Phytobacterium plantarum PCFS-60493 and application thereof in fermentation of low-sugar low-acid beverage

Abstract

The invention provides a plant lactobacillus PCFS-60493 and application of the plant lactobacillus PCFS-60493 in fermentation of low-sugar and low-acid beverages. The plant lactobacillus PCFS-60493 is preserved in China General Microbiological Culture Collection Center on November 25, 2025, and the preservation number is CGMCC No.36673; the strain can be used for preparing the low-sugar low-acid beverage, and the preparation method comprises the following steps: adding a proper amount of white sugar into fruit juice to enable the sugar degree to reach 15-25%, then adding a microbial preparation containing the plant lactobacillus PCFS-60493 into the fruit juice, and carrying out sealed standing fermentation at 37 + / -2 DEG C for 40-50 hours to obtain the low-sugar low-acid beverage. The strain can significantly reduce the content of total sugar and total acid in the beverage, degrade bitter components, and promote protein hydrolysis to generate amino acids to improve delicate flavor; the preparation process is simple, short in fermentation period and safe, realizes resource utilization of grape byproducts, is suitable for large-scale production of the high-polyphenol low-sugar fermented beverage, and has both economic and social benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology and fermented juice beverage processing, and particularly relates to a plant lactobacillus plantarum PCFS-60493 and application thereof in fermenting low-sugar and low-acid beverage. BACKGROUND

[0002] Juice beverage is a beverage prepared by adding water, sugar, pigment and other ingredients into juice liquid obtained by crushing, beating, juicing and filtering fruit as the main raw material. Juice has unique flavor and attractive color of fruit. With consumers preferring natural and healthy diet, juice beverage is favored by consumers. Grape planting industry is widely distributed, and grape varieties and scale have been expanding for many years, and production capacity is sufficient, providing excellent conditions for grape by-product processing. Grape has crisp sweet and juicy taste, high sugar content and rich nutrition, and is a high-quality raw material for beverage production.

[0003] Under the dual driving of global health awareness and increasing burden of metabolic diseases, the low-sugar functional food and beverage market is undergoing structural changes. The global functional beverage market is expected to exceed 2,080 billion US dollars in 2025, with low-sugar beverages accounting for the core increment, forming an industrial pattern of "beverage leading growth, food consolidating foundation". Excessive sugar intake can lead to many health problems such as tooth decay, obesity, diabetes, etc., so consumers are more pursuing "sugar control and reduction" when buying beverages. Low-sugar beverages have met this health demand well, and have rapidly swept the entire beverage market.

[0004] With the wide application of microbial fermentation technology, lactic acid bacteria fermented beverages have become a research hotspot in the beverage industry, such as lactic acid bacteria fruit and vegetable juice, lactic acid bacteria milk tea, lactic acid bacteria plant beverage, etc. Lactic acid bacteria fermented beverages can not only improve the flavor of beverages, but also increase many bioactive ingredients through microbial metabolism, but there are few related technologies of lactic acid bacteria fermented low-sugar and low-acid beverages on the market.

[0005] Therefore, it is of important market value and application prospect to screen lactic acid bacteria strains with the characteristics of reducing sugar content, total acid and optimizing flavor, and to develop corresponding beverage preparation process. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a plant lactobacillus plantarum PCFS-60493 and application thereof in fermenting low-sugar and low-acid beverage. The strain has the characteristics of reducing total acid and total sugar content in juice, reducing bitterness and astringency, and improving umami flavor. At the same time, the fermentation process is simple, the fermentation period is short, the cost is low, the efficiency is high, and it is suitable for the production of low-sugar and low-acid fermented beverage.

[0007] The present application is implemented as follows: A plant lactobacillus PCFS-60493, which is a plant lactobacillus (Lactobacillus plantarum) Lactiplantibacillus plantarum ) PCFS-60493, which was preserved in the China General Microbiological Culture Collection Center on November 18, 2025, at an address of No. 3, Beichen West Road, Chaoyang District, Beijing, with a preservation number of CGMCC No. 36673.

[0008] Further, an application of the plant lactobacillus PCFS-60493, the plant lactobacillus (Lactobacillus plantarum) Lactiplantibacillus plantarum ) PCFS-60493 in fermenting low-sugar and low-acid beverages.

[0009] Further, a method for preparing a low-sugar and low-acid beverage by using the plant lactobacillus (Lactobacillus plantarum) Lactiplantibacillus plantarum ) PCFS-60493, the method steps being as follows: adding an appropriate amount of white sugar to fruit juice to make the sugar degree reach 15-25%, and then adding a microbial preparation containing the plant lactobacillus PCFS-60493 to the fruit juice, and sealing and standing for fermentation at 37±2℃ for 40-50 h to obtain the low-sugar and low-acid beverage.

[0010] Further, the microbial preparation is a fermentation broth of the plant lactobacillus (Lactobacillus plantarum) Lactiplantibacillus plantarum ) PCFS-60493.

[0011] Further, the method steps are as follows: (1) Preparation of fruit juice: selecting fruits free of pests and diseases, with no damage on the surface and uniform color, washing and juicing, and reserving; (2) Dispensing: mixing fruit juice according to a water-material ratio of 1:2, and adding white sugar for dispensing to make the sugar degree reach 20%; (3) Sterilization and cooling: sterilizing the dispensed grape juice at 121±5℃ for 10-15 min, and naturally cooling to 35-42℃; (4) Inoculation and fermentation: inoculating the plant lactobacillus PCFS-60493 fermentation broth at an inoculation amount of 5%, and sealing and standing for fermentation at 37±2℃ for 48 h; (5) Post-treatment: filtering the fermentation broth to remove residues and precipitated particles to obtain the low-sugar and low-acid beverage.

[0012] Further, the fruit juice is grape juice.

[0013] Further, the preparation method of the plant lactobacillus PCFS-60493 fermentation broth is as follows: (1) Strain activation: using an inoculation loop to streak the plant lactobacillus PCFS-60493 on MRS medium, and placing in a constant-temperature incubator for culture at 37±2℃ for 48 h; (2) Preparation of fermentation liquid: The activated Lactobacillus plantarum PCFS-60493 obtained in step (1) was inoculated into MRS medium and cultured statically at 37±2 ℃ for 48 h.

[0014] The present invention has the following advantages: Excellent strain characteristics: Lactobacillus plantarum PCFS-60493 can significantly reduce the total acid content (only 28.86g / L) and total sugar content (only 3.55g / 100g) in beverages, while degrading bitter and astringent components, promoting protein hydrolysis to generate amino acids, effectively reducing the bitterness and astringency of beverages, enhancing umami, and optimizing taste; The process has obvious advantages: the preparation process is simple, no complex equipment is required, the fermentation cycle is short, the fermentation process is safe and controllable, the production cost is low and the efficiency is high, making it suitable for large-scale production. Highly efficient resource utilization: It realizes the resource utilization of grape by-products, extends the grape industry chain, increases the added value of the grape industry, meets the needs of sustainable development of planting industry, and has both economic and social benefits. Attached Figure Description

[0015] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0016] Figure 1 This is a colony morphology diagram of *Lactobacillus plantarum* PCFS-60493 in an embodiment of the present invention. Detailed Implementation

[0017] The following will be combined with the appendix Figure 1 The technical solution of the present invention will be clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products. Example

[0018] I. Obtaining Lactobacillus plantarum PCFS-60493 The present invention is based on Lactobacillus plantarum ( Lactiplantibacillus plantarumPCFS-60493, a lactic acid bacteria isolated from the root of Vitis vinifera L. cv. Kyoho in Fu'an City, Fujian Province, was deposited on November 18, 2025 at the China General Microbiological Culture Collection Center, located at No. 1, Xili Beichen, Beijing, China, with the accession number CGMCC No. 36673. The strain PCFS-60493 can produce β-glucosidase (yielding 131.56 U / g) and tannase (yielding 16.06 mmol / min / mL). The fermentation broth of the strain PCFS-60493 can be used for the fermentation of grape juice, which can reduce the total sugar and total acid content in the grape juice, reduce the bitter and astringent taste, and improve the fresh taste, and can be used for the fermentation of low-sugar and low-acid grape juice. Another lactic acid bacteria (Lactiplantibacillus plantarum Lactiplantibacillus plantarum ) PCFS-49563) was used as a control strain.

[0019] 1. Isolation and screening of strain PCFS-60493 The surface impurities of the grape root were washed with tap water. 5 g of sample was weighed, soaked in 75% alcohol for 30 s, rinsed with sterile water once, soaked in 4% sodium hypochlorite solution for 10 min, rinsed with sterile water for 3 times, and then the water on the surface of the tissue was absorbed with sterile filter paper. The tissue was placed in a sterile mortar, ground, and then 45 mL of sterilized water was added to prepare a sample solution with a concentration of 10 -1 . 100 μL of sample solution with a concentration of 10 -1 and 10 -2 was spread on MRS medium, and incubated at 37°C for 2-3 d, and then single colonies were streaked. The colony morphological characteristics of the strain PCFS-60493 are shown in Figure 1 .

[0020] 2. Identification of strain PCFS-60493 (1) Extraction of DNA: 400 μL STE was added to a 1.5 mL EP tube, and a suitable amount of purified colonies was scraped from the plate with a bacteriological loop, mixed, and centrifuged at 8000 g for 2 min. The supernatant was discarded, and 200 μL TE Buffer and 100 μL Tris-saturated phenol (lower layer) were added to the centrifuge tube, vortexed for 60 s (oscillation with a floating plate), and centrifuged at 4°C, 13000 g for 5 min. 160 μL of the supernatant was transferred to a clean 1.5 mL EP tube, 40 μL TE Buffer was added to the EP tube, and 100 μL chloroform was added. Centrifuge at 4°C, 13000 g for 5 min. 140 μL of the supernatant was transferred to a clean 1.5 mL EP tube, 40 μL TE Buffer was added to the EP tube, and 100 μL chloroform was added. Centrifuge at 4°C, 13000 g for 5 min. 100 μL of the supernatant was transferred to a clean 1.5 mL EP tube, and the supernatant at this time contained purified DNA, which was stored at -20°C.

[0021] (2) PCR amplification PCR reaction system: ddH2O: 9.5 μL, Mix: 12 μL, each 1 μL of upstream and downstream primers, 1.5 μL of DNA template. PCR amplification conditions: 94 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s, 55 °C annealing for 60 s, 72 °C extension for 90 s, a total of 30 cycles, 72 °C extension for 10 min. The PCR product was run on gel, and the band product was sent to Platinum Biotechnology Co., Ltd. for sequencing. The sequencing results were compared by BLAST on the NCBI website. The 16S rDNA sequence of strain PCFS-60493 is shown as SEQ ID NO: 1.

[0022] II. Application of Lactiplantibacillus plantarum PCFS-60493 1. Preparation of fermentation broth of strain PCFS-60493 (1) Strain activation: Use an inoculation loop to streak Lactiplantibacillus plantarum PCFS-60493 on MRS medium, and place it in a constant temperature incubator at 37°C for 48 h; (2) Preparation of fermentation broth: inoculate the PCFS-60493 single colony obtained in step (1) into MRS medium, and incubate at 37°C for 48 h.

[0023] 2. Preparation of low-sugar low-acid beverage (1) Preparation of grape juice: Select grapes that are free of pests and diseases, have no surface damage, and have uniform color as test raw materials. After washing off the surface dirt and dust, transfer them to a cell wall breaking machine for juicing. When there are no large particles in the juicer, remove them and reserve them for use.

[0024] (2) Blending: The sugar content of the filtered grape juice is measured with a sugar meter, mixed according to the ratio of water to material of 1:2, and sugar is added for blending. After thorough mixing, the soluble solids content is 20% (i.e. the sugar content is 20%). The sugar content provides carbon source for the growth and reproduction of the strain, promotes the generation of enzymes (β-glucosidase, tannase) and metabolic products (organic acids, umami substances), and ensures the fermentation efficiency. During sugar metabolism, esters, alcohols and other aroma substances are also generated, enriching the flavor levels of the fruit juice. At the same time, it can regulate the fermentation speed (high or low sugar concentration will affect the activity of the strain), and also slightly improve the thick consistency of the fruit juice and improve the drinking taste.

[0025] (3) Sterilization and cooling: The blended grape juice is subjected to high-temperature sterilization at 121°C for 15 minutes, and then naturally cooled to about 40°C.

[0026] (4) Inoculation: When the grape juice is cooled to about 40°C, the expanded PCFS-60493 fermentation broth is inoculated into the fermentation tank at an inoculation amount of 5%, and incubated at 37°C for 48 hours.

[0027] (5) Centrifugal filtration: The fermented grape beverage is subjected to centrifugal filtration to remove residues and precipitated particles, and then detected.

[0028] 3. Performance testing (1) Total acid content determination: The total acid content is determined according to the method in the national standard "National Food Safety Standard Determination of Total Acids in Food" (GB 12456-2021). The grape fermentation broth sample (50 mL) is directly titrated with 0.1 mol / L NaOH standard solution, and the total acid mass concentration is calculated based on lactic acid.

[0029] (2) Total sugar content: The total sugar content is determined according to the method in the national standard "Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Food" (GB 5009.8-2016).

[0030] (3) Electronic tongue analysis 20 mL of grape fermentation broth is diluted with 80 mL of distilled water to 100 mL, stirred uniformly, and then transferred to a special beaker for electronic tongue analysis. The TS-SA402B Plus-EX electronic tongue system sensor array includes a standard reference electrode and multiple taste sensors, which can determine bitterness, astringency and umami.

[0031] 4. Comparative test A blank control group (grape juice without inoculation of the strain) and an inoculation of the same strain control group (inoculation of Lactobacillus plantarum PCFS-49563) were prepared and detected in the same way as described above, and the results are shown in Table 1 below. Table 1 Evaluation of indicators of fermented beverages

[0032] As can be seen from the above table, the grape juice sample fermented by Lactobacillus plantarum PCFS-60493 has a total acid content of 28.86 g / L, a total sugar content of 3.55 g / 100 g, a bitterness value of 1.05, an astringency value of 2.98, and a freshness value of -2.60, that is, the Lactobacillus plantarum PCFS-60493 of the present application is significantly superior to the blank control group and the control group of the same strain in reducing the total acid content and the total sugar content of the juice beverage and improving the flavor (reducing bitterness and astringency and improving freshness).

[0033] It can be seen that the Lactobacillus plantarum PCFS-60493 of the present application can significantly reduce the total acid and total sugar content of the fermented juice beverage; at the same time, the Lactobacillus plantarum PCFS-60493 can produce β-glucosidase (with a yield of 131.56 U / g) to decompose the bitter terpene glycosides (such as limonin glycoside) in the juice and release non-bitter terpene substances; it can also secrete tannase (with a yield of 16.06 mmol / min / mL) to hydrolyze astringent tannins and proanthocyanidins, reduce their binding force with the oral mucosa, reduce the bitterness and astringency of the juice beverage, and promote the hydrolysis of a part of the protein into amino acids to improve the freshness of the juice beverage, thereby obtaining a low-sugar and low-acid beverage; from the perspective of consumer preferences, bitter and astringent beverages are not easily accepted by consumers in the market, while fresh beverages are not common in the market, and the preparation method can improve the richness of the taste of the beverage and expand the market for the beverage. In addition, the preparation method of the present application has the advantages of simple fermentation process, short fermentation period, low cost, high efficiency, and is suitable for large-scale production. In summary, the present application has strong practicality, can promote the resource utilization of grape by-products, extend the grape industry chain, improve grape production capacity, meet the needs of sustainable development of the planting industry, and have economic and social benefits.

[0034] Although the specific embodiments of the present application are described above, those skilled in the art should understand that the specific examples described are only illustrative and are not intended to limit the scope of the present application, and equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present application should be covered within the scope of the claims of the present application.

Claims

1. A type of *Lactobacillus plantarum* PCFS-60493, characterized in that: The plant lactobacillus is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum PCFS-60493, this strain was deposited on November 18, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36673.

2. An application of *Lactobacillus plantarum* PCFS-60493 as described in claim 1, characterized in that: The plant lactobacillus ( Lactiplantibacillus plantarum Application of PCFS-60493 in fermented low-sugar and low-acid beverages.

3. A method using *Lactobacillus plantarum* as described in claim 1 (… Lactiplantibacillus plantarum The method for preparing low-sugar, low-acid beverages using PCFS-60493 is characterized by: The method steps are as follows: add an appropriate amount of white sugar to the juice to make the sugar content reach 15-25%, then add a microbial preparation containing Lactobacillus plantarum PCFS-60493 to the juice, seal and let it ferment at 37±2℃ for 40-50 hours to obtain a low-sugar and low-acid beverage.

4. The method for preparing a low-sugar, low-acid beverage as described in claim 3, characterized in that: The microbial preparation is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum Fermentation broth of PCFS-60493.

5. The method for preparing a low-sugar, low-acid beverage as described in claim 3, characterized in that: The method steps are as follows: (1) Preparation of juice: Select fruits that are free from pests and diseases, have no surface damage, and have a uniform color. Wash them and juice them for later use. (2) Preparation: Mix the fruit juice with water at a ratio of 1:2, add white sugar to adjust the sugar content to 20%; (3) Sterilization and cooling: Sterilize the prepared grape juice at 121±5℃ for 10-15 minutes, and then let it cool naturally to 35-42℃; (4) Inoculation and fermentation: Inoculate with 5% of the fermentation broth of *Lactobacillus plantarum* PCFS-60493, and let it ferment in a sealed container at 37±2℃ for 48 hours; (5) Post-processing: Filter the fermentation liquid to remove residues and precipitate particles, and you will get a low-sugar and low-acid beverage.

6. The method for preparing a low-sugar, low-acid beverage as described in claim 3 or 5, characterized in that: The juice is grape juice.

7. The method for preparing a low-sugar, low-acid beverage as described in claim 5, characterized in that: The preparation method of the fermentation broth of *Lactobacillus plantarum* PCFS-60493 is as follows: (1) Activation of strain: The strain of *Lactobacillus plantarum* PCFS-60493 was streaked onto MRS medium with an inoculation loop and placed in a constant temperature incubator and cultured at 37±2℃ for 48 h. (2) Preparation of fermentation liquid: The activated Lactobacillus plantarum PCFS-60493 obtained in step (1) was inoculated into MRS medium and cultured statically at 37±2 ℃ for 48 h.

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