CN112725209B

CN112725209B - Saccharomyces cerevisiae and leaven for efficiently utilizing maltotriose and application of saccharomyces cerevisiae and leaven in fermented grain beverage and malt PYF factor detection

Info

Publication number: CN112725209B
Application number: CN202110065027.9A
Authority: CN
Inventors: 李小燕; 赵凯; 李慧; 陈文波; 祁雪; 王宇翕
Original assignee: Cofco Malt Dalian Co ltd; Cofco Nutrition and Health Research Institute Co Ltd
Current assignee: Cofco Malt Dalian Co ltd; Cofco Nutrition and Health Research Institute Co Ltd
Priority date: 2021-01-18
Filing date: 2021-01-18
Publication date: 2021-08-17
Anticipated expiration: 2041-01-18
Also published as: CN112725209A

Abstract

The invention relates to the technical field of food processing, and discloses saccharomyces cerevisiae and a leaven for efficiently utilizing maltotriose and application thereof in fermented grain beverages and malt PYF factor detection. The preservation number of the saccharomyces cerevisiae is CGMCC No. 20489. The saccharomyces cerevisiae provided by the invention has the advantages of high maltotriose utilization rate and high fermentation speed, is a brewer's saccharomyces cerevisiae with high fermentation degree, high diacetyl reducing capability and high alcohol production capability, has moderate flocculation property, is sensitive to a PYF factor, and can be used for detecting the PYF factor of malt. The Saccharomyces cerevisiae can be cultured at high density, and can be used for preparing beer starter, with convenient application.

Description

Saccharomyces cerevisiae and leaven for efficiently utilizing maltotriose and application of saccharomyces cerevisiae and leaven in fermented grain beverage and malt PYF factor detection

Technical Field

The invention relates to the technical field of food processing, in particular to saccharomyces cerevisiae for efficiently utilizing maltotriose, a leavening agent, a preparation method of the leavening agent and the leavening agent prepared by the method, application of the saccharomyces cerevisiae or the leavening agent in preparation of fermented cereal beverage, a preparation method of the fermented cereal beverage, application of the saccharomyces cerevisiae or the leavening agent in PYF detection and a detection method of a malt PYF factor.

Background

Saccharomyces cerevisiae is a unicellular eukaryotic microorganism mainly used for budding propagation, the cell shape is oval, long oval (elliptical), the colony size on a YPD medium plate is 4-5 mm, the surface is smooth and convex, the aged surface is in a radioactive umbrella shape, the texture is wet, and the color is milky white. Fermenting in wort liquid culture medium to generate bubbles and foam on the surface, suspending thallus in the culture medium to be turbid, depositing thallus cells at the bottom of the container in the later stage of fermentation, and clarifying the fermentation liquid. The excellent saccharomyces cerevisiae can ferment grape juice at 25-30 ℃ to generate good fruit fragrance and wine fragrance. The optimum pH value of the fermentation is 4.0-4.5, the fermentation capacity is high, the sugar can be completely fermented, and the coagulation capacity and the rapid sedimentation capacity are good.

The saccharides capable of being fermented by the yeast are called "fermentable sugars", and the fermentable sugars and the fermentation sequence of the saccharomyces cerevisiae are glucose > fructose > sucrose > maltose > maltotriose. In the fermentation process, the metabolic laws and metabolic mechanisms of 5 fermentable sugars are different, and the efficient fermentation of beer requires that the sugars can be quickly and completely utilized. Saccharomyces cerevisiae is the most commonly used strain for producing beer, but the capability of utilizing maltotriose is poor, and the failure of effectively utilizing the maltotriose in the beer fermentation process is the main reason of high residual sugar content and slow fermentation of the finished beer.

Diacetyl is an important byproduct in the beer fermentation process, is an important substance influencing the beer flavor, is also a restrictive index of beer maturation, and is diacetyl as one of the main influencing factors for shortening the beer fermentation period. The diacetyl in beer is mainly synthesized by secreting alpha-Acetolactate (alpha-Acetolactate) produced by saccharomyces cerevisiae cells through a valine metabolic pathway to the outside of cells and performing non-enzymatic oxidative decarboxylation reaction.

Therefore, the speed and the utilization rate of the maltotriose by yeast and the diacetyl reducing capability of the yeast are the bottleneck of preparing beer by fermenting wort, and are the key points for improving the fermenting capability.

The Phenomenon of Yeast Flocculation (PYF) is ubiquitous in the beer brewing industry and is not effectively solved so far. The yeast is abnormally seriously (or early) flocculated in the fermentation process of the beer, so that the beer has flavor defects and incomplete fermentation at the end of the fermentation. Therefore, it is predicted that malt PYF is of critical importance to the brewing industry.

Disclosure of Invention

In order to achieve the purpose, the invention provides a saccharomyces cerevisiae for efficiently utilizing maltotriose, a leavening agent, a preparation method of the leavening agent and the leavening agent prepared by the method, application of the saccharomyces cerevisiae or the leavening agent in preparation of fermented cereal beverage, a preparation method of the fermented cereal beverage, application of the saccharomyces cerevisiae or the leavening agent in PYF detection and a detection method of a malt PYF factor. The saccharomyces cerevisiae provided by the invention has the advantages of high maltotriose utilization rate and high fermentation speed, is a brewer's saccharomyces cerevisiae with high fermentation degree, high diacetyl reducing capability and high alcohol production capability, is sensitive to a PYF factor, can be cultured at high density, is used for preparing a beer starter, and is convenient to use.

Therefore, the invention provides a Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the preservation number of CGMCC No.20489 in the first aspect.

In a second aspect, the present invention provides a starter culture comprising a Saccharomyces cerevisiae (Saccharomyces cerevisiae) as described above.

In a third aspect, the present invention provides a method for preparing a fermentation agent, the method comprising:

(1) fermenting and culturing the saccharomyces cerevisiae in a fermentation culture medium to ensure that the viable count reaches 10⁸More than cfu/mL to obtain a liquid leavening agent;

(2) mixing the liquid leaven obtained in the step (1) with a fermentation substrate to obtain a semi-liquid leaven;

(3) carrying out solid-liquid separation on the semi-liquid leavening agent obtained in the step (2) to obtain a concentrated or compressed leavening agent;

(4) adding a freeze-drying protective agent into the concentrated or compressed leavening agent obtained in the step (3), and adjusting the concentration of the viable bacteria to 10¹⁰More than cfu/mL, and drying the obtained mixed material to obtain the dry leaven.

In a fourth aspect, the present invention provides a starter prepared by the preparation method as described above.

In a fifth aspect the present invention provides the use of a saccharomyces cerevisiae as described above or a leavening agent as described above for fermenting a cereal beverage.

In a sixth aspect, the present invention provides a method for preparing a fermented cereal beverage, the method comprising: the saccharomyces cerevisiae as described above or the starter culture as described above is contacted with a fermentation substrate and fermented to prepare a fermented cereal beverage.

The seventh aspect of the present invention provides a fermented cereal beverage prepared by the above preparation method.

In an eighth aspect the invention provides the use of a saccharomyces cerevisiae as described above or a starter culture as described above in the assay of malt PYF.

Through the technical scheme of the invention, the following beneficial effects can be obtained:

1. safe and healthy, and low cost: the beer saccharomyces cerevisiae is a safe strain which is screened from the old flour yeast reserved in Shandong near-Yi farmers and can be used for food, and the method has no chemical addition, is green and natural, and is nutritional and healthy;

2. the beer brewing yeast maltotriose of the invention has high utilization rate, high fermentation speed, high fermentation degree, high diacetyl reducing capability, high alcohol producing capability and good flavor of fermented beer. Specifically, the beer residue brewed by the saccharomyces cerevisiae is low in sugar content, the utilization rate of maltotriose is up to 88.80%, the main fermentation time is 5 days, diacetyl is reduced to 0.05mg/mL after being stored for 6 days at 4 ℃, and the alcoholic strength reaches 7.00% vol.

3. The beer saccharomyces cerevisiae is moderate in flocculation property and sensitive to the malt PYF factor, and the stability of the malt PYF factor is high by detecting, the repeatability is less than or equal to 10%, and the repeatability is less than or equal to 10%.

4. The beer saccharomyces cerevisiae CGMCC No.20489 is used as a starter, is easy to culture and prepare, and has high viable bacteria concentration.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Biological preservation

The Saccharomyces cerevisiae provided by the invention is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) at 8-month and 6-month in 2020, the preservation number is CGMCC No.20489, the preservation address is No. 3 of the national institute of microbiology (CGMCC) of Naja district of Beijing.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 shows the colony morphology of Saccharomyces cerevisiae on a culture medium according to the present invention;

FIG. 2 shows the microscopic morphology of Saccharomyces cerevisiae of the present invention;

FIG. 3 shows flocculation behavior of yeast of the present invention.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In a first aspect, the invention provides a Saccharomyces cerevisiae (Saccharomyces cerevisiae) with the preservation number of CGMCC No. 20489.

The saccharomyces cerevisiae CGMCC No.20489 has the following properties:

(1) morphological characteristics: the yeast is round and oval, and the diameter is 1.5-3.0 μm; after growing on the surface of the YPD solid medium for 48 hours, colonies with the diameter of about 3.0mm can grow, and the colonies are glossy yellow white dots, have neat edges, are uniform in texture and are easy to pick.

(2) Sugar utilization rate: the utilization rate of sugar is high, and the utilization rate of maltotriose is up to 88.80%.

(3) Fermentation capacity: the fermentation speed is high, the main fermentation time is 5 days, and the fermentation degree is high (82.33%).

(4) Diacetyl-reducing ability: the diacetyl is reduced rapidly, and the diacetyl is reduced to 0.05mg/mL after being stored for 6 days at 4 ℃.

(5) Flavor: the beer contains flavor components such as ethyl acetate, ethyl caprylate, ethyl caproate and phenethyl alcohol, and gives the beer the flavor of fresh and sweet fruits. The fermented beer is clear and transparent and has pure taste.

(6) Flocculation property: the Benss value was 0.5 cm.

(7) PYF factor sensitivity: sensitive to PYF factor and discriminative for different PPF value.

The ITS1/ITS4 sequence of the Saccharomyces cerevisiae CGMCC No.20489 is shown in SEQ ID NO. 1, and the similarity reaches 99.9 percent when the sequence is compared with the sequence of NCBI Saccharomyces cerevisiae (Saccharomyces cerevisiae).

SEQ ID NO:1：

TTTTGAAAATGGATTTTTTTGTTTTGGCAAGAGCATGAGAGCTTTTACTGGGCAAGAAGACAAGAGATGGAGAGTCCAGCCGGGCCTGCGCTTAAGTGCGCGGTCTTGCTAGGCTTGTAAGTTTCTTTCTTGCTATTCCAAACGGTGAGAGATTTCTGTGCTTTTGTTATAGGACAATTAAAACCGTTTCAATACAACACACTGTGGAGTTTTCATATCTTTGCAACTTTTTCTTTGGGCATTCGAGCAATCGGGGCCCAGAGGTAACAAACACAAACAATTTTATTTATTCATTAAATTTTTGTCAAAAACAAGAATTTTCGTAACTGGAAATTTTAAAATATTAAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCAGGGGGCATGCCTGTTTGAGCGTCATTTCCTTCTCAAACATTCTGTTTGGTAGTGAGTGATACTCTTTGGAGTTAACTTGAAATTGCTGGCCTTTTCATTGGATGTTTTTTTTTCCAAAGAGAGGTTTCTCTGCGTGCTTGAGGTATAATGCAAGTACGGTCGTTTTAGGTTTTACCAACTGCGGCTAATCTTTTTTATACTGAGCGTATTGGAACGTTATCGATAAGAAGAGAGCGTCTAGGCGAACAATGTTCTTAAAGTTTGACCTCAAATCAGGTAGGAGTACCC

The saccharomyces cerevisiae CGMCC No.20489 is a safe strain which is screened from aged leaven left in Shandong near-Yi farmers and can be used for food.

The saccharomyces cerevisiae provided by the invention can generate a large amount of live saccharomyces cerevisiae strains through liquid culture, and the culture method is not particularly required as long as the saccharomyces cerevisiae can be proliferated, and for example, the culture method can be 10^6-8Inoculating the live bacteria of the saccharomyces cerevisiae into a saccharomyces cerevisiae culture medium with the inoculation amount of CFU/mL, and culturing for 8-24 hours at the temperature of 25-30 ℃ under aerobic conditions to obtain a culture solution. The culture medium for the yeast may be any medium known in the art suitable for the cultivation of Saccharomyces cerevisiae, and may be at least one of molasses, 5 Bee wort and YPD medium, for example.

In the present invention, the viable cells of saccharomyces cerevisiae in the culture solution can be further separated, and the method for separating is not particularly limited as long as the cells can be enriched from the culture solution, and for example, the separation can be achieved by a centrifugation and/or filtration method, and the conditions for centrifugation and filtration can be known conditions, and the present invention is not described herein again.

According to the present invention, the form of the leavening agent may not be particularly limited, and may be, for example, a liquid leavening agent, a semi-liquid leavening agent, a concentrated leavening agent, a compressed leavening agent, or a solid leavening agent. The solid microbial inoculum can be a dry microbial inoculum, for example, a freeze-dried microbial inoculum, a spray-dried microbial inoculum, and the like.

In a third aspect, the present invention provides a method for preparing a starter, comprising:

According to the present invention, in step (1), the fermentation medium may be any of various fermentation media conventionally used in the art for fermenting Saccharomyces cerevisiae, for example, YPD medium as described above, and various media suitable for fermenting Saccharomyces cerevisiae, such as wort medium and molasses medium, may be used.

According to the present invention, the conditions of the fermentation culture may be conventional conditions for yeast fermentation culture well known in the art, for example, the temperature of the fermentation culture may be 10-40 ℃.

According to the invention, in step (2), the fermentation substrate may differ according to the intended use of the fermentation inoculant, for example, when the fermentation inoculant is intended for beer fermentation, the fermentation substrate may be wort.

Wherein the amount of the fermentation substrate to be used is not particularly limited as long as the liquid fermentation agent obtained in step (1) can be converted into a semi-liquid fermentation agent.

According to the present invention, in the step (3), the solid-liquid separation method can refer to the conventional technical means in the field, for example, centrifugation, filtration and the like can be adopted, so long as the activity of the saccharomyces cerevisiae is not significantly affected. According to a preferred embodiment of the invention, the fermented broth is centrifuged to obtain a concentrated or compressed starter culture. The centrifugation can be performed, for example, in a refrigerated centrifuge at a speed of 5000-.

According to the present invention, in the step (4), the lyoprotectant may be any conventional cryoprotectant in the art, for example, at least one of skim milk powder, maltodextrin, trehalose, dextran, glycerin, and the like.

According to the present invention, preferably, before adding the lyoprotectant to the concentrated or compressed starter culture, the method further comprises washing the concentrated or compressed starter culture with a buffer. The buffer may be a buffer conventionally used in the art for washing the cells, and may be, for example, physiological saline or PBS buffer.

The drying method is not particularly limited, and may be, for example, freeze-drying, oven-drying, air-drying or spray-drying.

In a fifth aspect, the present invention provides the use of a saccharomyces cerevisiae as described above or a leavening agent as described above for fermenting a cereal beverage.

According to a preferred embodiment of the present invention, the fermented cereal beverage is a fermented wine, more preferably a fermented beer.

In the conventional production process for producing fermented cereal beverages, Saccharomyces cerevisiae CGMCC No.20489 can be inoculated into a fermentation substrate to be treated according to a conventional use method, and fermentation is carried out at a temperature and a pressure at which the Saccharomyces cerevisiae CGMCC No.20489 can be propagated.

The CGMCC No.20489 is added into the fermentation substrate, and the metabolite of the CGMCC No.20489 can make the fermented wine have excellent characteristics of certain appearance, fragrance and the like, thereby improving the sensory quality characteristics of the product.

In a sixth aspect, the present invention provides a method of preparing a fermented cereal beverage, the method comprising: the saccharomyces cerevisiae as described above or the starter culture as described above is contacted with a fermentation substrate and fermented to prepare a fermented cereal beverage.

According to the present invention, those skilled in the art can inoculate the saccharomyces cerevisiae or the leaven to the corresponding fermentation substrate according to the kind of the predetermined fermented cereal beverage, and after the fermentation is finished, the preparation of the corresponding fermented food is performed according to the conventional method.

According to a preferred embodiment of the present invention, Saccharomyces cerevisiae CGMCC No.20489 is inoculated into a fermentation substrate for beer fermentation according to a conventional use method, and fermentation is carried out at 20-40 ℃ to obtain a fermented beer.

Among them, the Saccharomyces cerevisiae CGMCC No.20489 provided by the invention has high utilization rate of maltotriose and high fermentation speed in fermentation substrate containing maltotriose, so that the fermentation substrate preferably contains maltotriose, for example, wort is taken as fermentation substrate.

In a seventh aspect, the present invention provides a fermented cereal beverage prepared by the preparation method as described above.

In an eighth aspect, the invention provides the use of a saccharomyces cerevisiae as described above or a starter culture as described above in the assay of the malt PYF factor.

Examples

The media formulations referred to in the following examples are as follows:

YPD medium (wt%): yeast powder (1%), peptone (2%), glucose (2%) and agar (2%), heating for dissolving, and autoclaving at 121 deg.C for 15-20 min.

Wort culture medium: barley malt was pulverized according to a ratio of 1: 4, keeping the mixture at 45 ℃ for 30min, at 64 ℃ for 40min and at 70 ℃ for 20min, filtering, adding agar (2 percent), and autoclaving at 115 ℃ for 15 min.

Molasses culture medium: diluting molasses to 68-10o B, adding yeast powder 2g/L and ammonium sulfate 0.5g/L, and autoclaving at 115 deg.C for 15 min.

Example 1

This example illustrates the isolation, purification and characterization of Saccharomyces cerevisiae CGMCC No.20489

The strain is as follows: the beer brewing yeast CGMCC No.20489 is separated from aged flour yeast reserved in Shandong near-Yi farmer homes and is preserved by the nutritional and health strain preservation center of the Chinese grain nutritional and health research institute.

The inventor collects old flour leaven from Shandong, Shanxi, etc., and dilutes the old flour leaven to 10 degrees by aseptic normal saline in a gradient way^-6Each dilution gradient was plated on WL and YPD plates and incubated at 28. + -. 1 ℃ for 72 h. And selecting colonies with different colony morphologies by an inoculating needle, and streaking on a WL plate and a YPD plate until single colonies are uniform in size and morphology.

Selecting the bacterial strain with round and oval cell morphology and budding reproduction. The strain separated to tentatively become the saccharomycetes is activated in an YPD liquid culture medium for 3 generations and then subjected to physiological and biochemical identification and molecular biological identification, and the growth, fermentation performance, appearance quality characteristics, texture characteristics, fermentation flavor substances, sensory quality and other aspects of the saccharomycetes are researched, and a strain of the saccharomyces cerevisiae is finally screened from a plurality of wild saccharomycetes through multiple rounds of research and demonstration.

1. Morphological identification

The selected Saccharomyces cerevisiae was cultured at 28. + -. 1 ℃ for 48 hours, and the colony was pale green, round, smooth on the surface, moist, and opaque on the WL medium as shown in FIG. 1. As shown in FIG. 2, the microscopic cell morphology of Saccharomyces cerevisiae was ovoid.

2. Physiological and biochemical identification

The screened strain CGMCC NO. is subjected to physiological and biochemical identification by using a French Merrier API identification system, and the identification result is shown in Table 1 below. The separated strain is Saccharomyces cerevisiae (Saccharomyces cerevisiae) through physiological and biochemical identification.

TABLE 1 physiological and biochemical identification experiment results of Saccharomyces cerevisiae of the present invention

[ note ]: in the table, "+" indicates that the biochemical reaction result is positive, and "-" indicates that the biochemical reaction result is negative.

3. Molecular identification

The ITS1/ITS4 of the separated strain is cloned and sequenced, the nucleotide sequence of the ITS1/ITS4 gene is shown as SEQ ID NO:1, the ITS1/ITS4 sequence of the strain is compared with the sequence of Saccharomyces cerevisiae, and the ITS1/ITS4 sequence of the strain has 99.9 percent of similarity with the Saccharomyces cerevisiae sequence.

SEQ ID NO:1：

The strain is identified as Saccharomyces cerevisiae (Saccharomyces cerevisiae) by combining the biochemical identification result.

The isolated strain is determined as Saccharomyces cerevisiae (Saccharomyces cerevisiae) and is preserved in China general microbiological culture Collection center, the preservation address is as follows: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, has a preservation number of CGMCC No.20489 and a preservation date of 2020, 8 months and 6 days.

Example 2

This example illustrates the study of the application of Saccharomyces cerevisiae in beer fermentation

Activating yeast with wort, inoculating yeast liquid into 12 ° P wort at an inoculation amount of 4%, fermenting at 20 deg.C, and finishing pre-fermentation (the main stage of fermentation, the yeast active period, during which most of fermentable sugar in wort is fermented and some main metabolites of yeast are produced in the period) (CO)₂The weight loss is less than 0.1 g/day to indicate the end of fermentation), post-fermentation (post-fermentation is also called wine storage, and aims to finish the final fermentation of residual sugar, increase the stability of beer and fill CO₂Fully precipitating protein and clarifying the liquor; removing diacetyl, aldehydes and H₂S, and the like, to promote the maturation; the wine is in a reduction state as much as possible, the oxygen content is reduced) and stored at 4 ℃ until the diacetyl content meets the national standard requirement.

1. Determination research on utilization rate of maltotriose of saccharomyces cerevisiae

1) Experimental Material

High performance liquid chromatography, column: aminex HPX-87H (300 mm. times.7.8 mm)

2) Experimental methods

Liquid phase conditions: column temperature: 65 ℃; the mobile phase was 0.005moL/LH2SO4 at a flow rate of 0.600 mL/min. The detector is a differential detector; the amount of sample was 20. mu.L.

The experimental results are as follows:

after the post fermentation is finished, the beer brewed by the saccharomyces cerevisiae has the maltotriose content of 1.23mg/mL, the maltotriose content in wort is 10.98mg/mL, and the conversion rate of the maltotriose is 88.80%.

2. Research on fermentation speed of saccharomyces cerevisiae

The fermentation rate was determined by means of weighing, CO₂The weight loss within 0.1 g/day indicates the end of the primary fermentation.

The experimental results are as follows:

beer brewed by the saccharomyces cerevisiae of the invention reaches the pre-fermentation end point on the 5 th day of fermentation.

3. Research on diacetyl reducing capability of saccharomyces cerevisiae

1) Experimental Material

Diacetyl distillation device, microplate reader and colorimetric cylinder

Organosilicon defoaming agent, concentrated hydrochloric acid and o-phenylenediamine

2) Experimental methods

The diacetyl distiller was installed and the steam generating flask was heated to boiling. After ventilation and preheating, a 25mL volumetric flask is placed at the outlet of a condenser to receive distillate (an ice bath is added), 1-2 drops of antifoaming agent are added into a 100mL graduated cylinder, 100mL of non-degassed wine sample which is pre-cooled to 5 ℃ is injected, the wine sample is quickly transferred into a distiller, and a funnel with a plug and a cover plug are washed by a small amount of water. Then sealing with water, distilling until the distillate is close to 25mL (distillation needs to be completed within 3 min), taking off the volumetric flask, reaching room temperature, adding water to constant volume, and shaking up.

Respectively sucking 10.0mL of distillate into two dry colorimetric tubes, adding 0.50mL of o-phenylenediamine solution into a first branch tube, adding no (blank) solution into a second branch tube, fully shaking uniformly, simultaneously placing in a dark place for 20-30 min, then adding 2mL of hydrochloric acid solution (4mol/L) into the first branch tube, adding 2.5mL of hydrochloric acid solution into the second branch tube, uniformly mixing, and measuring the absorbance of the mixture by using a 20mm quartz cuvette under the wavelength of 335nm and taking the blank as a reference.

The diacetyl content of the sample was calculated as follows:

X-5＝A-335×1.2

in the formula:

x-5-diacetyl content of sample in milligrams per liter (mg/L);

a335-absorbance of the sample at a wavelength of 335nm measured with a 20mm quartz cuvette;

1.2-conversion factor

The experimental results are as follows:

after 6 days of storage at 4 ℃ the diacetyl content of the beer brewed with the Saccharomyces cerevisiae of the present invention was reduced to 0.05 mg/mL.

4. Research on alcohol production capacity of saccharomyces cerevisiae

1) Experimental Material

Alcohol distillation device, water bath, density bottle, 1000mL Erlenmeyer flask, 100mL volumetric flask

2) Experimental methods

a) Sample preparation:

pouring about 300mL of wine sample which is kept at the constant temperature of 15-20 ℃ into a 1000mL conical flask, adding a rubber stopper, slightly shaking in a constant temperature chamber, opening the stopper, deflating (beginning to have a popping sound), and covering the stopper. The operation is repeated until no gas escapes. Filter with single layer medium speed dry filter paper (top of funnel cover surface glass).

b) And (3) distilling a sample:

a clean and dry 100mL volumetric flask is used, 100mL of sample (liquid temperature is 20 ℃) is accurately measured in a 500mL distillation flask, the volumetric flask is rinsed with 50mL of water for three times, the rinsing liquid is merged into the 500mL distillation flask, a plurality of zeolites (or glass beads) are added, a serpentine condenser tube is connected, the original volumetric flask for sampling is used as a receiver (an ice bath is added), cooling water is started (the temperature of the cooling water is preferably lower than 15 ℃), the distillation is slowly heated, and distillate is collected. When the scale is close, the volumetric flask is taken down, the stopper is covered, the volumetric flask is kept warm in a water bath at 20 ℃ for 30min, water is added to the scale, and the volumetric flask and the water are uniformly mixed for later use.

c) Measurement of sample solution

The density vial was cleaned and dried, and weighed with a thermometer and side port cap. Drying and weighing were repeated until constant weight (m). Taking down the bottle stopper with a thermometer, filling a constant-weight density bottle with water which is boiled and cooled to 15 ℃, inserting the bottle stopper with the thermometer (no air bubbles exist in the bottle), immediately immersing the bottle stopper into a constant-temperature water bath at 20.0 +/-0.1 ℃, quickly sucking liquid overflowing the side tube by using filter paper after the temperature of the content reaches 20 ℃ and is kept constant for 20min, enabling the liquid level of the side tube to be flush with the tube opening of the side tube, immediately covering the side hole cover, taking out the density bottle, wiping water liquid on the outer wall of the bottle by using the filter paper, and immediately weighing (m 1). Pouring out water, washing the density bottle with absolute ethyl alcohol and then with ether, drying (or drying in an oven), repeatedly washing the density bottle with the sample distillate for 3-5 times, and then filling. Weigh (m2) according to 4.2.2 procedures.

Density of sample at 20 ℃

Push type(1) Calculating, the air buoyancy correction value (A) is calculated according to equation (1):

in the formula:

-the density of the sample at 20 ℃ in grams per liter (g/L);

ρ₂₀-density of distilled water at 20 ℃ (998.20 g/L);

m2- -mass of density bottles and samples at 20 ℃ in grams (g);

m- -mass of density bottle in grams (g);

a-air buoyancy correction value;

m1- -20 deg.C density bottle and water, unit is gram (g);

ρ_μ- -density of dry air at 20 ℃ at 1013.25hPa (. apprxeq.1.2 g/L);

997.0- -difference between the density values of distilled water and dry air at 20 ℃ in grams per liter (g/L).

According to the density of the sample

Looking at appendix A, the alcohol content was determined as volume fraction "% vol".

The experimental results are as follows: after the post fermentation is finished, the alcoholic strength of beer brewed by the saccharomyces cerevisiae is 7.00 vol%.

5. True fermentation degree of Saccharomyces cerevisiae fermented beer

1) Determination of true concentration

The residue after removal of the alcohol by distillation (in a distillation flask of known mass) was cooled to 20 ℃ and water was added accurately to make 100.0g of the residue, followed by mixing.

The relative density of the residue was determined using a density bottle. The grams of extract (g/100g) in a 100g sample were determined. I.e. the true concentration of the beer.

2) Presentation of analytical results

Calculating the concentration of the original wort of the sample according to the formula (2) according to the measured alcoholic strength and the real concentration:

X1＝((A×2.0665+E)×100)/(100+A×1.0665)

in the formula:

x1- -concentration of wort in Bera plot or mass fraction

A-alcohol content mass fraction of sample,%;

e- -the true concentration mass fraction of the sample,%.

RDF＝100×(2.0665×A)/(2.0665×A+Z)

In the formula:

RDF — real fermentation mass fraction,%;

a represents the alcoholic strength mass fraction of the sample,%;

z-the true concentration of the sample in degrees of Berthogram or mass percent.

The experimental results are as follows:

after the end of the post-fermentation, the degree of true fermentation of the beer brewed with the Saccharomyces cerevisiae of the present invention was 82.33%.

6. Flavor substance composition of saccharomyces cerevisiae fermented beer

1) Experimental Material

An ultrasonic cleaning instrument,

50/30 μm DVB/CAR/PDMS solid phase micro extraction head and extraction handle: supelco, USA; 6890N-5973I GC-MS: agilent Inc. of USA

Organic filter membrane (0.25 μm), disposable syringe

2) Experimental methods

And inserting the solid-phase micro-extraction head into a sample inlet of a gas chromatograph, aging for 1h at 270 ℃, and carrying out a blank experiment until no chromatographic peak appears. And (2) putting the sample into a sample bottle, wherein the liquid filling amount is 1/3-1/2 of the volume of the whole sample bottle, adding 2g of sodium chloride, inserting the solid phase micro-extraction head into the sample bottle, extracting for 45min at 45 ℃, desorbing for 5min in a sample inlet of a gas chromatograph-mass spectrometer at 270 ℃, and carrying out sample injection analysis.

Gas Chromatography (GC) conditions: HP-INNOWAX MS type chromatographic column (30 m.times.250 μm.times.0.25 μm); the carrier gas is helium (He) and the flow rate is 1 mL/min; the initial column temperature is 50 ℃, the temperature is kept for 5min, the temperature is increased to 160 ℃ at the speed of 6 ℃/min, the temperature is kept for 3min, and the temperature is increased to 230 ℃ at the speed of 20 ℃/min; no split-flow sample introduction. Mass Spectrometry (MS) conditions: the ionization mode is an Electron Ionization (EI) source, the electron energy is 70eV, the emission current is 200 muA, the ion source temperature is 230 ℃, the quadrupole is 150 ℃, the full scanning mode is adopted, the scanning mass range is 50-550 amu, and no solvent delay exists.

The qualitative analysis method comprises the following steps: the spectra obtained by analysis were compared to standard spectra in the National Institute of Standards and Technology (NIST) library, and fractions with a degree of matching > 80% were retained.

The quantitative analysis method comprises the following steps: the relative contents of the components were calculated by peak area normalization, and the results of the beer fermentation experiments are shown in Table 2.

As shown in table 2, a total of 40 aroma components were identified. Wherein, the concentration of 16 esters is 477.32 ug/L; 10 kinds of alcohol with the concentration of 988.83 ug/L; 3 kinds of aldehyde with the concentration of 16.15 ug/L; 1 fatty acid with the concentration of 3.71 ug/L.

Esters are produced by acid and alcohol esterification reactions or by yeast metabolism, with different acyl-coa activities in the cells of different yeast strains. Ethyl caprylate, phenethylacetate, ethyl palmitate, ethyl acetate and ethyl decanoate endow the wine with better flavors of fruit aroma, flower aroma, cream and the like. The yeast provided by the invention has obvious advantages in the aspect of producing ester flavor substances. Alcohols are mainly ethanol, phenethyl alcohol and nerolidol, which impart a pleasant floral note to the wine. In beer brewed by the saccharomyces cerevisiae, the coconut aldehyde with coconut aroma when the beer is thick, almond aroma or peach flower aroma when the beer is diluted and the benzaldehyde providing nut aroma are detected.

The color, the fragrance and the taste are three factors which form the fermented wine, and the molecular species, the content, the mutual ratio and the sensory threshold of the fragrance components determine the fragrance of the fermented wine. The beer brewed by the saccharomyces cerevisiae provided by the invention has better flavor.

TABLE 2

7. Saccharomyces cerevisiae flocculation detection

1) Experimental Material

Constant-temperature water bath, centrifuge, calcium sulfate, glacial acetic acid, sodium acetate and 15/50mL centrifuge tube

2) Experimental methods

Equal amounts of yeast fermentation broth were collected in 50mL centrifuge tubes and centrifuged to collect yeast (2000rpm, 5min), 5g of yeast sample was weighed in a new 50mL centrifuge tube, added to 30mL of 0.051% calcium sulfate solution, washed and centrifuged (1000rpm, 5min), and the washing and centrifugation repeated. Accurately weighing 1.0g of the centrifugal yeast paste, placing the centrifugal yeast paste into a 15mL centrifugal tube with a conical bottom and a scale and a cover, adding 10mL acetic acid buffer solution (0.51g of hydrated calcium sulfate, 6.80g of sodium acetate and 4.05g of glacial acetic acid) containing calcium sulfate and pH 4.5 into the centrifugal tube, dissolving the mixture, and carrying out water dissolution and heat preservation for 20min at 20 ℃. Then fully shaking to enable the yeast to be suspended again, preserving the temperature for 10min at 20 ℃, and settling the sediment in milliliters at the bottom of the centrifugal tube cone to obtain the Benss value.

The results of the experiment are shown in FIG. 3 below, and it can be seen from FIG. 3 that the Saccharomyces cerevisiae of the present invention forms an explanatory precipitate of about 0.5cm, which is a moderately flocculating yeast.

8. Saccharomyces cerevisiae detection of malt PYF factor

1) Experimental Material

Saccharifying device, constant temperature incubator, centrifuge, 200mL colorimetric tube, malt, glucose, peptone, yeast extract, and sodium chloride

2) Experimental methods

a) The malt sample is made into the agreement wort by a collaborative method.

b) Sterilizing at 115 deg.C for 20min to obtain malt extract, standing, cooling overnight, and slowly pouring to obtain supernatant.

c) After yeast is expanded and cultured by YPD culture medium, yeast paste is obtained by centrifugation at 4000rpm for 5 min.

d) Slowly pouring the wort, weighing 200mL of the supernatant after standing by using a measuring cylinder, pouring the wort into a triangular flask, adding 10.00g of yeast paste, vibrating by using a horizontal oscillator at 150r/min for 10min, uniformly mixing, pouring into an EBC (electron beam copper) tube, and sealing the opening of the EBC tube by using an aluminum cap.

e) Placing in a constant temperature incubator at 23 ℃ and fermenting for 48 hours.

d) Marking the position 5cm below the liquid surface, extending the liquid transfer gun into the position 5cm below the liquid surface, quickly sucking 5mL of liquid, and measuring the number of suspended yeast.

6.22.6 calculation of results

Malt PYF value ═ number of yeast cells in the test sample/number of yeast cells in the standard malt sample × 100%.

The experimental results are as follows:

the saccharomyces cerevisiae provided by the invention is sensitive to PYF factors, can distinguish malts with different PYF values, and has stable detection result, repeatability is less than or equal to 10%, and repeatability is less than or equal to 15%.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

SEQUENCE LISTING

<110> Zhongliang Nutrition and health research institute Co., Ltd

COFCO Malt (Dalian) Co., Ltd.

<120> Saccharomyces cerevisiae and leaven for efficiently utilizing maltotriose, and fermented cereal beverage and malt PYF factor using the same

Applications in assays

<130> I64207COF

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 766

<212> DNA

<213> Saccharomyces cerevisiae

<400> 1

ttttgaaaat ggattttttt gttttggcaa gagcatgaga gcttttactg ggcaagaaga 60

caagagatgg agagtccagc cgggcctgcg cttaagtgcg cggtcttgct aggcttgtaa 120

gtttctttct tgctattcca aacggtgaga gatttctgtg cttttgttat aggacaatta 180

aaaccgtttc aatacaacac actgtggagt tttcatatct ttgcaacttt ttctttgggc 240

attcgagcaa tcggggccca gaggtaacaa acacaaacaa ttttatttat tcattaaatt 300

tttgtcaaaa acaagaattt tcgtaactgg aaattttaaa atattaaaaa ctttcaacaa 360

cggatctctt ggttctcgca tcgatgaaga acgcagcgaa atgcgatacg taatgtgaat 420

tgcagaattc cgtgaatcat cgaatctttg aacgcacatt gcgccccttg gtattccagg 480

gggcatgcct gtttgagcgt catttccttc tcaaacattc tgtttggtag tgagtgatac 540

tctttggagt taacttgaaa ttgctggcct tttcattgga tgtttttttt tccaaagaga 600

ggtttctctg cgtgcttgag gtataatgca agtacggtcg ttttaggttt taccaactgc 660

ggctaatctt ttttatactg agcgtattgg aacgttatcg ataagaagag agcgtctagg 720

cgaacaatgt tcttaaagtt tgacctcaaa tcaggtagga gtaccc 766

Claims

1. A Saccharomyces cerevisiae (Saccharomyces cerevisiae) is characterized in that the preservation number of the Saccharomyces cerevisiae is CGMCC No. 20489.

2. A starter culture comprising the Saccharomyces cerevisiae (Saccharomyces cerevisiae) according to claim 1.

3. The starter culture of claim 2, wherein the starter culture is a liquid starter culture, a semi-liquid starter culture, a concentrated starter culture, a compressed starter culture, or a solid starter culture.

4. A method for preparing a starter, the method comprising:

(1) the saccharomyces cerevisiae of claim 1 is fermented and cultured in a fermentation medium, and the viable count is 10⁸More than cfu/mL to obtain a liquid leavening agent;

5. Use of the saccharomyces cerevisiae according to claim 1 or the starter culture according to any of claims 2 to 3 for fermenting a cereal beverage.

6. Use according to claim 5, wherein the fermented cereal beverage is a fermented beer.

7. A method of preparing a fermented cereal beverage, the method comprising: contacting the saccharomyces cerevisiae of claim 1 or the starter culture of any of claims 2-3 with a fermentation substrate and fermenting to produce a fermented cereal beverage.

8. The method of claim 7, wherein the fermented cereal beverage is a fermented beer and the fermentation substrate comprises maltotriose.

9. A fermented cereal beverage produced by the production method according to claim 7 or 8.

10. Use of the saccharomyces cerevisiae of claim 1 or the starter culture of any of claims 2-3 in a PYF assay.