Fermentation product of lactic acid bacteria fermented bamboo leaves, application of fermentation product and beverage prepared from fermentation product

Abstract

The invention provides a fermentation product of lactic acid bacteria fermented bamboo leaves, application of the fermentation product and a beverage prepared from the fermentation product, and belongs to the field of biological fermentation. The fermentation product disclosed by the invention is a fermentation product obtained by fermenting bamboo leaves by utilizing lactobacillus plantarum R1; the lactobacillus plantarum R1 is a strain which is preserved in the China Center for Type Culture Collection, and the preservation number of the lactobacillus plantarum R1 is CCTCC NO: M 2022659. The fermentation product disclosed by the invention has excellent oxidation resistance and antibacterial property, and is superior to fermentation products obtained by fermenting bamboo leaf extract by other lactic acid bacteria. The cellulase is utilized to treat the bamboo leaves before fermentation, so that phenols, amino acids and aroma components of the fermentation product can be increased, and the taste of the fermentation product is improved. The bamboo leaf compound beverage with good taste is prepared from the fermentation product. The application of the bamboo leaves is expanded, the additional value of bamboo industry production is improved, and the method has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biological fermentation, and particularly relates to a fermentation product of lactic acid bacteria fermenting bamboo leaves, its uses, and a beverage prepared therefrom. Background Art

[0002] Bamboo leaves, the leaves of plants in the genus Phyllostachys of the Poaceae family, are natural plants that can be used both as medicine and food. However, bamboo leaves have always been regarded as waste, causing serious waste of agricultural biomass resources. So far, bamboo leaves have been proven to contain a variety of active ingredients and have positive biological and pharmacological activities, such as antioxidant, anti-inflammatory, anti-cancer, anti-apoptotic, and lipid-lowering effects. However, the development and application of bamboo leaves in the food field are insufficient, and the exertion of their "dual use of medicine and food" value is restricted. Therefore, developing foods using bamboo leaves as raw materials is an effective means to realize their pharmacological activities and edible value.

[0003] Lactic acid bacteria fermentation is a common food processing method with a large number of practical applications. Fermented foods obtained by inoculating lactic acid bacteria can not only endow foods with unique flavors but also promote the release of their physiological activities, which is beneficial to the extension of the dual use of medicine and food. However, how to improve the utilization value of bamboo leaves through lactic acid bacteria fermentation and expand the application of bamboo leaves in the food field requires further research. Summary of the Invention

[0004] The purpose of the present invention is to provide a fermentation product of lactic acid bacteria fermenting bamboo leaves, its uses, and a beverage prepared therefrom.

[0005] The present invention provides a bamboo leaf fermentation product, which is a fermentation product obtained by fermenting bamboo leaves with Lactobacillus plantarum R1; the Lactobacillus plantarum R1 is a strain preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M 2022659.

[0006] Further, the bamboo leaves when using Lactobacillus plantarum R1 to ferment bamboo leaves are bamboo leaf water extracts.

[0007] Further, the bamboo leaf water extract is a water extract prepared from the following raw materials in weight ratio: 1 - 5 parts of bamboo leaves, 0.1 - 1 part of chrysanthemum, 7 - 10 parts of granulated sugar.

[0008] Preferably, the bamboo leaf water extract is a water extract prepared from the following raw materials in weight ratio: 4 parts of bamboo leaves, 0.8 part of chrysanthemum, 7.2 parts of granulated sugar.

[0009] Further, the preparation method of the bamboo leaf fermentation product includes the following steps: (1) Take bamboo leaves, chrysanthemums, and granulated sugar. Dissolve the granulated sugar in water, immerse the bamboo leaves and chrysanthemums in the water for extraction, and filter or not filter after extraction to obtain a water extract of bamboo leaves. (2) Inoculate the seed liquid of Lactobacillus plantarum R1 into the water extract of bamboo leaves obtained in step (1) for fermentation to obtain the product.

[0010] Further, in step (1), the obtained water extract of bamboo leaves is enzymatically hydrolyzed with cellulase.

[0011] Further, In step (1), the concentration of bamboo leaves is 0.005 - 0.05 mg / ml; the concentration of chrysanthemums is 0.0001 - 0.005 mg / ml; the concentration of granulated sugar is 0.01 - 0.05 mg / ml; And / or, in step (1), the extraction temperature is 80 - 100 °C, and the extraction time is 30 - 60 min; And / or, in step (1), stir during extraction, and the stirring speed is 500 - 1000 r / min; And / or, in step (1), do not filter after extraction.

[0012] Further, in step (2), the fermentation temperature is 35 - 37 °C, the fermentation time is 24 - 72 h, and the density of the seed liquid of Lactobacillus plantarum R1 during fermentation is 10 8 ~ 10 10 CFU / mL, and the inoculation amount of the seed liquid of Lactobacillus plantarum R1 is 1 - 3%.

[0013] Further, Adjust the pH of the water extract of bamboo leaves to 4 - 6 during enzymatic hydrolysis with cellulase; And / or, the addition amount of cellulase during enzymatic hydrolysis with cellulase is 2 - 4%; And / or, the enzymatic hydrolysis temperature during enzymatic hydrolysis with cellulase is 30 - 50 °C, and the enzymatic hydrolysis time is 1 - 5 h; And / or, perform ultrasonic treatment during enzymatic hydrolysis with cellulase, the ultrasonic power is 60 - 100 W, and the ultrasonic time is 20 - 60 min.

[0014] Preferably, Adjust the pH of the water extract of bamboo leaves to 5 during enzymatic hydrolysis with cellulase; And / or, the addition amount of cellulase during enzymatic hydrolysis with cellulase is 3%; the enzymatic activity of cellulase is 16 U / mg; And / or, the enzymatic hydrolysis temperature during enzymatic hydrolysis with cellulase is 35 °C, and the enzymatic hydrolysis time is 3 h; And / or, perform ultrasonic treatment during enzymatic hydrolysis with cellulase, the ultrasonic power is 90 W, and the ultrasonic time is 40 min.

[0015] The present invention also provides the use of the aforementioned bamboo leaf fermentation product in the preparation of functional foods.

[0016] The present invention also provides a functional beverage, which is prepared from the aforementioned bamboo leaf fermentation product as the raw material and food-acceptable auxiliary materials.

[0017] Preferably, the functional beverage is homogenized after mixing the following raw and auxiliary materials in parts by weight per volume: 100 parts by volume of the aforementioned bamboo leaf fermentation product, 1 to 10 parts by weight of erythritol, and 0.1 to 1 part by weight of xanthan gum.

[0018] In the present invention, the bamboo leaf water extract refers to a mixture obtained by dissolving white granulated sugar in water and submerging bamboo leaves and a little chrysanthemum in water for a period of time. This mixture can either not filter the solid components therein or filter the solid components to obtain a solution.

[0019] The present invention has achieved the following beneficial effects: The present invention provides a fermentation product obtained by fermenting a bamboo leaf extract with an endogenous strain Lactobacillus plantarum R1 of pickled bamboo shoots. This fermentation product has excellent antioxidant and antibacterial properties, superior to those of fermentation products obtained by fermenting bamboo leaf extracts with other lactic acid bacteria. Treating the bamboo leaf extract before fermentation with cellulase can increase the phenolic, amino acid, and aroma components of the fermentation product, improving the taste of the fermentation product. A bamboo leaf composite beverage with a good taste is prepared using this fermentation product. The present invention expands the uses of bamboo leaves, increases the added value of bamboo industry production, and has good application prospects.

[0020] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made.

[0021] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a graph showing the change of pH during the fermentation of bamboo leaf water extract by lactic acid bacteria: A is the graph showing the change of pH during the fermentation of clarified bamboo leaf water extract by different lactic acid bacteria; B is the graph showing the change of pH during the fermentation of bamboo leaf water extract retaining the bamboo leaf and chrysanthemum base by different lactic acid bacteria.

[0023] Figure 2Graph of the change in total sugar during the fermentation of bamboo leaf aqueous extract by lactic acid bacteria: A is the graph of the change in total sugar during the fermentation of clarified plant bamboo leaf aqueous extract by different lactic acid bacteria; B is the graph of the change in total sugar during the fermentation of bamboo leaf aqueous extract retaining bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0024] Figure 3 Graph of the change in soluble protein during the fermentation of bamboo leaf aqueous extract by lactic acid bacteria: A is the graph of the change in soluble protein during the fermentation of clarified plant bamboo leaf aqueous extract by different lactic acid bacteria; B is the graph of the change in soluble protein during the fermentation of bamboo leaf aqueous extract retaining bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0025] Figure 4 Graph of the change in total phenols during the fermentation of bamboo leaf aqueous extract by lactic acid bacteria: A is the graph of the change in total phenols during the fermentation of clarified plant bamboo leaf aqueous extract by different lactic acid bacteria; B is the graph of the change in total phenols during the fermentation of bamboo leaf aqueous extract retaining bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0026] Figure 5 PCA graph of organic acids and free sugars during the fermentation of bamboo leaf aqueous extract by lactic acid bacteria: A is the score graph; B is the loading graph.

[0027] Figure 6 PCA graph of electronic nose analysis during the fermentation of bamboo leaf aqueous extract by lactic acid bacteria: A is the score graph; B is the loading graph.

[0028] Figure 7 PLS-DA analysis graph before and after the fermentation of bamboo leaf aqueous extract by four strains of lactic acid bacteria: A is the score graph; B is the 200-time permutation test graph; C is the VIP score graph.

[0029] Figure 8 Graph of the change in ABTS radical scavenging rate during the fermentation of bamboo leaf aqueous extract by lactic acid bacteria: A is the graph of the change in ABTS radical scavenging rate during the fermentation of clarified plant bamboo leaf aqueous extract by different lactic acid bacteria; B is the graph of the change in ABTS radical scavenging rate during the fermentation of bamboo leaf aqueous extract retaining bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0030] Figure 9 Graph of the change in DPPH radical scavenging ability during the fermentation of bamboo leaf aqueous extract by lactic acid bacteria: A is the graph of the change in DPPH radical scavenging ability during the fermentation of clarified plant bamboo leaf aqueous extract by different lactic acid bacteria; B is the graph of the change in DPPH radical scavenging ability during the fermentation of bamboo leaf aqueous extract retaining bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0031] Figure 10 Graph of the difference in antibacterial ability of bamboo leaf aqueous extract before and after fermentation by different lactic acid bacteria: A is the antibacterial circle graph of different experimental groups; B is the radar graph of the antibacterial circle diameter of different experimental groups, with the unit of mm and the outer diameter of the Oxford cup being 8 mm.

[0032] Figure 11 The figure shows the changes in total phenol content before and after co-fermentation of fungus and enzyme and single fermentation; in the figure, "**" statistically represents P <0.01, with extremely significant differences; A is the retained substrate, B is clarification; UH is unenzymolyzed, and Cel-EH is the cellulase enzymolysis treatment.

[0033] Figure 12 The figure shows the changes in phenolic compounds before and after fungus and enzyme fermentation of the aqueous extract of Phyllostachys heterocycla cv. Pubescens leaves; in the figure, "*" statistically represents P <0.05, "**" statistically represents P <0.01, "***" statistically represents P <0.001.

[0034] Figure 13 The figure shows the changes in amino acids before and after fungus and enzyme fermentation of the aqueous extract of Phyllostachys heterocycla cv. Pubescens leaves; in the figure, "*" statistically represents P <0.05, "**" statistically represents P <0.01, "***" statistically represents P <0.001.

[0035] Figure 14 The figure shows the total ion current chromatogram of GC-MA analysis before and after fungus and enzyme fermentation of the aqueous extract of Phyllostachys heterocycla cv. Pubescens leaves.

[0036] Figure 15 The figure shows the stacked chart of the number of types and peak areas of volatile aroma components before and after fungus and enzyme fermentation of the aqueous extract of Phyllostachys heterocycla cv. Pubescens leaves.

[0037] Figure 16 The figure shows the radar chart of the sensory evaluation of the fermentation broth: the odor score of the fermentation broth with the retained substrate is significantly higher than that of the clarified fermentation broth, and "*" represents P < 0.05, with significant differences statistically. Specific implementation manners

[0038] The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

[0039] Dendrocalamus latiflorus Munro ( Dendrocalamus latiflorus Munro ) bamboo leaves were collected from Longhanling Forest Farm, Liunan District, Liuzhou City, Guangxi Zhuang Autonomous Region; chrysanthemums (Chrysanthemi Flos) and granulated sugar are both commercially available; Lactobacillus plantarum R1 ( Lactobacillus plantarum.R1) is a strain preserved in this laboratory. It was preserved on May 18, 2022, at the China Center for Type Culture Collection (CCTCC) located within Wuhan University, Wuhan City, Hubei Province, China, with the preservation number CCTCC NO: M 2022659. At the same time, this strain is recorded in a Chinese patent with an application date of May 25, 2022, and an application number of 202210576703.3; Escherichia coli (Escherichia coli. ATCC 35218), Staphylococcus aureus ( Staphylococcus aureus. ATCC6538), Klebsiella pneumoniae ( Klebsiella pneumonia. ATCC 13883) are standard strains and can be obtained by purchase; Lactobacillus plantarum ( L. plantarum. Lp90), Lactobacillus bulgaricus ( L.bulgaricus. LB42), Lactobacillus acidophilus ( L. acidophilus. LA85) were purchased from Microkang Probiotics (Suzhou) Co., Ltd. and are all stored in the form of freeze-dried powder.

[0040] The fresh leaves of Dendrocalamus latiflorus used in this invention have high edible value. As a zongzi leaf, it is an important material for making zongzi and can also be used for brewing.

[0041] Example 1: Fermentation of bamboo leaves by Lactobacillus plantarum R1 to obtain fermentation products 1. Preparation of bamboo leaf aqueous extract Raw material pretreatment: Fresh bamboo leaves were picked from Liuzhou, Guangxi, and the pest-infected and discolored bamboo leaves were removed. After being washed clean, they were placed in an oven at 50°C for drying and ground by a crusher for 5 min until they passed through an 800-mesh sieve. Chrysanthemum is dried Hangju, and it is stored with the crushed bamboo leaves in a dry and cool place for later use.

[0042] Preparation of bamboo leaf aqueous extract: Take 4.0 g of bamboo leaves, 0.80 g of chrysanthemum, and 7.20 g of granulated sugar and soak them in 240 mL of distilled water. Stir magnetically at 900 r / min at 80°C for 30 min to obtain the bamboo leaf aqueous extract. This bamboo leaf aqueous extract can either be not filtered to obtain the bamboo leaf aqueous extract retaining the plant base or be filtered to obtain the clarified bamboo leaf aqueous extract. Both of these bamboo leaf aqueous extracts are heated in boiling water for 10 min for sterilization for subsequent operations.

[0043] 2. Preparation of seed liquid Lactobacillus plantarum R1 was stored at -80°C, taken out and thawed at room temperature, streaked on MRS agar medium, and a single colony with normal morphology and size was picked and inoculated into MRS broth medium for activation. After two generations of activation, liquid culture was carried out, and static culture was carried out at 37°C until it reached about 8 10 CFU / ml.

[0044] 3. Preparation of fermentation products by fermenting the bamboo leaf aqueous extract with Lactobacillus plantarum R1 The seed liquid of Lactobacillus plantarum R1 was centrifuged at 4000 r / min for 5 min, the culture medium was discarded, and it was dissolved in sterile water and vortexed to obtain a resuspension (the density of Lactobacillus plantarum R1 in the resuspension was 10 8 CFU / mL). The resuspension was inoculated into the aqueous extract of bamboo leaves (clarified aqueous extract of bamboo leaves or aqueous extract of bamboo leaves retaining the plant base) at an inoculation amount of 1% (v / v) and fermented. It was fermented statically at 37 °C for 72 h to obtain the fermentation product.

[0045] Example 2. The fermentation product was obtained by fermenting the aqueous extract of bamboo leaves enzymatically hydrolyzed by cellulase with Lactobacillus plantarum R1 1. Preparation of the aqueous extract of bamboo leaves The clarified aqueous extract of bamboo leaves or the aqueous extract of bamboo leaves retaining the plant base was prepared according to the method described in Example 1.

[0046] 2. Ultrasonic-assisted enzymatic hydrolysis of the aqueous extract of bamboo leaves with cellulase The pH of the aqueous extract of bamboo leaves obtained in Step 1 was adjusted to 5 using food-grade citric acid and baking soda. Subsequently, 3% (v / v) of cellulase (16 U / mg) was added, and it was enzymatically hydrolyzed at 35 °C for 3 h. Ultrasonic treatment was performed during enzymatic hydrolysis (ultrasonic power was 90 W, time was 40 min). After enzymatic hydrolysis was completed, the enzyme was inactivated by boiling water for 5 min and cooled to room temperature to obtain the aqueous extract of bamboo leaves to be fermented.

[0047] 3. Preparation of the seed liquid Same as Example 1.

[0048] 4. Preparation of the fermentation product by fermenting the aqueous extract of bamboo leaves with Lactobacillus plantarum R1 The aqueous extract of bamboo leaves obtained in Step 2 was taken and fermented according to the method described in Example 1 to obtain the fermentation product.

[0049] For the enzymatic hydrolysis conditions, the present invention screened the parameters of pH value (4, 4.5, 5, 5.5, 6), cellulase content (2, 2.5, 3, 3.5, 4%) (v / v), enzymatic hydrolysis temperature (30, 35, 40, 45, 50 °C), enzymatic hydrolysis time (1, 2, 3, 4, 5 h), ultrasonic power (60, 70, 80, 90, 100 W), and ultrasonic time (20, 30, 40, 50, 60 min), using the reducing sugar generated after enzymatic hydrolysis as an index. When screening, the basic conditions were a temperature of 40 °C, an enzymatic hydrolysis pH of 5, an enzymatic hydrolysis time of 3 h, a cellulase addition amount of 3.5% (160 U / mL), an ultrasonic time of 40 min, and an ultrasonic power of 80 W. The method for measuring the reducing sugar is as follows: (1) The reducing sugar content was determined by the DNS method. Preparation of DNS reagent: Take 162.5 mL of 2 mol / L sodium hydroxide solution, add 3.25 g of 3,5-dinitrosalicylic acid, then add 23 mL of glycerol. After complete dissolution, make up the volume to 500 mL.

[0050] (2) Preparation of the standard curve: Weigh a series of concentrations of glucose standard solutions into 10 mL colorimetric tubes, make up to 1 mL with distilled water, accurately add 2 mL of DNS reagent respectively, heat in a boiling water bath for 5 min, cool with running water, and make up to 8 mL with water. Measure the absorbance at a wavelength of 540 nm. The standard curve obtained is: (y = 0.3064x + 0.0577, R 2 = 0.9975).

[0051] (3) Determination of the reducing sugar content in the sample: Take 1 mL of the sample to be tested diluted by an appropriate multiple and 2 mL of DNS reagent and add them to a 10 mL colorimetric tube, place in a boiling water bath for 5 min, measure the absorbance value at a wavelength of 540 nm, and calculate the reducing sugar content in the sample according to the standard curve and the dilution factor.

[0052] Finally, according to the test results, it was determined that the optimal parameters were a pH value of 5, a cellulase addition amount of 3% (v / v) (16 U / mg), an enzymatic hydrolysis temperature of 35 °C, an enzymatic hydrolysis time of 3 h, an ultrasonic power of 90 W, and an ultrasonic time of 40 min. The reducing sugar content of the enzymatic hydrolysis product obtained was the highest, which was 2.30 ± 0.01 mg / mL.

[0053] Example 3: Preparation of a compound beverage from the fermentation product obtained by fermenting bamboo leaves with Lactobacillus plantarum R1 A compound beverage was prepared using the fermentation product obtained in Example 1 or Example 2. In this Example 3, the fermentation product prepared from the aqueous extract of bamboo leaves with the plant base retained in Example 2 was taken as an example.

[0054] Take 100 mL of the fermentation product, add 5 wt% of erythritol and 0.15 wt% of xanthan gum respectively, mix and then homogenize (the rotation speed of homogenization is 12000 r / min and the time is 3 min) to obtain a compound beverage, which is subjected to pasteurization and then stored.

[0055] The prepared compound beverage had excellent sensory evaluation, and the microbial indicators met the requirements of the "National Food Safety Standard Beverages" (GB 7101-2022).

[0056] The beneficial effects of the present invention are demonstrated by the following specific test examples.

[0057] Test Example 1: Study on different lactic acid bacteria for the fermentation product of bamboo leaves 1. Preparation of the aqueous extract of bamboo leaves Raw material pretreatment: Fresh bamboo leaves were picked from Liuzhou, Guangxi. Insect-damaged and discolored bamboo leaves were removed. After being washed clean, they were placed in an oven and dried at 50 °C, and then ground by a crusher for 5 min until they passed through an 800-mesh sieve. Chrysanthemums were dried Hangju and stored in a dry and cool place for later use with the crushed bamboo leaves.

[0058] Preparation of bamboo leaf water extract: Eight groups were prepared. In each group, 4.0 g of bamboo leaves, 0.80 g of chrysanthemums and 7.20 g of granulated sugar were immersed in 240 mL of distilled water, and magnetically stirred at 900 r / min at 80 °C for 30 min. Four groups of the sample solutions were filtered to obtain clear bamboo leaf water extracts, and the remaining four groups of sample solutions retained the plant matrix to obtain bamboo leaf water extracts with the plant matrix retained. The bamboo leaf water extracts were sterilized by heating with boiling water for 10 min.

[0059] 2. Preparation of seed liquid Lactobacillus plantarum R1 was stored at -80 °C. After being taken out, it was thawed at room temperature, streaked on MRS agar medium, and a single colony with normal morphology and size was picked and inoculated into MRS broth medium for activation. The freeze-dried powders of Lactobacillus plantarum Lp90, Lactobacillus bulgaricus LB42, and Lactobacillus acidophilus LA85 were directly placed in MRS broth medium for activation and rejuvenation. After all the strains were activated for two generations, they were cultured in liquid, and statically cultured at 37 °C until about 8 10 CFU / ml.

[0060] 3. Fermentation of bamboo leaf water extract by different lactic acid bacteria The seed liquids of four strains of lactic acid bacteria (Lactobacillus plantarum R1, Lactobacillus plantarum Lp90, Lactobacillus bulgaricus LB42, and Lactobacillus acidophilus LA85) were centrifuged at 4000 r / min for 5 min, the culture medium was discarded, dissolved in sterile water, and vortexed to obtain a resuspended solution (the density of Lactobacillus plantarum R1 in the resuspended solution was 10 8 CFU / mL). The resuspended solution was inoculated into the bamboo leaf water extract for fermentation at an inoculation amount of 1% (v / v). Each lactic acid bacteria resuspended solution was inoculated into two groups of bamboo leaf water extracts, namely the clear and the ones with the plant matrix retained, and statically fermented at 37 °C for 72 h, and samples were taken every 12 h. The sample solutions were centrifuged at 8000 r / min for 5 min to obtain the fermentation supernatant, and the corresponding indexes were measured.

[0061] 4. Index determination 4.1 Determination of pH value and color difference analysis The pH value during the fermentation of the bamboo leaf water extract was measured using a pH meter.

[0062] 2 mL of each group of sample solutions was added to a colorimetric cuvette, and the L* (brightness), a* (red-green degree), b* (yellow-blue degree) values were measured with a color difference analyzer, and the total color difference (ΔE It is calculated according to the following formula (1): (1) Wherein, L 0 *、a 0 *、b 0 * are the color parameter values of the standard sample solution respectively, L * The larger the value, the higher the brightness, a * The larger the value, the deeper the color tends to red (conversely, the deeper the green, the lighter the red), b * The larger the value, the deeper the color tends to yellow (conversely, the deeper the blue, the lighter the yellow).

[0063] 4.2 Determination of basic physical and chemical indexes (1)Determination of total sugar: It is determined by the phenol-sulfuric acid method. Add 0.5 mL of 5% phenol aqueous solution, 5 mL of concentrated sulfuric acid and 0.5 mL of the fermentation supernatant diluted 200 times, mix well, let stand and react for 5 min, place in a boiling water bath for 15 min, take out, cool to room temperature, and measure the absorbance at 490 nm. Calculate the total sugar content according to the previously obtained standard curve y = 4.3909x + 0.0103 (R 2 = 0.9964, showing a good linear relationship in the range of 0.01 - 0.1 mg / mL).

[0064] (2)Determination of soluble protein content: It is determined using a Coomassie Brilliant Blue G-250 method kit (Bradford method). Add 200 μL of Coomassie Brilliant Blue G-250 reagent and 20 μL of the fermentation supernatant to each well of a 96-well plate, mix well and react at room temperature for 5 min, measure the absorbance at 595 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific Inc., USA). Calculate the soluble protein content according to the previously obtained standard curve y = 0.0037x + 0.0004 (R 2 = 0.9936, showing a good linear relationship in the range of 5 - 25 μg / mL).

[0065] (3)Determination of total phenol content: Using gallic acid as the standard product, it is determined after appropriate modification of the Folin-Ciocalteu method. Add 0.5 mL of Folin-Ciocalteu reagent, 1 mL of 10% Na2CO3 aqueous solution and 1 mL of the fermentation supernatant, mix well, react in the dark at room temperature for 1 h, and measure the absorbance at 765 nm. According to the gallic acid standard curve y = 0.0177x - 0.0099 (R 2= 0.9979, and has a good linear relationship in the range of 2.5 - 30 μg / mL) to calculate the total phenol content.

[0066] 4.3 Determination of organic acid and free sugar indices Determination of organic acids: Determination was carried out using UPLC - PDA (Waters Technology Co., Ltd., USA). Chromatographic conditions: The chromatographic column was an AQUITY UPLC® BEH C18 chromatographic column (2.1 mm × 100 mm × 1.7 μm) (Waters, USA); the mobile phase was 5% acetonitrile aqueous solution (containing 0.1% phosphoric acid); flow rate: 0.15 mL / min; column temperature: 30 °C; ultraviolet detection wavelength: 210 nm; injection volume: 4 μL; isocratic elution. After the fermented supernatant obtained by centrifugation passed through a 0.22 μm aqueous filter membrane, it was diluted 20 times and directly injected for analysis. The organic acids in the fermentation broth were qualitatively and quantitatively analyzed by obtaining the retention time and standard curve through organic acid standards, and the results were expressed in μg / mL.

[0067] Determination of free sugars: Determination was carried out by HPLC - ELSD (Waters Technology Co., Ltd., USA). Chromatographic conditions: The chromatographic column was a ShimNex HE - NH2 chromatographic column (250 mm × 4.6 mm, 5 μm) (Shimadzu Corporation, Japan); the mobile phase was acetonitrile - water (85∶15); column temperature: 30 °C; flow rate: 1.0 mL / min; drift tube temperature: 80 °C; carrier gas volume flow rate: 2.0 mL / min; gain: 10; injection volume: 10 μL. The fermented supernatant was directly injected for analysis after passing through a 0.22 μm aqueous filter membrane. Qualitative and quantitative analysis was carried out by the external standard method, and the results were expressed in μg / mL.

[0068] 4.4 Electronic nose analysis The electronic nose used (PEN3, Airsense Analytics, Schwerin, Germany) was equipped with a metal oxide semiconductor (MOS) sensor array composed of 10 different MOS sensors. First, 20 mL of the fermented supernatant sample was collected in a 50 mL sample bottle, and left to stand for 30 min at a constant temperature (28 °C) for equilibration, and then the electronic nose probe was inserted for detection. The specific parameters for E - nose detection were a detection time of 90 s, a cleaning time of 300 s, a carrier gas speed of 400 mL / min, and an injection flow rate of 400 mL / min.

[0069] 4.5 Comprehensive evaluation of different lactic acid bacteria strains The partial least squares discriminant analysis model (PLS-DA) was used to effectively distinguish different experimental groups, and key indicators were screened according to the variable importance in the projection (VIP value) and the P value of the difference between groups. Based on the key indicators, comprehensive evaluation by entropy weight - technique for order preference by similarity to an ideal solution (EWM-TOPSIS) was carried out (Lu et al. 2024). The EWM-TOPSIS method can reduce the inherent subjectivity in weight allocation. The positive ideal solution (D⁺) and negative ideal solution (D⁻) correspond to the ideal solutions of the maximum and minimum attribute values of the samples, representing the advantages and disadvantages of all indicators. According to the relative distances between the evaluation indicators and the solutions, the four strains of lactic acid bacteria fermented and unfermented bamboo leaf aqueous extracts were ranked by distance to determine the strain with the best fermentation effect. Specifically: (2) Indicates calculating the entropy value for the indicator. Where n is the number of samples, m is the number of indicators, and p ij Is the normalized value of the i-th sample on the j-th indicator.

[0070] (3) Indicates the weight w of the j-th indicator j . Where d j Is the difference coefficient of the j-th indicator, and n is the total number of indicators.

[0071] (4) (5) Z + 、Z - Represent the positive ideal solution and negative ideal solution respectively. Where (6) Indicates calculating the Euclidean distance Ij between the positive and negative ideal solutions of each evaluation indicator. Where is the positive ideal solution of the i-th solution, and is the negative ideal solution of the i-th solution.

[0072] (7) Indicates the closeness coefficient C between the evaluation solution and the ideal value i . Where the range of C i Is [0, 1], and the higher the value of C i , the higher the comprehensive score. According to the comprehensive comparison ratio, the solution Si is the optimal choice.

[0073] 4.6 Determination of antioxidant indicators (1) Determination of DPPH scavenging ability The DPPH free radical scavenging ability kit was used to determine the changes in DPPH free radical scavenging ability during the fermentation process of different experimental groups. The fermentation supernatant after centrifugation was diluted 4 times and mixed with the reagent. The reaction was carried out at room temperature in the dark for 30 minutes. After centrifugation at 4000r / min for 5 minutes, 80% methanol was used as a blank control, and the absorbance of the reaction solution was measured at 517 nm. The standard curve was made by using the DPPH free radical scavenging rate and the standard Trolox concentration, and the DPPH free radical scavenging ability was calculated by the absorbance value. The results were expressed as the antioxidant Trolox equivalent.

[0074] (2) Determination of ABTS scavenging ability The ABTS free radical scavenging ability kit was used to determine the changes in total antioxidant capacity during the fermentation process of different experimental groups. The fermentation supernatant after centrifugation was mixed with the reagent and reacted at room temperature in the dark for 6 minutes. Distilled water was used as a blank control and the absorbance of the reaction solution was measured at 734 nm. The results were expressed as ABTS scavenging rate, and the calculation formula is as follows: (8) 4.7 Determination of antibacterial activity The total colony counts of three indicator bacteria, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 6538, and Klebsiella pneumoniae 18188, were adjusted to 10 7 CFU / mL. Using the Oxford cup method, add 30 mL of LB agar as the bottom layer in the culture dish and insert a sterile Oxford cup. Add the indicator bacteria solution to the LB agar cooled to 50°C at a ratio of 1% (v / v), mix well and pour into the plate immediately. After the culture medium is completely solidified, take out the Oxford cup and add 180 μL of the supernatant to be tested into the well. A total of 3 parallels were performed, with sterile saline as a control. Let the plate stand for 1 hour to allow the supernatant to fully diffuse, place it in a 37°C incubator for 12 hours, and measure the diameter of the inhibition zone in mm.

[0075] 4.8 Data Processing and Analysis SPSS 26.0 software was used for data processing, and Duncan test was used to calculate significant differences. P The difference was considered significant when the value was <0.05. OriginPro 2023 was used to draw the graph, and the experimental data were expressed as mean ± standard deviation.

[0076] 5. Results and Analysis 5.1 pH changes during fermentation During the lactic acid bacteria fermentation process, the fermentation degree of bamboo leaf water extract can be measured by pH changes. Figure 1It can be seen that the initial pH of the bamboo leaf aqueous extract is about 6.20, which is suitable for the growth of lactic acid bacteria. The lactic acid bacteria quickly enter the logarithmic growth phase, grow vigorously within the first 12 h, and grow steadily after 12 h, with a gentle change in pH. In addition, whether the bamboo leaf aqueous extract is clarified or the substrate is retained will have a certain impact on the decrease in pH. Among them, the lactic acid bacteria ferment more vigorously in the bamboo leaf aqueous extract with the plant substrate retained. In both environments, Lactobacillus plantarum R1 has the highest degree of fermentation.

[0077] 5.2 Color difference analysis before and after fermentation The color of the bamboo leaf aqueous extract is an important parameter, reflecting the changes in the chromogenic components during the fermentation process and affecting the sensory quality of the beverage and the choice of consumers. Table 1 shows the differences in the color attributes of the lactic acid bacteria-fermented bamboo leaf aqueous extract. As can be seen from Table 1: Compared with the clarified bamboo leaf aqueous extract, the a * 、 b * values of the experimental groups after fermentation were all significantly reduced ( P <0.05), and the L * values were all significantly increased ( P <0.05), indicating that the lactic acid bacteria fermentation caused the red color of the sample solution to fade and tend to be greenish, the yellow color to fade, and the brightness to increase, and the changes in the fermented product with the substrate retained were more obvious. According to the formula calculation, the color difference value △E was found. The greater the color difference, the greater the color change, the more it tended to be bright green, and the better the visual sensory experience. And retaining the plant substrate was beneficial to the vigorous growth of lactic acid bacteria and more conducive to the fermentation process.

[0078] Table 1. Differences in color attributes of lactic acid bacteria-fermented bamboo leaf aqueous extract Note: Different letters in the same column indicate significant differences (P < 0.05).

[0079] 5.3 Changes in total sugar and soluble protein (1) Changes in total sugar The carbon source in the bamboo leaf aqueous extract is mainly sucrose in white granulated sugar, which can slow down the inhibition of the accumulated lactic acid. During the growth and fermentation process of lactic acid bacteria, the lactic acid bacteria decompose sucrose to release glucose and fructose, and at the same time utilize the monosaccharides for their own metabolism, resulting in a decrease in the total sugar content. As Figure 2 can be seen, in the bamboo leaf aqueous extract with tea residues retained, the decrease in the total sugar content is greater, about 50%. Among them, the consumption of the carbon source by the commercial strains LA85, LB42, Lp90, and the endogenous strain R1 is not very different, which are 47.75%, 52.73%, 46.72%, and 49.57% respectively. In the clarified bamboo leaf aqueous extract, the utilization degree of the carbon source by each strain is lower.

[0080] (2) Changes in Soluble Proteins Lactic acid bacteria can utilize proteins as nitrogen sources, degrade them into polypeptides and amino acids, which are more easily absorbed by the human body, and at the same time enrich the product flavor. As Figure 3 shown, during the fermentation process, the soluble protein content in the clarified bamboo leaf aqueous extract decreased from about 60.00 μg / mL to 30.0 - 40.00 μg / mL. The soluble protein content in the bamboo leaf aqueous extract with tea residues retained had a greater decrease, and the endogenous strain R1 and the commercial strain consumed nitrogen sources to a similar extent. After 3 days of fermentation, the soluble protein contents in the bamboo leaf aqueous extracts fermented by LA85, LB42, Lp90, and R1 were 18.81 ± 0.38, 21.92 ± 2.48, 24.35 ± 3.25, and 19.62 ± 1.64 μg / mL, respectively.

[0081] 5.4 Changes in Total Phenols Phenolic compounds usually exist in the form of glycosides and have poor absorption, but microbial biotransformation can improve their bioaccessibility and biological activity. As Figure 4 shown, in the clarified bamboo leaf compound tea, the total phenol content increased rapidly in the first 12 h of fermentation, probably because the lactic acid bacteria had strong metabolism and hydrolyzed conjugated phenols to release free phenols. After 36 h, the change in the total phenol content was slow. After 3 days of fermentation, the total phenol contents in the bamboo leaf compound teas fermented by LA85, LB42, Lp90, and R1 increased by 6.46%, 5.71%, 9.10%, and 14.35%, respectively. In the bamboo leaf compound tea with tea residues retained, the total phenol content decreased from 360 - 390 μg / mL to 300 - 340 μg / mL, probably because lactic acid bacteria transformed phenolic substances under stress to maintain growth.

[0082] 5.5 Changes in Organic Acids and Free Sugars The PCA model was used to perform dimensionality reduction processing on the variables, analyze the correlation and separation of each group of variables, and determine the important variables. As Figure 5 shown, the experimental groups and the control group were completely separated, indicating that lactic acid bacteria fermentation significantly affected the organic acids and free sugars in the samples. The experimental groups deviated towards the negative axis of PC1, and lactic acid, sucrose, fructose, and glucose were the characteristic components for separation. Malic acid, citric acid, succinic acid, oxalic acid, and sucrose decreased after fermentation and were positively correlated with the first component; lactic acid had a relatively high absolute value of the loading coefficient on the negative part of PC1 with fructose and glucose and was negatively correlated with the first component. Among them, the oxalic acid content decreased by 22.27 - 41.75% after fermentation, which was beneficial to the degradation of antinutritional factors.

[0083] After 3 days of fermentation with commercial strains LA85, LB42, Lp90 and the endogenous strain Lactobacillus plantarum R1, the lactic acid contents in the aqueous extracts of bamboo leaves retaining tea residues were 3.43±0.02, 3.35±0.01, 2.96±0.00, 3.39±0.00 mg / mL respectively, all higher than those in the clarified aqueous extracts of bamboo leaves after fermentation, and the lactic acid taste was mild and long-lasting.

[0084] 5.6 Changes in the electronic nose The aroma characteristics of the aqueous extracts of bamboo leaves after fermentation were detected by an electronic nose. From Figure 6 it can be seen that the experimental groups after fermentation were completely separated from the unfermented control group, indicating that fermentation significantly affected the aroma components of the aqueous extracts of bamboo leaves. The experimental groups were also completely separated from each other. The experimental group retaining tea residues moved further in the positive axis direction of PC1, with greater changes in aroma characteristics.

[0085] Load analysis showed that the positions of sensors W2W (aromatic compounds and organic sulfides), W1S (methane), W5S (nitrogen oxides), W2S (alcohols and some aromatic compounds) and W3S (alkanes) deviated significantly from the origin, with high load coefficients, indicating that these 5 sensors played an important role in distinguishing the changes in the aroma components of the aqueous extracts of bamboo leaves after fermentation.

[0086] 5.7 Isolation and comprehensive evaluation of different lactic acid bacteria strains (1)PLS-DA analysis PLS-DA analysis was performed on the above index data. From Figure 7 it can be seen that in the PLS-DA model, the sample groups after fermentation were significantly deviated from the untreated blank group, and the fermented samples retaining tea residues moved further, indicating that tea residues promoted the fermentation of lactic acid bacteria.

[0087] The VIP values and P values (VIP>1, P<0.05) indicated that components such as total phenols, oxalic acid, lactic acid, and fructose had a key impact on the aroma components with higher detection responses by the electronic nose, such as alkanes, nitrogen oxides, and aromatic alcohols.

[0088] After 200 permutation tests, the test value R2 was always higher than Q2, and both R2 and Q2 were always higher than the original values on the left (R2 0.108, Q2 -0.649), indicating that the model would not overfit and had strong applicability and predictability.

[0089] (2)EWM-TOPSIS comprehensive evaluation The EWM-TOPSIS comprehensive evaluation method combines entropy weight and TOPSIS method, and has been widely used in many fields due to its objectivity and practicality.

[0090] The fermentation of four strains of lactic acid bacteria in the aqueous extract of bamboo leaves for 3 days involves multiple indicators. The effects of different lactic acid bacteria on different indicators vary in strength. The comprehensive evaluation by EWM-TOPSIS helps to determine the most ideal strain. The weights (w) of each indicator are calculated by formula (3). As can be seen from Table 2, fructose (13.9840%), total phenols (10.7094%), and W5S (10.4548%) have relatively high weights among all indicators, and these weights help to reduce the inherent subjectivity of the TOPSIS method.

[0091] As can be seen from Table 3, Lactobacillus plantarum R1 has the best effect in fermenting the aqueous extract of bamboo leaves, with a ranking of 1 (the comprehensive score is 0.644). Lactobacillus plantarum R1 is determined to be the best fermentation strain.

[0092] Table 2. Summary of the weight calculation results based on the entropy weight method Table 3. Comprehensive evaluation results of EWM-TOPSIS 5.8 Changes in antioxidant capacity (1) Changes in ABTS scavenging ability Antioxidants play an important role in human health, used to reduce the risk of diseases, protect the body from various diseases caused by oxidative damage (such as diabetes, cancer, and neurodegenerative diseases), and can also control the oxidation process and prevent the decline in food quality caused by free radical reactions. Free radicals are one of the main causes of oxidative stress and can trigger various degenerative diseases, such as cancer, coronary heart disease, and vascular diseases.

[0093] The antioxidant capacity of the sample was determined by the ABTS free radical scavenging activity. The results showed that the fermentation of lactic acid bacteria could significantly improve the antioxidant capacity of the aqueous extract of bamboo leaves. As Figure 8 can be seen: within 0 h to 12 h, the ABTS free radical scavenging rate increased rapidly; within 12 h to 72 h, the ABTS free radical scavenging rates of the clarified strong aqueous extract of bamboo leaves remained above 95%. The scavenging rates of LA85, LB42, Lp90, and R1 after 3 days of fermentation were 97.74±0.01, 97.74±0.01, 97.63±0.01, and 97.72±0.32 %, respectively; the ABTS free radical scavenging rate of the strong aqueous extract of bamboo leaves retaining the filter residue decreased slightly. Among them, the ABTS scavenging rate of the Lp90 strain after 3 days of fermentation was the lowest, at 85.91±0.053 %, and the ABTS scavenging rate of the R1 strain was the highest, at 94.1±0.304 %, still above 90%.

[0094] (2) Changes in DPPH scavenging ability The DPPH scavenging ability is expressed in terms of the antioxidant Trolox equivalent. AsFigure 9 It can be seen that fermentation by different lactic acid bacteria can enhance the DPPH scavenging ability of the aqueous extract of bamboo leaves, increasing it from less than 10 μg Trolox / mL before fermentation to more than 140 μg Trolox / mL. After 3 days of fermentation, the DPPH scavenging abilities of the aqueous extracts of bamboo leaves retaining the substrate were 140.71±0.36, 147.36±0.21, 146.76±0.21, and 160.75±0.51 μg Trolox / mL respectively. Compared with the aqueous extracts of bamboo leaves retaining the substrate, the DPPH scavenging ability of the clarified aqueous extracts of bamboo leaves was stronger. The DPPH scavenging abilities of the clarified aqueous extracts of bamboo leaves after 3 days of fermentation by strains LA85, LB42, Lp90, and R1 were 193.81±0.21, 212.80±0.21, 211.22±0.21, and 211.42±0.48 μg Trolox / mL respectively. However, regardless of whether the aqueous extract of bamboo leaves retained the substrate or was clarified, the DPPH scavenging ability after fermentation by strain R1 was excellent and better than that of other lactic acid bacteria strains.

[0095] The above experimental results show that: the aqueous extract of bamboo leaves after fermentation by lactic acid bacteria has stronger antioxidant ability, and Lactobacillus plantarum R1 has more advantages in enhancing the antioxidant ability of the aqueous extract of bamboo leaves.

[0096] 5.9 Differences in antibacterial ability In this experiment, the Oxford cup method was used to evaluate the antibacterial activity by measuring the diameter of the inhibition zone. The results showed that the fermentation products of the aqueous extract of bamboo leaves after fermentation by lactic acid bacteria all showed certain inhibitory activity (inhibition zone diameter > 8 mm). Figure 10 It can be seen that: the unfermented aqueous extract of bamboo leaves (No-fermented) did not produce an obvious inhibition zone against the three pathogenic bacteria, indicating that its antibacterial activity was low. However, obvious inhibition zones appeared in most of the experimental groups after 3 days of fermentation by lactic acid bacteria, and the inhibition zones shown by the clarified aqueous extracts of bamboo leaves after fermentation by the same strain were larger than those of the experimental groups retaining the plant substrate.

[0097] In the antibacterial experiment against Escherichia coli ATCC 35218, except for the aqueous extract of bamboo leaves retaining the substrate after fermentation by Lactobacillus acidophilus, antibacterial properties were expressed in the other experimental groups. After fermentation by LA85, LB42, Lp90, and R1, the diameters of the inhibition zones of the clarified aqueous extracts of bamboo leaves were 12.04±0.25, 18.11±0.66, 23.24±0.60, and 24.86±0.35 mm respectively, while the diameters of the inhibition zones of the aqueous extracts of bamboo leaves retaining the substrate were 8.00, 13.58±0.17, 16.16±0.50, and 21.63±0.29 mm respectively. The antibacterial effect of the fermentation product of strain R1 was the best.

[0098] The fermentation product of strain R1 also has the strongest inhibitory ability against Klebsiella pneumoniae, and the diameters of the inhibition zones of its fermentation broth and the fermentation product of the bamboo leaf aqueous extract retaining the substrate are 19.3±0.12 and 22.00±0.36 mm, respectively. In the bamboo leaf aqueous extract retaining the substrate, no obvious inhibition zone was formed in the experimental groups fermented by LA85 and LB42, while that of Lp90 was 13.70±0.62 mm. In the clarified bamboo leaf aqueous extract, the diameters of the inhibition zones of the fermentation products of LA85, LB42 and Lp90 were 12.12±0.69, 19.66±0.41 and 21.48±0.94 mm, respectively, all of which were inferior to the fermentation product of strain R1.

[0099] Except for LA85, the experimental groups after fermentation all had obvious inhibitory ability against Staphylococcus aureus. The sizes of the inhibition zones of the clarified bamboo leaf aqueous extracts fermented by LB42, Lp90 and R1 were 18.25±0.40, 19.31±1.32 and 22.75±0.30 mm, respectively. The fermentation of the bamboo leaf aqueous extract retaining the filter residue produced inhibition zones of 19.27±1.08, 17.56±0.69 and 14.10±0.12 mm against Staphylococcus aureus.

[0100] Generally speaking, regardless of whether the filter residue is retained or not, the fermentation products of the bamboo leaf aqueous extract fermented by Lactobacillus plantarum R1 have good antibacterial activity, and the antibacterial activity is better than that of the other lactic acid bacteria.

[0101] It can be seen from the above experiments that: the fermentation degree of the bamboo leaf aqueous extract fermented by Lactobacillus plantarum R1 is the highest and the effect is the most ideal. Moreover, fermentation significantly enhances the antioxidant and antibacterial abilities of the bamboo leaf aqueous extract. The antioxidant ability and antibacterial effect of the fermentation product obtained by fermenting the bamboo leaf aqueous extract with Lactobacillus plantarum R1 are better than those of the fermentation products obtained by fermenting the bamboo leaf aqueous extract with other lactic acid bacteria.

[0102] Test Example 2: Cellulase-assisted fermentation of Lactobacillus plantarum R1 to solve phenolic loss and comparison of retaining filter residue and clarification The fermentation product prepared according to the method and results described in Example 1 (Lactobacillus plantarum R1, fermented bamboo leaf aqueous extract retaining the plant substrate and clarification) was optimized according to the method described in Example 2 (ultrasonic combined with cellulase enzymolysis, Lactobacillus plantarum R1, bamboo leaf aqueous extract retaining the plant substrate bamboo leaf aqueous extract), and the fermentation products of retaining filter residue and clarification were further compared.

[0103] 1. Determination of total phenol content The determination was carried out according to the method described in Test Example 1.

[0104] 2. Determination of phenolic compounds Phenols in the sample solution were adsorbed and purified using macroporous resin D101. Pretreatment: The D101 packing was soaked in 90% aqueous ethanol solution for 24 hours, and 5 g was weighed and put into a 100 mL Erlenmeyer flask. Adsorption: 50 mL of the sample solution was added, and it was treated on a shaker at 25°C and 120 r / min for 4 hours. Washing: The sample solution was poured out, and the resin was washed with distilled water. Desorption: 50 mL of 30% aqueous ethanol solution was added, and it was treated at 25°C and 120 r / min for 4 hours. The desorbed solution was collected, vacuum concentrated and then freeze-dried, and the dried powder was stored at -20°C.

[0105] The phenols in the sample solution after enrichment were determined using ultra-high performance liquid chromatography equipped with a diode array detector and an AQUITY UPLC® BEH C18 chromatographic column (2.1 mm × 50 mm × 1.7 μm) (Waters, USA). Elution conditions: The mobile phase was gradient elution (Table 4), ultrapure water plus 0.1% ( v / v ) acetic acid (mobile phase A), acetonitrile plus 0.1% ( v / v ) acetic acid (mobile phase B), the detection wavelength was 270 nm, the column temperature of the analytical column was 20 °C, the flow rate of the mobile phase was 0.5 mL / min, the injection volume was 10 μL, and the sample passed through a 0.22 μm filter membrane. Qualitative and quantitative analysis was carried out by the external standard method.

[0106] Table 4. UPLC elution program 3. Determination of free amino acids The types and contents of free amino acids in the sample solution were analyzed by an automatic amino acid analyzer. Before injection, to precipitate proteins and polypeptides in the sample solution to avoid interference, 4% sulfosalicylic acid solution in equal proportion was added to the fermentation supernatant and vortexed, mixed evenly, and left standing at 4°C for 1 h. Then it was centrifuged at 15000 r / min for 15 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane and then injected for analysis. The results were expressed as mg / mL.

[0107] 4. Identification of volatile components by GC-MS Extraction of volatile flavor components by low-temperature freezing liquid-liquid extraction (LTF-LLI): 5 mL of the sample solution was vortex-mixed with 3 mL of dichloromethane, placed upright in the refrigerator, frozen at -20 °C for 3 h, taken out and thawed, centrifuged at 6000 r / min for 6 min, and the lower organic phase was aspirated and dehydrated with anhydrous Na2SO4, and then passed through a 0.45 μm organic phase filter membrane for analysis. Chromatographic conditions: DB-5MS Ultra Inert chromatographic column (30 m × 250 μm × 0.25 μm); injection port temperature 280 °C; injection volume 1 μL; splitless injection; flow rate 1.0 mL / min; programmed temperature rise: held at 50 °C for 2 min, heated to 240 °C at a rate of 4 °C / min, and held for 5 min. Mass spectrometry conditions: EI source; electron energy 70 eV; ion source temperature 230 °C; transfer line temperature 280 °C; solvent delay 3 min; scanning range: m / z 50 - 450; carrier gas is high-purity helium. The database for comparing and identifying substances is Masshunter.

[0108] 5. Sensory evaluation Sensory evaluation was carried out to determine the differences in sensory attributes among samples fermented by various methods. The sensory evaluation was carried out in a sensory panel room at 25 ± 1 °C, and each sensory attribute of the fermentation broth was evaluated using Table 5. The members of the sensory panel were 10 non-expert students (5 females and 5 males), who received training before the evaluation. The fermented aqueous extracts of bamboo leaves were randomly coded with different numbers and provided to the panel members, who scored them according to color, odor, tissue state, taste, sourness and sweetness, and overall acceptability (calculated as the average of the scores of the previous five indicators).

[0109] Table 5. Scoring criteria for sensory evaluation Note: 17 - 20 is "excellent", 13 - 16 is "good", 9 - 12 is "barely acceptable", ≤8 is "rejected".

[0110] 6. Data processing and analysis SPSS 26.0 software was used for data processing, and Duncan's test was applied to calculate the significant differences. P < 0.05 indicates the existence of significant differences. OriginPro 2023 was used for plotting, and the experimental data were expressed as mean ± standard deviation.

[0111] 7. Results 7.1 Changes in total phenol content From Figure 11It can be seen that at the beginning of fermentation, the total phenol contents of the untreated and enzymatically hydrolyzed bamboo leaf aqueous extracts were 358.33±3.80, 336.63±7.80, 354.66±2.59, and 360.49±0.40 μg / mL, respectively. After 3 days of fermentation of the enzymatically hydrolyzed bamboo leaf aqueous extracts, the total phenol contents increased significantly. The total phenol contents of the bamboo leaf aqueous extracts with retained residues and clarified ones increased by 22.94% and 24.76% respectively, reaching 436.02±3.39 and 442.16±3.29 μg / mL, which were significantly higher than those of the experimental groups with only fermentation ( P <0.01).

[0112] Subsequently, the fermentation products of the clarified and residue-retained bamboo leaf aqueous extracts assisted by cellulase in the fermentation of Lactobacillus plantarum R1 were studied.

[0113] 7.2 Increase in gallic acid, chlorogenic acid, and luteolin As Figure 12 can be seen, during the 3-day fermentation process, the content of gallic acid increased by 112.79% and 82.94% respectively from 1.17±0.00 μg / mL before fermentation, possibly due to the hydrolysis of tannins in bamboo leaves and chrysanthemums and the hydroxylation and methylation of p-hydroxybenzoic acid. The content of chlorogenic acid increased significantly after fermentation, increasing by 6.39% and 12.08% in the clarified and residue-retained samples respectively. Contrary to the examples where the content of chlorogenic acid decreased significantly due to the production of esterase by most bacteria, Lactobacillus plantarum R1, an endogenous strain in pickled bamboo shoots, was proven to be able to increase the content of chlorogenic acid. The content of luteolin increased from 0.82±0.05 μg / mL to 1.29±0.01 and 1.03±0.02 μg / mL, possibly involving the conversion of complex polyphenols into flavonoids.

[0114] 7.3 Increase in sweet and hydrophobic amino acids and decrease in bitter amino acids As Figure 13 can be seen, during the whole fermentation process, the amino acids characterized by bitterness decreased significantly. Leucine (Leu) decreased from 9.00±0.00 μg / mL to 0.667±0 and 4.33±0.47 μg / mL after fermentation with retained residues and clarified samples respectively, phenylalanine (Phe) decreased from 22±0 μg / mL to 0.666666667±0 and 0 μg / mL, and tyrosine (Tyr) decreased from 8.25±0 μg / mL to 0. As hydrophobic amino acids, the increase in the contents of threonine (Thr) and valine (Val) is one of the reasons for the high antioxidant activity of the fermented bamboo leaf aqueous extracts. After 3 days of fermentation, the content of threonine increased from 0 to 1.33±0 and 2.00±0 μg / mL after fermentation with retained residues and clarified samples respectively, and the content of valine increased from 1.5±0 μg / mL to 16±0 and 18.67±0 μg / mL.

[0115] In addition, the change of free amino acids will affect the flavor and aroma of the water extract of bamboo leaves. Proline (Pro), as a sweet amino acid, can be used to enhance the flavor of beverages and is a common food flavor. After 3 days of fermentation, the content of proline in the water extract of bamboo leaves with retained residue and clarified fermentation increased from 22.63±0.18 μg / mL to 54.33±0.47 μg / mL and 45.33±0.00 μg / mL respectively.

[0116] 7.4 Enrichment of Volatile Aroma Components The liquid-liquid cryogenic extraction method combined with GC-MS was used to detect volatile flavor substances. The peaks in the total ion current chromatogram represent the volatile substances in the water extract of bamboo leaves ( Figure 14 ). Lactobacillus ferments in the water extract of bamboo leaves and produces a variety of volatile components through metabolic activities, giving the fermented product a unique odor. Before fermentation, the odor of the water extract of bamboo leaves was not obvious, and only 10 volatile components were detected. After fermentation, the types of volatile substances increased significantly ( Figure 15 ). There were 38 kinds in the water extract of bamboo leaves with retained residue and 33 kinds in the clarified one. After fermentation, alcohols, ketones, acids and esters became the main components. In the clarified water extract of bamboo leaves, 8, 6, 5 kinds increased respectively and 1 kind decreased, accounting for 72.73% of the total content; in the water extract of bamboo leaves with retained residue, the proportion reached 74.55%, with 9, 7, 6 and 3 more kinds than those before fermentation. A total of 55 volatile components were detected during the fermentation process, including alcohols, acids, phenols, olefins, aldehydes, esters, ketones, alkanes and furans. Many components were produced after fermentation and were related to the unique aroma. Among them, esters, alcohols and ketones had obvious odors such as floral and fruity, and acids had strong odors and low thresholds. The fermentation of the water extract of bamboo leaves by Lactobacillus plantarum R1 can not only highlight the flavor of the plant itself, but also form a variety of special flavor substances, and the water extract of bamboo leaves fermented with retained residue helps to accumulate more flavor components.

[0117] 7.5 Sensory Evaluation Results of Fermented Liquor Ten teachers and students from the School of Food Science and Technology conducted sensory evaluations on the fermented liquor with retained residue and clarified fermentation liquor to evaluate sensory attributes and acceptability. The data obtained from the survey were presented in the form of a radar chart to evaluate the attributes of aroma, acidity, color, texture, flavor and overall acceptability ( Figure 16 ). Compared with the clarified fermented liquor, it was observed that the fermented liquor with retained residue had higher scores in terms of olfaction and overall acceptance, especially in terms of odor ( P(<0.05), while other sensory attributes are relatively close. For the fermented liquid with residue retained, the responses of all attributes except sourness are above 12 (expressed as barely acceptable). Regarding the acceptability of aroma and acidity, preferences and rejections for the fermented liquid with residue retained were expressed, with average values of 7.73 and 19.09 respectively. In contrast, for the same attributes, the values shown by the clarified fermented liquid were 8.00 and 13.57 respectively, indicating that its sourness was also rejected, while the aroma was not as appealing.

[0118] Generally speaking, lactic acid bacteria ferment well in the aqueous extract of bamboo leaves, and the sensory score of the aroma produced by fermenting with residue retained is higher.

[0119] As can be seen from Test Example 2: Using cellulase-assisted fermentation of Lactobacillus plantarum R1 can improve the phenolic, amino acid, and aroma components of the fermentation product of the aqueous extract of bamboo leaves, and the sensory score of the substrate retained is higher. While achieving excellent antioxidant and antibacterial properties of the fermentation product, the taste of the fermentation product is improved.

[0120] In summary, the present invention provides a fermentation product obtained by fermenting bamboo leaf extract with the endogenous strain Lactobacillus plantarum R1 of pickled bamboo shoots. This fermentation product has excellent antioxidant and antibacterial properties, superior to the fermentation products obtained by fermenting bamboo leaf extract with other lactic acid bacteria. Treating the bamboo leaf extract before fermentation with cellulase can increase the phenolic, amino acid, and aroma components of the fermentation product, and improve the taste of the fermentation product. A bamboo leaf composite beverage with a good taste is prepared using this fermentation product. The present invention expands the uses of bamboo leaves, increases the added value of bamboo industry production, and has good application prospects.

Claims

1. A bamboo leaf fermentation product, characterized in that: It is a fermentation product obtained by fermenting bamboo leaves with Lactobacillus plantarum R1; the Lactobacillus plantarum R1 is a strain preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M2022659.

2. The bamboo leaf fermentation product according to claim 1, characterized in that: When using Lactobacillus plantarum R1 to ferment bamboo leaves, the bamboo leaves are bamboo leaf water extract.

3. The bamboo leaf fermentation product according to claim 2, characterized in that: The bamboo leaf water extract is a water extract prepared from raw materials with the following weight ratios: 1 - 5 parts of bamboo leaves, 0.1 - 1 part of chrysanthemum, and 7 - 10 parts of granulated sugar.

4. The bamboo leaf fermentation product according to claim 3, wherein: The preparation method of the bamboo leaf fermentation product includes the following steps: (1) Take bamboo leaves, chrysanthemum, and granulated sugar, dissolve the granulated sugar in water, immerse the bamboo leaves and chrysanthemum in water for extraction, and filter or not filter after extraction to obtain bamboo leaf water extract; (2) Inoculate the Lactobacillus plantarum R1 seed liquid into the bamboo leaf water extract obtained in step (1) for fermentation to obtain the product.

5. The fermented product of bamboo leaves according to claim 4, characterized in that: In step (1), the obtained bamboo leaf water extract is enzymatically hydrolyzed with cellulase.

6. The bamboo leaf fermentation product according to claim 4, wherein: In step (1), the concentration of bamboo leaves is 0.005 - 0.05 mg / ml; the concentration of chrysanthemum is 0.0001 - 0.005 mg / ml; the concentration of granulated sugar is 0.01 - 0.05 mg / ml; and / or, in step (1), the extraction temperature is 80 - 100 °C, and the extraction time is 30 - 60 min; and / or, in step (1), stirring is performed during extraction, and the stirring speed is 500 - 1000 r / min; and / or, in step (1), filtration is not performed after extraction.

7. The fermented product of bamboo leaves according to claim 4, characterized in that: In the step (2), the fermentation temperature is 35~37 °C, the fermentation time is 24~72 h, and the density of the Lactobacillus plantarum R1 seed liquid during fermentation is 10 8 ~ 10 10 CFU / mL, and the inoculation amount of the Lactobacillus plantarum R1 seed liquid is 1~3%.

8. The bamboo leaf fermentation product according to claim 5, wherein: When enzymatically hydrolyzing with cellulase, the pH of the bamboo leaf water extract is adjusted to 4 - 6; and / or, when enzymatically hydrolyzing with cellulase, the addition amount of cellulase is 2 - 4%; and / or, when enzymatically hydrolyzing with cellulase, the enzymatic hydrolysis temperature is 30 - 50 °C, and the enzymatic hydrolysis time is 1 - 5 h; and / or, when enzymatically hydrolyzing with cellulase, ultrasonic treatment is performed, the power of ultrasonic is 60 - 100 W, and the ultrasonic time is 20 - 60 min.

9. Use of the bamboo leaf fermentation product according to any one of claims 1 - 8 in the preparation of functional foods.

10. A functional beverage, characterized in that: It is prepared from the bamboo leaf fermentation product according to any one of claims 1 - 8 as the raw material, plus food - acceptable excipients.

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