Green tea beverage and complex bacteria fermentation method

Abstract

The application provides a kind of green tea beverage and compound bacteria fermentation method, belong to fermentation tea drink technical field.The application provides a kind of method for preparing green tea beverage by compound bacteria fermentation, uses green tea soup as raw material, supplements carbon source and then utilizes compound bacteria to carry out fermentation, and the green tea beverage can be obtained by mixing fermentation tea soup and stabilizer.The green tea beverage of the application has high evaluation and high nutrient content.The green tea beverage of the application is a kind of live bacteria type compound bacteria fermented green tea beverage, has mellow fruity aroma, sweet and sour taste, and bright orange yellow color, which widens the type of fermented tea beverage.

Description

A green tea beverage and a fermentation method using compound bacteria Technical Field

[0001] This invention belongs to the field of fermented tea beverage technology, specifically relating to a green tea beverage and a method for fermenting with compound bacteria. Background Technology

[0002] Green tea is the largest tea category in my country in terms of both production and consumption, and is widely loved for its pleasant aroma and numerous health benefits. According to data from the China Tea Marketing Association, in 2022, green tea production reached 1.8594 million tons, accounting for 58.3% of total tea production. Domestic sales reached 1.311 million tons, exports reached 313,900 tons, and unsold sales amounted to 234,500 tons, representing 12.61% of total green tea production that year. As the storage time of unsold green tea increases, environmental conditions such as temperature, humidity, oxygen, and light cause changes in its composition, leading to a decline in its sensory qualities, including color, aroma, and taste. Therefore, it is necessary to find effective ways to utilize aged tea, improve its sensory flavor, and enhance its quality.

[0003] In terms of products, aged green tea can be extracted to obtain tea liquor, fermented to obtain fermented liquid, and further blended to make fermented tea beverages. Utilizing bacteria or fungi to ferment tea, during the fermentation process, microorganisms transform tea polyphenols into other components such as catechin derivatives, flavonoids and their glycosides, phenolic acids, alkaloids, and terpenoids. This results in fermentation products with health benefits such as preventing hypertension, anti-oxidation, lowering body lipids, alleviating metabolic syndrome, and promoting intestinal function.

[0004] Currently, there are many fermented tea beverages on the market, but relatively few products are developed using aged green tea as a raw material. Furthermore, the types of fermentation strains used are limited, and there is a lack of relevant technology for developing compound-strain fermented tea beverages. Therefore, developing green tea fermented beverages that meet market demand and offer health benefits aims to further enrich the variety of tea beverages and increase the added value of aged green tea. Summary of the Invention

[0005] The purpose of this invention is to provide a green tea beverage and a method for fermenting with compound bacteria. Through fermentation with compound bacteria, a live bacteria fermented green tea beverage containing tea polyphenols is obtained, thereby increasing the added value of green tea.

[0006] This invention provides a method for preparing green tea beverage by fermentation with compound bacteria, comprising the following steps: (1) soaking green tea in water for a certain period of time, then filtering and collecting the tea soup;

[0007] (2) Mix the tea soup from step (1) with a carbon source to make a fermentation base liquid, inoculate the fermentation base liquid with fermentation compound bacteria, and ferment at a constant temperature to obtain fermented tea soup.

[0008] The fermentation complex is selected from at least two of Lactobacillus plantarum, Lactobacillus paracasei, Saccharomyces cerevisiae, and Koudriazwiecki.

[0009] (3) After mixing the fermented tea soup from step (2) with the stabilizer, sterilize it to obtain the green tea beverage.

[0010] Preferably, the green tea in step (1) includes dried new tea or aged tea.

[0011] Preferably, the mass-to-volume ratio of the green tea to the water is 1g:50-150mL;

[0012] The water temperature is 75–100℃, and the soaking time is 2–15 minutes.

[0013] Preferably, step (2) further includes sterilizing the fermentation base liquid before inoculation;

[0014] The carbon source in the fermentation base liquid is selected from at least one of reducing sugars.

[0015] Preferably, the mass of the carbon source is 3% to 9% of the volume of the tea infusion.

[0016] Preferably, the inoculum amount of the fermentation complex bacteria in step (2) is 2%.

[0017] Preferably, in step (2), when Lactobacillus paracasei and Kouridazvipichia are used to form the fermentation complex, the volume ratio of Lactobacillus paracasei to Kouridazvipichia is 1:3 to 3:1.

[0018] OD of the bacterial cultures of *Lactobacillus paracasei* and *Pichia kudrica* 600 The values ​​are all between 0.5 and 0.8.

[0019] Preferably, the temperature of the constant temperature fermentation in step (2) is 35℃~37℃, and the time of the constant temperature fermentation is 30h.

[0020] Preferably, the stabilizer in step (3) includes carrageenan and / or xanthan gum.

[0021] The present invention also provides a green tea beverage prepared using the above method.

[0022] Beneficial effects: This invention provides a method for preparing green tea beverages by fermentation with compound bacteria. Green tea infusion is used as raw material, and after supplementing with a carbon source, it is fermented with compound bacteria. The fermented tea infusion is mixed with a stabilizer to obtain the green tea beverage.

[0023] The green tea beverage described in this invention has received high praise and has a high content of nutrients, such as a sensory score of 88 points and an OD (Oxygen Demand) of [missing information]. 600The pH values ​​were 0.606±0.04, 3.67±0.01, total acid content was 1.06±0.02 g / L, free amino acid content was 180.55±0.52 mg / L, tea polyphenol content was 694.60±0.87 mg / L, and reducing sugar content was 44.36±0.17 g / L. The green tea beverage described in this invention is a live-culture compound-fermented green tea beverage, possessing a rich fruity aroma, a pleasantly sweet and sour taste, and a bright orange-yellow color, thus broadening the variety of fermented tea beverages.

[0024] Biological Preservation Information

[0025] Pichia kudriavzevii, strain number MZ5, was deposited on October 12, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 28610. Attached Figure Description

[0026] Figure 1 is a bar chart of the biomass of fermentation strains in fermented tea infusion;

[0027] Figure 2 is a bar chart showing the effect of different bacterial strains on the content of free amino acids in fermented tea infusion;

[0028] Figure 3 is a bar chart showing the effect of different strains of bacteria on the content of tea polyphenols in fermented tea infusion;

[0029] Figure 4 is a bar chart showing the effect of different bacterial strains on the reducing sugar content of fermented tea infusion;

[0030] Figure 5 is a bar chart showing the effects of different microbial strains on the sensory evaluation of fermented tea infusion;

[0031] Figure 6 is a bar chart showing the effect of different compound ratios on biomass in fermented tea infusion;

[0032] Figure 7 is a bar chart showing the effect of different compound combinations on the free amino acid content of fermented tea infusion;

[0033] Figure 8 is a bar chart showing the effect of different compound ratios on the content of tea polyphenols in fermented tea infusion;

[0034] Figure 9 is a bar chart showing the effect of different compound ratios on the reducing sugar content of fermented tea infusion;

[0035] Figure 10 is a bar chart showing the effect of different compound ratios on the sensory evaluation of fermented tea infusion;

[0036] Figure 11 shows the radar images of different fermented teas obtained by the 14 sensors of the electronic nose;

[0037] Figure 12 shows the PCA analysis results for different fermentation groups. Detailed Implementation

[0038] This invention provides a method for preparing green tea beverage by fermentation with compound bacteria, comprising the following steps: (1) soaking green tea in water for a certain period of time, then filtering and collecting the tea soup;

[0039] (2) Mix the tea soup from step (1) with a carbon source to make a fermentation base liquid, inoculate the fermentation base liquid with fermentation compound bacteria, and ferment at a constant temperature to obtain fermented tea soup.

[0040] The fermentation complex is selected from at least two of Lactobacillus plantarum, Lactobacillus paracasei, Saccharomyces cerevisiae, and Koudriazwiecki.

[0041] (3) After mixing the fermented tea soup from step (2) with the stabilizer, sterilize it to obtain the green tea beverage.

[0042] This invention uses green tea as the initial raw material. The green tea is soaked in water for a certain period of time, and then the tea infusion is collected by filtration. The green tea used in this invention can be commercial green tea, such as dried new tea or unsold or slow-moving aged tea. The embodiments use aged tea as an example, but this should not be considered the entire scope of protection of this invention. The green tea is soaked in water, with the preferred mass-to-volume ratio of green tea to water being 1g:50-150mL, more preferably 1g:100mL; the preferred water temperature is 75-100℃, more preferably 85℃; and the preferred soaking time is 2-15 minutes, more preferably 10 minutes.

[0043] After collecting the tea infusion, this invention mixes the tea infusion with a carbon source to prepare a fermentation base liquid. A fermentation complex of bacteria is then inoculated into the fermentation base liquid, and fermentation is carried out at a constant temperature to obtain fermented tea infusion. The carbon source of this invention preferably includes at least one reducing sugar, more preferably at least one selected from sucrose, fructose, and glucose, and most preferably glucose. The preferred mass of the carbon source added is 3% to 9% of the volume of the tea infusion, more preferably 6%.

[0044] In this invention, the fermentation base liquid is sterilized before being inoculated with fermentation compound bacteria. The sterilization is preferably high-temperature sterilization, and more preferably sterilization at 121°C for 15 minutes. The fermentation complex bacteria of this invention are selected from at least two of Lactobacillus plantarum, Lactobacillus paracasei, Saccharomyces cerevisiae, and Pichia kudriavzevii, preferably including Lactobacillus plantarum (CICC 22846), Lactobacillus plantarum (CICC V3), Saccharomyces cerevisiae (CICC 32268), Lactobacillus paracasei, and Pichia kudriavzevii MZ5. Lactobacillus plantarum 22846, Lactobacillus plantarum V3, and Saccharomyces cerevisiae 32268 are all derived from the China Industrial Microbial Culture Collection Center, and Lactobacillus paracasei is obtained from lactic acid bacteria beverages. In the examples, Lactobacillus paracasei CICC is used. 20241; *Pichia kudriazina* MZ5 was obtained from fish sauce and its preservation number is CGMCC No. 28610. The inoculum amount of the fermentation complex strain described in this invention is preferably 2%. Furthermore, when the fermentation complex strain consists of *Lactobacillus paracasei* and *Pichia kudriazina* MZ5, the volume ratio of the *Lactobacillus paracasei* to *Pichia kudriazina* MZ5 is preferably 1:3 to 3:1, more preferably 1:2 to 2:1, and most preferably 1:1. The OD of the *Lactobacillus paracasei* and *Pichia kudriazina* bacterial cultures described in this invention is... 600 The values ​​are preferably 0.5 to 0.8, and more preferably 0.645 ± 0.02. In this invention, after inoculation with the fermentation complex bacteria, isothermal fermentation is performed, preferably at a temperature of 37°C, and preferably for 30 hours.

[0045] After obtaining the fermented tea infusion, the present invention adds a stabilizer to the fermented tea infusion, and then sterilizes it at high temperature to obtain the green tea beverage. The preferred mass-to-volume ratio of the stabilizer to the fermented tea infusion is 1 g:1000 mL. The stabilizer preferably includes carrageenan and / or xanthan gum, and when a mixture of carrageenan and xanthan gum is used as the stabilizer, the preferred mass ratio of carrageenan to xanthan gum is 1:1.

[0046] The present invention also provides a green tea beverage prepared using the above method.

[0047] In this embodiment of the invention, Lactobacillus paracasei and Pichia kudricazvichid are fermented in a 1:1 (v / v) ratio, and carrageenan and xanthan gum are added to the tea infusion at a 1:1 (m / m) ratio after fermentation. The combined fermentation promotes the growth of Lactobacillus paracasei and retains tea polyphenols to a greater extent. The resulting fermented tea has a sweet and sour taste, a bright orange-yellow color, a mellow fruity aroma, and a uniform texture. Furthermore, the green tea beverage obtained by using Lactobacillus paracasei and Pichia kudricazvichid in combined fermentation has a better flavor than the fermented beverage obtained by using Lactobacillus paracasei alone.

[0048] To further illustrate the present invention, the following detailed description of a green tea beverage and a compound bacteria fermentation method provided by the present invention is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] Using green tea infusion as the research object, *Lactobacillus plantarum* 22846 (abbreviated as 22846), *Lactobacillus paracasei*, *Lactobacillus plantarum* V3 (abbreviated as LpV3), *Saccharomyces cerevisiae* 32268 (abbreviated as 32268), and *Pichia kudrica* MZ5 (abbreviated as Pichia kudrica MZ5) were inoculated into tea infusion with a glucose content of 1.5% at a 2% inoculation rate. *Lactobacillus plantarum* was fermented at 37℃ and yeast at 30℃ for 30 hours. After fermentation, 1 / 1000 carrageenan was added as a stabilizer to obtain the fermentation broth. The OD of the fermentation broth was then analyzed. 600 Dominant lactic acid bacteria and dominant yeast strains were screened based on pH, acidity, free amino acid content, tea polyphenol content, reducing sugar content, and sensory evaluation.

[0051] Next, the bacterial culture was fermented in ratios of 1:1, 1:2, 1:3, 2:1, and 3:1 to screen for the optimal ratio. Orthogonal experiments were then conducted to screen the optimal formula based on the type of sugar (glucose, sucrose, fructose), the amount of sugar added (3%, 6%, 9%), and the type of stabilizer (carrageenan, xanthan gum, carrageenan:xanthan gum = 1:1).

[0052] 1. Materials and reagents

[0053] Sun-dried green tea, from the organic management base of Senquan Tea Factory in Lüchun County, Yunnan Province. It has been aged for one year.

[0054] Food-grade glucose, food-grade sucrose, food-grade fructose, Zhejiang Yinuo Biotechnology Co., Ltd.; carrageenan, analytical grade, BASF (China) Co., Ltd.; xanthan gum, analytical grade, Beijing Solarbio Technology Co., Ltd.

[0055] 2. Experimental Methods

[0056] 2.1 Activation of microbial strains

[0057] (1) Activation of Lactobacillus plantarum

[0058] The purchased *Lactobacillus plantarum* 22846 and *Lactobacillus plantarum* V3 lyophilized powders were aseptically inoculated into MRS broth medium and activated at 37°C. After activation, the powders were inoculated onto blank MRS slant agar and incubated at 37°C for 48 hours. The slant agar was then stored at 4°C. The selected *Lactobacillus paracasei* was inoculated onto blank MRS slant agar and incubated at 37°C for 48 hours. The slant agar was then stored at 4°C.

[0059] Take the slant culture stored in the refrigerator, activate it by slant culture, and aseptically inoculate it into MRS broth medium. Incubate at 37°C. After incubation, adjust the initial OD. 600 It reached 0.645±0.02.

[0060] (2) Activation of yeast

[0061] Saccharomyces cerevisiae 32268 and Pichia pastoris MZ5 lyophilized powder were aseptically inoculated into YPD liquid medium. After activation at 30°C, they were inoculated onto blank YPD medium slant and cultured at 30°C for 48 hours. The slant was then stored in a refrigerator at 4°C.

[0062] Take the slant culture stored in the refrigerator, activate it by slant culture, and aseptically inoculate it into YPD broth medium. Incubate at 30℃. After the culture is completed, adjust the initial OD600 to 0.645±0.02.

[0063] 2.2 Preparation of Tea Infusion

[0064] Weigh out green tea according to a tea-to-water ratio of 1g:100mL, soak the green tea in ultrapure water at 85℃ for 10 minutes, add sugar of the appropriate mass fraction, and sterilize at 121℃ for 15 minutes.

[0065] 2.3 Screening of bacterial strains

[0066] Under aseptic conditions, *Lactobacillus plantarum* 22846, *Lactobacillus paracasei*, *Lactobacillus plantarum* V3, *Saccharomyces cerevisiae* 32268, and *Pichia pastoris* MZ5 were inoculated at a rate of 2% (v / v) into tea infusion containing 1.5% glucose. The lactic acid bacteria were cultured at 37°C for 30 hours, and the yeasts were cultured at 30°C for 30 hours. After fermentation, 1 / 1000 of sterilized carrageenan was added to prepare a live-culture tea beverage.

[0067] 2.4 Screening of compound ratios

[0068] Using lactic acid bacteria and yeast (v:v) in ratios of 1:1, 1:2, 1:3, 2:1, and 3:1, and with an inoculum size of 2% (v:v), the mixture was inoculated into green tea infusion containing 1.5% glucose and fermented at 37°C for 30 hours. After fermentation, 1 / 1000 of sterilized carrageenan was added to prepare a live-culture tea beverage.

[0069] 2.5 Formula Optimization

[0070] The optimal compound ratio was used for inoculation, with an inoculation amount of 2%, a fermentation temperature of 37℃, and a fermentation time of 30 hours. Three-factor, three-level experiments were conducted according to Tables 1 and 2.

[0071] Table 1. Three Factors and Three Levels Table

[0072]

[0073] Table 2 Orthogonal Design Table

[0074]

[0075] 2.6 Preparation of the test solution

[0076] Centrifuge the fermentation broth at 4000 r / min for 10 min at low temperature, and the supernatant is the test solution.

[0077] 2.7 Analytical Methods

[0078] 2.7.1 Determination of biomass in fermentation broth

[0079] The absorbance of the fermentation liquid was measured directly at a wavelength of 600 nm, with unfermented tea soup as a control.

[0080] 2.7.2 Determination of pH and total acidity

[0081] The total acid (calculated as lactic acid) in the fermentation broth was determined according to GB 12456-2021, "Determination of Total Acid in Food by Acid-Base Titration Method", with unfermented tea infusion as a control.

[0082] 2.7.3 Determination of Free Amino Acids

[0083] The content of free amino acids in fermented tea was determined in accordance with GB / T 8314-2013 "Determination of Total Free Amino Acids in Tea", with unfermented tea liquor as a control.

[0084] 2.7.4 Determination of Tea Polyphenols

[0085] The content of tea polyphenols in fermented tea was determined according to GB / T 21733-2008 "Tea Beverages - Determination of Tea Polyphenols in Tea Beverages", with unfermented tea infusion as a control.

[0086] 2.7.5 Determination of reducing sugars

[0087] The OD was determined using the 3,5-dinitrosalicylic acid (DNS method). The fermentation broth was diluted 100-fold, and 1 mL of the diluted solution was added to 1 mL of 3,5-dinitrosalicylic acid. The solution was then boiled in a water bath for 5 minutes, cooled, and 4 mL of ultrapure water was added. 540 Use unfermented tea infusion as a control.

[0088] 2.7.6 Sensory evaluation

[0089] A sensory evaluation panel of 12 people scored the appearance, aroma and taste of fermented green tea. The scoring criteria are shown in Tables 3 and 4.

[0090] Table 3 Sensory evaluation criteria for green tea infusion fermented with a single strain

[0091]

[0092]

[0093] Table 4 Sensory evaluation criteria for fermented green tea infusion using compound microbial strains

[0094]

[0095] 2.8 Data Processing

[0096] All experiments were performed in triplicate, and the average value was calculated. The content of various components was analyzed by plotting using Origin 2021 software, and the significance of the orthogonal experiments was analyzed using IBM SPSS Statistics 25.

[0097] Different lowercase letters in the same row of the table indicate significant differences, and different lowercase letters in the same graph also indicate significant differences.

[0098] 3. Experimental Results and Analysis

[0099] 3.1 Screening of fermentation strains

[0100] Figure 1 shows the biomass of various fermentation strains in the fermented tea infusion. 32268 grows slowly in the tea infusion, and its OD value after fermentation is [not specified]. 600 The OD value is only 0.128, possibly because the glucose concentration in the fermented tea infusion is too low, preventing the brewer's yeast from initiating fermentation. (OD value of MZ5 fermented tea) 600 The maximum value was 0.499, indicating that MZ5 grew rapidly in the tea infusion. The OD values ​​of 22846 fermented tea and LpV3 fermented tea were also discussed. 600There were no significant differences between them, at 0.306 and 0.292, respectively. OD of Lactobacillus paracasei fermented tea 600 The value is 0.273.

[0101] The effects of different bacterial strains on the pH and acidity of fermented tea infusion are shown in Table 5. The pH of each fermentation group was lower than that of the control group, while the total acidity was higher. Lactic acid bacteria had a greater acid-producing capacity than yeast, leading to a significant decrease in pH and a significant increase in acidity in the fermentation broth. The acid-producing capacity ranking among lactic acid bacteria was 22846 > LpV3 > Lactobacillus paracasei, while among yeast, the ranking was MZ5 > 32268.

[0102] Table 5. Effects of different microbial strains on pH and acidity of fermented tea infusion.

[0103]

[0104] The effects of different bacterial strains on the free amino acid content of fermented tea infusion are shown in Figure 2. Compared with unfermented tea infusion, the free amino acid content in fermented tea with 22846, LpV3, and Lactobacillus paracasei showed varying degrees of increase, rising by 55.02%, 75.51%, and 53.5%, respectively. However, there was no significant difference in free amino acid content between fermented tea with 22846 and LpV3. Conversely, the free amino acid content in fermented tea with 32268 and MZ5 decreased, decreasing by 2.60% and 23.30%, respectively. The 32268 strain, due to its slow growth in the tea infusion, consumed almost no amino acids, resulting in no significant difference in free amino acid content between the fermented tea and the control group.

[0105] The effects of different microbial strains on the polyphenol content of fermented tea infusion are shown in Figure 3. The polyphenol content of unfermented tea infusion was 703.15 mg / L. The polyphenol content of fermented tea infusion decreased in all cases, with a reduction of 5.00% to 9.58%. This indicates that lactic acid bacteria and yeast consume only a small portion of the polyphenols during fermentation, effectively ensuring the antioxidant function of the tea beverage. Furthermore, the slight reduction in the polyphenol content helps to reduce the bitterness of the tea infusion and improve its flavor quality.

[0106] The effects of different bacterial strains on the reducing sugar content of fermented tea infusions are shown in Figure 4. The reducing sugar content in all fermented beverages was lower than that in the control group. Specifically, the reducing sugar content in 32268 fermented tea did not decrease significantly, while the reducing sugar content in lactic acid bacteria fermented tea decreased by 4.54%–8.55%. The reducing sugar content in MZ5 fermented tea decreased significantly, by 42.62%. This is related to the OD values ​​of various bacterial strains in the tea infusion after fermentation. 600 The values ​​correspond.

[0107] Figure 5 shows the influence of different microbial strains on the sensory evaluation of fermented tea infusion. Fermented teas containing 22846, *Lactobacillus paracasei*, and LpV3 were grouped together for comparison. Due to excessively low glucose addition, the teas were overly acidic and received low scores. The 22846 and *Lactobacillus paracasei* fermented teas were orange-red in color, while the LpV3 fermented tea was pale yellow. *Lactobacillus paracasei* fermented tea had the highest sensory evaluation score of 73.67, because it added a milky aroma to the lactic acid bacteria fermentation aroma and had a moderate acidity. The other two strains had ordinary lactic acid bacteria flavors, and their excessive acid production led to a heavy acidity. Comparing the two yeast strains, MZ5 fermented tea scored significantly higher than 32268 fermented tea by 70 points. This is because 32268 did not grow well in the tea infusion, resulting in a weak fermented aroma and a darker color, while MZ5 grew well, producing a rich fermented aroma and an orange-red color.

[0108] Based primarily on sensory evaluation, and considering other indicators, the preferred lactic acid bacteria were identified as *Lactobacillus paracasei*, and the preferred yeast was identified as MZ5. *Lactobacillus paracasei* fermented tea had the highest sensory evaluation score, the highest free amino acid content, and the lowest tea polyphenol content, but the largest difference between it and other fermented teas was only 4.68%. MZ5 exhibited a greater growth advantage than 32268, and its fermented tea had a higher sensory evaluation score than *Saccharomyces cerevisiae* tea.

[0109] 3.2 Optimization of bacterial culture compound ratio

[0110] Different compound ratios (V) 副干酪乳杆菌 V 库德毕赤酵母菌MZ5 The effect of the compound ratio on the biomass in fermented tea infusion is shown in Figure 6. When the compound ratio is 1:1, 2:1, 1:2, and 1:3, the OD in each fermented tea infusion is as follows: 600 There were no significant differences, with values ​​of 0.459, 0.475, 0.469, and 0.474 respectively. The OD values ​​of fermented tea at a blend ratio of 3:1 were... 600 The minimum value was 0.447, which was lower than the OD value of Lactobacillus paracasei fermentation alone. 600 Large. Based on the pH and acidity analysis in Table 6, as the proportion of *Lactobacillus paracasei* in the formula gradually increased, the pH of the fermented tea gradually decreased and the acidity gradually increased. Compared with single fermentation with *Lactobacillus paracasei* (pH 4.00, acidity 0.60 g / L), the pH and acidity of each compound fermented tea showed both decreases and increases (pH between 3.63 and 3.89, total acid between 0.82 and 0.98 g / L). This indicates that MZ5 can promote the growth and reproduction of *Lactobacillus paracasei* and acid production.

[0111] Table 6. Effects of different blending ratios on pH and acidity of fermented tea infusion.

[0112]

[0113] The effects of different compound ratios on the free amino acid content of fermented tea infusion are shown in Figure 7. Compared with the control group, the free amino acid content of each fermentation group increased. There was no significant difference in free amino acid content for compound ratios of 1:1, 2:1, and 3:1, which were 334.87, 336.64, and 340.15 mg / L, respectively. The free amino acid content was significantly higher than that for compound ratios of 1:2 and 1:3, with no significant difference between 1:2 and 1:3, which were 274.60 and 276.40 mg / L, respectively. During fermentation, *Lactobacillus paracasei* produced more amino acids than MZ5, and this effect was more pronounced when the amount of *Lactobacillus paracasei* in the compound ratio was not less than that of yeast.

[0114] The effect of different blending ratios on the polyphenol content of fermented tea infusion is shown in Figure 8. Compared with the control group, the polyphenol content of each fermentation group decreased by 7.22% to 19.15%. The polyphenol content decreased the least when the blending ratio was 1:1, with a value of 615.87 mg / L.

[0115] The effect of different compound ratios on the reducing sugar content of fermented tea infusion is shown in Figure 9. Compared with the control group, the reducing sugar content of each compound fermentation group decreased, with the decrease ranging from 21.66% to 30.17%. The higher the proportion of yeast in the compound ratio, the more reducing sugar is consumed.

[0116] Figure 10 shows the effect of different blending ratios on the sensory evaluation of fermented tea. The highest sensory evaluation score (76.33 points) was achieved with a 1:1 blending ratio. This 1:1 blend exhibited a rich fruity aroma, with harmonious aromas from lactic acid bacteria and yeast fermentation, a complex flavor, and a bright orange-yellow color. The fruity aroma is likely due to the fruity notes produced by the co-fermentation of yeast and lactic acid bacteria, including 2-phenylethanol, benzyl alcohol, and ethyl octanoate. There were no significant differences in sensory evaluation scores for blending ratios of 2:1 and 1:2 (73 and 71 points respectively). Similarly, there were no significant differences in scores for blending ratios of 3:1 and 1:3 (66.67 and 67 points respectively). Fermented teas with a high proportion of *Lactobacillus paracasei* in their blends exhibited an overly strong lactic acid bacteria fermentation aroma, a more acidic taste, and a relatively darker color. Conversely, fermented teas with a high proportion of MZ5 in their blends showed an overly strong alcoholic flavor and a relatively darker color.

[0117] Based primarily on sensory evaluation, and taking other indicators into consideration, the optimal blending ratio is V. 副干酪乳杆菌 V 库德毕赤酵母 =1:1.

[0118] 3.3 Optimization of Compound Fermented Tea Formula

[0119] Table 6 shows that the factors affecting the quality of compound fermented tea, in descending order, are: sugar content (B) > sugar type (A) > stabilizer type (C). Through this three-factor, three-level orthogonal experiment, the optimal combination of levels for compound fermented tea was determined to be A1B2C3, namely, glucose, sugar content of 6%, and a 1:1 ratio of carrageenan and xanthan gum as stabilizers. Under these conditions, the sensory evaluation score of the fermented tea was 88 points, and the OD... 600 The values ​​were 0.6056±0.037, pH was 3.67, total acid content was 1.06±0.09 g / L, free amino acid content was 180.55±0.52 mg / L, tea polyphenol content was 694.60±0.87 mg / L, and reducing sugar content was 44.36±0.17 g / L.

[0120] The inter-subject effects tests of the orthogonal experiment are shown in Table 7. The differences in items A and B were extremely significant (P < 0.01), while the difference in item C was not significant (P > 0.05). Therefore, the type and amount of sugar added have a greater impact on sensory evaluation than the type of stabilizer. Furthermore, comparing the F-values ​​of each factor, the F-value of A is less than that of B, indicating that the amount of sugar added has a greater impact on sensory evaluation than the type of sugar. Therefore, the order of influence of each formulation factor on sensory evaluation is: amount of sugar added > type of sugar > type of stabilizer.

[0121] Table 6. Orthogonal Experiment Table and Results Analysis

[0122]

[0123]

[0124] Table 7. Inter-subject effects test

[0125]

[0126] aR 2 =0.851 (adjusted R) 2 =0.806)

[0127] Note: P < 0.05 indicates a significant difference, and P < 0.01 indicates a highly significant difference.

[0128] In summary, under optimal conditions, namely, the fermentation strains are Lactobacillus paracasei and Pichia pastoris MZ5, with a ratio of V... 副干酪乳杆菌 V 库德毕赤酵母 =1:1; the amount of glucose and sugar added is 6%, and the stabilizer is a compound stabilizer m. 卡拉胶 :m 黄原胶 =1:1, the sensory evaluation score of the compound fermented tea obtained by fermentation with this formula is 88, OD 600The pH value was 0.606±0.04, the total acid content was 3.67, the free amino acid content was 1.058±0.09 g / L, the free amino acid content was 180.55±0.52 mg / L, the tea polyphenol content was 694.60±0.87 mg / L, and the reducing sugar content was 44.36±0.17 g / L. This compound fermented tea has a pleasantly sweet and sour taste, a bright orange-yellow color, a mellow fruity aroma, and a uniform texture.

[0129] Example 2

[0130] Electronic nose analysis of the fermented tea beverage prepared in Example 1

[0131] During data collection for fermented tea beverages fermented with different strains, the strong response of a particular sensor to a specific type of characteristic gas allowed for the identification of the main volatile gas types emitted during sample analysis. Table 8 shows the 14 different metal oxide sensors and their corresponding aroma types. For sample differentiation analysis, the characteristic values ​​of the 14 sensors were extracted, and principal component analysis (PCA) was then used as the differentiation method.

[0132] Table 8. Types of electronic nose sensors and their representative compounds

[0133]

[0134]

[0135] In this experiment, the signal value was taken as 50 seconds after the response curve stabilized. The response signals of 14 sensors for fermented tea beverages from different strains are shown in Table 9. Among the two different fermentation groups, sensors 1 and 8 had higher response signals. As shown in Table 8, sensor 1 is sensitive to aromatic compounds, and sensor 8 is sensitive to ammonia compounds, indicating that each fermentation group contains aromatic compounds, ammonia compounds, and other substances. The response signals of sensors 2, 4, 5, 7, and 11 showed significant differences, which may play an important role in analyzing fermented tea fermented by three different strains.

[0136] Table 9. Sensor response signals of 14 different strains of fermented tea beverages.

[0137]

[0138]

[0139] As shown in Figure 11, sensors 1 and 8 in the *Lactobacillus paracasei* fermentation group exhibited the strongest signal responses; while sensors 1, 4, and 8 in the combined fermentation group of *Pichia pastoris* MZ5 and *Lactobacillus paracasei* showed the strongest signal responses. The signal responses of sensors 1-14 in the *Pichia pastoris* MZ5 and *Lactobacillus paracasei* fermentation groups were all stronger than those in the *Lactobacillus paracasei* fermentation group, indicating that fermentation using *Pichia pastoris* MZ5 combined with *Lactobacillus paracasei* is more beneficial for enhancing the flavor of fermented tea beverages compared to fermentation with *Lactobacillus paracasei* alone. Specifically, the signal values ​​of sensors 1, 2, 4, and 8 significantly increased after combined fermentation, indicating that combined fermentation is conducive to the production of aromatic compounds, nitrogen oxides, organic esters, terpenes, and amines, and enhancing the production of these substances contributes to a richer flavor in fermented tea beverages.

[0140] Figure 12 shows the PCA analysis results of fermented teas fermented with different microbial strains. In the correlation matrix model, the contribution rate of the first principal component was 92.3%, and the contribution rate of the second principal component was 7.3%. The sum of the contribution rates of the first and second principal components was 99.6%, indicating that there are significant differences in the volatile aromas between tea fermented with Lactobacillus paracasei alone and tea fermented with Lactobacillus paracasei and MZ5 compound. The first principal component played the most crucial role, and the electronic nose was able to accurately identify and distinguish them.

[0141] There are significant differences in the volatile aromas between tea fermented solely with *Lactobacillus paracasei* and tea fermented with a combination of *Lactobacillus paracasei* and MZ5. Compared to fermentation with *Lactobacillus paracasei* alone, fermentation using a combination of *Pichia pastoris* and *Lactobacillus paracasei* MZ5 is more effective in enhancing the flavor of fermented tea beverages. It promotes the production of aromatic compounds, nitrogen oxides, organic esters, terpenes, and amines, resulting in a richer flavor profile in the fermented tea.

[0142] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing green tea beverage by fermentation with compound bacteria, characterized in that, The process includes the following steps: (1) soaking green tea in water for a certain period of time, then filtering and collecting the tea liquor; the mass-to-volume ratio of green tea to water is 1g:50~150mL; the water temperature is 75~100℃, and the soaking time is 2~15min; (2) mixing the tea liquor obtained in step (1) with a carbon source to make a fermentation base liquid, inoculating the fermentation base liquid with a fermentation complex bacteria, and fermenting at a constant temperature to obtain fermented tea liquor; the carbon source is selected from at least one of sucrose, fructose, and glucose; the mass of the carbon source is 3%~9% of the volume of the tea liquor; the fermentation complex bacteria consists of Lactobacillus paracasei CICC 20241 and Pichiakudriavzevii MZ5; the preservation number of Pichiakudriavzevii MZ5 is CGMCC. No. 28610; the volume ratio of the *Lactobacillus paracasei* and *Pichia kudriazina* bacterial suspension is 1:3 to 3:1; the OD of the bacterial suspension of *Lactobacillus paracasei* and *Pichia kudriazina*... 600 The values ​​are all 0.5~0.8; the inoculation amount of the fermentation compound bacteria is 2%; the temperature of the constant temperature fermentation is 35℃~37℃, and the time of the constant temperature fermentation is 30h; (3) the fermented tea soup in step (2) is mixed with the stabilizer and then sterilized to obtain the green tea beverage.

2. The method according to claim 1, characterized in that, The green tea mentioned in step (1) includes dried new tea or aged tea.

3. The method according to claim 1, characterized in that, Step (2) also includes sterilizing the fermentation base liquid before inoculation.

4. The method according to claim 1, characterized in that, The stabilizer in step (3) includes carrageenan and / or xanthan gum.

5. A green tea beverage prepared by the method according to any one of claims 1 to 4.

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