Method for improving volatile flavor compounds of kiwi fruit wine

Abstract

The invention discloses a method for improving volatile flavor compounds of kiwi fruit wine, and belongs to the field of fruit wine brewing. 127 strains are obtained through screening of a TTC culture medium, the 127 strains are screened to obtain a pichia kuchii FPW7-6, compared with AQ, fruit wine prepared by fermentation of kiwi fruits through the pichia kuchii FPW7-6 shows higher fruit flavor, sweet taste, sour taste and flower flavor, the content of beta-damascone in the kiwi fruit wine reaches 3.4 micrograms / L, the content of beta-damascone in the kiwi fruit wine reaches 3.4 micrograms / L, and the content of beta-damascone in the kiwi fruit wine reaches 3.4 micrograms / L; the content of linalool is 338.3 [mu] g / L, the content of ethyl 2-methylbutyrate is 117.7 [mu] g / L, the content of gamma-butyrolactone is 254.0 [mu] g / L, and the content of phenethyl alcohol is 63265.6 [mu] g / L. The pichia kuchiana FPW7-6 is suitable for fermentation of kiwi fruit wine and can make the kiwi fruit wine have a better flavor.

Description

Technical Field

[0001] The invention relates to a method for improving volatile flavor compounds in kiwi fruit wine, and belongs to the field of fruit wine brewing. Background Art

[0002] Kiwi fruit is sweet and delicious, and is an excellent raw material for brewing fruit wine. Traditionally, kiwi fruit wine is mainly fermented with Saccharomyces cerevisiae. For example, the patent application with publication number CN103160405A discloses a wild kiwi fruit wine and a brewing method thereof, which is obtained by preparing kiwi fruit pulp, using Saccharomyces cerevisiae, and undergoing primary fermentation, secondary fermentation of juice and aging of juice wine. Another example is the patent with publication number CN102776098A, which discloses a kiwi fruit wine and a brewing method thereof, which is brewed by kiwi fruit, white sugar, potassium bicarbonate, sulfur dioxide, pectinase and Saccharomyces cerevisiae through kiwi fruit pulp preparation, primary fermentation, squeezing and separation, secondary fermentation, aging and other process steps. However, the kiwi fruit wine prepared by Saccharomyces cerevisiae has a bland flavor and is not distinctive. The kiwi fruit wine fermentation lacks a yeast strain that specifically improves the flavor of the fruit wine. Therefore, screening out non-brewery yeasts with fast fermentation speed and enhanced volatile flavor compounds in kiwifruit wine is of great significance to improving the quality and value of fruit wine. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for improving the volatile flavor compounds of kiwi wine, aiming to solve the technical problem that the existing kiwi wine fermentation lacks a yeast strain that can specifically improve the flavor of the wine.

[0004] The first technical solution provided by the present invention is a strain of Pichia kudriavzevii FPW7-6, which has been deposited in the China Center for Type Culture Collection on July 1, 2024, with a preservation number of CCTCC NO: M 20241440.

[0005] The second technical solution provided by the present invention is a microbial preparation containing Pichia coli FPW7-6 described in the first technical solution.

[0006] In certain embodiments, the amount of Pichia kuseri FPW7-6 added to the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0007] Furthermore, the amount of Pichia kusneri FPW7-6 added to the microbial preparation is at least 1×10 8 CFU / mL or 1×108 CFU / g.

[0008] The third technical solution provided by the present invention is a fermentation agent, which contains the Pichia coli FPW7-6 described in the first technical solution or the microbial preparation described in the second technical solution.

[0009] In certain embodiments, the amount of Pichia kuseri FPW7-6 added to the starter is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] Furthermore, the amount of the Pichia kusneri FPW7-6 added to the starter is at least 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0011] The fourth technical solution provided by the present invention is the use of Pichia coli FPW7-6 described in the first technical solution, or the microbial preparation described in the second technical solution, or the third fermentation agent in fruit wine brewing.

[0012] In some embodiments, the fruit wine is kiwi fruit wine.

[0013] The fifth technical solution provided by the present invention is a method for brewing kiwi wine, which comprises fermenting the Pichia coli FPW7-6 described in the first technical solution, the microbial preparation described in the second technical solution, or the starter described in the third technical solution with kiwi juice to prepare kiwi wine.

[0014] In certain embodiments, the fermentation parameters are: fermentation temperature is 20-30° C., and fermentation time is not less than 48 hours.

[0015] The sixth technical solution provided by the present invention is a method for increasing the volatile flavor compounds of kiwi fruit wine, wherein the method comprises fermenting kiwi fruit juice using the Pichia coli FPW7-6 described in the first technical solution, the microbial preparation described in the second technical solution, or the starter described in the third technical solution.

[0016] In certain embodiments, in the fermentation system, the sugar content of the kiwifruit juice is 20-22%.

[0017] In certain embodiments, the fermentation parameters are: fermentation temperature is 20-30° C., and fermentation time is not less than 48 hours.

[0018] In certain embodiments, the volatile flavor compounds include β-damascenone, linalool, ethyl 2-methylbutyrate, γ-butyrolactone, and phenylethanol.

[0019] The technical effects of the present invention are as follows:

[0020] The invention obtains 127 strains through TTC culture medium screening, and obtains a Pichia kuckeri FPW7-6 strain by screening the 127 strains. The fruit wine prepared by fermenting kiwi fruit with the Pichia kuckeri FPW7-6 has higher fruity, sweet, sour and floral tastes than AQ. In the kiwi fruit wine, the content of beta-damascenone reaches 3.4 μg / L, the content of linalool is 338.3 μg / L, the content of ethyl 2-methylbutyrate is 117.7 μg / L, the content of gamma-butyrolactone is 254.0 ug / L, the content of phenylethanol is 63265.6 μg / L, and the Pichia kuckeri FPW7-6 is suitable for the fermentation of kiwi fruit wine, and can make it produce better flavor.

[0021] Biomaterial Deposit Certificate A strain of Pichia kummerii ( Pichia kudriavzevii )FPW7-6, taxonomic name Pichia kudriavzevii FPW7-6 (Pichia pastoris kudriavzi FPW7-6) was deposited in the China Center for Type Culture Collection on July 1, 2024. The deposit address is: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO:M 20241440. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TTC staining (a) and colony morphology (b) of some strains.

[0024] Figure 2 Sensory evaluation of different kiwi wines.

[0025] Figure 3 The morphological characteristics of the FPW7-6 yeast strain. a: Single colony morphology on TTC solid medium cultured at 28°C for 48 hours; b: Colony morphology under a microscope.

[0026] Figure 4 Phylogenetic tree of FPW7-6 yeast based on neighbor-joining analysis of ITS rDNA sequence data.

[0027] Figure 5 Figure 1 shows the aroma pleasantness of kiwi wine fermented with different yeasts (Figure A) and the content of important aroma compounds; B: β-damascenone; C: linalool; D: ethyl 2-methylbutyrate; E: γ-butyrolactone; F: phenylethanol. DETAILED DESCRIPTION

[0028] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0029] Test method:

[0030] 1. The determination of total acid, ascorbic acid and reducing sugar content shall refer to the national standard GB / T 15038-2006 "General Analysis Method for Grape and Fruit Wine".

[0031] 2. The pH value is measured by an acidity meter.

[0032] 3. Total phenols were determined by the folin phenol colorimetric method: Preparation of standard curve: 0, 20, 40, 60, 80, 100 μL of gallic acid were taken, and 1000, 980, 960, 940, 920, 900 μL of distilled water were added to prepare gallic acid solutions of different concentrations, 50 μL of gallic acid + 50 μL of folin phenol + 50 μL of sodium carbonate + 100 μL of distilled water, and the OD value was measured at 765 nm.

[0033] Experimental group: 50 μL sample + 50 μL folin phenol + 50 μL 7.5% sodium carbonate + 100 μL distilled water, let stand for 30 min, and measure the OD value at 765 nm.

[0034] 4. Alcohol content is measured using a handheld alcohol meter.

[0035] 5. The sugar content is measured by a handheld saccharimeter.

[0036] 6. Headspace solid phase microextraction (HS-SPME): HS-SPME combined with gas chromatography-mass spectrometry (GC-MS) (Agilent 8890BGC, 5977B MS; Agilent Technologies, Santa Clara, CA, USA) was used to analyze the volatile compounds produced at the end of the kiwi wine fermentation process. 5 mL of sample was accurately aspirated and 1 mL of NaCl saturated solution was added to a 15 mL SPME flask. Then 25 μL of toluene-d8 (2.024 mg / L) and 50 μL of ethyl acetate-d3 (19.7 mg / L) were added as internal standards, sealed and equilibrated at 40 °C for 15 min, and the aged extraction head was inserted and extracted at a stirring speed of 300 rpm / min for 30 min. After the extraction, the injector was inserted into the headspace bottle (depth 22 mm), placed at 40 °C for 10 min, and the volatile substances were collected for gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS conditions: A DB-5MS column (60 m × 250 μm, film thickness 0.25 μm; Agilent, Santa Clara, CA, USA) was used with helium as the carrier gas at a flow rate of 1.0 mL / min. The initial column temperature was 40 °C, maintained for 1 min, and then increased to 230 °C at a rate of 4 °C / min and maintained for 15 min. Mass spectrometry analysis was performed under the conditions of ion source temperature of 230 °C, mass range of m / z 29-500, and electron ionization energy of 70 eV. The identification of volatile compounds involved the comparison of mass spectra and retention indices (Rl). The relative residual times of volatile compounds were calculated based on the linear retention times of the n-alkane (C, Con DB-WAX) column using the modified Kovats method.

[0037] 7. Qualitative and quantitative analysis of aroma substances: Match the detected unknown compounds with the mass spectral library of the National Institute of Standards and Technology (NIST) and the Wiley Library, and select substances with high matching degree as effective aroma components for qualitative comparison. Use the internal standard method for semi-quantitative analysis. Using toluene-d8 and ethyl acetate-d3 as internal standards, calculate the mass concentration of the component to be tested by comparing the peak area ratio of each component with the internal standard and correcting it.

[0038] The raw materials used in the examples are:

[0039] 1. Angel Wine Yeast (Saccharomyces cerevisiae, abbreviated as AQ): Angel Yeast Co., Ltd.

[0040] 2. Kiwifruit (MHT), loquat (PP), mulberry (SS), blueberry (LM), sunshine rose (PTX), and jasmine grape (TZ) were purchased from Guida Fresh Fruit Shop in Lao Chaoyang Village, Huaxi District (Guiyang, Guizhou Province), and commercial pickled pepper (PJ) was purchased from Huimin Fresh Supermarket in Lao Chaoyang Village, Huaxi District (Guiyang, Guizhou Province).

[0041] 3. YPD liquid medium (100 mL): weigh 1 g of yeast extract, 2 g of tryptone, and 2 g of glucose, add distilled water to make up to 100 mL, and sterilize at 121°C for 15 min before use.

[0042] 4. Kiwifruit juice YPD liquid medium (100 mL): weigh 1 g of yeast extract and 2 g of tryptone, add kiwifruit juice with a sugar content of 20% to make up to 100 mL, sterilize at 121°C for 15 min and set aside.

[0043] 5. Yeast extract peptone dextrose agar (YPD) solid culture medium (100 mL): Weigh 4.9 g of sample, add 100 mL of distilled water, heat to dissolve, and sterilize at 115°C for 30 min before use.

[0044] 6. TTC medium (100 mL): Weigh 3.3 g of TTC nutrient agar, add 100 mL of distilled water, stir and heat until completely dissolved, and sterilize at 121°C for 15 min.

[0045] Example 1

[0046] 1. Preliminary screening by TTC method

[0047] In a clean bench, wash the surface of the purchased fruits and pickled peppers with YPD liquid culture medium, add the obtained bacterial solution into a shaking tube (3-5mL), centrifuge at 5000rpm for 5min, pour out the supernatant, and dilute to 10% by adding sterile water. -3 and 10 -4 Gradient. Pipette 20 μL of each onto the TTC solid medium for yeast screening, culture in a 28°C constant temperature incubator for 48-72 hours, observe the color of the medium, pick out single colonies with good growth to streak TTC plates for purification, and observe the colony morphology.

[0048] 2. Dulbecco's tubule rescreening

[0049] The strains obtained by the initial screening of TTC method were inoculated into YPD medium for activation, and the inoculation amount was 2% (1×10 8 CFU / mL) was inoculated into 5 mL kiwifruit juice YPD liquid medium equipped with a Dulbecco's tube and cultured at 28°C and 50 rpm / min for 72 h. The bubble generation was observed and recorded every 24 h, and the strain number with excellent gas production performance was recorded.

[0050] 127 strains were screened by TTC medium, and the gas production of these strains and AQ strains in the Dulbecco's tubules was summarized and compared for 36h fermentation (as shown in Table 1). Among them, when the MHT-6-11, MHT-8-49, MHT-8-58, MHT-8-66, PP-2-16, PP-2-25, PP-5-3, FPW7-6, PP-7-12, PJ-I-5, PJ-I-6, TZ-1-9, TZ-1-11, TZ-2-1, LM-1-4, LM-1-5 and AQ strains were cultured to 24 hours, it was observed that the Dulbecco's tubules were 1 / 3 full of gas, indicating that these strains have rapid gas production capabilities. At 48h of culture, strains such as MHT-1-8, MHT-6-16, MHT-8-42, PP-2-5, and PP-3-1 began to produce gas, but the volume of gas was small; at 72h, the tubules of MHT-8-49, MHT-8-66, PP-2-16, PP-2-25, PP-5-3, FPW7-6, PJ-I-5, PJ-I-6, LM-1-4, and AQ were full of gas and completely floated on the liquid surface. Therefore, 10 yeast strains with strong gas production ability suitable for kiwi fruit wine fermentation were obtained through preliminary screening.

[0051] Table 1 Gas production of Dulbecco's tubules of some strains at 72 h

[0052]

[0053] Note: “-” means no gas is produced in the Dulbecco’s tubules, “+” means the gas volume is less than 1 cm, “++” means the gas volume is 1.5 to 2 cm, and “+++” means the gas volume is 2 to 3 cm.

[0054] After kiwi wine was fermented with a single inoculation of 10 bacterial strains, the aroma characteristics of the kiwi wine were evaluated by 10 trained winemaking professionals aged between 24 and 35 years old. In the sensory experiment, the samples were randomly coded and randomly assigned. The descriptive definitions of the six aroma attributes for evaluating the aroma of kiwi wine are: fruity, alcoholic, sweet, acidic, medicinal, floral, and herbal. The tasters scored the intensity of the aroma characteristics: 0 = no aroma characteristics, 1 = very weak, 2-weak, 3 = medium, 4-strong, 5 = very strong. Each test wine sample was analyzed twice. The sensory results showed that AQ and 5 strains of non-brewer's yeast had higher sensory evaluation scores. The results are as follows Figure 2As shown. It can be seen that the FPW7-6 and PP-5-3 groups showed higher fruity, sweet, sour and floral flavors compared to AQ. LM-1-4 showed obvious acidic taste characteristics, but its fruity and sweet aroma intensity was not significant, and excessive acidity had a negative impact on the flavor of the wine samples. M-8-49 had different chemical characteristics, but the overall aroma intensity was low. In summary, FPW7-6 fermented kiwi wine improved the sensory quality of kiwi wine to a certain extent and had certain market sales prospects.

[0055] 3. Morphological identification of high gas-producing yeast strains

[0056] Observe the colony morphology and color on the culture medium, and observe the cell morphology of single colonies with typical characteristics of yeast under a microscope.

[0057] 1. Strain morphological identification results

[0058] According to the color and morphology of the colony, a single colony was picked and placed on a slide. The characteristics of the picked cells were observed under a 100x microscope. The microscopic examination results of the FPW7-6 strain are as follows: Figure 3 shown.

[0059] 2. Results of molecular identification of strains

[0060] The ITS1 and ITS4 gene regions of four non-Saccharomyces strains were amplified. Sequences of selected strains representing potential species were obtained from GenBank and multiple alignments were performed. Phylogenetic analysis was performed using MEGA7 software. Figure 4 As shown in the figure, we identified FPW7-6 as Pichia kudriavzevii, which has the highest homology with Pichia kudriavzevii (100%) and is in the same evolutionary branch as Pichia kudravzevii SA-NS2 (OR672915). Many studies have shown that non-brew yeast can improve the quality of kiwi wine. Therefore, it is of great significance to use Pichia kudriavzevii FPW7-6 as a fermentation strain for kiwi wine.

[0061] Example 2

[0062] The PP-5-3 strain, FPW7-6 strain, M-8-49 strain, LM-1-4 strain and AQ in Example 1 were inoculated into YPD medium for activation, and then kiwi wine was prepared. The specific method is as follows:

[0063] 1. Select 80-90% ripe kiwifruit, wash, peel, squeeze the juice, and sterilize to obtain the raw material.

[0064] 2. Add potassium pyrosulfite (65 mg) and pectinase (500 mg) to the raw materials, perform enzymolysis in a constant temperature water bath at 35°C for 2 h, obtain kiwifruit juice, adjust the sugar content, add glucose to make the sugar content reach 20·Brix, and sterilize.

[0065] 3. Inoculate strain FPW7-6 into YPD liquid culture medium at a 4% (v / v) inoculation rate, culture at 28°C, 280rpm / min for 8h, then inoculate into sterilized kiwifruit pulp at 2%, perform pre-fermentation (fermentation in fermentation room, 20-22°C, 7d), collect the supernatant by centrifugation for post-fermentation (fermentation, 20-22°C, 10d), sterilize, centrifuge, collect the supernatant (i.e. kiwifruit wine), seal and bottle.

[0066] First, the physical and chemical indicators of kiwi wine were tested. The results are shown in Table 2. The detection of physical and chemical indicators is crucial in the production of kiwi wine. It ensures the quality and flavor of the wine, evaluates the nutritional components (such as vitamin C and total phenols), and improves the stability and shelf life of the wine (such as the regulation of pH and total acid). In addition, the physical and chemical properties directly affect consumer acceptance, provide a scientific basis for product optimization and market positioning, and help improve the market competitiveness of the product.

[0067] Table 2 Determination of physicochemical indexes of kiwi wine fermented by strains Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), and data with any identical lowercase letters or no letters indicate no significant differences (P>0.05).

[0068]

[0069] The aroma pleasantness of the above fruit wines was scored, and the aroma pleasantness of PP-5-3, FPW7-6, M-8-49 and LM-1-4 strains was significantly higher than that of AQ( Figure 5 A), among which FPW7-6 had an aroma pleasantness of 3.4, which was 30.8% higher than AQ. Then, the volatile compounds of these five yeast strains were quantitatively analyzed, and the contents of five common compounds in fruit wine were compared. β-Damascenone is an important terpene ketone compound in fruit wine, with the aroma properties of apple and sweet aroma. The aroma threshold in fruit wine is 0.05μg / L, which is Figure 5 B shows that the contents in the fruit wine prepared by FPW7-6 strain were 3.4 μg / L, which is 2.8 times that of AQ. Figure 5C is the quantitative result of linalool, which has the aroma of flowers. Its value in fruit wine is 15 μg / L. The content of linalool in fruit wine prepared by FPW7-6 strain is 338.3 μg / L, which is significantly higher than the content of linalool in AQ and 1.4 times that of AQ. Ethyl 2-methylbutyrate has a typical fruity aroma. The content of linalool in fruit wine prepared by FPW7-6 strain is 117.7 μg / L, which is 1.3 times that of AQ ( Figure 5 D). γ-butyrolactone has the aroma properties of butter and caramel. The content of γ-butyrolactone in the fruit wine prepared by FPW7-6 strain is 254.0 μg / L, which is 1.6 times that of AQ ( Figure 5 E). Phenylethanol has a rose scent. The content in AQ is 24255.6μg / L, and the content in FPW7-6 is 63265.6μg / L. FPW7-6 is significantly higher than AQ, which is 2.6 times that of AQ ( Figure 5 F).

[0070] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Pichia kudriavzevii FPW7-6, characterized in that: The Pichia pastoris FPW7-6 was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number CCTCC NO: M20241440.

2. A microbial preparation containing the Pichia coli FPW7-6 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The amount of Pichia kusei FPW7-6 added to the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A leavening agent, characterized in that The fermentation agent contains the Pichia kurowii FPW7-6 according to claim 1 or the microbial preparation according to claim 2 or 3.

5. Use of the Pichia coli FPW7-6 according to claim 1, or the microbial preparation according to claim 2 or 3, or the starter according to claim 4 in fruit wine brewing.

6. A method for brewing kiwi fruit wine, characterized in that: The method comprises fermenting the Pichia coli FPW7-6 described in claim 1, or the microbial preparation described in claim 2 or 3, or the starter described in claim 4 with kiwi fruit juice to prepare kiwi fruit wine.

7. The method according to claim 6, characterized in that The fermentation temperature is 20-30°C and the fermentation time is not less than 48 hours.

8. A method for increasing the volatile flavor compounds of kiwi fruit wine, characterized in that: The method comprises fermenting kiwifruit juice using the Pichia coli FPW7-6 described in claim 1, or the microbial preparation described in claim 2 or 3, or the starter described in claim 4.

9. The method according to claim 8, characterized in that In the fermentation system, the sugar content of the kiwifruit juice is 20-22%; the fermentation parameters are as follows: the fermentation temperature is 20-30° C., and the fermentation time is not less than 48 hours.

10. The method according to claim 8, characterized in that The volatile flavor compounds include β-damascenone, linalool, ethyl 2-methylbutyrate, γ-butyrolactone and phenylethanol.

Images