Fermentation product of bamboo leaves fermented by lactic acid bacteria, use thereof, and beverage prepared therefrom

Abstract

The present invention provides a fermentation product of bamboo leaves fermented by lactic acid bacteria, its use and a beverage prepared therefrom, and belongs to the field of biological fermentation. The fermentation product of the present invention 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 a preservation number of CCTCC NO: M 2022659. The fermentation product of the present invention has excellent antioxidant and antibacterial properties, and is superior to fermentation products obtained by fermenting bamboo leaf extracts with other lactic acid bacteria. Treating the bamboo leaves 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 the fermentation product. The present invention expands the use of bamboo leaves, increases the added value of bamboo industry production, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biological fermentation, and particularly relates to a fermentation product of bamboo leaves fermented by lactic acid bacteria, a use thereof, and a beverage prepared therefrom. Background Art

[0002] Bamboo leaves, the leaves of the genus Bambusa in the Poaceae family, are a natural plant with both medicinal and edible uses. However, bamboo leaves, a product of bamboo, have long been considered waste, resulting in a significant waste of agricultural biomass resources. Bamboo leaves have been shown to contain a variety of active ingredients with positive biological and pharmacological activities, such as antioxidant, anti-inflammatory, anti-cancer, anti-apoptotic, and lipid-lowering properties. However, their development and application in the food industry is insufficient, hindering their potential as both a medicinal and edible food. Therefore, developing foods using bamboo leaves as an ingredient is an effective means of realizing their pharmacological and edible properties.

[0003] Lactic acid fermentation is a common food processing method with numerous practical applications. Fermented foods inoculated with lactic acid bacteria can impart unique flavors and promote the release of physiological activities, potentially extending their potential as both medicinal and edible. However, further research is needed to explore how to enhance the utility of bamboo leaves through lactic acid fermentation and expand their application in the food industry. Summary of the Invention

[0004] The present invention aims to provide a fermentation product of bamboo leaves fermented by lactic acid bacteria, its use and a beverage prepared therefrom.

[0005] The 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 a preservation number of CCTCC NO: M 2022659.

[0006] Furthermore, the bamboo leaves fermented by Lactobacillus plantarum R1 are bamboo leaf water extracts.

[0007] Furthermore, the bamboo leaf water extract is a water extract prepared from raw materials in the following weight ratio:

[0008] 1-5 parts of bamboo leaves, 0.1-1 part of chrysanthemum, and 7-10 parts of white sugar.

[0009] Preferably, the bamboo leaf aqueous extract is an aqueous extract prepared from raw materials in the following weight ratio:

[0010] 4 parts of bamboo leaves, 0.8 parts of chrysanthemums, and 7.2 parts of white sugar.

[0011] Furthermore, the preparation method of the bamboo leaf fermentation product comprises the following steps:

[0012] (1) Take bamboo leaves, chrysanthemums and white sugar, dissolve the white sugar in water, immerse the bamboo leaves and chrysanthemums in water for extraction, and filter or not filter after extraction to obtain bamboo leaf water extract;

[0013] (2) Inoculating Lactobacillus plantarum R1 seed liquid into the bamboo leaf water extract obtained in step (1) and fermenting to obtain the product.

[0014] Furthermore, in the step (1), the obtained bamboo leaf water extract is hydrolyzed with cellulase.

[0015] Furthermore,

[0016] In the 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 white sugar is 0.01-0.05 mg / ml;

[0017] And / or, in step (1), the extraction temperature is 80-100°C and the extraction time is 30-60 minutes;

[0018] And / or, in step (1), the extraction is performed with stirring at a speed of 500-1000 r / min;

[0019] And / or, in step (1), no filtration is performed after extraction.

[0020] Furthermore, in the step (2), the fermentation temperature is 35-37°C, the fermentation time is 24-72 hours, and the density of the Lactobacillus plantarum R1 seed liquid during fermentation is 10 8 ~ 10 10 CFU / mL, and the inoculation amount of Lactobacillus plantarum R1 seed liquid was 1~3%.

[0021] Furthermore,

[0022] During the enzymatic hydrolysis with cellulase, the pH of the bamboo leaf water extract is adjusted to 4-6;

[0023] And / or, the amount of cellulase added during the enzymatic hydrolysis with cellulase is 2-4%;

[0024] And / or, the enzymatic hydrolysis temperature during the enzymatic hydrolysis with cellulase is 30-50° C., and the enzymatic hydrolysis time is 1-5 h;

[0025] And / or, ultrasound is performed during the enzymatic hydrolysis with cellulase, the ultrasound power is 60-100 W, and the ultrasound time is 20-60 min.

[0026] Preferably,

[0027] During the enzymatic hydrolysis of the cellulase, the pH of the bamboo leaf water extract is adjusted to 5;

[0028] And / or, the amount of cellulase added during the cellulase enzymolysis is 3%; the enzymatic activity of the cellulase is 16 U / mg;

[0029] And / or, the enzymatic hydrolysis temperature during the cellulase hydrolysis is 35° C. and the enzymatic hydrolysis time is 3 h;

[0030] And / or, ultrasound is performed during the cellulase enzymolysis, the ultrasound power is 90W, and the ultrasound time is 40 minutes.

[0031] The present invention also provides use of the aforementioned bamboo leaf fermentation product in preparing functional foods.

[0032] The present invention also provides a functional beverage, which is prepared by taking the above-mentioned bamboo leaf fermentation product as raw material and adding auxiliary materials acceptable to food.

[0033] Preferably, the functional beverage is prepared by homogenizing the following raw materials and auxiliary materials in parts by weight and volume:

[0034] 100 parts by volume of the aforementioned bamboo leaf fermentation product, 1-10 parts by weight of erythritol, and 0.1-1 part by weight of xanthan gum.

[0035] In the present invention, the bamboo leaf water extract refers to a mixture obtained by dissolving white sugar in water and immersing bamboo leaves and a small amount of chrysanthemum in water for a period of time. The solid components in the mixture may not be filtered, or the solid components may be filtered to obtain a solution.

[0036] The present invention has achieved the following beneficial effects:

[0037] The present invention provides a fermentation product obtained by fermenting bamboo leaf extract using the endogenous strain Lactobacillus plantarum R1. This fermentation product has excellent antioxidant and antibacterial properties, surpassing those obtained by fermenting bamboo leaf extracts with other lactic acid bacteria. Treating the pre-fermented bamboo leaf extract with cellulase increases the phenolic, amino acid, and aroma components of the fermentation product, improving its taste. This fermentation product can be used to prepare a bamboo leaf composite beverage with a good taste. This invention expands the uses of bamboo leaves, increases the added value of bamboo industry production, and has promising application prospects.

[0038] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0039] The following is a further detailed description of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-mentioned content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 These are graphs showing changes in pH during the fermentation of bamboo leaf water extracts by lactic acid bacteria: A is a graph showing changes in pH during the fermentation of clarified plant bamboo leaf water extracts by different lactic acid bacteria; B is a graph showing changes in pH during the fermentation of bamboo leaf water extracts with retained bamboo leaves and chrysanthemum bases by different lactic acid bacteria.

[0041] Figure 2 These are graphs showing changes in total sugar content during the fermentation of bamboo leaf water extracts by lactic acid bacteria: A shows changes in total sugar content during the fermentation of clarified bamboo leaf water extracts by different lactic acid bacteria; B shows changes in total sugar content during the fermentation of bamboo leaf water extracts with retained bamboo leaves and chrysanthemum bases by different lactic acid bacteria.

[0042] Figure 3 These are graphs showing changes in soluble protein during the fermentation of bamboo leaf water extract by lactic acid bacteria: A is a graph showing changes in soluble protein during the fermentation of clarified plant bamboo leaf water extract by different lactic acid bacteria; B is a graph showing changes in soluble protein during the fermentation of bamboo leaf water extract with retained bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0043] Figure 4 These are graphs showing changes in total phenols during the fermentation of bamboo leaf water extract by lactic acid bacteria: A is a graph showing changes in total phenols during the fermentation of clarified plant bamboo leaf water extract by different lactic acid bacteria; B is a graph showing changes in total phenols during the fermentation of bamboo leaf water extract with retained bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0044] Figure 5 This is the PCA diagram of organic acids and free sugars during the fermentation of bamboo leaf water extract by lactic acid bacteria: A is the score diagram; B is the loading diagram.

[0045] Figure 6 This is the PCA diagram of the electronic nose analysis during the lactic acid bacteria fermentation of bamboo leaf water extract: A is the score diagram; B is the loading diagram.

[0046] Figure 7 These are the PLS-DA analysis graphs of four lactic acid bacteria before and after fermentation of bamboo leaf water extract: A is the score graph; B is the 200-time permutation test graph; C is the VIP score graph.

[0047] Figure 8 These are graphs showing changes in ABTS free radical scavenging rates during the fermentation of bamboo leaf water extracts by lactic acid bacteria: A is a graph showing changes in ABTS free radical scavenging rates during the fermentation of clarified bamboo leaf water extracts by different lactic acid bacteria; B is a graph showing changes in ABTS free radical scavenging rates during the fermentation of bamboo leaf water extracts with retained bamboo leaves and chrysanthemum bases by different lactic acid bacteria.

[0048] Figure 9 The graphs show the changes in DPPH radical scavenging ability during the fermentation of bamboo leaf water extract by lactic acid bacteria: A shows the changes in DPPH radical scavenging ability during the fermentation of clarified plant bamboo leaf water extract by different lactic acid bacteria; B shows the changes in DPPH radical scavenging ability during the fermentation of bamboo leaf water extract with retained bamboo leaves and chrysanthemum base by different lactic acid bacteria.

[0049] Figure 10 Figure 3 shows the difference in antibacterial ability of bamboo leaf water extracts before and after fermentation with different lactic acid bacteria: A is the inhibition zone diagram of different test groups; B is the radar diagram of the inhibition zone diameters of different test groups, in mm, and the outer diameter of the Oxford cup is 8 mm.

[0050] Figure 11 The graph shows the changes in total phenol content before and after bacterial enzyme cooperative fermentation and fermentation alone; in the graph, “**” represents statistically P <0.01, the difference is extremely significant; A means retaining the base, B means clarifying; UH means unenzymatically hydrolyzed, Cel-EH means hydrolyzed with cellulase.

[0051] Figure 12 The graph shows the changes in phenolic compounds in bamboo leaf water extract before and after bacterial enzyme fermentation; in the graph, “*” represents statistically P <0.05, “**” represents statistical significance P <0.01, “***” means statistically P <0.001.

[0052] Figure 13 The graph shows the changes in amino acids in bamboo leaf water extract before and after bacterial enzyme fermentation; in the graph, “*” represents statistically P <0.05, “**” represents statistical significance P <0.01, “***” means statistically P <0.001.

[0053] Figure 14 This is the total ion current chromatogram of GC-MA analysis of bamboo leaf water extract before and after enzyme fermentation.

[0054] Figure 15 This is a stacked graph of the number of species and peak areas of volatile aroma components in bamboo leaf water extract before and after enzyme fermentation.

[0055] Figure 16 This is a radar chart of the sensory evaluation of the fermentation broth: the odor score of the retained base fermentation broth was significantly higher than that of the clarified fermentation broth, and * represents P < 0.05, which is statistically significant. DETAILED DESCRIPTION

[0056] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0057] Mazhu ( Dendrocalamus latiflorus Munro ) bamboo leaves were collected from Longhanling Forest Farm, Liunan District, Liuzhou City, Guangxi Zhuang Autonomous Region; chrysanthemum (Hangzhou chrysanthemum) and white sugar were commercially available; Lactobacillus plantarum R1 ( Lactobacillus plantarum. R1) is a strain preserved in our laboratory and was deposited in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, Hubei, China on May 18, 2022, with the deposit number CCTCC NO: M 2022659. This strain is also described in a Chinese patent application with application number 202210576703.3 filed on May 25, 2022; Escherichia coli (ATCC 35218), Staphylococcus aureus ( Staphylococcus aureus. ATCC6538), Klebsiella pneumoniae ( Klebsiella pneumonia. ATCC 13883) is a standard strain and can be purchased; Lactobacillus plantarum ( L. plantarum. Lp90), Lactobacillus bulgaricus ( L.bulgaricus. LB42), Lactobacillus acidophilus ( L. acidophilus. LA85) was purchased from Weikang Probiotics (Suzhou) Co., Ltd. and stored in the form of freeze-dried powder.

[0058] The bamboo leaves used in the present invention are leaves of the bamboo, which have high edible value, are important materials for making rice dumplings as rice dumplings leaves, and can also be used for brewing wine.

[0059] Example 1: Fermentation of bamboo leaves by Lactobacillus plantarum R1 to obtain fermentation products

[0060] 1. Preparation of bamboo leaf water extract

[0061] Raw material pretreatment: Fresh bamboo leaves were harvested from Liuzhou, Guangxi. Insect pests and discolored leaves were removed. After cleaning, they were oven-dried at 50°C and crushed in a crusher for 5 minutes until they passed an 800-mesh sieve. Chrysanthemums were dried from Hangzhou and stored in a cool, dry place with the chopped bamboo leaves.

[0062] Preparation of bamboo leaf aqueous extract: 4.0 g of bamboo leaves, 0.80 g of chrysanthemum flowers, and 7.20 g of white sugar were soaked in 240 mL of distilled water. The mixture was stirred at 80°C and 900 rpm for 30 min. This extract was either left unfiltered to retain the plant base or filtered to obtain a clarified extract. Both extracts were sterilized by boiling in water for 10 min for subsequent use.

[0063] 2. Preparation of seed solution

[0064] Lactobacillus plantarum R1 was frozen at -80°C, taken out and thawed at room temperature, streaked onto MRS agar medium, and single colonies with normal morphology and size were 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 10 8 CFU / ml or so.

[0065] 3. Fermentation of bamboo leaf water extract by Lactobacillus plantarum R1 to prepare fermentation products

[0066] The Lactobacillus plantarum R1 seed solution was centrifuged at 4000 r / min for 5 min, the culture medium was discarded, and the solution was dissolved in sterile water and vortexed to obtain a resuspension (the density of Lactobacillus plantarum R1 in the resuspension was 10 8 The resuspension was inoculated into bamboo leaf water extract (clarified bamboo leaf water extract or bamboo leaf water extract with plant base retained) at an inoculum size of 1% (v / v) for fermentation. The fermentation was allowed to proceed at 37 °C for 72 h to obtain the fermentation product.

[0067] Example 2: Fermentation of bamboo leaf water extract by cellulase hydrolysis by Lactobacillus plantarum R1 to obtain fermentation product

[0068] 1. Preparation of bamboo leaf water extract

[0069] According to the method described in Example 1, a clear bamboo leaf water extract or a bamboo leaf water extract retaining a plant base is prepared.

[0070] 2. Ultrasound-assisted cellulase enzymatic hydrolysis of bamboo leaf water extract

[0071] The pH of the bamboo leaf aqueous extract obtained in step 1 was adjusted to 5 using food-grade citric acid and baking soda. 3% (v / v) cellulase (16 U / mg) was then added and enzymatic hydrolysis was carried out at 35°C for 3 hours, accompanied by ultrasonication at 90 W for 40 minutes. After enzymatic hydrolysis, the enzyme was inactivated in boiling water for 5 minutes, and the extract was cooled to room temperature to obtain the fermented bamboo leaf aqueous extract.

[0072] 3. Preparation of seed solution

[0073] Same as Example 1.

[0074] 4. Fermentation of bamboo leaf water extract by Lactobacillus plantarum R1 to prepare fermentation products

[0075] The bamboo leaf water extract obtained in step 2 was fermented according to the method described in Example 1 to obtain a fermentation product.

[0076] Regarding enzymatic hydrolysis conditions, the present invention screened parameters such as pH value (4, 4.5, 5, 5.5, 6), cellulase content (2, 2.5, 3, 3.5, 4%) (v / v), hydrolysis temperature (30, 35, 40, 45, 50°C), 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 indicator. The screening conditions were based on a temperature of 40°C, a hydrolysis pH of 5, a hydrolysis time of 3 h, a cellulase addition of 3.5% (160 U / mL), an ultrasonic time of 40 min, and an ultrasonic power of 80 W. The reducing sugar determination method is as follows:

[0077] (1) Determine the reducing sugar content using 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, and then add 23 mL of glycerol. After fully dissolving, adjust the volume to 500 mL.

[0078] (2) Preparation of standard curve: Weigh a series of glucose standard solutions into a 10 mL colorimetric tube, add distilled water to 1 mL, accurately add 2 mL of DNS reagent, heat in a boiling water bath for 5 min, cool in running water, and add water to 8 mL. Measure the absorbance at a wavelength of 540 nm. The standard curve is obtained: (y = 0.3064x + 0.0577, R 2 = 0.9975).

[0079] (3) Determination of reducing sugar content in samples: Take 1 mL of the sample to be tested and 2 mL of DNS reagent diluted appropriately, add them to a 10 mL colorimetric tube, place it in a boiling water bath for 5 min, measure the absorbance at a wavelength of 540 nm, and calculate the reducing sugar content in the sample based on the standard curve and the dilution multiple.

[0080] Finally, according to the test results, the optimal parameters were determined to be pH 5, cellulase (16 U / mg) addition amount of 3% (v / v), enzymatic hydrolysis temperature of 35°C, enzymatic hydrolysis time of 3h, ultrasonic power of 90W, and ultrasonic time of 40min. The enzymatic hydrolysis product obtained had the highest reducing sugar content of 2.30±0.01 mg / mL.

[0081] Example 3: Preparation of a compound beverage using the fermentation product obtained by fermenting bamboo leaves with Lactobacillus plantarum R1

[0082] The compound beverage was prepared using the fermentation product prepared in Example 1 or Example 2. In Example 3, the fermentation product prepared using the bamboo leaf water extract retaining the plant base in Example 2 was used as an example.

[0083] 100 mL of the fermentation product was taken, and 5 wt% of erythritol and 0.15 wt% of xanthan gum were added respectively. The mixture was mixed and homogenized (the homogenization speed was 12000 r / min and the time was 3 min) to obtain a composite beverage. The composite beverage was pasteurized and then stored.

[0084] The prepared compound beverage has excellent sensory evaluation, and its microbial indicators meet the requirements of the "National Food Safety Standard Beverages" (GB 7101-2022).

[0085] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0086] Experimental Example 1: Study on the fermentation products of bamboo leaves by different lactic acid bacteria

[0087] 1. Preparation of bamboo leaf water extract

[0088] Raw material pretreatment: Fresh bamboo leaves were harvested from Liuzhou, Guangxi. Insect pests and discolored leaves were removed. After cleaning, they were oven-dried at 50°C and crushed in a crusher for 5 minutes until they passed an 800-mesh sieve. Chrysanthemums were dried from Hangzhou and stored in a cool, dry place with the chopped bamboo leaves.

[0089] Preparation of bamboo leaf water extract: Eight groups were prepared. In each group, 4.0 g of bamboo leaves, 0.80 g of chrysanthemum and 7.20 g of white sugar were soaked in 240 mL of distilled water. The samples were magnetically stirred at 900 r / min at 80°C for 30 min. Four groups of samples were filtered to obtain clarified bamboo leaf water extracts. The remaining four groups of samples retained the plant base to obtain bamboo leaf water extracts with retained plant base. The bamboo leaf water extracts were sterilized by heating them with boiling water for 10 min.

[0090] 2. Preparation of seed solution

[0091] Lactobacillus plantarum R1 was frozen at -80°C, taken out and thawed at room temperature, streaked onto MRS agar medium, and single colonies of normal morphology and size were picked and inoculated into MRS broth medium for activation. Lyophilized powders of Lactobacillus plantarum Lp90, Lactobacillus bulgaricus LB42, and Lactobacillus acidophilus LA85 were directly placed into MRS broth medium for activation and rejuvenation. All strains were activated for two generations and then cultured in liquid culture at 37°C for 10 8 CFU / ml or so.

[0092] 3. Bamboo leaf water extract fermented by different lactic acid bacteria

[0093] The seed liquid of four lactic acid bacteria (Lactobacillus plantarum R1, Lactobacillus plantarum Lp90, Lactobacillus bulgaricus LB42 and Lactobacillus acidophilus LA85) was centrifuged at 4000 r / min for 5 min, the culture medium was discarded, the solution was dissolved in sterile water, and the suspension was vortexed to obtain a resuspension (the density of Lactobacillus plantarum R1 in the resuspension was 108 CFU / mL) and inoculated the resuspension at a 1% (v / v) inoculum into bamboo leaf aqueous extract for fermentation. Each lactic acid bacteria resuspension was inoculated into two separate bamboo leaf aqueous extracts, one clarified and one retaining the plant matrix. Fermentation was allowed to proceed at 37°C for 72 hours, with samples collected every 12 hours. The samples were centrifuged at 8000 rpm for 5 minutes to obtain the fermentation supernatant, from which the corresponding indices were measured.

[0094] 4. Index determination

[0095] 4.1 pH determination and color difference analysis

[0096] The pH value of the bamboo leaf water extract during fermentation was measured using a pH meter.

[0097] Add 2 mL of each sample solution into the cuvette and measure the L* (brightness), a* (redness), b* (yellow-blue) value, total color difference ( ΔE ) is calculated according to the following formula (1):

[0098] (1)

[0099] in, L 0 *、a 0 *、b 0 * are the color parameter values ​​of the standard solution, L * The larger the value, the higher the brightness. a * The larger the value, the darker the red (conversely, the darker the green, the lighter the red). b * The larger the value, the darker the yellow (conversely, the darker the blue, the lighter the yellow).

[0100] 4.2 Determination of basic physical and chemical indicators

[0101] (1) Determination of total sugar: The phenol-sulfuric acid method was used. 0.5 mL of 5% phenol aqueous solution, 5 mL of concentrated sulfuric acid, and 0.5 mL of fermentation supernatant diluted 200 times were added, mixed, and allowed to react for 5 min. The mixture was then placed in a boiling water bath for 15 min. After removal, the mixture was cooled to room temperature and the absorbance at 490 nm was measured. According to the previously obtained standard curve y = 4.3909x + 0.0103 (R 2 = 0.9964, with a good linear relationship in the range of 0.01-0.1 mg / mL) to calculate the total sugar content.

[0102] (2) Determination of soluble protein content: Coomassie Brilliant Blue G-250 assay kit (Bradford method) was used for determination. 200 μL of Coomassie Brilliant Blue G-250 reagent and 20 μL of fermentation supernatant were added to each well of a 96-well plate, mixed and reacted at room temperature for 5 min, and the absorbance was measured at 595 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific, USA). According to the pre-obtained standard curve y = 0.0037x + 0.0004 (R 2 = 0.9936, with a good linear relationship in the range of 5-25 μg / mL) to calculate the soluble protein content.

[0103] (3) Determination of total phenol content: Gallic acid was used as the standard and the Folin-phenol method was used with appropriate modifications. 0.5 mL of Folin-phenol reagent, 1 mL of 10% Na2CO3 aqueous solution, and 1 mL of fermentation supernatant were added and mixed. The mixture was allowed to react in the dark at room temperature for 1 h and the absorbance at 765 nm was measured. According to the gallic acid standard curve y = 0.0177x - 0.0099 (R 2 = 0.9979, with a good linear relationship in the range of 2.5-30 μg / mL) to calculate the total phenol content.

[0104] 4.3 Determination of organic acid and free sugar indexes

[0105] Organic acids were determined using UPLC-PDA (Waters Technologies, Inc., USA). Chromatographic conditions included an AQUITY UPLC® BEH C18 column (2.1 mm × 100 mm × 1.7 μm) (Waters, USA); the mobile phase consisted of 5% acetonitrile in water (containing 0.1% phosphoric acid); a flow rate of 0.15 mL / min; a column temperature of 30°C; UV detection at 210 nm; an injection volume of 4 μL; and isocratic elution. The fermentation supernatant obtained by centrifugation was filtered through a 0.22 μm aqueous filter, diluted 20-fold, and directly injected for analysis. Organic acids in the fermentation broth were qualitatively and quantitatively determined using retention times and calibration curves derived from organic acid standards. Results are expressed in μg / mL.

[0106] Free sugars were determined by HPLC-ELSD (Waters Technologies, Inc., USA). Chromatographic conditions included a ShimNex HE-NH2 column (250 mm × 4.6 mm, 5 μm) (Shimadzu Corporation, Japan); the mobile phase consisted of acetonitrile-water (85:15); column temperature: 30°C; flow rate: 1.0 mL / min; drift tube temperature: 80°C; carrier gas flow rate: 2.0 mL / min; gain: 10; and injection volume: 10 μL. The fermentation supernatant was filtered through a 0.22 μm aqueous filter and directly injected for analysis. Qualitative quantification was performed using an external standard method, and results are expressed in μg / mL.

[0107] 4.4 Electronic nose analysis

[0108] The electronic nose (PEN3, Airsense Analytics, Schwerin, Germany) was equipped with a metal oxide semiconductor (MOS) sensor array consisting of 10 different MOS sensors. First, a 20 mL sample of fermentation supernatant was collected in a 50 mL vial and allowed to equilibrate at a constant temperature (28°C) for 30 minutes before insertion of the electronic nose probe for detection. The specific parameters for the E-nose detection were a 90 s detection time, a 300 s cleaning time, a 400 mL / min carrier gas flow rate, and a 400 mL / min injection flow rate.

[0109] 4.5 Comprehensive evaluation of different lactic acid bacteria strains

[0110] Partial least squares discriminant analysis (PLS-DA) was used to effectively distinguish between experimental groups. Key indicators were identified based on projected importance variables (VIP values) and intergroup differences (P values). These key indicators were then evaluated using the Entropy Weighted-Top-Solution Similarity Ranking Technique (EWM-TOPSIS) method (Lu et al. 2024). The EWM-TOPSIS method mitigates the inherent subjectivity in assigning weights. The positive ideal solution (D⁺) and the negative ideal solution (D⁻) correspond to ideal solutions with the maximum and minimum attribute values, respectively, representing the performance of all indicators. Four lactic acid bacteria strains were then ranked based on the relative distances between the evaluation indicators and the solutions, comparing the results to those of untreated bamboo leaf extract to identify the strain with the best fermentation performance. Specifically, the following were used:

[0111] (2)

[0112] Indicates the calculation of entropy value for the indicator. Among them, 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 index.

[0113] (3)

[0114] Expressed as the weight w of the jth indicator j Among them, d j is the coefficient of variation of the jth indicator, and n is the total number of indicators.

[0115] (4)

[0116] (5)

[0117] Z + 、Z - Represent positive ideal solutions and negative ideal solutions respectively.

[0118] (6)

[0119] It is expressed as 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.

[0120] (7)

[0121] Expressed as the closeness coefficient C between the evaluation solution and the ideal value i Among them, C i The range is [0,1], and C i The higher the value of , the higher the comprehensive score. According to the comprehensive comparison ratio, plan Si is the best choice.

[0122] 4.6 Determination of antioxidant index

[0123] (1) Determination of DPPH scavenging ability

[0124] A DPPH radical scavenging assay was used to determine changes in DPPH radical scavenging activity during fermentation in different experimental groups. The fermentation supernatant after centrifugation was diluted fourfold and mixed with the reagent. The mixture was reacted at room temperature in the dark for 30 minutes. After centrifugation at 4000 rpm for 5 minutes, the absorbance of the reaction solution was measured at 517 nm using 80% methanol as a blank control. A standard curve was constructed by comparing the DPPH radical scavenging rate with the concentration of the standard Trolox. The DPPH radical scavenging activity was calculated from the absorbance, and the results were expressed as Trolox equivalents.

[0125] (2) Determination of ABTS scavenging ability

[0126] An ABTS free radical scavenging assay was used to determine changes in total antioxidant capacity during fermentation in different experimental groups. The centrifuged fermentation supernatant was mixed with the reagent and allowed to react 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, calculated as follows:

[0127] (8)

[0128] 4.7 Determination of antibacterial activity

[0129] The total colony count of three indicator bacteria, Escherichia coli ATCC 35218, Staphylococcus aureus ATCC 6538, and Klebsiella pneumoniae 18188, was adjusted to 10 7 Approximately 100 CFU / mL. Using the Oxford cup method, add 30 mL of LB agar as a base layer to a culture dish and insert a sterile Oxford cup. Add the indicator bacteria solution at a ratio of 1% (v / v) to the LB agar cooled to 50°C, mix thoroughly, and immediately pour onto the plate. After the culture medium has completely solidified, remove the Oxford cup and add 180 μL of the supernatant to be tested to the well. Perform three parallel assays, using sterile saline as a control. Let the plate stand for 1 hour to allow the supernatant to fully diffuse. Then, place the plate upright in a 37°C incubator and incubate for 12 hours. Measure the diameter of the inhibition zone (in mm).

[0130] 4.8 Data Processing and Analysis

[0131] SPSS 26.0 software was used for data processing, and Duncan test was used to calculate significant differences. P A significant difference was considered when the difference was < 0.05. Graphs were drawn using OriginPro 2023, and all experimental data were expressed as mean ± standard deviation.

[0132] 5. Results and Analysis

[0133] 5.1 pH changes during fermentation

[0134] During the lactic acid bacteria fermentation process, the fermentation degree of bamboo leaf water extract can be measured by pH changes. Figure 1 The initial pH of the bamboo leaf extract was around 6.20, which was suitable for the growth of lactic acid bacteria. Lactobacillus quickly entered the logarithmic growth phase, with vigorous growth within the first 12 hours. After 12 hours, the growth of lactic acid bacteria stabilized, and the pH changed gently. Furthermore, whether the bamboo leaf extract was clarified or retained in a base had a certain impact on the pH drop. Lactic acid bacteria fermented more vigorously in the bamboo leaf extract with a plant base. In both environments, Lactobacillus plantarum R1 achieved the highest fermentation level.

[0135] 5.2 Color difference analysis before and after fermentation

[0136] The color of bamboo leaf water extract is an important parameter, reflecting the changes in color components during the fermentation process, affecting the sensory quality of the beverage and the consumer's choice. Table 1 shows the color attribute differences of bamboo leaf water extracts fermented by lactic acid bacteria. As shown in Table 1, compared with the clarified bamboo leaf water extract, the experimental group after fermentation has a a * 、 b * The values ​​were significantly decreased ( P <0.05), and L * The values ​​were significantly increased ( P <0.05), indicating that the fermentation of lactic acid bacteria causes the red color of the sample to fade and turn green, the yellow color to fade and the brightness to increase, and the changes in the fermentation products that retain the base are more obvious. The color difference is calculated according to the formula △E It was found that the greater the color difference, the greater the color change, the brighter the green, the better the visual perception, and retaining the plant base is conducive to the vigorous growth of lactic acid bacteria and is more conducive to the fermentation process.

[0137] Table 1. Differences in color properties of aqueous extracts of bamboo leaves fermented with lactic acid bacteria

[0138]

[0139] Note: Different letters in the same column indicate significant differences (P < 0.05).

[0140] 5.3 Changes in total sugar and soluble protein

[0141] (1) Changes in total sugar

[0142] The carbon source in the bamboo leaf water extract is mainly sucrose in white sugar, which can slow down the inhibition of accumulated lactic acid. During the growth and fermentation of lactic acid bacteria, lactic acid bacteria release glucose and fructose by breaking down sucrose and use monosaccharides for their own metabolism, thus reducing the total sugar content. Figure 2 As can be seen, the total sugar content in the bamboo leaf extract containing tea residue decreased significantly, by approximately 50%. Carbon source consumption by the commercial strains LA85, LB42, and Lp90 was similar to that of the endogenous strain R1, at 47.75%, 52.73%, 46.72%, and 49.57%, respectively. In the clarified bamboo leaf extract, carbon source utilization by each strain was even lower.

[0143] (2) Changes in soluble protein

[0144] Lactic acid bacteria can use protein as a nitrogen source and degrade it into peptides and amino acids, which are more easily absorbed by the human body and enrich the flavor of the product. Figure 3The results showed that during the fermentation process, the soluble protein content of the clarified bamboo leaf water extract decreased from about 60.00 μg / mL to 30.0-40.00 μg / mL. The soluble protein content in the bamboo leaf water extract that retained the tea residue decreased even more significantly. The nitrogen source consumption of the endogenous strain R1 was similar to that of the commercial strain. After 3 days of fermentation, the soluble protein contents of the bamboo leaf water 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.

[0145] 5.4 Changes in total phenols

[0146] Phenolic compounds usually exist in the form of glycosides and are poorly absorbed, but microbial biotransformation can increase their bioaccessibility and bioactivity. Figure 4 In the clarified bamboo leaf compound tea, total phenolic content increased rapidly during the first 12 hours of fermentation, likely due to active lactic acid bacteria metabolism, which hydrolyzed bound phenols to release free phenols. After 36 hours, total phenolic content changed slowly. After three days of fermentation, the total phenolic content of bamboo leaf compound tea fermented with LA85, LB42, Lp90, and R1 increased by 6.46%, 5.71%, 9.10%, and 14.35%, respectively. In bamboo leaf compound tea fermented with tea residue, total phenolic content decreased from 360-390 μg / mL to 300-340 μg / mL, likely due to the lactic acid bacteria converting phenolic compounds to maintain growth under stress.

[0147] 5.5 Changes in organic acids and free sugars

[0148] The PCA model is used to reduce the dimension of variables, analyze the correlation and separation of each group of variables, and determine the important variables. Figure 5 As can be seen, the experimental and control groups were completely separated, indicating that lactic acid fermentation significantly affected the organic acids and free sugars in the samples. The experimental group deviated toward the negative axis of PC1, with lactic acid, sucrose, fructose, and glucose as the characteristic components of separation. Malic acid, citric acid, succinic acid, oxalic acid, and sucrose decreased after fermentation and were positively correlated with the first component. Lactic acid, fructose, and glucose had high absolute values ​​of loading coefficients on the negative side of PC1 and were negatively correlated with the first component. Oxalic acid content decreased by 22.27-41.75% after fermentation, facilitating the degradation of anti-nutritional factors.

[0149] After 3 days of fermentation with commercial strains LA85, LB42, Lp90 and endogenous strain Lactobacillus plantarum R1, the lactic acid contents of the bamboo leaf water extracts retaining tea residues were 3.43±0.02, 3.35±0.01, 2.96±0.00, and 3.39±0.00 mg / mL, respectively, which were all higher than those of the clarified bamboo leaf water extracts after fermentation, and the lactic acid had a mild and long-lasting taste.

[0150] 5.6 Changes in Electronic Noses

[0151] The aroma characteristics of bamboo leaf water extract after fermentation were detected by electronic nose. Figure 6 As can be seen, the fermented experimental group was completely separated from the unfermented control group, indicating that fermentation significantly affects the aroma components of the bamboo leaf aqueous extract. The experimental groups were also completely separated from each other, with the group retaining tea residue shifting further toward the positive axis of PC1 and exhibiting greater changes in aroma characteristics.

[0152] 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) were significantly deviated from the origin and had higher load coefficients, indicating that these five sensors play an important role in distinguishing the changes in aroma components of bamboo leaf water extract after fermentation.

[0153] 5.7 Isolation and Comprehensive Evaluation of Different Lactic Acid Bacteria Strains

[0154] (1) PLS-DA analysis

[0155] The above indicator data were analyzed by PLS-DA. Figure 7 It can be seen that in the PLS-DA model, the fermented samples in each group deviated significantly from the untreated blank group, and the fermented samples with tea residues moved farther, indicating that tea residues promoted lactic acid bacteria fermentation.

[0156] The VIP value and P value (VIP>1, P<0.05) showed that total phenols, oxalic acid, lactic acid, fructose and other components played a key role in the aroma components with higher electronic nose detection response values, such as alkanes, nitrogen oxides, and aromatic alcohols.

[0157] After 200 permutation tests, the test value R2 is always higher than Q2, and both R2 and Q2 are always higher than the original values ​​on the left (R2 0.108, Q2 -0.649), indicating that the model will not be overfitted and has strong applicability and predictability.

[0158] (2) EWM-TOPSIS comprehensive evaluation

[0159] The EWM-TOPSIS comprehensive evaluation method combines entropy weight and TOPSIS method. Due to its objectivity and practicality, it has been widely used in many fields.

[0160] The three-day fermentation of four lactic acid bacteria strains in bamboo leaf water extract involved multiple indicators. Different lactic acid bacteria had different strengths and weaknesses in their effects on different indicators. Comprehensive evaluation using EWM-TOPSIS can help determine the most ideal strain. The weight (w) of each indicator was calculated using formula (3). As shown in Table 2, fructose (13.9840%), total phenols (10.7094%), and W5S (10.4548%) had higher weights among the indicators. These weights help reduce the inherent subjectivity of the TOPSIS method.

[0161] As shown in Table 3, Lactobacillus plantarum R1 had the best effect in fermenting bamboo leaf water extract, ranking 1 (comprehensive score of 0.644), confirming that Lactobacillus plantarum R1 was the best fermentation strain.

[0162] Table 2. Summary of weight calculation results based on entropy weight method

[0163]

[0164] Table 3. EWM-TOPSIS comprehensive evaluation results

[0165]

[0166] 5.8 Changes in Antioxidant Capacity

[0167] (1) Changes in ABTS scavenging ability

[0168] Antioxidants play a vital role in human health, reducing the risk of disease by protecting the body from oxidative damage, which can lead to a variety of conditions (such as diabetes, cancer, and neurodegenerative diseases). They also control the oxidation process, preventing free radical reactions that can degrade food quality. Free radicals are one of the main causes of oxidative stress, which can trigger a variety of degenerative diseases, such as cancer, coronary heart disease, and vascular disease.

[0169] The antioxidant capacity of the samples was determined by ABTS free radical scavenging activity. The results showed that lactic acid bacteria fermentation can significantly improve the antioxidant capacity of bamboo leaf water extract. Figure 8 The results showed that the ABTS free radical scavenging rate increased rapidly from 0 h to 12 h. From 12 h to 72 h, the ABTS free radical scavenging rate of the clarified water 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 water extract of bamboo leaves with the residue retained decreased slightly. Among them, the ABTS scavenging rate of Lp90 strain after 3 days of fermentation was the lowest, which was 85.91±0.053%. The ABTS scavenging rate of strain R1 was the highest, which was 94.1±0.304%, still above 90%.

[0170] (2) Changes in DPPH scavenging ability

[0171] The DPPH scavenging capacity is expressed as the antioxidant Trolox equivalent. Figure 9 The results showed that fermentation with different lactic acid bacteria enhanced the DPPH scavenging capacity of bamboo leaf water extracts, increasing the concentration of DPPH from less than 10 μg Trolox / mL before fermentation to over 140 μg Trolox / mL. After 3 days of fermentation, the DPPH scavenging capacities of the bamboo leaf water extracts with the substrate retained 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 bamboo leaf water extracts with the substrate retained, the clarified bamboo leaf water extracts exhibited even greater DPPH scavenging capacity. The DPPH scavenging capacities of the clarified bamboo leaf water extracts with strains LA85, LB42, Lp90, and R1 after 3 days of fermentation 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 bamboo leaf water extract retained the base or was clarified, the DPPH scavenging ability of strain R1 after fermentation was excellent and better than that of other lactic acid bacteria strains.

[0172] The above experimental results show that the bamboo leaf water extract after lactic acid bacteria fermentation has stronger antioxidant capacity, and Lactobacillus plantarum R1 has a greater advantage in enhancing the antioxidant capacity of bamboo leaf water extract.

[0173] 5.9 Differences in Antibacterial Ability

[0174] This experiment used the Oxford cup method to evaluate the antibacterial activity by measuring the diameter of the inhibition zone. The results showed that the fermentation products of the bamboo leaf water extract after lactic acid bacteria fermentation showed a certain inhibitory activity (inhibition zone diameter > 8 mm). Figure 10 The results show that the unfermented bamboo leaf water extract did not produce a clear zone of inhibition against the three pathogens, indicating low antibacterial activity. However, most of the experimental groups fermented with lactic acid bacteria for 3 days showed a clear zone of inhibition, and the clarified bamboo leaf water extract fermented with the same strains showed a larger zone of inhibition than the experimental group retaining the plant substrate.

[0175] In the antibacterial activity test against Escherichia coli ATCC 35218, all experimental groups, except the bamboo leaf extract fermented with Lactobacillus acidophilus and retaining the substrate, exhibited antibacterial activity. The inhibition zone diameters of the clarified bamboo leaf extracts fermented with LA85, LB42, Lp90, and R1 were 12.04±0.25, 18.11±0.66, 23.24±0.60, and 24.86±0.35 mm, respectively, while those with the bamboo leaf extract retaining the substrate were 8.00, 13.58±0.17, 16.16±0.50, and 21.63±0.29 mm, respectively. The product fermented with strain R1 exhibited the greatest antibacterial activity.

[0176] The fermentation product of strain R1 also had the strongest inhibitory activity against Klebsiella pneumoniae. The diameters of the inhibition zones of the fermentation products of the clarified and retained bamboo leaf water extracts were 19.3±0.12 and 22.00±0.36 mm, respectively. In the retained bamboo leaf water extract, no obvious inhibition zones were formed after fermentation of LA85 and LB42, while that of Lp90 was 13.70±0.62 mm. In the clarified bamboo leaf water extract, the diameters of the inhibition zones of LA85, LB42, and Lp90 after fermentation were 12.12±0.69, 19.66±0.41, and 21.48±0.94 mm, respectively, all significantly lower than those of the fermentation product of strain R1.

[0177] Except for LA85, the fermented experimental groups all had significant inhibitory ability against Staphylococcus aureus. The inhibition zone sizes of the clarified bamboo leaf water extracts after fermentation of LB42, Lp90 and R1 were 18.25±0.40, 19.31±1.32 and 22.75±0.30 mm, respectively. The bamboo leaf water extracts with the residue retained produced inhibition zones of 19.27±1.08, 17.56±0.69 and 14.10±0.12 mm against Staphylococcus aureus after fermentation.

[0178] In summary, regardless of whether the filter residue is retained or not, the fermentation products of bamboo leaf water extract fermented by Lactobacillus plantarum R1 have good antibacterial activity, and the antibacterial activity is better than that of other lactic acid bacteria.

[0179] The above experiments show that fermenting bamboo leaf water extract with Lactobacillus plantarum R1 achieves the highest degree of fermentation and the most ideal results. Furthermore, fermentation significantly enhances the antioxidant and antibacterial capabilities of the bamboo leaf water extract. The fermentation product obtained with Lactobacillus plantarum R1 exhibits superior antioxidant and antibacterial properties to those obtained with other lactic acid bacteria.

[0180] Experimental Example 2: Cellulase-Assisted Lactobacillus plantarum R1 Fermentation to Solve Phenolic Loss and Comparison of Residue Retention and Clarification

[0181] The fermentation product prepared according to the method and results described in Example 1 (Lactobacillus plantarum R1, retained plant substrate and clarified fermented bamboo leaf water extract) was optimized by the method described in Example 2 (fiber ultrasound combined with cellulose enzyme hydrolysis, Lactobacillus plantarum R1, retained plant substrate bamboo leaf water extract) and further compared with the retained filter residue and clarified fermentation products.

[0182] 1. Determination of total phenol content

[0183] The measurement was carried out according to the method described in Test Example 1.

[0184] 2. Determination of phenolic compounds

[0185] Use macroporous resin D101 to purify phenols from the sample by adsorption. Pretreatment: Soak D101 filler in 90% ethanol / water for 24 hours, then weigh 5 g into a 100 mL Erlenmeyer flask. Adsorption: Add 50 mL of sample solution and shake at 25°C, 120 rpm, for 4 hours. Washing: Discard the sample solution and rinse the resin with distilled water. Desorption: Add 50 mL of 30% ethanol / water and shake at 25°C, 120 rpm, for 4 hours. Collect the desorbed solution, vacuum concentrate, and freeze-dry. Store the dried powder at -20°C.

[0186] The phenolic compounds enriched in the sample were determined using ultra-high performance liquid chromatography equipped with a diode array detector and an AQUITY UPLC® BEH C18 column (2.1 mm × 50 mm × 1.7 μm) (Waters, USA). Elution conditions: the mobile phase was a gradient elution (Table 4), ultrapure water with 0.1% ( v / v ) of acetic acid (mobile phase A), acetonitrile plus 0.1% ( v / v ) in acetic acid (mobile phase B). The detection wavelength was 270 nm, the analytical column temperature was 20 °C, the mobile phase flow rate was 0.5 mL / min, the injection volume was 10 μL, and the sample was filtered through a 0.22 μm filter. Qualitative quantification was performed using an external standard method.

[0187] Table 4. UPLC elution program

[0188]

[0189] 3. Determination of free amino acids

[0190] The types and contents of free amino acids in the sample solution were analyzed using a fully automated amino acid analyzer. Before injection, to precipitate proteins and peptides in the sample solution to prevent interference, an equal proportion of 4% sulfosalicylic acid solution was added to the fermentation supernatant. The mixture was vortexed and mixed thoroughly, and then allowed to stand at 4°C for 1 hour. The supernatant was then centrifuged at 15,000 rpm for 15 minutes, and the supernatant was filtered through a 0.22 μm aqueous filter membrane before injection and analysis. Results are expressed in mg / mL.

[0191] 4. GC-MS identification of volatile components

[0192] Volatile flavor components were extracted using low-temperature frozen liquid-liquid extraction (LTF-LLI): 5 mL of sample was vortexed with 3 mL of dichloromethane, placed upright in a freezer, and frozen at -20°C for 3 h. The mixture was then thawed and centrifuged at 6000 rpm for 6 min. The lower organic phase was aspirated and dehydrated with anhydrous sodium sulfate (Na2SO4) and filtered through a 0.45 μm organic filter for analysis. Chromatographic conditions included a DB-5MS Ultra Inert column (30 m × 250 μm × 0.25 μm), an inlet temperature of 280°C, an injection volume of 1 μL, splitless injection, and a flow rate of 1.0 mL / min. The temperature program was: 50°C for 2 min, then increased at 4°C / min to 240°C, where it was held for 5 min. Mass spectrometry conditions included an EI source, an electron energy of 70 eV, an ion source temperature of 230°C, a transfer line temperature of 280°C, a solvent delay of 3 min, a scan range of m / z 50–450, and high-purity helium as the carrier gas. Substances were identified using the Masshunter database.

[0193] 5. Sensory evaluation

[0194] A sensory evaluation was conducted to determine the differences in sensory attributes between samples fermented using various methods. The sensory evaluation was conducted in a sensory panel room at 25 ± 1°C, and the fermented broths were evaluated for each sensory attribute using Table 5. The sensory panelists consisted of 10 non-expert students (5 female and 5 male) who received training prior to the evaluation. The fermented bamboo leaf aqueous extracts were randomly coded with different numbers and presented to the panelists, who rated them based on color, aroma, texture, mouthfeel, sweetness and sourness, and overall acceptability (calculated as the average of the scores for the previous five indicators).

[0195] Table 5. Sensory evaluation scoring criteria

[0196]

[0197] Note: 17-20 is "excellent", 13-16 is "good", 9-12 is "marginal", and ≤8 is "rejected".

[0198] 6. Data processing and analysis

[0199] SPSS 26.0 software was used for data processing, and Duncan's test was used to calculate significant differences. P < 0.05 was considered significant. OriginPro 2023 was used for plotting, and experimental data are expressed as mean ± standard deviation.

[0200] 7. Results

[0201] 7.1 Changes in total phenol content

[0202] Depend on Figure 11 It can be seen that at the beginning of fermentation, the total phenolic content of the untreated bamboo leaf water extract and the enzymatically hydrolyzed bamboo leaf water extract was 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, the total phenolic content of the enzymatically hydrolyzed bamboo leaf water extract increased significantly. The retained filter residue and clarified bamboo leaf water extract increased by 22.94 and 24.76%, reaching 436.02±3.39 and 442.16±3.29 μg / mL, respectively, which was significantly higher than that of the fermentation group ( P <0.01).

[0203] The following studies were conducted on the fermentation products of bamboo leaf water extract with the assistance of cellulase to clarify and retain the base of Lactobacillus plantarum R1.

[0204] 7.2 Increase of Gallic Acid, Chlorogenic Acid, and Luteolin

[0205] Depend on Figure 12 During the three-day fermentation, gallic acid increased by 112.79% and 82.94% from the pre-fermentation level of 1.17±0.00 μg / mL, respectively. This is likely due to the hydrolysis of tannins in bamboo leaves and chrysanthemum flowers, as well as the hydroxylation and methylation of p-hydroxybenzoic acid. Chlorogenic acid significantly increased after fermentation, increasing by 6.39% and 12.08% in clarified and retained tea leaves, respectively. Contrary to the esterase production observed in most bacterial fermentations, which significantly reduces its content, the endogenous strain Lactobacillus plantarum R1 was shown to increase chlorogenic acid. Luteolin increased from 0.82±0.05 μg / mL to 1.29±0.01 and 1.03±0.02 μg / mL, possibly due to the conversion of complex polyphenols into flavonoids.

[0206] 7.3 Increase in Sweet and Hydrophobic Amino Acids and Decrease in Bitter Amino Acids

[0207] Depend on Figure 13Throughout the fermentation process, bitter amino acids decreased significantly. Leucine (Leu) decreased from 9.00±0.00 μg / mL to 0.667±0 and 4.33±0.47 μg / mL after the retained residue and clarified fermentation, respectively. Phenylalanine (Phe) decreased from 22±0 μg / mL to 0.666666667±0 and 0 μg / mL, respectively. Tyrosine (Tyr) decreased from 8.25±0 μg / mL to 0. Increased levels of hydrophobic amino acids, threonine (Thr) and valine (Val), contributed to the high antioxidant activity of the fermented bamboo leaf aqueous extract. After 3 days of fermentation, threonine increased from 0 to 1.33±0 and 2.00±0 μg / mL after the retained residue and clarified fermentation, respectively. Valine increased from 1.5±0 μg / mL to 16±0 and 18.67±0 μg / mL, respectively.

[0208] In addition, changes in free amino acids can affect the flavor and aroma of bamboo leaf aqueous extract. Proline (Pro), a sweet amino acid that can be used to enhance the taste of beverages and is a common edible flavoring, increased from 22.63±0.18 μg / mL to 54.33±0.47 and 45.33±0.00 μg / mL after 3 days of fermentation, respectively, after retained filtration and clarified fermentation.

[0209] 7.4 Richness of Volatile Aroma Components

[0210] Liquid-liquid freeze extraction combined with GC-MS was used to detect volatile flavor compounds. The peaks in the total ion current chromatogram represent the volatile compounds in the bamboo leaf water extract ( Figure 14 Lactic acid bacteria ferment in bamboo leaf water extract, producing a variety of volatile components through metabolic activities, giving the fermented product a unique smell. Before fermentation, the bamboo leaf water extract had no obvious smell, and only 10 volatile components were detected; after fermentation, the types of volatile substances increased significantly ( Figure 15 ), the bamboo leaf extract with the residue retained contained 38 compounds, while the clarified extract contained 33 compounds. After fermentation, alcohols, ketones, acids, and esters became the primary components. The clarified extract increased by 8, 6, and 5 compounds, respectively, and decreased by 1, accounting for 72.73% of the total content. The extract with the residue retained accounted for 74.55% of the total, with 9, 7, 6, and 3 more compounds than the unfermented extract. A total of 55 volatile components were detected during the fermentation process, including alcohols, acids, phenols, alkenes, aldehydes, esters, ketones, alkanes, and furans. Many of these components, produced after fermentation, are associated with unique aromas. Esters, alcohols, and ketones possess distinct floral and fruity aromas, while acids have a strong odor and a low threshold. Fermentation of bamboo leaf extracts with Lactobacillus plantarum R1 not only highlights the inherent flavor of the plant but also produces a variety of specialized flavor compounds. Fermentation with the residue retained, however, helps accumulate more flavor compounds.

[0211] 7.5 Sensory evaluation results of fermentation broth

[0212] Ten students and faculty from the School of Food Science and Technology conducted sensory evaluations of the retained residue and clarified fermentation broth to assess sensory attributes and acceptability. The data obtained from the survey were presented in the form of radar charts to evaluate the attributes of aroma, acidity, color, texture, flavor, and overall acceptability ( Figure 16 ). Compared to the clarified fermentation broth, the fermentation broth that retained the filter residue was observed to have higher scores in terms of olfaction and overall acceptability, especially for the smell ( P <0.05), while other sensory attributes were relatively similar. For the fermentation broth with the residue retained, responses for all attributes, except sourness, were above 12 (indicating marginal acceptance). Regarding the acceptability of aroma and acidity, the fermentation broth with the residue retained showed a preference and rejection, with mean values ​​of 7.73 and 19.09, respectively. In contrast, for the same attributes, the clarified fermentation broth displayed values ​​of 8.00 and 13.57, indicating that its sourness was also rejected, while its aroma was less favorable.

[0213] In general, lactic acid bacteria fermented well in bamboo leaf water extract, and the aroma sensory score produced by retaining the residue for fermentation was higher.

[0214] From Experimental Example 2, it can be seen that the use of cellulase-assisted Lactobacillus plantarum R1 fermentation can improve the phenolic, amino acid and aroma components of the bamboo leaf water extract fermentation product, retain the base sensory score higher, and improve the taste of the fermentation product while achieving excellent antioxidant and antibacterial properties of the fermentation product.

[0215] In summary, the present invention provides a fermentation product obtained by fermenting bamboo leaf extract using the endogenous strain Lactobacillus plantarum R1 of bamboo shoots. The fermentation product has excellent antioxidant and antibacterial properties, which are superior to 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, thereby improving the taste of the fermentation product. A bamboo leaf composite beverage with a good taste is prepared using the fermentation product. The present invention expands the use 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: The fermentation product is 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 a preservation number of CCTCC NO: M2022659; The preparation method of the bamboo leaf fermentation product comprises the following steps: (1) Take bamboo leaves, chrysanthemums and white sugar, dissolve the white sugar in water, immerse the bamboo leaves and chrysanthemums in water for extraction, and filter or not filter after extraction to obtain bamboo leaf water extract; (2) inoculating Lactobacillus plantarum R1 seed solution into the bamboo leaf water extract obtained in step (1) and fermenting to obtain; In step (1), the weight ratio of bamboo leaves, chrysanthemums and white sugar is: 1-5 parts of bamboo leaves, 0.1-1 parts of chrysanthemums, and 7-10 parts of white sugar; In step (2), the fermentation temperature is 35-37°C, the fermentation time is 24-72 hours, and the density of the Lactobacillus plantarum R1 seed liquid during fermentation is 10 8 ~ 10 10 CFU / mL, and the inoculation amount of Lactobacillus plantarum R1 seed liquid was 1~3%.

2. The bamboo leaf fermentation product according to claim 1, wherein: In the step (1), the obtained bamboo leaf water extract is hydrolyzed with cellulase; During the enzymatic hydrolysis with cellulase, the pH of the bamboo leaf water extract is adjusted to 4-6; When the cellulase is used for enzymatic hydrolysis, the amount of cellulase added is 2-4%; The enzymatic hydrolysis temperature during the cellulase hydrolysis is 30-50°C, and the enzymatic hydrolysis time is 1-5 hours; During the enzymatic hydrolysis with cellulase, ultrasound is performed with a power of 60-100W and a duration of 20-60 minutes.

3. The bamboo leaf fermentation product according to claim 1, wherein: In the 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 white 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 minutes; And / or, in step (1), the extraction is performed with stirring at a speed of 500-1000 r / min; And / or, in step (1), no filtration is performed after extraction.

4. Use of the bamboo leaf fermentation product according to any one of claims 1 to 3 in preparing a beverage.

5. A beverage characterized by: The fermented bamboo leaf product is prepared by taking the bamboo leaf fermentation product according to any one of claims 1 to 3 as a raw material and adding auxiliary materials acceptable to food.

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