Highly resistant saccharomyces cerevisiae strains and their use

Abstract

This invention belongs to the field of microbial technology, specifically relating to a *Saccharomyces cerevisiae* strain with high sugar content and resistance to multiple preservatives, and its applications. The specific *Saccharomyces cerevisiae* strain is *Saccharomyces cerevisiae* AMCC 33043, with accession number CCTCC NO: M 2026601. This strain exhibits excellent fermentation activity in applications involving high sugar content and the addition of multiple preservatives such as calcium propionate, encapsulated sorbic acid, ε-polylysine, and whey yeast, enabling rapid fermentation and wide applicability. This invention also includes a method for preparing highly active yeast products from the aforementioned strain, and its application in fermented foods and other fields.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Saccharomyces cerevisiae with high sugar content and multiple preservative resistance, and its application. Background Technology

[0002] Saccharomyces cerevisiae, as an excellent microbial starter culture, plays a vital role in the food industry and is widely used in the production of fermented grain products such as rice and flour. Industrial production pursues high efficiency and low cost, which requires yeast to adapt to different application scenarios and quickly leaven dough. However, when the sugar concentration of the dough is high (e.g., 25%), its osmotic pressure directly affects the yeast's fermentation performance, leading to insufficient gas production, slow fermentation, and a sharp decline in production efficiency.

[0003] Furthermore, as one of the most consumed staple foods globally, flour products have seen continuous expansion in industrial production scale, making extended shelf life and wider distribution channels core demands for industry development. To inhibit microbial contamination and delay product spoilage, multiple preservatives are often added during flour product production to enhance preservation effects. This is a crucial means of ensuring food quality and safety and reducing storage and logistics losses. However, while the addition of preservatives effectively inhibits the growth of unwanted bacteria and extends product shelf life, it inevitably suppresses yeast fermentation activity, which directly determines dough fluffiness, flavor compound formation, and final product quality. Therefore, maintaining efficient yeast fermentation within a preservative-containing system becomes the core contradiction in balancing the preservation needs of flour products with product quality.

[0004] Existing technologies have disclosed some strains and screening methods capable of tolerating high osmotic pressure and preservatives. For example, patent document CN117165455B provides a *Saccharomyces cerevisiae* strain that exhibits resistance to high sugar osmotic pressure, cold osmotic shock, and organic acids, but only its resistance to calcium propionate was evaluated. Patent document CN120682955A provides a *Saccharomyces cerevisiae* strain that demonstrates excellent activity in different concentrations of high sugar and with the addition of high concentrations of organic acids and their salts, but only its resistance to calcium propionate was evaluated. Patent document CN107142224A discloses a baker's yeast with osmotic pressure tolerance and intrinsic resistance to weak organic acids, involving a sugar concentration of 15%-25% and a calcium propionate concentration of 0.4%. In summary, most existing technologies focus on the strain's tolerance to a single preservative (calcium propionate), without evaluating the strain's tolerance to multiple preservatives. Furthermore, existing inventions lack a systematic screening and evaluation framework for strains, and the preservative resistance of yeast strains varies considerably, making it difficult to meet the personalized needs of different flour product manufacturing processes.

[0005] Therefore, in order to meet the increasingly demanding application requirements of users in the baking industry, it is urgent to develop yeast strain products with high sugar content and high resistance to multiple preservatives. These products can effectively reduce the damage to yeast cells caused by adverse factors in the application environment, improve yeast fermentation efficiency, help the company quickly enter the market during the critical period of developing long-shelf-life baking, and enhance the company's overall strength and market influence. Summary of the Invention

[0006] To address the problems existing in the prior art, the present invention aims to provide a high-performance brewing yeast strain and its application method. This strain has high sugar content and high tolerance to multiple preservatives, which can effectively reduce the damage to yeast cells caused by adverse factors in the application environment and improve yeast fermentation efficiency.

[0007] The specific technical solution of the present invention is as follows: Technical Solution 1: A brewing yeast with high sugar content and multiple preservative resistance, characterized in that the brewing yeast is brewing yeast AMCC 33043 ( Saccharomyces cerevisiae AMCC 33043 was deposited at the China Center for Type Culture Collection on April 3, 2026, with accession number CCTCCNO: M 2026601.

[0008] Technical Solution 2: The brewing yeast according to Technical Solution 1, characterized in that the net dry weight of the brewing yeast reaches 90%-110% of any parent strain, wherein the any parent strain is brewing yeast AMCC 31557 with preservation number CCTCC NO: M20231789 or brewing yeast AMCC31248 with preservation number CCTCC NO: M 20211686.

[0009] Technical Solution 3: The brewing yeast according to Technical Solution 1 or 2, characterized in that the net dry weight of the brewing yeast reaches 95%-110% of any parent strain, wherein the any parent strain is brewing yeast AMCC 31557 with preservation number CCTCC NO: M20231789 or brewing yeast AMCC31248 with preservation number CCTCC NO: M 20211686.

[0010] Technical Solution 4: The brewing yeast according to any one of technical solutions 1-3, characterized in that the brewing yeast is obtained by sexual spore hybridization using brewing yeast AMCC 31557 and brewing yeast AMCC 31248 as parent strains.

[0011] Technical Solution 5: A yeast milk, characterized in that it comprises the brewing yeast as described in any one of Technical Solutions 1-4.

[0012] Technical Solution 6: The yeast milk according to Technical Solution 5, characterized in that the yeast milk is prepared by a method including the following steps: culturing the brewing yeast, performing solid-liquid separation and washing to obtain the yeast milk. Preferably, the culture time is 15-24 hours and the culture temperature is 28-32℃. More preferably, the brewing yeast is cultured in a liquid culture medium, wherein the liquid culture medium comprises, by weight, 1-5% yeast extract, 1-5% peptone, 1-5% glucose, and the remainder is water.

[0013] Technical Solution 7: A microbial agent, characterized in that it comprises the brewing yeast as described in any one of Technical Solutions 1-4.

[0014] Technical Solution 8: The microbial agent according to Technical Solution 7 is characterized in that the microbial agent is active dry yeast.

[0015] Technical Solution 9: The microbial agent according to Technical Solution 7 or 8 is characterized in that the microbial agent is prepared by a method including the following steps: culturing the brewing yeast, and then separating, washing, filtration, pressure filtration and drying.

[0016] Technical Solution 10: The application of the brewing yeast of any one of Technical Solutions 1-4, or the yeast milk of any one of Technical Solutions 5 or 6, or the microbial agent of any one of Technical Solutions 7-9 in the food field, preferably, the food field includes the field of pasta fermentation, more preferably, the pasta fermentation field includes the preparation of one or more of sweet bread, fancy dinner rolls, hand-torn bread, Danish pastries and premixed powders containing preservatives.

[0017] Technical Solution 11: The application of the yeast milk described in Technical Solution 5 or 6 in dough preparation, wherein the fermentation activity of the brewer's yeast AMCC 33043 in the yeast milk in the heavy sugar dough system reaches 95%-120% of the fermentation activity of any parent strain, wherein the fermentation activity refers to the total volume of carbon dioxide gas produced by the fermentation of raw dough obtained through the heavy sugar dough system at 30°C by the brewer's yeast AMCC33043 in the yeast milk; the sugar mass percentage of the heavy sugar dough system is 20-40%. The parent strain is either Saccharomyces cerevisiae AMCC31557 with accession number CCTCC NO: M 20231789 or Saccharomyces cerevisiae AMCC 31248 with accession number CCTCC NO: M 20211686.

[0018] Technical Solution 12: According to the application described in Technical Solution 11, wherein the fermentation activity of the brewer's yeast AMCC 33043 in the yeast milk in the heavy sugar dough system reaches 100%-120% of the fermentation activity of the parent strain.

[0019] Technical Solution 13: The application of the yeast milk described in Technical Solution 5 or 6 in dough preparation, wherein the brewer's yeast AMCC 33043 in the yeast milk achieves a fermentation activity of 95%-120% of any parent strain in a high-sugar dough system containing 0-0.6% by mass of preservative A, wherein the fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained through the high-sugar dough system containing 0-0.6% by mass of preservative A at 30°C by the brewer's yeast AMCC 33043 in the yeast milk; the sugar mass percentage of the high-sugar dough system is 20-40%. Alternatively, the brewer's yeast AMCC 33043 in the yeast milk may achieve a fermentation activity of 95%-120% of any parent strain in a heavy sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B. The fermentation activity refers to the total volume of carbon dioxide gas produced by fermenting raw dough obtained from the heavy sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B at 30°C using the brewer's yeast AMCC 33043 in the yeast milk. The sugar content of the heavy sugar dough system is 20-40% by mass. Among them, any one of the parental strains is either Saccharomyces cerevisiae AMCC31557 with accession number CCTCC NO: M 20231789 or Saccharomyces cerevisiae AMCC 31248 with accession number CCTCC NO: M 20211686. Wherein, preservative A is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; and preservative B is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives. Preferably, the organic acid salt preservative is selected from calcium propionate, the organic acid preservative is selected from encapsulated sorbic acid, and the polypeptide preservative is selected from ε-polylysine. More preferably, the preservative A is calcium propionate, and the preservative B is sorbic acid encapsulated.

[0020] Technical Solution 14: According to the application described in Technical Solution 13, the brewer's yeast AMCC 33043 in the yeast milk achieves 100%-120% of the fermentation activity of any parent strain in a high-sugar dough system containing 0-0.6% by mass of preservative A. Alternatively, the brewer's yeast AMCC 33043 in the yeast milk can achieve a fermentation activity of 100%-120% of any parent strain in a heavy sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B.

[0021] Technical Solution 15: The application of the microbial agent described in any one of Technical Solutions 7-9 in the preparation of dough, wherein the fermentation activity of the brewer's yeast AMCC 33043 in the microbial agent in the heavy sugar dough system reaches 95%-120% of the fermentation activity of any parent strain, wherein the fermentation activity refers to the total volume of carbon dioxide gas produced by the fermentation of raw dough obtained through the heavy sugar dough system by the brewer's yeast in the active dry yeast at 30°C; the sugar mass percentage of the heavy sugar dough system is 20-40%.

[0022] Technical Solution 16: The application of the microbial agent according to Technical Solution 15 in the preparation of dough, wherein the fermentation activity in the heavy sugar dough system reaches 100%-120% of the fermentation activity of any parent strain.

[0023] Technical Solution 17: The application of the microbial agent described in any one of Technical Solutions 7-9 in dough preparation, wherein the fermentation activity of the brewer's yeast AMCC 33043 in the microbial agent in a high-sugar dough system containing 0-0.6% by mass of preservative A reaches 95%-120% of the fermentation activity of any parent strain, wherein the fermentation activity refers to the total volume of carbon dioxide gas produced by the fermentation of raw dough obtained through the high-sugar dough system containing 0-0.6% by mass of preservative A at 30°C by the brewer's yeast AMCC 33043 in the microbial agent; the sugar mass percentage of the high-sugar dough system is 20-40%. Alternatively, the fermentation activity of the brewer's yeast AMCC 33043 in the aforementioned inoculum in a high-sugar dough system containing 0-0.2% preservative B reaches 95%-120% of the fermentation activity of any parent strain. Here, fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained from the high-sugar dough system containing 0-0.2% preservative B at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Alternatively, the fermentation activity of the brewer's yeast AMCC 33043 in the aforementioned inoculum in a high-sugar dough system containing 0-0.015% by mass of preservative C reaches 95%-120% of the fermentation activity of any parent strain. Here, fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained from the high-sugar dough system containing 0-0.015% by mass of preservative C at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Alternatively, the fermentation activity of the brewer's yeast AMCC 33043 in the aforementioned inoculum in a high-sugar dough system containing 0-0.3% by mass of preservative D reaches 95%-120% of the fermentation activity of any parent strain. Here, fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained through the high-sugar dough system containing 0-0.3% by mass of preservative D at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned inoculum may achieve a fermentation activity of 95%-120% of the fermentation activity of any parent strain in a high-sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B. The fermentation activity refers to the total volume of carbon dioxide gas produced by fermenting raw dough obtained from the high-sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned inoculum, in a high-sugar dough system containing 0-0.2% by mass of preservative A, 0-0.2% by mass of preservative B, 0-0.015% by mass of preservative C, and 0-0.3% by mass of preservative D, achieves a fermentation activity of 95%-120% of the fermentation activity of any parent strain. The fermentation activity refers to the total volume of carbon dioxide gas produced by fermenting raw dough obtained from the high-sugar dough system containing 0-0.2% by mass of preservative A, 0-0.2% by mass of preservative B, 0-0.015% by mass of preservative C, and 0-0.3% by mass of preservative D at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Among them, any one of the parental strains is either Saccharomyces cerevisiae AMCC31557 with accession number CCTCC NO: M 20231789 or Saccharomyces cerevisiae AMCC 31248 with accession number CCTCC NO: M 20211686. Wherein, preservative A is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; preservative B is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; preservative C is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; and preservative D is a preservative with natural antibacterial and freshness-preserving functions. Preferably, the organic acid salt preservative is selected from calcium propionate; the organic acid preservative is encapsulated sorbic acid; the polypeptide preservative is ε-polylysine; and the preservative with natural antibacterial and preservative functions is whey baking powder. More preferably, the preservative A is calcium propionate, the preservative B is encapsulated sorbic acid, the preservative C is ε-polylysine, and the preservative D is whey baking powder.

[0024] Technical Solution 18: According to the application described in Technical Solution 17, the brewing yeast AMCC33043 in the microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.6% preservative A by mass, reaching 100%-120% of the fermentation activity of any parent strain. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.2% preservative B, reaching 100%-120% of the fermentation activity of any parent strain. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.015% preservative C, reaching 100%-120% of the fermentation activity of any parent strain. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.3% by mass of preservative D, reaching 100%-120% of the fermentation activity of any parent strain. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned microbial agent may exhibit fermentation activity reaching 100%-120% of the fermentation activity of any parent strain in a high-sugar dough system simultaneously containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B. Alternatively, the brewing yeast AMCC 33043 in the microbial agent may exhibit fermentation activity in a heavy sugar dough system containing 0-0.2% by mass of preservative A, 0-0.2% by mass of preservative B, 0-0.015% by mass of preservative C, and 0-0.3% by mass of preservative D, reaching 100%-120% of the fermentation activity of any parent strain.

[0025] Technical Solution 19: A dough, characterized in that it comprises the brewing yeast described in any one of Technical Solutions 1-4, or the yeast milk described in Technical Solutions 5 or 6, or the inoculant described in Technical Solutions 7-9.

[0026] Technical Solution 20: The dough according to Technical Solution 19, characterized in that the dough is any one of the following doughs: (1) Heavy sugar dough, (2) Heavy sugar dough containing 0-0.6% of preservative A relative to the weight of flour, (3) Heavy sugar dough containing 0-0.2% of preservative B relative to the weight of flour, (4) Heavy sugar dough containing 0-0.015% of preservative C relative to the weight of flour, (5) Heavy sugar dough containing 0-0.3% of preservative D relative to the weight of flour, (6) Heavy sugar dough containing both 0-0.2% of preservative A and 0-0.2% of preservative B relative to the weight of flour, or (7) Heavy sugar dough containing both 0-0.2% of preservative A, 0-0.2% of preservative B, 0-0.015% of preservative C, and 0-0.3% of preservative D relative to the weight of flour. The sugar content in the sugar-rich dough is 20-40% by mass. Wherein, preservative A is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; preservative B is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; preservative C is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; and preservative D is a preservative with natural antibacterial and freshness-preserving functions. Preferably, the organic acid salt preservative is selected from calcium propionate; the organic acid preservative is encapsulated sorbic acid; the polypeptide preservative is ε-polylysine; and the preservative with natural antibacterial and preservative functions is whey baking powder. More preferably, the preservative A is calcium propionate, the preservative B is encapsulated sorbic acid, the preservative C is ε-polylysine, and the preservative D is whey baking powder.

[0027] Technical Solution 21: A method for preparing the dough described in Technical Solution 19 or 20, characterized in that it includes the following steps: adding the brewing yeast described in any one of Technical Solutions 1-4, or the yeast milk described in Technical Solutions 5 or 6, or the inoculant described in Technical Solutions 7-9 to the dough and fermenting it.

[0028] Technical Solution 22: A method for preparing a baked product, characterized in that the dough described in Technical Solution 19 or 20 or the dough prepared by the method described in Technical Solution 21 is baked to obtain the baked product.

[0029] Technical Solution 23: The preparation method according to Technical Solution 22 is characterized in that the baked product is one or more of sweet bread, fancy dinner rolls, hand-torn bread, and Danish pastry.

[0030] Beneficial effects of this invention: (1) The *Saccharomyces cerevisiae* strain of the present invention exhibits good tolerance to high sugar content and multiple preservatives, maintaining stable growth and metabolic activity even under multiple environmental stresses. It is tolerant to common compound preservative systems on the market, such as those composed of calcium propionate, encapsulated sorbic acid, ε-polylysine, and whey yeast. This effectively broadens the practical application scenarios and scope of the strain. Attached Figure Description

[0031] Figure 1 This is a colony diagram of Saccharomyces cerevisiae AMCC 33043.

[0032] Figure 2 This is a growth curve diagram of Saccharomyces cerevisiae AMCC 33043.

[0033] Microbial strain preservation information The brewing yeast AMCC 33043 provided by this invention ( Saccharomyces cerevisiae AMCC 33043 was deposited at the China Center for Type Culture Collection on April 3, 2026, with accession number CCTCC NO: M 2026601. The depository address is: Wuhan University, Wuhan, China, 430072, China. Detailed Implementation

[0034] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely explained below in conjunction with specific embodiments. It should be noted that the content of the specific embodiments is only a specific implementation and explanation of the technical solutions of the present invention, and should not be construed as a limitation on the scope of protection of the present invention.

[0035] The first aspect of this invention provides a brewer's yeast strain with high sugar content and high resistance to multiple preservatives, wherein the brewer's yeast strain is brewer's yeast AMCC 33043 ( Saccharomyces cerevisiae AMCC 33043 was deposited at the China Center for Type Culture Collection on April 3, 2026, with accession number CCTCC NO: M 2026601.

[0036] In some embodiments, the net dry weight of the brewer's yeast strain reaches 90%-110% of that of any parent strain, preferably 95%-110%.

[0037] In some embodiments, the yeast milk of the brewer's yeast strain has one or more of the following fermentation activities: (1) the fermentation activity in a heavy sugar dough system reaches 95%-120% of the fermentation activity of any parent strain, preferably 100%-120%; (2) the fermentation activity in a heavy sugar dough system containing 0-0.6% by mass of preservative A reaches 95%-120% of the fermentation activity of any parent strain, preferably 100%-120%; and (3) the fermentation activity in a heavy sugar dough system containing both 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B reaches 95%-120% of the fermentation activity of any parent strain, preferably 100%-120%. Wherein, the preservative A is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; The preservative B is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; More preferably, the organic acid salt preservative is selected from calcium propionate; the organic acid preservative is selected from encapsulated sorbic acid; and the polypeptide preservative is selected from ε-polylysine.

[0038] In some embodiments, the active dry yeast of the brewing yeast strain has one or more of the following fermentation activities: (1) fermentation activity in a heavy sugar dough system reaches 95%-120% of the fermentation activity of any parent strain, preferably 100%-120%; (2) fermentation activity in a heavy sugar dough system containing 0-0.6% by mass of preservative A reaches 95%-120% of the fermentation activity of any parent strain, preferably 100%-120%; (3) fermentation activity in a heavy sugar dough system containing 0-0.2% by mass of preservative B reaches 95%-120% of the fermentation activity of any parent strain. (3) The fermentation activity of a parent strain reaches 95%-120%, preferably 100%-120%, in a heavy sugar dough system containing 0-0.015% C preservative by mass. (4) The fermentation activity of a parent strain reaches 95%-120%, preferably 100%-120%, in a heavy sugar dough system containing 0-0.3% D preservative by mass. (5) The fermentation activity of a parent strain reaches 95%-120%, preferably 100%-120%, in a heavy sugar dough system containing 0-0.2% D preservative by mass. The fermentation activity of the heavy sugar dough system containing 0-0.2% B by mass reaches 95%-120% of the fermentation activity of any parent strain, preferably 100%-120%; and (7) the fermentation activity of the heavy sugar dough system containing 0-0.2% A by mass, 0-0.2% B by mass, 0-0.015% C by mass and 0-0.3% D by mass reaches 95%-120% of the fermentation activity of any parent strain, preferably 100%-120%; Wherein, preservative A is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; preservative B is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives; and preservative C is selected from any one of organic acid preservatives, organic acid salt preservatives, or polypeptide preservatives. More preferably, the organic acid salt preservative is selected from calcium propionate; the organic acid preservative is encapsulated sorbic acid; and the polypeptide preservative is ε-polylysine.

[0039] The preservative D is a preservative with natural antibacterial and preservation functions. More preferably, the preservative with natural antibacterial and preservative functions is whey yeast powder.

[0040] In some embodiments, when the yeast strain and the parent strain ferment to the same height in the same dough, the fermentation time of the yeast strain is shorter than that of the parent strain.

[0041] The second aspect of this invention discloses a Saccharomyces cerevisiae strain, which is obtained by sexual spore hybridization using Saccharomyces cerevisiae AMCC31557 and Saccharomyces cerevisiae AMCC 31248 as parents.

[0042] The third aspect of the present invention discloses a fermentation product obtained by fermentation using the brewer's yeast strain described in the first aspect of the present invention.

[0043] The fourth aspect of this invention discloses a microbial agent comprising the Saccharomyces cerevisiae strain described in the first aspect of this invention.

[0044] The fifth aspect of this invention discloses the application of the brewing yeast strain described in the first aspect of this invention, or the fermentation product described in the third aspect of this invention, or the microbial agent described in the fourth aspect of this invention in the food field.

[0045] In some embodiments, the food field includes a pasta fermentation field; the pasta fermentation field includes preparing one or more of the following: sweet bread, fancy rolls, pull-apart bread, Danish pastries, and premixed powders containing preservatives.

[0046] The sixth aspect of the present invention discloses a dough comprising the brewer's yeast strain described in the first aspect of the present invention, or the fermentation product described in the third aspect of the present invention, or the inoculant described in the fourth aspect of the present invention.

[0047] The seventh aspect of this invention discloses a method for preparing the dough described in the sixth aspect of this invention, comprising: The dough is fermented by adding the brewing yeast strain described in the first aspect of the present invention, or the fermenting agent described in the third aspect of the present invention, or the inoculant described in the fourth aspect of the present invention.

[0048] In some embodiments, the dough is any one of the following: (1) a heavy sugar dough; (2) a heavy sugar dough containing 0-0.6% of preservative A relative to the mass of flour; (3) a heavy sugar dough containing 0-0.2% of preservative B relative to the mass of flour; (4) a heavy sugar dough containing 0-0.015% of preservative C relative to the mass of flour; (5) a heavy sugar dough containing 0-0.3% of preservative D relative to the mass of flour; (6) a heavy sugar dough containing both 0-0.2% of preservative A and 0-0.2% of preservative B relative to the mass of flour; or (7) a heavy sugar dough containing both 0-0.2% of preservative A, 0-0.2% of preservative B, 0-0.015% of preservative C, and 0-0.3% of preservative D relative to the mass of flour.

[0049] The eighth aspect of the present invention discloses a baking product comprising the dough described in the sixth aspect of the present invention or the dough prepared by the method of the seventh aspect of the present invention.

[0050] In some embodiments, the baked product is one or more of sweet bread, fancy dinner rolls, pull-apart bread, and Danish pastry.

[0051] In this invention, the term PCR (Polymerase Chain Reaction) refers to a molecular biology technique that rapidly amplifies specific DNA fragments in vitro, often referred to as the "copy machine of molecular biology." It can amplify trace amounts of target DNA fragments millions, even billions, of times in a short period, thereby enabling the detection and analysis of trace amounts of nucleic acids.

[0052] The term TTC refers to 2,3,5-triphenyltetrazolium chloride.

[0053] The term DMSO refers to dimethyl sulfoxide.

[0054] The term CCTCC refers to the China Center for Type Culture Collection.

[0055] The preservative in this invention is a conventional commercially available preservative known in the art. The whey yeast powder in this invention itself has certain natural antibacterial and preservative functions. Through the organic acids, active peptides and other metabolites produced by fermentation, it inhibits spoilage microorganisms and extends the shelf life of food, and can be regarded as a type of preservative.

[0056] In some specific embodiments, this invention discloses a *Saccharomyces cerevisiae* strain, namely *Saccharomyces cerevisiae* AMCC 33043, which was deposited at the China Center for Type Culture Collection (CCTCC) on April 3, 2026, with accession number CCTCC NO: M 2026601. This *Saccharomyces cerevisiae* strain of the present invention is tolerant to common compound preservative systems on the market, exhibiting good tolerance to multiple preservative systems formed by combining calcium propionate, encapsulated sorbic acid, ε-polylysine, whey yeast, and other preservatives. This strain of the present invention is adaptable to high-sugar systems, maintaining stable growth and metabolic activity even under the environmental stress of high sugar and compound preservatives, and can survive stably and grow and metabolize normally in such systems. The strain of the present invention possesses excellent overall environmental tolerance and can be applied to complex sugar-containing food fermentation systems without requiring additional adjustments to the food formula to adapt to the yeast, simplifying the food production process.

[0057] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0058] In some embodiments of the present invention, the sugar content of the heavy sugar dough system is 20-40% (including but not limited to dough systems with sugar contents of 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, and 40%). In some preferred embodiments, the sugar content of the heavy sugar dough system is 20-35%. In some more preferred embodiments, the sugar content of the heavy sugar dough system is 20-30%. Particularly preferred is the sugar content of the heavy sugar dough system, which is 23-26%. The sugar in the heavy sugar dough system is exogenously added sucrose.

[0059] It should be noted that the x% sugar, x% calcium propionate, or x% sorbic acid involved in the dough in the embodiments of the present invention refers to the mass ratio of sugar to flour, or calcium propionate to flour, or sorbic acid to flour in the dough being x:100.

[0060] Unless otherwise stated, all reagents / instruments used in the embodiments of this invention are conventional commercially available products. Information on the sources of experimental reagents used in this invention is shown in Table 1, and information on the sources of experimental instruments used in this invention is shown in Table 2.

[0061]

[0062]

[0063] In this embodiment of the invention, YPD solid culture medium was used to activate various Saccharomyces cerevisiae strains. The YPD solid culture medium formula, by mass percentage, is: 1% yeast extract, 2% peptone, 2% glucose, and 2% agar. (The calculation method is as follows: for 100g of water, the corresponding yeast extract is 100*1%=1g, and so on, for 2g of peptone, 2g of glucose, and 2g of agar, and the calculation method below is the same).

[0064] In this embodiment of the invention, YPD liquid culture medium was used to culture various Saccharomyces cerevisiae strains. The YPD liquid culture medium formula, by mass percentage, is: 1% yeast extract, 2% peptone, and 2% glucose.

[0065] In this embodiment of the invention, the growth curves of the prepared single-spore and heterozygous strains were screened using a heavy sugar composite preservative screening medium. The formulation of the heavy sugar composite preservative screening medium, by mass percentage, is: 25% sucrose, 1% yeast extract, 2% peptone, 0.2% calcium propionate, and 0.1% potassium sorbate.

[0066] The yeast extract powder (model: FM888) used in the examples contains the following vitamins: vitamin B1 2.3 ppm, vitamin B2 38.8 ppm, vitamin B5 115.0 ppm, vitamin B6 18.0 ppm, vitamin B7 7.9 ppm, vitamin B9 26.7 ppm, vitamin B12 0.23 (ug / 100g), choline 3206.0 ppm, inositol 1577.7 ppm, and niacin 328.0 ppm.

[0067] The trace elements contained in the yeast extract powder (model: FM888) used in the examples are as follows: potassium 31911.66 mg / kg, sodium 5738.91 mg / kg, calcium 355.25 mg / kg, magnesium 2673.59 mg / kg, zinc 80.94 mg / kg and iron 80.21 mg / kg.

[0068] The yeast extract (model: FM888) used in the examples contained 35.1% free amino acids. And hydrolyzed amino acids 61.21%.

[0069] The free amino acid content is as follows, based on the weight of the yeast extract: free aspartic acid 1.6%, free threonine 2.1%, free serine 1.7%, free glutamic acid 6.7%, free glycine 1.2%, free alanine 4.2%, free cysteine ​​0.1%, free valine 2.7%, free methionine 0.8%, free isoleucine 2.2%, leucine 3.5%, free tyrosine 0.9%, free phenylalanine 1.8%, free lysine 2.3%, free histidine 0.5%, free arginine 2.0%, and free proline 0.8%.

[0070] The hydrolyzed amino acid content is as follows: based on the weight of the yeast extract, hydrolyzed aspartic acid 6.23%, hydrolyzed threonine 2.71%, hydrolyzed serine 2.73%, hydrolyzed glutamic acid 12.33%, hydrolyzed glycine 2.74%, hydrolyzed alanine 5.17%, hydrolyzed cysteine ​​0.61%, hydrolyzed valine 3.84%, hydrolyzed methionine 0.84%, hydrolyzed isoleucine 3.65%, hydrolyzed leucine 4.72%, hydrolyzed tyrosine 1.65%, hydrolyzed phenylalanine 2.68%, hydrolyzed lysine 4.63%, hydrolyzed histidine 1.19%, hydrolyzed arginine 3.30%, and hydrolyzed proline 2.19%.

[0071] The *Saccharomyces cerevisiae* AMCC 33043 obtained in Example 1 of this invention was deposited at the China Center for Type Culture Collection (CCTCC) on April 3, 2026, with accession number CCTCC NO: M 2026601. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072.

[0072] The brewing yeast AMCC 31248 in this invention ( Saccharomyces cerevisiae AMCC 31248 was deposited at the China Center for Type Culture Collection (CCTCC) on December 29, 2021, with accession number CCTCC NO: M20211686, at Wuhan University, Wuhan, China, postal code 430072. It is also described in patent application CN117165460B.

[0073] The *Saccharomyces cerevisiae* strain AMCC 31557 of this invention was deposited on September 25, 2023, at the China Center for Type Culture Collection (CCTCC), accession number CCTCCNO: M20231789, address: Wuhan University, Wuhan, China, postcode: 430072. It has been described in patent application CN120682955A.

[0074] Example 1: Strain Construction This invention uses *Saccharomyces cerevisiae* strains AMCC 31557 and AMCC 31248 as parents to induce sporulation under the same conditions. The sporulation medium was formulated as follows (by weight percentage): 1% potassium acetate, 0.1% yeast extract, 0.05% glucose, and 2% agar, with the remainder being water. Haploids of both parental strains were prepared using a yeast micromanipulator and cultured at 30°C. The number of spores was recorded (the survival rate and serotyping of individual spores of the parental strains are shown in Table 3). After colony growth, the individual spores of parental strain AMCC 31248 were named dan1-dan40, and those of parental strain AMCC 31557 were named dan41-dan88. PCR identification was performed on these 88 surviving individual spores to determine their MATa / α serotyping. The mating type identification method was as follows: Using MAT-a, MAT-α, and MAT-F as primers, colony PCR was performed on the above-mentioned surviving single spores. The PCR program was: 94℃ pre-denaturation for 10 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s, for 30 cycles, followed by a final extension at 72℃ for 10 min. The amplified products were detected by 1.5% (1.5 g / 100 mL) agarose gel electrophoresis. A band at 404 bp confirmed the mating type as α, and a band at 544 bp confirmed the mating type as a. Specific results are shown in Table 3. Primer sequences are as follows: MAT-a(5'-ACTCCACTTCAAGTAAGAGTTTG-3')(SEQIDNO.1); MAT-α(5'-GCACGGAATATGGGACTACTTCG-3')(SEQIDNO.2); MAT-F(5'-AGTCACATCAAGATCGTTTATGG-3')(SEQIDNO.3); Survival rate (%) = (Number of surviving spores / Number of spores placed) * 100%;

[0075] The prepared single spores were inoculated into test tubes containing YPD liquid medium and cultured overnight at 30°C with shaking. They were then inoculated into 100-well microplates containing heavy sugar composite preservative screening medium. BioscreenC was used to detect the OD600 value of each strain in real time. The culture temperature was set to 30°C, the culture time to 48 h, and the measurement wavelength to 600 nm. Data were measured every 30 min. A growth curve was plotted with time (h) on the x-axis and OD600 value on the y-axis. Growth efficiency was analyzed based on the growth curve results. The formula for calculating growth efficiency is as follows: Growth efficiency = (OD2 - OD1) / (t2 - t1); OD1: The OD600 value of the strain at time t1; OD2: The OD600 value of the strain at time t2; t1: The start time of the logarithmic growth phase; t2: The end of the logarithmic growth phase.

[0076] Haploid strains with growth advantage in the screening medium of heavy sugar compound preservative were selected, and the top five single spores with the highest growth efficiency were selected (the growth efficiency and serotype of each strong spore are shown in Table 4). After activation culture, single spores of different parents and different serotypes were inoculated into test tubes containing YPD liquid medium and cultured overnight at 30°C with shaking. The bacterial solution was diluted and spread on YPD solid plates and cultured at 30°C. The colony growth was observed, and larger colonies were selected, marked and named.

[0077]

[0078] After activating and culturing the colonies marked on the above plates, typing and sporulation tests were performed. The screening criteria were: strains with double bands on electrophoresis and ascospores observed under a microscope after sporulation culture were identified as heterozygous strains.

[0079] Example 2: Growth Efficiency Determination The constructed hybrid strains were inoculated into a screening medium containing a high-sugar composite preservative and cultured at 30°C for 48 hours. The OD600 values ​​of each strain at different time points were measured using a Bioscreen C fully automated growth curve analyzer. Growth curves were plotted with time (h) on the x-axis and the corresponding OD600 values ​​on the y-axis to analyze and calculate growth efficiency. This screening step ultimately selected the top 40 strains based on their overall growth efficiency.

[0080] The growth curve of the Saccharomyces cerevisiae AMCC 33043 strain is shown below. Figure 2 As shown in the figure, it can be seen that the strain can grow rapidly in the screening medium of heavy sugar compound preservative. Table 5 shows the growth efficiency of the parent strain and the Saccharomyces cerevisiae AMCC 33043 strain. It can be seen that the growth efficiency of the obtained Saccharomyces cerevisiae AMCC 33043 strain is significantly higher than that of the parent strains Saccharomyces cerevisiae AMCC 31248 strain and Saccharomyces cerevisiae AMCC31557 strain.

[0081]

[0082] Example 3: TTC staining to detect cell viability After activating and culturing the preferred heterozygous strains obtained in Example 2, 500 μL of bacterial culture was taken, centrifuged, and the supernatant was discarded. An equal volume of pressure medium (25% sugar + 0.1% calcium propionate + 0.1% potassium sorbate) containing a final concentration of 0.4% TTC was added for resuspending. The culture was incubated at 30°C and 180 rpm for 3 hours. 200 μL of the bacterial culture was then used to measure OD600 using a microplate reader. The remaining bacterial culture was centrifuged, and the supernatant was discarded. The bacterial cells were resuspended in an equal volume of DMSO, centrifuged, and the supernatant was measured for OD486. The OD486 / OD600 ratio for each strain was calculated, and the heterozygous strain with the highest ratio was selected as the preferred strain. This screening step ultimately ranked the top 20 strains based on their OD486 / OD600 ratios.

[0083] TTC is a chromogenic agent; it is colorless in itself but can be reduced to red TTF by respiratory chain dehydrogenases in living cells. TTF is insoluble in water and, after extraction with organic solvents, its absorbance at a specific wavelength reflects the hydrogen transport capacity of the cell membrane electron transport chain. The deeper the color and the higher the absorbance, the stronger the cell viability. By detecting the cell viability of various hybrid strains under pressure culture conditions, their tolerance to environmental stress can be reflected to some extent. Based on this, superior hybrid strains that can tolerate high sugar content and complex preservatives can be further screened.

[0084] The OD486 / OD600 ratios of each strain are shown in Table 6. It can be seen that the OD486 / OD600 ratio of the Saccharomyces cerevisiae AMCC 33043 strain in the TTC-containing pressure medium is 0.322, which is significantly higher than that of the parental strains Saccharomyces cerevisiae AMCC31557 and Saccharomyces cerevisiae AMCC 31248. This indicates that the strain has good tolerance to high sugar content and compound preservatives and can maintain high cell activity under high environmental pressure conditions.

[0085]

[0086] The OD486 / OD600 ratios of other preferred heterozygous strains obtained in Example 2 were between 0.084 and 0.440.

[0087] Example 4: Yeast Milk Fermentation Activity Detection The preferred hybrid strains obtained in Example 3 were subjected to yeast milk fermentation experiments. The net dry weight of the hybrid strains and the fermentation activity of their yeast milk in various dough systems were used as screening indicators. The hybrid strains were inoculated into YPD liquid fermentation medium and cultured overnight (approximately 20 hours) at 30°C with shaking. After centrifugation and washing, the yeast milk was obtained. The mass of the yeast milk was measured, which is the biomass. The moisture content was detected using a rapid moisture meter. The net dry weight (g / L) and relative percentage (%) of the yeast milk for each strain were calculated using the following formula: Net dry weight (g / L) = mass of yeast milk × (1 - moisture %); Net dry weight percentage (%) = (net dry weight of heterozygous new strain / net dry weight of parent strain) * 100%; The collected yeast milk strains were tested for fermentation activity in different dough systems, including System 1: 25% sugar dough system, System 2: 25% sugar + 0.2% calcium propionate + 0.2% embedded sorbic acid dough system, and System 3: 25% sugar + 0.6% calcium propionate dough system. The proportions of each ingredient in different dough systems (calculated with flour mass as 100%) are shown in Table 7. The required amount of yeast milk to be added for each strain was calculated and weighed according to the moisture content of each strain. Flour, salt, sugar, and water were weighed according to the dough formulas shown, and the raw dough was prepared by mixing them evenly in a dough mixer. The total volume of carbon dioxide gas produced by yeast fermentation in the raw dough prepared in the systems shown in Table 7 at 30℃ was directly measured using an SJA fermentation apparatus, which is the fermentation activity of the strain. The results are expressed in milliliters (mL). The total gas production of 70g dough for 2 hours was measured for System 1, and the total gas production of 70g dough for 3 hours was measured for Systems 2 and 3.

[0088]

[0089] The relative percentage of dough fermentation activity of each strain compared to its parent strain can be calculated using the following formula: Dough fermentation activity relative percentage (%) = (Dough fermentation activity of heterozygous new strain / Dough fermentation activity of parent strain) * 100% As shown in Table 8, the dry biomass of the parental strains *Saccharomyces cerevisiae* AMCC 31557 and AMCC 31248 were 14.40 g / L and 13.66 g / L, respectively. The dry biomass of strain *Saccharomyces cerevisiae* AMCC 33043 reached 14.71 g / L, which is 2.2% higher than that of parental strain *Saccharomyces cerevisiae* AMCC 31557 and 7.7% higher than that of parental strain *Saccharomyces cerevisiae* AMCC 31248.

[0090]

[0091] The net dry weight of other preferred heterozygous strains obtained in Example 3 was between 12.37 and 16.27 g / L, which was 80.2% to 105.6% of the parent strain Saccharomyces cerevisiae AMCC 31557.

[0092] As shown in Table 9, using the parent strain AMCC 31557, which has higher fermentation activity, as the control strain, the fermentation activity of Saccharomyces cerevisiae AMCC33043 strain was 13.0% higher than that of the parent strain AMCC 31557 in the 25% sugar dough system, 13.3% higher in the 25% sugar + 0.2% calcium propionate + 0.2% sorbic acid embedded dough system, and 16.6% higher in the 25% sugar + 0.6% calcium propionate dough system.

[0093]

[0094] The activity of the other preferred heterozygous strains obtained in Example 3 for the 25% sugar dough system was 78.6%–128.9% of that of the parent strain Saccharomyces cerevisiae AMCC 31557; the activity of the 25% sugar + 0.2% calcium propionate + 0.2% sorbic acid-encapsulated dough system was 75.4%–125.7% of that of the parent strain Saccharomyces cerevisiae AMCC 31557; and the activity of the 25% sugar + 0.6% calcium propionate dough system was 79.2%–131.1% of that of the parent strain Saccharomyces cerevisiae AMCC 31557.

[0095] Example 5: Detection of Fermentation Activity of Active Dry Yeast The selected strains screened in Example 4 were scaled up and cultured in a 45L fermenter. The resulting active dry yeast was obtained through separation, washing, filtration, pressure filtration, and drying. Flour, salt, sugar, water, and active dry yeast were weighed according to the dough formulas shown in Table 10 to prepare different dough systems. The total amount of carbon dioxide produced, i.e., the fermentation activity of the active dry yeast in the corresponding dough system, was determined using the SJA method. The results were expressed in milliliters (mL). Different dough systems included: System 1: 25% sugar dough system (total gas production of 280g dough over 2 hours); System 2: 25% sugar + 0.6% calcium propionate dough system (total gas production of 280g dough over 3 hours); System 3: 25% sugar + 0.2% sorbic acid-encapsulated dough system (total gas production of 280g dough over 3 hours); and System 4: 25% sugar + 0.015% ε-polylysine dough system (total gas production of 280g dough over 3 hours). System 5: 25% sugar + 0.3% whey baking powder dough system, the total gas production of 280g dough over 3 hours was measured; System 6: 25% sugar + 0.2% calcium propionate + 0.2% encapsulated sorbic acid dough system, the total gas production of 70g dough over 3 hours was measured; System 7: 25% sugar + 0.2% calcium propionate + 0.2% encapsulated sorbic acid + 0.015% ε-polylysine + 0.3% whey baking powder dough system, the total gas production of 70g dough over 3 hours was measured.

[0096]

[0097] The brewing yeast strain AMCC 33043 showed no abnormalities during fermentation and sample preparation, and all physicochemical indicators were normal. As shown in Table 11, using the parent strain AMCC 31557, which has more superior traits, as the control strain, the active dry yeast prepared from the brewing yeast strain AMCC 33043 provided by this invention showed significant advantages in fermentation activity in various dough systems, indicating that it has excellent tolerance to high sugar content and multiple preservatives.

[0098]

[0099] Other strains of dry yeast did not exhibit outstanding fermentation activity in various dough systems, which was lower than that of the parent strain, Saccharomyces cerevisiae AMCC 31557, and thus did not meet the optimal selection criteria.

[0100] Example 6 Application Test The superior strain of active dry yeast was evaluated through application testing, and the formula used is shown in Table 12. The parent strain AMCC 31557, with superior performance, was used as the control strain. Fermentation time was used as the indicator; a shorter fermentation time indicated faster rise in the dough system, stronger yeast strain tolerance, and more advantageous characteristics. Specifically, the fermentation time refers to the time taken after preparing the dough according to the formula in Table 12, 400g of dough was shaped and placed in a proofing box at 38±1℃ and humidity controlled at 85-90%. The 400g dough rose to the height where the top was level with the top edge of the loaf pan.

[0101]

[0102] Table 13 shows the percentage data of fermentation time of Saccharomyces cerevisiae AMCC 33043 strain relative to the control strain. The results show that the fermentation speed of Saccharomyces cerevisiae AMCC 33043 strain in formulation 1 was significantly better than that of the control strain AMCC31557, with a 10.1% advantage in fermentation time. This indicates that Saccharomyces cerevisiae AMCC 33043 strain has excellent tolerance to high sugar content and multiple preservatives, making it highly suitable for long-shelf-life baking. In formulation 2, the fermentation time of Saccharomyces cerevisiae AMCC 33043 strain was 3% longer than that of the control strain, indicating that this strain has stronger tolerance and higher cell viability in high-oil, high-sugar systems, making it suitable for use in high-oil, high-sugar baked goods.

[0103] The resulting heterozygous strain AMCC 33043 was named Saccharomyces cerevisiae AMCC33043.

[0104] The target strain described in this invention is *Saccharomyces cerevisiae* strain AMCC 33043, and its colony morphology on YPD solid plates is shown in the figure below. Figure 1 As shown, a single colony is a slightly convex spherical shape in the center, milky white, with a relatively loose texture, easily picked up by the inoculation loop, with a smooth and relatively dry surface and neat edges.

[0105]

[0106] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0107] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not intended to limit the present invention. Any improvements made by those skilled in the art on this basis that do not highlight substantive features or make significant progress should fall within the protection scope of the present invention.

Claims

1. A brewing yeast with high sugar content and high resistance to multiple preservatives, characterized in that, The brewing yeast is brewing yeast AMCC 33043 ( Saccharomyces cerevisiae AMCC 33043 was deposited at the China Center for Type Culture Collection on April 3, 2026, with accession number CCTCC NO: M 2026601.

2. The brewing yeast according to claim 1, characterized in that, The net dry weight of the brewing yeast reaches 90%-110% of that of any parent strain, wherein the parent strain is brewing yeast AMCC 31557 with accession number CCTCC NO: M 20231789 or brewing yeast AMCC 31248 with accession number CCTCC NO: M 20211686.

3. The brewing yeast according to claim 1, characterized in that, The net dry weight of the brewing yeast reaches 95%-110% of that of any parent strain, wherein the parent strain is brewing yeast AMCC 31557 with accession number CCTCC NO: M 20231789 or brewing yeast AMCC 31248 with accession number CCTCC NO: M 20211686.

4. The brewing yeast according to any one of claims 1-3, characterized in that, The brewing yeast was obtained by sexual spore hybridization using brewing yeast AMCC 31557 and brewing yeast AMCC 31248 as parent strains.

5. A yeast milk, characterized in that, It contains the brewing yeast according to any one of claims 1-4.

6. The yeast milk according to claim 5, characterized in that, The yeast milk is prepared by a method including the following steps: culturing the brewing yeast, separating the solid and liquid components, and washing to obtain the yeast milk.

7. A microbial agent, characterized in that, It contains the brewing yeast according to any one of claims 1-4.

8. The microbial agent according to claim 7, characterized in that, The inoculant is active dry yeast.

9. The microbial agent according to claim 8, characterized in that, The microbial agent is prepared by a method including the following steps: culturing the brewing yeast, followed by separation, washing, filtration, pressure filtration and drying.

10. The use of the brewing yeast of any one of claims 1-4, or the yeast milk of any one of claims 5 or 6, or the inoculant of any one of claims 7-9, in the preparation of sweet bread, fancy dinner rolls, pull-apart bread, Danish pastries, or premixed powders containing preservatives.

11. The use of the yeast milk according to claim 5 or 6 in the preparation of dough, wherein, The fermentation activity of the brewer's yeast AMCC 33043 in the yeast milk in the heavy sugar dough system reaches 95%-120% of the fermentation activity of any parent strain. Here, fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained through the heavy sugar dough system at 30°C using the brewer's yeast AMCC 33043 in the yeast milk. The sugar mass percentage of the heavy sugar dough system is 20-40%. The parent strain is either Saccharomyces cerevisiae AMCC 31557 with accession number CCTCC NO: M 20231789 or Saccharomyces cerevisiae AMCC 31248 with accession number CCTCC NO: M 20211686.

12. The application according to claim 11, wherein, The fermentation activity of the brewer's yeast AMCC 33043 in the yeast milk in the heavy sugar dough system reached 100%-120% of the fermentation activity of the parent strain.

13. The use of the yeast milk according to claim 5 or 6 in the preparation of dough, wherein, The brewer's yeast AMCC 33043 in the yeast milk exhibits fermentation activity of 95%-120% of any parent strain in a high-sugar dough system containing 0-0.6% by mass of preservative A. This fermentation activity refers to the total volume of carbon dioxide gas produced by fermenting raw dough obtained from the high-sugar dough system containing 0-0.6% by mass of preservative A at 30°C using the brewer's yeast AMCC33043 in the yeast milk. The sugar content of the high-sugar dough system is 20-40% by mass. Among them, any one of the parental strains is *Saccharomyces cerevisiae* AMCC 31557 with accession number CCTCC NO: M 20231789 or *Saccharomyces cerevisiae* AMCC 31248 with accession number CCTCC NO: M 20211686. The preservative A is calcium propionate.

14. The use of the yeast milk according to claim 5 or 6 in the preparation of dough, wherein, The brewer's yeast AMCC 33043 in the yeast milk exhibits fermentation activity of 95%-120% of any parent strain in a heavy sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B. The fermentation activity refers to the total volume of carbon dioxide gas produced by fermenting raw dough obtained from the heavy sugar dough system (containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B) at 30°C using the brewer's yeast AMCC 33043 in the yeast milk. The sugar content of the heavy sugar dough system is 20-40% by mass. Among them, any one of the parental strains is *Saccharomyces cerevisiae* AMCC 31557 with accession number CCTCC NO: M 20231789 or *Saccharomyces cerevisiae* AMCC 31248 with accession number CCTCC NO: M 20211686. Wherein, preservative A is calcium propionate, and preservative B is encapsulated sorbic acid.

15. The application according to claim 13, wherein, The brewer's yeast AMCC 33043 in the yeast milk exhibits a fermentation activity of 100%-120% of that of any parent strain in a heavy sugar dough system containing 0-0.6% by mass of preservative A.

16. The application according to claim 14, wherein, The brewing yeast AMCC 33043 in the yeast milk achieves a fermentation activity of 100%-120% of that of any parent strain in a heavy sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B.

17. The use of the microbial agent according to claim 8 or 9 in the preparation of dough, wherein, The fermentation activity of the brewer's yeast AMCC 33043 in the microbial agent in the heavy sugar dough system reaches 95%-120% of the fermentation activity of any parent strain. The fermentation activity refers to the total volume of carbon dioxide gas produced by the fermentation of raw dough made by the heavy sugar dough system at 30°C by the brewer's yeast in the active dry yeast. The sugar mass percentage of the heavy sugar dough system is 20-40%.

18. The application of the microbial agent according to claim 17 in the preparation of dough, wherein, The fermentation activity in the heavy sugar dough system reached 100%-120% of the fermentation activity of any parent strain.

19. The use of the microbial agent according to claim 8 or 9 in the preparation of dough, wherein, The brewer's yeast AMCC 33043 in the microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.6% preservative A, reaching 95%-120% of the fermentation activity of any parent strain. This fermentation activity refers to the total volume of carbon dioxide gas produced by fermenting raw dough obtained from the high-sugar dough system containing 0-0.6% preservative A at 30°C using the brewer's yeast AMCC33043 in the microbial agent. The sugar content of the high-sugar dough system is 20-40% by mass. Alternatively, the fermentation activity of the brewer's yeast AMCC 33043 in the aforementioned inoculum in a high-sugar dough system containing 0-0.2% preservative B reaches 95%-120% of the fermentation activity of any parent strain. Here, fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained from the high-sugar dough system containing 0-0.2% preservative B at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Alternatively, the fermentation activity of the brewer's yeast AMCC 33043 in the aforementioned inoculum in a high-sugar dough system containing 0-0.015% preservative C reaches 95%-120% of the fermentation activity of any parent strain. Here, fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained from the high-sugar dough system containing 0-0.015% preservative C at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Alternatively, the fermentation activity of the brewer's yeast AMCC 33043 in the aforementioned inoculum in a high-sugar dough system containing 0-0.3% by mass of preservative D reaches 95%-120% of the fermentation activity of any parent strain. Here, fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained through the high-sugar dough system containing 0-0.3% by mass of preservative D at 30°C using the brewer's yeast AMCC 33043 in the aforementioned inoculum; the sugar content of the high-sugar dough system is 20-40% by mass. Among them, any one of the parental strains is *Saccharomyces cerevisiae* AMCC 31557 with accession number CCTCC NO: M 20231789 or *Saccharomyces cerevisiae* AMCC 31248 with accession number CCTCC NO: M 20211686. Wherein, preservative A is calcium propionate, preservative B is encapsulated sorbic acid, preservative C is ε-polylysine, and preservative D is whey baking powder.

20. The use of the microbial agent according to claim 8 or 9 in the preparation of dough, wherein, The brewer's yeast AMCC 33043 in the inoculum agent exhibits fermentation activity reaching 95%-120% of the fermentation activity of any parent strain in a high-sugar dough system containing 0-0.2% (w / w) of preservative A and 0-0.2% (w / w) of preservative B. The fermentation activity refers to the total volume of carbon dioxide gas produced by fermenting raw dough obtained from the high-sugar dough system (containing 0-0.2% (w / w) of preservative A and preservative B) at 30°C using the brewer's yeast AMCC 33043 in the inoculum agent. The sugar content of the high-sugar dough system is 20-40% by mass. Among them, any one of the parental strains is *Saccharomyces cerevisiae* AMCC 31557 with accession number CCTCC NO: M 20231789 or *Saccharomyces cerevisiae* AMCC 31248 with accession number CCTCC NO: M 20211686. Wherein, preservative A is calcium propionate, and preservative B is encapsulated sorbic acid.

21. The use of the microbial agent according to claim 8 or 9 in the preparation of dough, wherein, The brewer's yeast AMCC 33043 in the inoculum, in a heavy sugar dough system containing 0-0.2% by mass of preservative A, 0-0.2% by mass of preservative B, 0-0.015% by mass of preservative C, and 0-0.3% by mass of preservative D, achieves 95%-120% of the fermentation activity of any parent strain. The fermentation activity refers to the total volume of carbon dioxide gas produced by fermentation of raw dough obtained from the heavy sugar dough system containing 0-0.2% by mass of preservative A, 0-0.2% by mass of preservative B, 0-0.015% by mass of preservative C, and 0-0.3% by mass of preservative D at 30°C using the brewer's yeast AMCC 33043 in the inoculum; the sugar percentage of the heavy sugar dough system is 20-40% by mass. Among them, any one of the parental strains is *Saccharomyces cerevisiae* AMCC 31557 with accession number CCTCC NO: M 20231789 or *Saccharomyces cerevisiae* AMCC 31248 with accession number CCTCC NO: M 20211686. Wherein, preservative A is calcium propionate, preservative B is encapsulated sorbic acid, preservative C is ε-polylysine, and preservative D is whey baking powder.

22. The application according to claim 19, wherein, The brewer's yeast AMCC 33043 in the microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.6% preservative A, reaching 100%-120% of the fermentation activity of any parent strain. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.2% preservative B, reaching 100%-120% of the fermentation activity of any parent strain. Alternatively, the brewer's yeast AMCC 33043 in the aforementioned microbial agent exhibits fermentation activity in a high-sugar dough system containing 0-0.015% preservative C, reaching 100%-120% of the fermentation activity of any parent strain. Alternatively, the fermentation activity of the brewing yeast AMCC 33043 in the microbial agent in a heavy sugar dough system containing 0-0.3% by mass of preservative D reaches 100%-120% of the fermentation activity of any parent strain.

23. The application according to claim 20, wherein, The brewing yeast AMCC 33043 in the microbial agent exhibits fermentation activity in a heavy sugar dough system containing 0-0.2% by mass of preservative A and 0-0.2% by mass of preservative B, reaching 100%-120% of the fermentation activity of any parent strain.

24. The application according to claim 21, wherein, The brewing yeast AMCC 33043 in the microbial agent exhibits fermentation activity in a heavy sugar dough system containing 0-0.2% by mass of preservative A, 0-0.2% by mass of preservative B, 0-0.015% by mass of preservative C, and 0-0.3% by mass of preservative D, reaching 100%-120% of the fermentation activity of any parent strain.

25. A type of dough, characterized in that, It comprises the brewing yeast of any one of claims 1-4, or the yeast milk of any one of claims 5 or 6, or the inoculum of any one of claims 7-9.

26. The dough according to claim 25, characterized in that, The dough is any one of the following types of dough: (1) Heavy sugar dough, (2) Heavy sugar dough containing 0-0.6% of preservative A relative to the weight of flour, (3) Heavy sugar dough containing 0-0.2% of preservative B relative to the weight of flour, (4) Heavy sugar dough containing 0-0.015% of preservative C relative to the weight of flour, or (5) Heavy sugar dough containing 0-0.3% of preservative D relative to the weight of flour. The sugar content in the sugar-rich dough is 20-40% by mass. Wherein, preservative A is calcium propionate, preservative B is encapsulated sorbic acid, preservative C is ε-polylysine, and preservative D is whey baking powder.

27. The dough according to claim 25, characterized in that, The dough is a high-sugar dough containing both preservative A (0-0.2% by weight of flour) and preservative B (0-0.2% by weight of flour). The sugar content in the sugar-rich dough is 20-40% by mass. Wherein, preservative A is calcium propionate, and preservative B is encapsulated sorbic acid.

28. The dough according to claim 25, characterized in that, The dough is a high-sugar dough containing preservative A (0-0.2% of flour weight), preservative B (0-0.2% of flour weight), preservative C (0-0.015% of flour weight), and preservative D (0-0.3% of flour weight). The sugar content in the sugar-rich dough is 20-40% by mass. Wherein, preservative A is calcium propionate, preservative B is encapsulated sorbic acid, preservative C is ε-polylysine, and preservative D is whey baking powder.

29. A method for preparing dough according to any one of claims 25-28, characterized in that, The method includes the following steps: adding the brewing yeast of any one of claims 1-4, or the yeast milk of any one of claims 5 or 6, or the inoculant of any one of claims 7-9 to the dough and fermenting it.

30. A method for preparing a baked product, characterized in that, The dough prepared by any one of claims 25-28 or by the method of claim 29 is baked to obtain a baked product.

31. The preparation method according to claim 30, characterized in that, The baked products are one or more of the following: sweet bread, fancy dinner rolls, pull-apart bread, and Danish pastries.

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