Phytobacterium atratus PCFS-60502 and application thereof in fermentation of high-polyphenol low-sugar beverage

Abstract

The invention provides an Artegulat lactobacillus plantarum PCFS-60502 and application thereof in fermentation of high-polyphenol low-sugar beverage, the strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 18, 2025, and the preservation number is CGMCC No.36674. The invention further provides a preparation method of the Artegulat lactobacillus plantarum PCFS-60502 and application of the Artegulat lactobacillus plantarum PCFS-60502. The strain can be used for preparing the high-polyphenol low-sugar 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 Aradicatrata lactobacillus PCFS-60502 into the fruit juice, and carrying out sealed standing fermentation at 37 + / -2 DEG C for 40-50 hours to obtain the high-polyphenol low-sugar beverage. The strain can significantly improve the total phenol content in the beverage, reduce the total sugar content, degrade bitter components, and promote protein hydrolysis to generate amino acids so as to improve the 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] This invention relates to the field of microbial technology and fruit juice fermentation processing, specifically to a strain of Lactobacillus argyrate PCFS-60502 and its application in fermented high-polyphenol, low-sugar beverages. Background Technology

[0002] Fruit juice beverages are made primarily from fruits, processed through crushing, pulping, juicing, and filtering to obtain fruit juice, which is then blended with water, sugar, coloring, and other ingredients. Fruit juice possesses the unique flavor and appealing color of the fruit. As consumers increasingly favor natural and healthy diets, fruit juice beverages are gaining popularity. The grape cultivation industry is widespread, and after years of development, grape varieties and scale have continuously expanded, resulting in ample production capacity and providing excellent conditions for processing grape by-products. Grapes are crisp, sweet, juicy, high in sugar, and rich in nutrients, making them a high-quality raw material for beverage production.

[0003] As consumers pay increasing attention to health, the market for high-sugar beverages, fruit juices, and tea drinks is facing decline. The beverage industry is undergoing significant changes and is showing a trend of diversified development. Types of beverages such as those with reduced sugar content, nutritional value, health benefits, and environmental friendliness have emerged. Consumers' growing focus on the flavor and health benefits of beverages is also placing higher demands on manufacturers.

[0004] In the era of health and wellness, plant-based fermented products have become popular worldwide. With the widespread 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 juices, lactic acid bacteria milk tea, and lactic acid bacteria plant-based beverages. Lactic acid bacteria fermented tea beverages can not only improve the flavor of drinks but also increase many bioactive components through microbial metabolism. However, there are relatively few technologies on the market related to lactic acid bacteria fermented high-polyphenol, low-sugar beverages.

[0005] Therefore, screening lactic acid bacteria strains that can efficiently increase polyphenol content, reduce sugar content, and optimize flavor, and developing corresponding beverage preparation processes, has significant market value and application prospects. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus argyrate PCFS-60502 and its application in fermented high-polyphenol and low-sugar beverages. This strain has the characteristics of increasing the polyphenol content in fruit juice, reducing the total sugar content, reducing bitterness and enhancing umami. At the same time, its fermentation process is simple, with a short fermentation cycle, low cost and high efficiency, and is suitable for the production of high-polyphenol and low-sugar fermented beverages.

[0007] This invention is implemented as follows: A type of Lactobacillus argyrutilis PCFS-60502, wherein the Lactobacillus argyrutilis is Lactobacillus argyrutilis ( Lactiplantibacillus argentoratensisPCFS-60502, this strain was deposited on November 18, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36674.

[0008] Furthermore, the application of the aforementioned *Lactobacillus argyrutilatus* PCFS-60502, wherein the *Lactobacillus argyrutilatus* ( Lactiplantibacillus argentoratensis Application of PCFS-60502 in fermented high-polyphenol low-sugar beverages.

[0009] Furthermore, a method for preparing a high-polyphenol, low-sugar beverage using the aforementioned *Lactobacillus argyrophylla* PCFS-60502 is provided, comprising the following steps: adding an appropriate amount of white sugar to the fruit juice to achieve a sugar content of 15-25%, then adding a microbial preparation containing *Lactobacillus argyrophylla* PCFS-60502 to the fruit juice, and sealing and allowing it to ferment at 37±2℃ for 40-50 hours to obtain the high-polyphenol, low-sugar beverage.

[0010] Furthermore, the microbial preparation is *Lactobacillus argentulatus* (…). Lactiplantibacillus from Argentorate Fermentation broth of PCFS-60502.

[0011] Furthermore, 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 argyrate PCFS-60502, seal and let ferment at 37±2℃ for 48h; (5) Post-processing: Filter the fermentation liquid to remove residues and precipitate particles, and you will get a high-polyphenol, low-sugar beverage.

[0012] Furthermore, the juice is grape juice.

[0013] Furthermore, the preparation method of the *Lactobacillus argentifer* PCFS-60502 fermentation broth is as follows: (1) Activation of strain: The *Lactobacillus argyrulatus* PCFS-60502 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 argitulatus PCFS-60502 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: The strain exhibits excellent characteristics: Lactobacillus argituratii PCFS-60502 can significantly increase the total phenol content in beverages (up to 208.12±4.28mg / 100mL), greatly reduce the total sugar content (only 2.93±0.43g / 100g), and at the same time degrade bitter components and promote protein hydrolysis to generate amino acids, effectively reducing the bitterness of beverages, enhancing umami, and optimizing the 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 argitulatus* PCFS-60502 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 argituratii PCFS-60502 The present invention is based on Lactobacillus argituratii ( Lactiplantibacillus argentoratensisPCFS-60502 is a lactic acid bacterium isolated from the roots of 'Xiahei' grapes in Fu'an City, Fujian Province. It was deposited on November 18, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36674. *Lactobacillus tularensis* PCFS-60502 produces β-glucosidase (yield of 159.75 U / g) and tanninase (yield of 22.92 mmol / min / mL). Fermentation broth from this strain can increase the polyphenol content, reduce the total sugar content, decrease bitterness, and enhance umami flavor in grape juice. It also has a short fermentation cycle, making it suitable for fermenting high-polyphenol, low-sugar juices. Simultaneously, another lactic acid bacterium isolated from the roots of 'Xiahei' grapes in Fu'an City, Fujian Province, *Lactobacillus thuringiensis* (also known as *Lactobacillus thuringiensis*), is also present. Lactiplantibacillus argentoratensis PCFS-40121 was used as a control strain.

[0019] 1. Isolation and screening of strain PCFS-60502 Rinse the grape roots with tap water to remove impurities. Weigh 5 g of sample, first soak in 75% alcohol for 30 seconds, rinse once with sterile water, then soak in 4% sodium hypochlorite solution for 10 minutes, rinse three times with sterile water, and finally blot dry with sterile filter paper. Place the tissue in a sterile mortar, grind it, and add 45 mL of sterile water to prepare a solution with a concentration of 10%. -1 Take 100 μL of the sample solution with a concentration of 10... -1 and 10 -2 The sample solution was spread onto MRS medium and incubated at 37°C for 2-3 days. Single colonies were then picked and streaked onto plates. The colony morphology of strain PCFS-60502 is as follows: Figure 1 As shown.

[0020] 2. Identification of strain PCFS-60502 (1) DNA extraction: Add 400 μL of STE to a 1.5 mL EP tube, scrape an appropriate amount of purified colonies from the plate using a scraper loop, mix well, and centrifuge at 8000 g for 2 min. Discard the supernatant, add 200 μL of TE Buffer and 100 μL of Tris-saturated phenol (remove the lower layer) to the centrifuge tube, vortex for 60 s (using a float plate for shaking), and centrifuge at 13000 g for 5 min at 4°C. Transfer 160 μL of supernatant to a clean 1.5 mL EP tube, add 40 μL of TE Buffer, and add 100 μL of chloroform. Centrifuge at 13000 g for 5 min at 4°C. Transfer 140 μL of supernatant to a clean 1.5 mL EP tube, add 40 μL of TE Buffer, and add 100 μL of chloroform. Centrifuge at 13000 g for 5 min at 4°C. Transfer 100 μL of the supernatant to a clean 1.5 mL EP tube containing purified DNA and store at -20°C.

[0021] (2) PCR amplification: PCR reaction system: ddH2O: 9.5 μL, Mix: 12 μL, forward and reverse primers: 1 μL each, DNA template: 1.5 μL. 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, for a total of 30 cycles, followed by a 72 °C extension for 10 min. PCR products were run on a gel, and products with bands were sent to Platinum Biotech Co., Ltd. for sequencing. Sequencing results were compared using BLAST on the NCBI website. The 16S rDNA sequence of strain PCFS-60502 is shown in SEQ ID NO:1.

[0022] II. Application of Lactobacillus argituratii PCFS-60502 1. Preparation of fermentation broth for strain PCFS-60502 (1) Strain activation: Inoculate *Lactobacillus argentulatus* (Lactobacillus argentulatus) with an inoculation loop. Lactiplantibacillus from Argentorate PCFS-60502 was streaked onto MRS medium and incubated in a constant temperature incubator at 37 ℃ for 48 h. (2) Preparation of fermentation liquid: The PCFS-60502 single colony obtained in step (1) was inoculated into MRS medium and cultured statically at 37°C for 48 h.

[0023] 2. Preparation of high-polyphenol, low-sugar beverages (1) Grape juice preparation: Select grapes that are free from pests and diseases, have no surface damage, and have a uniform color as the test raw materials. After washing away the dirt and dust on the surface, transfer them to a blender for juicing. When there are no large particles in the blender, take them out for later use.

[0024] (2) Preparation: Mix the grapes according to a water-to-grape ratio of 1:2, add white sugar and mix thoroughly. Then, use a saccharimeter to measure the sugar content of the filtered grape juice to make it 20%. This sugar content provides a carbon source for the growth and reproduction of the strain, while promoting the production of enzymes (β-glucosidase, tanninase) and metabolites (organic acids, umami substances), ensuring fermentation efficiency. During sugar metabolism, esters, alcohols and other aroma substances are also produced, enriching the flavor profile of the juice. At the same time, it can regulate the fermentation speed (too high or too low sugar concentration will affect the activity of the strain), and can also slightly increase the viscosity of the juice, improving the drinking taste.

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

[0026] (4) Inoculation and fermentation: When the grape juice is cooled to about 40°C, the fermentation broth of the propagated Lactobacillus argyrate PCFS-60502 is inoculated into the fermenter at a rate of 5% and then cultured at a constant temperature of 37°C for 48 h.

[0027] (5) Centrifugal filtration: The fermented grape beverage is centrifuged and filtered to remove residues and precipitate particles, and then tested.

[0028] 3. Performance Testing (1) Determination of total phenol content: Accurately pipette 200 μL of fermentation broth sample, add 800 μL of ethanol solution containing 2% formic acid, mix well, and place in an ultrasonic instrument (power 100%, temperature 30 ℃) for 45 min. Centrifuge at 12000 r / min for 10 min, and transfer the supernatant to a new 1.5 mL centrifuge tube for testing. Pipe 100 μL of total phenol extract, add 250 μL of 0.2 mol / L Folin-Ciocalteu, react for 4 min, add 200 μL of 7.5% sodium carbonate solution, shake well, incubate at room temperature for 30 min, centrifuge, and take the supernatant to measure the absorbance at a wavelength of 760 nm. Use the serial dilution of gallic acid standard to construct a standard curve, and the regression equation is y=12.064x+0.0167, R 2 =0.999, and the total phenol content in the sample was calculated based on the regression equation.

[0029] (2) Total sugar content: The total sugar content was determined by referring to the method in the national standard food "Determination of fructose, glucose, sucrose, maltose and lactose" (GB 5009.8-2016).

[0030] (3) Electronic tongue analysis: Take 20 mL of grape fermentation broth, add 80 mL of distilled water to dilute to 100 mL, stir well and transfer 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 to measure bitterness and umami.

[0031] 4. Comparative Experiment A blank control group (grape juice without bacterial strain inoculation) and a control group inoculated with the same bacterial strain (inoculated with Lactobacillus argyroderma PCFS-40121) were set up and tested according to the same method described above. The results are shown in Table 1 below: Table 1 Evaluation of Fermented Beverage Indicators

[0032] As shown in the table above, the total phenol content of the grape juice sample fermented with Lactobacillus argentaria PCFS-60502 was 208.12±4.28 mg / 100ml, the total sugar content was 2.93±0.43 g / 100g, the bitterness value was 1.06, and the umami value was -2.46. That is, Lactobacillus argentaria PCFS-60502 of the present invention is significantly better than the blank control group and the control group of similar strains in terms of increasing the total phenol content, reducing the total sugar content, and improving the flavor (reducing bitterness and increasing umami) of fruit juice beverages.

[0033] As can be seen, the *Lactobacillus argentifer* PCFS-60502 strain of this invention can significantly increase the total phenolic content and decrease the total sugar content in fermented fruit juice beverages. This strain can produce β-glucosidase (yield of 159.75 U / g), decompose bitter terpene glycosides (such as limonene glycosides) in fruit juice, and release non-bitter terpenoids, thereby reducing the bitter components in fruit juice beverages. It can also secrete tanninase (yield of 22.92 mmol / min / mL), hydrolyze astringent tannins and proanthocyanidins, reduce their binding force with the oral mucosa, reduce the astringency and bitterness in fruit juice beverages, and promote the hydrolysis of some proteins into amino acids to enhance the umami flavor of fruit juice beverages, resulting in high-polyphenol, low-sugar beverages. From the perspective of consumer preferences, bitter beverages are not easily accepted by consumers in the market, while umami beverages are not common in the market. This preparation method can improve the richness of the beverage's taste and broaden the beverage market. In addition, the preparation method of this invention has a simple fermentation process, short fermentation cycle, low cost, and high efficiency, making it suitable for large-scale production. In summary, this invention is highly practical, can promote the resource utilization of grape by-products, extend the grape industry chain, increase grape production capacity, meet the needs of sustainable development in the planting industry, and has both economic and social benefits.

[0034] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A strain of *Lactobacillus argituratus* PCFS-60502, characterized in that: The *Lactobacillus argentulatus* is *Lactobacillus argentulatus* (… Lactiplantibacillus argentoratensis PCFS-60502, this strain was deposited on November 18, 2025 at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36674.

2. An application of *Lactobacillus argituratii* PCFS-60502 as described in claim 1, characterized in that: The Lactobacillus argentulatus ( Lactiplantibacillus argentoratensis Application of PCFS-60502 in fermented high-polyphenol low-sugar beverages.

3. A method for preparing a high-polyphenol, low-sugar beverage using *Lactobacillus argyrophylla* PCFS-60502 as described in claim 1, characterized in that: 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 argyrophylla PCFS-60502 to the juice, seal and let it ferment at 37±2℃ for 40-50 hours to obtain a high-polyphenol low-sugar beverage.

4. The method for preparing a high-polyphenol, low-sugar beverage as described in claim 3, characterized in that: The microbial preparation is *Lactobacillus arginulatus* (… Lactiplantibacillus argentoratensis Fermentation broth of PCFS-60502.

5. The method for preparing a high-polyphenol, low-sugar 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 cool it naturally to 35-42℃; (4) Inoculation and fermentation: Inoculate with 5% of the fermentation broth of Lactobacillus argyrate PCFS-60502, seal and let ferment at 37±2℃ for 48h; (5) Post-processing: Filter the fermentation liquid to remove residues and precipitated particles, and you will get a high-polyphenol, low-sugar beverage.

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

7. The method for preparing a high-polyphenol, low-sugar beverage as described in claim 5, characterized in that: The preparation method of the fermentation broth of Lactobacillus argyrate PCFS-60502 is as follows: (1) Activation of strain: The *Lactobacillus argyrulatus* PCFS-60502 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 argitulatus PCFS-60502 obtained in step (1) was inoculated into MRS medium and cultured statically at 37±2 ℃ for 48 h.

Images