CN114196558A - Extraction method and application of salt-tolerant yeast - Google Patents
Extraction method and application of salt-tolerant yeast Download PDFInfo
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- CN114196558A CN114196558A CN202111649106.0A CN202111649106A CN114196558A CN 114196558 A CN114196558 A CN 114196558A CN 202111649106 A CN202111649106 A CN 202111649106A CN 114196558 A CN114196558 A CN 114196558A
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Abstract
The invention relates to a method for extracting salt-tolerant yeast and application, adopting a part which is 5 to 10cm below a sauce mash and is not stirred in a fermentation tank as a sample, preparing A, B culture medium, wherein the component of the culture medium A is 1 to 10g of glucose, 0.1 to 2g of yeast powder, 0.1 to 1g of polypeptone, 0.1 to 2g of potassium dihydrogen phosphate, 1 to 8g of agar powder and 100g of water, the component of the culture medium B is a mixture of soy sauce, salt and water, uniformly mixing A, B, adding the mixture into the sample to prepare suspension, and obtaining the salt-tolerant yeast through flat-plate separation and purification; the extraction of the yeast is realized by the method, and after the yeast is subjected to enlarged culture, the produced alcohol has good aroma, special sweet taste and distinguishing taste; the salt-tolerant yeast is subjected to expanded culture and added into soy sauce mash as a dominant bacterium to participate in the fermentation process, so that the beany flavor of the soy sauce can be effectively reduced, the special fragrance of the salt-tolerant yeast can enrich the flavor of the soy sauce, and the soy sauce has better discrimination than other soy sauce.
Description
Technical Field
The invention belongs to the technical field of soy sauce preparation, and particularly relates to an extraction method and application of salt-tolerant yeast.
Background
The soy sauce can improve the color, the fragrance and the taste of food, is popular with people, is one of indispensable condiments in daily life, and has good nutritional value. In the preparation process of the soy sauce, the yeast plays an important role in the preparation process, so that the soy sauce generates special aroma and is a main source of sauce aroma.
However, most of the soy sauce in the current market has strong beany flavor, and the soy sauce flavor which is thin and thin is often covered, so that the soy sauce has single function, and therefore, an extraction method and application of salt-tolerant yeast are provided to solve the problems.
Disclosure of Invention
In view of the problems that soy sauce produced by the prior art has heavy beany flavor, the flavor of the soy sauce is monotonous, and the soy sauce is often covered by the beany flavor, the invention provides an extraction method of salt-tolerant yeast, and the specific extraction method comprises the following steps:
selecting a sample: taking a part of unstirred sauce mash which is 5 to 10 centimeters downwards from the surface as a sample in a fermentation tank for later use;
preparing a culture medium: taking the prepared culture medium A for later use;
taking 10-30g of raw soy sauce, 25-30g of salt and 70-90g of water from the soy sauce pool, and filling the raw soy sauce, the salt and the water into a conical flask marked as a culture medium B for standby;
and (3) disinfection treatment: sealing the culture medium A, B respectively, sterilizing in autoclave at 121 deg.C for no more than 5min, and placing in 70 deg.C water bath;
preparing a reagent: preparing and sterilizing a plurality of 9mL test tubes of 15% saline and a plurality of 90g conical bottles of 15% saline in advance, and cooling for later use;
preparing a culture dish: pouring the mixture of the culture medium B in the conical flask into the culture medium A in the conical flask, uniformly stirring the culture medium A on a magnetic stirrer, then moving the culture medium A to a super-clean workbench, quickly pouring the culture medium A into a marked culture dish when the culture medium A is hot beside the flame of an alcohol lamp, opening a culture dish cover, opening a fan to accelerate solidification of the culture medium B, cooling and drying the culture medium B, and then covering the culture dish cover for later use;
preparing a suspension: adding 10g of the soy sauce mash sample into a conical flask containing 90g of 15% salt water, and mixing to obtain 10g of soy sauce mash sample-1Suspension a; further, the suspension a1ml was aspirated and dropped into a test tube containing 9ml of 15% saline solution, and the mixture was diluted to 10-2Suspension b, prepared by the same method 10-3Suspension c, 10-4Suspension d;
inoculating a culture dish: respectively sucking 0.1ml of the suspension a, the suspension b, the suspension c and the suspension d and respectively smearing the suspension on a marked culture dish flat plate, preparing a parallel sample for each sample liquid, putting the four culture dishes into a group after the parallel samples are prepared, and inversely placing the groups in an incubator for constant-temperature culture at 30 ℃;
selecting strains: continuously culturing for 6 days, observing growth condition of yeast colony, recording, selecting culture dish with good growth vigor of yeast, selecting colony, streaking and purifying on new culture dish, and culturing in incubator;
selecting the yeast with better growth vigor after purification, inoculating the yeast into a prepared preservation culture test tube, placing the culture test tube in an incubator at 30 ℃, and after four to five days of culture, transferring the yeast to a refrigerator at 6 ℃ for cold preservation when the bacterial colony of the yeast grows well, thus completing the preparation of yeast extraction.
The components of the culture medium A are 1-10g of glucose, 0.1-2g of yeast powder, 0.1-1g of polypeptone, 0.1-2g of monopotassium phosphate, 1-8g of agar powder, 70-90g of water and one stirring rotor.
The stirring piece is made of tetrachloroethylene material and does not react with other materials.
In the yeast extraction process, a plurality of prepared test tubes of 9ml of 15% saline water, a plurality of silica gel plugs covered, a plurality of conical flasks of 90g of 15% saline water, a plurality of tinfoil seals, a plurality of pipettes, a plurality of culture dishes, a plurality of coating rods and a plurality of medicine spoons are required, and the whole process is placed in an autoclave for sterilization at 121 ℃ for 15 minutes.
High-temperature disinfection: high-temperature sterilization is carried out by adopting an autoclave.
The fermentation tank manufacturing steps comprise:
firstly, starter propagation: mixing 8-14 tons of wheat flour, 8-14 tons of soybean meal and 20-40kg of yeast according to a proportion, and then sending the mixture to a disc yeast making chamber, adjusting oxygen supply, humidity and temperature through a fan air conditioning system in the yeast making process, controlling the temperature of the materials to be not more than 35 ℃, and culturing for about 40 hours.
② mash preparation: adding 30-40 tons of cooling salt water with the concentration of 15% -25% into the finished koji, and sending the finished koji into a fermentation tank to prepare 60 tons of sauce mash.
The application of salt tolerant yeast comprises performing amplification culture on the above extracted yeast, adding yeast solution obtained by amplification culture into a fermentation tank, and fermenting and brewing with soy sauce mash.
And (3) yeast expanding culture step:
first amplification culture: inoculating with alcohol lamp on clean bench, burning the inoculating needle with outer flame, cooling slightly, picking out yeast strain on the preservation culture medium, inoculating into liquid culture medium C prepared in advance, and determining to inoculate several rings of strains according to actual conditions. And (3) ventilating the liquid culture medium C for culture, continuously culturing for three days, observing the conditions in the culture medium, and finishing the enlarged culture when rich and fine white foams overflow from a culture medium bottle and are accompanied with strong wine aroma and special fragrant sweet taste. At this time, a great amount of yeast is deposited at the bottom of the bottle.
And (3) second amplification culture: based on the steps, secondary expanding culture of the yeast is continued in the processing workshop, and the yeast is added into the fermentation tank after the secondary expanding culture is completed, so that the fermentation dominant bacteria are established to participate in the fermentation process until the soy sauce mash lightens the beany flavor and generates special aroma.
The preparation method of the liquid culture medium C comprises the following steps: placing 350g glucose, 400g salt, 350g raw juice of soy sauce (obtained by fermenting, squeezing, cooling, precipitating, sterilizing, precipitating, and filtering common soy sauce mash) in 5L conical flask, adding water to 3500ml, sealing with tin foil, sterilizing at 121 deg.C for 15 min, sterilizing, and cooling.
The yeast solution added: 1 ton (yeast count: 2.0X 10)8More than one/ml).
The stirring mode of the fermentation tank is as follows:
firstly, each batch of materials is completely fed, and stirring is carried out for 10 minutes for 1 time.
② 2 to 6 days, stirring for 20 minutes for 1 time every other day.
③ 10 to 60 days, stirring for 15 minutes 1 time per week.
Fourthly, on day 60, stirring for 30min for 1 time when yeast is added, and keeping for 3 days.
Fifthly, stirring for 10min for 1 time every 5 days for 60-120 days.
Sixthly, on the 120 th day, after the sauce mash is fully dissolved, stirring for 5min for 1 time every 15 days.
And seventhly, stirring is carried out before material taking each time, and the stirring is mainly uniform.
And stirring the mixture before squeezing and pumping the mixture, wherein the stirring is mainly uniform.
Fermentation tank temperature:
the initial stage of mash preparation is kept at a low temperature below 15 ℃.
Secondly, heating is started in 20-30 days, and is stopped when the temperature reaches 28 ℃ in 50-60 days.
③ controlling the temperature in the main fermentation period to be between 28 and 32 ℃ and not more than 32 ℃, and recording.
Fourthly, beginning to cool about 120 days until reaching about 30 ℃ until the next working procedure.
Taking materials from the sauce mash, wherein sampling is carried out before adding yeast, and the other sampling time is 1 day, 30 days, 90 days, 120 days and 180 days until the next procedure.
Compared with the prior art, the invention has the beneficial effects that:
the invention realizes the extraction work of the saccharomycetes by the method, the extracted saccharomycetes generate alcohol with good aroma, no pungent smell, special fragrant and sweet taste and relatively distinctive taste after amplification culture, and the alcohol is subjected to secondary amplification culture and added into sauce mash in a proper amount to be determined as fermentation dominant bacteria, so that the beany flavor of the soy sauce can be effectively reduced in the subsequent fermentation production process of the soy sauce, the special aroma of the soy sauce can enrich the flavor of the soy sauce, and the soy sauce has better distinctiveness compared with other soy sauce.
Drawings
FIG. 1 is an electronic nose data radar chart of five different soy sauce
FIG. 2 is a PCA analysis chart of five different soy sauces
FIG. 3 shows the comparison of the types and relative contents of five soy sauce esters
FIG. 4 shows the comparison of the types and relative contents of five kinds of soy sauce alcohol substances
FIG. 5 shows the comparison of the types and relative contents of five kinds of acids in soy sauce
FIG. 6 shows comparison of types and relative contents of five kinds of aldehydes in soy sauce
FIG. 7 is a 26SrDNA sequencing sequence diagram of the yeast of the present invention
FIG. 8 is a table of specific flavor components of soy sauce to which the present invention is applied
FIG. 9 is a table showing the characteristic of unique aroma components of soy sauce portions after application of the present invention
Detailed Description
The technical solution of the present invention will be described in detail and fully with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated by reference herein in their entirety. In case of conflict, the present specification, including definitions, will control.
All percentages, parts, ratios, etc., are by weight unless otherwise indicated; additional instructions include, but are not limited to, "wt%" means weight percent, "mol%" means mole percent, "vol%" means volume percent.
The materials, methods, and examples described herein are illustrative only and not intended to be limiting unless otherwise specified. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
The present invention provides:
a method for extracting salt-tolerant yeast comprises the following steps:
selecting a sample: taking a part of unstirred sauce mash which is 5 to 10 centimeters downwards from the surface as a sample in a fermentation tank for later use;
preparing a culture medium: taking the prepared culture medium A for later use;
taking 10-30g of raw soy sauce, 25-30g of salt and 70-90g of water from the soy sauce pool, and filling the raw soy sauce, the salt and the water into a conical flask marked as a culture medium B for standby;
and (3) disinfection treatment: sealing the culture medium A, B respectively, sterilizing in autoclave at 121 deg.C for no more than 5min, and placing in 70 deg.C water bath;
preparing a reagent: preparing and sterilizing a plurality of 9mL test tubes of 15% saline and a plurality of 90g conical bottles of 15% saline in advance, and cooling for later use;
preparing a culture dish: pouring the mixture of the culture medium B in the conical flask into the culture medium A in the conical flask, uniformly stirring the culture medium A on a magnetic stirrer, then moving the culture medium A to a super-clean workbench, quickly pouring the culture medium A into a marked culture dish when the culture medium A is hot beside the flame of an alcohol lamp, opening a culture dish cover, opening a fan to accelerate solidification of the culture medium B, cooling and drying the culture medium B, and then covering the culture dish cover for later use;
preparing a suspension: adding 10g of the soy sauce mash sample into a conical flask containing 90g of 15% salt water, and mixing to obtain 10g of soy sauce mash sample-1Suspension a; further, the suspension a1ml was aspirated and dropped into a test tube containing 9ml of 15% saline solution, and the mixture was diluted to 10-2Suspension b, prepared by the same method 10-3Suspension c, 10-4Suspension d;
inoculating a culture dish: respectively sucking 0.1ml of the suspension a, the suspension b, the suspension c and the suspension d and respectively smearing the suspension on a marked culture dish flat plate, preparing a parallel sample for each sample liquid, putting the four culture dishes into a group after the parallel samples are prepared, and inversely placing the groups in an incubator for constant-temperature culture at 30 ℃;
selecting strains: continuously culturing for 6 days, observing growth conditions of yeast colonies, recording, selecting a culture dish with good growth vigor of the yeast, selecting the colonies, placing the colonies on a new culture dish, streaking, purifying, and placing in an incubator for culturing;
selecting the yeast with better growth vigor after purification, inoculating the yeast into a prepared preservation culture test tube, placing the culture test tube in an incubator at 30 ℃, and after four to five days of culture, transferring the yeast to a refrigerator at 6 ℃ for cold preservation when the bacterial colony of the yeast grows well, thus completing the preparation of yeast extraction.
The components of the culture medium A are 1-10g of glucose, 0.1-2g of yeast powder, 0.1-1g of polypeptone, 0.1-2g of monopotassium phosphate, 1-8g of agar powder, 70-90g of water and one stirring rotor.
The stirring piece is made of tetrachloroethylene material and does not react with other materials.
The yeast powder is soy sauce yeast.
In the yeast extraction process, a plurality of prepared test tubes of 9ml of 15% saline water, a plurality of silica gel plugs covered, a plurality of conical flasks of 90g of 15% saline water, a plurality of tinfoil seals, a plurality of pipettes, a plurality of culture dishes, a plurality of coating rods and a plurality of medicine spoons are required, and the whole process is placed in an autoclave for sterilization at 121 ℃ for 15 minutes.
High-temperature disinfection: high-temperature sterilization is carried out by adopting an autoclave.
The fermentation tank manufacturing steps comprise:
firstly, starter propagation: mixing 8-14 tons of wheat flour, 8-14 tons of soybean meal and 20-40kg of yeast according to a proportion, and then sending the mixture to a disc yeast making chamber, adjusting oxygen supply, humidity and temperature through a fan air conditioning system in the yeast making process, controlling the temperature of the materials to be not more than 35 ℃, and culturing for about 40 hours.
② mash preparation: adding 30-40 tons of cooling salt water with the concentration of 15% -25% into the finished koji, and conveying the finished koji into a fermentation tank to prepare 60 tons of sauce mash.
The application of salt tolerant yeast comprises performing amplification culture on the above extracted yeast, adding yeast solution obtained by amplification culture into a fermentation tank, and fermenting and brewing with soy sauce mash.
And (3) yeast expanding culture step:
first amplification culture: inoculating with alcohol lamp on clean bench, burning the inoculating needle with outer flame, cooling slightly, picking out yeast strain on the preservation culture medium, inoculating into liquid culture medium C prepared in advance, and determining to inoculate several rings of strains according to actual conditions. And (3) ventilating the liquid culture medium C for culture, continuously culturing for three days, observing the conditions in the culture medium, and finishing the enlarged culture when rich and fine white foams overflow from a culture medium bottle and are accompanied with strong wine aroma and special fragrant sweet taste. At this time, a great amount of yeast is deposited at the bottom of the bottle.
And (3) second amplification culture: based on the steps, secondary expanding culture of the yeast is continued in the processing workshop, and the yeast is added into the fermentation tank after the secondary expanding culture is completed, so that the fermentation dominant bacteria are established to participate in the fermentation process until the soy sauce mash lightens the beany flavor and generates special aroma.
The preparation method of the liquid culture medium C comprises the following steps: placing 350g glucose, 400g salt, 350g raw juice of soy sauce (obtained by fermenting, squeezing, cooling, precipitating, sterilizing, precipitating, and filtering common soy sauce mash) in 5L conical flask, adding water to 3500ml, sealing with tin foil, sterilizing at 121 deg.C for 15 min, sterilizing, and cooling.
The yeast solution added: 1 ton (yeast count: 2.0X 10)8More than one/ml).
The stirring mode of the fermentation tank is as follows:
firstly, each batch of materials is completely fed, and stirring is carried out for 10 minutes for 1 time.
② 2 to 6 days, stirring for 20 minutes for 1 time every other day.
③ 10 to 60 days, stirring for 15 minutes 1 time per week.
Fourthly, on day 60, stirring for 30min for 1 time when yeast is added, and keeping for 3 days.
Fifthly, stirring for 10min for 1 time every 5 days for 60-120 days.
Sixthly, on the 120 th day, after the sauce mash is fully dissolved, stirring for 5min for 1 time every 15 days.
And seventhly, stirring is carried out before material taking each time, and the stirring is mainly uniform.
And stirring the mixture before squeezing and pumping the mixture, wherein the stirring is mainly uniform.
Fermentation tank temperature:
the initial stage of mash preparation is kept at a low temperature below 15 ℃.
Secondly, heating is started in 20-30 days, and is stopped when the temperature reaches 28 ℃ in 50-60 days.
③ controlling the temperature in the main fermentation period to be 28-32 ℃ and not to exceed 32 ℃, and recording.
Fourthly, beginning to cool about 120 days until reaching about 30 ℃ until the next working procedure.
Taking materials from the sauce mash, wherein sampling is carried out before adding yeast, and the other sampling time is 1 day, 30 days, 90 days, 120 days and 180 days until the next procedure.
The method realizes the extraction work of the salt-tolerant yeast and the batch expanded culture of the salt-tolerant yeast, and the extracted yeast can emit aromatic hydrocarbons and esters in the expanded culture process, so that the yeast can have unique flavor in the subsequent preparation process of the soy sauce, and the generation of beany flavor can be effectively reduced.
Example 1
Based on the mode, the specific extraction method of the salt-tolerant yeast comprises the following steps:
s1 sample selection: taking a part of unstirred sauce mash which is 5 to 10 centimeters downwards from the surface as a sample in a fermentation tank for later use;
preparation of S2 culture medium: filling 10g of glucose, 2g of yeast powder, 1g of polypeptone, 2g of monopotassium phosphate and 6g of agar powder into a conical flask, adding water to 100g, placing one stirring piece, and marking as a culture medium A for later use;
taking 30g of raw soy sauce, 24.5g of salt and 70g of water from a soy sauce pool, and filling the raw soy sauce, the salt and the water into a conical flask marked as a culture medium B for later use;
s3 sterilization treatment: respectively sealing the culture medium A, B, sterilizing in autoclave at 121 deg.C for 5min, and placing in 70 deg.C water bath;
s4: preparing a reagent: preparing and sterilizing a plurality of 9mL test tubes of 15% saline and a plurality of 90g conical flasks of 15% saline in advance, and cooling for later use;
preparation of S5 culture dish: pouring the mixture of the culture medium B in the conical flask into the culture medium A in the conical flask, uniformly stirring the culture medium A on a magnetic stirrer, then moving the culture medium A to a super-clean workbench, quickly pouring the culture medium A into a marked culture dish when the culture medium A is hot beside the flame of an alcohol lamp, opening a culture dish cover, opening a fan to accelerate solidification of the culture medium B, cooling and drying the culture medium B, and then covering the culture dish cover for later use;
s6 suspension preparation: adding 10g of the soy sauce mash sample into a conical flask containing 90g of 15% salt water, and mixing to obtain 10g of soy sauce mash sample-1Suspension a; further, the suspension a1ml was aspirated and dropped into a test tube containing 9ml of 15% saline solution, and the mixture was diluted to 10-2Suspension b, prepared by the same method 10-3Suspension c, 10-4Suspension d;
s7 Petri dish inoculation: respectively sucking 0.1ml of the suspension a, the suspension b, the suspension c and the suspension d, and respectively smearing the suspension a, the suspension b, the suspension c and the suspension d on a marked culture dish flat plate, preparing a parallel sample for each sample liquid, putting the four culture dishes into a group after the parallel samples are prepared, inverting the group of four culture dishes into a culture box, and culturing at the constant temperature of 30 ℃;
s8 selecting strains: continuously culturing for 6 days, observing growth conditions of yeast colonies, recording, selecting a culture dish with good growth vigor of the yeast, selecting the colonies, placing the colonies on a new culture dish, carrying out plate streaking and purification, and placing the colonies in an incubator for culture;
s9, selecting and purifying the yeast with better growth vigor, inoculating the yeast into a prepared preservation culture test tube, placing the yeast in a 30 ℃ incubator for culture, transferring the yeast to a 6 ℃ refrigerator for cold preservation after four to five days of culture and when the bacterial colony of the yeast grows well, and finishing the extraction and preparation of the yeast.
Example 2
Based on the mode, the specific extraction method of the salt-tolerant yeast comprises the following steps:
s1 sample selection: taking a part of unstirred sauce mash which is 5 to 10 centimeters downwards from the surface as a sample in a fermentation tank for later use;
s2 preparation of culture medium, namely, filling 6g of glucose, 1g of yeast powder, 0.4g of polypeptone, 1g of monopotassium phosphate and 4g of agar powder into a conical flask, adding water to 100g, and placing one stirring piece to mark as the culture medium A for later use;
taking 20g of raw soy sauce, 26.5g of salt and 80g of water from a soy sauce pool, and filling the raw soy sauce, the salt and the water into a conical flask marked as a culture medium B for later use;
s3 sterilization treatment: respectively sealing the culture medium A, B, sterilizing in autoclave at 121 deg.C for 5min, and placing in 70 deg.C water bath;
preparing an S4 reagent: preparing and sterilizing a plurality of 9mL test tubes of 15% saline and a plurality of 90g conical flasks of 15% saline in advance, and cooling for later use;
preparation of S5 culture dish: pouring the mixture of the culture medium B in the conical flask into the culture medium A in the conical flask, uniformly stirring the culture medium A on a magnetic stirrer, then moving the culture medium A to a super-clean workbench, quickly pouring the culture medium A into a marked culture dish when the culture medium A is hot beside the flame of an alcohol lamp, opening a culture dish cover, opening a fan to accelerate solidification of the culture medium B, cooling and drying the culture medium B, and then covering the culture dish cover for later use;
s6 suspension preparation: adding 10g of the soy sauce mash sample into a conical flask containing 90g of 15% salt water, and mixing to obtain 10g of soy sauce mash sample-1Suspension a; further, the suspension a1ml was aspirated and dropped into a test tube containing 9ml of 15% saline solution, and the mixture was diluted to 10-2Suspension b, prepared by the same method 10-3Suspension c, 10-4Suspension d;
s7 Petri dish inoculation: respectively sucking 0.1ml of the suspension a, the suspension b, the suspension c and the suspension d, and respectively smearing the suspension a, the suspension b, the suspension c and the suspension d on a marked culture dish flat plate, preparing a parallel sample for each sample liquid, putting the four culture dishes into a group after the parallel samples are prepared, inverting the group of four culture dishes into a culture box, and culturing at the constant temperature of 30 ℃;
s8 selecting strains: continuously culturing for 6 days, observing growth conditions of yeast colonies, recording, selecting a culture dish with good growth vigor of the yeast, selecting the colonies, placing the colonies on a new culture dish, carrying out plate streaking and purification, and placing the colonies in an incubator for culture;
s9, selecting and purifying the yeast with better growth vigor, inoculating the yeast into a prepared preservation culture test tube, placing the yeast in a 30 ℃ incubator for culture, transferring the yeast to a 6 ℃ refrigerator for cold preservation after four to five days of culture and when the bacterial colony of the yeast grows well, and finishing the extraction and preparation of the yeast.
Example 3
S1 sample selection: taking a part of unstirred sauce mash which is 5 to 10 centimeters downwards from the surface as a sample in a fermentation tank for later use;
s2 preparing culture medium, namely putting 2g of glucose, 0.1g of yeast powder, 0.1g of polypeptone, 0.1g of monopotassium phosphate and 3.5g of agar powder into a conical flask, adding water to 100g, putting one stirring piece, and marking as the culture medium A for later use;
taking 10g of raw soy sauce, 28g of salt and 90g of water from a soy sauce pool, and filling the raw soy sauce, the salt and the water into a conical flask marked as a culture medium B for later use;
s3 sterilization treatment: respectively sealing the culture medium A, B, sterilizing in autoclave at 121 deg.C for 5min, and placing in 70 deg.C water bath;
preparing an S4 reagent: preparing and sterilizing a plurality of 9mL test tubes of 15% saline and a plurality of 90g conical flasks of 15% saline in advance, and cooling for later use;
preparation of S5 culture dish: pouring the mixture of the culture medium B in the conical flask into the culture medium A in the conical flask, uniformly stirring the culture medium A on a magnetic stirrer, then moving the culture medium A to a super-clean workbench, quickly pouring the culture medium A into a marked culture dish when the culture medium A is hot beside the flame of an alcohol lamp, opening a culture dish cover, opening a fan to accelerate solidification of the culture medium B, cooling and drying the culture medium B, and then covering the culture dish cover for later use;
s6 suspension preparation: adding 10g of the soy sauce mash sample into a conical flask containing 90g of 15% salt water, and mixing to obtain 10g of soy sauce mash sample-1Suspension a; further, the suspension a1ml was aspirated and dropped into a test tube containing 9ml of 15% saline solution, and the mixture was diluted to 10-2Suspension b, prepared by the same method 10-3Suspension c, 10-4Suspension d;
s7 Petri dish inoculation: respectively sucking 0.1ml of the suspension a, the suspension b, the suspension c and the suspension d, and respectively smearing the suspension a, the suspension b, the suspension c and the suspension d on a marked culture dish flat plate, preparing a parallel sample for each sample liquid, putting the four culture dishes into a group after the parallel samples are prepared, inverting the group of four culture dishes into a culture box, and culturing at the constant temperature of 30 ℃;
s8 selecting strains: continuously culturing for 6 days, observing growth conditions of yeast colonies, recording, selecting a culture dish with good growth vigor of the yeast, selecting the colonies, placing the colonies on a new culture dish, carrying out plate streaking and purification, and placing the colonies in an incubator for culture;
s9, selecting and purifying the yeast with better growth vigor, inoculating the yeast into a prepared preservation culture test tube, placing the yeast in a 30 ℃ incubator for culture, transferring the yeast to a 6 ℃ refrigerator for cold preservation after four to five days of culture and when the bacterial colony of the yeast grows well, and finishing the extraction and preparation of the yeast.
The yeast extracted in the embodiment 2 of the invention is selected for application culture and amplification culture, yeast liquid prepared by amplification culture is added into a fermentation tank to participate in the whole subsequent fermentation process, and finally the prepared soy sauce is named as good taste; meanwhile, the fragrance components are compared with the Fenoer, Qian He, Zhenjin and Zhujiang Qiao soy sauce on the market.
Volatile odor characteristic test of soy sauce of one brand and different brands
Comparative materials: five kinds of soy sauce: respectively comprising Qian He (I), Vanoel (II), good taste (III), Zhenjin (IV) and Zhujiang bridge (V) soy sauce.
The measuring instrument is as follows: PEN3 electronic nose: germany Airsense company for the measurement of the volatile odor profile test for the electronic nose analysis of soy sauce.
The test method comprises the following steps:
a: analyzing a soy sauce volatile odor characteristic test by an electronic nose;
accurately measuring 20mL of soy sauce samples of different varieties in 50mL centrifuge tubes respectively, standing for 30min at 25 ℃, and detecting by adopting a headspace sampling method through an electronic nose. Measurement conditions of the electronic nose: the self-cleaning time of the sensor is 150s, the zero-setting time of the sensor is 5s, the sample preparation time is 5s, the sample determination time is 120s, the sample determination interval time is 1s, the automatic dilution is 0, and the sample introduction flow rate is 300 mL/min.
b: the mode identification method is characterized in that the mode identification is completed based on a WinMuster software platform;
in the data acquisition process of the sample, as each sensor responds to a certain characteristic gas intensely, which type of the main volatile gas of the sample is can be determined, and 10 different metal oxide sensors and corresponding aroma types thereof are shown in table 1. In the test, the characteristic values of 10 sensors are extracted, and a Principal Component Analysis (PCA) method is adopted as a distinguishing analysis method.
TABLE 1-1 Sensors and their corresponding fragrance types
And (3) test results:
TABLE 1-2 resistance ratio of 10 sensors for different soy sauce (G0/G is air resistance)
As shown in fig. 1, radar mapping analysis is performed on the electronic nose test results of different soy sauce, and it is observed that G/G0 values of different sensors are different for each sample, and soy sauce No. iii has obvious difference with other samples, curve coincidence degree is not obvious, and difference is obviously distributed in sensors W1S, W2S, W1W and W5S, and mainly represents methyl compounds, alcohols, inorganic sulfides and volatile substances containing nitrogen oxide compounds respectively.
As shown in fig. 2, Principal Component Analysis (PCA) can observe that, in the correlation matrix mode: the first principal component discrimination contribution ratio is 98.33%, the second principal component discrimination contribution ratio is 1.42%, and the sum of the two principal component discrimination contribution ratios is 99.75%, which shows that the two principal components already substantially represent the main information characteristics of the sample. And the farther the sample is from the origin, the greater the difference, so there is a significant difference from sample to sample in the graph, and there is no significant overlap between each sample. Therefore, soy sauce No. iii was different from the other samples.
Second, measuring color difference of soy sauce of different brands
The soy sauce dilution of five varieties is measured by 10 times, and a JZ-350 colorimeter is adopted and manufactured by Shenzhen Jinqin Instrument and Equipment Co.
TABLE 1-3 color difference analysis results for different brands of soy sauce
L: represents lightness, a: represents the red/green value
From the L and A values, the color of No. III soy sauce is darker than that of other soy sauce, and then the soy sauce is No. I.
The volatile aroma component analysis reports of the three or five kinds of soy sauce comprise
TABLE 2-1 (I) Soy sauce fragrance ingredient Table
TABLE 2-2 (II) Soy sauce fragrance ingredient Table
TABLE 2-3 (III) Soy sauce fragrance ingredient Table
TABLE 2-4 (IV) Soy sauce fragrance ingredient Table
TABLE 2-5 (V) Soy sauce fragrance ingredient Table
As shown in FIG. 7, the 26SrDNA sequencing sequence diagram of the yeast of the present invention shows that the detection data of the present invention are all from the detection center for microbial analysis in Guangdong province.
As shown in fig. 8, the soy sauce of the present invention has unique aroma components;
as shown in FIG. 9, the soy sauce portion of the present invention has unique aroma characteristics.
After the analysis of the components is combined, the extracted saccharomycetes generate good alcohol fragrance after the enlarged culture, are not pungent and have special fragrant and sweet taste, and are relatively distinctive taste. The soybean sauce is expanded to be cultured and added into sauce mash in a proper amount to participate in the fermentation process, and is determined to be fermentation dominant bacteria, so that the beany flavor can be reduced, the special fragrance of the soybean sauce can enrich the flavor of the soybean sauce, and the soybean sauce has better discrimination compared with other soybean sauce.
The above disclosure is only illustrative of the embodiments of the present invention, and is not intended to limit the present invention in any way, and any methods and materials similar or equivalent to those described herein can be used in the method of the present invention. The preferred embodiments and materials described herein are exemplary only, and the embodiments of the present application are not intended to be limited thereto, since modifications and variations of the disclosed embodiments may occur to those skilled in the art and are intended to be included within the scope of the appended claims. The above sequence numbers are for illustrative purposes only and do not represent the relative merits of the implementation scenario. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention, without departing from the technical solution of the present invention, still belong to the protection scope of the technical solution of the present invention.
Sequence listing
<110> good taste food Co., Ltd
<120> extraction method and application of salt-tolerant yeast
<140> 202111649106.0
<141> 2021-12-30
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<170> SIPOSequenceListing 1.0
<210> 1
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Claims (9)
1. The extraction method of the salt-tolerant yeast is characterized by comprising the following steps:
selecting a sample: taking a part of unstirred sauce mash which is 5 to 10 centimeters downwards from the surface as a sample in a fermentation tank for later use;
preparing a culture medium: taking the prepared culture medium A for later use;
taking 10-30g of raw soy sauce, 25-30g of salt and 70-90g of water from the soy sauce pool, and filling the raw soy sauce, the salt and the water into a conical flask marked as a culture medium B for later use;
and (3) disinfection treatment: respectively sealing the culture medium A, B, sterilizing in autoclave at 121 deg.C for 5min, and placing in 70 deg.C water bath;
preparing a reagent: preparing and sterilizing a plurality of 9mL test tubes of 15% saline and a plurality of 90g conical flasks of 15% saline in advance, and cooling for later use;
preparing a culture dish: pouring the mixture of the culture medium B in the conical flask into the culture medium A in the conical flask, uniformly stirring the culture medium A on a magnetic stirrer, then moving the culture medium A to a super-clean workbench, quickly pouring the culture medium A into a marked culture dish when the culture medium A is hot beside the flame of an alcohol lamp, opening a culture dish cover, opening a fan to accelerate solidification of the culture medium B, cooling and drying the culture medium B, and then covering the culture dish cover for later use;
preparing a suspension: adding 10g of the soy sauce mash sample into a conical flask containing 90g of 15% salt water, and mixing to obtain 10g of soy sauce mash sample-1Suspension a; further, the suspension a1ml was aspirated and dropped into a test tube containing 9ml of 15% saline solution, and the mixture was diluted to 10-2Suspension of turbid urineLiquid b, preparation of the same 10-3Suspension c, 10-4Suspension d;
inoculating a culture dish: respectively sucking 0.1ml of the suspension a, the suspension b, the suspension c and the suspension d and respectively smearing the suspension on a marked culture dish flat plate, preparing a parallel sample for each sample liquid, forming a group of four culture dishes after the parallel samples are finished, inversely placing the groups of four culture dishes into an incubator, and culturing at the constant temperature of 30 ℃;
selecting strains: continuously culturing for 6 days, observing growth conditions of yeast colonies, recording, selecting a culture dish with good growth vigor of the yeast, selecting the colonies, carrying out plate streaking and purification on a new culture dish, and placing the culture dish in an incubator for culture;
selecting the yeast with better growth after purification, inoculating the yeast into a prepared preservation culture test tube, placing the culture tube in an incubator at 30 ℃ for culture, transferring the yeast to a refrigerator at 6 ℃ for cold storage after the yeast colony grows well after four to five days of culture, and completing the yeast extraction and preparation.
2. The method for extracting the salt-tolerant yeast according to claim 1, wherein the culture medium A comprises 1-10g of glucose, 0.1-2g of yeast powder, 0.1-1g of polypeptone, 0.1-2g of monopotassium phosphate, 1-8g of agar powder, 70-90g of water and one stirring rotor.
3. The method for extracting salt-tolerant yeast according to claim 2, wherein the stirring piece is made of tetrachloroethylene.
4. The method for extracting salt-tolerant yeast according to claim 1, wherein the yeast powder is soy sauce yeast.
5. The method for extracting the salt-tolerant yeast according to claim 1, wherein in the yeast extraction process, a plurality of prepared test tubes with 9ml of 15% saline, a plurality of silica gel plugs, a plurality of conical bottles with 90g of 15% saline, a plurality of tinfoil seals, a plurality of pipettes, a plurality of culture dishes, a plurality of coating rods and a plurality of spoons are required, and all the test tubes, the pipette plugs, the coating rods and the spoons are required to be placed in an autoclave for sterilization at 121 ℃ for 15 minutes.
6. The method for extracting salt-tolerant yeast according to claim 1, wherein the preparation step of the moromi in the fermentation tank comprises the following steps:
firstly, starter propagation: mixing wheat flour, steamed bean pulp and koji in proportion, and feeding into a disc koji making chamber, wherein the amount of oxygen supply, humidity and temperature are adjusted by a fan air conditioning system during koji making.
② mash preparation: adding quantitative cooling saline water into the yeast, and sending the raw mash into a fermentation tank to obtain the sauce mash.
7. The application of salt-tolerant yeast is characterized in that the extracted yeast is subjected to expanded culture, and yeast liquid prepared by the expanded culture is added into a fermentation tank to be fermented and brewed together with soy sauce mash.
8. The application of the salt-tolerant yeast according to claim 7, wherein the fermentation tank is stirred in the following way:
firstly, each batch of materials is completely fed, and stirring is carried out for 10 minutes for 1 time.
② 2 to 6 days, stirring for 20 minutes for 1 time every other day.
③ 10 to 60 days, stirring for 15 minutes 1 time per week.
Fourthly, on day 60, stirring for 30min for 1 time when yeast is added, and keeping for 3 days.
Fifthly, stirring for 10min for 1 time every 5 days in 60-120 days.
Sixthly, on the 120 th day, after the sauce mash is fully dissolved, stirring for 5min for 1 time every 15 days.
9. The use of the salt tolerant yeast of claim 7, wherein the fermentor temperature is set at 28-32 ℃.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030129277A1 (en) * | 2001-12-21 | 2003-07-10 | Kikkoman Corporation | Method of brewing soy sauce |
| CN103589653A (en) * | 2013-11-26 | 2014-02-19 | 江南大学 | Zygosaccharomyces rouxii and its application |
| CN108285874A (en) * | 2017-01-09 | 2018-07-17 | 江苏恒顺沭阳调味品有限公司 | It is a kind of to make the culture of saccharomyces soya and its adding method during soy sauce |
| CN108315271A (en) * | 2018-04-09 | 2018-07-24 | 佛山市海天(高明)调味食品有限公司 | One primary yeast and its application |
| CN108330074A (en) * | 2017-10-24 | 2018-07-27 | 张晨 | Purposes and aromatizing process of the abnormal dimension gram Durham saccharomycete T1 in soy sauce flavouring |
| CN109280625A (en) * | 2018-10-11 | 2019-01-29 | 千禾味业食品股份有限公司 | A kind of Lu Shi Zygosaccharomyces S96 and its application |
| CN111423988A (en) * | 2020-04-01 | 2020-07-17 | 广东美味鲜调味食品有限公司 | Soybean sauce brewing method for reducing content of free tyrosine in sauce mash by adding halotolerant bacteria |
| CN113322193A (en) * | 2021-05-18 | 2021-08-31 | 华南理工大学 | Salt-tolerant yeast for increasing content of Sotolone in soy sauce and application thereof |
| CN113637596A (en) * | 2021-08-24 | 2021-11-12 | 广东海天创新技术有限公司 | Saccharomyces cerevisiae ZB421 and application thereof |
-
2021
- 2021-12-30 CN CN202111649106.0A patent/CN114196558A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030129277A1 (en) * | 2001-12-21 | 2003-07-10 | Kikkoman Corporation | Method of brewing soy sauce |
| CN103589653A (en) * | 2013-11-26 | 2014-02-19 | 江南大学 | Zygosaccharomyces rouxii and its application |
| CN108285874A (en) * | 2017-01-09 | 2018-07-17 | 江苏恒顺沭阳调味品有限公司 | It is a kind of to make the culture of saccharomyces soya and its adding method during soy sauce |
| CN108330074A (en) * | 2017-10-24 | 2018-07-27 | 张晨 | Purposes and aromatizing process of the abnormal dimension gram Durham saccharomycete T1 in soy sauce flavouring |
| CN108315271A (en) * | 2018-04-09 | 2018-07-24 | 佛山市海天(高明)调味食品有限公司 | One primary yeast and its application |
| CN109280625A (en) * | 2018-10-11 | 2019-01-29 | 千禾味业食品股份有限公司 | A kind of Lu Shi Zygosaccharomyces S96 and its application |
| CN111423988A (en) * | 2020-04-01 | 2020-07-17 | 广东美味鲜调味食品有限公司 | Soybean sauce brewing method for reducing content of free tyrosine in sauce mash by adding halotolerant bacteria |
| CN113322193A (en) * | 2021-05-18 | 2021-08-31 | 华南理工大学 | Salt-tolerant yeast for increasing content of Sotolone in soy sauce and application thereof |
| CN113637596A (en) * | 2021-08-24 | 2021-11-12 | 广东海天创新技术有限公司 | Saccharomyces cerevisiae ZB421 and application thereof |
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