A kind of Fabianseber Lindner yeast CfM-2 and its application in winemaking

Abstract

The present invention discloses a Fabienseiberlindner yeast CfM-2 and its application in brewing, belonging to the field of microbial fermentation and food technology. The present invention separates and purifies 5 strains of Fabienseiberlindner yeast from a variety of characteristic yellow wine koji in the main yellow wine producing areas of China, screens these 5 yeasts, and finally obtains the strain CfM-2 with excellent fermentation performance, tolerance and aroma production characteristics in beer wort. The strain can tolerate at least 100ppm of iso-α acid and 5%vol of ethanol, produces high-quality aroma substances such as acetate, and the fruity aroma of brewed beer is prominent. Fermentation of 12.0°P wort can produce low-alcohol beer with an ethanol concentration of 0.59%vol, and fermentation of 6.0°P wort can obtain non-alcoholic beer with an ethanol concentration of 0.42%vol. The strain CfM-2 of the present invention reduces the ethanol content of beer, improves the taste of beer, increases flavor diversity, and gives beer regional characteristics, laying a foundation for the development of new alcoholic beverages.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation and food technology, and in particular relates to Fabianseber Lindner yeast CfM-2 and application thereof in winemaking. Background Art

[0002] Beer is an alcoholic beverage made from malt, hops, water and yeast. It is one of the three ancient wines in the world, along with rice wine and grape wine. With the improvement of consumption level and consumption capacity, people are no longer satisfied with the existing beer styles and flavors, and are beginning to pursue a richer and more diverse drinking experience. Moreover, as living standards improve, the number of people with chronic diseases and sub-health has increased significantly, people's health awareness has gradually increased, and they are paying more and more attention to the nutritional and health benefits of food. Therefore, the main development direction of the beer industry in the future will focus on more personalized new flavor beers and non-alcoholic beer and low-alcohol beer that conform to the concept of health (non-alcoholic beer and low-alcohol beer are beers with ethanol content below 0.5%vol and 2.5%vol, respectively).

[0003] At present, most beers are fermented by a single yeast strain, among which Saccharomyces is the most widely used. The most commonly used brewing yeast and Pasteur yeast in beer brewing are both of the genus Saccharomyces. Non-brewer yeast is yeast other than the genus Saccharomyces. In recent years, some non-brewer yeasts have been innovatively used in beer brewing to improve the quality of beer. The rich and diverse non-brewer yeast species provide more possibilities for the innovation of beer varieties and styles, and promote the diversified development of the beer industry. However, the fermentation performance and alcohol tolerance of non-brewer yeast are generally not as good as brewer yeast. If the fermentation performance of the strain is too poor, it may cause premature fermentation stagnation, affecting the flavor and ethanol concentration of the beer. Hop bitter acids (α acids and β acids) are natural antibacterial agents, among which α acids will isomerize to form iso-α acids with better solubility during the boiling process of wort, thereby improving the antibacterial effect. In addition, antibacterial substances such as ethanol will be produced during the fermentation of wort. Under the stress of high concentrations of ethanol or iso-α acids, the growth and metabolism of some non-brewer yeast strains will be inhibited. Therefore, strains with poor fermentation performance and tolerance cannot be used to brew beer, and the performance of strains to enhance the aroma of beer when fermenting wort is also one of the issues that need to be considered. Summary of the invention

[0004] In order to solve the above technical problems, the present invention collects Fabianseber Lindner yeast from rice wine koji, and obtains a strain CfM-2 with the best performance through fermentation performance screening, iso-α acid tolerance screening, ethanol tolerance screening, aroma preference screening, etc., providing a new choice for the development of beer.

[0005] The invention provides a Cyberlindnera fabianii yeast, which is named as Cyberlindnera fabianii Cf M-2 and has a deposit number of CGMCC No.31027.

[0006] The present invention provides a culture containing the above-mentioned Lindnerella Fabiensis yeast or a processed product thereof.

[0007] Another object of the present invention is to provide a food composition comprising the above-mentioned Fabianseber Lindner yeast, culture or processed product thereof.

[0008] Preferably, a wine-containing product contains the above-mentioned culture or a processed product thereof.

[0009] Furthermore, the preparation of the alcohol-containing product comprises the step of fermenting wort using the Fabian Seber Lindner yeast.

[0010] Furthermore, the wort may be of any concentration, preferably 6.0-18.0°P, such as 6.0°P, 7.0°P, 8.0°P, 9.0°P, 10.0°P, 11.0°P, 12.0°P, 13.0°P, 14.0°P, 15.0°P, 16.0°P, 17.0°P, 18.0°P, etc., including but not limited to the values ​​listed above.

[0011] A microbial agent comprising the above-mentioned Fabianseber Lindner yeast.

[0012] Furthermore, the microbial agent is a liquid agent.

[0013] Furthermore, the microbial agent is a solid agent.

[0014] Another object of the present invention is to provide the use of the above-mentioned Fabianseberlindner yeast, culture or its processed product, food composition, microbial agent, etc. in the preparation of alcohol-containing products.

[0015] Furthermore, the wine includes but is not limited to beer, such as low-alcohol beer, non-alcoholic beer, etc.

[0016] The above technical solution of the present invention has the following advantages compared with the prior art:

[0017] The present invention provides a non-brew yeast derived from yellow wine koji. The strain can tolerate at least 100ppm of iso-α acid and 5%vol of ethanol, can metabolize glucose and sucrose in wort, cannot metabolize fructose, maltose and maltotriose, and can produce 0.59%vol of ethanol by fermenting wort with a temperature of 12.0°P. The strain has good aroma production characteristics, can improve the levels of acetic acid ester compounds such as ethyl acetate, phenylethyl acetate, isoamyl acetate, isobutyl acetate, furfuryl acetate, etc. in beer, and the beer brewed by the strain has a prominent fruity aroma. In addition, the strain also has the potential to brew non-alcoholic beer, and can also obtain non-alcoholic beer with an ethanol concentration of 0.42%vol by fermenting wort with a temperature of 6.0°P. Compared with Lutheran yeast commercially used for brewing non-alcoholic beer, the ester content of the non-alcoholic beer product fermented by the strain is significantly improved. The strain provided by the present invention has a very broad application prospect in the field of beer brewing.

[0018] Biomaterial Deposit

[0019] Cyberlindnera fabianii CfM-2 was deposited in the General Microbiology Center of China Microorganism Culture Collection on June 20, 2024, with the deposit number CGMCC No.31027, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0021] Figure 1 The colonies of CfM-2 grown on WL nutrient medium.

[0022] Figure 2 Aroma sensory evaluation results for CfM-2 brewed beers and controls. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0024] The experimental method involved in the present invention is as follows:

[0025] Yeast activation and wort preparation:

[0026] Pick a single yeast colony, inoculate it into YPD liquid medium, and culture it in a shaking incubator at 28°C for 48 hours for expansion. Use an automatic yeast counter to measure the yeast concentration of each bacterial liquid sample. Dilute the light ale concentrated wort appropriately, boil it for 20 minutes for sterilization, and make the original wort concentration 12.0°P.

[0027] Preparation of 12.0°P pale ale wort:

[0028] Dilute the pale ale concentrated wort appropriately, heat to boiling, then add 0.5g / L of Citra hops and boil for 60 minutes. After boiling, the original wort concentration is 12.0°P, pH value is 5.30, and bitterness value is about 20IBU.

[0029] Fermentation performance screening:

[0030] Add 10 mL of sterile wort to a 15 mL centrifuge tube with a cap and inoculate with yeast solution to make the yeast concentration in the wort 1×10 7 After the wort samples were cultured at 20°C for 10 days, the sugar content of each sample was measured with a saccharimeter and recorded, and the fermentation degree of each sample was calculated. Each experiment was repeated 3 times.

[0031]

[0032] Iso-alpha acid tolerance screening:

[0033] 10 mL of sterile wort with iso-α acid concentrations of 0 ppm, 50 ppm, and 100 ppm (equivalent to International Bitterness Units, IBU) was added to 15 mL centrifuge tubes with lids, and yeast solution was inoculated to make the initial yeast concentration of the wort 1×10 7 After the wort was cultured at 20°C for 3 days, the yeast concentration of each sample was measured and recorded. Each experiment was repeated 3 times.

[0034] Ethanol tolerance screening:

[0035] Add 10 mL of sterile wort with 0%, 5%, and 10% ethanol concentrations to 15 mL centrifuge tubes with lids, and inoculate with yeast solution to make the initial yeast concentration of the wort 1×10 7 After the wort was cultured at 20°C for 3 days, the yeast concentration of each sample was measured and recorded. Each experiment was repeated 3 times.

[0036] Aroma preference screening:

[0037] Add 30 mL of sterile wort to a 50 mL centrifuge tube with a cap and inoculate with yeast solution to make the initial yeast concentration of the wort 1×10 7After the wort was incubated at 20°C for 10 days, all samples were numbered with a random three-digit code and poured into a transparent tasting cup. Ten sensory evaluators (5 men and 5 women) who had received beer sensory evaluation training smelled the wort fermentation liquid and rated the aroma of the fermentation liquid on a 10-point intensity level according to their preferences. The average score was taken as the final score.

[0038] Data processing and statistical analysis:

[0039] Data processing was performed using Excel 2019, SPSS 21.0 and other software. Data significance analysis was performed using one-way analysis of variance and Duncan method, with p < 0.05 indicating a significant difference.

[0040] Sugar composition determination:

[0041] After fermentation, the sugar composition in beer samples and wort was determined by liquid chromatography.

[0042] Preparation and pretreatment of standard solutions: prepare 20 g / L fructose, 20 g / L glucose, 20 g / L sucrose, 40 g / L maltose, and 20 g / L maltotriose solutions, and dilute them 2 times, 4 times, 8 times, and 16 times. All standard solutions are sterilized by filtration through a 0.22 μm water filter membrane.

[0043] Sample pretreatment: Degas the sample by ultrasonic for 15 min, dilute appropriately, and filter through a 0.22 μm water filter to sterilize.

[0044] Mobile phase pretreatment: prepare the mobile phase in the ratio of acetonitrile: water = 85:15, filter through a 0.45 μm organic filter membrane for sterilization, and mix thoroughly by ultrasonication for 15 min.

[0045] Chromatographic conditions: The detector is a differential refractive index detector, the column temperature is set to 40°C, the detector temperature is 40°C, the flow rate is 1.5 mL / min, the elution time is 16 min, and the injection volume is 10 μL. A standard curve is drawn based on the results of the standard solution for compound quantification.

[0046] Ethanol production characteristics determination:

[0047] Sample pretreatment: Ultrasonic degassing of beer sample for 15 min, 4 mL was taken into an extraction bottle, 50 μL of n-propanol was added, and a magnetic stirrer was placed.

[0048] Preparation and pretreatment of standard solution: pipette 20 μL, 60 μL, 100 μL, 140 μL, and 180 μL of ethanol into the extraction bottle respectively, add 50 μL of n-propanol as the internal standard, make up to 4 mL with deionized water and add a magnetic stirrer.

[0049] Extraction method: The extraction bottle was magnetically stirred (100 rpm) in a 70°C water bath and balanced for 10 minutes. The solid phase extraction head was inserted into the extraction bottle to extract the volatile components for 20 minutes. After the extraction, the extraction head was inserted into the gas phase injection port for injection and analysis for 5 minutes.

[0050] Gas chromatography conditions: DB-INOWAX column (30m x 0.25mm, 0.5μm), carrier gas is high-purity nitrogen, nitrogen flow rate: 1mL / min, sample port temperature 250℃, injection mode is splitless, desorption time is 5min. The heating program is the initial temperature of 50℃, maintained for 1min, and then increased to 200℃ at 20℃ / min.

[0051] The ethanol standard curve was drawn based on the determination results of the standard solution for compound quantification.

[0052] Volatile flavor compounds detection:

[0053] Determination of volatile flavor compounds in wort and beer samples without yeast inoculation by headspace solid phase microextraction coupled with gas chromatography-mass spectrometry.

[0054] Sample pretreatment: Take 4 mL of sample into an extraction bottle, add 5 μL of internal standard solution (prepared by adding 2 μL of 2-octanol to 1.2 mL of anhydrous ethanol), put in a magnetic stirrer (100 rpm), and place in a water bath at 40°C for 20 min. Then insert the extraction needle and adsorb for 40 min in a 40°C water bath.

[0055] Chromatographic conditions: desorption for 5 min after injection, injection port temperature of 250 °C, helium column flow rate of 0.8 mL / min.

[0056] The chromatographic program was set to hold at 50 °C for 1 min, then gradually increase to 170 °C at 3 °C / min, hold for 3 min, and finally increase to 215 °C at 7 °C / min, hold for 5 min. The mass spectrometry conditions were set to ionization energy of 70 eV, ion source temperature of 200 °C, and transfer line temperature of 280 °C.

[0057] Example 1 Screening and preservation of strains

[0058] (1) Obtaining Fabianseberlindner's yeast from rice wine koji

[0059] 16 kinds of yellow wine koji with different characteristics were selected from the five major yellow wine producing areas of Hubei, Zhejiang, Fujian, Jiangxi and Jiangsu, from which Fabian Seberlindner yeast was screened. Specifically, chloramphenicol was dissolved in ethanol and prepared into a 50 mg / mL solution. YPD liquid culture medium was prepared, 0.2% sodium propionate was added to inhibit mold growth, chloramphenicol solution was added at a final concentration of 100 μg / mL to inhibit bacterial growth, sterilized and cooled. Different wine koji were crushed in a mortar, added to the culture medium at 0.05 g / mL, and cultured in a shaker at 28°C and 200 r / min for 24 hours. The WL nutrient agar medium powder was heated and dissolved, and then cooled and poured after sterilization. The yellow wine koji culture solution was moderately diluted, spread on the WL nutrient agar medium plate, and cultured at 28°C for three days. WL nutrient medium is a yeast selective medium, and different types of yeast will grow colonies of different colors and shapes on this medium. Non-Saccharomyces cerevisiae colonies were selected based on color and morphology, and streaked and purified three times to obtain a single strain of non-Saccharomyces cerevisiae, and the colony color and morphology were recorded. The strains were identified by 26S rDNA sequence analysis, and 5 strains of Cyberlindnera fabianii were isolated from 16 traditional Chinese rice wine kojis for subsequent screening.

[0060] (2) Fermentation performance screening

[0061] In order to evaluate the fermentation performance of five strains of Cyberlindnerafabianii in wort, they were inoculated into pale ale wort with an original wort concentration of 12.0°P. After 10 days, the residual sugar content of the wort fermentation liquid was measured and the fermentation degree was calculated. The results are shown in Table 1.

[0062] Table 1 Fermentation degree of wort fermentation liquid of Fabian Seber Lindner yeast strain

[0063]

[0064] The lower the wort fermentation degree, the worse the fermentation performance of the strain. Among the five yeast strains, only CfM-2 had a fermentation degree of more than 15%, and its fermentation performance was significantly better than the other strains.

[0065] (3) Iso-α acid tolerance screening

[0066] Five strains of Fabianseber Lindner yeast were incubated in worts with iso-α acid concentrations of 0 ppm, 50 ppm, and 100 ppm (which can be converted into International Bitterness Units, IBU, with corresponding bitterness values ​​of 0 IBU, 50 IBU, and 100 IBU). The initial yeast concentration was 1.00 × 10 7CFU / mL, the original wort concentration was 12.0°P. The yeast concentration was measured after culturing at 20°C for 3 days, and the results are shown in Table 2.

[0067] Table 2 Growth of strains in worts with different iso-α acid concentrations

[0068]

[0069] Note: Different letters in the same row of the same strain represent significant differences (P<0.05).

[0070] The results showed that except for CfF-6, the other four yeast strains were able to grow normally, and there was no significant difference in yeast concentration and wort without iso-α acid. In contrast, the growth of CfF-6 was inhibited to a certain extent in wort with 50ppm and 100ppm. In addition, the yeast concentration of CfM-2 in 0ppm and 50ppm wort was the highest among all strains, and in 100ppm wort, it was almost indistinguishable from Cf C-9 with the highest concentration, indicating that this strain can tolerate iso-α acid at a concentration of at least 100ppm, and its tolerance to iso-α acid is stronger than that of other strains.

[0071] (4) Ethanol tolerance screening

[0072] The yeast concentrations of the five strains of Fabianseber Lindner yeast were measured after being cultured in worts with ethanol concentrations of 0% vol, 5% vol, and 10% vol for 3 days. The results are shown in Table 3.

[0073] Table 3 Growth of strains in worts with different ethanol concentrations

[0074]

[0075] Note: Different letters in the same row of the same strain represent significant differences (P<0.05).

[0076] The above results show that all strains can grow normally in wort without ethanol, and the yeast concentration is similar to the initial inoculation concentration (1.00×10 7 Among all strains, Cf M-2 and CfC-9 had higher yeast concentrations in wort at 0%vol and 5%vol, indicating that they can tolerate 5%vol ethanol, and their ethanol tolerance is stronger than that of other strains. When the ethanol concentration was increased to 10%vol, CfM-2 showed better tolerance than CfC-9.

[0077] (5) Aroma preference screening

[0078] The aroma preference scores of the wort fermentation broths of the five strains of Fabian Seber Lindner's yeast are shown in Table 4. The aroma preference score of CfM-2 was the highest among all the strains, indicating that its fermentation produced the best aroma.

[0079] Table 4 Aroma preference scores of wort fermentation broth of strains

[0080]

[0081]

[0082] In summary, CfM-2 has the best fermentation performance, iso-α acid tolerance, and aroma performance among the five strains of Fabianseber Lindner yeast, and has strong ethanol tolerance. Therefore, CfM-2 is considered to be the most suitable strain for beer brewing and has been sent for preservation.

[0083] Example 2 Fermentation characteristics of CfM-2 strain in beer

[0084] The ability of non-brew yeast to synthesize volatile flavor compounds such as esters and alcohols can improve the aroma of beer, and its metabolism of sugars and ethanol in wort is also crucial. In order to explore the fermentation characteristics of Fabianseber Lindner's yeast CfM-2 in wort, it was inoculated into 12.0°P pale ale wort. The initial yeast inoculation amount in wort was 1×10 7 CFU / mL, cultured at 20°C until fermentation was complete (sugar content did not change for 3 days), and then stored at 4°C for one week. The sugar composition in the beer was measured to evaluate the sugar metabolism characteristics of the strain, the ethanol concentration was measured to evaluate the ethanol production characteristics, and the volatile flavor substance content was measured and the aroma sensory evaluation was performed to evaluate the aroma production characteristics. Saccharomyces cerevisiae Sc US-05 and Lutheran yeast Sl 31238 were used as controls. Each treatment was repeated 3 times. The results are as follows:

[0085] (1) Metabolic sugar characteristics

[0086] Table 5 Sugar composition in wort before and after fermentation (g / L)

[0087]

[0088] Note: Different letters in the same row represent significant differences (P<0.05), and “ / ” means no difference was detected.

[0089] The sugar composition in the wort before fermentation and after CfM-2 fermentation was determined, and the results are shown in Table 5. Cf M-2 can utilize almost all of the glucose and sucrose in the wort, but cannot utilize fructose, maltose, and maltotriose.

[0090] (2) Ethanol production characteristics

[0091] After the fermentation of the strain was completed, the ethanol concentration in the wort was determined to be 0.59% vol.

[0092] (3)Fragrance-producing characteristics

[0093] The volatile flavor substances in the wort were determined, and it was found that CfM-2 could significantly increase the levels of acetate compounds such as ethyl acetate, phenylethyl acetate, isoamyl acetate, isobutyl acetate, and furfuryl acetate compared with the control brewer's yeast Sc US-05 and Lutheran yeast Sl 31238.

[0094] Table 6 Content of some volatile flavor substances in CfM-2 fermented beer and control (μg / L)

[0095]

[0096] Organize evaluators to conduct sensory evaluation on the aroma of fermented beer products (full score 10 points for each item), the results are as follows Figure 2 As shown in the figure, the aroma preference, fruity aroma, and floral aroma of CfM-2 beer were highly rated, with the fruity aroma being particularly prominent, exceeding 7 points.

[0097] Example 3 Preparation of non-alcoholic beer by CfM-2 strain

[0098] In the national standard "Beer" GB 4927-2008, non-alcoholic beer (NAB) is defined as beer with an alcohol content of less than or equal to 0.5% vol and an original wort concentration of greater than or equal to 3.0°P. At present, most of the non-alcoholic beer on the market is made by physical dealcoholization of ordinary beer, but during the dealcoholization process, many volatile flavor substances in the beer also evaporate along with the ethanol, affecting the overall flavor of the beer.

[0099] In this example, Fabian Seber Lindner yeast CfM-2 was used to ferment 6.0°P wort to produce non-alcoholic beer. The specific process is to appropriately dilute the pale ale concentrated wort, heat it to boiling, then add 0.5g / L Citra hops, boil it for 60 minutes, and after boiling, the original wort concentration is 6.0°P, the pH value is 5.00, and the bitterness value is about 20IBU. After the wort is cooled, CfM-2 (yeast inoculation concentration is 1×10 7 CFU / mL), cultured at 20°C until fermentation was complete (sugar content did not change for 3 days), and stored at 4°C for one week. The beer obtained by this process was evaluated, and the beer fermented by pure strain Lutheran yeast S1 31238 was used as a control. The experimental results are as follows:

[0100] (1) Ethanol metabolism

[0101] The ethanol concentration of the non-alcoholic beer brewed by this process is 0.42% vol, which meets the requirements of non-alcoholic beer.

[0102] (2) Metabolism of volatile flavor substances

[0103] The content of volatile flavor substances in non-alcoholic beer was determined, and it was found that compared with the control group, the content of esters such as ethyl acetate, ethyl propionate, isobutyl acetate, isoamyl acetate, and phenylethyl acetate in the non-alcoholic beer fermented by CfM-2 was significantly increased (as shown in Table 7), and the total ester content was 9888.16 μg / L, which was higher than that of the control group (2285.58 μg / L).

[0104] Table 7 Content of some volatile flavor substances in non-alcoholic beer and control (μg / L)

[0105]

[0106] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A strain of Lindner's yeast, characterized in that Lindnerella fabianseberii Cyberlindnera fabianii ) Cf M-2, the deposit number is CGMCC No. 31027.

2. A culture comprising the Fabianseber Lindner yeast of claim 1.

3. A food composition, characterized in that Contains the Fabianseberlindner yeast according to claim 1 or the culture according to claim 2.

4. A microbial agent, characterized in that: The method comprises the Fabianseber Lindner yeast according to claim 1.

5. The microbial agent according to claim 4, characterized in that: The microbial agent is a liquid agent or a solid agent.

6. Use of the Fabianseberlindner yeast according to claim 1, the culture according to claim 2, the food composition according to claim 3, or the microbial agent according to claim 4 or 5 in the preparation of an alcoholic product, characterized in that: The wine is beer.

7. The use according to claim 6, characterized in that: The beer includes low-alcohol beer or non-alcohol beer.

8. A product containing alcohol, characterized in that: A food composition comprising the culture according to claim 2 or the food composition according to claim 3.

9. A method for preparing a wine-containing product, characterized in that: The method comprises the step of fermenting wort using the Fabianseber Lindner yeast described in claim 1 or the microbial agent described in claim 4 or 5.

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