A walnut and goat milk double protein yoghurt and a preparation method thereof

Abstract

The application discloses walnut goat milk double-protein yoghourt and a preparation method thereof, and belongs to the technical field of yoghourt processing. The walnut goat milk double-protein yoghourt is prepared by the following steps: walnut protein is modified by using papain; and then the modified walnut protein is compounded with reconstituted goat milk. The walnut protein has rich and complete amino acid content and has extremely high nutritional value, and is a plant protein with great development potential. The walnut protein modified by papain overcomes the problems of low solubility and poor functional characteristics of plant proteins, and plays an important role in improving the quality of the walnut goat milk double-protein yoghourt.

Description

Technical Field

[0001] This invention belongs to the field of yogurt processing technology, specifically relating to a walnut and goat milk double-protein yogurt and its preparation method. Background Technology

[0002] In recent years, with people's pursuit of a healthy lifestyle, the market demand for protein has been increasing, while the price of animal protein has been rising year by year. Therefore, the development and utilization of widely available and low-cost plant proteins has become a key focus in the food industry. The nutrition and taste provided by animal protein cannot be completely replaced by plant protein, and plant protein also cannot fully supplement the essential amino acids required by the human body. Dual-protein refers to edible protein sources obtained by combining natural, high-quality plant and animal proteins, represented by plant proteins such as soy protein and animal proteins such as cow's milk protein, according to their nutritional efficacy and precise interactions. The research and development of dual-protein products is attracting increasing attention. Currently, there is limited research both domestically and internationally on the preparation of yogurt using plant protein and goat's milk as the main raw materials, and the development of related products has significant market competitiveness.

[0003] Fermented goat milk has a unique flavor, and its nutritional and health benefits surpass those of raw milk. Compared to fermented cow milk, fermented goat milk contains higher levels of minerals such as Ca, Zn, Fe, Cu, and Se, as well as fatty acids such as lauric acid, oleic acid, and linoleic acid. A high-animal-protein diet increases the burden on the kidneys, and high cholesterol intake can lead to cardiovascular disease. Moderate intake of plant protein helps reduce the risk of cardiovascular disease. Therefore, developing and promoting dual-protein fermented milk made primarily from plant protein and goat milk can not only enrich the variety of high-nutritional-value fermented milk on the market but also promote the integrated and coordinated development of modern agriculture, the food industry, and nutrition and health.

[0004] Walnut protein contains 18 amino acids, including all eight essential amino acids required by the human body, and is particularly high in arginine, glutamic acid, and aspartic acid. The amino acid composition of walnut protein is well-balanced; except for methionine, it conforms to the FAO / WHO recommendations for adults. Histidine, valine, leucine, and isoleucine all exceed the FAO / WHO recommendations for preschool children aged 2-5 years. Furthermore, after enzymatic hydrolysis with proteases, walnut protein can produce bioactive peptides with various physiological functions, including inhibiting xanthine oxidase, inhibiting angiotensin-converting enzyme, and possessing antioxidant properties, and is also more easily digested and absorbed. Therefore, walnut protein has high development and utilization value.

[0005] There is limited research on the preparation of double-protein yogurt using walnut protein and sheep milk fermentation in the current technology. Most double-protein yogurt is prepared using cow milk and soy protein or whey protein as raw materials. This situation is far from meeting consumers' choices and needs for nutritious and healthy foods. Summary of the Invention

[0006] The purpose of this invention is to provide a walnut and goat milk double-protein yogurt and its preparation method, in order to solve the technical problem that the nutritional value and flavor of existing double-protein yogurts are far from meeting consumers' choices and needs for nutritious and healthy foods.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention discloses a method for preparing walnut and goat milk double-protein yogurt, comprising the following steps:

[0009] Modified walnut protein was prepared using walnut protein solution and papain as raw materials.

[0010] Modified walnut protein and reconstituted sheep milk were mixed to obtain a mixture; white sugar, xanthan gum, β-cyclodextrin and sucralose were added to the mixture, and homogenization and sterilization were carried out in sequence to obtain mixed raw milk.

[0011] Fermentation starters are added to the mixed raw milk for fermentation. After fermentation, fermented milk is obtained.

[0012] After post-processing the fermented milk, walnut goat milk double protein yogurt was obtained.

[0013] Furthermore, the steps for preparing modified walnut protein using walnut protein solution and papain as raw materials are as follows:

[0014] Walnut protein was mixed with water to obtain a walnut protein solution; papain was added to the walnut protein solution for enzymatic hydrolysis, and then the supernatant was collected by centrifugation and dried to obtain modified walnut protein.

[0015] Furthermore, the pH of the walnut protein solution was adjusted to 7 using NaOH or HCl, and the mass ratio of walnut protein to water was 1:15; the enzymatic hydrolysis time was 90 min; and the ratio of walnut protein solution to papain was 100 mL: 4 g.

[0016] Further, papain was added to the walnut protein solution for enzymatic hydrolysis, and the prepared walnut protein hydrolysate was subjected to enzyme inactivation treatment at 95-100℃ for 10-15 minutes. Then, the supernatant was collected by centrifugation and dried to obtain modified walnut protein.

[0017] Furthermore, the mass ratio of the modified walnut protein to the reconstituted sheep milk protein is 1:(1-9).

[0018] Furthermore, in every 100g of mixed raw milk, the amount of added white sugar is 5% to 10%, the amount of added xanthan gum is 0.20%, the amount of added β-cyclodextrin is 0.06% to 0.10%, and the amount of added sucralose is 0.005% to 0.02%.

[0019] Furthermore, the sterilization treatment is performed at a temperature of 90–95°C for a time of 10–15 minutes.

[0020] Furthermore, the fermentation is carried out under aseptic conditions, at a temperature of 42–45°C, for a time of 4–9 hours;

[0021] The amount of fermentation bacteria added to each 100g of mixed raw milk is 0.05% to 0.30%.

[0022] Furthermore, the fermentation strain is a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

[0023] The present invention also discloses a walnut goat milk double protein yogurt prepared by the above preparation method.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention discloses a method for preparing walnut and goat milk double-protein yogurt. The method involves modifying walnut protein with papain, followed by compounding the modified walnut protein with reconstituted goat milk to prepare the walnut and goat milk double-protein yogurt. Walnut protein is rich in a complete range of amino acids, possessing extremely high nutritional value and representing a plant protein with significant development potential. The papain-modified walnut protein overcomes the common problems of low solubility and poor functional properties found in plant proteins, significantly improving its solubility, nutritional value, and functional properties such as antioxidant and blood pressure-lowering effects. This plays a crucial role in enhancing the quality of the walnut and goat milk double-protein yogurt.

[0026] This invention also discloses a walnut goat milk double-protein yogurt prepared by the above preparation method. Compared with the prior art, it has the advantages of good sensory quality, strong nutritional and health benefits, and high storage stability. The main features are that the walnut goat milk double-protein yogurt has a moderate sweet and sour taste and a unique flavor. It has potential effects such as anti-oxidation, blood sugar reduction, and anti-thrombosis. During cold storage, the degree of post-acidification is small and the number of live lactic acid bacteria decreases slowly. Attached Figure Description

[0027] Figure 1 The process flow diagram for preparing walnut goat milk double-protein yogurt according to the present invention is shown below;

[0028] Figure 2 The changes in the number of viable lactic acid bacteria in walnut-fed goat milk double-protein yogurt during storage;

[0029] Figure 3 Changes in the in vitro antioxidant activity of walnut-sheep milk double-protein yogurt during storage;

[0030] Among them: (a) - Changes in the DPPH free radical scavenging capacity of yogurt during storage; (b) - Changes in the hydroxyl free radical scavenging capacity of yogurt during storage; (c) - Changes in the reducing power of yogurt during storage;

[0031] Figure 4 The changes in α-glucosidase and α-amylase inhibitory activities in walnut goat milk double-protein yogurt during storage;

[0032] Figure 5 Changes in thrombin inhibitory activity in walnut-fed sheep milk double-protein yogurt during storage;

[0033] Figure 6 The effect of the ratio of two proteins on the pH of yogurt;

[0034] Figure 7 The effect of the ratio of two proteins on the acidity of yogurt;

[0035] Figure 8 The effect of the ratio of two proteins on the viscosity of yogurt;

[0036] Figure 9 The effect of fermentation time on the pH and acidity of yogurt;

[0037] Figure 10 The effect of fermentation time on the viscosity of yogurt;

[0038] Figure 11 The effect of added white sugar on the pH of yogurt;

[0039] Figure 12 The effect of added white sugar on the acidity of yogurt;

[0040] Figure 13 The effect of added white sugar on the viscosity of yogurt;

[0041] Figure 14 The effect of the amount of fermentation starter culture added on the pH of yogurt;

[0042] Figure 15 The effect of the amount of fermentation strain added on the acidity of yogurt;

[0043] Figure 16 The effect of the amount of fermentation bacteria added on the viscosity of yogurt;

[0044] Figure 17 The response surface plot shows the effect of the interaction of various factors on the acidity of yogurt.

[0045] Among them: (a) - the effect of the interaction between the ratio of two proteins and fermentation time on the acidity of yogurt; (b) - the effect of the interaction between the ratio of two proteins and the amount of inoculum added on the acidity of yogurt; (c) - the effect of the interaction between the ratio of two proteins and the amount of white sugar added on the acidity of yogurt; (d) - the effect of the interaction between fermentation time and the amount of inoculum added on the acidity of yogurt; (e) - the effect of the interaction between fermentation time and the amount of white sugar added on the acidity of yogurt; (f) - the effect of the interaction between the amount of inoculum added and the amount of white sugar added on the acidity of yogurt.

[0046] Figure 18 The response surface plot shows the effect of the interaction of various factors on the viscosity of the yogurt.

[0047] Among them: (a) - the effect of the interaction between the ratio of two proteins and fermentation time on the viscosity of yogurt; (b) - the effect of the interaction between the ratio of two proteins and the amount of inoculum added on the viscosity of yogurt; (c) - the effect of the interaction between the ratio of two proteins and the amount of white sugar added on the viscosity of yogurt; (d) - the effect of the interaction between fermentation time and the amount of inoculum added on the viscosity of yogurt; (e) - the effect of the interaction between fermentation time and the amount of white sugar added on the viscosity of yogurt; (f) - the effect of the interaction between the amount of inoculum added and the amount of white sugar added on the viscosity of yogurt. Detailed Implementation

[0048] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0049] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0050] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0051] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0052] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0053] like Figure 1 As shown, this invention provides a method for preparing walnut and goat milk double-protein yogurt, comprising the following steps:

[0054] Step 1: Mix walnut protein and distilled water in a certain mass ratio to obtain a walnut protein solution. Adjust the pH of the solution to 7.0. Add papain to the walnut protein solution and hydrolyze for 90 minutes. Heat to inactivate the enzyme. Adjust the pH of the hydrolysis system to 7.0. Centrifuge to collect the supernatant and dry to obtain modified walnut protein.

[0055] Step 2: Mix the modified walnut protein prepared in Step 1 and the filtered reconstituted sheep milk according to the protein ratio of walnut protein to sheep milk, add white sugar, xanthan gum, β-cyclodextrin and sucralose, homogenize and sterilize, and then cool to room temperature to obtain mixed raw milk; wherein, the protein ratio of modified walnut protein to sheep milk is 1:(1~9).

[0056] The addition amount of white sugar is 5-10% / 100g of mixed raw milk; the addition amount of xanthan gum is 0.20% / 100g of mixed raw milk; the addition amount of β-cyclodextrin is 0.06-0.10% / 100g of mixed raw milk; and the addition amount of sucralose is 0.005-0.02% / 100g of mixed raw milk.

[0057] Step 3: Under aseptic conditions, add fermentation starter to the mixed raw milk prepared in Step 2 and ferment at 42-45℃ for 4-9 hours to obtain fermented milk; wherein, the amount of starter added is 0.05-0.30% / 100g mixed raw milk;

[0058] Step 4: Cool the fermented milk from Step 3 to room temperature and refrigerate at 4°C for 12 hours to obtain walnut goat milk double protein yogurt.

[0059] Preferably, in step 1, the mass ratio of walnut protein to distilled water is 1:15, the reagent used to adjust the pH of the walnut protein solution to 7.0 is NaOH or HCl, the volume ratio of walnut protein solution to papain is 100:4, the enzymatic hydrolysis temperature is 55℃, and when the enzymatic hydrolysis time reaches 90 min, the walnut protein hydrolysate is immediately subjected to enzyme inactivation treatment at 95℃ for 15 min, and the supernatant is collected after centrifugation at 10,000×g for 15 min.

[0060] Preferably, the drying method used in this step is vacuum freeze drying.

[0061] Preferably, in step 2, while ensuring that the total protein content of the raw milk is 4%, the mass ratio of modified walnut protein to sheep milk protein is 1:5 to 9.

[0062] Preferably, in this step, the amount of white sugar added is 5-7% / 100g of raw milk, the amount of β-cyclodextrin added is 0.06% / 100g of raw milk, and the amount of sucralose added is 0.01% / 100g of raw milk.

[0063] Preferably, the sterilization temperature in this step is 90°C and the sterilization time is 10 minutes.

[0064] Preferably, in step 3, the fermentation strain is a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and the strain addition amount is 0.15–0.25% / 100g of mixed raw milk.

[0065] Preferably, the fermentation time in this step is 5 to 7 hours.

[0066] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0067] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0068] The experimental materials and reagents used in the examples are shown in Table 1, and the equipment is shown in Table 2.

[0069] Table 1 Experimental Materials and Reagents

[0070]

[0071] Table 2 Experimental Instruments and Equipment

[0072]

[0073]

[0074] Example 1

[0075] A method for preparing walnut-sheep milk double-protein yogurt includes the following steps:

[0076] Step 1: Mix walnut protein and distilled water at a mass ratio of 1:15. Adjust the pH of the walnut protein solution to 7.0 with NaOH (0.5M). Place the solution in a 55℃ water bath. When the temperature of the walnut protein solution reaches 55℃, add papain (4%, w / v) for enzymatic hydrolysis. Throughout the enzymatic hydrolysis process, monitor the pH value of the hydrolysis system with a pH meter. Maintain the pH value at 7.0 by adding NaOH (0.5M). After 90 minutes of enzymatic hydrolysis, immediately place the hydrolysis system in a 95℃ water bath for 15 minutes to inactivate the papain. Adjust the pH of the hydrolysis system to 7.0, centrifuge at 10,000×g for 15 minutes, collect the supernatant, and freeze-dry to obtain modified walnut protein.

[0077] Step 2: While ensuring the total protein content of the raw milk is 4% and the ratio of modified walnut protein to sheep milk protein is 1:9, mix the reconstituted sheep milk and modified walnut protein to obtain a mixture. Add 6.6% white sugar, 0.20% xanthan gum (pre-gelatinized), β-cyclodextrin (0.06%, 0.08%, 0.10%), and sucralose (0.005%, 0.010%, 0.015%, 0.020%) to the mixture and mix well. Preheat the mixture to 60℃ and then shear homogenize it for 4 minutes at a shearing speed of 16000 r / min. Then sterilize it in a 90℃ water bath for 10 minutes, remove it, and cool it to 42℃ to obtain the mixed raw milk.

[0078] Step 3: Under aseptic conditions, inoculate the mixed raw milk with a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus as the fermentation starter, with an addition amount of 0.18%. After thorough stirring, the inoculated raw milk is immediately filled into sterilized bottles and fermented in a 42℃ constant temperature incubator for 7 hours to obtain fermented milk.

[0079] Step 4: Refrigerate the fermented milk at 4℃ for 12 hours to obtain walnut goat milk double protein yogurt.

[0080] The sensory quality of the walnut and goat milk double-protein yogurt obtained under different conditions in Example 1 was evaluated. According to Table 7, 19 combinations of β-cyclodextrin and sucralose were added to the mixed raw milk and fermented. After maturation, the optimal combination was selected from the 19 groups of double-protein yogurts through sensory evaluation.

[0081] Table 7. Design of β-cyclodextrin and sucralose combination

[0082]

[0083] Ten students from the College of Food Science with basic sensory evaluation knowledge were selected to conduct sensory evaluations of the color, taste, aroma, and texture of the walnut goat milk double protein yogurt. The specific scoring criteria are shown in Table 8, and the sensory evaluation results are shown in Table 9.

[0084] Table 8 Sensory Evaluation Criteria

[0085]

[0086]

[0087] Table 9 Sensory Evaluation Results

[0088]

[0089] According to Table 9, the total sensory evaluation scores of the 19 samples were ranked as follows: 2 > 10 > 7 = 8 > 6 > 4 = 16 > 9 > 15 > 12 > 14 > 3 > 5 > 1 > 11 > 17 = 13 > 18 > 19. When 0.005% sucralose was added alone, the sensory score was lower than that of the other sucralose-added groups. Furthermore, increasing the addition amount to 0.02% also resulted in the double-protein yogurt being too sweet, affecting its taste. When β-cyclodextrin was added alone, the sensory scores of all three groups of double-protein yogurt were low, especially when the addition amount was increased to 0.1%, the sensory evaluation was the lowest (73.70). This may be because adding too much β-cyclodextrin would excessively mask the sweetness and sourness of the yogurt, resulting in a poor taste and flavor, and affecting the quality of the yogurt. The walnut-goat milk double-protein yogurt prepared by properly combining the two had the best quality. When the addition amount of sucralose was 0.01% and the addition amount of β-cyclodextrin was 0.06%, the sensory score of the double-protein yogurt was the highest, at 78.50.

[0090] The viable lactic acid bacteria count, in vitro bioactivity, and storage stability of the walnut goat milk double-protein yogurt with the highest sensory score in Example 1 were studied. Two control groups were used: yogurt fermented with xanthan gum, β-cyclodextrin, and sucralose (M+T group) and yogurt fermented without xanthan gum, β-cyclodextrin, and sucralose (Control group). The changes in the viable lactic acid bacteria count of the walnut goat milk double-protein yogurt stored at 4℃ for 21 days were investigated. The results are shown in… Figure 2 .

[0091] Depend on Figure 2It was found that the total number of lactic acid bacteria in all three groups of yogurt decreased with prolonged storage time. Within 1–5 days, the rate of decrease in viable bacteria count in the M+T+P group was lower than that in the M+T and Control groups, and the viable bacteria count was also lower in the M+T+P group. After 5 days of storage, the viable bacteria count in the M+T+P group of double-protein yogurt was actually higher than that in the two control groups. This may be attributed to the modified WP providing the double-protein yogurt with abundant glutamic acid, leucine, glycine, and other amino acids necessary for lactic acid bacteria growth, which helps maintain bacterial activity. After 21 days of storage, the viable lactic acid bacteria count in the Control group decreased to 8.19 log CFU / mL, in the M+T group to 8.39 log CFU / mL, and in the M+T+P group to 8.40 log CFU / mL. The decrease in viable lactic acid bacteria count in the M+T and M+T+P groups was relatively smaller, but the viable lactic acid bacteria count in all three groups of yogurt was higher than the 6 log CFU / mL specified in the national standard GB 19302-2010.

[0092] The in vitro antioxidant activity (DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, and reducing power), hypoglycemic activity (α-glucosidase inhibitory activity and α-amylase inhibitory activity), and antithrombotic activity (thrombin inhibitory activity) of the walnut goat milk double-protein yogurt with the highest sensory score in Example 1 were investigated. The in vitro antioxidant activity of the walnut goat milk double-protein yogurt stored at 4°C for 1 day and 21 days was studied, and the results are shown in… Figure 3 .

[0093] Depend on Figure 3 The results showed that the DPPH and hydroxyl radical scavenging abilities and reducing power of the M+T+P group yogurt on day 1 of storage (with an added walnut protein concentration of 2.28 mg / mL) were significantly higher than those of the two control groups (P<0.05), at 62.07%, 82.16%, and 0.65, respectively. With prolonged storage, the DPPH radical scavenging ability and reducing power of all three groups of yogurt significantly decreased (P<0.05). The DPPH radical scavenging abilities of the Control group, M+T group, and M+T+P group decreased by 13.41%, 17.50%, and 13.75%, respectively, and the reducing power decreased by 0.193, 0.17, and 0.24, respectively. Conversely, the hydroxyl radical scavenging ability of all three groups significantly increased with prolonged storage (P<0.05), and the hydroxyl radical scavenging ability was superior to the DPPH radical scavenging ability. This may be because the antioxidant biopeptides in the yogurt are more sensitive to hydroxyl radicals. During storage, the antioxidant peptides in yogurt are further degraded into amino acids or small peptides that do not have antioxidant activity, resulting in a decrease in the DPPH free radical scavenging capacity and reducing power of yogurt.

[0094] This study investigated the α-glucosidase and α-amylase inhibitory activities of walnut-fed goat milk double-protein yogurt stored at 4℃ for 1 day and 21 days. The results are shown in... Figure 4 .

[0095] Depend on Figure 4 It can be seen that with the extension of storage time, the α-glucosidase inhibition rate of the Control group, M+T group and M+T+P group all increased significantly during the storage period (P<0.05), increasing by 4.87%, 7.45% and 7.15% respectively. Moreover, the α-glucosidase inhibition activity of the yogurt in the M+T+P group with added modified walnut protein was significantly higher than that of the two control groups (P<0.05).

[0096] All three groups of yogurt exhibited α-amylase inhibitory activity, which significantly decreased with prolonged storage time (P<0.05). The α-amylase inhibitory activity of the M+T+P group decreased by 8.09%, while the α-amylase inhibitory activity of the Control and M+T groups decreased substantially, by 26.17% and 28.80%, respectively. During storage, the inhibitory activity of the M+T+P group was significantly higher than that of the two control groups (P<0.05).

[0097] The study investigated the thrombin inhibitory activity of walnut-based goat milk double-protein yogurt stored at 4℃ for 1 day and 21 days. The results are shown in... Figure 5 .

[0098] Depend on Figure 5 It was found that all three groups of yogurt exhibited effective thrombin inhibitory activity, which may be related to the fermentation process. Studies have shown that after fermentation, the starter culture can alter the fatty acid composition of polar lipids in dairy products through lipolysis and fatty acid biosynthesis, thereby achieving higher antithrombotic activity. With prolonged storage time, the thrombin inhibitory activity of the Control group, M+T group, and M+T+P group of yogurt all decreased significantly (P<0.05), decreasing by 19.31%, 21.49%, and 17.36%, respectively. However, during storage, the thrombin inhibitory activity of the M+T+P group of yogurt remained higher than that of the two control groups.

[0099] Exploration Example 1

[0100] Similar to Example 1, the various schemes in this example all maintain a total protein content of 4% in the raw milk and a fermentation temperature of 42°C as the basic fermentation conditions, with pH, ​​acidity, and viscosity as indicators. The differences are that the fermentation times are 4h, 5h, 6h, 7h, 8h, and 9h, the amount of white sugar added is 5%, 6%, 7%, 8%, 9%, and 10%, the ratio of dual proteins (modified walnut protein and goat milk protein) is 1:1, 1:3, 1:5, 1:7, and 1:9, and the amount of bacterial strain added is 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, and 0.30%, ultimately yielding walnut and goat milk dual-protein yogurt under different conditions.

[0101] The quality of walnut and goat milk double-protein yogurt obtained under different conditions in Example 1 was studied. The effects of different double-protein ratios on the pH, acidity, and viscosity of the walnut and goat milk double-protein yogurt were investigated. The results are shown in the figure. Figure 6 , Figure 7 and Figure 8 .

[0102] Depend on Figure 6 It can be seen that, with the extension of fermentation time, the pH of yogurt prepared with different ratios of double protein exhibits the same trend. The lower the proportion of modified walnut protein, the lower the pH of the double-protein yogurt. This may be because, with the increase of the proportion of sheep milk protein, the lactose content in the raw milk increases, the reducing sugars available to lactic acid bacteria increase, the lactic acid bacteria reproduction rate increases, and the acid production increases. Figure 7 It can be seen that the acidity changes of yogurt prepared with different protein ratios show roughly the same trend during fermentation. From 0 to 4 hours, acidity increases rapidly; from 4 to 8 hours, acidity increases slowly; and from 8 to 10 hours, the acidity change tends to stabilize. As the proportion of modified walnut protein decreases, the acidity of the yogurt gradually increases, and the pH decreases accordingly. Figure 8 As shown, different ratios of the two proteins can cause changes in the viscosity of the two-protein yogurt. With a decrease in the proportion of modified walnut protein, the viscosity of the yogurt gradually increases. This may be because a reduction in the amount of modified walnut protein increases the probability of protein collision and cross-linking in the milk, thereby promoting the formation of the yogurt gel structure. There were no significant differences in pH, acidity, and viscosity changes between yogurts prepared with two-protein ratios of 1:7 and 1:9 (P>0.05). Considering all factors, a two-protein ratio of 1:7 is preferred.

[0103] This study investigated the effects of different fermentation times on the pH, acidity, and viscosity of walnut-fed sheep milk double-protein yogurt. The results are shown in... Figure 9 and Figure 10 .

[0104] Depend on Figure 9 It can be seen that the pH of double-protein yogurt gradually decreases with the extension of fermentation time. For example... Figure 10As shown, the acidity of the double-protein yogurt increases with fermentation time, but the rate of increase in acidity tends to level off as fermentation time continues. The yogurt reaches its fermentation endpoint at a fermentation time of 6 hours. Figure 10 It can be seen that the viscosity of the yogurt first increases and then decreases as fermentation progresses. This may be because prolonged fermentation increases the acid production of lactic acid bacteria, causing excessive contraction of casein, which in turn disrupts the texture of the yogurt and results in a decrease in viscosity. There was no significant difference in viscosity change between the two-protein yogurt at 6 and 7 hours of fermentation (P>0.05), and the yogurt reached its fermentation endpoint at 6 hours. Considering all factors, a fermentation time of 6 hours is the preferred choice.

[0105] This study investigated the effects of different amounts of added white sugar on the pH, acidity, and viscosity of walnut-based goat milk double-protein yogurt. The results are shown in... Figure 11 , Figure 12 and Figure 13 .

[0106] Depend on Figure 11 It can be seen that during fermentation, the pH value of double-protein yogurt with different proportions of added white sugar showed a consistent trend. During fermentation from 0 to 4 hours, the pH decreased rapidly; after 4 hours, the pH decreased slowly and tended to stabilize. Figure 12 It can be seen that the acidity of double-protein yogurt with different proportions of added white sugar showed a consistent trend during fermentation. When the fermentation time exceeded 6 hours, the acidity decreased slowly. When the added white sugar was 6% and 7%, the acidity of the yogurt reached a relatively high level, indicating that a low amount of white sugar was insufficient for fermentation, while an excessively high amount would inhibit lactic acid bacteria activity, leading to a slower fermentation rate. However, the added white sugar at 6% and 7% had little effect on the acidity and pH of the yogurt. Figure 13 As shown, the viscosity of the yogurt gradually increases with the increase of added white sugar. This may be because white sugar provides a carbon source for lactic acid bacteria fermentation, promotes fermentation and the formation of metabolites, and promotes protein aggregation, thus resulting in a good viscosity. Considering both health and economic costs, a white sugar addition of 6% was chosen as the basis for further research.

[0107] This study investigated the effects of different strains of bacteria added on the pH, acidity, and viscosity of walnut-fed goat milk double-protein yogurt. The results are shown in... Figure 14 , Figure 15 and Figure 16 .

[0108] Depend on Figure 14 and Figure 15It can be seen that the pH of the double-protein yogurt gradually decreases with increasing fermentation time, and tends to stabilize after 8 hours of fermentation. The acidity of the yogurt during fermentation corresponds to the change in pH. The yogurt has the lowest acidity when the inoculum is 0.05%. With increasing inoculum, the logarithmic growth phase of the lactic acid bacteria is significantly shortened, the acid production capacity is enhanced, the fermentation time is shortened, and the acidity of the yogurt increases accordingly. When the inoculum is 0.20%, 0.25%, and 0.30%, the double-protein yogurt reaches the fermentation endpoint after 6 hours. Figure 16 It can be seen that the amount of starter culture added has a relatively small impact on the viscosity of double-protein yogurt. The yogurt viscosity is lowest when the starter culture content is 0.05%, and highest when it reaches 0.20%. When the starter culture content exceeds 0.20%, excessive proliferation of lactic acid bacteria leads to the accumulation of metabolic products, resulting in over-fermentation, over-acidification, and a deterioration in the texture of the yogurt. Taking all factors into consideration, a starter culture content of 0.20% was ultimately chosen.

[0109] Based on the single-factor experiments, and according to the Box-Behnken experimental design principle, Design Expert 8.0.6 software was used to design experiments on the double protein ratio, fermentation time, white sugar addition, and inoculum addition of walnut-goat milk double protein yogurt. With acidity and viscosity as response values, a 4-factor, 3-level response surface experiment was designed to determine the optimal fermentation conditions. The factors and levels are shown in Table 3, and the response surface experiment design and results are shown in Table 4.

[0110] Table 3. Factors and Levels in Response Surface Experiment

[0111]

[0112] Table 4 Response Surface Experimental Design and Results

[0113]

[0114]

[0115] The data in Table 4 were analyzed using Deign Expert 8.0.6 software. A quadratic multinomial regression model for acidity (Y1) and viscosity (Y2) was established, with the protein-to-soil ratio (A), fermentation time (B), inoculum addition amount (C), and white sugar addition amount (D) as factors of consideration. The equations are as follows:

[0116] Y1=73.50+1.15A+0.9236B+0.4861C+0.9722D+0.4375AB-0.0417AC-

[0117] 0.6250AD+0.0417BC+0.3333BD+0.3750CD-0.1458A 2-0.5417B 2 -0.9479C 2 -1.18D 2 ;

[0118] Y2=1136.70+52.10A+32.37B+25.51C+1.63D-

[0119] 1.37AB+15.42AC+60.67AD-153.58BC+51.83BD-57.79CD-48.27A 2 -65.19B 2 -65.27C 2 -58.77D 2 ;

[0120] Table 5 shows the analysis of variance for the acidity regression model. As shown in Table 5, the regression model is highly significant (P < 0.01), the lack-of-fit term is not significant (P = 0.3595 > 0.05), and the model's coefficient of determination R0 is [value missing]. 2 = 0.9546, adjusted coefficient of determination R 2 adj =0.9092, indicating that the experimental model has a high degree of fit, sufficient fit, small error, and high reliability. Therefore, it is entirely feasible to conduct analysis and prediction on the process optimization experiment of double-protein yogurt using the model. Furthermore, as shown in Table 5, the linear terms in the model have a highly significant effect on acidity (P<0.01), the interaction term AD has a significant effect (P<0.05), and the remaining interaction terms have no significant effect (P>0.05). The quadratic term B... 2 C 2 D 2 All had extremely significant effects (P<0.01).

[0121] Table 5. Analysis of Variance for Acidity Regression Model

[0122]

[0123]

[0124] Note: "*" indicates a significant difference (P<0.05), and "**" indicates an extremely significant difference (P<0.01).

[0125] Table 6 shows the analysis of variance for the viscosity regression model. As can be seen from the table, the regression model is highly significant (P<0.01); the lack-of-fit term is not significant (P=0.3710>0.05), and the model's coefficient of determination R0 is... 2 =0.9500, adjusted coefficient of determination R 2 adj=0.9000, indicating that the experimental data fits the regression mathematical model well, and the above model can be used to analyze and study the process optimization of double-protein yogurt effectively. The order of influence of each factor on viscosity is: double protein ratio > fermentation time > amount of inoculum added > amount of white sugar added. The linear terms A and B, the interaction terms AD, BC, BD and CD, and all quadratic terms in the model have extremely significant effects on the viscosity of double-protein yogurt (P<0.01), the linear term C has a significant effect on viscosity (P<0.05), and the other terms have no significant effect (P>0.05).

[0126] Table 6. Analysis of Variance for Viscosity Regression Model

[0127]

[0128] Note: "*" indicates a significant difference (P<0.05), and "**" indicates an extremely significant difference (P<0.01).

[0129] To visually represent the effects of the interactions between four factors—the ratio of protein to solids (A), fermentation time (B), inoculum quantity (C), and sugar content (D)—on acidity, a three-dimensional surface plot of the interaction effects on acidity was created. (See attached image.) Figure 17 .Depend on Figure 17 (c) It can be seen that the slope of the response surface of the interaction term AD is steep, indicating that the interaction between the ratio of double protein and the amount of added white sugar has a significant effect on the acidity of double-protein yogurt. The slopes of the other response surfaces are relatively gentle, indicating that the interaction terms AB, AC, BC, BD, and CD are not significant. A three-dimensional surface plot of the effect of the interaction of each factor on viscosity is shown in [reference needed]. Figure 18 . Figure 18 The response surfaces (c), (d), (e), and (f) have a large longitudinal span, a steep slope, and the bottom-projected contour plots are close to ellipses, indicating that the interaction between AD, BC, BD, and CD has a significant effect on viscosity. Figure 18 The slope of the response surfaces in (a) and (b) is relatively small, indicating that the interaction between the two factors has no significant effect on viscosity, which is consistent with the results of the analysis of variance.

[0130] The optimal fermentation conditions for double-protein yogurt, determined through regression model analysis, were: a double-protein ratio of 1:9, a fermentation time of 7.00 h, a microbial inoculum addition of 0.18%, and a white sugar addition of 6.63%. The theoretical acidity was 74.84°T, and the viscosity was 1204.00 mPa·s. Based on actual conditions, the white sugar addition was adjusted to 6.60%. Three experiments under the modified optimal fermentation conditions verified that the acidity of the double-protein yogurt was 74.50°T, and the viscosity was 1280.33 mPa·s, which is close to the theoretical results.

[0131] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing walnut and goat milk double-protein yogurt, characterized in that, Includes the following steps: Modified walnut protein was prepared using walnut protein solution and papain as raw materials. Modified walnut protein and reconstituted sheep milk were mixed to obtain a mixture; white sugar, xanthan gum, β-cyclodextrin and sucralose were added to the mixture, and homogenization and sterilization were carried out in sequence to obtain mixed raw milk. Fermentation starters are added to the mixed raw milk for fermentation. After fermentation, fermented milk is obtained. After post-processing the fermented milk, walnut goat milk double protein yogurt was obtained; The steps for preparing modified walnut protein using walnut protein solution and papain as raw materials are as follows: Walnut protein was mixed with water to obtain a walnut protein solution; papain was added to the walnut protein solution for enzymatic hydrolysis, followed by centrifugation, collection of supernatant, and drying to obtain modified walnut protein; The pH of the walnut protein solution was adjusted to 7 using NaOH or HCl, and the mass ratio of walnut protein to water was 1:15; the enzymatic hydrolysis time was 90 min; and the ratio of walnut protein solution to papain was 100 mL: 4 g. In every 100g of mixed raw milk, the added amount of white sugar is 5%~10%, xanthan gum is 0.20%, β-cyclodextrin is 0.06%~0.10%, and sucralose is 0.005%~0.02%. The fermentation is carried out under aseptic conditions, at a temperature of 42-45°C, for a time of 4-9 hours. The amount of fermentation bacteria added per 100g of mixed raw milk is 0.05%~0.30%; The mass ratio of the modified walnut protein to the reconstituted sheep milk protein is 1:(1~9). The fermentation strain is a mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.

2. The method for preparing a walnut and goat milk double-protein yogurt according to claim 1, characterized in that, After adding papain to the walnut protein solution for enzymatic hydrolysis, the prepared walnut protein hydrolysate was subjected to enzyme inactivation treatment at 95~100℃ for 10~15 min. Subsequently, the supernatant was collected by centrifugation and dried to obtain modified walnut protein.

3. The method for preparing a walnut-sheep milk double-protein yogurt according to claim 1, characterized in that, The sterilization process is carried out at a temperature of 90-95°C for 10-15 minutes.

4. A walnut and goat milk double-protein yogurt, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 3.

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