A bacterial enzyme-assisted debittered pea protein beverage and its preparation method

Abstract

The present invention discloses a kind of bacterial enzyme synergistic debittering pea protein beverage and preparation method thereof, belong to the field of food processing technology.The method adopts pea protein as raw material, utilizes alkaline protease, flavor protease enzymolysis and plant lactobacillus, thermophilic streptococcus, Lactobacillus helveticus composite bacterial fermentation synergistic effect, compared with using lactic acid bacteria fermentation debittering time alone is shorter, overall debittering efficiency is higher, sensory evaluation is better, overall acceptance is high; The pea-related odor-related volatile compounds of the prepared product include hexanal, 2 nonanone, (E, E) 2,4 nonadienal, 2 amylfurans, (E, E) 2,4 decadienal etc. are reduced or completely eliminated.The debittering pea protein beverage provided by the present invention and preparation method thereof are conducive to broadening pea protein utilization approach and promoting the development of my country's pea protein beverage industry.

Description

Technical Field

[0001] The invention relates to a bacterial enzyme-assisted debittered pea protein beverage and a preparation method thereof, belonging to the technical field of food processing. Background Art

[0002] Peas are one of the world's major legumes, widely used as a commercial protein source due to their excellent yield, availability, and low price. Peas themselves are rich in protein, but their nutritional profile also includes significant amounts of starch and dietary fiber. Pea protein, a highly purified protein extracted from peas, is rich in essential amino acids such as leucine, isoleucine, and valine, which are important for muscle repair and growth. Pea protein and its specific peptide components also possess antioxidant, antihypertensive, cholesterol-lowering, and gut microbiome-regulating properties. Currently, pea protein applications are concentrated in food supplements and emulsifiers, with pea protein beverages being scarce. Compared to pea-based beverages, beverages made with pea protein offer benefits for cardiovascular health due to their high protein content, helping to lower cholesterol and blood pressure. Furthermore, their richness in specific amino acids and proteins makes them beneficial for post-exercise recovery, muscle growth, and as a protein supplement for vegetarians.

[0003] Due to issues such as pea protein's poor solubility and poor nutritional absorption, pea protein resources are underutilized, impacting the overall performance and development potential of the pea protein beverage market. Existing solutions use enzymatic hydrolysis (commonly using endo-enzymes) to improve its solubility and nutritional absorption. However, pea protein beverages prepared using endo-enzyme hydrolysis have a distinct bitterness that is difficult to accept. The hydrophobic amino acids at the ends of the peptide chain are the primary cause of the bitterness. Existing debittering methods include masking, enzymatic methods, and selective separation. Masking masks the bitterness of pea protein by adding flavoring agents such as sweeteners and flavors. This method is simple to operate, but may alter the original flavor of the product, and long-term consumption of additives may have adverse health effects. Enzymatic methods further treat pea protein with specific enzyme preparations to alter its molecular structure or remove the hydrophobic amino acids at the ends of the peptide chain. However, the selectivity and efficiency of enzymes are often difficult to control, and debittering efficiency needs to be improved. Selective separation separates the bitter components from the non-bitter components in pea protein through physical or chemical means. Although this method can theoretically accurately remove bitterness, it is complex to operate and has high requirements for equipment.

[0004] In addition, the presence of off-flavors described as "beany" or "green and grassy" is a major factor affecting consumer acceptance of pea protein products. Low-bitterness, off-flavor pea protein is rarely available in the market and is expensive, hindering its large-scale application in production. Therefore, a method for preparing a low-bitterness, off-flavor pea protein beverage using common pea protein available on the market as a raw material was invented to reduce or eliminate volatile compounds with pea-related off-flavors, including hexanal, furfural, 2-nonanone, (E,E)-2,4-nonadienal, 2-pentylfuran, (E,E)-2,4-decadienal, etc. This method is conducive to broadening the utilization of pea protein, increasing the market value of pea protein, and filling the gap in the pea protein beverage market. Summary of the Invention

[0005] In order to overcome the defects of the prior art, the present invention provides a pea protein beverage that is debittered by bacterial enzymes and a preparation method thereof. The method uses ordinary pea protein circulating on the market as raw material, adds alkaline protease and flavor protease, and inoculates lactic acid bacteria for fermentation. The fermentation conditions are strictly controlled to produce a pea protein beverage. The volatile compounds with pea-related odors, including hexanal, furfural, 2-nonanone, (E,E)-2,4-nonadienal, 2-pentylfuran, (E,E)-2,4-decadienal, etc., are reduced or eliminated, and the bitterness is low, which is conducive to broadening the utilization of pea protein and filling the gap in pea protein beverages. The specific technical solution is as follows:

[0006] The first object of the present invention is to provide a method for preparing a pea protein beverage debittered by bacterial enzyme synergism, comprising the following steps:

[0007] (1) adding water to pea protein powder and stirring to dissolve to obtain a pea protein solution;

[0008] (2) adding alkaline protease to the pea protein solution obtained in step (1) and stirring to perform a first-stage enzymatic hydrolysis, and heating to inactivate the enzyme;

[0009] (3) adding flavor protease to the solution obtained in step (2) and stirring, performing a two-stage enzymatic hydrolysis, heating to inactivate the enzyme, and sterilizing to obtain a pea protein hydrolyzate;

[0010] (4) mixing Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus to obtain a mixed bacterial suspension, inoculating the mixed bacterial suspension into the pea protein hydrolysate obtained in step (3), culturing and fermenting, and then sterilizing to obtain a fermentation broth;

[0011] (5) The pH of the fermentation liquid obtained in step (4) is adjusted to neutral, edible oil and edible salt are added, and homogenization and sterilization are performed to obtain the pea protein beverage.

[0012] In one embodiment, the ratio of the pea protein powder to water in step (1) is 2% to 5% (w / w); preferably, the ratio of the pea protein powder to water is 2% (w / w).

[0013] In one embodiment, the stirring condition in step (1) is stirring at 800-1200 rpm for 1.5-2.5 hours; preferably, the stirring condition is stirring at 1000 rpm for 2 hours.

[0014] In one embodiment, the alkaline protease in step (2) is alkaline protease Alcalase 2.4L FG.

[0015] In one embodiment, the amount of alkaline protease added is 0.5‰ to 1.5‰ (based on the mass of pea protein powder); preferably, the amount of alkaline protease added is 1.5‰ (based on the mass of pea protein powder).

[0016] In one embodiment, the enzymatic hydrolysis conditions in step (2) are a temperature of 45-55°C, a time of 1-2 hours, and a stirring speed of 800-1200 rpm; preferably, the enzymatic hydrolysis conditions are a temperature of 50°C, a time of 1 hour, and a stirring speed of 1000 rpm.

[0017] In one embodiment, the enzyme inactivation conditions in step (2) and step (3) are a temperature of 95 to 100° C. and a time of 8 to 12 minutes; preferably, the enzyme inactivation conditions are a temperature of 95° C. and a time of 10 minutes.

[0018] In one embodiment, the flavor protease in step (3) is flavor protease Flavourzyme 1000L.

[0019] In one embodiment, the added amount of the flavor protease is 0.5‰ to 4.5‰ (based on the mass of the pea protein powder); preferably, the added amount is 3‰ (based on the mass of the pea protein powder).

[0020] In one embodiment, the enzymatic hydrolysis conditions in step (3) are a temperature of 45-55°C, a time of 1-2 hours, and a stirring speed of 800-1200 rpm; preferably, the enzymatic hydrolysis conditions are a temperature of 50°C, a time of 1 hour, and a stirring speed of 1000 rpm.

[0021] In one embodiment, the sterilization condition in step (3) is a temperature of 100-120° C. and a time of 10-20 min; preferably, the sterilization condition is a temperature of 110° C. and a time of 15 min.

[0022] In one embodiment, the Lactobacillus plantarum in step (4) is Lactobacillus plantarum CICC 22703; the Streptococcus thermophilus is Streptococcus thermophilus CICC 20375; and the Lactobacillus helveticus is Lactobacillus helveticus CICC 20275.

[0023] In one embodiment, the mixed bacterial suspension is obtained by mixing Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus in a ratio of (0.8-1.2):(0.8-1.2):(0.8-1.2) in terms of viable bacteria count; preferably, the ratio is 1:1:1.

[0024] In one embodiment, the total viable count of the mixed bacterial suspension in step (4) is (1-3)×10 6 CFU / mL; preferably, the total viable count is 3×10 6 CFU / mL.

[0025] In one embodiment, the inoculation amount of the bacterial suspension is 2% to 6% (based on the mass of the pea protein hydrolysate); preferably, the inoculation amount is 3% (based on the mass of the pea protein hydrolysate).

[0026] In one embodiment, the culture and fermentation conditions in step (4) are 32-42° C. for 3-5 hours; preferably, the culture and fermentation conditions are 37° C. for 3.5 hours.

[0027] In one embodiment, the sterilization conditions in step (4) are a temperature of 80-120° C. and a time of 10-15 min; preferably, the sterilization conditions are a temperature of 110° C. and a time of 15 min.

[0028] In one embodiment, the method of adjusting the pH to neutral in step (5) is to use sodium bicarbonate.

[0029] In one embodiment, the added amount of the edible oil is 0.5% to 1.5% (w / w); preferably, the added amount of the edible oil is 1% (w / w); preferably, the edible oil is rapeseed oil.

[0030] In one embodiment, the added amount of the edible salt is 0.4% to 0.8% (w / w); preferably, the added amount of the edible salt is 0.6% (w / w).

[0031] In one embodiment, the homogenization treatment conditions in step (5) are a rotation speed of 7500 to 8500 U / min and a time of 10 to 20 min; preferably, the homogenization treatment conditions are a rotation speed of 8000 U / min and a time of 15 min.

[0032] In one embodiment, the sterilization treatment in step (5) is performed at a temperature of 100-120° C. for 10-20 minutes; preferably, the sterilization treatment is performed at a temperature of 110° C. for 15 minutes.

[0033] A second object of the present invention is to provide a pea protein beverage, which is prepared by the above-mentioned preparation method.

[0034] Beneficial effects of the present invention:

[0035] The present invention provides a pea protein beverage debittered by bacterial enzymes and a preparation method thereof. The method uses pea protein as a raw material, adds flavor protease, and inoculates lactic acid bacteria for fermentation to produce a pea protein beverage with a low bitterness value. The pea protein beverage and preparation method of the present invention have the following advantages:

[0036] (1) The raw material of the beverage of the present invention is pea protein, which has a high protein content compared to beverages made from peas. Beverages with high protein content are beneficial to cardiovascular health, post-exercise recovery, and muscle growth;

[0037] (2) The synergistic effect of flavor protease hydrolysis and three types of lactic acid bacteria fermentation resulted in higher overall debittering efficiency, shorter debittering time than using lactic acid bacteria fermentation alone, significantly reduced bitterness, better sensory evaluation, and high overall acceptance;

[0038] (3) The method of the present invention can produce a pea protein beverage without adding additional sweeteners, flavors, and other flavoring substances;

[0039] (4) The enzymes or bacteria added to the pea protein beverage preparation method of the present invention are common materials on the market and are low-cost; the preparation method does not require special means and is simple to operate;

[0040] (5) In the pea protein beverage of the present invention, unpleasant flavor substances such as hexanal, furfural, 2-nonanone, (E,E)-2,4-nonadienal, 2-pentylfuran, and (E,E)-2,4-decadienal that cause off-flavor are significantly reduced or eliminated.

[0041] The debittered pea protein beverage and its preparation method provided by the present invention are beneficial for broadening the utilization of pea protein, increasing the market value of pea protein, and filling the gap in the pea protein beverage market. Furthermore, they provide a valuable theoretical basis for further research on pea protein beverages and can promote the development of my country's pea protein beverage industry. DETAILED DESCRIPTION

[0042] In the following examples, unless otherwise specified, the methods used are conventional methods, and the reagents used can be purchased through commercial channels. Unless otherwise specified, the ratios involved are all weight ratios.

[0043] 1. The sources of materials involved in the following examples and comparative examples are as follows:

[0044] Pea protein powder was purchased from Shandong Jianyuan Biotechnology Co., Ltd.;

[0045] Alcalase 2.4L FG, Neutrase 0.8L, and Novozym 37071 were purchased from Novozymes Enzymes (Denmark).

[0046] Flavorzyme 1000L and Protana Prime were purchased from Novozymes Enzymes, Denmark; Flavorzyme PR EXO-L was purchased from Shanghai Maikaize Ecological Technology Co., Ltd.

[0047] Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375, Lactobacillus helveticus CICC 20275, Lactobacillus casei CICC 20286, Lactobacillus rhamnosus CICC 6001, and Lactococcus lactis CICC 23610 were purchased from China Industrial Microbiological Culture Collection Center;

[0048] Edible salt was purchased from Jiangsu Yinbao Salt Industry Co., Ltd.;

[0049] Rapeseed oil was purchased from COFCO Jiayue (Tianjin) Co., Ltd.;

[0050] Sodium bicarbonate was purchased from Jingshan Jijiang Food Co., Ltd.

[0051] MRS solid medium and MRS liquid medium were purchased from Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0052] 2. The determination methods involved in the following examples and comparative examples are as follows:

[0053] 1. Solubility:

[0054] The sample was dissolved in deionized water to 1 g / 100 mL, stirred separately at 25°C for 30 min, and centrifuged at 4000 × g for 15 min at 4°C. The protein content of the supernatant fraction and the total protein content of the sample were determined by Kjeldahl method.

[0055] The solubility calculation formula is as follows:

[0056] Solubility (%) = protein content in supernatant / total protein content × 100%

[0057] 2. Degree of hydrolysis:

[0058] Preparation of 0.02 g / mL ninhydrin solution: Accurately weigh 1.00 g of ninhydrin and 0.04 g of stannous chloride, dissolve in hot water, filter, cool to room temperature, dilute to volume in a 50 mL brown volumetric flask, and store in a dark place.

[0059] To prepare a glycine standard curve: Weigh 50 mg of glycine and dissolve it in a 100 mL volumetric flask to a concentration of 500 μg / mL. Perform a gradient dilution to 100, 125, 150, 175, 200, and 225 μg / mL. Add 1 mL of each standard solution (using 1 mL of distilled water as a blank), 1 mL of sodium phosphate buffer (pH 8.0), and 1 mL of ninhydrin solution to a 25 mL colorimetric tube. Heat in a boiling water bath for 15 minutes. After cooling, dilute to 25 mL and measure absorbance at 570 nm. Linearly regress the measured absorbance against the corresponding concentration to generate the standard curve.

[0060] Determination of sample hydrolysis degree: Add 1 mL of diluted hydrolyzate (using 1 mL of distilled water as a blank control), 1 mL of sodium phosphate buffer (pH 8.0), and 1 mL of 0.02 g / mL ninhydrin solution to a 25 mL colorimetric tube. Heat in a boiling water bath for 5 minutes. After cooling, dilute to 25 mL and measure absorbance at 570 nm. Calculate the amino acid concentration of the sample using a glycine standard curve.

[0061] The calculation formula of degree of hydrolysis (DH) is as follows:

[0062] DH(%)=(ρ PPH ×V PPH ×D PPH -ρ PPI ×V PPI ×D PPI ) / m×N×10 6

[0063] Where: ρ PPH and ρ PPI are the mass concentrations of free amino groups in the hydrolyzate and PPI solution, μg / mL; V PPH and V PPI are the volumes of hydrolyzate and PPI solution, mL; D PPH and D PPI are the dilution multiples of the hydrolyzate and PPI solution, respectively; m is the mass of the raw material, g; N is the nitrogen content of the raw material, %.

[0064] 3. Free amino acids:

[0065] The free amino acid content was determined by high-performance liquid chromatography (HPLC) using an Agilent 1100 HPLC system. The mobile phase A (pH 7.2) consisted of 27.6 mmol / L sodium acetate-triethylamine-tetrahydrofuran (volume ratio: 500:0.11:2.5) and the mobile phase B (pH 7.2) consisted of 80.9 mmol / L sodium acetate-methanol-acetonitrile (volume ratio: 1:2:2). The column was an Agilent Hypersil ODS column (5 μm, 4.0 mm × 250 mm). The elution program was gradient elution: 8% B (92% A) at 0 min, 50% B (50% A) at 17 min, 100% B (0% A) at 20.1 min, and 0% B (100% A) at 24 min. The flow rate was 1.0 mL / min. The column temperature was 40°C. The UV detection wavelength was 338 nm.

[0066] The sweet amino acids involved in the present invention include: serine, histidine, glycine, threonine, proline, and alanine;

[0067] The bitter amino acids involved in the present invention include: arginine, valine, methionine, isoleucine and leucine.

[0068] 4. Organic acids:

[0069] Add 1 mL of sample to a 10 mL volumetric flask and adjust to volume with 1% phosphoric acid solution. After mixing, let it stand for a while, centrifuge (10,000 × g, 15 min, 25 ° C), extract the supernatant, and filter through a 0.45 μm organic membrane. Use HPLC to detect the content of organic acids. HPLC uses a C18 chromatographic column (250 × 4.6 mm), the column temperature is set to 30 ° C, and detection is performed at a UV wavelength of 210 nm. The mobile phase is 0.1% phosphoric acid solution and methanol (97.5:2.5, v / v) with an isothermal elution rate of 1 mL / min and an injection volume of 5 μL.

[0070] The organic acids involved in the present invention include: malic acid, lactic acid, acetic acid, citric acid, succinic acid, fumaric acid, propionic acid and butyric acid.

[0071] 5. Determination of bitterness value (electronic tongue):

[0072] The electronic tongue intelligent taste analysis system was used to objectively evaluate the bitter taste characteristics of the samples. To ensure stable and reliable data acquisition, the system was calibrated and self-tested before each measurement. The sample was prepared into a 0.05g / mL solution in deionized water and placed in a measuring cup. 30M KCl and 0.3M tartaric acid were used as reference solutions. The test sequence and cleaning procedures were then configured. The bitterness intensity was quantified by the bitterness sensor (CO0) of the electronic tongue system.

[0073] 6. Volatile flavor substance content:

[0074] HS-SPME conditions: Accurately take 5 g of sample and place it in a 20 mL headspace vial, add 10 μL of internal standard (4-methyl-2-pentanol, 50 mg / L), put it in the rotor and quickly tighten the lid, place the extraction vial in a 60°C water bath for 7 minutes, insert the extraction head into the extraction vial, and adsorb while magnetically stirring for 30 minutes.

[0075] GC-MS conditions: GC conditions: A DB-WAX column (30 m × 0.25 mm, 0.25 μm) was used. Temperature program: Initial temperature 40°C, hold for 3 min; then increase the temperature to 100°C at 6°C / min; finally increase the temperature to 230°C at 10°C / min and hold for 7 min; splitless injection. MS conditions: EI ion source, ion source temperature 230°C, interface temperature 250°C, electron energy 70 eV, scan range m / z 33-500, acquisition mode Scan.

[0076] Qualitative and quantitative analysis of flavor compounds: Volatile compounds can be identified qualitatively by comparing the results retrieved from mass spectrometry databases (NIST and WILEY databases) with standard compounds. Quantitative analysis is performed using the internal standard method, comparing the chromatographic peak areas of the internal standard and the test compound, and calculating the test compound content in μg / L.

[0077] 7. Sensory evaluation:

[0078] A sensory evaluation panel of 20 students, 10 males and 10 females, was selected after training on aroma and flavor characteristics. They rated the flavor, aroma, mouthfeel, and overall acceptability of the pea protein beverages. The highest and lowest scores for each metric were excluded when calculating the average. During the scoring process, the students rinsed their mouths with purified water between samples. The scores for each sensory evaluation metric are shown in Table 1.

[0079] Table 1 Sensory indicators of pea protein beverages

[0080]

[0081] Example 1: Screening of proteases for hydrolyzing pea protein

[0082] The specific steps are as follows:

[0083] 1. Mix pea protein powder and water at a ratio of 2% (w / w) and stir thoroughly for 2 hours. Add 1.5‰ of alkaline protease Alcalase 2.4L FG (based on the mass of pea protein powder). Place the pea protein solution at 50°C and stir for 1 hour for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat at 95°C for 10 minutes to inactivate the enzyme. Determine the solubility and degree of hydrolysis of the pea protein powder.

[0084] 2. The specific implementation method is the same as step 1, except that the protease is adjusted to 0.8L of neutral protease Neutrase.

[0085] 3. The specific implementation method is the same as step 1, except that the protease is adjusted to the hydrolytic protease Novozym 37071.

[0086] Experimental results:

[0087] The solubility and hydrolysis degree results of pea protein powder in steps 1 to 3 are as follows:

[0088] When pea protein powder is hydrolyzed with alkaline protease Alcalase 2.4L FG, its solubility is 78% and the degree of hydrolysis is 9.28%.

[0089] When pea protein powder was hydrolyzed using Neutrase 0.8L, its solubility was 56% and the degree of hydrolysis was 4.53%;

[0090] When pea protein powder is hydrolyzed using the hydrolyzing protease Novozym 37071, its solubility is 58% and the degree of hydrolysis is 5.77%.

[0091] The results showed that the solubility and hydrolysis degree of pea protein powder hydrolyzed by alcalase were the highest, at 78% and 9.28%, respectively. The following examples all used alcalase 2.4L FG to prepare pea protein beverages.

[0092] Example 2: Preparation of lactic acid bacteria debittered pea protein beverage

[0093] The specific steps are as follows:

[0094] (1) Stir and dissolve:

[0095] The mixture was stirred at 1000 rpm for 2 h at a ratio of pea protein powder to water = 2% (w / w) to obtain a pea protein solution;

[0096] (2) Enzymatic hydrolysis:

[0097] After adding 1.5‰ of alkaline protease Alcalase 2.4L FG (based on the mass of pea protein powder) to the pea protein solution obtained in step (1), the mixture was placed at 50°C and stirred at 1000 rpm for 1 hour for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was heated at 95°C for 10 minutes to inactivate the enzyme to obtain a pea protein hydrolyzate;

[0098] (3) Preparation of fermentation agent:

[0099] Under sterile conditions, the Lactobacillus plantarum CICC 22703 glycerol tube was activated: inoculated into MRS liquid culture medium and cultured at 37°C for 12 hours. After successful activation, the culture was repeated at 37°C for 12 hours. The bacterial solution was picked with an inoculation loop and placed on a test tube slope made of MRS solid culture medium for culture for 48 hours. A single colony of Lactobacillus plantarum obtained on the slope was picked and cultured in MRS liquid culture medium at 37°C for 12 hours. From the liquid culture medium, 1% (v / v) was taken and expanded in MRS liquid culture medium (culture conditions were: 37°C, 12 hours). The obtained bacterial solution was centrifuged at 4°C at a speed of 5000r / min for 10 minutes, the supernatant was discarded and the bacterial mud was retained. The bacterial mud was repeatedly washed with sterile saline for 3 times, and finally it was redissolved with an equal volume of saline to obtain a viable bacterial count of 3×10 6 CFU / mL of Lactobacillus plantarum CICC 22703 bacterial suspension;

[0100] According to the above method, the viable bacterial counts were 3×10 6 CFU / mL of Streptococcus thermophilus CICC20375 bacterial suspension, Lactobacillus helveticus CICC 20275 bacterial suspension, Lactobacillus casei CICC 20286 bacterial suspension, Lactobacillus rhamnosus CICC 6001 bacterial suspension, and Lactococcus lactis CICC 23610 bacterial suspension;

[0101] (4) Inoculation and fermentation:

[0102] The Lactobacillus plantarum CICC 22703 bacterial suspension, the Streptococcus thermophilus CICC 20375 bacterial suspension, the Lactobacillus helveticus CICC 20275 bacterial suspension, the Lactobacillus casei CICC 20286 bacterial suspension, the Lactobacillus rhamnosus CICC6001 bacterial suspension, and the Lactococcus lactis CICC 23610 bacterial suspension obtained in step (3) were added to the pea protein hydrolysate obtained in step (2) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), and the mixture was shaken and placed in a 37° C. incubator for 6 h. After the fermentation was completed, the mixture was sterilized at 110° C. to obtain a fermentation broth;

[0103] (5) Preparation of beverages:

[0104] Sodium bicarbonate was added to the fermentation broth obtained in step (4) to adjust the pH of the system to neutral, 1% (w / w) rapeseed oil and 0.6% (w / w) edible salt were added, and homogenization and sterilization were performed at a homogenization speed of 8000 U / min and a time of 15 min, and a sterilization temperature of 110° C. and a time of 15 min;

[0105] Debittered pea protein beverages 1 to 6 were obtained respectively (corresponding to the bacterial suspensions of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375, Lactobacillus helveticus CICC 20275, Lactobacillus casei CICC20286, Lactobacillus rhamnosus CICC 6001, and Lactococcus lactis CICC 23610 in step (4)).

[0106] Here are the results:

[0107] Table 2 Preparation of lactobacillus debitterized pea protein beverage

[0108]

[0109] Table 3 Volatile flavor substance content of lactic acid bacteria debitterized pea protein beverage (μg / L)

[0110]

[0111]

[0112] ND means not detected, the same below.

[0113] Compared to the pea protein beverage 22 (bitterness value 10.22) obtained in Comparative Example 1 using only alkaline protease hydrolysis without lactic acid bacteria fermentation, the lactic acid bacteria-debittered pea protein beverage had a significant debittering effect and significantly improved sensory evaluation; the free amino acid content increased by 13% to 28%; and the lactic acid bacteria fermentation produced organic acids, which improved the overall flavor of the beverage (Table 2). In addition, Lactobacillus plantarum had the best debittering effect, with a bitterness value of 5.09.

[0114] Compared with the pea protein beverage 22 obtained by only using alkaline protease hydrolysis without lactic acid bacteria fermentation in Comparative Example 1, the undesirable flavor substance furfural was eliminated in the beverage debittered by Lactobacillus plantarum, and the new good flavor substance (E)-2-nonenal was generated; the content of undesirable flavor substances such as hexanal, 2-nonanone, and 2-pentylfuran was reduced, and the content of good flavor substances such as hexanoic acid and 1-hexanol was increased (Table 3).

[0115] Example 3: Preparation of flavor protease-debittered pea protein beverage

[0116] The specific steps are as follows:

[0117] (1) Stir and dissolve:

[0118] The mixture was stirred at 1000 rpm for 2 h at a ratio of pea protein powder to water = 2% (w / w) to obtain a pea protein solution;

[0119] (2) One-stage enzymatic hydrolysis:

[0120] After adding 1.5‰ of alkaline protease Alcalase 2.4L FG (based on the mass of pea protein powder) to the pea protein solution obtained in step (1), the mixture was placed at 50°C and stirred at 1000 rpm for 1 hour to perform a first-stage enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was heated at 95°C for 10 minutes to inactivate the enzyme;

[0121] (3) Two-stage enzymatic hydrolysis:

[0122] To the solution obtained in step (2), 3‰ of Flavourzyme 1000L, Protana Prime, or PR EXO-L (based on the mass of pea protein powder) was added, and the mixture was stirred at 50°C and 1000 rpm for 1 hour to perform a two-stage enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the mixture was heated at 95°C for 10 minutes to inactivate the enzyme, and then sterilized at 110°C for 15 minutes to obtain a pea protein hydrolysate.

[0123] (4) Preparation of beverages:

[0124] Sodium bicarbonate was added to the pea protein hydrolysate obtained in step (3) to adjust the pH of the system to neutral, and 1% (w / w) rapeseed oil and 0.6% (w / w) edible salt were added, and homogenization and sterilization were performed. The homogenization speed was 8000 U / min and the time was 15 min. The sterilization temperature was 110° C. and the time was 15 min.

[0125] Debittered pea protein beverages 7 to 9 were obtained respectively (corresponding to the flavor protease Flavourzyme 1000L, flavor protease Protana Prime, and flavor protease PR EXO-L in step (3)).

[0126] Here are the results:

[0127] Table 4 Preparation of flavor protease-debittered pea protein beverage

[0128]

[0129] Compared to the pea protein beverage 22 (bitterness value 10.22) obtained in Comparative Example 1 using only alkaline protease hydrolysis without flavor protease, the pea protein beverage debittered with flavor protease showed a significant debittering effect. Pea protein beverage 7 debittered with flavor protease Flavourzyme 1000L had a lower bitterness value and a higher sensory score; the free amino acid content was increased by 352% to 362% (Table 4). The following examples all used Flavourzyme 1000L to prepare pea protein beverages.

[0130] Example 4: Preparation of debittered pea protein beverage using flavor protease and a single bacterial strain

[0131] The specific steps are as follows:

[0132] (1) Stir and dissolve:

[0133] The mixture was stirred at 1000 rpm for 2 h at a ratio of pea protein powder to water = 2% (w / w) to obtain a pea protein solution;

[0134] (2) One-stage enzymatic hydrolysis:

[0135] After adding 1.5‰ of alkaline protease Alcalase 2.4L FG (based on the mass of pea protein powder) to the pea protein solution obtained in step (1), the mixture was placed at 50°C and stirred at 1000 rpm for 1 hour to perform a first-stage enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the enzyme was heated at 95°C for 10 minutes to inactivate the enzyme;

[0136] (3) Two-stage enzymatic hydrolysis:

[0137] Add 3‰ of Flavourzyme 1000L (based on the mass of pea protein powder) to the solution obtained in step (2), and stir thoroughly at 1000 rpm at 50°C for 1 hour to perform a second-stage enzymatic hydrolysis. After the enzymatic hydrolysis is completed, heat at 95°C to inactivate the enzyme for 10 minutes, and sterilize at 110°C for 15 minutes to obtain a pea protein hydrolyzate.

[0138] (4) Preparation of fermentation agent:

[0139] Under sterile conditions, the Lactobacillus plantarum CICC 22703 glycerol tube was activated: inoculated into MRS liquid culture medium and cultured at 37°C for 12 hours. After successful activation, the culture was repeated at 37°C for 12 hours. The bacterial solution was picked with an inoculation loop and placed on a test tube slope made of MRS solid culture medium for culture for 48 hours. A single colony of Lactobacillus plantarum obtained on the slope was picked and cultured in MRS liquid culture medium at 37°C for 12 hours. From the liquid culture medium, 1% (v / v) was taken and expanded in MRS liquid culture medium (culture conditions were: 37°C, 12 hours). The obtained bacterial solution was centrifuged at 4°C at a speed of 5000r / min for 10 minutes, the supernatant was discarded and the bacterial mud was retained. The bacterial mud was repeatedly washed with sterile saline for 3 times, and finally it was redissolved with an equal volume of saline to obtain a viable bacterial count of 3×10 6 CFU / mL of Lactobacillus plantarum CICC 22703 bacterial suspension.

[0140] According to the above method, the viable bacterial counts were 3×10 6CFU / mL of Streptococcus thermophilus CICC20375 bacterial suspension, Lactobacillus helveticus CICC 20275 bacterial suspension, Lactobacillus casei CICC 20286 bacterial suspension, Lactobacillus rhamnosus CICC 6001 bacterial suspension, and Lactococcus lactis CICC 23610 bacterial suspension;

[0141] (5) Inoculation and fermentation:

[0142] The bacterial suspensions of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375, Lactobacillus helveticus CICC 20275, Lactobacillus casei CICC 20286, Lactobacillus rhamnosus CICC 6001, and Lactococcus lactis CICC 23610 obtained in step (4) were added to the pea protein hydrolysate obtained in step (3) in an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), the mixtures were shaken and placed in a 37° C. incubator for 3.5 h. After fermentation, the mixtures were sterilized at 110° C. to obtain a fermentation broth.

[0143] (6) Preparation of beverages:

[0144] Sodium bicarbonate was added to the fermentation broth obtained in step (5) to adjust the pH of the system to neutral, 1% (w / w) rapeseed oil and 0.6% (w / w) edible salt were added, and homogenization and sterilization were performed at a homogenization speed of 8000 U / min and a time of 15 min, and a sterilization temperature of 110° C. and a time of 15 min;

[0145] 10 to 15 of debitterized pea protein beverages were obtained respectively (corresponding to the bacterial suspensions of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375, Lactobacillus helveticus CICC 20275, Lactobacillus casei CICC20286, Lactobacillus rhamnosus CICC 6001, and Lactococcus lactis CICC 23610 in step (5)).

[0146] Here are the results:

[0147] Table 5 Preparation of flavor protease and single strain synergistic debittered pea protein beverage

[0148]

[0149] Table 6 Volatile flavor compound content (μg / L) of pea protein beverage debittered by flavor protease and single strain

[0150]

[0151]

[0152] Compared with the pea protein beverage debittered only with lactic acid bacteria in Example 2, the pea protein beverage debittered with flavor protease and a single strain had a better debittering effect, the bitterness value was further reduced, and the sensory evaluation was improved; the free amino acid content was increased by 281% to 364% compared with Example 2; the organic acid content was increased by 50% to 100% compared with Example 2, and the overall flavor and acceptance were enhanced (Table 5); the content of undesirable flavor substances such as hexanal, 2-nonanone, and (E,E)-2,4-decadienal was further reduced (Table 6).

[0153] In addition, flavor protease and Lactobacillus plantarum synergistically debittered pea protein (beverage 10), compared with beverages 11 to 15, had the lowest bitterness value, significantly increased sweet amino acid content, and the highest sensory score.

[0154] Example 5: Preparation of debittered pea protein beverage using flavor protease and multi-strain composite bacteria

[0155] 1. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0156] The Lactobacillus plantarum CICC 22703 bacterial suspension obtained in step (4) and the Streptococcus thermophilus CICC 20375 bacterial suspension were mixed in a volume ratio of 1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0157] A debittered pea protein beverage 16 was obtained.

[0158] 2. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0159] The thermophilic Streptococcus CICC 20375 bacterial suspension obtained in step (4) and the Lactobacillus helveticus CICC 20275 bacterial suspension were mixed in a volume ratio of 1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0160] Obtain debittered pea protein beverage 17.

[0161] 3. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0162] The Lactobacillus plantarum CICC 22703 bacterial suspension obtained in step (4) and the Lactobacillus helveticus CICC 20275 bacterial suspension were mixed in a volume ratio of 1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0163] A debittered pea protein beverage 18 is obtained.

[0164] 4. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0165] The bacterial suspension of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375 and Lactobacillus helveticus CICC 20275 obtained in step (4) were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0166] Obtain debittered pea protein beverage 19.

[0167] Here are the results:

[0168] Table 7 Organic acid content of flavor protease and multi-strain composite strain synergistic debittering pea protein beverage (mg / L)

[0169] Organic acid content Pea protein drink16 Pea protein drink17 Pea protein drink18 Pea protein drink19 Malic acid 766.25 761.23 738.62 781.62 lactic acid 16.28 15.33 17.21 18.25 Acetic acid 1879.22 1887.52 1883.87 1860.10 citric acid 156.58 160.49 158.54 152.67 Succinic acid 240.45 248.87 265.57 258.29 Fumaric acid 0.37 0.43 0.40 0.36 Propionic acid 561.17 569.07 576.98 537.46 Butyric acid 1532.50 1499.72 1524.31 1544.80 Total organic acid 5152.82 5142.66 5165.5 5153.55

[0170] Table 8 Free amino acid content of flavor protease and multi-strain composite strain debittered pea protein beverage (mg / L)

[0171]

[0172]

[0173] Table 9 Preparation of flavor protease and multi-strain composite strain synergistic debittering pea protein beverage

[0174] Pea protein drink16 Pea protein drink17 Pea protein drink18 Pea protein drink19 Bitterness 2.24 2.13 1.99 0.56 Sensory score (10 points) 9.39 9.23 9.15 9.81

[0175] Table 10 Volatile flavor compound content (μg / L) of pea protein beverage debittered by flavor protease and multi-strain composite bacteria

[0176]

[0177]

[0178] Overall, compared with the pea protein beverage debittered by the flavor protease and a single strain in Example 4, the organic acid content of the pea protein beverage debittered by the flavor protease and a multi-strain composite strain was increased by 16% to 39% (Table 7) compared with Example 4, and the overall flavor and acceptance were enhanced; the free amino acid content was increased by 2% to 12% compared with Example 4 (Table 8); the bitterness value was further reduced, and the sensory evaluation was further improved (Table 9); the content of undesirable flavor substances such as hexanal, 2-nonanone, and (E,E)-2,4-decadienal was further reduced (Table 10).

[0179] Among them, compared with beverages 16 to 18, pea protein beverage 19 had a moderate total amount of organic acids and did not bring a noticeable sour taste under the conditions of overall flavor improvement; the free amino acid content increased the most, the bitterness value was the lowest, and the sensory score was the highest. In addition, undesirable flavor substances that cause bitterness or off-flavor, such as hexanal, furfural, 2-nonanone, 2-pentylfuran, and (E,E)-2,4-decadienal, were significantly reduced or eliminated (Table 10). Therefore, the pea protein beverage debittered by the combination of flavor protease and Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus had the lowest bitterness value, the highest sensory score, and the best debittering effect.

[0180] Example 6: Preparation of debittered pea protein beverage using flavor protease and multi-strain composite bacteria

[0181] The specific implementation is the same as that of Example 4, except that the adjustment step (5) is:

[0182] The bacterial suspension of Lactobacillus plantarum CICC 22703, the bacterial suspension of Streptococcus thermophilus CICC 20375, and the bacterial suspension of Lactobacillus helveticus CICC 20275 obtained in step (4) were mixed in a volume ratio of 2:2:1 or 7:7:1 to obtain two mixed bacterial suspensions (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0183] Debittered pea protein beverages 20 and 21 were obtained (corresponding to a suspension volume ratio of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus of 2:2:1 or 7:7:1, respectively).

[0184] Here are the results:

[0185] Table 11 Preparation of bacterial enzyme-assisted debittered pea protein beverage

[0186]

[0187] Table 12 Volatile flavor compound content of pea protein beverage synergized with bacterial enzymes (μg / L)

[0188]

[0189]

[0190] Compared with the pea protein beverage 19 in Example 5 (the volume ratio of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus suspension was 1:1:1), the pea protein beverages 20 and 21 prepared by fermenting the Lactobacillus plantarum CICC 22703 suspension, Streptococcus thermophilus CICC 20375 suspension, and Lactobacillus helveticus CICC 20275 suspension in a volume ratio of 2:2:1 or 7:7:1 were inferior to the pea protein beverage 19 in debittering effect, sensory score, and overall acceptance (Table 11); the contents of undesirable flavor substances such as hexanal, 2-nonanone, and (E,E)-2,4-decadienal were also higher than those of the pea protein beverage 19 (Table 12).

[0191] Therefore, when flavor protease and lactic acid bacteria synergistically debitter pea protein, the lactic acid bacteria suspension is prepared by Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus in a volume ratio of 1:1:1. The resulting pea protein beverage has the best debittering effect and the highest sensory evaluation.

[0192] Comparative Example 1: Preparation of pea protein beverage

[0193] The specific steps are as follows:

[0194] (1) Stir and dissolve:

[0195] The mixture was stirred at 1000 rpm for 2 h at a ratio of pea protein powder to water = 2% (w / w) to obtain a pea protein solution;

[0196] (2) Enzymatic hydrolysis:

[0197] After adding 1.5‰ of alkaline protease Alcalase 2.4L FG (based on the mass of pea protein powder) to the pea protein solution obtained in step (1), the mixture was placed at 50°C and stirred for 1 hour for enzymolysis. After the enzymolysis was completed, the enzyme was heated at 95°C for 10 minutes to inactivate the enzyme to obtain a pea protein enzymatic hydrolyzate;

[0198] (3) Preparation of beverages:

[0199] Sodium bicarbonate was added to the pea protein hydrolysate obtained in step (2) to adjust the pH of the system to neutral, 1% (w / w) rapeseed oil and 0.6% (w / w) edible salt were added, and homogenization and sterilization were performed at a homogenization speed of 8000 U / min for 15 min and a sterilization temperature of 110° C. for 15 min;

[0200] A pea protein beverage 22 is obtained.

[0201] The results are shown in Table 13 and Table 14:

[0202] Table 13 Preparation of pea protein beverage

[0203]

[0204] Table 14 Volatile flavor content of pea protein beverage (μg / L)

[0205]

[0206] Comparative Example 2: Preparation of debittered pea protein beverage using flavor protease and multi-strain composite bacteria

[0207] 1. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0208] The bacterial suspension of Lactobacillus plantarum CICC 22703, the bacterial suspension of Streptococcus thermophilus CICC 20375, and the bacterial suspension of Lactococcus lactis CICC 23610 obtained in step (4) were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0209] Obtain debittered pea protein beverage 23.

[0210] 2. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0211] The bacterial suspension of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375 and Lactobacillus casei CICC 20286 obtained in step (4) were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension (3×10 6CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0212] A debittered pea protein beverage 24 is obtained.

[0213] 3. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0214] The bacterial suspension of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375 and Lactobacillus rhamnosus CICC 6001 obtained in step (4) were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0215] Obtain debittered pea protein beverage 25.

[0216] 4. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0217] The bacterial suspension of Lactobacillus plantarum CICC 22703, Lactobacillus helveticus CICC 20275 and Lactococcus lactis CICC 23610 obtained in step (4) were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0218] A debittered pea protein beverage 26 was obtained.

[0219] 5. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0220] The bacterial suspension of Lactobacillus plantarum CICC 22703, Lactobacillus helveticus CICC 20275 and Lactobacillus casei CICC 20286 obtained in step (4) were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension (3×10 6CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0221] Obtain debittered pea protein beverage 27.

[0222] 6. The specific implementation method is the same as that of Example 4, except that the adjustment step (5) is:

[0223] The bacterial suspension of Lactobacillus plantarum CICC 22703, Lactobacillus helveticus CICC 20275 and Lactobacillus rhamnosus CICC 6001 obtained in step (4) were mixed in a volume ratio of 1:1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), the mixed bacterial suspension was added to the pea protein hydrolysate obtained in step (3) at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate), shaken and placed in a 37°C incubator for 3.5h. After the fermentation was completed, it was sterilized at 110°C.

[0224] A debittered pea protein beverage 28 is obtained.

[0225] Here are the results:

[0226] Table 15 Preparation of flavor protease and multi-strain composite strain synergistic debittering pea protein beverage

[0227]

[0228] Table 16 Volatile flavor compound content (μg / L) of pea protein beverage debittered by flavor protease and multi-strain composite strains

[0229]

[0230]

[0231] Compared with the pea protein beverage debittered by the synergistic use of flavor protease and a composite strain of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375, and Lactobacillus helveticus CICC 20275 in Example 5, the pea protein beverage obtained in steps 1 to 5 of this comparative example had a higher bitterness value and a lower sensory score (Table 15); the content of undesirable flavor substances such as hexanal, 2-nonanone, and (E,E)-2,4-decadienal was relatively high (Table 16).

[0232] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a pea protein beverage debittered by bacterial enzymes, characterized in that: The following steps are involved: (1) adding water to pea protein powder and stirring to dissolve to obtain a pea protein solution; the ratio of the pea protein powder to water is 2% to 5% w / w; (2) adding alkaline protease to the pea protein solution obtained in step (1) and stirring, performing a first-stage enzymatic hydrolysis, and heating to inactivate the enzyme, wherein the amount of alkaline protease added is 0.5‰ to 1.5‰ based on the mass of the pea protein powder; (3) adding flavor protease to the solution obtained in step (2) and stirring, performing a two-stage enzymatic hydrolysis, heating to inactivate the enzyme, and sterilizing to obtain a pea protein hydrolyzate, wherein the amount of flavor protease added is 0.5‰ to 4.5‰ based on the mass of the pea protein powder; (4) Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus were mixed in a volume ratio of 1:1:1, 2:2:1, or 7:7:1 to obtain a mixed bacterial suspension, wherein the total viable bacterial count of the mixed bacterial suspension was (1-3)×10 6 CFU / mL, inoculate the mixed bacterial suspension into the pea protein hydrolysate obtained in step (3), culture and ferment, and then sterilize to obtain a fermentation broth; (5) The pH of the fermentation liquid obtained in step (4) is adjusted to neutral, edible oil and edible salt are added, and homogenization and sterilization are performed to obtain the pea protein beverage.

2. The preparation method according to claim 1, characterized in that The amount of flavor protease added in step (3) is 3‰ based on the mass of pea protein powder.

3. The preparation method according to claim 1, characterized in that The plant lactobacillus is plant lactobacillus CICC22703, the thermophilic streptococcus is thermophilic streptococcus CICC 20375, and the lactobacillus helveticus is lactobacillus helveticus CICC20275.

4. The preparation method according to claim 1, characterized in that The total viable count of the mixed bacterial suspension in step (4) is 3×10 6 CFU / mL.

5. The preparation method according to claim 1, characterized in that The inoculation amount of the bacterial suspension in step (4) is 2% to 6% based on the mass of the pea protein hydrolysate.

6. The preparation method according to claim 1, characterized in that The amount of edible oil added in step (5) is 0.5% to 1.5% w / w; the amount of edible salt added is 0.4% to 0.8% w / w.

7. A pea protein beverage, characterized in that: The pea protein beverage is prepared by the preparation method according to any one of claims 1 to 6.

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