Application of lactobacillus paracasei in reducing quinic acid content of fruit wine and preparation method of fruit wine

Abstract

The invention provides application of lactobacillus paracasei in reducing quinic acid content of fruit wine and a preparation method of the fruit wine, and relates to the technical field of fermentation. The research of the inventor finds that the lactobacillus paracasei with the preservation number of CGMCCNO: 1.9089 has the effect of remarkably reducing the quinic acid content in the blueberry juice; the lactobacillus paracasei is added for co-fermentation in the process of preparing the fruit wine through fermentation of the saccharomyces cerevisiae, so that the quinic acid content in the fruit wine is remarkably reduced, the flavor of the fruit wine is improved, the taste is improved, and the sensory comfort is improved.

Description

Technical Field

[0001] This invention relates to the field of fermentation technology, and in particular to the application of Lactobacillus paracasei in reducing the quinic acid content of fruit wine and a method for preparing fruit wine. Background Technology

[0002] Blueberry wine is a processed product made primarily from blueberries through yeast fermentation. It not only retains the maximum amount of nutrients from blueberries but also produces a large number of bioactive substances through fermentation. Rich in phenolic compounds, alcohols, esters, proanthocyanidins, and other functional substances, it is hailed as "liquid gold" and "oral cosmetics for humans." The wine has a ruby ​​red color and is clear and free of cloudiness. Its aroma combines the natural fruitiness of blueberries with a rich, mellow fragrance derived from fermentation, resulting in a uniquely smooth and palatable taste that appeals to consumers.

[0003] Lactobacillus is a group of beneficial bacteria widely found in humans and nature. It produces lactic acid through the fermentation of sugars and is an anaerobic or facultative anaerobic bacterium. It promotes fermentation, inhibits the growth of harmful bacteria, and regulates fermentation flavor, and is commonly found in fermented foods. Lactobacillus is a species of microorganism other than yeast present during the brewing process. Different lactobacilli can impart unique characteristics to fruit wines that cannot be produced by brewing yeast; *Lactobacillus paracasei* is one such example. However, lactobacilli primarily perform lactic acid fermentation and have relatively weak fermentation power in alcoholic fermentation. In blueberry wine brewing, they cannot complete fermentation independently and need to be co-fermented with brewing yeast to achieve complete fermentation. This not only increases the difficulty of process control but may also affect the yeast's ability to produce and metabolize aroma and flavor compounds.

[0004] Organic acids are crucial substances affecting the flavor and free radical scavenging ability of blueberry wine, playing a key role in its sensory characteristics and antioxidant capacity. Organic acids derived from the fruit raw materials are the main organic acids in blueberry wine. Different raw materials, different types and compositions of organic acids are the main reasons for the significant differences in taste between various blueberry wines. When the organic acids in blueberry wine are in a reasonable balance of sweetness and acidity, the wine can be refreshing and stimulating. Quinic acid is a unique alicyclic organic acid found in high amounts in higher plants such as cinchona bark, coffee seeds, blueberries, and apples. It is present as a major organic acid in blueberries from Guizhou. Excessive quinic acid content in fruit wine can lead to unpleasant taste, reduced sensory comfort, and gastrointestinal discomfort. Regulating the prominent quinic acid content in blueberries is of great significance for improving the flavor of blueberry wine.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide the application of Lactobacillus paracasei in reducing the quinic acid content of fruit wine, thereby solving the aforementioned technical problems.

[0007] The second objective of this invention is to provide a method for preparing fruit wine.

[0008] To achieve the above objectives, the following technical solution is adopted:

[0009] In one aspect, the present invention provides the application of Lactobacillus paracasei in reducing the quinic acid content of fruit wine. Lactobacillus paracasei is added during the fermentation process of fruit wine for mixed fermentation, thereby reducing the quinic acid content in the resulting fruit wine.

[0010] The Lactobacillus paracasei is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No:1.9089.

[0011] It should be noted that the Lactobacillus paracasei in this invention is an existing strain, see CN202210310973.X, which was purchased by the inventor.

[0012] As a further technical solution, the fruit wine fermentation is carried out using brewing yeast.

[0013] As a further technical solution, the brewing yeast is red fruit yeast.

[0014] As a further technical solution, the fruit wine includes blueberry wine.

[0015] Secondly, the present invention provides a method for preparing fruit wine, comprising the following steps:

[0016] Inoculate the fruit juice with brewer's yeast for fermentation. After fermentation for 6-8 days, inoculate with Lactobacillus paracasei and continue fermentation to prepare fruit wine.

[0017] The Lactobacillus paracasei is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No:1.9089.

[0018] As a further technical solution, the brewing yeast is red fruit yeast;

[0019] The inoculation amount of the brewing yeast is 0.2‰-0.3‰ of the juice mass.

[0020] As a further technical solution, the inoculation volume of the *Lactobacillus paracasei* bacterial solution is 5%-7% of the fruit juice volume, and the viable bacteria concentration in the *Lactobacillus paracasei* bacterial solution is 2×10⁻⁶. 7 -6×10 7 cfu / mL.

[0021] As a further technical solution, the fermentation temperature is 24.5-26℃;

[0022] The total fermentation time is 15-17 days.

[0023] As a further technical solution, the method for preparing the fruit juice includes: sequentially processing the fruit through pulping, enzymatic hydrolysis, solid-liquid separation, concentration, and sterilization to obtain the fruit juice;

[0024] Preferably, the enzymatic hydrolysis involves treating the fruit pulp with pectinase and cellulase.

[0025] Preferably, the initial residual sugar content of the fruit juice is 236-253 g / L.

[0026] As a further technical solution, the fruit wine includes blueberry wine.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The inventors discovered that Lactobacillus paracasei with accession number CGMCC No:1.9089 has a significant effect on reducing the quinic acid content in blueberry juice. Furthermore, when Lactobacillus paracasei is added to the process of preparing fruit wine by fermenting with Saccharomyces cerevisiae for co-fermentation, the quinic acid content in the fruit wine is significantly reduced, while the flavor of the fruit wine is enhanced, the taste is improved, and the sensory comfort is increased. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 Examples and comparative examples show the quinic acid content in the raw materials. Detailed Implementation

[0031] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0032] In one aspect, the present invention provides the application of Lactobacillus paracasei in reducing the quinic acid content of fruit wine. Lactobacillus paracasei is added during the fermentation process of fruit wine for mixed fermentation, thereby reducing the quinic acid content in the resulting fruit wine.

[0033] The Lactobacillus paracasei is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No:1.9089.

[0034] It should be noted that the Lactobacillus paracasei in this invention is an existing strain, see CN202210310973.X, which was purchased by the inventor.

[0035] The inventors discovered that the aforementioned *Lactobacillus paracasei* significantly reduces the quinic acid content in blueberry juice; furthermore, adding *Lactobacillus paracasei* for co-fermentation during the fermentation of fruit wine with *Saccharomyces cerevisiae* significantly reduces the quinic acid content in the fruit wine.

[0036] In some alternative embodiments, the fruit wine fermentation is carried out using brewer's yeast.

[0037] In some alternative embodiments, the brewing yeast is red fruit yeast, such as Enartis red fruit yeast.

[0038] In some alternative implementations, the fruit wine includes, but is not limited to, blueberry wine, or other fruit wines well known to those skilled in the art.

[0039] Secondly, the present invention provides a method for preparing fruit wine, comprising the following steps:

[0040] Inoculate the fruit juice with brewer's yeast for fermentation. After fermentation for 6-8 days, inoculate with Lactobacillus paracasei and continue fermentation to prepare fruit wine.

[0041] The Lactobacillus paracasei is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No:1.9089.

[0042] This preparation method is simple and can significantly reduce the quinic acid content in fruit wine while improving its flavor, taste, and sensory comfort.

[0043] In some alternative embodiments, the brewing yeast is red fruit yeast, such as Enartis-China red fruit yeast.

[0044] The inoculation amount of the brewing yeast is 0.2‰-0.3‰ of the juice mass.

[0045] In some optional embodiments, the inoculation volume of the *Lactobacillus paracasei* culture is 5%-7% of the fruit juice volume, and the viable cell concentration in the *Lactobacillus paracasei* culture is 2 × 10⁻⁶. 7 cfu / mL, 5×10 7 cfu / mL or 6×10 7 cfu / mL, OD of Lactobacillus paracasei culture 600 The value is 0.8-1.0.

[0046] In some alternative embodiments, the fermentation temperature may be, for example, but not limited to, 24.5°C, 25°C, or 26°C;

[0047] The total fermentation time can be, for example, but is not limited to, 15 days, 16 days, or 17 days.

[0048] In some optional embodiments, the method for preparing the fruit juice includes: sequentially processing the fruit through pulping, enzymatic hydrolysis, solid-liquid separation, concentration, and sterilization to obtain the fruit juice.

[0049] The enzymatic hydrolysis includes: mixing pectinase and cellulase in a 1:1 weight ratio to obtain a compound enzyme; adding the compound enzyme to the pulped fruit pulp at a concentration of 250-300 ppm; and cyclically hydrolyzing for 0.5-2 hours or more until the pectin test is negative; preferably, the enzymatic hydrolysis temperature is 50-55℃.

[0050] The solid-liquid separation adopts a three-stage clarification process, including: pre-decanter separation, post-decanter separation, and disc separator; preferably, the rotation speed of the pre-decanter separation is 2500 rpm / min; the rotation speed of the post-decanter separation is 3200 rpm / min; the pressure of the disc separator is ≤0.5MPa; the flow rate of the disc separator is ≤5t / h; and the rotation speed of the disc separator is 6000-8000 rpm / min.

[0051] Concentration is achieved through evaporation concentration, which includes: subjecting the blueberry juice, which has undergone three-stage clarification, to triple-effect low-temperature concentration; preferably, the temperature of the first-effect concentration is 70-85℃; preferably, the temperature of the second-effect concentration is 50-75℃; preferably, the temperature of the third-effect concentration is 25-55℃.

[0052] In some alternative embodiments, the fruit is further subjected to a hot soaking treatment before pulping, the hot soaking treatment lasting for 30 minutes at a temperature of 51-56°C.

[0053] In some alternative embodiments, the initial residual sugar content of the juice is 236-253 g / L.

[0054] In some alternative embodiments, the fermentation is terminated with a sugar content of 12-18 g / L and an alcohol content of 12-14% vol.

[0055] In some alternative implementations, the process of terminating fermentation includes the following steps:

[0056] (1) Add potassium sorbate to stop fermentation; (2) Cross-flow filtration; (3) Mixing; (4) Freezing and gelling; (5) Cross-flow filtration again; (6) Terminal filtration.

[0057] In some alternative implementations, the fruit wine includes, but is not limited to, blueberry wine, or other fruit wines well known to those skilled in the art.

[0058] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0059] The various measurement methods used in the following examples and comparative examples are as follows:

[0060] Method for determining the total sugar content of blueberry wine: Performed in accordance with GB / T 15038.

[0061] Determination of Quinic Acid Content in Blueberry Wine: Following GB / T 32783-2016, ultra-high performance liquid chromatography (UHPLC) was used to determine the quinic acid content in optimized blueberry fermented wine. The blueberry wine was diluted 5 times, centrifuged (12000 r / min, 10 min), filtered through a 0.22 μm organic filter membrane, and then loaded for analysis. The chromatographic system was an Agilent 1290 UHPLC system, with a Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 50 μm), a UV spectrophotometer (UV), a detection wavelength of 210 nm, an injection volume of 2 μL, a mobile phase of 0.12% phosphoric acid aqueous solution-2% methanol, isocratic elution, a flow rate of 0.8 mL / min, and a column temperature of 20 °C.

[0062] The method for determining the alcohol content in the fermentation broth shall be in accordance with GB / T 15038.

[0063] The preservation information of the microbial strains used in the following examples is as follows:

[0064] Lactobacillus paracasei, preservation number CGMCCNo:1.9089. Saccharomyces cerevisiae: Product name: Active Yeast (Indice Yeast Red Fruit Flavor); Batch number: 248232; Country of origin: Italy; Manufacturer: Aisaike Co., Ltd.; Distributor: Beijing Borui Zhiheng Technology Development Co., Ltd.

[0065] Example 1: Blueberry wine produced by sequential co-fermentation of *Saccharomyces cerevisiae* and *Lactobacillus paracasei*.

[0066] I. Preparation of blueberry raw materials before fermentation:

[0067] 1.1 Raw material pretreatment: Select fresh, disease-free, odorless, and pollution-free blueberries from the mountainous areas of southeastern Guizhou. After washing, freeze them at 20℃ to -16℃ and store them as reserve raw materials.

[0068] 1.2 Hot extraction and pulping: Before hot extraction, thaw the frozen fruit stored in step 1.1, hot extract for 30 minutes at a temperature of 55°C, and then pulp the hot-extracted blueberries to obtain blueberry pulp.

[0069] 1.3 Enzymatic hydrolysis: Pectinase and cellulase are mixed in a 1:1 ratio to obtain a compound enzyme, and the compound enzyme is added to the blueberry pulp obtained in step 1.2 at a concentration of 300 ppm. The temperature is controlled at 55℃, and the enzymatic hydrolysis is carried out for more than 4 hours until the pectin test is negative.

[0070] 1.4 Three-stage clarification (pre-decanalization, post-decanalization, and disc separation): First, the slurry after enzymatic hydrolysis in step 1.3 is subjected to pre-decanalization at a rotation speed of 2500 rpm / min; then, post-decanalization is performed at a rotation speed of 3200 rpm / min; finally, disc separation is performed at a pressure ≤0.5 MPa, a flow rate ≤5 t / h, and a rotation speed of 6000-8000 rpm / min.

[0071] 1.5 Concentration and Evaporation: The clarified blueberry juice obtained in step 1.4 is concentrated at a triple-effect low temperature. The first effect concentration temperature is 75°C, the second effect concentration temperature is 65°C, and the third effect concentration temperature is 50°C. The sugar content of the final concentrated blueberry juice is controlled to be 60°Bx, and it is aseptically filled into aseptic bags.

[0072] 1.6. Sterilization: Dilute the concentrated blueberry juice obtained in step 1.5 and add a small amount of decolorized and deacidified concentrated apple juice to control the initial residual sugar to 236-253 g / L. Then sterilize at 125℃ for 8 seconds to obtain sterilized blueberry juice.

[0073] II. Preparation of Seed Liquid

[0074] Activate the red fruit aroma brewing yeast (Saccharomyces cerevisiae) in the following proportion (activate the active dry yeast with 10 times its weight of chlorine-free softened water at 35-40℃, stirring to avoid clumping);

[0075] The culture temperature for *Lactobacillus paracasei* was 37°C. *Lactobacillus paracasei* was inoculated into MRS liquid medium for scale-up culture until the bacterial concentration reached an OD value. 600 The value was 0.8, resulting in a Lactobacillus paracasei seed culture (viable count controlled at 5 × 10⁸). 7 (cfu / mL).

[0076] III. Blueberry Wine Fermentation

[0077] 3.1 Fermentation: After activating the red fruit yeast powder from step two at a mass ratio of 0.2-0.3‰, add it to the blueberry juice obtained in step one and brew blueberry wine in a 2.5L system. Control the fermentation temperature and ferment at a low temperature of 24.5-26℃ for 7 days. On the 7th day of fermentation, add the activated Lactobacillus paracasei from step two at a volume ratio of 5-7% to the fermentation liquid on the 7th day. Then continue fermentation at 24.5℃ for 8-10 days to obtain the fermented base wine.

[0078] 3.2 Clarification: Add bentonite as a clarifying agent to the fermented wine obtained in step 3.1, clarify at a low temperature of 5°C for 24-48 hours, filter, and obtain blueberry fermented wine.

[0079] Comparative Example 1

[0080] The difference from Example 1 is that Lactobacillus paracasei was not added during the fermentation process.

[0081] Test case

[0082] (1) Results of changes in residual sugar content and alcohol content in each fermentation broth

[0083] During the fermentation preparation of blueberry wine using the methods described in Example 1 and Comparative Example 1, specifically on days 0, 7, and 15 of fermentation, the residual sugar content and alcohol content in the fermentation broth were monitored, and the results are shown in Table 1 below:

[0084] Table 1. Results of changes in residual sugar content and alcohol content in each fermentation broth.

[0085]

[0086] According to the results presented in Table 1 above, after 15 days of fermentation, the residual sugar in the fermentation broth of *Saccharomyces cerevisiae* and *Lactobacillus paracasei* was only 17.21±0.29 g / L, which meets the residual sugar requirements for semi-dry fruit wine; and the alcohol content in the fermentation broth reached 12.50±0.23% vol, which is higher than that of commercial strains of *Saccharomyces cerevisiae* fermented alone.

[0087] (2) Results of quinic acid degradation in each group during blueberry wine fermentation

[0088] The quinic acid content in the blueberry wine prepared in Example 1 and Comparative Example 1, as well as in the blending raw juice, was determined. The results are shown in Table 2 and... Figure 1 As shown:

[0089] Table 2. Results of organic acid content in blueberry raw materials and finished blueberry wines obtained from each group of fermentation.

[0090]

[0091] According to the results presented in Table 2, the blueberry wine raw material (the blended blueberry juice) has a high quinic acid content, resulting in a prominent tart flavor in the blueberry wine. Therefore, there is an urgent need to develop methods to reduce quinic acid. Further analysis of the results in Table 2 revealed that after fermentation, the quinic acid content in the co-fermentation group of *Lactobacillus paracasei* and *Lactobacillus rubrum* was 7558.3 ​​mg / L, which was 22.1% lower than that in the blueberry wine raw material. This indicates that differences exist in the metabolic pathways of the microbial strains due to different fermentation methods.

[0092] (3) Sensory evaluation

[0093] Sensory evaluation of the blueberry wines prepared in the examples and comparative examples included the following steps:

[0094] Using a weighted analysis method, a sensory evaluation system for fermented blueberry wine was established, focusing on its appearance, taste, aroma, and aftertaste. The evaluation criteria are shown in Table 3. The sensory evaluation was conducted by a panel of 20 trained members (10 women and 10 men, aged 20 to 30 years). The sensory evaluation result is the average score obtained from the 20 participants' sensory evaluations, as shown in Table 4.

[0095] Table 3 Sensory Evaluation Standards for Fermented Blueberry Wine

[0096]

[0097] Table 4 Sensory ratings for examples and comparative examples

[0098]

[0099] Based on the scoring results, it can be concluded that Example 1 has a higher sensory score, indicating that the blueberry wine obtained by this fermentation method and the addition of microorganisms has a better sensory comfort.

[0100] In summary, adding Lactobacillus paracasei to alter the fermentation process can reduce the quinic acid content in blueberry juice raw materials. Applying it to the brewing process of blueberry wine can also reduce the difficulty and cost of blueberry wine preparation, improve the sensory comfort of blueberry wine, alleviate problems such as sourness and astringency, and enhance the application value of rabbiteye blueberry raw materials.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of *Lactobacillus paracasei* in reducing the quinic acid content of fruit wine, characterized in that... Adding Lactobacillus paracasei during the fruit wine fermentation process for mixed fermentation reduces the quinic acid content in the resulting fruit wine. The Lactobacillus paracasei is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:1.9089.

2. The application according to claim 1, characterized in that, The fruit wine is fermented using brewer's yeast.

3. The application according to claim 2, characterized in that, The brewing yeast is *Saccharomyces cerevisiae*.

4. The application according to claim 1, characterized in that, The fruit wines include blueberry wine.

5. A method for preparing fruit wine, characterized in that, Includes the following steps: Inoculate the fruit juice with brewer's yeast for fermentation. After fermentation for 6-8 days, inoculate with Lactobacillus paracasei and continue fermentation to prepare fruit wine. The Lactobacillus paracasei is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No:1.9089.

6. The preparation method according to claim 5, characterized in that, The brewing yeast is red fruit yeast; The inoculation amount of the brewing yeast is 0.2‰-0.3‰ of the juice mass.

7. The preparation method according to claim 5, characterized in that, The inoculation volume of the *Lactobacillus paracasei* culture was 5%-7% of the fruit juice volume, and the viable cell concentration in the *Lactobacillus paracasei* culture was 2×10⁻⁶. 7 -6×10 7 cfu / mL.

8. The preparation method according to claim 5, characterized in that, The fermentation temperature is 24.5-26℃; The total fermentation time is 15-17 days.

9. The preparation method according to claim 5, characterized in that, The method for preparing the fruit juice includes: sequentially processing the fruit through pulping, enzymatic hydrolysis, solid-liquid separation, concentration, and sterilization to obtain the fruit juice; Preferably, the enzymatic hydrolysis involves treating the fruit pulp with pectinase and cellulase. Preferably, the initial residual sugar content of the fruit juice is 236-253 g / L.

10. The preparation method according to claim 5, characterized in that, The fruit wines include blueberry wine.

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