A strain of brettanomyces custersii and its application in fermented beverages

Abstract

The application discloses a strain of apple-flavor Saccharomyces dielmannii and application thereof in fermented drinks, and belongs to the fields of microorganisms, biotechnology and food. The Saccharomyces dielmannii with a preservation number of CCTCCM2022046 is screened and separated from apples, and is preserved in the China Center for Type Culture Collection on January 10, 2022. The strain can produce phenyl ethyl acetate with apple-like fruit aroma, rose flower aroma and honey-like bottom aroma when fermented alone. When the strain is mixed with Angelus yeast to ferment apple drinks, the content of phenyl ethyl acetate, a representative component of apple aroma, in the drinks also increases with the increase of the inoculation amount of the Saccharomyces dielmannii. The strain can be used as a typical apple-flavor strain to ferment drinks, and has a wide prospect in the fields of fruit wine, fruit vinegar and other food.

Description

Technical Field

[0001] This invention relates to an apple-scented yeast and its application in apple cider and apple cider vinegar, belonging to the fields of microbiology, biotechnology, and food. Background Technology

[0002] Cider is a product of apple juice fermented by yeast, and it is the world's second most popular fruit wine after grape wine. Apple cider vinegar is a nutritious and flavorful fruit vinegar product made from apples through two stages: alcoholic fermentation and acetic acid fermentation. Its flavor and nutritional value are superior to ordinary vinegar.

[0003] Among the many factors influencing the quality of fermented products, the interaction of raw material variety, fermentation process, fermentation equipment, and fermentation microorganisms determines the product's quality. Once the fruit variety, fermentation process, and fermentation equipment are determined, the yeast strain selected for high-quality fermented beverages becomes crucial. Compared to similar products from abroad, apple fermented beverages produced in my country often suffer from insufficient aroma and a lack of typical apple aromas. Therefore, focusing on breeding brewing yeast strains capable of producing typical fruity and floral flavor compounds is a critical issue that urgently needs to be addressed in the deep processing of apple fermented products.

[0004] Non-saccharifying yeasts are yeasts that produce little or no alcohol during fruit wine fermentation. Also known as aroma-producing or ester-producing yeasts, their metabolites play a crucial role in the aroma, flavor, and other sensory characteristics of fermented products. Commonly used non-saccharifying yeasts in fruit winemaking include *Klebsiella pneumoniae*, *Candida albicans*, *Morchella maltosa*, *Hanamitratus*, *Schizosaccharizoa*, and *Zygosaccharizoa*. Although non-saccharifying yeasts have weak fermentation capabilities and may not even be able to complete fermentation independently, the metabolites they produce during fermentation can enhance the aroma and quality of the fermented product. Therefore, it is now common practice to use mixed fermentation of saccharifying and non-saccharifying yeasts to increase the aroma of fruit wines. Simultaneously, the flavor compounds and flavor precursors produced by non-saccharifying and saccharifying yeasts during the alcoholic fermentation stage can also provide better flavor for further acetic acid fermentation to produce related vinegar products. Summary of the Invention

[0005] This invention obtains a strain of *Delkirk's yeast* that produces an apple aroma from apples. This strain can be used in the fermentation production of fermented beverages such as apple cider and apple cider vinegar to enhance the typical apple flavor.

[0006] This invention provides a strain of *Cytomyces delta-Kjeldahl* NJSYGA2021, which was deposited at the China Center for Type Culture Collection on January 10, 2022, with accession number CCTCC M 2022046.

[0007] In one embodiment, the *Delkirk spore-forming yeast* was screened and identified from apples, and it is capable of producing [products with...].

[0008] Phenethyl acetate has an apple-like, rose-like, and honey-like aroma.

[0009] This invention also provides the application of an apple-scented yeast strain in fermented beverages.

[0010] In one implementation, the fermented beverage includes apple cider vinegar and apple wine.

[0011] In one embodiment, the application of the strain in fermented beverages includes its use in the fermentation of cider and apple cider vinegar.

[0012] Beneficial effects:

[0013] The *Cyclocarya davidii* strain CCTCCM 2022046 obtained in this invention was screened from apples and possesses the ability to produce phenethyl acetate, a typical apple-like fruit aroma, making it a suitable strain for enhancing the apple and floral aromas of fermented products. When *Cyclocarya davidii* is co-fermented with Angel Yeast active dry yeast to produce apple cider, and further inoculated with acetic acid bacteria to ferment apple cider vinegar, the fermented products contain phenethyl acetate, which cannot be produced by Angel Yeast alone during fermentation. Furthermore, the phenethyl acetate content in the fermented products increases with the increase in the amount of *Cyclocarya davidii* inoculated. In summary, the *Cyclocarya davidii* strain can be applied in the brewing, vinegar-making, and other food industries to enhance or highlight the flavor characteristics of food. Attached Figure Description

[0014] Figure 1 The colony morphology of *Cyclocarya da Vincis* on YPD solid medium is shown in Example 1 of this invention.

[0015] Figure 2 The cell morphology of *Cyclocarya dalcis* from Example 1 of this invention is shown under a conventional optical microscope.

[0016] Figure 3 This indicates the results of the sequencing of *Cytomyces davidii* 26S from Example 1 of this invention compared on NCBI.

[0017] Figure 4 This is a phylogenetic tree of the 26S rDNA sequence of *Cytomyces deltaiculatus* from Example 1 of the present invention.

[0018] Figure 5 This is a total ion diagram of volatile flavor compounds in apple cider fermented by Angel Yeast.

[0019] Figure 6Total ion diagram of volatile flavor compounds in cider fermented by a mixture of Dale Kelley's spore-forming yeast NJSYGA2021 and Angel yeast.

[0020] Figure 7 This is a total ion diagram of volatile flavor compounds in apple cider vinegar produced by mixed fermentation of Angel yeast and acetic acid bacteria.

[0021] Figure 8 Total ion diagram of volatile flavor compounds in apple cider vinegar fermented by a mixture of Dale Kelley's Spore-bearing Saccharomyces NJSYGA 2021, Angel Yeast, and Acetic Acid Bacteria. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0023] The following detailed description illustrates the specific implementation method:

[0024] Example 1 Screening and identification of apple-scented yeast

[0025] Culture medium:

[0026] Malt extract liquid culture medium: 130.0 g / L malt extract powder, 0.1 g / L chloramphenicol, pH 5.6 ± 0.2 (25℃).

[0027] YPD solid medium: peptone 20.0 g / L, glucose 20.0 g / L, yeast extract powder 10.0 g / L, agar 20.0 g / L, natural pH.

[0028] The screening steps for the *DellKip* spore-forming yeast are as follows:

[0029] Step 1: Enrichment: Wash the apples and wipe their surface with 75% alcohol. Peel and chop the apples in a sterile environment and put them into a sterile conical flask containing malt extract liquid culture medium. Incubate at 28°C in a constant temperature shaker at 120 r / min for 24 h to obtain the enriched solution.

[0030] Step 2: Separation: The enrichment solution was serially diluted, and the bacterial solutions with dilutions of 10⁻⁵, 10⁻⁶, and 10⁻⁷ were spread onto YPD solid medium and incubated at 25-28℃ for 48-72 hours.

[0031] Step 3: Purification: Observe the colony morphology, smell the colony aroma and examine under a microscope. Select single colonies with yeast characteristics and perform multiple streak purifications.

[0032] Step 4: Preservation: After microscopic examination confirms that the yeast is a pure culture, the culture is preserved at -80℃ with glycerol.

[0033] Furthermore, the identification steps for the aforementioned *Delkirk spore-forming yeast* are as follows:

[0034] Step 1: Colony morphology observation: After 48-72 hours of incubation on YPD solid medium, the colonies are round, cream-colored, glossy, with neat edges, smooth surface, opaque, viscous, and easy to pick up;

[0035] Step 2: Individual morphological observation: After picking a single colony and preparing a water-soaked slide, observe it under a regular optical microscope. It appears to be oval-shaped.

[0036] Step 3: Molecular biological identification was performed on the obtained pure yeast strains. Yeast-specific classification primers NL1 and NL417 were used to amplify the 26S rDNA fragments of the strains, and the results were detected by gel electrophoresis. Sequencing was then performed to determine the species of the screened yeast. The strains were deposited at the China Center for Type Culture Collection.

[0037] The yeast species and preservation number is Torulaspora delbrueckii CCTCCM2022046.

[0038] The process for identifying yeast species is as follows:

[0039] 1. Homology identification of 26S rDNA sequence of isolated strains: Extraction of yeast DNA: ① Yeast DNA was extracted...

[0040] After activation, the bacteria were inoculated into YPD liquid medium and cultured on a shaker at 25°C for 24 hours. 1 mL of the bacterial culture was then transferred to a 1.5 mL EP tube.

[0041] ① Centrifuge at 12000 rpm for 1 min, remove the supernatant, and collect the bacterial cells; ② Add 500 μL of sterile water, mix thoroughly, centrifuge at 12000 rpm for 1 min, and remove as much supernatant as possible; ③ Resuspend the cells in 500 μL of DNA extraction buffer, add 10 μL of lysozyme (final concentration 100 mg / mL), vortex to mix, and incubate at 55℃ for 10 min; ④ Add 10 μL of snailase (final concentration 100 mg / mL), vortex to mix for 30 s, and incubate at 37℃ for 30 minutes. min; ⑤ Add 25 μL of 10% SDS and vortex to mix, incubate at 60℃ for 1 h until the solution becomes clear, then add 250 μL of sterile water; ⑥ Add 500 μL of phenol-chloroform-isoamyl alcohol (25:24:1), mix well, centrifuge at 13000 rpm for 2 min, take the supernatant, transfer to a new centrifuge tube, and repeat step ⑥ twice; ⑦ Add 500 μL of isopropanol, mix well, centrifuge at 13000 rpm for 1 min, and transfer the supernatant to a new centrifuge tube; ⑧ Add 500 μL of isopropanol, mix well, incubate at -20℃ for 20 min until DNA precipitation occurs, centrifuge at 12000 rpm for 5 min, and remove the supernatant; ⑨ Wash the DNA twice with 500 μL of 75% ethanol (centrifuge at 12000 rpm for 2 min) to remove as much liquid as possible, air dry until there is no alcohol smell, add 50 μL of sterile double-distilled water, and incubate at 65℃ for 1 h. The next step after sending the sample to the Chengdu branch of Beijing Qingke Biotechnology Co., Ltd. for identification is to perform PCR amplification, electrophoresis detection, and sequencing of the 26S rDNA.

[0042] 2. Data Analysis: Compare the amplified sequences with the NCBI database to identify sequences identical to those of the cloned daughter sequences.

[0043] The sequence with the highest primacy was used to construct the phylogenetic tree and determine its taxonomic position. The MEGA7 software used the neighbor-joining method to construct the phylogenetic tree. The phylogenetic tree was constructed using the neighbor-joining algorithm and 1000 Bootstrap calculations. The results are as follows: Figure 4 As shown.

[0044] Example 2: Application of Dalkistrodon halys NJSYGA 2021 in cider production

[0045] In another embodiment of this application, the application of Dalkistrodon halys spore-forming yeast NJSYGA2021 in cider production involves using Dalkistrodon halys spore-forming yeast and commercially available Angel wine active dry yeast as inoculum, which are then inoculated into fresh apple juice for mixed fermentation, followed by flavor testing.

[0046] The specific application method is as follows: Apples are used as raw material. They are peeled, cored, and pulped (with sterile water added at a 1:1 mass ratio and mixed well). Ascorbic acid and pectinase are then added to adjust the initial pH and sugar content of the apple juice to 3.5 and 15°Brix, respectively. The mixture is then sterilized at 116℃ for 20 minutes. Dalck's yeast and Angel wine active dry yeast are prepared into bacterial suspensions, and inoculated simultaneously into sterilized fresh apple juice fermentation bottles at inoculation rates of 12%–13% and 6%–7% (both at a concentration of 10⁶ CFU / mL), respectively. Fermentation is carried out at 25–28℃ for 7–8 days. The control group is inoculated only with commercially available Angel wine active dry yeast (inoculation rate of 6%–7%, concentration of 10⁶ CFU / mL).

[0047] The total ion spectroscopy of volatile flavor compounds in apple cider fermented with Angel Yeast and Dalmatian Saccharomyces cerevisiae NJSYGA2021 are shown in the attached figures in the instruction manual. Figure 5 , Figure 6 .

[0048] The volatile flavor compounds content of each group of cider is shown in Table 1. Table 1 Volatile flavor compounds (mg / L) of each group of cider

[0049] Angie NJSYGA 2021 NJSYGA 2021: Angel (12% : 6%) n-Propanol 3.73 0.26 0.42 Isobutanol 1.51 0.39 0.36 2,3-Butanediol 0.80 - - Isoamyl alcohol 21.02 4.85 4.79 n-Hexyl alcohol - 0.09 - Phenylacetyl alcohol 15.89 10.34 8.15 3-Ethoxypropanol - 0.24 0. 12 Acetaldehyde 3.60 0.75 - 3-Hydroxy-2-Butanone 0.52 - - acetic acid 1.81 0.07 0.08 Cyclopropanecarboxylic acid - - - hexanoic acid - - - Sour 8.56 - 0.51 Decanoic acid 1.71 - - Hexyl formate - - - Ethyl acetate 10.29 - 1.23 heptyl acetate - 3.31 - Phenylacetyl acetate - 0.66 0.54 Phenylacetyl propionate - - - Phenylacetyl isobutyrate - - - Ethyl hexanoate 11.65 0.06 0.26 Ethyl octanoate 20.81 0.21 1.08 Ethyl decanoate 19.26 - 0.49 Ethyl lauryl acid 2.08 - - ethyl 9-decenoate - - - 2-Methylbutyric acid-2-phenylethyl ester - 0.15 - Ethylene oxide - - 0.92

[0050] As shown in Table 1, *Cyclocarya paliurus* NJSYGA 2021 can produce substances that *Angelica pubescens* cannot.

[0051] Raw phenethyl acetate has an apple-like fruity aroma. According to the volatile flavor compound content of the various groups of cider fermented according to Example 2, the cider fermented solely by *Cytosaccharomyces da Kjeldahl* NJSYGA 2021 contained 0.66 mg / L of phenethyl acetate; the cider produced by co-fermentation of *Cytosaccharomyces da Kjeldahl* NJSYGA 2021 and commercially available Angel yeast contained 0.54 mg / L of phenethyl acetate. Therefore, inoculating with *Cytosaccharomyces da Kjeldahl* NJSYGA 2021 can impart a typical apple aroma to the fermented product.

[0052] Example 3: Application of Dalmatian Flocculation Yeast NJSYGA 2021 in Apple Cider Vinegar Production

[0053] In another embodiment of this application, the application of Dalkistrodon halys spore-forming yeast NJSYGA2021 in apple cider vinegar production involves using Dalkistrodon halys spore-forming yeast and commercially available Angel wine active dry yeast as inoculum, inoculating fresh apple juice for alcoholic fermentation, and when the alcohol content reaches 5-6% vol, inoculating commercially available acetic acid bacteria for acetic acid fermentation until the fermentation is completed, and then performing flavor testing.

[0054] The specific application method is as follows: Using apples as raw material, peel and core them, then pulp them (add sterile water at a 1:1 mass ratio and mix well). Add ascorbic acid and pectinase to adjust the initial pH and sugar content of the apple juice to 3.5 and 15°Brix, respectively. Sterilize at 116℃ for 20 minutes. Prepare bacterial suspensions of *Delkirk's yeast* and *Angel Wine Active Dry Yeast* separately. Inoculate these suspensions simultaneously into sterilized fresh apple juice fermentation bottles at inoculation rates of 12%–13% and 6%–7% (both concentrations of 10⁶ cfu / mL), respectively. Seal and ferment at 25–28℃ until the alcohol content reaches 5%–6% vol. Then, inoculate with commercially available acetic acid bacteria at an inoculation rate of 0.3g–0.4g / 100mL and carry out acetic acid fermentation at 30–32℃ and 180–200 r / min until fermentation is complete. The control group was inoculated with commercially available Angel yeast (inoculation amount of 6%–7%, concentration of 106 cfu / mL) during the alcoholic fermentation stage, with all other conditions being the same.

[0055] The total ions of volatile flavor compounds in apple cider vinegar fermented with Angel Yeast and acetic acid bacteria, and with Dalky Keck's Spore-bearing Saccharomyces NJSYGA 2021 and Angel Yeast and acetic acid bacteria respectively are shown in the attached figures in the instruction manual. Figure 7 , Figure 8 .

[0056] The content of volatile flavor compounds in each group of apple cider vinegar is shown in Table 2.

[0057] Table 2. Volatile flavor compounds (mg / L) of apple cider vinegar in each group

[0058] Angel + Acetic Acid Bacteria NJSYGA2021 + Angel + Acetic Acid Bacteria benzyl alcohol 0.81 2.34 Diisobutylmethanol 0.11 - Phenylacetyl alcohol 119.63 156.75 n-Propanol 2.29 0.50 3-Ethoxypropanol 0.04 0.76 n-Butanol 0.68 0.05

[0059] Table 2. Volatile flavor compounds in apple cider vinegar for each group (Continued Table 2)

[0060] 2-Butanol 0.01 - 2-Ethylbutanol 0.18 - 2,3-Butanediol 0.71 1.45 (2R,3R)-(-)-2,3-Butanediol 0.71 0.24 Isoamyl alcohol 68.31 40.71 n-Hexyl alcohol 0.68 0.15 6-Methyl-1-heptanol 0.22 - Isooctyl alcohol 0.27 0.02 (S)-(+)-6-methyl-1-octanol 1.46 0.35 1-Nonol 0.20 0.09 alpha-terpineol 0. 11 - furfural 0.06 - 3-Furfural 0.19 - benzaldehyde 8.57 15.55 Acetaldehyde 12.18 12.00 Acetaldehyde 3.39 2.04 Butyraldehyde 0.28 - 5-Methylfuranaldehyde 0.51 0.23 4-Pentenal - 0.04 3-Hydroxy-2-Butanone 12.91 2.75 Pentylcyclopentanene - 0.47 acetic acid 3.01 2.50 Propylmalonic acid 0.82 - Isobutyric acid 0.71 0.24 hexanoic acid 6.53 - DL-A-hydroxyisohexanoic acid 0.10 - Sour 29. 11 - Decanoic acid 9.06 - Ethyl acetate 0.79 1.03 Ethyl lactate 0.8 0.47

[0061] Table 2. Volatile flavor compounds in apple cider vinegar for each group (Continued Table 2)

[0062] L-(-)-Ethyl lactate 0.58 0.16 Ethyl hexanoate 0.79 0. 12 2-Phenylacetyl ester - 0.17 Phenylacetyl acetate 4.38 6.44 Phenylacetyl propionate - 2.01 Diethyl succinate 2.45 1.60 3-Chloro-,2-Phenylacetyl propionate - 0.45 Ethyl pyruvate 5.22 - Ethyl octanoate 1.17 - Ethyl decanoate 2.51 3.49 Ethyl phenylacetate 1.22 0.66 Ethyl lauryl acid 0.96 - Valeric acid, 2-hydroxy-4-methyl-ethyl ester - 0. 14 Butyl butyrate 0.06 - Butyl formate 0.02 - Hexyl formate 0.09 0.03 Propionic acid, 2-methyl-,3-hydroxy-2,4,4-trimethylpentyl ester 0.01 -- 2-Methylpropyl 2-hydroxydecanoate 0.02 0.02 glycidyl acetate 0.06 0.02 phenol 2.58 1.25 2,6-Di-tert-butyl-p-cresol 0.58 - 2,5-Di-tert-butylphenol 0.37 0.21 3,5-Di-tert-butylphenol - 0.06 2,4-Di-tert-butylphenol 2.59 0. 12 Ethylene oxide 0.27 - 1(1-ethoxyethoxy)-pentane 0.03 0. 12 1,3-Dioxolane,2,4,5-Trimethyl- 16.20 13.66 1-Heptene 0.15 0.01 N,N-Dimethylpropionamide - 2.55

[0063] Table 2 shows that the phenethyl acetate content, which is characteristic of apple aroma, in apple cider vinegar fermented by a mixture of *Cytosaccharomyces cerevisiae* NJSYGA 2021, *Saccharomyces cerevisiae*, and acetic acid bacteria is 6.44 mg / L, higher than the 4.38 mg / L in apple cider vinegar fermented by a mixture of *Saccharomyces cerevisiae* and acetic acid bacteria. This indicates that *Cytosaccharomyces cerevisiae* NJSYGA 2021 can enhance the apple-like aroma of apple cider vinegar, highlighting the typical apple-like flavor of the fermented product.

[0064] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific structures and / or characteristics in the solutions are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A yeast strain that produces an apple-scented aroma, characterized in that, The yeast strain is *Saccharomyces cerevisiae* (Delkirk spore-forming yeast). Torulaspora delbruecki NJSYGA 2021 was deposited at the China Center for Type Culture Collection on January 10, 2022, with accession number CCTCC M 2022046.

2. The application of the apple-scented yeast strain as described in claim 1, characterized in that, The application of yeast in the fermentation of fermented beverages.

3. The application of the apple-scented yeast strain as described in claim 2, characterized in that, Fermented beverages include apple cider vinegar and apple wine.

4. The application of the apple-scented yeast strain as described in claim 2, characterized in that, The yeast can increase the content of phenethyl acetate in cider and apple cider vinegar, thereby enhancing the typical apple-like aroma in fermented beverages.

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