme |
| CN112625928A (en) * | 2021-01-15 | 2021-04-09 | 江南大学 | Hansenula polymorpha strain capable of increasing wine brewing aroma |
| CN117264789A (en) * | 2023-09-05 | 2023-12-22 | 云南大学 | Hansenula polymorpha strain and application thereof in preparation of blueberry fruit wine |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694371A (en) * | 2015-02-06 | 2015-06-10 | 丽水学院 | Citrus fruit vinegar prepared by composite strain mixed fermentation and preparation method thereof |
| CN106434264A (en) * | 2016-11-14 | 2017-02-22 | 天津科技大学 | Method for strengthening traditional solid fermentation of vinegar by mixed agent and application of mixed agent |
| CN111607621A (en) * | 2020-05-08 | 2020-09-01 | 晶叶(银川)生物科技有限公司 | Yeast capable of producing rose fragrance and application of yeast in Lingwu jujube enzyme |
| CN112625928A (en) * | 2021-01-15 | 2021-04-09 | 江南大学 | Hansenula polymorpha strain capable of increasing wine brewing aroma |
| CN117264789A (en) * | 2023-09-05 | 2023-12-22 | 云南大学 | Hansenula polymorpha strain and application thereof in preparation of blueberry fruit wine |
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