Saccharomyces marxianus yf51-1 and its application in semi-dry fermented whey wine

Abstract

This invention relates to the field of microbial technology, specifically to Kluyveromyces martensii YF51-1 and its application in semi-dry fermented whey wine. This strain can efficiently ferment lactose and galactose, while also exhibiting a high ABTS scavenging rate. The fermentation broth contains a high amount of amino acid nitrogen. Under conditions where sucrose is present, the amino acid nitrogen content in the sweetened whey fermentation broth is significantly higher than that in the unsweetened whey fermentation broth. Through process optimization, the optimal fermentation conditions for strain YF51-1 were obtained, ultimately producing a semi-dry whey fermented wine with a rich aroma and a sweet and sour taste, achieving an amino acid nitrogen content of 0.337 g / L, significantly higher than existing technologies. Through optimization of the freeze-drying process, a freeze-drying protectant suitable for strain YF51-1 was derived. This protectant consists of mannose, corn steep liquor powder, lysine, and calcium chloride. After optimization, this protectant demonstrates good protective effect on the strain, with a survival rate of up to 97.22%.

Description

Kluyveromyces macrocarpa YF51-1 and its application in semi-dry fermented whey wine Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Kluyveromyces martensii YF51-1 and its application in semi-dry fermented whey wine. Background Technology

[0002] Whey wine, a type of milk wine, is an alcoholic beverage made primarily from whey liquid and whey powder through fermentation and other processes. Most cheese companies in my country have small production volumes, resulting in relatively low whey emissions. However, directly discharging whey would waste resources and pollute the environment. Extracting whey powder, whey protein, or lactose from whey liquid requires expensive equipment and technology, and does not utilize all the components of whey. Whey contains approximately 95% water, 3%-4% lactose, 0.3%-0.7% whey protein, 1% lactic acid, and <0.2% citric acid. It also contains various vitamins, minerals, and trace elements. Fermenting whey to make wine allows for the full utilization of its nutrients, reducing resource waste.

[0003] Guangxi is a buffalo milk producing region. Studies have found that buffalo milk has more comprehensive nutrition, with a nutritional value far exceeding that of Holstein milk. Compared to other animal milks, it has higher levels of milk fat, protein, and ash, and its mineral content, such as calcium, phosphorus, and magnesium, is also significantly higher. In whey wine fermentation, ordinary brewing yeasts cannot utilize lactose. After years of strain screening, current technology has found that most Kluyveromyces species have a high lactose conversion rate, especially Kluyveromyces martensii, which can efficiently ferment lactose to produce alcohol. Therefore, Kluyveromyces martensii is often selected as the fermenting agent for whey wine. However, during our strain screening process, we found that due to physiological characteristics, not all Kluyveromyces martensii species can utilize lactose; some cannot. Therefore, to enrich the fermentation flavor of whey wine, it is necessary to screen strains to identify Kluyveromyces martensii strains that can efficiently ferment whey.

[0004] Semi-dry fermented whey wine has advantages such as a sweet and sour taste, a mellow body, and a good taste, and has been popular in recent years. In order to improve the taste and fermentation effect, during the fermentation of semi-dry whey wine, we often need to add sucrose. During the research process, we found that after adding sucrose, the ability of most Kluyveromyces marxianus to utilize protein will decrease, and the content of amino acid nitrogen will decrease; in addition, the most important thing in the taste of semi-dry whey wine is the conversion of aroma substances. In order to improve the aroma of the wine body, in addition to aging, adding aroma-producing yeast is also a direction for improving the preparation process. Therefore, we consider screening Kluyveromyces marxianus strains with aroma, amino acid nitrogen content in the fermentation broth, and fermentation ability as the preferred indicators. At the same time, in order to effectively preserve the strains, we also consider preparing the strains into freeze-dried powder. During the preparation process of the freeze-dried powder, the selection of the freeze-drying protectant is particularly crucial. Therefore, how to carry out freeze-drying protection on the strains is also a technical problem to be studied in this application. Summary of the Invention

[0005] In view of the above content, it is necessary to screen Kluyveromyces marxianus strains with excellent fermentation ability and effectively improved aroma from whey raw materials, and use these strains to ferment whey to produce semi-dry fermented whey wine, effectively improving the quality of fermented whey wine. In addition, in order to effectively preserve the strains, it is also necessary to select a freeze-drying protectant to produce freeze-dried powder.

[0006] To achieve the above object, the present invention has screened a new strain: Kluyveromyces marxianus strain YF51-1, whose taxonomic name is: Kluyveromyces marxianus YF51-1, and the Chinese taxonomic name is: Kluyveromyces marxianus YF51-1, and the preservation number is GDMCC NO: 65780; this strain is preserved in the Guangdong Provincial Microbiology Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025.

[0007] The present invention also includes a bacterial agent containing the Kluyveromyces marxianus strain YF51-1.

[0008] The present invention also includes a freeze-dried powder containing the Kluyveromyces marxianus strain YF51-1.

[0009] Furthermore, the protectant of the freeze-dried powder is composed of 30-70 g / L of mannose, 50-70 g / L of corn steep liquor powder, 5-15 g / L of lysine, and 0.5-1.5 g / L of calcium chloride.

[0010] Furthermore, the protectant of the freeze-dried powder consists of 70 g / L mannose, 50 g / L corn steep liquor powder, 5 g / L lysine and 1.5 g / L calcium chloride.

[0011] The present invention also includes a method for preparing the freeze-dried powder, the method comprising: enriching and culturing the Kluyveromyces marxianus strain YF51-1 to obtain the fermentation broth of strain YF51-1; then using sterile water to precipitate the fermentation broth of strain YF51-1 to prepare a yeast suspension; then mixing the yeast suspension with a protective solution at a volume ratio of 1:1, dispensing, pre-freezing, vacuum freeze-drying, and sealing for storage to obtain the final product.

[0012] The present invention also includes the application of Kluyveromyces marxianus strain YF51-1 in the preparation of semi-dry fermented whey wine.

[0013] The present invention also includes a method for preparing semi-dry fermented whey wine using the Kluyveromyces marxianus strain YF51-1, the method comprising the following steps:

[0014] (1) Ingredients: After filtering the cheese whey to remove excess protein and impurities, add white sugar and stir until completely dissolved;

[0015] (2) Pasteurization;

[0016] (3) Inoculation and fermentation: Inoculate with Kluyveromyces martensii YF51-1 and incubate for fermentation;

[0017] (4) Fining: After fermentation, add activated bentonite and stir evenly. Let it stand to fin the wine sample and make it clear.

[0018] (5) Filtration: Use a filter to remove fermentation substrates such as proteins and cells from the fermentation broth to obtain a clear fermentation broth, which is then bottled.

[0019] Furthermore, the mass fraction of white sugar in step (1) is 120 g / L; the fermentation temperature in step (3) is 30 °C.

[0020] The present invention has the following beneficial effects:

[0021] 1. The strain YF51-1 of the present invention was isolated by the research group from buffalo milk. This strain can efficiently ferment lactose and galactose. In addition, this strain also has a relatively high ABTS scavenging rate, the fermentation broth contains more amino acid nitrogen, and under the condition of the presence of sucrose, the content of amino acid nitrogen in the fermented whey with added sugar increases and is significantly higher than that in the sugar-free whey fermented broth, which is very suitable for the production of semi-dry whey wine; through process optimization, the optimal fermentation conditions of strain YF51-1 were obtained, and finally a semi-dry whey fermented wine with rich aroma, sweet and sour taste was produced. Its amino acid nitrogen can reach 0.337 g / L, which is significantly higher than 0.09 g / L in the literature and the national standard requirement of 0.05 g / L for milk wine.

[0022] 2. Through the optimization of the freeze-dried powder process, a freeze-drying protectant suitable for strain YF51-1 was obtained. This protectant is composed of mannose, corn steep powder, lysine and calcium chloride. After optimization, the protectant has a good protective effect on the strain, and the survival rate can reach 97.22%. Brief Description of the Drawings

[0023] Figure 1 is the colony morphology diagram of strain YF51-1.

[0024] Figure 2 is the phylogenetic tree diagram of strain YF51-1.

[0025] Figure 3 is the microscopy diagram of strain YF51-1.

[0026] Biological Material Deposit Information

[0027] The strain information deposited in this application is as follows: Kluyveromyces marxianus strain YF51-1, its taxonomic name is: Kluyveromyces marxianus YF51-1, and the Chinese taxonomic name is: Kluyveromyces marxianus YF51-1. The deposit number is GDMCC NO: 65780; this strain is deposited in the Guangdong Provincial Culture Collection of Microorganisms, address: 5th Floor, Building ⅤⅨ, No. 100, Xianlie Middle Road, Guangzhou. The deposit date is January 14, 2025. Detailed Embodiments

[0028] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0029] Any feature disclosed in this specification (including any additional claims, abstract) is, unless otherwise stated, only an example of a series of equivalent or similar features.

[0030] Example 1:

[0031] This example describes the isolation and identification of Kluyveromyces martensii YF51-1.

[0032] 1. Isolation of strain YF51-1:

[0033] Solid culture medium: WLN medium

[0034] Liquid culture medium: YPD (1% glucose, 1% peptone, 0.5% beef extract)

[0035] Isolation steps: Dilute the sample with sterile physiological saline using a gradient. Take 100 μL of the appropriate gradient dilution and spread it on a WLN plate. Incubate at 30 ℃ for 24-48 h. If the colony growth on the plate is too low, the sample needs to be enriched. This can be done by directly adding the sample to YPD broth and culturing for 24-48 h, then taking the culture medium, diluting it further, spreading it on a plate, and observing the growth.

[0036] Select plates with good growth, moderate colony density, and clearly visible single colonies for isolation. Pile single colonies onto fresh plates for purification. After purification for at least three generations, when the plates are free of contaminants and the colony morphology and size are uniform, the plates can be cultured in liquid culture and then stored in glycerol.

[0037] 2. Identification of strain YF51-1: ① Morphological identification: The specific operation was as follows: The morphological observation of this strain on PDA plate medium is shown in Figure 1. The colony diameter is 3-5 mm, white, oval, with a raised, dry, smooth surface and neat edges. The cells were observed under a microscope, and the results are shown in Figure 3. They are oval and have obvious spores, which is consistent with the morphological characteristics of yeast budding reproduction; ② The YF51-1 strain obtained above was identified by 28S rDNA sequencing. After comparative analysis with NCBI, the sequence obtained by the strain of this invention is closely related to Kluyveromyces marxianus, with homology reaching 99.5%. The phylogenetic tree is shown in Figure 2. Based on the morphological identification, this strain was classified and named Kluyveromyces marxianus.

[0038] Example 2:

[0039] This example illustrates the screening of strain YF51-1 and the method for processing whey wine using this strain, as detailed below:

[0040] I. Construction and strain screening of buffalo milk yeast strain bank, as detailed below:

[0041] 1. Sample collection for yeast isolates: Fresh milk samples were collected from various buffalo ranches in Guangxi, aseptically packaged, and refrigerated; solid dairy products such as mature cheese made from buffalo milk were collected and isolated.

[0042] 2. Study on the fermentation performance of buffalo milk yeast:

[0043] Yeast fermentable sugar detection: The sugars in buffalo milk are mainly lactose, galactose and other sugars that are difficult for yeast to utilize. For the screening of dairy yeast, yeast nitrogen source basal (YNK) medium was used as a supplement to yeast nitrogen source to determine the degradation of lactose, galactose and lactic acid by yeast.

[0044] Preparation of test culture medium: Yeast nitrogen source basal (YNK) 6.8 g / L, sterile filtration, filtrate to prepare 1% concentrations of sterile lactose, galactose and lactic acid, respectively. 1% glucose was used as a positive control and pure YNK culture medium was used as a negative control. The medium was placed in sterile fermentation tubes, and an inverted Durham tube was placed in each tube to remove air from the Durham tube. The gas production of sugar during yeast fermentation was observed.

[0045] 3. Screening of superior buffalo whey fermentation yeast:

[0046] Divide the cheese whey into two portions. Add 5% purified water to one portion and 5% (w / w) white sugar to the other portion. After complete dissolution, pasteurize. Inoculate the two pasteurized whey portions with yeast and ferment at 37°C for 48-72 hours. Stop fermentation when there is no obvious fermentation and refrigerate at 4°C until testing.

[0047] Aroma and pH measurement of fermentation broth: The aroma of each fermentation broth was compared and recorded by smelling; the pH of the fermentation broth was measured using a pH meter.

[0048] Total ester content determination in fermentation broth: The total ester content in fermentation broth was determined according to the method of CHEN et al. Take 1 mL of fermentation broth after sterilization, add 9 mL of deionized water, add 1-2 drops of phenolphthalein, and titrate with 0.1 mol / L NaOH standard solution (C) until pink. Add 10 mL of 0.1 mol / L NaOH standard solution (Va) and saponify overnight at 30℃. Then titrate with 0.1 mol / L H2SO4 standard solution (Cl) in the reverse direction until the red color just disappears. Record the volume percentage of H2SO4 solution consumed. Use blank culture medium as a control. The total ester content, calculated using ethyl acetate, is shown below:

[0049] Total esters (g / L) = (C×Va - C1×Vb)×88.12÷1.0÷1000

[0050] In the formula: 88.12-molar mass fraction of ethyl acetate

[0051] ABTS Antioxidant Activity Assay: Following the method of Abdel-Hamid et al. (2019), the antioxidant activity of fermented milk samples was determined using the ABTS method. 50 µL of a mixture of probiotic fermented milk supernatant and 200 µL of ABTS working solution was injected into each well of a 96-well microplate reader. After incubation in the dark for 30 min, absorbance was measured at 405 nm using a microplate reader (Epoch 2, BioTek, Guangzhou, China). Antioxidant Activity

[0052] The calculation formula is as follows:

[0053] ABTS clearance rate % = 100 ×100

[0054] In the formula, “A sample” is the absorbance of the mixture of ABTS working solution and sample, and “A control” is the mixture of ABTS working solution and water, without sample.

[0055] Determination of amino acid nitrogen in whey fermentation broth: Refer to the method for determination of amino acid nitrogen in the national standard GB / T 23546-2009 for milk wine.

[0056] HPLC organic acid detection of whey fermentation broth: Agilent HPLC system 1260, Wech C18 column, mobile phase 0.01 mol / L NH4H2PO4 (dissolved in 3% methanol aqueous solution), adjusted to pH 2.3 with phosphoric acid, flow rate 0.8 ml / min, column temperature 30℃, detection wavelength 210 nm; the types of organic acids detected included tartaric acid, D-malic acid, L-malic acid, lactic acid, acetic acid, citric acid, and succinic acid.

[0057] 4. Experimental Results:

[0058] (1) Screening of yeast physicochemical indicators: Eighteen yeast strains were screened and molecularly identified. The results of fermentable sugars and gas production of the strains are shown in Table 1.

[0059] Table 1 Summary of yeast fermentable sugars and gas production

[0060]

[0061] Table 1 shows that 18 yeast strains were screened in the experiment, including *Kluyveromyces marxianus* (K. marxianus), *Pichia kudriavzevii* (P. kudriavzevii), *Cyberlindnera jadinii* (C. jadinii), *Geotrichumpandrosion* (G. pandrosion), *Candida parapsilosis* (C. parapsilosis), and *Candida tropicalis* (Candida tropicalis). The study investigated the fermentation of sugars and gas production by *C. tropicalis* and *Pichiacactophila* (P. cactophila). Results showed that some strains of *Kluyveromyces martensii* could ferment lactose and galactose, with some producing gas during lactose fermentation; *P. kudriavzevii* could not ferment lactose and galactose, but could ferment lactic acid; *C. jadinii* could not ferment lactose and galactose, but some strains could ferment lactic acid; *C. tropicalis* could ferment lactose, but with relatively weak fermentation ability; and *P. cactophila* could ferment both lactose and lactic acid, producing gas during lactose fermentation.

[0062] (2) Screening of aroma-producing whey fermentation yeast: The main nutrients in whey are lactose, whey protein and milk fat, with lactose having the highest content. Sweetened whey contains sucrose, which is a sugar that yeast can generally ferment. All yeasts were inoculated with both whey and sweetened whey at the same time, and the physicochemical properties of their fermentation broth are shown in Table 2.

[0063] Table 2 Physicochemical properties of whey fermentation broth from strains

[0064]

[0065] Table 2 shows that the ABTS scavenging rate was measured after the fermentation broth was diluted 5 times. Among the strains, YF51-1, YF54-8, YF54-1, YM20-1A, F52-4, F55①-4, F55①-5, F52-6, YM19-6, and YM51-1 exhibited high antioxidant levels in the whey fermentation broth, all exceeding 60%, while the ABTS scavenging rate of the blank whey was 34.732%. This indicates that the antioxidant activity of these strains increased after whey fermentation. The antioxidants in whey mainly originate from proteins, peptides, and polysaccharides, which may indicate that these strains have the ability to degrade proteins or synthesize other active substances. The antioxidant activity of the sweetened whey fermentation broth was significantly lower than that of the unsweetened whey fermentation broth. This may be due to the increased yeast metabolic pathways in the presence of sucrose, leading to the breakdown of some active substances by the produced enzymes. Comparison of antioxidant activities among strains showed that strain YF51-1 exhibited the highest antioxidant activity in sweetened whey, reaching 45.487%.

[0066] Amino acid nitrogen content is significant for the quality, flavor, and nutritional value of milk wine. It not only reflects the degree of fermentation but also contributes to its unique flavor profile. A higher amino acid nitrogen content indicates a greater quantity of amino acids in the milk wine, thus enhancing its nutritional value. Moderate consumption of milk wine containing a certain amount of amino acid nitrogen can help supplement the body's amino acid needs, playing a positive role in maintaining normal physiological functions and promoting metabolism. For fermentation strains, amino acid nitrogen content represents their protein-degrading capacity. In unsweetened whey, strain F52-6 had the highest amino acid nitrogen content, reaching 0.540 g / L. However, the amino acid nitrogen content of this strain decreased in sweetened whey, indicating that strain F52-6 is not suitable for fermenting semi-dry whey. In sweetened whey, the strain with the highest amino acid nitrogen content was *Kluyveromyces martensii* YF51-1, which had an amino acid nitrogen content of only 0.175 g / L in unsweetened whey. This indicates that the protein degradation ability of this strain is better in the presence of sucrose. This may be because *Kluyveromyces martensii* can secrete acidic proteases. The optimal pH for this enzyme is 3.0-5.0. In the presence of sucrose, the glycolysis pathway is enhanced, and the pH of the fermentation broth decreases rapidly. Below 5, the protease activity increases, exhibiting a strong ability to decompose β-lactoglobulin and α-lactalbumin in whey. Therefore, the amino acid nitrogen content in sweetened whey fermentation broth is significantly higher than that in unsweetened whey fermentation broth. *Kluyveromyces martensii* is the preferred strain for fermenting semi-dry whey.

[0067] Based on the combined antioxidant and amino acid nitrogen content of the fermentation broth, 11 strains were screened for ester content detection, aroma screening, and organic acid content detection. The results are shown in Tables 3 and 4.

[0068] Table 3. Ester content and aroma description of whey fermentation broth from strains

[0069]

[0070] Table 3 shows that strains 1 and 5-11 have relatively high ester content in their whey fermentation broth, both exceeding 14 g / L. However, sensory examination revealed that while these strains exhibited a pronounced aroma in sweetened whey, the aroma intensity was not directly proportional to the ester content. Strains with better aroma performance include YF51-1, YM51-1, and F51-4, all possessing pleasant fruity and floral aromas. Yeast fermentation of whey primarily produces volatile esters such as ethyl acetate, ethyl butyrate, and ethyl hexanoate, which, at appropriate concentrations, can contribute rich fruity and floral flavors to the wine. Strain F52-6 has a high amino acid nitrogen content, but its aroma performance is weaker.

[0071] Table 4. Organic acid content (g / L) in whey fermentation samples as determined by liquid chromatography.

[0072]

[0073] As shown in Table 4, acetic acid is a volatile organic acid with a pungent taste, usually produced by yeast metabolism or spoilage of the fermentation broth. In fermented wines, the volatile acid content is generally required to be no higher than 2 g / L, otherwise it will bring unpleasant flavors to the wine. The liquid phase organic acid detection results show that only strains YF51-1, YM51-1, and F51-1 among the screened yeasts do not produce acetic acid. The remaining strains produce acetic acid, with most exceeding 2 g, and some approaching 2 g, which is detrimental to the flavor of fermented whey wine. Lactic acid is a mild-tasting organic acid that can improve the flavor of fermented wines. The lactic acid content of strain YF51-1 is 1.544 g / L, strain YM51-1 is 3.678 g / L, while strain F51-1 does not produce lactic acid, and this strain has a high malic acid production. Malic acid is a strong-tasting acid with astringent properties and a long-lasting sour taste. In wine, it is often converted into lactic acid to improve the wine's mouthfeel. Based on the aroma description of this strain, it can be found that the fermentation liquid has a distinctly sour taste and is not suitable as a strain for whey wine fermentation.

[0074] Based on the comprehensive evaluation of the strain's aroma, ester yield, antioxidant activity, and amino acid peptide nitrogen content, strain YF51-1 was ultimately selected as the whey wine fermentation yeast, and the fermentation process was optimized.

[0075] II. Optimization of whey wine fermentation process:

[0076] Optimization of whey wine fermentation conditions: Pasteurized whey with a sugar content of 80 g / L was inoculated with YF51-1 and fermented at three different temperatures: 20℃, 30℃, and 37℃. Pasteurized whey with sugar additions of 40 g / L, 80 g / L, and 120 g / L was inoculated with YF51-1 and fermented at 30℃. After fermentation, the reducing sugar, acidity, pH, and distillation alcohol content of the fermentation broth were measured, and sensory evaluation was performed. The optimal fermentation conditions for the strain were determined. The physicochemical indicators of the fermentation broth are shown in Table 5, and the sensory and aroma evaluations are shown in Table 6.

[0077] Table 5 Effects of different temperatures on fermented whey wine

[0078]

[0079] From a physicochemical perspective, the fermentation broth at 20℃ had a slightly higher residual sugar content, lower alcohol content, and a slightly higher pH. This is likely because *Kluyveromyces martensii* is a thermotolerant yeast; multiple studies have shown that it still has strong alcohol production capacity under high temperatures (approximately 50℃). In the experiment, the fermentation capacity of this yeast at 20℃ was significantly lower than that at 30℃ and 37℃, which also indirectly indicates the heat tolerance of this strain. The alcohol content and residual reducing sugar content of the 30℃ and 37℃ samples were similar, indicating that the fermentation capacity of this strain was comparable at these two temperatures. The amino acid nitrogen content of the samples increased with higher temperature, indicating that the protein degradation capacity of this strain increased with increasing temperature. However, sensory evaluation showed that the 30℃ fermentation sample had a significant advantage in aroma expression, with more pronounced fruity and floral aromas. Therefore, 30℃ was chosen as the final fermentation temperature for whey wine.

[0080] Table 6 Sensory Evaluation and Aroma Description

[0081]

[0082] The effects of different sugar additions on the physicochemical properties of the fermentation broth are shown in Table 7:

[0083] Table 7 Effects of different sugar contents on fermented whey wine

[0084]

[0085] The amount of whey sugar added was also optimized, and the physicochemical results are shown in Table 7. The three samples showed little difference in pH and reducing sugar content, but significant differences in distillation alcohol content and acidity, indicating that the amount of sugar added has a significant impact on yeast fermentation. Regarding the amino acid nitrogen content, the higher the amount of sugar added, the higher the amino acid nitrogen content in the liquor, indicating that the protein degradation ability of this strain increases with the increase of white sugar addition. In terms of aroma and sensory evaluation, the sample with 120 g / L of added sugar performed better, with a rich aroma and a sweet and sour taste, making it a high-quality semi-dry whey fermented liquor. Its amino acid nitrogen content reached 0.337 g / L, significantly higher than the literature's 0.09 g / L and the national standard requirement for milk wine of 0.05 g / L.

[0086] The final processing method for whey wine obtained from strain YF51-1 is as follows:

[0087] 1. Ingredients: After filtering the cheese whey to remove excess protein and impurities, add 120g / L of white sugar in proportion and stir until completely dissolved.

[0088] 2. Pasteurization: Heat to 95℃ and pasteurize for 10 minutes, then cool to 30℃ for later use.

[0089] 3. Inoculation and fermentation: Inoculate with Kluyveromyces macrocarpa YF51-1 to achieve a bacterial concentration of 10. 6 ~10 7 Fermentation was carried out at 30°C with cfu / mL until the pH of the fermentation broth dropped below 4.1 and the solid content dropped below 3%, at which point fermentation was stopped.

[0090] 4. Fining: Cool the fermentation liquid to below 10℃, add 1% bentonite that has been activated for 24 hours, stir well, let stand for 48 hours, and then fin the wine sample to make it clear.

[0091] 5. Filtration: Use a filter to remove fermentation substrates such as proteins and cells from the fermentation broth to obtain a clear fermentation broth, which is then bottled.

[0092] Example 3:

[0093] This embodiment studies the process of preparing Kluyveromycin YF51-1 into lyophilized powder:

[0094] (1) Enrichment culture of yeast: After activation, Kluyveromyces martensii YF51-1 was inoculated into NYTB fermentation medium to obtain yeast fermentation broth. The NYTB fermentation medium consisted of 8g beef extract, 5g yeast extract, 10g trehalose, and 1000ml distilled water at natural pH.

[0095] (2) Preparation of yeast suspension: The yeast fermentation broth obtained in step (1) is placed in a centrifuge cup, centrifuged, and the precipitate is washed with sterile water. Then, the precipitate is used to prepare a yeast suspension with sterile water. The viable count of the suspension is 10. 6 -10 9 cfu / mL available;

[0096] (3) Add freeze-drying protectant: Select the following components as protectant, prepare the protectant solution according to the mass concentration, and mix the protectant solution with the yeast suspension at a volume ratio of 1:1 to obtain the mixture;

[0097] ①Carbohydrates: Sucrose, trehalose, maltose, lactose and mannose, each single protective agent is prepared into a solution with a final concentration of 50g / L.

[0098] ② Protein preservatives: skim milk powder, beef extract, corn syrup powder and peptone, each single preservative is prepared into a solution with a final concentration of 50g / L.

[0099] ③ Amino acid protective agents: Lysine, cysteine ​​and monosodium glutamate, each single protective agent is prepared into a solution with a final concentration of 10 g / L.

[0100] ④ Trace element protective agents: Sodium chloride, potassium dihydrogen phosphate and calcium chloride, each single component protective agent is prepared into a solution with a final concentration of 1g / L.

[0101] (4) Dispensing: Pour the mixture prepared in step (3) into a plate with a diameter of 9cm, so that the thickness is about 0.5cm;

[0102] (5) Pre-freezing: Seal the packaged flat plates with plastic wrap and place them in a -80℃ constant temperature refrigerator for 3 hours;

[0103] (6) Vacuum freeze drying: Quickly transfer the pre-frozen plate to a freeze dryer pre-cooled to below -30°C, turn on the vacuum pump, set the cold trap temperature of the freeze dryer to -50°C, and freeze dry under vacuum for 36 hours;

[0104] (7) Sealing and preservation: The freeze-dried plates are quickly transferred to the ultra-clean workbench and sealed and preserved with plastic wrap and sealing film.

[0105] After staining the lyophilized bacterial powder with a combination of SYTO9 and PI staining reagent for 15 min, flow cytometry was used for analysis. The survival rate was then calculated using the following formula: Survival rate = (Number of viable cells in lyophilized powder / Number of viable cells before lyophilization) × 100%. The survival rate results are shown in Table 7.

[0106] Table 7. Effects of various protectants on the survival rate of yeast YF51-1

[0107]

[0108] Table 7 shows that among carbohydrate protectants, the effects on the survival rate of yeast YF51-1, from highest to lowest, are: mannose (76.25%) > lactose (23.11%) > maltose (15.23%) > trehalose (12.36%) > sucrose (12.07%); among protein protectants, the effects on the survival rate of yeast YF51-1, from highest to lowest, are: corn steep liquor powder (66.32%) > beef extract (12.62%) > skim milk powder. (6.97%) > Peptone (1.68%); Among amino acid protectants, the effects on the survival rate of yeast YF51-1 from high to low are: lysine (65.78%) > cysteine ​​(21.03%) > monosodium glutamate (1.25%); Among trace element protectants, the effects on the survival rate of yeast YF51-1 from high to low are: calcium chloride (70.36%) > potassium dihydrogen phosphate (13.58%) > sodium chloride (1.36%).

[0109] Therefore, we selected mannose, corn steep liquor powder, lysine, and calcium chloride for further orthogonal experiments, using survival rate as the indicator, to study whether the composite protectant had a promoting effect on the freeze-drying protection of yeast strain YF51-1, as shown in Tables 8 and 9:

[0110] Table 8. Orthogonal experimental factor levels for different mass percentages of preservative liquid components

[0111]

[0112] The orthogonal experimental results are shown in Table 9:

[0113] Table 9. Results of Orthogonal Experiments

[0114]

[0115] As shown in Table 9, the combination of mannose, corn steep liquor powder, lysine, and calcium chloride significantly affected the survival rate of strain YF51-1. When the freeze-drying protectant concentrations were adjusted according to experimental groups 1, 4, 7, and 9, the survival rate of strain YF51-1 decreased, even falling below the survival rate of lysine (65.78%) (as shown in Table 7). Conversely, when the freeze-drying protectant concentrations were adjusted according to experimental groups 2, 3, 5, and 8, the survival rate of strain YF51-1 significantly increased, exceeding the required concentrations. Table 7 shows the survival rate of mannose (76.25%). After compounding with the freeze-drying protectant concentration of experimental group 6, the survival rate of strain YF51-1 did not increase significantly and was close to the survival rate of mannose (76.25%) in Table 7. Therefore, we believe that the freeze-drying protectant composed of the following components—30-70 g / L mannose, 50-70 g / L corn steep liquor powder, 5-15 g / L lysine, and 0.5-1.5 g / L calcium chloride—will greatly improve the survival rate of strain YF51-1. The optimal freeze-drying protectant composition was found in Experiment 8, which consisted of the following components: 70 g / L mannose, 50 g / L corn steep liquor powder, 5 g / L lysine, and 1.5 g / L calcium chloride. The survival rate range showed that corn steep liquor powder > lysine > calcium chloride > mannose. This indicates that among the freeze-dried powders, corn steep liquor powder had the greatest impact on the survival rate of strain YF51-1, followed by lysine, then calcium chloride, with mannose having the least impact.

[0116] Therefore, considering all factors, the optimal method for preparing the lyophilized powder of strain YF51-1 in this application is as follows:

[0117] (1) Enrichment culture of yeast: After activation, Kluyveromyces martensii YF51-1 was inoculated into NYTB fermentation medium to obtain yeast fermentation broth. The NYTB fermentation medium consisted of 8g beef extract, 5g yeast extract, 10g trehalose, and 1000ml distilled water at natural pH.

[0118] (2) Preparation of yeast suspension: The yeast fermentation broth obtained in step (1) is placed in a centrifuge cup, centrifuged, and the precipitate is washed with sterile water. Then, the precipitate is used to prepare a yeast suspension with sterile water. The viable count of the suspension is 10. 6 -10 9 cfu / mL available;

[0119] (3) Adding freeze-drying protectant: Select the following components as protectant, prepare the protectant solution according to the mass concentration, and mix the protectant solution with the yeast suspension at a volume ratio of 1:1 to obtain the mixture; wherein, the protectant consists of the following components: 30-70 g / L of mannose, 50-70 g / L of corn steep liquor powder, 5-15 g / L of lysine and 0.5-1.5 g / L of calcium chloride;

[0120] (4) Dispensing: Pour the mixture prepared in step (3) into a plate with a diameter of 9cm, so that the thickness is about 0.5cm;

[0121] (5) Pre-freezing: Seal the packaged flat plates with plastic wrap and place them in a -80℃ constant temperature refrigerator for 3 hours;

[0122] (6) Vacuum freeze drying: Quickly transfer the pre-frozen plate to a freeze dryer pre-cooled to below -30°C, turn on the vacuum pump, set the cold trap temperature of the freeze dryer to -50°C, and freeze dry under vacuum for 36 hours;

[0123] (7) Sealing and preservation: The freeze-dried plates are quickly transferred to the ultra-clean workbench and sealed and preserved with plastic wrap and sealing film.

[0124] In summary, the *Kluyveromyces martensii* strain YF51-1, screened by the applicant, can efficiently ferment lactose and galactose, while also exhibiting a high ABTS scavenging rate. The fermentation broth contains a high amount of amino acid nitrogen; under the presence of sucrose, the amino acid nitrogen content in the sweetened whey fermentation broth is significantly higher than that in the unsweetened whey fermentation broth. Through process optimization, the optimal fermentation conditions for strain YF51-1 were obtained, ultimately producing a semi-dry whey fermented wine with a rich aroma and a sweet and sour taste, achieving an amino acid nitrogen content of 0.337 g / L, significantly higher than existing technologies. Through optimization of the freeze-drying process, a suitable freeze-drying protectant for strain YF51-1 was derived. This protectant consists of mannose, corn steep liquor powder, lysine, and calcium chloride. After optimization, this protectant demonstrates excellent protection for the strain, achieving a survival rate of 97.22%.

[0125] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Kluyveromyces marxianus strain YF51-1, with accession number GDMCCNO:65780.

2. An inoculum containing the Kluyveromyces marxianus strain YF51-1 as described in claim 1.

3. A freeze-dried powder containing the Kluyveromyces marxianus strain YF51-1 as described in claim 1.

4. The freeze-dried powder according to claim 3, characterized in that, The freeze-dried powder also includes a protectant, which consists of 30-70 g / L of mannose, 50-70 g / L of corn steep liquor powder, 5-15 g / L of lysine and 0.5-1.5 g / L of calcium chloride.

5. The freeze-dried powder according to claim 4, characterized in that, The freeze-dried powder's protective agent consists of 70 g / L mannose, 50 g / L corn steep liquor powder, 5 g / L lysine, and 1.5 g / L calcium chloride.

6. A method for preparing the freeze-dried powder as described in claim 4, characterized in that, The method is as follows: after enriching and culturing the Kluyveromyces marxianus strain YF51-1, the fermentation broth of strain YF51-1 is obtained. Then, the fermentation broth of strain YF51-1 is precipitated with sterile water to prepare a yeast suspension. The protective agent is prepared into a protective solution according to the mass concentration. Then, the yeast suspension and the protective solution are mixed at a volume ratio of 1:1, dispensed, pre-frozen, vacuum freeze-dried, and sealed for storage.

7. The application of Kluyveromyces marxianus strain YF51-1 as described in claim 1 in the preparation of semi-dry fermented whey wine.

8. A method for preparing semi-dry fermented whey wine using Kluyveromyces marxianus strain YF51-1 as described in claim 1, characterized in that, The method includes the following steps: (1) Ingredient preparation: After filtering the cheese whey to remove excess protein and impurities, add white sugar and stir until completely dissolved; (2) Pasteurization; (3) Inoculation and fermentation: Inoculate with Kluyveromyces martensii YF51-1 and keep warm for fermentation; (4) Fining: After fermentation, add activated bentonite and stir evenly, let stand to fin, and make the wine sample clear; (5) Filtration: Use a filter to remove fermentation substrates such as protein and bacteria from the fermentation liquid to obtain a clear fermentation liquid, and bottle it.

9. The method according to claim 8, characterized in that, The mass fraction of white sugar in step (1) is 120 g / L; the fermentation temperature in step (3) is 30 °C.

10. The method according to claim 8, characterized in that, The cheese whey is buffalo cheese whey.

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