Pure plant-based walnut fermented milk and preparation method thereof

Abstract

The invention provides pure plant-based walnut fermented milk and a preparation method thereof, the main raw materials of the fermented milk are walnut milk and a mixed strain, the mixed strain is compounded by lactobacillus bulgaricus, streptococcus thermophilus and lactobacillus plantarum according to a ratio of 1: 1: 1, and the fermentation process flow is optimized. The process conditions are as follows: the material-to-liquid ratio is 1: 5 (kg / L), the addition amount of sucrose is 7%, the addition amount of strains is 4.5%, the fermentation temperature is 36 DEG C, and the fermentation time is 12 hours. The pure plant-based walnut fermented milk prepared by the preparation method has fine and smooth mouth feel and natural and rich walnut fragrance, the problems of rough mouth feel, poor flavor and the like of the plant-based walnut fermented milk are solved, the sensory score is improved from 68.4 to 86.8, and meanwhile, the hardness (75%), cohesiveness (17.6%), elasticity (33.9%), adhesiveness (140.9%) and chewiness (167.1%) of the fermented milk are improved. The product is completely based on plant-based raw materials, does not contain animal-derived components, and is more in line with the concepts of health and environmental protection.

Description

Technical Field

[0001] The present invention relates to the field of food and fermentation technology, and in particular to a pure plant-based walnut fermented milk and a preparation method thereof. Background Art

[0002] Plant fermented milk is a dairy substitute made from plant raw materials (such as soybeans, almonds, coconuts, etc.) through a fermentation process. During the fermentation process, probiotics such as lactic acid bacteria ferment the sugars in the plant raw materials into lactic acid, forming a fermented dairy product with a sour taste and rich texture. Unlike traditional animal dairy products, plant fermented milk does not contain animal ingredients and is suitable for vegetarians, lactose intolerant patients and people with allergies. In addition, plant fermented milk can also promote intestinal health by regulating intestinal flora and providing probiotics, and has certain health functions. In recent years, with the improvement of global health awareness and the rise of vegetarianism and environmentalism, plant-based diets have gradually become a popular trend. Therefore, as an emerging food category, plant fermented milk has a wide market demand and huge application prospects. With the improvement of consumers' health awareness and the popularization of environmental protection concepts, it is particularly important to study the production process of plant fermented milk, which has important production and practical significance.

[0003] Pure plant-based fermented milk is difficult to ferment because it is different from the animal protein system. Plant protein is difficult to form a stable three-dimensional network structure, and thus difficult to form a stable gel system. The water phase is severely separated, the texture is poor, the odor is bad, more aldehydes form a grassy smell, it is easy to oxidize to produce bad color, and the taste is not delicate. This is attributed to the fact that the fermentation bacteria cannot effectively use walnut pulp for fermentation. Therefore, plant fermented milk is currently mostly fermented in a mixed way, but dairy-based fermented milk and walnuts have their own unique substances, especially animal protein and plant protein are quite different, and the flavors produced belong to two different systems. Mixed fermentation is difficult to highlight their respective characteristics.

[0004] At present, the fermentation strains of plant-based fermented milk are mainly Lactobacillus bulgaricus and Streptococcus thermophilus, which are commonly used in dairy yogurt. The two are mutually beneficial. The formic acid, pyruvic acid, folic acid, etc. produced by Streptococcus thermophilus can be used by Lactobacillus bulgaricus. At the same time, the free amino acids and peptides produced by Lactobacillus bulgaricus during the fermentation process can be used by Streptococcus thermophilus. Studies have shown that the current research on probiotic walnut fermented milk mainly focuses on mixed fermentation with traditional fermentation strains. The lactobacilli used as probiotics today include Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus casei. Traditional solutions are mostly limited to improving product formulas and optimizing processing technology, while research on screening suitable fermentation strains is relatively lacking.

[0005] At present, there are few studies on the use of walnuts as plant-based raw materials to completely replace dairy raw materials to prepare plant-based fermented milk. Therefore, the development of pure plant-based walnut fermented milk has significant nutritional and health advantages. Walnuts are rich in healthy fats, plant proteins, dietary fiber and antioxidants. After fermentation, they can provide rich probiotics, which are helpful for intestinal health, immune regulation and weight loss management. At the same time, walnut fermented milk does not contain lactose, which is suitable for people with lactose intolerance, vegetarians and allergies. Its production process has little impact on the environment, which is in line with the trend of sustainable development. In addition, the unique flavor of walnuts and the taste after fermentation give them strong application prospects. Summary of the invention

[0006] In view of the shortcomings of the existing research on screening suitable fermentation strains for pure plant fermentation matrices and plant-based fermented milk processing technology, the present application provides a bacteria screening method and a method for preparing pure plant-based walnut fermented milk to solve the problems of poor structural stability, poor flavor, poor texture, and poor taste of pure plant-based walnuts.

[0007] On the one hand, the present application provides a pure plant-based walnut fermented milk, the components of which include: walnut kernels, water, sucrose, and bacteria; the solid-liquid ratio of walnut kernels to water is 1:5 (kg / L); the added amount of sucrose is 7%, and the added amount of bacteria is 4.5%.

[0008] Furthermore, the bacterial strains are: Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus plantarum; and the compound ratio of the bacterial strains is: 1:1:1.

[0009] Furthermore, the fermentation temperature of the strain is 36° C., and the fermentation time is 12 h.

[0010] On the other hand, the present application also provides a method for preparing pure plant-based walnut fermented milk, the preparation method comprising the following steps:

[0011] 1) filtering the processed walnut kernels using a 100-mesh filter to obtain a puree;

[0012] 2) Add sucrose to the walnut puree obtained in 1) to obtain a mixed walnut puree; add sucrose in an amount of 4%, heat the mixed walnut puree to 45° C., and stir for 10 minutes; homogenize and sterilize for later use; obtain a sterile walnut puree;

[0013] 3) cooling the sterile stock in 2) to 35-40° C., adding the mixed bacteria into the stock, stirring evenly; and then fermenting at 36° C. for 12 hours; the mixed bacteria are Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus plantarum in a ratio of 1:1:1;

[0014] 4) Refrigerated ripening: Store at 4°C for 24 hours to ripen and produce acid;

[0015] The above-mentioned pure plant-based walnut fermented milk is obtained.

[0016] Furthermore, the processing steps are specifically as follows: select dry walnuts with full appearance, no mildew and no insect infestation, remove the shell to obtain walnut kernels, soak the walnut kernels in boiling water for 10 minutes, and remove the seed coat; add pure water according to the solid-liquid ratio to grind the walnut kernels; and obtain the processed walnut kernels.

[0017] Furthermore, the solid-liquid ratio was 1:5 (kg / L); the refining speed was 2000 rpm, and the time was 20 min; and the process was repeated 3 times.

[0018] Furthermore, the specific operation of homogenization in 2) is: homogenization at 25 MPa for 3 minutes, and cycle 3 times.

[0019] Furthermore, the specific operation of sterilization in 2) is: the raw pulp is sterilized at a temperature of 85° C., and the sterilization time is 20 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The above and other objects, features and advantages of the present application will become more apparent by describing in more detail the exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0021] Figure 1 This is a schematic diagram of the process flow of pure plant-based walnut fermented milk;

[0022] Figure 2 This is a schematic diagram of the acidity comparison of different strains after 20 hours of fermentation;

[0023] Figure 3 Schematic diagram for investigating the effect of solid-liquid ratio on water holding capacity and sensory scores;

[0024] Figure 4 Schematic diagram for investigating the effect of sucrose addition on water holding capacity and sensory scores;

[0025] Figure 5 Schematic diagram for investigating the effect of fermentation temperature on water holding capacity and sensory scores;

[0026] Figure 6 Schematic diagram for investigating the effect of fermentation time on water holding capacity and sensory scores;

[0027] Figure 7 Schematic diagram for investigating the effect of bacterial inoculation amount on water holding capacity and sensory scores;

[0028] Figure 8 The response surface and contour diagram to investigate the effects of fermentation temperature and fermentation time on sensory scores;

[0029] Fig. 9The response surface and contour diagram to investigate the effects of fermentation temperature and bacterial inoculation amount on sensory scores;

[0030] Fig.10 The response surface and contour diagram to investigate the effects of fermentation time and bacterial inoculation amount on sensory scores;

[0031] Beneficial effects:

[0032] 1. The present invention prepares pure plant-based walnut fermented milk by screening out suitable fermentation bacteria to ferment walnut pulp, and at the same time determines the optimal fermentation process conditions, thereby alleviating the problems of rough taste and poor flavor of the original walnut fermented milk, and the sensory score is improved from 68.4 to 86.8. At the same time, the hardness, cohesion, elasticity, viscosity and chewiness of the walnut fermented milk are significantly improved, and the improvement ranges are 75%, 17.6%, 33.9%, 140.9% and 167.1% respectively. The developed pure plant-based walnut fermented milk product has a delicate taste, rich aroma and stable structure.

[0033] 2. The preparation method of the present invention has a simple process, is convenient and efficient to operate, and the production cost of the required fermentation matrix raw materials is low. It has a certain promoting effect on the utilization of walnut resources and satisfying consumers' diversified choices for pure plant-based fermented milk, and provides a certain theoretical basis for the subsequent compounding of the starter of pure plant-based fermented milk. DETAILED DESCRIPTION

[0034] The optional embodiments of the present invention will be described in more detail below. Although the optional embodiments of the present invention are described, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0035] Plant-based fermented milk is different from dairy yogurt. It is not appropriate to prepare it according to the fermentation process of dairy yogurt. It should be fermented according to the properties of walnut pulp and the fermentation conditions required for screening suitable bacteria. The fermentation process should be explored and optimized to maximize the quality of pure plant-based walnut fermented milk.

[0036] Using walnut pulp as the fermentation substrate, by screening suitable lactic acid bacteria strains for fermentation and determining the optimal formula ratio and processing technology, we improved a series of problems of pure plant-based walnut fermented milk such as poor flavor, poor texture and poor taste. We developed a pure plant-based fermented milk drink with unique flavor, good texture, healthy nutrition, which provides certain theoretical support for the development of domestic pure plant-based fermented milk drinks with special flavors, and also lays the foundation for the development of excellent plant-based fermentation strains.

[0037] For the sake of clarity, the following is described in detail by the following Example 1. Unless otherwise specified, the test materials and reagents used in the following Example 1 are purchased from the market.

[0038] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0039] Example 1 Selection of strains

[0040] This embodiment is to screen strains with high fermentation activity and strong acid production capacity for this application, and determine the strains required for the composite starter of the pure plant-based walnut fermented milk of this application. The screened strains include: Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus paracasei, and Lactobacillus acidophilus. Specifically, the following steps are carried out:

[0041] Step 1: Bacteria Activation

[0042] The -80℃ preserved bacterial strains (all the above bacterial strains) in the laboratory were restored to room temperature, and 1 mL was taken from each strain and inoculated into 9 mL of MRS liquid culture medium that had been sterilized in advance and cooled to room temperature, and cultured at 37℃ for 24 hours. Repeat the subculture three times, and the viable bacterial count reached more than 109 CFU / mL for use.

[0043] Step 2: Fermentation index determination

[0044] 1) Water holding capacity: Take 10-15 mL of fermented walnut fermented milk sample, weigh the initial mass of the sample, centrifuge at 10000 r / min for 15 min, let it stand for 10 min, pour off the supernatant, weigh the mass of the remaining sediment, and calculate the water holding capacity of the fermented milk according to the following formula.

[0045] Water holding capacity calculation formula:

[0046] 2) Viscosity: Take the fermented walnut fermented milk, break the emulsion and return to room temperature, and measure the viscosity of the sample at 30 seconds using a Brookfield DV3T viscometer with rotor No. 3 and a speed of 60 RPM.

[0047] 3) Coagulation time: The activated strains are fermented according to the preparation method of a pure plant-based walnut fermented milk provided in the present application (the treated walnut kernels are added with pure water according to the solid-liquid ratio for grinding; the ground walnut kernels are filtered using a 100-mesh filter to obtain a stock paste; sucrose is added to the stock paste to obtain a mixed stock paste; adding: the amount of sucrose added is 4%, the mixed walnut paste is heated to 45°C, and stirred for 10 minutes; homogenized and sterilized for standby use; aseptic stock paste is obtained; the aseptic stock paste is cooled to 35-40°C, the mixed strains are added to the stock paste, and stirred evenly, the mixed strains are Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus plantarum in a ratio of 1:1:1; and then fermented at 36°C for 12 hours; refrigerated post-ripening: stored at 4°C for 24 hours for post-ripening and acid production) until coagulation occurs, and the coagulation time is recorded.

[0048] 4) Fermentation endpoint time: The fermentation endpoint is when the pH of the fermented milk reaches 4.5, and the fermentation endpoint time is recorded.

[0049] 5) Acidity at the end of fermentation: The acidity of fermented milk was measured according to the phenolphthalein indicator method in GB 5009.239-2016 “National Food Safety Standard - Determination of Acidity of Foods”, and the acidity at the end of fermentation was recorded.

[0050] 6) Determination of acid production capacity of bacterial strains: After activating the 11 strains to be tested, inoculation and fermentation were carried out according to the above-mentioned fermentation process. Samples were taken every 2 hours to measure the acidity and the acid production curve was drawn.

[0051] Table 1 Determination results of fermentation indexes of different strains

[0052]

[0053] As can be seen from Table 1, the coagulation time and fermentation end time of Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus casei and Lactobacillus paracasei are relatively slow, between 15 and 18 hours and 18 and 21 hours respectively, and the acidity at the fermentation end is also low, between 30 and 40 ° T. According to the "National Group Standard for Plant Protein Beverage and Plant Yogurt Standard", the acidity of plant-based fermented milk should be greater than 30 ° T. It can be seen that their fermentation ability for walnut puree is poor. Bulgarian Lactobacillus and thermophilic Streptococcus showed strong fermentation acid production ability, followed by fermented Lactobacillus, rhamnosus Lactobacillus, plant Lactobacillus, helveticus and Lactobacillus acidophilus also showed good fermentation acid production ability, although not as good as Bulgarian Lactobacillus and thermophilic Streptococcus, but generally fermented milk is multi-strain compounded, which is compounded with strains with strong fermentation ability, and then has a positive effect on the texture and flavor of fermented milk.

[0054] Depend on Figure 2It can be seen that except for Lactobacillus bulgaricus, whose acidity began to increase rapidly at the 6th hour, other strains increased rapidly at 8 to 14 hours, and the acidity growth basically stabilized after 14 hours. From the beginning to the end of fermentation, the acidity of Lactobacillus bulgaricus increased from 12.7°T to 74.6°T, and the acidity increased by 63.7°T, with the strongest acid production capacity. The acidity of Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus fermentum, and Lactobacillus helveticus rhamnosus also increased by 61.8°T, 57.6°T, 55.9°T, 51.7°T, 44.3°T, and 43.7°T, respectively. In contrast, the acidity growth of Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus casei and Lactobacillus paracasei was smaller, only 36.9°T, 31.5°T, 30.6°T and 28.1°T respectively. As the acid production was too slow and the acid production was low, the fermentation cycle was long, which could easily lead to poor quality and product contamination. Therefore, they are not suitable as fermentation strains.

[0055] According to Table 3 and Figure 2 According to the results, Lactobacillus bulgaricus with the strongest acid-producing ability and Streptococcus thermophilus with the strongest viscosity-producing ability were finally selected as the basic fermentation strains, while Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus and Lactobacillus acidophilus were selected as the pending matching strains, in order to further carry out the optimal strain combination fermentation experiment.

[0056] Step 3: Fermentation screening of dominant bacterial strains

[0057] According to the fermentation index in the above step 2, lactic acid bacteria with good fermentation performance of walnut puree were selected for three-strain combination screening, and 4.5% inoculation amount was inoculated into the sterilized walnut puree at a strain concentration ratio of 1:1:1, and fermented at 36°C for 12 hours. After fermentation, it was placed in a 4°C refrigerator for refrigeration and ripening for 24 hours, and then texture testing and sensory evaluation were performed to select the best strain combination with good texture and the best sensory evaluation.

[0058] 1) Texture detection: Full texture determination of fermented milk TPA: The texture analyzer test mode selects full texture test, the probe is TA / 36R, the speed before, during and after the test is 1mm / s, and the trigger force is 4g.

[0059] 2) Sensory evaluation: The sensory evaluation of pure plant-based walnut fermented milk was conducted in accordance with the provisions of GB 19302-2010 "National Food Safety Standard Fermented Milk". A sensory evaluation panel consisting of 10 personnel who had passed professional tasting training scored the fermented milk based on four indicators: visual level - color (20 points), taste level - flavor (20 points), olfactory level - smell (30 points), and tactile level - tissue state (30 points). The total score was 100 points. The specific sensory scoring standards are shown in Table 2.

[0060] Table 2 Sensory evaluation criteria for pure plant-based walnut fermented milk

[0061]

[0062]

[0063] Example 2 Optimization of the fermentation process of pure plant-based walnut fermented milk

[0064] Key points of operation: The appropriate bacterial strain combination selected in Example 1 is used as the fermentation strain of pure plant-based walnut fermented milk to prepare fermented milk. The process route is as follows: Figure 1 As shown. On this basis, single factor and response surface experiments were conducted on the material-liquid ratio, fermentation temperature, fermentation time, bacterial inoculation amount, and sucrose addition to determine the optimal value of each parameter. Finally, the physical, chemical, and microbiological indicators of the fermented milk product were tested.

[0065] Step 1: Process Optimization

[0066] Taking water holding capacity and sensory score as evaluation indicators, the solid-liquid ratio, sucrose addition, fermentation temperature, fermentation time and bacterial inoculation amount were set as single factors, and five experimental levels were set for each single factor. The effects of solid-liquid ratio (1:2, 1:4, 1:6, 1:8, 1:10), sucrose addition (1%, 2%, 3%, 4%, 5%), fermentation temperature (32℃, 34℃, 36℃, 38℃, 40℃), fermentation time (8h, 10h, 12h, 14h, 16h) and bacterial inoculation amount (1.5%, 3%, 4.5%, 6%, 7.5%) on the quality of pure plant-based walnut fermented milk were investigated in turn.

[0067] like Figure 3 As shown, when the solid-liquid ratio is 1:1-1:9 (kg / L), the water holding capacity and sensory evaluation score of the prepared samples are higher, and when it is 1:5 (kg / L), the sensory evaluation score is the highest. Therefore, the optimal solid-liquid ratio is 1:5 (kg / L);

[0068] like Figure 4 As shown, when the sucrose addition amount is 1% to 9%, the water holding capacity and sensory evaluation score of the prepared samples are higher, and the sensory evaluation score is the highest when it is 7%. Therefore, the optimal sucrose addition amount is 7%;

[0069] like Figure 5 As shown, the water holding capacity and sensory evaluation score of the samples prepared when the fermentation temperature is 32-40°C are higher, and the sensory evaluation score is the highest at 36°C. Therefore, the optimal fermentation temperature is 36°C;

[0070] like Figure 6 As shown, the water holding capacity and sensory evaluation score of the samples prepared when the fermentation time is 8 to 16 hours are higher, and the sensory evaluation score is the highest when it is 8 hours. Therefore, the optimal fermentation time is 8 hours;

[0071] like Figure 7 As shown, when the inoculation amount of the bacterial strain is 1.5% to 7.5%, the water holding capacity and sensory evaluation score of the prepared sample are higher, and when it is 4.5%, the sensory evaluation score is the highest. Therefore, the most suitable inoculation amount of the bacterial strain is 4.5%;

[0072] Step 2: Response surface optimization experiment

[0073] Based on the results of the single factor experiment, the fermentation time, fermentation temperature and inoculation amount of pure plant-based walnut fermented milk were used as independent variables. The Design-Expert-Box-Behnken program software was used to perform response surface analysis under the conditions of 3 factors and 3 levels to optimize the optimal fermentation process formula of pure plant-based walnut fermented milk. The Box-Behnken test factor levels are shown in Table 3.

[0074] Table 3 Box-Behnken test factors and levels

[0075]

[0076] Example 3 Preparation of pure plant-based walnut fermented milk

[0077] Step 1: Prepare pure plant-based walnut fermented milk

[0078] According to the conditions selected in the above-mentioned embodiment 1 and embodiment 2, pure plant-based walnut fermented milk is prepared, and the specific steps are as follows:

[0079] 1) adding pure water to the treated walnut kernels according to the material-liquid ratio for grinding;

[0080] 2) filtering the walnut kernels after grinding in 1) through a 100-mesh filter to obtain raw pulp;

[0081] 3) Add sucrose to the walnut puree obtained in 2) to obtain a mixed walnut puree; add sucrose in an amount of 4%, heat the mixed walnut puree to 45° C., and stir for 10 minutes; homogenize and sterilize for later use; obtain a sterile walnut puree;

[0082] 4) cooling the sterile stock in 3) to 35-40° C., adding the mixed bacteria into the stock, stirring evenly; and then fermenting at 36° C. for 12 hours; the mixed bacteria are Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus plantarum in a ratio of 1:1:1;

[0083] 5) Refrigerated ripening: Store at 4°C for 24 hours to ripen and produce acid;

[0084] The above-mentioned pure plant-based walnut fermented milk is obtained.

[0085] Furthermore, the steps of processing walnut kernels are specifically as follows: selecting dry walnuts with full appearance, no mildew, and no insect infestation, removing the shell to obtain walnut kernels, soaking the walnut kernels in boiling water for 10 minutes, and removing the seed coat.

[0086] Further, the material-liquid ratio in 1) was 1:5 (kg / L), and the refining was repeated 3 times at 2000 rpm for 20 min.

[0087] Further, filtering is performed in 2).

[0088] Furthermore, the specific operation of homogenization in 3) is: homogenization at 25 MPa for 3 minutes, and cycle 3 times.

[0089] Furthermore, the specific operation of sterilization in 3) is: the raw pulp is sterilized at a temperature of 85° C., and the sterilization time is 20 minutes.

[0090] Step 2: Determination of physical, chemical and microbiological indicators of pure plant-based walnut fermented milk

[0091] The pure plant-based walnut fermented milk prepared in step one was tested.

[0092] The pH of fermented milk is determined using a pH meter; the protein content is measured according to the Kjeldahl nitrogen method in GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Foods"; the number of viable lactic acid bacteria is determined in accordance with GB 4789.35-2016 "National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination"; Escherichia coli is determined in accordance with GB 4789.3-2016 "National Food Safety Standard Food Microbiology Examination Coliform Count"; pathogenic bacteria are determined in accordance with GB 29921.3-2021 "National Food Safety Standard Limits of Pathogenic Bacteria in Pre-packaged Foods".

[0093] result

[0094] 1) As shown in Table 4, the texture parameters of fermented milk fermented by the strain combination (L1+L2+L5) are the best among all combinations, especially hardness and viscosity. Comprehensive comparison shows that the strain combination (L1+L2+L5) has the best effect on the texture of pure plant-based walnut fermented milk.

[0095] Table 4 Effects of different bacterial strain combinations on the texture of walnut fermented milk

[0096]

[0097] Note: L1 represents Lactobacillus bulgaricus; L2 represents Streptococcus thermophilus; L3 represents Lactobacillus fermentum; L4 represents Lactobacillus rhamnosus; L5 represents Lactobacillus plantarum; L6 represents Lactobacillus helveticus; L7 represents Lactobacillus acidophilus. Different letters in the shoulder represent significant differences.

[0098] 2) As shown in Table 5, different bacterial strain combinations have little effect on the color and smell of fermented milk, but have a significant effect on the taste and texture of fermented milk. The bacterial strain combination (L1+L2+L5) performs very well in terms of the taste and texture of fermented milk. Based on the results of Tables 4 and 5, the optimal fermentation bacterial strain combination was finally determined to be: Lactobacillus bulgaricus, Streptococcus thermophilus and Lactobacillus plantarum.

[0099] Table 5 Sensory scores of walnut fermented milk with different bacterial strain combinations

[0100]

[0101] 3) Establish regression model and variance analysis

[0102] On the basis of the single factor experimental results, three factors including fermentation temperature (X1), fermentation time (X2) and bacterial inoculation amount (X3) were selected, and the sensory score (Y) was used as the response value. The response surface test was used to optimize the processing technology of walnut fermented milk. The experimental design and results are shown in Table 6, and the variance analysis is shown in Table 7.

[0103] The data in Table 6 were subjected to multiple regression fitting using Design-Expert 8.0.6.1 software, and the quadratic multiple regression equation was obtained: Y = 89.28 + 1.42X1 + 0.36X2 + 2.71X3 - 0.67X1X2 + 1.33X1X3 - 0.10X2X3 - 5.11X 12 -5.19X 22 -7.34X 32

[0104] According to the variance analysis results in Table 7, the P value in the quadratic multiple regression model of sensory scores in this test is <0.0001, which shows that the regression model is extremely significant (P <0.01). 12 , X 22 and X 32 The interaction between them was extremely significant (P < 0.01), which had a greater impact on the sensory score of pure plant-based walnut fermented milk; the interaction term X1X3 had a significant impact on the results (P < 0.05); the interaction terms X1X2 and X2X3 had no significant impact on the results (P > 0.05), which had a smaller impact on the sensory score of pure plant-based walnut fermented milk.

[0105] Table 6 Box-Behnken test design and test results

[0106]

[0107]

[0108] Table 7 Results of variance analysis of regression model

[0109]

[0110] Note: * indicates significant effect on the results (P < 0.05); ** indicates extremely significant effect on the results (P < 0.01).

[0111] 4) The impact of interactions among factors

[0112] The response surface diagram and contour diagram of the interaction of fermentation temperature, fermentation time, and bacterial inoculation amount on the sensory score of pure plant-based walnut fermented milk are shown in Figure 8-10 The surface of the interaction term X1X3 has a larger inclination and a steeper slope, and the contour lines are elliptical and dense, indicating that the interaction is significant, which is consistent with the results of the variance analysis.

[0113] 5) Analysis of physical, chemical and microbiological indicators of pure plant-based walnut fermented milk

[0114] As shown in Table 8, the acidity was 76.4 ± 2.2 °T, the protein content was 4.16 ± 0.15 g / 100 g, and the lactic acid bacteria content was 1.68 × 10 9 CFU / g, no Escherichia coli and pathogenic bacteria were detected, and the physical and chemical indicators and microbiological indicators all met the relevant requirements of the standard T / WSJD12-2020 "Plant Protein Beverage Plant Yogurt".

[0115] Table 8 Physical and chemical indicators and microbiological indicators of pure plant-based walnut fermented milk

[0116]

[0117] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A pure plant-based walnut fermented milk, characterized in that: The components of the fermented milk include: walnut kernels, water, sucrose, and bacterial strains; the material-liquid ratio of the walnut kernels to water is 1:5 (kg / L), the added amount of sucrose is 7%, and the added amount of bacterial strains is 4.5%.

2. The pure plant-based walnut fermented milk according to claim 1, characterized in that: The bacterial species are: Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus plantarum; the compounding ratio of the bacterial species is: 1:1:

1.

3. The pure plant-based walnut fermented milk according to claim 1, characterized in that: The fermentation temperature of the strain is 36° C. and the fermentation time is 12 h.

4. A method for preparing pure plant-based walnut fermented milk, characterized in that: The preparation method comprises the following steps: 1) filtering the processed walnut kernels using a 100-mesh filter to obtain a puree; 2) Add sucrose to the walnut pulp obtained in 1) to obtain a mixed walnut pulp; add sucrose in an amount of 4%, heat the mixed walnut pulp to 45° C., and stir for 10 minutes; homogenize and sterilize for later use; obtain a sterile walnut pulp; 3) cooling the sterile stock in 2) to 35-40° C., adding the mixed bacteria into the stock, stirring evenly; and then fermenting at 36° C. for 12 hours; the mixed bacteria are Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus plantarum in a ratio of 1:1:1; 4) Post-ripening in cold storage: Store at 4°C for 24 hours to allow post-ripening and acid production; Obtain the pure plant-based walnut fermented milk as described in claim 1.

5. The preparation method according to claim 4, characterized in that: The specific steps of the treatment in 1) are: selecting dry walnuts with full appearance, no mildew and no insect infestation, removing the shell to obtain walnut kernels, soaking the walnut kernels in boiling water for 10 minutes, and removing the seed coat; adding pure water according to the solid-liquid ratio to grind the walnut kernels; and obtaining the treated walnut kernels.

6. The preparation method according to claim 5, characterized in that: The solid-liquid ratio is 1:5 (kg / L); the refining speed is 2000 rpm, and the time is 20 min; and this process is repeated 3 times.

7. The preparation method according to claim 4, characterized in that: The specific operation of homogenization in the above 2) is: homogenization for 3 minutes at 25 MPa, and cycled 3 times.

8. The preparation method according to claim 4, characterized in that: The specific operation of sterilization in 2) is: the original paste is sterilized at a temperature of 85° C., and the sterilization time is 20 minutes.

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