Bacteria-enzyme synergistic debitterizing pea protein beverage and preparation method thereof

Abstract

The invention discloses a bacterium-enzyme synergistic debitterizing pea protein beverage and a preparation method thereof, and belongs to the technical field of food processing. According to the method, pea protein is adopted as a raw material, enzymolysis of alkaline protease and flavourzyme and fermentation of composite strains of lactobacillus plantarum, streptococcus thermophilus and lactobacillus helveticus are utilized to achieve a synergistic effect, and compared with fermentation by independently using lactic acid bacteria, debitterizing time is shorter, overall debitterizing efficiency is higher, sensory evaluation is better, and overall acceptability is high; pea-related peculiar smell-related volatile compounds, including hexanal, 2-nonanone, (E, E)-2, 4-nonadienal, 2-amyl furan, (E, E)-2, 4-decadienal and the like, of the prepared product are reduced or completely eliminated. The debittered pea protein beverage and the preparation method thereof provided by the invention are beneficial to widening the utilization way of pea protein and promoting the development of the pea protein beverage industry in China.

Description

Technical Field

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

[0002] Pea is one of the main legumes in the world. Due to its excellent yield, availability and low price, pea is widely used as a source of commercial protein. Pea itself is rich in protein, but its nutritional composition also contains a lot of starch and dietary fiber. Pea protein is a high-purity protein extracted from peas, rich in essential amino acids such as leucine, isoleucine and valine, which play an important role in muscle repair and growth. Pea protein and its specific peptide components also have antioxidant, anti-hypertensive, cholesterol-lowering and intestinal flora regulation effects. At present, the application of pea protein is concentrated in food supplements, emulsifiers, etc., and pea protein beverage varieties are scarce. Compared with beverages made of peas, beverages made of pea protein are beneficial to cardiovascular health due to their high protein content and help lower cholesterol and blood pressure; they are also rich in specific amino acids and proteins, which are beneficial for post-exercise recovery, muscle growth or as a protein supplement for vegetarians.

[0003] Due to the poor solubility of pea protein and the difficulty of nutrition being absorbed by the human body, the pea protein resources are not fully utilized, which affects the overall performance and development potential of the pea protein beverage market. The existing solution uses enzymatic hydrolysis (commonly used endonucleases) to improve its solubility and other characteristics and nutrient absorption rate, but the pea protein beverage prepared by endonuclease hydrolysis has a significant bitter taste and is difficult to be accepted by people. The hydrophobic amino acids at both ends of the peptide chain are the main cause of the bitterness. The existing debittering methods mainly include masking method, enzyme method, selective separation method, etc. The masking method is to mask the bitterness of pea protein by adding sweeteners, flavors and other flavor substances. This method is simple to operate, but it may change the original flavor of the product, and long-term consumption of additives may have adverse effects on health; the enzyme method is to further process pea protein using specific enzyme preparations to change its molecular structure or remove the hydrophobic amino acids at both ends of the peptide chain. However, the selectivity and efficiency of enzymes are often difficult to control, and the debittering efficiency needs to be improved; the selective separation method is to separate the bitter components from the non-bitter components in pea protein by physical or chemical means. Although this method can theoretically accurately remove bitterness, it is complex to operate and has high requirements on equipment.

[0004] In addition, the presence of off-flavors described as "bean flavor" or "green and grass flavor" is the main factor affecting consumers' acceptance of pea protein products, while low-bitterness, odor-free pea protein is less circulated in the market and is expensive, which is not conducive to its large-scale application in production. Therefore, using ordinary pea protein circulating in the market as a raw material, a method for preparing a low-bitterness, odor-free pea protein beverage is invented, which reduces or eliminates 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., which is conducive to broadening the utilization of pea protein, improving 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 bacterial enzyme-coordinated debittered pea protein beverage and a preparation method thereof. The method uses common 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 of 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 scheme is as follows:

[0006] The first object of the present invention is to provide a method for preparing a debitterized pea protein beverage synergistically prepared by bacterial enzymes, 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), stirring, performing 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 two-stage enzymatic hydrolysis, heating to inactivate the enzyme, and sterilizing to obtain pea proteinase 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 liquid;

[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 95-100° C. and a time of 8-12 min; preferably, the enzyme inactivation conditions are a temperature of 95° C. and a time of 10 min.

[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) of viable bacteria; 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 h; preferably, the culture and fermentation conditions are 37° C. for 3.5 h.

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

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

[0029] In one embodiment, the edible oil is added in an amount of 0.5% to 1.5% (w / w); preferably, the edible oil is added in an amount of 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 7500-8500 U / min and a time of 10-20 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 to 120° C. for 10 to 20 minutes; preferably, the sterilization treatment is performed at a temperature of 110° C. for 15 minutes.

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

[0034] Beneficial effects of the present invention:

[0035] The present invention provides a pea protein beverage de-bittered by bacterial enzymes and a preparation method thereof, wherein pea protein is used as a raw material, flavor protease is added, and lactic acid bacteria are inoculated for fermentation to produce a pea protein beverage with a low bitterness value. The pea protein beverage of the present invention and the preparation method thereof 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 a higher overall debittering efficiency, a shorter debittering time than using lactic acid bacteria fermentation alone, a significantly lower bitterness value, better sensory evaluation, and a high overall acceptance;

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

[0039] (4) The enzymes or bacteria added in 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, undesirable flavor substances that cause off-flavor, such as hexanal, furfural, 2-nonanone, (E,E)-2,4-nonadienal, 2-pentylfuran, and (E,E)-2,4-decadienal, are significantly reduced or eliminated.

[0041] The debittered pea protein beverage and the preparation method thereof provided by the present invention are conducive to broadening the utilization of pea protein, improving the market value of pea protein, and filling the gap of pea protein beverages. At the same time, it provides 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 all 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 Novozyme 37071 were purchased from Novozymes Enzyme Company, Denmark.

[0046] Flavor protease Flavourzyme 1000L and flavor protease Protana Prime were purchased from Novozymes Enzyme, Denmark; Flavor protease 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 Administration 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 embodiments and comparative examples are as follows:

[0053] 1. Solubility:

[0054] The samples were 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 samples were determined by the 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 0.02g / mL ninhydrin solution: Accurately weigh 1.00g of ninhydrin and 0.04g of stannous chloride, dissolve in hot water, filter and cool to room temperature, make up to volume in a 50mL brown volumetric flask, and store away from light.

[0059] Drawing of glycine standard curve: Weigh 50 mg of glycine and dissolve it in a 100 mL volumetric flask to a mass concentration of 500 μg / mL. Perform gradient dilution with a concentration gradient of 100, 125, 150, 175, 200 and 225 μg / mL. Take 1 mL of each concentration standard solution (use 1 mL of distilled water as a blank control), 1 mL of sodium phosphate buffer (pH 8.0), and 1 mL of ninhydrin solution and add them to a 25 mL colorimetric tube. Heat in a boiling water bath for 15 minutes, cool and dilute to 25 mL, and measure the absorbance at a wavelength of 570 nm. Linearly regress the measured absorbance value with the corresponding concentration to obtain the standard curve.

[0060] Determination of sample hydrolysis degree: Take 1 mL of diluted hydrolyzate (use 1 mL of distilled water as blank control), 1 mL of sodium phosphate buffer (pH 8.0), and 1 mL of 0.02 g / mL ninhydrin solution and add them to a 25 mL colorimetric tube, heat in a boiling water bath for 5 min, cool and dilute to 25 mL, and measure the absorbance at 570 nm. Calculate the amino acid concentration of the sample by comparing with the glycine standard curve.

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

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

[0063] Where: 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, the instrument model was Agilent 1100 high performance liquid chromatography system, mobile phase A (pH = 7.2): 27.6mmol / L sodium acetate - triethylamine - tetrahydrofuran (volume ratio of 500: 0.11: 2.5), mobile phase B (pH = 7.2): 80.9mmol / L sodium acetate - methanol - acetonitrile (volume ratio of 1: 2: 2); Agilent Hypersil ODS column (5μm, 4.0mm × 250mm); gradient elution was adopted, and the elution program was: 0min 8% B (92% A), 17min 50% B (50% A), 20.1min 100% B (0% A), 24min 0% B (100% A); the flow rate was 1.0mL / min; the column temperature was 40 ° C; the ultraviolet detection wavelength was 338nm.

[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 acid:

[0069] Add 1mL of sample to a 10mL volumetric flask and dilute to volume with 1% phosphoric acid solution. After mixing, let it stand for a while, centrifuge (10000×g, 15min, 25℃), extract the supernatant, and filter through a 0.45μm organic membrane. Use HPLC to detect the content of organic acids. HPLC uses a C18 chromatographic column (250×4.6mm), the column temperature is set to 30℃, and the detection is at a wavelength of 210nm. 0.1% phosphoric acid solution and methanol (97.5:2.5, v / v) are used as the mobile phase for isothermal elution, the flow rate is 1mL / min, and the injection volume is 5μL.

[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 taste characteristics of the bitter taste of the sample. To ensure the stability and reliability of data acquisition, the system was calibrated and self-checked before each sample measurement. The sample was prepared into a 0.05g / mL solution with deionized water and placed in a measuring cup, and 30M KCl and 0.3M tartaric acid were used as reference solutions. The detection sequence and cleaning procedure were then set. Among them, 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 bottle, 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 bottle in a 60 ° C water bath for 7 min, insert the extraction head into the extraction bottle, and adsorb while magnetically stirring for 30 min.

[0075] GC-MS conditions: GC conditions: Use DB-WAX column (30m×0.25mm, 0.25μm). Temperature program: Start temperature 40℃, hold for 3min; then heat to 100℃ at 6℃ / min; finally heat to 230℃ at 10℃ / min, hold for 7min; non-split injection. MS conditions: ion source EI source, ion source temperature 230℃, interface temperature 250℃, electron energy 70ev, scan range m / z 33-500, acquisition mode Scan.

[0076] Qualitative and quantitative analysis of flavor substances: Volatile substances can be qualitatively analyzed by comparing the results retrieved from the mass spectrometry database (NIST and WILEY database) with standard compounds. The internal standard method is used for quantitative analysis, and the chromatographic peak areas of the internal standard and the tested compound are compared to calculate the content of the tested compound according to μg / L.

[0077] 7. Sensory evaluation:

[0078] A sensory evaluation panel was formed by 20 students who had been trained on aroma and taste characteristics, including 10 males and 10 females. The taste, aroma, mouthfeel and overall acceptance of pea protein beverages were scored. When calculating the average of each indicator, we excluded the highest and lowest scores in each indicator. During the scoring process, the mouth was rinsed with purified water between different samples. The scores of each indicator of sensory evaluation 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. Pea protein powder: water = 2% (w / w) ratio was fully stirred for 2h, 1.5‰ of alkaline protease Alcalase 2.4L FG (based on the mass of pea protein powder) was added, and the pea protein solution was placed at 50°C and stirred for 1h for enzymolysis. After the enzymolysis was completed, the enzyme was heated at 95°C for 10min to inactivate the enzyme. The solubility and hydrolysis degree of pea protein powder were measured.

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

[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 by 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 by neutral protease Neutrase 0.8L, its solubility was 56% and the degree of hydrolysis was 4.53%;

[0090] When pea protein powder is hydrolyzed by 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 alkaline protease were the highest, which were 78% and 9.28% respectively. The following examples all used alkaline protease 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) Stirring and dissolving:

[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 enzymolysis. After the enzymolysis was completed, the mixture was heated at 95°C for 10 minutes to inactivate the enzyme, thereby obtaining a pea protein enzymatic 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, it was repeatedly cultured at 37°C for 12 hours, and the bacterial solution was picked up with an inoculation loop and placed on a test tube slope made of MRS solid culture medium for 48 hours. A single colony of Lactobacillus plantarum obtained on the slope was picked up and cultured in MRS liquid culture medium at 37°C for 12 hours. From the liquid culture medium, 1% (v / v) was taken for expansion culture in MRS liquid culture medium (culture conditions: 37°C, 12 hours), and 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, and the bacterial mud was repeatedly washed with sterile saline for 3 times, and finally it was redissolved with equal volume of saline to obtain a viable 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 bacterial suspensions of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375, Lactobacillus helveticus CICC 20275, Lactobacillus casei CICC 20286, Lactobacillus rhamnosus CICC6001 and Lactococcus lactis CICC 23610 obtained in step (3) are 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 mixtures are shaken and placed in a 37° C. incubator for 6 h. After the fermentation is completed, the mixtures are sterilized at 110° C. to obtain a fermentation liquid.

[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] The results are as follows:

[0107] Table 2 Preparation of lactic acid bacteria debittered 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 with the pea protein beverage 22 (bitterness value 10.22) obtained by only using alkaline protease enzymolysis without lactic acid bacteria fermentation in comparative example 1, the lactic acid bacteria debittered pea protein beverage has a significant debittering effect, and the sensory evaluation is also significantly improved; the free amino acid content is increased by 13% to 28%; lactic acid bacteria fermentation produces organic acids, which improves the overall flavor of the beverage (Table 2). In addition, Lactobacillus plantarum has the best debittering effect, and the beverage bitterness value is 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 good flavor substance (E)-2-nonenal was newly 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) Stirring and dissolving:

[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 solution was heated at 95°C for 10 minutes to inactivate the enzyme;

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

[0122] Add 3‰ flavor protease Flavourzyme 1000L, flavor protease Protana Prime or flavor protease PR EXO-L (based on the mass of pea protein powder) to the solution obtained in step (2), stir thoroughly at 50°C and 1000 rpm for 1 hour to perform two-stage enzymolysis. After the enzymolysis is completed, heat at 95°C to inactivate the enzyme for 10 minutes, and sterilize at 110°C for 15 minutes to obtain 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 respectively, and homogenization and sterilization were performed. The homogenization speed was 8000 U / min, the time was 15 min, and the sterilization temperature was 110° C., and the time was 15 min.

[0125] De-bittered 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) respectively).

[0126] The results are as follows:

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

[0128]

[0129] Compared with the pea protein beverage 22 (bitterness value 10.22) obtained by enzymolysis with alkaline protease but not flavor protease in comparative example 1, the pea protein beverage debittered by flavor protease has a significant debittering effect, among which the pea protein beverage 7 debittered by flavor protease Flavourzyme 1000L has a lower bitterness value and a higher sensory score; the free amino acid content is increased by 352% to 362% (Table 4). The following examples all use flavor protease 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) Stirring and dissolving:

[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 solution was heated at 95°C for 10 minutes to inactivate the enzyme;

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

[0137] Add 3‰ flavor enzyme Flavourzyme 1000L (based on the mass of pea protein powder) to the solution obtained in step (2), stir thoroughly at 50°C and 1000rpm for 1h to perform a second-stage enzymolysis. After the enzymolysis is completed, heat at 95°C to inactivate the enzyme for 10min, and sterilize at 110°C for 15min 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, it was repeatedly cultured at 37°C for 12 hours, and the bacterial solution was picked up with an inoculation loop and placed on a test tube slope made of MRS solid culture medium for 48 hours. A single colony of Lactobacillus plantarum obtained on the slope was picked up and cultured in MRS liquid culture medium at 37°C for 12 hours. From the liquid culture medium, 1% (v / v) was taken for expansion culture in MRS liquid culture medium (culture conditions: 37°C, 12 hours), and 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, and the bacterial mud was repeatedly washed with sterile saline for 3 times, and finally it was redissolved with equal volume of saline to obtain a viable 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) are 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), and the mixture is shaken and placed in a 37° C. incubator for 3.5 h. After the fermentation is completed, the mixture is sterilized at 110° C. to obtain a fermentation liquid;

[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] The debittered pea protein beverages 10 to 15 (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) respectively) were obtained.

[0146] The results are as follows:

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

[0148]

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

[0150]

[0151]

[0152] Compared with the pea protein beverage debittered only with lactic acid bacteria in Example 2, the pea protein beverage debittered by flavor protease and a single strain has a better debittering effect, the bitterness value is further reduced, and the sensory evaluation is improved; the free amino acid content is increased by 281% to 364% compared with Example 2; the organic acid content is increased by 50% to 100% compared with Example 2, and the overall flavor and acceptance are enhanced (Table 5); the content of undesirable flavor substances such as hexanal, 2-nonanone, (E,E)-2,4-decadienal is 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 bacterial suspension of Lactobacillus plantarum CICC 22703 and the bacterial suspension of Streptococcus thermophilus CICC 20375 obtained in step (4) were mixed at a volume ratio of 1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize 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 and the Lactobacillus helveticus CICC 20275 bacterial suspension obtained in step (4) were mixed at a volume ratio of 1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize at 110°C.

[0160] The debittered pea protein beverage 17 was obtained.

[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 and the Lactobacillus helveticus CICC 20275 bacterial suspension obtained in step (4) were mixed at a volume ratio of 1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize 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, 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 1:1:1 to obtain a mixed bacterial suspension (3×10 6 CFU / mL), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize at 110°C.

[0166] The debittered pea protein beverage 19 was obtained.

[0167] The results are as follows:

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

[0171]

[0172]

[0173] Table 9 Preparation of flavor protease and multi-strain composite strains to de-bitter 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 substance content of pea protein beverage debittered by flavor protease and multi-strain composite bacteria (μg / L)

[0176]

[0177]

[0178] In general, 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% (Table 8) compared with Example 4; 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, (E,E)-2,4-decadienal was further reduced (Table 10).

[0179] Among them, compared with beverages 16 to 18, the total amount of organic acid in pea protein beverage 19 is moderate, and under the condition of overall flavor improvement, it does not bring obvious sourness; the free amino acid content is increased the most, the bitterness value is the lowest, and the sensory score is the highest. In addition, undesirable flavor substances that cause bitterness or odor, such as hexanal, furfural, 2-nonanone, 2-pentylfuran, (E,E)-2,4-decadienal, etc., are significantly reduced or eliminated (Table 10). Therefore, the pea protein beverage debittered by flavor protease in combination with Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus helveticus has 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), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize at 110°C.

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

[0184] The results are as follows:

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

[0186]

[0187] Table 12 Volatile flavor substance content of pea protein beverage synergized by bacterial enzyme debitterization (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 bacterial suspension of Lactobacillus plantarum CICC 22703, Streptococcus thermophilus CICC 20375, and Lactobacillus helveticus CICC 20275 in a volume ratio of 2:2:1 or 7:7:1 were not as good as the pea protein beverage 19 in terms of 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 a suspension of 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) Stirring and dissolving:

[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 mixture was heated at 95°C for 10 minutes to inactivate the enzyme, thereby obtaining 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), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize at 110°C.

[0209] A debittered pea protein beverage 23 was obtained.

[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, the bacterial suspension of Streptococcus thermophilus CICC 20375, and the bacterial suspension of 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), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize 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, the bacterial suspension of Streptococcus thermophilus CICC 20375, and the bacterial suspension of 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), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize at 110°C.

[0215] A debittered pea protein beverage 25 is obtained.

[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, the bacterial suspension of Lactobacillus helveticus CICC 20275, 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), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize 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, the bacterial suspension of Lactobacillus helveticus CICC 20275, and the bacterial suspension of 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), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize at 110°C.

[0221] A debittered pea protein beverage 27 was obtained.

[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, the bacterial suspension of Lactobacillus helveticus CICC 20275, and the bacterial suspension of 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), add the mixed bacterial suspension at an inoculum amount of 3% (based on the mass of the pea protein hydrolysate) to the pea protein hydrolysate obtained in step (3), shake well and place in a 37°C incubator for 3.5h. After the fermentation is completed, sterilize at 110°C.

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

[0225] The results are as follows:

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

[0227]

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

[0229]

[0230]

[0231] Compared with the pea protein beverage debittered by flavor protease and composite strains 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 has 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 is relatively high (Table 16).

[0232] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 debitterized pea protein beverage using 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; (2) adding alkaline protease to the pea protein solution obtained in step (1), stirring, performing a first-stage enzymatic hydrolysis, and heating to inactivate the enzyme; (3) adding flavor protease to the solution obtained in step (2) and stirring, performing two-stage enzymatic hydrolysis, heating to inactivate the enzyme, and sterilizing to obtain pea enzymatic hydrolyzate; (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 liquid; (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 ratio of pea protein powder to water in step (1) is 2% to 5% w / w.

3. The preparation method according to claim 1, characterized in that: The amount of alkaline protease added in step (2) is 0.5‰ to 1.5‰ based on the mass of pea protein powder.

4. The preparation method according to claim 1, characterized in that: The amount of flavor protease added in step (3) is 0.5‰ to 4.5‰ based on the mass of pea protein powder; preferably, the amount added is 3‰.

5. The preparation method according to claim 1, characterized in that: The mixed bacterial suspension in step (4) 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) of viable bacteria count.

6. 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.

7. The preparation method according to claim 1, characterized in that: 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.

8. 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.

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

10. A pea protein beverage, characterized in that: The pea protein beverage is prepared by the preparation method described in claims 1 to 9.

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