A monk fruit flavored dairy product and methods of making and using the same

Abstract

The present application relates to the technical field of microorganism, and particularly relates to a luohanguo flavor dairy product and a preparation method and application thereof, the present application adopts buffalo milk, luohanguo powder, paracasei and rhamnosus to prepare the flavor dairy product, it is found during the preparation process research that the luohanguo powder, paracasei and rhamnosus have good synergies, the luohanguo powder fermented dairy product prepared by the combination has good antioxidant function, through animal experiment, the product can effectively relieve the inflammation of DSS-induced mouse colon, and the effect of the luohanguo flavor fermented milk is close to that of positive drug, so the product is a good diet therapy product.

Description

[Technical Field]

[0001] This invention relates to the field of microbial technology, and in particular to a monk fruit flavored dairy product, its preparation method, and its application. [Background Technology]

[0002] Lactic acid bacteria fermentation has a wide range of applications in the food industry, most commonly in yogurt, cheese, and lactic acid bacteria beverages, demonstrating its broad application potential. The emergence of plant-based fermented milk caters to health needs and provides more diverse choices. However, its development also faces challenges, such as taste, texture, and market penetration, requiring continuous innovation and optimization from the perspectives of raw materials, processes, and functions. Animal-derived fermented milk, which dominates the mainstream market, is popular worldwide due to its unique taste and flavor, as well as numerous physiological functions. The fats and proteins in its raw materials further enrich the nutritional value of fermented milk. The probiotics—lactic acid bacteria—in fermented milk can regulate intestinal microbiota and maintain intestinal microbial balance. Lactic acid bacteria in fermented milk can reduce the lactose content in raw milk, helping the body digest lactose and thus alleviating lactose intolerance. It also has multiple benefits such as anti-aging, improving immunity, lowering cholesterol, and preventing cardiovascular disease, making fermented milk increasingly popular with consumers.

[0003] Domestic and international scholars have been researching probiotics (mainly lactic acid bacteria) and plant-based species. Probiotics, as a microbial preparation, benefit host health by improving the balance of intestinal microbiota, and their health benefits, such as regulating intestinal microbiota balance and enhancing immunity, are gradually being recognized by consumers. Meanwhile, plant-based foods also contain bioactive substances that enhance the balance of intestinal flora, playing an important role in regulating intestinal microecological balance, improving immunity, and alleviating gastrointestinal discomfort. Research on the proliferation of plant extracts has found that many plant extracts, which are both medicinal and edible, can promote the proliferation of most probiotics, increasing their growth and survival rate. Plant extracts contain abundant nutrients and active ingredients, some of which have a probiotic effect, including proteins, flavonoids, polyphenols, polysaccharides, and oligosaccharides. These can influence human health by stimulating the proliferation of probiotics such as lactic acid bacteria. Overall, plant-based lactic acid bacteria fermented foods not only effectively retain the rich vitamins, minerals, dietary fiber, and plant compounds of the plant raw materials, but also achieve a comprehensive nutritional enhancement after lactic acid bacteria fermentation.

[0004] Monk fruit (Siraitia grosvenorii) is a typical Chinese medicinal plant, mainly growing in southern China, and is one of the first plants to be used as both food and medicine. The fruit contains glycosides, as well as abundant fructose, fatty acids, flavonoids, and essential amino acids. Mogroside is a cucurbitane-type triglyceride, 300 times sweeter than white sugar, making it the best alternative. Mogroside exists in the forms of mogroside I, III, IV, V, and VI, but mogroside V (MGV) is the most abundant and best-studied form. Studies have reported that MGV has certain health-promoting effects, including anti-inflammatory, antioxidant, anti-aging, anti-tumor, and anti-diabetic effects. Furthermore, MGV can alleviate LPS-induced acute lung injury, ovalbumin-induced experimental allergic asthma, and non-alcoholic fatty liver disease. It can also regulate blood sugar levels and is currently widely used in the food industry as a sugar substitute. In my country, mogroside is now permitted as a food additive in all foods.

[0005] The following technical challenges still need to be addressed in fermented foods: 1) Low probiotic activity in the products. The mortality rate of probiotics in fermented milk is as high as 50%-80% during the shelf life, while foods containing live probiotics must ensure that the number of live bacteria is not less than 10 during the best consumption period. 6 1) cfu / mL(g). 2) Functionality is not significant; the main function is too broad and vague. To solve the above problems, the applicant considers starting from two aspects: early strain screening and process control. The applicant plans to organically combine mogrosides, probiotics, and buffalo milk to develop a new type of fermented milk, combining the probiotic functions of the three to develop a green and healthy product, hoping to solve the above problems. [Summary of the Invention]

[0006] In view of the above, in order to improve the product function and flavor of fermented dairy products, it is necessary to conduct research on different plant-based raw materials. Monk fruit is a specialty fruit of Guangxi with good medicinal and edible value. Research on monk fruit powder will be conducted to prepare a new type of fermented dairy product, which can improve the utilization rate of Guangxi fruits and enrich the flavor of buffalo fermented milk. This fermented dairy product has the characteristics of strong product functionality and unique flavor.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention also includes a method for preparing monk fruit flavored fermented milk, wherein the preparation method is as follows: fresh milk is heated as raw milk, monk fruit powder and white sugar are added, and the mixture is evenly mixed to obtain a fermentation base. The fermentation base is homogenized, sterilized and cooled, and then probiotics and a starter culture are added and mixed. The mixture is then fermented at a constant temperature and allowed to mature to obtain the final product.

[0009] The probiotics are obtained by mixing *Lactaseibacillus paracasei* L. casei 431 and *Lactobacillus rhamnosus* hsryfm 1301. The 1301 strain used in this application is a patented strain of Huangshi Group Co., Ltd., with accession number CGMCC NO. 8545. The *Lactaseibacillus paracasei* L. casei 431 used in this application was purchased from Chr. Hansen, product number 706147.

[0010] Furthermore, the mass ratio of *Lactaseibacillus paracasei* L. casei 431 to *Lactobacillus rhamnosus* hsryfm 1301 is 1-2:1-2, the amount of monk fruit powder added is 0.05%-0.20% of the fermentation base mass, the amount of white sugar added is 0-10% of the fermentation base mass, and the amount of starter culture added is 0.001-0.005% of the base mass.

[0011] Furthermore, the mass ratio of Lacticaseibacillus paracasei L.casei 431 to Lactobacillus rhamnosus hsryfm 1301 is 1:1, the amount of monk fruit powder added is 0.1% of the mass of the fermentation base, and the fermenting agent is fermenting agent 904, which is added at 0.003% of the mass of the fermentation base.

[0012] Furthermore, the raw milk is buffalo milk.

[0013] The present invention also includes the application of monk fruit powder in promoting the antioxidant and microbial growth effects of the monk fruit flavored fermented milk.

[0014] The present invention also includes the application of the monk fruit flavored fermented milk in the preparation of foods that relieve colitis.

[0015] The present invention has the following beneficial effects:

[0016] The combination of buffalo milk, monk fruit powder, and multiple probiotics works synergistically to give the product a functional and flavorful effect greater than the sum of its parts (1+1+1>3). Monk fruit powder enhances the proliferation of lactic acid bacteria in the combined probiotics and starter culture, which is crucial for live-culture fermented products. Simultaneously, monk fruit powder is rich in active substances such as glycosides, phenols, and ketones, which significantly improve the antioxidant properties of fermented milk and enhance or maintain its sensory characteristics. The combination of these three elements enriches the functionality and sensory flavor of the buffalo milk fermented milk. The organic combination of monk fruit glycosides and fermented milk makes the product sugar-free or low-sugar, meeting the needs of both healthy individuals and obese or diabetic patients, aligning with the concept of green consumption. Furthermore, the probiotic strains and monk fruit powder in this application exhibit a good synergistic effect. The resulting flavored fermented milk has been validated in mouse models, showing a good alleviating effect on colitis in mice. Therefore, this provides a broader research space for subsequent dietary therapy research on alleviating colitis. [Attached Image Description]

[0017] Figure 1 This is a graph showing the changes in the Disease Activity Index (DAI) of mice.

[0018] Figure 2 The graph shows the results of the analysis of colon length and weight / length in mice; in the graph, A represents colon length and B represents colon weight / length.

[0019] Figure 3 The graph shows the changes in spleen weight and spleen index in mice; in the graph, A represents spleen weight; and B represents liver index.

[0020] Figure 4 Image showing the results of H&E staining of mouse colon.

[0021] Figure 5 This is a graph showing the results of the pathological tissue damage scoring of the mouse colon.

[0022] Figure 6 The figure shows the expression levels of cytokines in mouse colon tissue; in the figure, A represents the expression level of IL-4, B represents the expression level of IL-10, C represents the expression level of IL-6, and D represents the expression level of TNF-α.

Detailed Implementation Methods

[0023] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0024] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0025] Example 1:

[0026] The effects of monk fruit powder on the proliferation, screening, and metabolism of lactic acid bacteria are as follows:

[0027] The plate count method was used, with lactic acid bacteria as the experimental strain and milk as the fermentation substrate. Exogenous monk fruit powder was added to skim milk medium (experimental group) for fermentation to evaluate lactic acid bacteria enrichment. High-purity monk fruit glycoside V was used, and functional components were determined based on the viable count, acidity, and antioxidant results of the fermented milk. The effects of the combination of monk fruit and probiotic 431 on the organic acids in fermented milk were studied. The antioxidant functions of plant-based probiotic fermented milk were compared, and the effects of monk fruit powder combined with multiple probiotic strains on the quality of fermented milk were investigated. Titration was used to determine titration acidity, and high-performance liquid chromatography (HPLC) was used to determine organic acids. The antioxidant activity assay included: a. determination of ABTS free radical scavenging activity; b. determination of hydroxyl free radical scavenging activity; and c. determination of reducing power. Specific experimental results are as follows:

[0028] 1. Effects of monk fruit powder on the proliferation and function of lactic acid bacteria:

[0029] The effects of monk fruit powder on the enrichment of single probiotics and starter cultures are shown in Table 1.

[0030] Table 1. Effects of Luo Han Guo powder on the enrichment of single probiotics and starter cultures.

[0031]

[0032] Note: Data are presented as mean ± standard deviation (n = 3). Different uppercase letters in the same row indicate significant differences (P < 0.05); different lowercase letters in the same column indicate significant differences (P < 0.05); the same applies below. The strain 1301 used in this application is a patented strain of Huangshi Group Co., Ltd., with accession number CGMCC NO. 8545. The *Lactobacillus paracasei* L. casei 431 used in this application was purchased from Chr. Hansen, product number 706147.

[0033] As shown in Table 1, monk fruit powder has a beneficial effect on both probiotics and starter cultures, with the most significant effect on Lactobacillus paracasei L. casei 431. The research group will focus on revealing the mechanism by which monk fruit powder enhances the function of probiotic 431 and fermented milk base, providing theoretical support for the subsequent development of fermented milk by combining the three components.

[0034] 2. The effect of monk fruit powder components on the functional quality of probiotic 431 milk base

[0035] Using the content of glycoside V in monk fruit powder as a conversion standard, the effects of glycoside V, the main active ingredient of monk fruit, on the viable count and functional quality of probiotic 431 lactone were investigated, as shown in Table 2.

[0036] Table 2. Effects of Luo Han Guo components on the milk-based functional quality of probiotic 431

[0037]

[0038] Table 2 shows that, regarding viable bacteria count, the fruit powder group increased with increasing addition amount, while the pure glycoside V group showed the opposite trend, indicating that low-dose glycoside V promoted the proliferation of 431, and the increased bacteria count was the result of the synergistic effect of multiple components. Regarding ABTS scavenging rate, the glycoside V group significantly increased with increasing addition amount (P < 0.05), but low concentrations of fruit powder had the same ABTS scavenging ability as high concentrations of glycoside V, indicating that the antioxidant components in monk fruit powder include other active ingredients besides glycosides, and that glycoside V is not the main active ingredient. Regarding titratable acidity, the fruit powder group increased with increasing addition amount, corresponding to the viable bacteria count trend, suggesting that monk fruit powder may enhance functionality by affecting the acidic environment of fermented milk. Overall, adding monk fruit powder can improve the viable bacteria and antioxidant function of probiotic 431 alone in the milk base. The next step will be to evaluate the effect of the combination of fermentation agents on improving the antioxidant function of fermented milk.

[0039] 3. The effect of monk fruit powder on organic acids in fermented milk

[0040] Previous experiments showed that the acidity of fermented milk increased with the addition of monk fruit powder, indicating that monk fruit powder may improve the function of fermented milk by improving the acidic environment. Therefore, using pH 4.6 as the endpoint, the changes in organic acids in fermented milk were examined in different combinations of monk fruit powder, Lactobacillus paracasei L. casei 431, and starter culture. See Table 3 for details.

[0041] Table 3. Changes in organic acids in different fermentation combinations

[0042]

[0043] Lactic acid bacteria utilize fermentable sugars to produce more than a dozen organic acids, which can reflect the degree of fermentation in yogurt. Among them, lactic acid, citric acid, and malic acid mainly regulate the acidity of fermented milk, enhance flavor, improve digestibility and absorption, and inhibit harmful microorganisms. Table 3 shows that lactic acid levels increased with fermentation time in all groups. The levels in the monk fruit powder + 431 + starter culture group and the monk fruit powder + starter culture group were significantly higher than the control group at all fermentation points (P < 0.05), indicating that monk fruit powder significantly promotes the metabolism of lactic acid bacteria to produce the main organic acid, lactic acid. Citric acid showed a trend of first increasing and then decreasing in both monk fruit powder groups, indicating that the addition of monk fruit powder enhanced the symbiotic relationship of lactic acid bacteria, promoting their metabolism of citric acid. Furthermore, the citric acid content in the monk fruit powder group and the probiotic 431 group was significantly higher than the control group at the same fermentation stage (P < 0.05), also contributing to the promotion of lactic acid bacteria metabolism. Malic acid was only produced in the monk fruit powder group, contributing a unique and pleasant sour taste to the product. This indicates that the combination of monk fruit powder and 431 can significantly enrich the content and types of organic acids in fermented milk, and improve the overall quality of organic acids in fermented milk.

[0044] 4. The effect of monk fruit powder and probiotic combination on enhancing the antioxidant capacity of fermented milk base.

[0045] Table 4. Effects of plant-based probiotic combinations on enhancing the antioxidant capacity of fermented milk.

[0046]

[0047]

[0048] Table 4 shows that the hydroxyl radical scavenging effect of the monk fruit + 431 + fermentation agent group was significantly higher than that of other groups (P < 0.05), reaching 86.73%. In terms of reducing power, the monk fruit powder group was significantly better than the passion fruit peel group (P < 0.05). Overall, the addition of monk fruit powder and probiotic compound fermentation agent significantly improved the antioxidant capacity of fermented milk.

[0049] 5. The effect of monk fruit powder combined with multiple strains of probiotics on the quality of fermented milk.

[0050] To further improve the quality of fermented milk, we will study the effect of monk fruit powder combined with multiple probiotic strains on the quality of fermented milk. As mentioned above, monk fruit powder has a promoting effect on the growth of probiotics 431 and 1301. Now, we will combine the three with a fermentation agent to prepare fermented milk.

[0051] Table 5. Effects of Luo Han Guo powder combined with probiotics on the quality of fermented milk.

[0052]

[0053] Table 5 shows that the viable bacteria count and ABTS clearance rate of the fermented milk in the monk fruit powder group and the probiotic group were significantly higher than those in the unadded group, indicating that monk fruit powder and probiotics enhance the function of fermented milk. Among the monk fruit powder groups, the quality indicators of the monk fruit powder + 431 + 1301 + starter culture group were significantly higher than the other two groups, with each indicator reaching the highest value. Based on this, the process of monk fruit and probiotic flavored fermented milk will be optimized by combining monk fruit powder, probiotics 431 and 1301, and the starter culture.

[0054] Example 2:

[0055] This embodiment studies the optimization of the fermented buffalo milk product process with monk fruit flavor. The specific methods are as follows:

[0056] Fresh, antibiotic-free buffalo milk was used as the raw material. The protein content was standardized to 4.0%, and the mixture was heated to 60-65°C. A portion of this buffalo milk was then extracted and mixed with 0.05-0.2% monk fruit powder and 0-10% white sugar (by weight of the total raw milk). The mixture was stirred until completely dissolved and then combined with the remaining buffalo milk to obtain the base material. This base material was homogenized at 15-20 MPa and 60-70°C, heated to 90°C, and then kept at that temperature for 10 minutes for pasteurization. The pasteurized base material was cooled to 40-42°C. Then, *Lactobacillus paracasei* L. casei 431 and *Lactobacillus rhamnosus* hsryfm were used. Mix 1301 as probiotics and add fermentation agent at a weight percentage of 0.001-0.005% of the base material. Stir and mix evenly to obtain inoculated milk. Then, put the obtained inoculated milk into cups, seal them, and place them at a constant temperature of 38-44℃ for fermentation. Stop fermentation when the acidity reaches 70-75°T or the pH value is 4.4-4.8 to obtain the initial product of Luo Han Guo extract flavored fermented milk. After refrigeration at 2-6℃ for 24 hours, it is ready.

[0057] Table 6. Optimization of Single-Factor Experiments for Luo Han Guo Flavored Fermented Milk

[0058]

[0059]

[0060] Table 6 shows that the antioxidant capacity of fermented milk significantly increased with increasing monk fruit powder addition (P < 0.05), exhibiting dose-dependent behavior. Sensory evaluation showed an initial increase followed by a decrease, with the 0.2% group significantly lower than other groups. This indicates that low addition levels can compensate for the deficiency of white sugar, giving the fermented milk a more palatable sweet and sour taste. However, after exceeding 0.15%, the sensory score of the fermented milk significantly decreased, possibly due to a longer-lasting aftertaste causing discomfort. Overall, the texture decreased with increasing monk fruit powder addition. Except for hardness, cohesiveness, and adhesiveness, where the 0.2% group was significantly lower than other groups, there were no significant differences in other addition levels. This indicates that monk fruit powder has a certain impact on the texture of fermented milk, making the product softer. This is because the sweet glycosides repel casein molecules in the milk during the gelation process of lactic acid bacteria fermentation, thereby inhibiting the aggregation of casein and leading to a decrease in the texture indicators of fermented milk. Comprehensive analysis shows that fermented milk produced with monk fruit powder added at amounts of 0.05%, 0.10%, and 0.15% exhibits better overall quality.

[0061] Among different types of starter cultures, the fermented milk of group 904 showed significantly better ABTS removal rate and adhesion than X11 (P<0.05), and significantly better sensory evaluation, adhesion and cohesion than 1.0 (P<0.05). Considering all factors, starter culture 904 was selected for subsequent optimization.

[0062] In the probiotic ratio (431:1301), the ABTS scavenging rate of the fermented milk group with probiotic 1301 added was significantly higher than that of the group with probiotic 431 added alone (P<0.05). The cohesiveness, elasticity, chewiness, and water-holding capacity of the fermented milk in the compound group were higher than those in the single probiotic group, indicating that the synergistic fermentation of multiple strains can improve the antioxidant activity of fermented milk. In summary, fermented milk with probiotic ratios (431:1301) of 1:1, 1:2, and 2:1 has better overall quality.

[0063] Among the different amounts of starter culture 904, the ABTS scavenging rate of fermented milk in the 0.003%, 0.004%, and 0.005% groups was significantly higher than that in the 0.002% group, but the elasticity was significantly lower. The hardness and water-holding capacity of the 0.004% and 0.005% groups were higher than those of the other groups. In summary, fermented milk produced with 0.003%, 0.004%, and 0.005% of starter culture 904 showed better overall quality.

[0064] There was no significant difference in ABTS clearance rate among the different groups of fermented milk with different amounts of added white sugar. The sensory evaluation of fermented milk in the 4% and 6% groups was significantly higher than that in the 0% group (P<0.05). This is because mogrosides, as a sweetener, add a certain amount of sweetness to fermented milk. Adding an appropriate amount of white sugar can improve the taste of fermented milk. The preferred amount of added white sugar is 6% for subsequent optimization.

[0065] The starter cultures 904, 1.0, and X11 were all purchased from Chr. Hansen of Denmark.

[0066] Based on the single-factor experiments, considering sensory evaluation and ABTS clearance rate, the following four factors are closely related to solving production problems: the amount of monk fruit powder added, the amount of fermenting agent 904 added, and the probiotic ratio (431:1301). An orthogonal experiment was conducted using these factors, with the amount of white sugar added fixed at 6%. The experimental results are shown in Table 7.

[0067] Table 7. Results of the orthogonal experiment and range analysis of Luo Han Guo flavored fermented milk.

[0068]

[0069] As shown in Table 7, the range analysis of ABTS clearance rate shows that the order of importance of the factors is: amount of monk fruit powder added > probiotic ratio (431:1301) > amount of fermenting agent 904 added. The effect of the amount of monk fruit powder added is the most significant (P<0.05), while the effects of the other two factors are not significant. Considering the overall cost, the optimal condition is A3B1C1. The range analysis of sensory evaluation also shows that the order of importance of the factors is: amount of monk fruit powder added > amount of fermenting agent 904 added > probiotic ratio (431:1301). The effect of the amount of monk fruit powder added is the most significant (P<0.05), while the effects of the other two factors are not significant. Considering the overall cost, the optimal condition is A2B1C1. Considering that the taste of fermented milk is the primary concern for consumers when purchasing ordinary food, the optimal combination is A2B1C1, which means that the amount of monk fruit powder added is 0.1%, the amount of starter culture 904 added is 0.003%, and the ratio of probiotics (431:1301) is 1:1.

[0070] Based on the above conditions, this application adopted the optimal process A2B1C1 for production experiments. The results showed that the ABTS removal rate was 66.11±0.39%, and the sensory score was 86±7 points, which was higher than the combination in the orthogonal experiment. The product had a delicate taste and a combination of milky and monk fruit aroma. It is possible to develop a fermented milk product with excellent monk fruit flavor using monk fruit powder. The preferred process conditions are A2B1C1.

[0071] Example 3:

[0072] This example studies the quality changes of fermented dairy products made from monk fruit powder during storage:

[0073] The product is prepared using the optimal process for monk fruit powder flavored fermented milk, with a consistent probiotic ratio (431:1301 = 1:1), and the added probiotics are divided into three orders of magnitude for live bacteria count, namely 1×10⁻⁶. 8 CFU / g (Probiotic Additives Formula 1), 1×10 7 CFU / g (Probiotic Additives Formula 2), 1×106 The CFU / g (probiotic addition formula 3) was used as a control, and the quality changes of fermented milk under refrigeration conditions over 21 days were evaluated. See Table 8 for details.

[0074] Table 8. Quality Changes of Flavored Fermented Milk During Storage Period

[0075]

[0076] Note: Different lowercase letters: significant differences within the same column (P < 0.05); different uppercase letters: significant differences within the same row (P < 0.05).

[0077] Regarding the total viable count, bacterial activity was significantly affected by storage time and the type of fermented milk (P<0.05). Throughout the storage period, the viable count of fermented milk on day 1 ranged from 8.64 to 9.06 μg CFU / g, and decreased in all groups by day 21, attributed to substrate competition. Among the three different orders of magnitude formulations, there was no significant difference in the viable count of the two plant probiotic fermented milks on day 1 (P>0.05), indicating that the addition of probiotics had little effect on the total bacterial count at the fermentation endpoint. However, as storage time progressed, the viable count in the fermented milk was positively correlated with the amount of probiotics added, indicating a synergistic promoting effect between probiotics and starter cultures. Under the same storage time, except for Luo Han Guo powder fermented milk formula 3, which had a lower viable count than ordinary fermented milk on day 21, the viable counts of other Luo Han Guo powder probiotic fermented milk groups were higher than those of ordinary fermented milk. Moreover, many groups showed a significant increase in bacteria after day 7 (P < 0.05). This indicates that adding Luo Han Guo powder and probiotics during storage can maintain and enhance the activity of lactic acid bacteria in fermented milk. Other studies have shown that the addition of plant-based materials and probiotics is beneficial to increasing the viable count of fermented milk.

[0078] In the ABTS scavenging rate, the antioxidant activity of each group generally showed a decreasing and then increasing trend. After the 7th day, the monk fruit powder fermented milk was higher than that of ordinary fermented milk. This may be because the plant substrate itself is rich in antioxidants and can promote the proliferation and metabolism of lactic acid bacteria to produce more antioxidant peptides, which can maintain stable antioxidant activity. As the fermentation time progresses, the antioxidants are decomposed by lactic acid bacteria, resulting in a decrease in the antioxidant activity of the fermented milk. As the fermentation time progresses, the number of antioxidant peptides increases, which improves the antioxidant activity of the fermented milk.

[0079] In the titration of acidity, the acidity of each group of fermented milk increased with the extension of storage time, all showing post-acidification. However, the degree of post-acidification of Luo Han Guo powder fermented milk was close to that of ordinary fermented milk, indicating that it did not aggravate the degree of post-acidification.

[0080] In the sensory evaluation, the optimal tasting period for each group was 7-14 days. Under the same storage time, there was no significant difference in the sensory evaluation of the fermented milk among the groups (P>0.05), indicating that the unique flavor of the fermented milk imparted by the exogenous addition of monk fruit powder can be appreciated by the evaluators.

[0081] In summary, during the storage period, the monk fruit powder probiotic fermented milk exhibited significantly higher viable cell counts and antioxidant activity than ordinary fermented milk, while its sensory characteristics were not significantly different from ordinary fermented milk. This indicates that the fermented milk product demonstrates significant functionality and stable quality during storage, making it suitable for widespread application.

[0082] Example 4:

[0083] This embodiment studies the safety and functional verification of fermented dairy products fortified with probiotics using monk fruit powder in animal models.

[0084] Based on the conclusions of Examples 1-3, the monk fruit probiotic fermented milk exhibits excellent antioxidant capacity. Antioxidant capacity helps scavenge free radicals, reduce oxidative stress, and thus help suppress inflammatory responses. Literature studies have shown that monk fruit, *Lactobacillus paracasei*, and *Lactobacillus rhamnosus* possess anti-inflammatory effects due to their inherent components and properties. Therefore, this study investigated the alleviating effect and potential mechanism of monk fruit powder probiotic fermented milk on diglucan sulfate (DSS)-induced colitis in mice. The above-mentioned monk fruit powder flavored fermented milk (MH) and low-added monk fruit powder flavored fermented milk (ML) were selected, and a normal control group (NC), a DSS group (MC), and a positive control group (SC) were established. Details are as follows:

[0085] 1. Changes in the Disease Activity Index (DAI) in mice:

[0086] DAI is an important indicator of inflammation; the higher the value, the more severe the inflammation. Figure 1 As shown, the DAI score in the NC group was close to zero, the lowest value, indicating that the mice were in good health during the experiment. The DAI score in the MC group was higher than that in the NC group, indicating that the inflammation model was successfully established. The DAI of the four groups of mice drinking DSS showed a trend of first increasing and then decreasing, indicating that the mice developed inflammation but it was alleviated. Among them, the fermented milk in two groups showed an inflection point on day 8, which was 2 days earlier than the MC group and the value was lower, indicating that the fermented milk can effectively prevent the occurrence of DSS-induced colitis in mice.

[0087] 2. Analysis of colon length and weight / length in mice

[0088] DSS-induced colitis shortens the colon in mice; therefore, colon length is one indicator of the degree of inflammation in DSS-induced colitis. Figure 2As shown in Figure A, the normal colon length of mice in the NC group was 9.66 cm, while the MC group showed the most severe inflammation, with the shortest colon length at 8.77 cm. There was no significant difference in colon length between the ML and MC groups, nor between the MH and SC groups. The colon weight-to-length ratio was used to evaluate the degree of colonic edema in mice; a higher ratio indicated more severe edema and inflammation. Figure 2 As shown in B, the ratios in each group were significantly lower than those in the MC group (80.71, P < 0.05), indicating that the MC group mice had the most severe colonic edema. After treatment with the drug and fermented milk, the edema in the colitis mice was significantly improved. The monk fruit powder fermented milk (ML and MH groups) showed effects similar to those of the drug.

[0089] 3. Analysis of spleen weight and spleen index in mice

[0090] The spleen is an important immune organ. External stimuli activate and proliferate lymphocytes and NK cells in the spleen, leading to splenomegaly. The weight and size of the spleen can reflect the body's immune function to some extent. Figure 3 As shown in Figure A, the spleen weight in the MC group was significantly greater than that in other groups (P < 0.05), indicating the greatest weight gain and most severe inflammation. There were no significant differences between the ML and MH groups and the SC group (P > 0.05), while the NC group had a significantly lower spleen weight than the SC group (P < 0.05), suggesting that monk fruit powder fermented milk helps maintain a normal spleen weight. The spleen index is the ratio of spleen weight to body weight; a lower ratio indicates a weaker immune response to some extent. Figure 3 As shown in Figure B, compared with other groups, the spleen index of mice in the MC group showed a significant increasing trend (P<0.05), indicating that DSS may stimulate the proliferation of immune cells in the spleen of colitis mice, thereby activating the immune response pathway. The spleen index of mice in the group receiving the monk fruit powder probiotic fermented milk and the drug group (SC group) by gavage was significantly lower than that of the MC group (P<0.05), indicating that the fermented milk and the drug can improve splenomegaly in colitis mice. In conclusion, the fermented milk of this invention can alleviate splenic edema in colitis mice.

[0091] 4. Observation of H&E pathological staining and tissue damage scoring of mouse colon

[0092] The integrity of colonic tissue structure is a direct indicator of the severity of colonic inflammation. Tissue damage scoring criteria include inflammation, lesion depth, degree of crypt destruction, and lesion extent. After H&E staining, pathological changes were observed and photographed under a microscope. Each group of mice was stained with H&E at a 40 μm microscope. Figure 4 A1- Figure 4 E1) and 100μm ( Figure 4 A2- Figure 4 E2, Figure 4 A3- Figure 4 E3) Two methods were used to observe the whole and the parts, and the results were as follows: Figure 4 As shown in the figure, the colonic epithelial cells in the NC group are intact, with regular crypt structures, goblet cells, complete mucosal epithelium, and neatly arranged glands. The tissue damage score is 0.25. In contrast, the epithelial structure in the MC group is severely damaged, with the lesion area reaching approximately 10% (indicated by the black arrows in B2 and B3), and the tissue damage score is 3.25. Figure 5 The SC group showed significantly higher levels of improvement than the NC group (P < 0.05). Both the SC and fermented milk groups showed varying degrees of relief from colitis symptoms, and their tissue damage scores were significantly different from those of the MC group (P < 0.01). The lesion relief in the ML and MH groups was similar to that in the SC group; see details below. Figure 4 The corresponding arrows indicate this. In summary, the fermented milk of this invention can alleviate colitis in mice.

[0093] The above conclusions indicate that monk fruit probiotic fermented milk can significantly and effectively prevent colonic shortening in mice with colitis, alleviate splenic edema, significantly alleviate tissue damage, and effectively maintain the integrity of the mucosal structure. The monk fruit powder probiotic fermented milk of this invention exhibits effects equivalent to this drug.

[0094] 5. Changes in inflammatory cytokines in the mouse colon

[0095] Cytokines play a crucial role in the pathogenesis of colitis. An imbalance between the secretion of anti-inflammatory cytokines and pro-inflammatory cytokines, resulting from a decrease in the secretion of these two types of factors, is one of the pathogenic mechanisms of inflammation. IL-4 and IL-10 are effective anti-inflammatory cytokines that can inhibit antigen transduction and the release of pro-inflammatory cytokines, thereby alleviating mucosal inflammation. IL-6 and TNF-α are pro-inflammatory cytokines that can coordinate the inflammatory response by activating various cells.

[0096] like Figure 6 As shown in A and B, although the expression level of IL-4 in the MC group was not significantly different from that in the NC group, it increased, which is speculated to be due to the activation of the non-specific immune system by intestinal inflammation in mice, thus regulating its function. The IL-10 content in the SC, ML, and MH groups was significantly higher than that in the MC group (P<0.05), showing a marked upregulation. This indicates that Luo Han Guo powder fermented milk can increase the secretion of anti-inflammatory cytokines, thereby alleviating intestinal inflammation in mice and improving their intestinal defense capabilities.

[0097] like Figure 6As shown in C and D, compared with the NC group, the MC group mice showed significantly increased levels of pro-inflammatory factors IL-6 and TNF-α (P<0.05), indicating that intestinal inflammation induced the secretion of multiple inflammatory factors. Compared with the MC group, the levels of the two pro-inflammatory factors in other groups were significantly decreased (P<0.05), indicating that drug and fermented milk intervention can reduce the secretion of inflammatory cytokines and effectively alleviate intestinal inflammation in mice. Specifically, there was no significant difference in IL-6 expression among the NC, ML, and MH groups (P<0.05); there was no significant difference in TNF-α levels among the NC, SC, and ML groups, and no significant difference in the regulatory level between the ML and MH groups (P>0.05). In summary, fermented milk intervention in colitis mice can regulate the secretion of pro-inflammatory factors to varying degrees, alleviating intestinal damage in mice. The effect of Luo Han Guo fermented milk is close to that of a positive drug, which is corroborated by the results of other indicators.

[0098] The above indicates that monk fruit powder fermented milk can alleviate intestinal inflammation by regulating anti-inflammatory and pro-inflammatory factors.

[0099] In summary, this invention uses buffalo milk, monk fruit powder, and compound probiotics to prepare flavored dairy products. The 1301 strain of the compound probiotics is a probiotic strain owned by the applicant. During the research on the preparation process, it was found that monk fruit powder and compound probiotics have a good synergistic effect. The monk fruit powder fermented dairy product prepared by this combination has good functionality. Furthermore, animal experiments showed that this product has a good alleviating effect on colitis in mice.

[0100] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for preparing a fermented milk with a Momordica grosvenori flavor, characterized by, The preparation method is as follows: after fresh cow milk is heated as raw milk, monk fruit powder and white granulated sugar are added, and after being uniformly mixed, a fermentation base is obtained; after the fermentation base is homogenized, sterilized and cooled, probiotics and leavening agent are added and mixed, constant temperature fermentation and post-ripening are carried out, and the fermented milk is obtained; The probiotic bacteria are obtained by mixing Lactobacillus paracasei (CNCM Lacticaseibacillus paracasei ) L. casei 431 and Lactobacillus rhamnosus (CNCM Lactobacillus rhamnosus ) hsryfm 1301; the preservation number of the Lactobacillus rhamnosus (CNCM Lactobacillus rhamnosus ) hsryfm 1301 is CGMCC NO. 8545. The Lactobacillus paracasei ( Lacticaseibacillus paracasei L. casei 431 and Lactobacillus rhamnosus ( Lactobacillus rhamnosus The mass ratio of hsryfm 1301 is 1-2:1-2. The amount of monk fruit powder added is 0.1%-0.15% of the mass of the fermentation base, the amount of white sugar added is 0-7% of the mass of the fermentation base, and the amount of fermentation agent added is 0.003-0.005% of the mass of the base. The raw milk is buffalo milk.

2. The method for preparing monk fruit flavored fermented milk according to claim 1, characterized in that, the Lactobacillus paracasei (L. paracasei) Lacticaseibacillus paracasei ) L. casei 431 and Lactobacillus rhamnosus (L. rhamnosus) Lactobacillus rhamnosus The mass ratio of L. paracasei (L. paracasei) 431 and L. rhamnosus (L. rhamnosus) hsryfm 1301 is 1:1, the added amount of monk fruit powder is 0.1% of the mass of the fermentation base, and the starter is starter 904, and the added amount is 0.003% of the mass of the fermentation base.

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