Preparation method of coagulation type pure walnut-based yoghurt

Abstract

This invention belongs to the field of plant protein modification and processing technology, specifically relating to a set-type pure walnut-based plant-based yogurt and its preparation method. The yogurt uses peeled walnut kernels as the main raw material, and is produced through protein glutaminase enzymatic hydrolysis, enzyme inactivation, centrifugation, homogenization, and sterilization, followed by inoculation with a commercial starter culture, fermentation, and cold-curing. The method of this invention uses protein glutaminase to treat walnut milk, effectively improving the solubility of walnut protein, significantly improving the water-holding capacity and whey separation rate of the walnut yogurt, and optimizing its texture. Through screening different commercial starter cultures, a suitable starter culture for preparing walnut yogurt was determined, increasing the acidity and viable cell count of the walnut yogurt.

Description

Technical Field

[0001] This invention relates to a set-type pure walnut-based yogurt and its preparation method, specifically to a method for preparing plant-based yogurt using walnuts, known as the "longevity fruit" and "brain-boosting fruit," as raw material, belonging to the field of plant protein modification and processing technology. Background Technology

[0002] Plant-based yogurt has seen a steady increase in market share in recent years due to its cholesterol-free and lactose-free nature, meeting the needs of people with hyperlipidemia, milk allergies, environmentalists, and animal welfare enthusiasts. It also helps alleviate pressure on the global livestock industry and aligns with the current trend of developing healthy and green foods. Therefore, research into developing plant-based foods using plant-based ingredients instead of animal-based ingredients has gradually become a hot topic.

[0003] Walnuts are rich in high-quality protein and oils, with a complete and balanced range of amino acids. They are particularly high in monounsaturated and polyunsaturated fatty acids, lecithin, vitamins, and minerals, making them highly nutritious. Currently, most plant-based yogurts on the market use soybeans, peas, coconuts, and almonds as raw materials; pure plant-based set yogurt made from walnuts is relatively rare. Fermenting walnuts with lactic acid bacteria to make yogurt not only preserves the nutrients in walnuts but also serves as a carrier for probiotics, regulating the balance of the human gut microbiota. Furthermore, it enriches the variety of plant-based foods, broadening consumer choices.

[0004] Currently, walnuts contain 20%-25% protein, with gluten being the most abundant. This results in poor solubility of walnut protein, limiting its application in food processing. Furthermore, unlike cow's milk, walnut milk lacks lactose and contains only certain amounts of sucrose and glucose. This may lead to weak growth and reproduction of lactic acid bacteria in the walnut milk matrix, resulting in limited acid production and difficulty in survival. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a set-type pure walnut-based yogurt and its preparation method. The purpose is to solve the technical problems in the existing process of preparing yogurt using walnuts as raw materials, such as the poor solubility of walnut protein, the inability to form a stable walnut milk system, the lack of lactose in walnut milk leading to weak growth and reproduction of lactic acid bacteria in the walnut milk matrix, and the lack of suitable walnut fermentation agents.

[0006] The first technical solution provided by this invention is a method for preparing a set-type pure walnut-based yogurt, as detailed below:

[0007] A method for preparing set-type pure walnut-based yogurt involves enzymatically hydrolyzing walnut milk using protein glutaminase, then inactivating the enzyme, centrifuging, homogenizing, and sterilizing the milk, followed by inoculation with a starter culture, fermentation, and refrigeration for maturation to obtain set-type pure walnut-based yogurt.

[0008] In some embodiments, the walnut milk is made by grinding peeled walnut kernels with water into a paste.

[0009] Furthermore, the ratio of peeled walnut kernels to water is 1:3, and the grinding time is 4 minutes (2 minutes / time).

[0010] In some embodiments, the enzymatic hydrolysis method for the walnut milk is as follows: the amount of protein glutaminase added (E / S) is 0.1% to 0.3% (0.67 U / g to 2.02 U / g), the hydrolysis temperature is 60°C, and the time is 15 min to 65 min.

[0011] In some embodiments, the enzyme inactivation temperature for the enzymatic hydrolysis of walnut milk is 95°C, and the time is 2 minutes.

[0012] In some embodiments, the walnut milk is centrifuged at 2000 rpm for 5 minutes.

[0013] In some embodiments, the homogenization conditions for the walnut milk are 200 bar, 2 cycles, and a homogenization temperature of 50-60°C.

[0014] In some embodiments, the sterilization conditions for the walnut milk are: sterilization temperature of 65°C for 30 minutes, or sterilization temperature of 72°C for 15 seconds.

[0015] In some embodiments, the starter culture is inoculated at an addition rate of 0.2%-1.0% (w / v), and the starter culture includes one or more of the following: Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium lactis, Bifidobacterium amphotericum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium animalis, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus reuteri, Bifidobacterium breve, Lactobacillus paracasei, Lactobacillus casei, Lactococcus lactis, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus helveticus, Lactobacillus johnsonii, Bifidobacterium longum, and Bifidobacterium adolescentis.

[0016] In some embodiments, the fermenting agent is one or more of direct-inoculation fermenting agent I, direct-inoculation fermenting agent II, direct-inoculation fermenting agent III, direct-inoculation fermenting agent IV, and direct-inoculation fermenting agent V.

[0017] The direct-inoculation starter I contains Lactobacillus bulgaricus and Streptococcus thermophilus.

[0018] The direct-inoculation starter II contains Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium lactis, Bifidobacterium amphotericum, and Bifidobacterium infantis.

[0019] The direct-inoculation fermentation agent III contains Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium lactis, Bifidobacterium animalis, Lactobacillus fermentum, Lactobacillus salivarius, and Lactobacillus reuteri.

[0020] The direct-inoculation starter IV contains Lactobacillus bulgaricus, Streptococcus thermophilus, Bifidobacterium lactis (AD011, BI-07, HN019, CP-9), Bifidobacterium breve Bv-889, Bifidobacterium infantis BLI-02, Bifidobacterium animalis BB-12, Lactobacillus plantarum 299V, Lactobacillus paracasei MP137, Lactobacillus salivarius AP-32, Lactococcus lactis (Lactobacillus lactis subsp. lactis, Lactobacillus fat subsp. diacetyl), Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus fermentum (CECT5716, TSF331), Lactobacillus reuteri GL-104, Lactobacillus rhamnosus (F-1, MP108, HN001, bv-77), Lactobacillus plantarum (LPL28, GLP1), Lactobacillus paracasei GL-156, Lactobacillus casei CS-773, Lactobacillus helveticus RE-78, Lactobacillus johnsonii MH-68, and Lactobacillus acidophilus TYCA06.

[0021] Direct-inoculation starter culture V contains *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, *Bifidobacterium lactis* (U9, U2), *Bifidobacterium animalis* (A16, A6S, A8, BLA-25), *Lactobacillus paracasei* (L9s, L55, L56, L62, LPA-33, R14), *Lactobacillus rhamnosus* (LL23, T8), *Lactobacillus reuteri* (LR09, LRE-01, LRE-02), *Lactobacillus plantarum* (CR12, CD71, LPL-01, LPL-26, LPL-606), and *Lactobacillus fermentum* C. D61, Lactobacillus acidophilus (RA15, CD81), Lactobacillus casei (CD21, CD22, CD23, W1-1), Lactobacillus salivarius (LS86, CD41, CD42, CD43, CD44, CD45), Bifidobacterium longum (BB68S, BB65, CD02, CD04, CD11), Bifidobacterium breve (CD12, BBR-154), Bifidobacterium bifidum (DB03, DB35), and Bifidobacterium adolescentis (BQ23, CD65, CD03, BBD-66).

[0022] In some embodiments, the fermentation conditions are: a fermentation temperature of 42°C and a fermentation time of 6-8 hours.

[0023] In some embodiments, the refrigeration temperature is 4°C and the time is 12 hours.

[0024] The present invention also provides a second technical solution, which is a set-type pure walnut-based yogurt, prepared by the method described in the first technical solution.

[0025] The advantages and benefits of the method of the present invention are as follows:

[0026] (1) The method for preparing set-type walnut yogurt provided by the present invention utilizes protein glutaminase (PG) treatment to improve the solubility of walnut protein and stabilize the walnut milk system without adding stabilizers or thickeners.

[0027] (2) The method for preparing set-type walnut yogurt provided by the present invention selects a starter suitable for fermenting sugar-free walnut yogurt from different direct-inoculation starter cultures, which can achieve an acidity of 93.06°T and a viable count of 10. 7 CFU / g;

[0028] (3) Compared with existing walnut-based yogurt, the present invention significantly improves the water-holding capacity of yogurt, with a maximum water-holding capacity of 87.48%, and reduces the whey separation rate, with a minimum whey separation rate of 1.91%. Therefore, it can obtain pure walnut-based yogurt with excellent coagulation performance. Attached Figure Description

[0029] Figure 1 The effect of starter culture on the pH of unsweetened yogurt;

[0030] Figure 2 The effect of starter culture on the acidity of sugar-free yogurt;

[0031] Figure 3 This is the SDS-PAGE electrophoresis image from Example 2;

[0032] Figure 4 These are actual photos of unsweetened walnut yogurt and sweetened walnut yogurt. Detailed Implementation

[0033] The specific operation of the present invention will be described in detail below with reference to the embodiments.

[0034] 1. Measurement of the textural properties of yogurt

[0035] The texture properties of walnut yogurt were determined using a TA.XTPlus texture analyzer from SMS (UK). The method was yogurt back extrusion, and the test probe was a P25 (cylindrical). The probe descent speed before testing was 1 mm / s; the testing speed was 0.5 mm / s; the probe return speed after testing was 1 mm / s; and the strain level was 30%. The texture properties of the walnut yogurt were analyzed using Texture Expert Exceed data analysis software, and the following four parameters were obtained: hardness, consistency, cohesiveness, and viscosity index. Each sample was measured in triplicate.

[0036] 2. Measurement of water-holding capacity

[0037] Weigh 15g of walnut milk into a 50mL centrifuge tube and ferment for 6 hours. Store at 4℃ for at least 12 hours, then centrifuge (4000rpm, 10min, 4℃). Weigh the separated whey and calculate its water-holding capacity using the following formula.

[0038]

[0039] Where W represents the weight of the centrifuge tube (g), W1 represents the total weight of the sample (g), and W2 represents the weight of the sample after centrifugation to remove whey. Each sample was measured in triplicate.

[0040] 3. Measurement of whey separation rate

[0041] Weigh 15g of walnut milk into a (model) box and ferment for 6 hours. After refrigerating at 4℃ for 12 hours, take it out, tilt the box at 45°, carefully suck out the whey and weigh it.

[0042]

[0043] Where W represents the weight of the yogurt fermentation box (g), W1 represents the total weight of the sample (g), and W2 represents the weight of the sample after whey removal (g). Each sample was measured in triplicate.

[0044] 4. Measurement of viable bacteria count

[0045] The determination of viable lactic acid bacteria count was carried out in accordance with GB 4789.2-2016 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count".

[0046] 5. Protein glutaminase

[0047] Trade name: Protein-glutaminase "Amano" 500

[0048] Source: Amano Enzyme Co., Ltd., Japan

[0049] Enzyme activity: 673 U / g

[0050] Enzyme activity is defined as the ability of an enzyme to catalyze a chemical reaction. Enzyme activity can be expressed as the rate of a chemical reaction it catalyzes under certain conditions. The higher the reaction rate catalyzed by the enzyme, the higher its activity; the lower the reaction rate, the lower its activity. Therefore, measuring enzyme activity is essentially measuring the rate of an enzyme-catalyzed reaction.

[0051] Enzyme activity measurement methods: Enzyme activity can be determined in two ways: one is by measuring the time required to complete a certain amount of reaction, and the other is by measuring the amount of chemical reaction catalyzed by the enzyme per unit time. Measuring enzyme activity essentially involves measuring the increase in product or decrease in substrate, and the specific measurement method for the enzyme-catalyzed reaction is determined primarily by the physical or chemical properties of the product or substrate. The main measurement methods include chemical analysis, spectrophotometry, gas chromatography, pH measurement, and polarographic determination of oxygen and hydrogen peroxide.

[0052] 6. Protein content detection methods:

[0053] The test was conducted according to the "GB 5009.5-2016 National Food Safety Standard - Determination of Protein in Food".

[0054] 7. SDS-PAGE:

[0055] Dilute the sample to a protein concentration of 2 mg / mL. Take 40 μL of the solution and place it in a 1.5 mL centrifuge tube. Mix the solution with the sample loading solution at a 4:1 ratio, boil for 10 min, and then cool. Inject 15 μL of sample into each well. First, concentrate the sample using 80V. After all the sample has entered the lower separating gel, increase the voltage to 120V and stop when the sample is 0.5 cm from the bottom edge. Stain the gel with Coomassie Brilliant Blue for 30 min, then wash 1-2 times with destaining buffer. Change the eluent every 6 hours until the bands are clear.

[0056] 8. Information on the direct-inoculation fermentation agents involved in the following embodiments is shown in Table 1.

[0057] Table 1 Information on 5 types of direct-inoculation fermentation agents

[0058]

[0059]

[0060] Direct-inoculation starter I is YO-MIX 900, purchased from Danix; direct-inoculation starter II is a household yogurt starter of Bifidobacterium type, purchased from Kunshan Baishengyou Biotechnology Co., Ltd.; direct-inoculation starter III, direct-inoculation starter IV, and direct-inoculation starter V are respectively Youbit 10-strain, 30-strain, and 50-strain yogurt starter powders, all purchased from Qingdao Youbit Biotechnology Co., Ltd.

[0061] Example 1:

[0062] Peeled walnut kernels and water were juiced and ground into a slurry at a ratio of 1:3 for 4 minutes (divided into two 2-minute intervals) to obtain walnut milk. Protein glutaminase (E / S) of 0.2% (1.35 U / g) was added to the walnut milk, and enzymatic hydrolysis was performed at 60℃ for 40 minutes. The enzyme was then inactivated at 95℃ for 2 minutes. After cooling, the mixture was centrifuged at 2000 rpm for 5 minutes, and the supernatant was homogenized twice at 20 MPa. The mixture was then sterilized at 72℃ for 15 seconds. After cooling to approximately 40℃, direct-inoculation starter cultures I, II, III, IV, and V were inoculated at an inoculation rate of 0.2% (w / v). The fermentation temperature was 42℃, and the fermentation time was 8 hours. The experimental results of the fermentation characteristics and textural parameters of the resulting set-type sugar-free walnut yogurt are shown in the table below. Figure 1 , 2 See Table 2. The water-holding capacity, whey separation rate, and viable bacteria count of set-type sugar-free walnut yogurt are shown in Table 2.

[0063] Depend on Figure 1 and Figure 2 It was observed that within the first 2 hours of fermentation, the pH decrease and acidity increase of the direct-inoculation starter culture V were relatively slow, accelerating after 2 hours. This indicates that the starter culture needs time to adapt after being inoculated into walnut milk, allowing for better growth, reproduction, and the metabolism of nutrients to produce acid. After 8 hours of fermentation, the unsweetened walnut yogurt prepared with direct-inoculation starter culture V had the lowest pH (4.28) and the highest titratable acidity (93.06°T). This demonstrates that the growth of direct-inoculation starter culture V in unsweetened walnut milk was superior to the other four starter cultures.

[0064] Table 2. Effects of different starter cultures on the textural properties of unsweetened walnut yogurt

[0065]

[0066] Table 2 shows that different starter cultures have varying effects on the textural properties of unsweetened walnut yogurt. Direct-inoculation starter culture V exhibits the greatest hardness and consistency, followed by direct-inoculation starter cultures I and II. Direct-inoculation starter culture III has the lowest hardness and consistency, indicating that it cannot adapt well to the unsweetened walnut milk matrix. Therefore, direct-inoculation starter culture V can not only produce walnut yogurt with good curdling effect but also impart higher consistency to unsweetened walnut yogurt.

[0067] Table 3. Effects of different starter cultures on water-holding capacity, whey separation rate, and viable bacterial count of unsweetened walnut yogurt.

[0068]

[0069] Different starter cultures had varying effects on the water-holding capacity, whey separation rate, and viable bacteria count of unsweetened walnut yogurt. Water-holding capacity characterizes the ability of a yogurt sample's gel network structure to bind water after centrifugation; the more whey separated, the worse the water-holding capacity. Whey separation rate characterizes the spontaneous whey separation on the surface of set yogurt under natural conditions; excessive whey separation is undesirable and reduces consumer acceptance. Higher water-holding capacity and lower whey separation rate indicate better water binding in the microstructure of the set yogurt and a more homogeneous and stable system. Table 3 shows that direct-inoculation starter culture V had the highest water-holding capacity and the lowest whey separation rate under natural conditions, both significantly improved compared to sweetened walnut yogurt. Meanwhile, without added sugar, the lactic acid bacteria in the direct-inoculation starter V showed the best growth and the highest number of live bacteria. This may be because the direct-inoculation starter V contains a variety of lactic acid bacteria, which can utilize the sugars already present in the walnut milk under sugar-free conditions and promote each other's growth.

[0070] Example 2:

[0071] The protein content of the enzymatically hydrolyzed walnut milk and the supernatant after centrifugation of the un-enzymatically hydrolyzed walnut milk in Example 1 were detected and analyzed by SDS-PAGE. The results are shown in Table 4.

[0072] Table 4 Protein content of samples (unit: g / 100g)

[0073]

[0074] From Table 4 and Figure 3 It can be seen that the protein content in unenzymatically hydrolyzed walnut milk is 1.07g / 100g, with the protein subunits mainly distributed at 10-15kDa, 25kDa, and 50-75kDa. After treatment with PG enzyme, the protein content is 2.84g / 100g. The content of walnut protein subunits near 10-15kDa, 25-35kDa, and 45-55kDa increases, while the content of subunits near 75kDa decreases. It is evident that PG enzyme has a very significant effect on improving the solubility of walnut protein.

[0075] Comparative Example 1:

[0076] Using the method of Example 1, the difference is that 8% sucrose was added to the upper liquid of the walnut milk enzymatic hydrolysate, and the results are shown in Table 5.

[0077] Table 5. Effects of different starter cultures on water-holding capacity, whey separation rate, and viable bacterial count of sweetened walnut yogurt.

[0078]

[0079]

[0080] As shown in Table 5, walnut yogurt with added sucrose exhibited a significant decrease in water-holding capacity and a marked increase in whey separation rate, while also showing a decrease in viable bacteria count. Figure 4 In images a and b, the left side shows unsweetened walnut yogurt, and the right side shows sweetened walnut yogurt. Figure 4 In diagram a, the red arrow indicates that the whey separation in unsweetened walnut yogurt is less than that in sweetened yogurt. Figure 4 In diagram b, as shown by the red arrow, sweetened walnut yogurt tends to flow more when inverted. Therefore, the addition of sucrose affects the water-holding capacity and whey separation rate of walnut yogurt, thus affecting its coagulation degree.

[0081] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the examples described above. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for preparing a sugar-free, set-type pure walnut-based yogurt, characterized in that, Walnut milk is made by grinding peeled walnut kernels with water. The walnut milk is then enzymatically hydrolyzed using protein glutaminase. After enzyme inactivation, centrifugation, homogenization, and sterilization, it is inoculated with a starter culture, fermented, and then refrigerated for maturation to obtain a set-type pure walnut-based yogurt. The specific enzymatic hydrolysis method for the walnut milk is as follows: the amount of protein glutaminase added (E / S) is 0.67 U / g~2.02 U / g, the hydrolysis temperature is 60 ℃, and the time is 15 minutes. The fermentation process takes 0.2%-1.0% of the sample and is carried out over a period of 65 minutes. The fermentation agent contains *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, *Bifidobacterium lactis* U9, *Bifidobacterium lactis* U2, *Bifidobacterium animalis* A16, *Bifidobacterium animalis* A6S, *Bifidobacterium animalis* A8, *Bifidobacterium animalis* BLA-25, *Lactobacillus paracasei* L9s, *Lactobacillus paracasei* L55, *Lactobacillus paracasei* L56, *Lactobacillus paracasei* L62, *Lactobacillus paracasei* LPA-33, *Lactobacillus paracasei* R14, *Lactobacillus rhamnosus* LL23, *Lactobacillus rhamnosus* T8, *Lactobacillus reuteri* LR09, *Lactobacillus reuteri* LRE-01, *Lactobacillus reuteri* LRE-02, *Lactobacillus plantarum* CR12, *Lactobacillus plantarum* CD71, *Lactobacillus plantarum* LPL-01, *Lactobacillus plantarum* LPL-26, and other plant-based bacteria. Lactobacillus LPL-606, Lactobacillus fermentum CD61, Lactobacillus acidophilus RA15, Lactobacillus acidophilus CD81, Lactobacillus casei CD21, Lactobacillus casei CD22, Lactobacillus casei CD23, Lactobacillus casei W1-1, Lactobacillus salivarius LS86, CD41, Lactobacillus salivarius CD42, Lactobacillus salivarius CD43, Lactobacillus salivarius CD44, Lactobacillus salivarius CD45, Bifidobacterium longum BB68S, Bifidobacterium longum BB65, Bifidobacterium longum CD02, Bifidobacterium longum CD04, Bifidobacterium longum CD11, Bifidobacterium breve CD12, Bifidobacterium breve BBR-154, Bifidobacterium bifidum DB03, Bifidobacterium bifidum DB35, and Bifidobacterium adolescentis BQ23, Bifidobacterium adolescentis CD65, Bifidobacterium adolescentis CD03, and Bifidobacterium adolescentis BBD-66.

2. The method for preparing a sugar-free, set-type pure walnut-based yogurt according to claim 1, characterized in that, The fermentation conditions are: fermentation temperature of 42 ℃ and fermentation time of 6-8 h.

3. A sugar-free, set-type pure walnut-based yogurt, characterized in that, It is a sugar-free set-type pure walnut-based yogurt prepared using the method described in any one of claims 1-2.

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