Process for the preparation of cider having a high content of ethyl butyrate

Abstract

The present application relates to the technical field of brewing, and particularly provides a preparation method of cider with high ethyl butyrate content. The method is to brew cider by mixed fermentation of Saccharomyces cerevisiae and Torulaspora delbrueckii. The Torulaspora delbrueckii is preferably Torulaspora delbrueckii SN-6, which was preserved in the China General Microbiological Culture Collection Center on November 7, 2022, and the preservation number is CGMCC No. 26064. The total ester content and ethyl butyrate content of the cider prepared by the method are significantly improved, the total sugar content is significantly reduced, the taste and aroma quality are both good, and the market prospect is broad.

Description

Technical Field

[0001] This invention relates to the field of brewing technology, specifically to a method for preparing cider with a high ethyl butyrate content. Background Technology

[0002] Esters are important flavor compounds in alcoholic beverages such as beer and cider, especially C2-C10 ethyl esters, which can enhance the overall flavor, impart fruity aromas, and make the body fuller and more harmonious. Based on carbon chain length, esters can be divided into two main categories: one is fat-soluble acetate esters, including ethyl acetate, isobutyl acetate, and isoamyl acetate, which are rapidly secreted into the fermentation broth through the cell membrane after production; the other is fatty acid ethyl esters with a carbon chain length of three or more carbons, including ethyl butyrate, ethyl hexanoate, ethyl decanoate, and ethyl octanoate, whose extracellular secretion rate decreases with increasing carbon chain length. Fat-soluble acetate esters are the most abundant type, but they are easily degraded, leading to a significant decrease in their content after a period of storage. Fatty acid ethyl esters, while making up a smaller proportion of the total ester content, play a crucial role in the flavor of the beverage, especially those containing 6-12 carbons, which have a rich and long-lasting aroma.

[0003] Ethyl butyrate has the molecular formula C6H. 12 O2, with six carbon atoms, is a type of short- and medium-chain fatty acid ethyl ester found in high amounts in wine. Ethyl butyrate is permitted as an edible flavoring in GB 2760-2014, possessing a kiwi-like aroma and widely used in edible and cosmetic flavorings to blend various fruit-flavored flavors; it is present in the flavor compounds of wine, therefore increasing its content can improve the quality of the wine.

[0004] Brewing yeast, commonly used in the brewing industry, has extremely high alcohol fermentation efficiency. However, due to its lack of acyl-CoA, the enzyme corresponding to ethyl butyrate, and the absence of highly specific acyltransferases, its ability to produce ethyl butyrate is low. Furthermore, it lacks the metabolic pathway for ethyl butyrate production, meaning it produces almost no ethyl butyrate itself. Therefore, alcoholic beverages fermented solely with brewing yeast often suffer from a shallow body and bland flavor. Combining aroma-producing yeasts with brewing yeast for use in alcoholic beverage production is a research hotspot in this field. Summary of the Invention

[0005] In view of the defects and shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing cider with a high ethyl butyrate content, and the cider prepared by this method has a richer aroma, so as to solve the problem of bland flavor in existing cider.

[0006] This invention relates to a method for preparing cider, which involves simultaneously adding Torulaspora delbrueckii and Saccharomyces cerevisiae to apple juice for cider fermentation.

[0007] The aforementioned *Cyclocarya delklai*, preferably *Cyclocarya delklai* SN-6, was deposited on November 7, 2022, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 26064.

[0008] The preservation number of the brewing yeast is ATCC 9763.

[0009] The method for preparing cider includes the following steps:

[0010] (1) Apple juice preparation:

[0011] Select ripe apples without rot or spoilage, wash, sort and cut them into pieces, put them in an antioxidant solution, soak for 15 minutes, take them out, drain the water and juice them to obtain apple pulp; add pectinase to the apple pulp, enzymatically hydrolyze at 40℃ for 3 hours, and filter to obtain clear apple juice.

[0012] (2) Activation and scale-up culture of the strain:

[0013] Saccharomyces cerevisiae ATCC 9763 and Saccharomyces delta-Kjeldahl SN-6 were respectively inoculated into YPD medium for activation; after 24 h of shaking culture at 180 r / min at 25℃, they were transferred to 200 mL of sterilized apple juice and incubated at 20℃ for 72 h to obtain seed culture of the two yeasts.

[0014] (3) Apple juice fermentation:

[0015] Saccharomyces cerevisiae ATCC 9763 seed culture and Saccharomyces delta-Kästmann SN-6 seed culture were simultaneously inoculated into sterilized apple juice at a volume ratio of 3%, and fermented in a constant temperature incubator at 20℃ for 15 days to obtain apple wine.

[0016] The antioxidant solution described in step (1) contains 1.5 g / L citric acid and 50 mg / L isoascorbic acid.

[0017] The pectinase addition ratio in step (1) is 700 mg / L.

[0018] Beneficial effects of the present invention

[0019] Compared with single-strain fermentation, the cider produced by the mixed fermentation of Saccharomyces cerevisiae ATCC 9763 and Saccharomyces delta-Kjeldahl SN-6 in this invention exhibits superior physicochemical properties and sensory evaluation. Specifically, the total ester content and ethyl butyrate content are significantly increased, reaching 124.520 mg / L and 5.301 mg / L, respectively, while the total sugar content is significantly reduced. The aroma and taste scores are also higher, with a richer fruity aroma and a total score of 90.41. The flavor is significantly improved, achieving unexpected technical results.

[0020] The method described in this invention fully leverages the advantages of multi-strain synergistic fermentation, effectively improving the taste and aroma of cider, and has broad market prospects. Attached Figure Description

[0021] Figure 1 This is a colony morphology diagram of *Cyclophorus deltaeni* SN-6.

[0022] Figure 2 Morphological image of an individual SN-6 cell of *Cytomyces delta-Kais*. Detailed Implementation

[0023] To better understand and implement the invention, the following detailed description is provided with reference to embodiments; the embodiments are only used to explain the invention and are not intended to limit the scope of the invention.

[0024] The *Cyclocarya pallida* TD1 used in the experimental examples of this invention was purchased from the China Industrial Microbiological Culture Collection Center (CICC 1004); *Cyclocarya pallida* TD2 was purchased from the China Industrial Microbiological Culture Collection Center (CICC 33295); *Cyclocarya pallida* TD3 was purchased from the China General Microbiological Culture Collection Center (CGMCC 2.145); and *Saccharomyces cerevisiae* was purchased from the United States Culture Collection Center (ATCC 9763).

[0025] The YPD culture medium formulation is as follows in the examples below:

[0026] Yeast extract 10g / 1000mL, peptone 20g / 1000mL, glucose 20g / 1000mL, chloramphenicol 2g / 1000mL, autoclave at 115℃ for 15min, at natural pH, then add 20g / 1000mL of agar to the solid.

[0027] The malt extract culture medium formulations in the following examples are as follows:

[0028] Weigh 130.1g / 1000mL of malt extract powder and 0.1g / 1000mL of chloramphenicol, heat to dissolve, adjust the pH to 6.4±0.2, dispense into test tubes, and autoclave at 121℃ for 15min for later use.

[0029] The analytical methods for detecting aroma substances such as ethyl butyrate in the sample are as follows:

[0030] Headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS) was employed. 5 mL of the sample was accurately measured and placed in a headspace vial containing 0.5 g NaCl. 10 μL of 50 mg / L n-butanol was added as an internal standard. A 50 / 30 μm CAR / DVB / PDMC extraction head was inserted, and extraction was performed at 45 °C for 45 min. The extraction head was then desorbed at 250 °C for 3 min through the GC inlet before GC-MS analysis. The temperature program was as follows: 40 °C for 2 min, increased to 80 °C at 5 °C / min, then increased to 280 °C at 10 °C / min and held for 3 min, finally increased to 300 °C at 5 °C / min and held for 1 min. The carrier gas (He) flow rate was 1.0 mL / min, splitless. An EI ion source of 70 eV at 230 °C was used; the ion source temperature was 200 °C; and the mass scan range was 50–600 m / z. The internal standard method was used for quantitative analysis of compounds.

[0031] The present invention will be further described below with reference to specific embodiments.

[0032] Example 1: Screening and Identification of *Cytomyces del Kernella* SN-6

[0033] This invention isolates and screens a fragrance-producing yeast from naturally fermented milk curds collected in Yining City, Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region.

[0034] The colony morphology of the aroma-producing yeast is as follows: Figure 1 As shown, the cell morphology is as follows Figure 2 As shown.

[0035] Based on a comprehensive analysis of the strain's physiological and biochemical characteristics and molecular biological identification results, the strain was ultimately identified as a *Cyclophorus deltaea* strain and named *Cyclophorus deltaea* SN-6.

[0036] (Torulaspora delbrueckii SN-6).

[0037] The applicant deposited the aforementioned Torulaspora delbrueckii SN-6 with the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) on November 7, 2022, with accession number CGMCC No. 26064.

[0038] The *DellKip* spore-forming yeast SN-6 provided by this invention possesses excellent aroma-producing properties, which can impart a rich fruity flavor to beer during the brewing and fermentation process. After fermentation, the total ester content in the beer is 84.79 mg / L, the total alcohol content is 64.36 mg / L, the total ketone content is 18.98 mg / L, and the organic acid content is 31.80 mg / L. Among them, the aroma-producing substances mainly include butyl butyrate (with an apple aroma), isoamyl acetate (with a fruity aroma), phenylethyl alcohol (with a rose aroma), and n-pentanol, etc. The contents of butyl butyrate, phenylethyl alcohol, and n-pentanol are as high as 25.22 mg / L, 15.87 mg / L, and 29.65 mg / L, respectively, achieving unexpected technical effects.

[0039] The aforementioned *Cyclocarya dalcis* SN-6 strain can utilize glucose, sucrose, soluble starch, maltose, citric acid, xylooligosaccharides, lactose, ethanol, and glycerol as carbon sources for growth, and ammonium oxalate as a nitrogen source. Its maximum tolerance concentrations to anhydrous ethanol, acetic acid, and NaCl are 9%, 0.6%, and 12%, respectively. The optimal growth pH is 3, the optimal glucose concentration is 30%, and the maximum tolerance concentration is 40%.

[0040] The *Saccharomyces cerevisiae* strain SN-6 exhibits the strongest ester production capacity at a fermentation temperature of 12–16°C, with 12°C being the most favorable temperature for cell growth and ester production. It also produces higher ester content at initial fermentation pH values ​​of 4–6, with an optimal initial fermentation pH of 5.

[0041] The beer prepared by fermentation with the aforementioned Dale Kelvin SN-6 yeast exhibits good fermentation degree (2.09%), sugar content (10.91 Bx), alcohol content (1.73%), total acid content (1.52 mg / L), diacetyl content (0.0389 mg / L), richer flavor compounds, and a high total ester content (106.46 mg / L). It also boasts a more ideal alcohol-to-ester ratio of 4.16:1, making it less likely to cause a "headache" after drinking, achieving unexpected technical results.

[0042] Example 2: Analysis of the ability of *Cytomyces delta-Kjeldahl* SN-6 to produce ethyl butyrate.

[0043] The strains selected in this embodiment are *Cyclophorus deltaea* (TD1, TD2, TD3) and *Cyclophorus deltaea* SN-6 obtained by screening in this invention, totaling 4 strains.

[0044] 1. Ability to produce ethyl butyrate in YPD medium

[0045] Single colonies of the four strains were picked and transferred to 25 mL test tubes containing 10 mL of YPD medium. The cultures were incubated at 25°C with shaking at 180 rpm for 24 h under natural pH conditions to obtain seed culture solutions. The prepared seed culture solutions were then inoculated at a rate of 5% into 200 mL of YPD medium and incubated at 20°C with shaking at 160 rpm for 48 h. The ethyl butyrate content in the fermentation broth of each strain was determined using GC-MS, and the results are shown in Table 1.

[0046] Table 1. Ethyl butyrate content (mg / L) of *Cyclocarya paliurus* in YPD medium.

[0047] strains TD1 TD2 TD3 SN-6 content 0.083 0.056 0.072 1.560

[0048] As shown in Table 1, the ethyl butyrate yield of *Cytosacchariformis deltaegi* SN-6 provided by this invention is the highest, reaching 1.560 mg / L, which is significantly higher than that of other *Cytosacchariformis deltaegi* species, achieving unexpected technical results.

[0049] 2. Ability to produce ethyl butyrate in malt extract medium

[0050] Single colonies of the four strains were picked and transferred to 25 mL test tubes containing 10 mL of malt extract medium. The cultures were incubated at 25°C with shaking at 180 rpm for 24 h under natural pH conditions to obtain seed culture solutions. The prepared seed culture solutions were then inoculated at a rate of 5% into 200 mL of malt extract medium and incubated at 20°C with shaking at 160 rpm for 48 h. The ethyl butyrate content in the fermentation broth of each strain was determined using GC-MS, and the results are shown in Table 2.

[0051] Table 2. Content (mg / L) of ethyl butyrate produced by *Cyclophorus dauricus* in malt extract medium.

[0052] strains TD1 TD2 TD3 SN-6 content 0.262 0.169 0.177 5.431

[0053] As shown in Table 2, the ethyl butyrate yield of *Cytosacchariformis SN-6* provided by this invention is the highest, reaching 5.431 mg / L, which is significantly higher than that of other *Cytosacchariformis SN-6* species, achieving unexpected technical results.

[0054] Example 3: Analysis of the optimal fermentation conditions for *Cytomyces delta-Kjeldahl* SN-6

[0055] Activated *Cyclocarya dalcis* SN-6 was inoculated into YPD medium. The total ester content and OD value at 560 nm were measured every 12 hours from 0 to 72 hours under different condition gradients. The total ester content curve was plotted with the blank medium at 0 hours of inoculation as a control to determine the optimal culture conditions.

[0056] The single-factor experiment was set as follows: the main fermentation temperature was set to 4℃, 8℃, 12℃, 16℃, 20℃, 24℃, and 28℃; the pH value of the fermentation broth was controlled to 2, 3, 4, 5, 6, 7, and 8, respectively.

[0057] 1. Optimal fermentation temperature of Dale Kelley's SN-6 yeast

[0058] The *Cyclocarya pallida* SN-6 strain exhibits the strongest ester production capacity at fermentation temperatures of 12–16°C, with 12°C being the most favorable temperature for cell growth and ester production. During the first two days of fermentation, this strain produces very little ester; from days 2–3, the ester content increases rapidly, reaching its peak at day 4.

[0059] 2. Optimal fermentation pH of Dale Kelvin SN-6

[0060] The *Cyclocarya dalcis* SN-6 strain produces higher ester content at initial fermentation pH values ​​of 4–6, with the optimal initial fermentation pH being 5. The ester production rate of this strain significantly increases on day 3 of fermentation, reaching a stable growth phase after 3 days, at which point the total ester content reaches its peak.

[0061] Example 4: Application of mixed fermentation of *Saccharomyces cerevisiae* SN-6 and *Saccharomyces cerevisiae* in cider production.

[0062] (1) Apple juice preparation: Select ripe apples without rot or spoilage, wash, sort and cut them into pieces, put them into a mixed antioxidant solution containing 1.5 g / L citric acid and 50 mg / L isoascorbic acid, soak for 15 min, take them out and drain the water and juice them to obtain apple pulp; add pectinase at a ratio of 700 mg / L, enzymatically hydrolyze at 40℃ for 3 h, filter to obtain clear apple juice; divide the apple juice into 200 mL portions and sterilize at 115℃ for 15 min;

[0063] (2) Activation and expansion culture of strains: Saccharomyces cerevisiae ATCC 9763 and Saccharomyces delta-Kjeldahl SN-6 were inoculated into YPD medium for activation; after 24 h of shaking culture at 180 r / min at 25℃, they were transferred to 200 mL of sterilized apple juice and incubated at 20℃ for 72 h to obtain seed culture of the two yeast strains.

[0064] (3) Apple juice fermentation: The seed liquid of Saccharomyces cerevisiae ATCC 9763 and the seed liquid of Saccharomyces delta-Kjeldahl SN-6 were simultaneously inoculated into sterilized apple juice at a volume ratio of 3% and fermented in a constant temperature incubator at 20℃ for 15 days to obtain apple wine.

[0065] Cider was fermented using single strains of Saccharomyces cerevisiae ATCC 9763 and Saccharomyces delta-Kästmannii SN-6, respectively, with other procedures as above, serving as a control group.

[0066] The total ester content, ethyl butyrate content, alcohol content, total sugar content, and pH of the cider were measured, and the sensory evaluation of the cider was performed. The results are shown in Tables 3 and 4.

[0067] Table 3. Results of the determination of physicochemical indicators of cider.

[0068]

[0069] Table 4 Sensory Evaluation Results of Cider

[0070]

[0071] As shown in Tables 3 and 4, compared with single-strain fermentation, the cider produced by the mixed fermentation of Saccharomyces cerevisiae and Saccharomyces delta-Kjeldahl SN-6 in this invention has better physicochemical indicators and sensory evaluation. Among them, the total ester content and ethyl butyrate content are significantly increased, reaching 124.520 mg / L and 5.301 mg / L respectively, while the total sugar content is significantly reduced. The aroma and taste scores are higher, with a total score of 90.41. The fruit aroma is rich and the flavor is significantly improved, achieving unexpected technical results.

[0072] The method described in this invention fully leverages the advantages of multi-strain synergistic fermentation, effectively improving the taste and aroma of cider, and has broad market prospects.

Claims

1. A method for preparing cider with a high ethyl butyrate content, characterized in that, The method includes using *Delkirk's yeast* (… Torulaspora delbrueckii ) and brewer's yeast ( Saccharomyces cerevisiae Apple juice is added simultaneously for the fermentation and brewing of cider; the preservation number of the Dale Kelley's spore-forming yeast is CGMCC No. 26064, and the preservation number of the brewing yeast is ATCC 9763. A method for preparing cider with a high ethyl butyrate content, characterized by the following steps: (1) Apple juice preparation: Select ripe apples without rot or spoilage, wash, sort and cut them into pieces, put them in an antioxidant solution, soak for 15 minutes, take them out, drain the water and juice them to obtain apple pulp; add pectinase to the apple pulp, enzymatically hydrolyze at 40℃ for 3 hours, and filter to obtain clear apple juice. (2) Activation and scale-up culture of the strain: Saccharomyces cerevisiae and Saccharomyces delta-Kjeldahl were inoculated into YPD medium and activated. After culturing at 25°C with shaking at 180 r / min for 24 h, they were transferred to 200 mL of sterilized apple juice and incubated at 20°C for 72 h to obtain seed cultures of the two yeasts. (3) Apple juice fermentation: Saccharomyces cerevisiae seed culture and Saccharomyces delta-Kästmannianus seed culture were simultaneously inoculated into sterilized apple juice at a volume ratio of 3%, and fermented in a constant temperature incubator at 20℃ for 15 days to obtain apple wine.

2. The method as described in claim 1, characterized in that, The antioxidant solution in step (1) of the method contains 1.5 g / L citric acid and 50 mg / L isoascorbic acid.

3. The method as described in claim 1 or 2, characterized in that, The pectinase addition ratio in step (1) of the method is 700 mg / L.

4. A cider, characterized in that, The cider is prepared by the method described in any one of claims 1-3.

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