Hanseniaspora uvarum sp.MF1 and application thereof in fermentation of mulberry wine

Abstract

The invention discloses hansenula polymorpha strain hanseniaspora sp. MF1 and application of the hanseniaspora polymorpha strain hanseniaspora sp. MF1 in fermentation of mulberry wine, and belongs to the technical field of food biology. According to the invention, a strain of hanseniaspora uvarum sp.MF1 is screened from mulberry fermentation liquor, and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the hanseniaspora uvarum sp.MF1 is CCTCC NO: M 20232181. According to the compound fermentation method, after main fermentation of saccharomyces cerevisiae is completed, the MF1 strain is sequentially inoculated for secondary fermentation, so that aroma components of the mulberry fruit wine can be remarkably optimized, the proportion of alcohols and esters is generally increased, and the flavor of the mulberry fruit wine is improved; damascenone, 4-terpene alcohol, 2-undecylenal and other high-value characteristic aroma substances are more specifically increased, and the content of n-valeric acid, isovaleric acid and other bad flavor acids is reduced, so that the mulberry wine presents unique and harmonious composite flower and fruit aroma.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, and more specifically to a strain of *Hansenula polymorpha* found in grape juice. Hanseniaspora sp. MF1 and its application in fermented mulberry wine. Background Technology

[0002] Mulberry, also known as mulberry fruit, is a fruit of the mulberry plant (Morus alba) in the Moraceae family. Mulberry L. Mulberry is the mature aggregate fruit of the mulberry plant. It is rich in sugars, organic acids, vitamins, minerals, and functional active ingredients such as anthocyanins and resveratrol, possessing high nutritional and health value. Mulberry wine, made from freshly squeezed mulberry juice through fermentation, represents an important direction for expanding mulberry processing methods and increasing product added value.

[0003] Currently, the industrial production of mulberry wine mainly relies on pure brewing yeast ( Saccharomyces yeast Single fermentation is used. While this process is highly efficient in producing alcohol, the resulting products generally suffer from a lack of complex aroma layers and flavor profiles, failing to capture the unique and complex aromas of mulberries. To improve this situation, researchers began exploring mixed fermentation using non-brewing yeasts. Existing technologies indicate that, for example, grape juice contains Hansenula polysaccharide (Hansenula polysaccharide). Hansenia sporatum grapes Kluyveromycin ( Pichia kluyveri Co-fermentation with non-brewing yeasts, such as mixed fermentation with brewing yeast, can increase the content of basic aroma substances such as esters (e.g., ethyl octanoate and ethyl decanoate) and alcohols (e.g., phenylethyl alcohol) in the fermentation liquid, thereby making the aroma of the wine more intense and full-bodied, with floral and fruity notes.

[0004] However, the aroma-enhancing effect of the aforementioned mixed-culture fermentation technology based on common non-brewing yeast still has significant limitations, which constitutes a defect in the existing technology that urgently needs to be improved:

[0005] The "homogenization" and "fundamental" defects of added aroma substances: Existing technologies for aroma enhancement primarily focus on common fatty acid ethyl esters and simple alcohols. These substances are "basic aroma" components commonly produced by many aroma-producing yeasts. While they can increase the "quantity" of aroma, they are insufficient to create a highly distinctive "quality" of aroma. Consequently, the resulting fruit wines easily fall into a situation of flavor homogeneity, lacking a memorable and unique style.

[0006] A key deficiency lies in the lack of "specific generation" of high-value aroma components: Existing common wine strains and processes cannot specifically produce or significantly enhance those high-value aroma compounds that are crucial to the flavor of high-quality fruit wines. These include terpenoids (such as damascene) that contribute complex aromas like rose, fruit, and tobacco; monoterpenols (such as 4-terpenols) that impart woody and spicy notes; and aldehydes (such as 2-undecenal) that provide fresh, diffuse aromas. The absence or low concentration of these substances results in insufficient complexity, elegance, and layering of the wine's aroma, making it difficult to meet the flavor standards of high-quality fruit wines.

[0007] The inadequacy of "directional shaping" of aroma profiles: Current technologies primarily focus on increasing the total amount of aroma, lacking the ability to "directionally shape" and "precisely control" the aroma profile. An ideal fermentation technology should, like blending spices, synergistically promote the generation of beneficial characteristic aromas while suppressing undesirable flavors, thereby creating a harmonious, balanced, and unique overall flavor profile. This is a goal that current conventional mixed-culture fermentation technologies have not yet effectively achieved.

[0008] Therefore, selecting a yeast strain that can not only enhance aroma in general but also specifically metabolize and produce high-value characteristic aroma components, thereby shaping a unique, complex and elegant floral and fruity aroma in a targeted and synergistic way, and combining it with a suitable fermentation process, has become a key technological innovation breakthrough for improving the sensory quality and market competitiveness of mulberry wine and other specialty fruit wines. Summary of the Invention

[0009] In view of this, the present invention provides a strain of *Hansenula polymorpha* from grape juice. Hanseniaspora sp. MF1 and its application in fermented mulberry wine.

[0010] To solve the above-mentioned technical problems, this application adopts the following technical solution: The primary objective of this application is to provide: a grape juice containing Hansenula polymorpha. Hanseniaspora sp. .MF1, the grape juice contains Hansenula polymorpha. Hanseniaspora sp. .MF1 is deposited at the China Center for Type Culture Collection (CCTCC) on November 10, 2023, with accession number CCTCC NO: M 20232181.

[0011] One object of this application is to provide: the grape juice contains Hansenula polymorpha. Hanseniaspora sp. Application of MF1 in the fermentation preparation of mulberry wine.

[0012] One object of this application is to provide: a compound fermentation method for improving the flavor and aroma components of mulberry wine, comprising the following steps: S1. Preparation of mulberry juice: Wash fresh mulberries, extract juice, filter, add sodium bisulfite at 80-100 mg / L, and let stand for 30-40 min; S2. Adjust the juice composition: Add white sugar to the mulberry juice to make its soluble solids content reach 22-23%, adjust the pH to 3.5-4.0, sterilize and set aside; S3. Inoculation with brewer's yeast: Inoculate the activated brewer's yeast into the adjusted mulberry juice at a concentration of 0.1–0.15 g / L and ferment at 25–30°C for 72–96 h; S4. Inoculate grape juice with Hansenula polymorpha. Hanseniaspora sp. MF1: ​​Grape juice with accession number CCTCC NO:M 20232181 contained Hansenula polymorpha. Hanseniaspora sp. After MF1 activation, it is inoculated into the fermentation broth of step S3 at a volume fraction of 10-20%, so that the fermentation broth contains... Hanseniaspora sp. MF1 concentration reached 1×10 6 ~2×10 6 CFU / mL, continue fermentation at 25-28℃ for 20-24 days; S5. Filter and bottle the fermentation liquid to obtain mulberry wine.

[0013] As a preferred technical solution, the filtration in step S1 uses a 200-mesh filter screen.

[0014] As a preferred technical solution, the pH in step S2 is adjusted using lemon juice; the sterilization conditions are maintained at 105℃ for 15-20 min.

[0015] As a preferred technical solution, the activation method of the brewing yeast in step S3 is as follows: add the yeast to warm sugar water with a sugar content of 5% and activate it in a water bath at 35-39°C for 30 min.

[0016] As a preferred technical solution, the grape juice in step S4 contains Hansenula polymorpha yeast. Hanseniaspora sp. The activation method for MF1 is as follows: inoculate it into slant culture medium and incubate it at 25-30℃ for 24-36 h.

[0017] Another object of this application is to provide the application of the above method in increasing the content of alcohols, esters and characteristic aroma substances in mulberry wine.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention obtains a specific aroma-producing yeast, *Hansenula polymorpha* MF1 (accession number: CCTCC NO: M 20232181), from mulberry fermentation broth, and innovatively employs a process of sequentially inoculating this strain after the primary fermentation of brewer's yeast, effectively improving the sensory quality and flavor complexity of mulberry wine.

[0019] Specifically, compared to fermentation with pure brewing yeast (Comparative Example 1), the method of the present invention: Significantly improved sensory quality: The resulting mulberry wine received the highest score (89.5 points) in blind tasting, with rich and harmonious floral and fruity aromas and a superior taste.

[0020] Optimize aroma composition: Increased key beneficial aroma compounds: In particular, significantly increased the content of trans-2-nonen-1-ol (from 0.00% to 12.39%) and phenethyl acetate (from 5.47% to 9.41%), as well as other floral and fruity aroma components, and generated a number of important aroma compounds that were not detected in Comparative Example 1, such as 4-terpene alcohol, damascene, and 2-undecenal.

[0021] Reduces unpleasant acidic substances: Effectively reduces the relative content of acidic substances such as valeric acid, isovaleric acid, and caprylic acid, which may cause unpleasant odors.

[0022] Verification of process uniqueness: Compared with the process of simultaneously inoculating brewer's yeast and MF1 (Comparative Example 2), the sequential inoculation method adopted in this invention can more effectively promote the generation of aroma substances such as alcohols and esters and inhibit the production of excessive acid, proving the technical necessity of this specific inoculation sequence.

[0023] In summary, the strains and compound fermentation process provided by this invention can directionally improve the aroma spectrum of mulberry wine, endowing it with a unique and harmonious floral and fruity flavor, and have a clear effect on enhancing aroma and quality. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1 This invention screened an aroma-producing yeast from the fermentation broth of mulberries: Mulberries were placed in sterile physiological saline and shaken overnight. After serial dilution, 200 μL of the sample was plated on PDA medium for culture. Strains with different colony morphologies were selected for 16S / 18S / ITS amplicon sequencing. The sequences were compared with known bacterial sequences to obtain a strain of *Hansenula polymorpha* from grape juice, named... Hanseniaspora sp. .MF1, the grape juice contains Hansenula polymorpha. Hanseniaspora sp. .MF1 is deposited at the China Center for Type Culture Collection (CCTCC); address: Wuhan University, Wuhan, China; deposit date: November 10, 2023; accession number: CCTCC NO: M 20232181.

[0026] Example 2 A compound fermentation method for improving the flavor and aroma components of mulberry wine includes the following steps: S1. Preparation of mulberry juice: Select fresh and intact mulberries, wash and juice them, filter through a 200-mesh sieve, add sodium bisulfite at 80 mg / L to the mulberry juice, and let it stand for 30 minutes. S2. Adjust the juice composition: Add white sugar to the juice until the soluble solids reach 23%, adjust the pH to 3.5 with lemon juice, dispense into containers, and sterilize at 105℃ for 15 min for later use; S3. Inoculation with brewer's yeast: Dissolve 10g of brewer's yeast (ATCC9763, purchased from Nanchang Chenfeng Technology Co., Ltd., Shanghai Preservation Biotechnology Center) in 200ml of 5% warm sugar water, stir evenly, and incubate at 38℃ for 30min. Inoculate the activated brewer's yeast into the prepared fruit juice at an inoculation rate of 0.1g / L, and ferment at 28℃ for 72h. S4. Inoculate grape juice with Hansenula polymorpha: Inoculate grape juice with Hansenula polymorpha... Hanseniaspora sp. MF1 was inoculated onto prepared YPD agar slant medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 18 g / L agar, pH natural or adjusted to 6.0), and cultured at 28℃ for 24 h. Cell counts were then performed using a hemocytometer. Activated *Hansenula polymorpha* spores was inoculated into the fermentation broth at a volume fraction of 12% (1.2 × 10⁻⁶). 6 CFU / mL, v / v), fermented at 26℃ for 22 days; S5. Filtration and filling: The fermentation broth is filtered through diatomaceous earth and then filled.

[0027] Example 3 A compound fermentation method for improving the flavor and aroma components of mulberry wine includes the following steps: S1. Preparation of mulberry juice: Select fresh and intact mulberries, wash and juice them, filter through a 200-mesh sieve, add sodium bisulfite at 80 mg / L to the mulberry juice, and let it stand for 30 minutes. S2. Adjust the juice composition: Add white sugar to the juice until the soluble solids reach 22%, adjust the pH to 3.8 with lemon juice, dispense into containers, and sterilize at 105℃ for 15 min for later use; S3. Inoculation with brewer's yeast: Dissolve 10g of brewer's yeast in 200ml of 5% warm sugar water and stir evenly. Incubate at 35℃ for 30 minutes. Inoculate the activated brewer's yeast into the prepared fruit juice at an inoculation rate of 0.12g / L and ferment at 25℃ for 84 hours. S4. Inoculate grape juice with Hansenula polymorpha: Inoculate grape juice with Hansenula polymorpha... Hanseniaspora sp. MF1 was inoculated onto the prepared slant culture medium and cultured at 25°C for 30 h, followed by hemocytometer counting. Activated *Hansenula polymorpha* spores were inoculated into the fermentation broth at a volume fraction of 18% (1.8 × 10⁻⁶). 6 CFU / mL, v / v), fermented at 25℃ for 20 days; S5. Filtration and filling: The fermentation broth is filtered through diatomaceous earth and then filled.

[0028] Example 4 A compound fermentation method for improving the flavor and aroma components of mulberry wine includes the following steps: S1. Preparation of mulberry juice: Select fresh and intact mulberries, wash and juice them, filter through a 200-mesh sieve, add sodium bisulfite at 80 mg / L to the mulberry juice, and let it stand for 30 minutes. S2. Adjust the juice composition: Add white sugar to the juice until the soluble solids reach 22%, adjust the pH to 4.0 with lemon juice, dispense into containers, and sterilize at 105℃ for 20 min for later use; S3. Inoculation with brewer's yeast: Dissolve 10g of brewer's yeast in 200ml of 5% warm sugar water and stir evenly. Incubate at 39℃ for 30 minutes. Inoculate the activated brewer's yeast into the prepared fruit juice at an inoculation rate of 0.15 g / L and ferment at 30℃ for 96 hours. S4. Inoculate grape juice with Hansenula polymorpha: Inoculate grape juice with Hansenula polymorpha... Hanseniaspora sp. MF1 was inoculated onto the prepared slant culture medium and cultured at 30℃ for 36 h, followed by hemocytometer counting. Activated *Hansenula polymorpha* from grape juice was inoculated into the fermentation broth at a volume fraction of 20% (2.0 × 10⁻⁶). 6 CFU / mL, v / v), fermented at 28℃ for 24 days; S5. Filtration and filling: The fermentation broth is filtered through diatomaceous earth and then filled.

[0029] Comparative Example 1 Similar to Example 1, except that the grape juice was not inoculated with Hansenula polysaccharide, that is, fermented for 25 days after inoculation with Saccharomyces cerevisiae.

[0030] Comparative Example 2 Similar to Example 1, except that grape juice was inoculated with Hansenula polysaccharide at the same time as the brewer's yeast, and fermentation lasted for 25 days.

[0031] To verify the sensory properties of the mulberry wines prepared in the different embodiments and comparative examples described above, the following experiments were conducted: Sensory evaluations were conducted on different mulberry wines. Following the "General Analytical Methods for Wines and Fruit Wines" (GB / T 15038-2006), a tasting panel of eight members comprehensively evaluated the color, taste, and aroma of three mulberry wines. A maximum score of 100 points was assigned, and the average score was used as the final evaluation result.

[0032] The results are as follows: There was no significant difference in color among the five mulberry wines. The mulberry wine of Example 2 had a stronger floral and fruity aroma and a more harmonious fragrance, scoring 89.5 points. This was followed by Examples 3 and 4. The sensory scores of Comparative Examples 1 and 2 were lower than those of the Examples.

[0033] Table 1 Evaluation of the taste of different mulberry wines

[0034] To further verify the flavor components of the mulberry wines prepared in the different embodiments and comparative examples described above, the following experiments were conducted: Volatile components in mulberry wine were determined using headspace solid-phase microextraction-gas chromatography (GC-MS). The instrument used was an Agilent 8890-5977C GC-MS. 5 mL of mulberry wine was pipetted into a 20 mL headspace vial, and the DVB / CAR / PDMS extraction head was inserted into the vial. Headspace extraction was performed at 50 °C for 30 min. The fiber head was then withdrawn, the extraction head was removed from the headspace vial, and the extraction head was quickly inserted into the GC-MS vaporization chamber. Desorption was performed at 280 °C for 3 min, while the instrument was simultaneously started to acquire data. Unknown substances were identified using the GC-MS built-in data processing system. Based on NIST mass spectrometry library comparison, results with a matching factor greater than 85 were selected. Quantitative analysis was performed using the area normalization method, and the relative percentage content of each component was calculated. The experimental results are shown in Table 1.

[0035] Table 2 Comparison of volatile components in different mulberry wines

[0036] Results Analysis: As shown in Table 2, compared with Comparative Example 1, the mulberry wines of Examples 2, 3, and 4 had relatively lower contents of valeric acid, isovaleric acid, and octanoic acid, while having relatively higher contents of phenethyl alcohol, trans-2-nonen-1-ol, ethyl acetate, phenethyl isobutyrate, phenethyl acetate, benzaldehyde, and 2-undecenal. Furthermore, substances not present in Comparative Example 1, such as 4-terpene alcohol, ethyl decanoate, dibutyl oxalate, ethyl hexanoate, acetophenone, damascene, and 2-undecenal, were also present. Compared with Comparative Example 2, the examples showed higher relative contents of alcohols and esters, and lower relative contents of acids. Among all experimental groups, Example 2 had the highest relative contents of trans-2-nonen-1-ol, phenethyl isobutyrate, phenethyl acetate, benzaldehyde, and 2-undecenal.

[0037] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0038] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A grape vine's juice contains Hansenula polymorpha yeast. Hanseniaspora sp. MF1, characterized in that, The grape juice contained Hansenula polymorpha. Hanseniaspora sp. MF1 is deposited at the China Center for Type Culture Collection (CCTCC) on November 10, 2023, with accession number CCTCC NO: M 20232181.

2. The grape juice containing Hansenula polymorpha according to claim 1 Hanseniaspora sp Application of MF1 in the fermentation preparation of mulberry wine.

3. A compound fermentation method for improving the flavor and aroma components of mulberry wine, characterized in that, Includes the following steps: S1. Preparation of mulberry juice: Wash fresh mulberries, extract juice, filter, add sodium bisulfite at 80-100 mg / L, and let stand for 30-40 min; S2. Adjust the juice composition: Add white sugar to the mulberry juice to make its soluble solids content reach 22-23%, adjust the pH to 3.5-4.0, sterilize and set aside; S3. Inoculation with brewer's yeast: Inoculate the activated brewer's yeast into the adjusted mulberry juice at a concentration of 0.1–0.15 g / L and ferment at 25–30°C for 72–96 h; S4. Inoculate grape juice with Hansenula polymorpha. Hanseniaspora sp MF1: ​​Grape juice containing Hansenula polymorpha with preservation number CCTCC NO: M20232181 was found to contain Hansenula polymorpha. Hanseniaspora sp After MF1 activation, it is inoculated into the fermentation broth of step S3 at a volume fraction of 10-20%, so that the fermentation broth contains... Hanseniaspora sp MF1 concentration reached 1×10 6 ~2×10 6 CFU / mL, continue fermentation at 25-28℃ for 20-24 days; S5. Filter and bottle the fermentation liquid to obtain mulberry wine.

4. The compound fermentation method according to claim 1, characterized in that, The filtration in step S1 uses a 200-mesh filter sieve.

5. The compound fermentation method according to claim 1, characterized in that, In step S2, the pH is adjusted using lemon juice; the sterilization conditions are 105°C for 15–20 min.

6. The compound fermentation method according to claim 1, characterized in that, The activation method of brewing yeast in step S3 is as follows: add yeast to warm sugar water with a sugar content of 5% and activate it in a water bath at 35-39℃ for 30 min.

7. The compound fermentation method according to claim 1, characterized in that, The grape juice mentioned in step S4 contains Hansenula polymorpha. Hanseniaspora sp The activation method for MF1 is as follows: inoculate it into slant culture medium and incubate it at 25-30℃ for 24-36 h.

8. The application of the method according to any one of claims 3 to 7 in increasing the content of alcohols, esters and characteristic aroma substances in mulberry wine.

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