Lactiplantibacillus plantarum strain glj2 and its use in the preparation of litchi-flavored dairy products

Abstract

The present application relates to the technical field of microorganism, and particularly relates to lactobacillus plantarum strain GLJ2 and its application in preparation of litchi flavor dairy product, the present application adopts buffalo milk, litchi juice and self-screened probiotic GLJ2 to prepare flavor dairy product, and it is found that litchi juice and lactobacillus plantarum GLJ2 have good synergies during preparation process research, the litchi juice fermented dairy product prepared by the combination has strong product functionality, has good antioxidant property and bacteria increasing efficacy, meanwhile, through animal experiment, the product also has good relieving effect on mouse colitis, and is a potential functional fermented milk suitable for colitis patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to Lactobacillus plantarum strain GLJ2 and its application in preparing litchi-flavored dairy products. BACKGROUND

[0002] In recent years, plant-based products have rapidly developed due to their healthy and nutritious image. The global plant-based market size has grown from $18 billion in 2013 to $22.9 billion in 2023, and 78% of Chinese consumers believe that a plant-based diet is a trend. Lactic acid bacteria fermentation has a wide variety of applications in the food industry, the most common of which include yogurt, cheese, and lactic acid bacteria beverages, demonstrating the broad application potential of lactic acid bacteria fermentation in the food industry. Pure vegetarianism and consumer preference for healthy products have driven the demand for plant-based fermented dairy products. With the upgrading of consumption, the concept of plant-based is becoming increasingly popular, and the emergence of plant-based fermented milk caters to the demand for health and provides more diversified choices. However, there are still some challenges in the development process, such as taste, texture, and popularity, and continuous innovation and optimization are needed from various angles such as raw materials, processes, and functions. Animal-derived fermented milk in the mainstream market is popular all over the world due to its unique taste and flavor as well as numerous physiological functions, and the fat and protein in its raw materials can further enrich the nutritional value of fermented milk. The probiotics-lactic acid bacteria in fermented milk can regulate the intestinal microbiota and maintain intestinal microbial balance. Lactic acid bacteria in fermented milk can reduce the lactose content in raw milk to help the human body digest lactose, thereby alleviating lactose intolerance. Anti-aging, improving immune function, reducing cholesterol, preventing cardiovascular disease, and other multiple functions make fermented milk increasingly popular among consumers.

[0003] Scholars at home and abroad have studied probiotics (mainly lactic acid bacteria) and plant sources. Probiotics, as a microbial preparation, can improve intestinal microbial balance and benefit host health. Its health benefits such as regulating intestinal microbial balance and enhancing immunity have gradually been recognized by consumers. Plant-based foods also have bioactive substances that can enhance the balance of intestinal flora, play an important role in regulating intestinal microecological balance, improving immunity, and reducing gastrointestinal discomfort. During the study of plant extract enrichment, it was found that many plant extracts that are both food and medicine can promote the enrichment of most probiotics and increase the survival rate of probiotics. Plant extracts contain rich nutrients and active ingredients, some of which have probiotic effects, including proteins, flavonoids, polyphenols, polysaccharides, and oligosaccharides. These components can stimulate the proliferation of probiotics such as lactic acid bacteria, thereby affecting human health. Overall, plant-based lactic acid bacteria fermented foods not only effectively retain the vitamins, minerals, dietary fiber, and plant compounds of plant raw materials, but also improve the nutrition of the fermented products.

[0004] Litchi is famous at home and abroad for its beautiful color, aroma, and taste, and is known as the "King of Fruit". Litchi juice is milky white, contains various vitamins, organic acids, sugars, proteins, and a large amount of free arginine and serine, and is rich in bioactive substances such as polyphenols and anthocyanins. The polyphenols in litchi pulp promote the biosynthesis of short-chain fatty acids during in vitro colon fermentation, indicating their role in regulating intestinal microbiota diversity. Since the altered metabolic pathways are also related to the barrier function of the intestinal flora, we speculate that litchi may have a positive effect on improving intestinal inflammation. At the same time, the inventors found that the nutritional ingredients rich in litchi juice can promote the growth of lactic acid bacteria, and the flavor of the fermented product can also be accepted by consumers.

[0005] However, litchi juice still faces the following technical problems when preparing fermented foods: Litchi juice is derived from plants, and when used for fermentation, it is found that not all lactic acid bacteria have good fermentation effects on substrates containing litchi juice due to the influence of active ingredients in plants. The technical problems of low probiotic activity, high mortality of probiotics in fermented milk during the shelf life, and insignificant functionality often exist in the fermented product. Therefore, it is necessary to start from the aspects of early strain screening and process control, select characteristic probiotics and process conditions for different plant raw materials, and develop green and healthy products for characteristic plants through strain selection and process optimization, which can well solve the above problems. SUMMARY

[0006] In view of the above, in order to improve the quality of fermented dairy products and enhance the functionality and flavor of the products, it is necessary to start from the aspects of early strain screening and process control, select characteristic probiotics and process conditions for litchi juice, and prepare a new type of fermented dairy product, thereby enhancing the functionality and unique flavor of the fermented dairy product.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is:

[0008] A plant Lactiplantibacillus plantarum strain GLJ2, with the preservation number GDMCC NO: 63168; the strain is preserved in the Guangdong Microbial Culture Collection Center, located at No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100, with a preservation date of February 14, 2023.

[0009] The present application also includes a litchi-flavored fermented milk containing the Lactiplantibacillus plantarum strain GLJ2.

[0010] The application also comprises a method for preparing the lychee-flavored fermented milk, which comprises the following steps: adding lychee juice and white granulated sugar into fresh milk as raw material milk after the raw material milk is heated, uniformly mixing to obtain a fermentation base, adding probiotics and a fermenting agent into the fermentation base after the fermentation base is homogenized, sterilized and cooled, constant-temperature fermentation and post-ripening.

[0011] The probiotics are obtained by mixing Lactiplantibacillus plantarum strain GLJ2 and Lactobacillus rhamnosus hsryfm 1301.

[0012] Further, the mass ratio of the Lactiplantibacillus plantarum strain GLJ2 and the Lactobacillus rhamnosus hsryfm 1301 is 1-2:1-2, the addition amount of the lychee juice is 5%-15% of the mass of the fermentation base, the addition amount of the white granulated sugar is 4%-8% of the mass of the fermentation base, and the addition amount of the fermenting agent is 0.002-0.005% of the mass of the base.

[0013] Further, the mass ratio of the Lactiplantibacillus plantarum strain GLJ2 and the Lactobacillus rhamnosus hsryfm 1301 is 1:1, the addition amount of the lychee juice is 10% of the mass of the fermentation base, and the addition amount of the white granulated sugar is 6% of the mass of the fermentation base.

[0014] The application also comprises the application of the lychee juice in promoting the lychee-flavored fermented milk in terms of antioxidation and bacteria increase.

[0015] The application also comprises the application of the Lactiplantibacillus plantarum strain GLJ2 in preparing food for relieving colitis.

[0016] The application has the following beneficial effects:

[0017] Buffalo milk, lychee juice, and multiple probiotics are compounded to complement each other to give the product a 1+1+1>3 functional and flavor effect. As can be seen from the examples, lychee juice has the effect of improving the proliferation of compounded probiotics and lactic acid bacteria in the starter culture, which is crucial for live bacteria type fermented products; meanwhile, lychee juice is rich in active substances such as phenols, which can significantly improve the antioxidant effect of fermented milk and at the same time improve the texture of fermented milk and enhance or maintain the sensory properties. The combination of the three enriches the function and sensory flavor of buffalo milk fermented milk. In addition, the probiotic strains and lychee juice of the present application have good synergistic effect, and the flavor fermented milk prepared is verified by mice, which has a good remission effect on colitis in mice. Therefore, it can provide a wider research space for subsequent food therapy research for relieving colitis. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a plate map of strain GLJ2.

[0019] Figure 2 It is a microscope map of strain GLJ2.

[0020] Figure 3 It is a result map of the value-added rate of different probiotics in lychee skim milk.

[0021] Figure 4 It is a result map of the change of the disease activity index (DAI) index of mice.

[0022] Figure 5 It is a result map of the analysis of the colon length and weight / length of mice; in the figure, A is the colon length, and B is the colon weight / length.

[0023] Figure 6 It is a result map of the change of the spleen weight and spleen index of mice; in the figure, A is the spleen weight, and B is the liver index.

[0024] Figure 7 It is a result map of the H&E staining of the colon of mice.

[0025] Figure 8 It is a result map of the pathological tissue damage score of the colon of mice.

[0026] Figure 9 It is a result map of the expression level of cytokines in the colon tissue of mice; in the figure, A is the IL-4 expression level, B is the IL-10 expression level, C is the IL-6 expression level, and D is the TNF-α expression level. DETAILED DESCRIPTION

[0027] All features disclosed in this specification, and / or all steps of any methods or processes disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0028] Any feature in the foregoing specification that is expressed as a particular combination of features, unless specifically stated otherwise, can be expressed or performed with reference to alternate combinations characterised by one or more features other than those features expressing the particular combination.

[0029] Example 1

[0030] The probiotic used in this example is Lactiplantibacillus plantarum strain GLJ2, which has a preservation number of GDMCC NO: 63168. The strain is preserved in the Guangdong Microbial Culture Collection Center, located at 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, with a preservation date of February 14, 2023.

[0031] The strain was isolated from mature cheese.

[0032] The morphological characteristics of the strain on the plate are shown in Figure 1 The strain colony is white, smooth edge, and semispherical in the middle; the microscopic image is shown in Figure 2 The strain is rod-shaped, mostly in pairs or single, of varying lengths, non-motile, and no spores under the microscope. The strain was identified as Lactiplantibacillus plantarum.

[0033] Example 2

[0034] The screening and metabolic effects of litchi juice on probiotics are as follows:

[0035] Lactic acid bacteria were used as probiotic strains, and milk base was used as the fermentation substrate. Litchi juice was added to the skim milk medium (experimental group) for fermentation, and the lactic acid bacteria were evaluated for their ability to increase the number of bacteria. The lactic acid bacteria strain was optimized. Multiple components of litchi were obtained by separation and purification, and the functional components were determined based on the number of viable probiotic bacteria and antioxidant results. The effect of litchi juice on the increase of probiotic GLJ2 complex starter was evaluated. The effect of litchi juice complexed with probiotic GLJ2 on the antioxidant capacity of fermented milk was evaluated. The effect of litchi juice complexed with multiple probiotic strains on the quality of fermented milk was studied. The number of viable bacteria was determined by plate count method, and titration acidity was determined by titration method. Antioxidant activity determination scheme: a. ABTS free radical scavenging activity determination; b. Hydroxyl radical scavenging activity determination; c. Reducing power determination; d. DPPH free radical scavenging capacity determination. The specific experimental results are as follows:

[0036] 1. Effect of litchi juice on the increase of probiotic milk base and function:

[0037] The effect of litchi juice on the increase of lactic acid bacteria is shown in Figure 3 Figure 3 ​As can be seen from the comparison of the viable bacteria count of skim milk without the addition of litchi juice, the proliferation rate of the other four strains is greater than 100% except for strain W218, which has a bacteria increasing effect. Among them, strain GLJ2 has the highest bacteria increasing rate in litchi juice milk base, close to 200%, and is the preferred strain GLJ2 for subsequent research.

[0038] The effect of litchi juice on the bacteria increasing of single patent lactic acid bacteria 1301 and fermentation agent is shown in Table 1. Litchi juice significantly increases the viable bacteria count of strain 1301 and fermentation agent strain in milk base (P<0.05), but the bacteria increasing effect is not as good as GLJ2. The mechanism of litchi juice on the bacteria increasing and milk base function improvement of single probiotic GLJ2 will be disclosed, which provides theoretical support for the subsequent research and development of fermented milk with three compounds.

[0039] Table 1 Effect of litchi juice on the bacteria increasing of single patent lactic acid bacteria and fermentation agent

[0040]

[0041] Note: The data is mean ± standard deviation (n=3). The same row mark different capital letters represent significant difference (P<0.05); the same column mark different small letters represent significant difference (P<0.05); the same below. The 1301 strain of the present application is a patent strain of Imperial Group Co., Ltd. The preservation number of the strain is: CGMCC NO. 8545.

[0042] 2, Effect of litchi juice components on the functional quality of strain GLJ2 milk base

[0043] Table 2 Effect of litchi juice components on the functional quality of strain GLJ2 milk base

[0044]

[0045] The effect of different component concentrations on the function of strain GLJ2 milk base was investigated with the content of polysaccharide and polyphenol in litchi juice as the conversion benchmark. As can be seen from Table 2, in terms of the effect on viable bacteria count, litchi mainly provides high-quality carbon and nitrogen sources and other nutrients, so that probiotics can efficiently utilize them for proliferation metabolism; at the same time, there are bacteriostatic substances such as polysaccharide and polyphenol in litchi, which can reduce pH value, improve antioxidant activity and inhibit harmful bacteria to promote the growth of probiotics. As can be seen from Table 2, the polysaccharide and polyphenol / flavonoids crude extract of litchi juice is positively correlated with the concentration of GLJ2 viable bacteria count, and the 2.0 times and 10% litchi juice group is the closest, which may play a major role in promoting proliferation. In terms of antioxidant activity, the DPPH clearance rate of polysaccharide group and 10% litchi juice group is significantly higher than that of other groups (P<0.5), and it is speculated that polysaccharide mainly plays an antioxidant role.

[0046] 3, Effect of litchi juice on the bacteria increasing of probiotic GLJ2 compound fermentation agent:

[0047] Table 3 Effect of litchi juice on the lactobacillus lactobase inoculation

[0048]

[0049] As shown in Table 3, compared with the group without adding litchi juice, the addition of litchi juice can increase the viable counts of total bacteria, bacilli and cocci in the lactobase, especially significantly promote the viable counts of total bacteria and cocci (P<0.5), indicating that the combination of litchi + GLJ2 + starter has synergistic inoculation effect, which is better than the combination of GLJ2 + starter.

[0050] 4. Effect of litchi juice and probiotic GLJ2 combination on the antioxidant capacity of fermented lactobase

[0051] Table 4 Effect of litchi juice and probiotic combination on the antioxidant effect of fermented milk

[0052]

[0053] As shown in Table 4, in general, probiotic fermented milk shows antioxidant activity due to the presence of bioactive peptides, while lactic acid bacteria and litchi belong to natural antioxidants, and the synergistic effect of the two can improve the antioxidant activity of the fermentation substrate. In the hydroxyl radical scavenging and reducing capacity effect, the litchi juice + GLJ2 + starter group is significantly higher than the ordinary starter fermented milk group, reaching 59.26% and 0.55, respectively. The addition of litchi juice and probiotics significantly improves the antioxidant effect of fermented milk.

[0054] 5. Effect of litchi juice and multiple probiotics on the quality of fermented milk

[0055] Table 5 Effect of litchi juice and probiotics on the quality of fermented milk

[0056]

[0057] As shown in the foregoing, litchi juice has a promoting effect on the inoculation of probiotics 431, 1301 and starter. Now the three are compounded to prepare fermented milk. As shown in Table 5, the viable count and ABTS clearance rate of fermented milk in the litchi juice and probiotic group are significantly higher than those in the group without adding, indicating that litchi juice and probiotics can improve the antioxidant function of fermented milk. In the litchi juice group, the quality indicators of the litchi juice + GLJ2 + 1301 + starter group are significantly higher than those of the other two groups. The indicators of fermented milk with litchi juice and multiple probiotics are the highest. Based on this, litchi juice, probiotics GLJ2 and 1301, and starter are compounded to optimize the litchi juice and probiotic flavor fermented milk process.

[0058] Example 3:

[0059] This example studies the process optimization of litchi juice flavor fermented buffalo milk products, and the specific method is as follows:

[0060] Fresh raw buffalo milk without antibiotics is used as raw milk, and the protein is standardized to 4.0%. The raw milk is heated to 60-65℃, and then 0-15% litchi juice and 2-8% white sugar are added to the raw milk, and the mixture is stirred and dissolved completely. Then the mixture is mixed with the remaining raw milk and stirred evenly to obtain a base material. The obtained base material is homogenized at 15-20MPa and 60-70℃, heated to 90℃, and then held for 10min for pasteurization. The pasteurized base material is cooled to 40-42℃, and then Lactiplantibacillus plantarum GLJ2 and Lactobacillus rhamnosus hsryfm 1301 are mixed as probiotics. Then, the probiotics are added at an amount of 0.2-0.6% of the base material, and a starter culture is added at an amount of 0.002-0.005% of the base material. The mixture is stirred and mixed evenly to obtain inoculated milk. Then, the inoculated milk is filled into cups, sealed, and fermented at 38-44℃ until the acidity reaches 70-75°T or the pH value reaches 4.4-4.8. The litchi juice flavored fermented milk is obtained, and is stored at 2-6℃ for 24h of post-ripening.

[0061] Table 6 Optimization of litchi juice flavored fermented milk by single factor experiment

[0062]

[0063]

[0064] From Table 6, in the litchi juice addition amount (starter 904), the ABTS clearance rate of the 10% and 15% groups was significantly higher than that of the other two groups (P<0.5). There were significant differences in flavor and overall acceptability, and the 5% group was significantly lower than the other groups, and the 15% group reached the highest, and the 10% and 15% groups had little difference in sensory indicators. It is shown that after adding a certain amount of litchi juice, the antioxidant and sensory quality of fermented milk can be significantly improved. Comprehensive analysis of the litchi juice addition amount of 10% and 15% shows that the comprehensive quality of the produced fermented milk is better, which can be used for subsequent optimization.

[0065] In the litchi juice addition amount (starter 1.0), the ABTS clearance rate increases with the increase of the addition amount, and the ABTS clearance rate of the 15% group is significantly higher than that of the other three groups (P<0.5). There is no significant difference in sensory indicators between groups, which shows that the addition of litchi juice has little effect on the sensory effect under the combination of starter 1.0.

[0066] The ABTS clearance rate of the test group with GLJ2:1301 ratio of 1:1 was the highest, and the appearance, flavor and texture of the 1:2 group were the highest. As shown in Table 1, litchi juice had a bacterium increasing effect on GLJ2 and 1301 strains. Comprehensive analysis showed that the compound probiotic bacteria were used for subsequent optimization.

[0067] The addition of white sugar had no significant effect on the ABTS clearance rate of each group, but had a significant effect on multiple sensory indicators including flavor, texture, overall acceptability and total score (P<0.5). With the increase of white sugar addition, the 2% group was significantly lower than other groups (P<0.5). Comprehensive analysis showed that the addition of white sugar was 4%, 6% and 8%, and the overall quality of the fermented milk produced was better, which could be used for subsequent optimization.

[0068] Among them, the ferment 904 and 1.0 were purchased from Denmark company.

[0069] On the basis of single factor experiment, considering the sensory evaluation and ABTS clearance rate, litchi juice addition, white sugar addition and probiotic bacteria ratio (GLJ2:1301) were used as influencing factors, and the total addition of probiotic bacteria was fixed. The addition of ferment 904 was 0.003%, and the sensory indicators and ABTS clearance rate of the product were evaluated. The orthogonal test results are shown in Table 7:

[0070] Table 7 Orthogonal test results of litchi flavor fermented milk and range analysis

[0071]

[0072]

[0073] As shown in Table 7, the range analysis of ABTS clearance rate showed that the main and secondary factors were litchi juice addition>probiotic bacteria ratio (GLJ2:1301)>white sugar addition, and the influence of litchi juice addition was the most significant (P<0.05) but not significant between 10% and 15% groups, and the influence of other two factors was not significant. The range analysis of sensory evaluation showed that the main and secondary factors were white sugar addition>litchi juice addition>probiotic bacteria ratio (GLJ2:1301), and the influence of white sugar addition was significant (P<0.05) but not significant between 6% and 8% groups, and the influence of other two factors was not significant. Considering the significance and cost factors, the optimal combination was A2B2C3, i.e. litchi juice 10%, white sugar 6% and probiotic bacteria ratio (GLJ2:1301) 1:1.

[0074] Based on the above conditions, in actual production, the optimal process of A2B2C3 combination was used for production test, and it was found that the ABTS clearance rate was 57.39±1.92%, and the sensory score was 90±9, which was higher than all the combinations of orthogonal experiment. The product had delicate taste, and combined with frankincense and litchi juice fruit aroma. A litchi juice flavor fermented milk product with uniform organization, unique litchi and milk taste and aroma, sweet and palatable, uniform organization and thick taste was developed. Therefore, the A2B2C3 process condition was selected for production.

[0075] Example 4:

[0076] This example studies the quality change of litchi juice fermented dairy products during storage period:

[0077] The optimal process of litchi juice flavor fermented milk was used to prepare the product, and under the premise of the same ratio of probiotics (GLJ2:1301 was 1:1), the addition was divided into three orders of magnitude of viable bacteria, i.e. 1×10 8 CFU / g (probiotic addition formula 1), 1×10 7 CFU / g (probiotic addition formula 2), and 1×10 6 CFU / g (probiotic addition formula 3), and the ordinary fermented milk without adding plant matrix and probiotics was used as control to evaluate the quality change of fermented milk within 21 days under cold storage condition, as shown in Table 8:

[0078] Table 8 Quality change of flavor fermented milk during storage period

[0079]

[0080] Note: Different lowercase letters: significant difference in the same column (P<0.05); different capital letters: significant difference in the same row (P<0.05).

[0081] The total viable count was significantly affected by storage time and type of fermented milk (P < 0.05). During the entire storage period, the viable count of the first day fermented milk was between 8.64-8.97 Lg CFU / g, and the viable count of each group decreased with the storage time, which was attributed to the substrate competition effect. The viable count of the litchi juice fermented milk increased significantly (P < 0.05), which might be related to the synergistic effect of lactic acid bacteria. Among the three different orders of magnitude of the formula, there was no significant difference in the viable count of the litchi juice probiotic fermented milk on the first day (P > 0.05), indicating that the addition of probiotics had little effect on the total bacterial count at the end of fermentation. However, with the storage time, the viable count of the fermented milk was positively correlated with the amount of probiotics added, indicating that probiotics and starter bacteria formed a synergistic effect. Under the same storage time, the litchi juice probiotic fermented milk group was higher than the ordinary fermented milk, and in many groups, it showed a significant increase in the seventh day (P < 0.05), indicating that the addition of litchi juice and probiotics could maintain the effect of improving the viability of lactic acid bacteria in fermented milk. Other scholars' studies have shown that the addition of plant base and probiotics can help to improve the viable count of fermented milk.

[0082] In the ABTS scavenging rate, the antioxidant activity of each group showed a downward trend, and the litchi juice probiotic fermented milk group decreased significantly before the seventh day (P > 0.05), and increased by 2-26% than the ordinary fermented milk on the 21st day. This might be because the litchi juice itself was rich in antioxidant substances, and could promote the proliferation and metabolism of lactic acid bacteria to produce more antioxidant peptides, which could maintain stable antioxidant activity. With the fermentation time, the antioxidant substances were decomposed by lactic acid bacteria, leading to the decrease of the antioxidant activity of fermented milk. With the increase of fermentation time, the number of antibacterial substances increased, improving the antioxidant activity of fermented milk. Among the three different orders of magnitude of the formula, the litchi juice fermented milk showed a dose-effect dependence of probiotics, and was higher than the ordinary fermented milk. As mentioned above, it showed that litchi juice and probiotics could improve the antioxidant activity of the product.

[0083] In titratable acidity and pH, the fermented milk of each group increased in acidity and decreased in pH with the extension of storage time, showing a post-acidification phenomenon. Among them, formula 1 showed a more serious post-acidification, which was not recommended for production. Under the same storage period, the litchi juice fermented milk was significantly higher than the ordinary fermented milk group (P < 0.05), indicating that it significantly improved the ability of lactic acid bacteria to decompose lactose to produce lactic acid, which was consistent with the conclusion of the viable count.

[0084] In sensory evaluation, the best tasting period of each group was 7-14 days, and there was no significant difference in the sensory evaluation of each group of fermented milk under the same storage time (P > 0.05), indicating that the unique flavor of fermented milk endowed by the exogenous addition of litchi juice could be loved by the evaluators.

[0085] In summary, during the storage period, the litchi juice probiotic group fermented milk has a significantly higher number of viable bacteria and antioxidant activity than ordinary fermented milk. Formulas 2 and 3 have a similar degree of post-acidification to ordinary fermented milk, and the sensory characteristics are not significantly different from ordinary fermented milk. The litchi flavor fermented milk product has significant functionality during the storage period and stable quality, and can be used for popularization and application.

[0086] Example 5:

[0087] This example studies the safety and functionality of litchi juice fortified probiotic functional fermented milk products in animal models

[0088] According to the conclusions of Examples 2-4, litchi juice probiotic fermented milk has excellent antioxidant capacity, which can help scavenge free radicals and reduce oxidative stress, thereby helping to inhibit inflammatory response. Combined with literature research, it is shown that litchi juice, plant lactobacillus and rhamnose lactobacillus have anti-inflammatory efficacy due to their own composition and characteristics. The relief effect of litchi juice probiotic fermented milk on dextran sodium sulfate (DSS)-induced colitis mice and the potential mechanism were studied, and the litchi juice flavor fermented milk (MT) optimized by the above process was selected, and a normal control group (NC), a DSS group (MC) and a positive control group (SC) were set. The specific process is as follows:

[0089] 1. Change of mouse disease activity index (DAI) index:

[0090] DAI is one of the important indicators reflecting the inflammatory condition, and the larger the value, the more severe the inflammation. As shown in Figure 4 , the DAI score of the NC group is close to zero, which is the lowest value, indicating that the mice are in good health during the experiment; the DAI score of the MC group is higher than that of the NC group, indicating that the inflammation modeling is successful. The DAI of the four groups of mice drinking DSS shows a trend of first rising and then falling, indicating that the mice have inflammation but are relieved, among which the inflection point of the fermented milk appears on the 8th day, which is 2 days earlier than that of the MC group and the value is lower, indicating that the fermented milk can effectively prevent the occurrence of DSS-induced colitis in mice.

[0091] 2. Analysis of colon length and weight / length of mice

[0092] DSS-induced colitis can make the colon of mice shorter, so the colon length is one of the indicators to reflect the degree of inflammation of DSS-induced colitis. As shown in Figure 5 A, the normal colon length of the NC group mice is 9.66 cm, while the MC group mice have the most severe inflammation, with the shortest colon length of 8.77 cm. The colon length of the MT group is 9.69 cm, which is significantly higher than that of the MC group (P<0.05), which can effectively promote the recovery of colon length of DSS acute colitis mice. The ratio of colon weight to length is used to evaluate the degree of edema of mouse colon, and the larger the ratio, the more severe the edema and inflammation. As shown in Figure 5BAs can be seen, the ratio of each group is significantly lower than that of the MC group (P<0.05), indicating that the colon edema of the MC group of mice is the most serious, and after the drug and fermented milk treatment, the colon edema of the colonitis mice is significantly improved, and it is worth mentioning that the MT group is significantly lower than the SC group. In summary, the fermented milk of litchi juice (MT group) can effectively prevent the colon of colonitis mice from shortening and edema.

[0093] 3, Analysis of mouse spleen weight and spleen index

[0094] The spleen is an important immune organ of the body, and external stimulation can activate lymphocytes and NK cells in the spleen and make them proliferate, leading to splenomegaly. The weight and index of the spleen can reflect the immune function of the body to a certain extent. Figure 6 A shows that the spleen weight of the MC group is significantly greater than that of the other groups (P<0.05), and the weight gain is the most and the inflammation is the most serious, and the NC group and the MT group are significantly lower than the SC group (P<0.05). The spleen index is the ratio of the weight of the spleen to the body weight, and the lower the ratio, the weaker the immune response to a certain extent. As shown in Figure 6 B, compared with other groups, the spleen index of the MC group of mice shows a significant increasing trend (P<0.05), indicating that DSS may stimulate the proliferation of immune cells in the spleen of colonitis mice, and then activate the immune response pathway. The spleen index of the litchi juice probiotic fermented milk group and the drug group (SC group) of mice is significantly lower than that of the MC group (P<0.05), and the MT group is significantly lower than the NC group and the SC group (P<0.05), indicating that fermented milk and drugs can improve the splenomegaly of colonitis mice, and the MT group is the best. In summary, the fermented milk of the present application can relieve the splenomegaly of colonitis mice.

[0095] 4, Observation of mouse colon H&E pathological staining and tissue damage score

[0096] The integrity of the colon tissue structure is a direct indicator of the severity of colon inflammation, and the tissue damage score standard includes inflammation, lesion depth, crypt destruction, and lesion range. After H&E staining, the pathological changes were observed under a microscope and photographed, and each group of mice was observed as a whole and locally in two forms of 40 μm and 100 μm, and the results are shown in Figure 7 As can be seen from the figure, the colon epithelial cells of the NC group are complete, the crypt structure is regular, the goblet cells are complete, the mucosal epithelium is complete, the glands are arranged in order, and the tissue damage score is 0.25. The epithelial structure of the MC group is severely damaged, the lesion range reaches about 10% (B2, B3 black arrow shown), and the tissue damage score is 3.25 Figure 8) was significantly higher than that of the NC group (P<0.05). The SC group and the fermented milk group both relieved the symptoms of colitis to different degrees, and the histological damage scores were significantly different from those of the MC group (P<0.01). Among them, the MT group significantly reduced the colonic tissue lesions, most of the intestinal epithelial cells were structurally complete, there was a complete crypt structure, a large number of goblet cells were arranged in an orderly and regular manner, only a small part of inflammatory infiltration (D2 arrow), and the histological score was 1.38, which was not significantly different from that of the NC group (P>0.05). In summary, the fermented milk of the application can relieve colitis in mice, and the fermented milk of litchi juice can significantly relieve tissue damage and effectively maintain the integrity of the mucosal structure.

[0097] The above conclusion shows that the fermented milk of litchi juice can significantly and effectively prevent the colon of colitis mice from shortening, relieve the splenic edema, significantly relieve the tissue damage, and effectively maintain the integrity of the mucosal structure.

[0098] 5, Changes of inflammatory cytokines in the colon of mice

[0099] Cytokines play an important role in the pathogenesis of colitis. The decrease in the secretion of anti-inflammatory cytokines and the excessive secretion of pro-inflammatory cytokines lead to the imbalance of the two types of factors, which is one of the pathogenesis of inflammation. IL-4 and IL-10 are effective anti-inflammatory cytokines, which can inhibit antigen transmission and the release of pro-inflammatory cytokines, thereby reducing mucosal inflammation. IL-6 and TNF-α are pro-inflammatory cytokines, which can coordinate the inflammatory response by activating various cells.

[0100] As shown in Figure 9 Although the expression amount of IL-4 of the MC group was not significantly different from that of the NC group, it showed an upward trend, which was speculated to be caused by the activation of the mouse intestinal inflammatory non-specific immune system to play a regulatory function. The content of IL-10 in the SC group was significantly higher than that in the MC group (P<0.05), and the up-regulation was obvious. It shows that the fermented milk of litchi can increase the secretion of anti-inflammatory cytokines, achieve the purpose of relieving the intestinal inflammation of mice, and improve the defense ability of the intestinal tract of mice.

[0101] As shown in Figure 9C, D showed that compared with the NC group, the pro-inflammatory factors IL-6 and TNF-α of the MC group were significantly increased (P<0.05), indicating that intestinal inflammation induced the secretion of various inflammatory factors. Compared with the MC group, the contents of the other two pro-inflammatory factors in the mice of other groups were significantly reduced (P<0.05), indicating that drug and fermented milk intervention can reduce the secretion of inflammatory cytokines and effectively alleviate the intestinal inflammation of mice. Among them, the expression amount of IL-6 in the MT group was the lowest, which was significantly lower than that in the NC group (P<0.05), and there was no significant difference between NC and SC; the content of TNF-α had no significant difference among NC, SC and MT groups (P>0.05). The above shows that fermented milk intervention in colitis mice can regulate the secretion of pro-inflammatory factors to different degrees, and relieve the intestinal injury of mice, and the fermented milk of litchi juice is better than the conventional fermented milk, which is confirmed by other index results.

[0102] The above shows that the fermented milk of litchi juice has good function of down-regulating pro-inflammatory factors and can achieve the purpose of relieving intestinal inflammation.

[0103] In summary, the application uses buffalo milk, litchi juice and self-screened probiotics GLJ2 and rhamnolactose lactobacillus 1301 to prepare flavored dairy products. It is found during the study of the preparation process that litchi juice and complex probiotics have good synergies, and the litchi juice fermented dairy product prepared by the combination has product functionality. At the same time, through animal experiments, it is found that the product has a good relieving effect on colitis in mice.

[0104] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but should not be understood as limiting the scope of the present application. It should be noted that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A method of preparing a lychee flavoured fermented milk, characterized in that, The method is: after the fresh milk is heated as raw material milk, lychee juice and white granulated sugar are added, and after being uniformly mixed, a fermentation base material is obtained, after the fermentation base material is homogenized, sterilized and cooled, probiotics and leavening agent are added and mixed, constant temperature fermentation, post-ripening are carried out, and the fermented milk is obtained; The probiotics are Lactobacillus plantarum ( Lactiplantibacillus plantarum ) strain GLJ2 and Lactobacillus rhamnosus ( Lactobacillus rhamnosus The mixture of hsryfm 1301 and Lactobacillus rhamnosus ( Lactobacillus rhamnosus The accession number for hsryfm 1301 is CGMCC NO. 8545; the *Lactobacillus plantarum* ( Lactiplantibacillus plantarum The preservation number of strain GLJ2 is GDMCC NO: 63168; The plant lactobacillus ( Lactiplantibacillus plantarum ) strain GLJ2 and Lactobacillus rhamnosus ( Lactobacillus rhamnosus The mass ratio of hsryfm 1301 is 1:

1. The amount of lychee juice added is 10% of the fermentation base mass, the amount of white sugar added is 6% of the fermentation base mass, and the amount of starter culture added is 0.002-0.005% of the base mass. The starter culture is Lactobacillus delbrueckii subsp. bulgaricus. (Lactobacillus delbrueckii subsp. bulgaricus ) and Streptococcus thermophilus subsp. ( Streptococcus salivarius subsp.thermophilus )composition.

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