Waxberry extract, composite functional fermented milk as well as preparation method and application of waxberry extract and composite functional fermented milk

Abstract

The invention relates to the technical field of food processing, and discloses a waxberry extract with an anti-inflammatory function as well as a preparation method and application thereof. The waxberry extract is a waxberry pulp ethanol extract, or at least one of a petroleum ether extract, an ethyl acetate extract, an n-butyl alcohol extract or an aqueous extract of the waxberry pulp ethanol extract, or any mixture of the petroleum ether extract, the ethyl acetate extract, the n-butyl alcohol extract and the aqueous extract. The waxberry extract can be used for preparing medicines, health-care products or foods for assisting in resisting inflammation, assisting in treating or relieving colitis injury or assisting in healing ulcerative colitis injury. The invention also provides a functional dairy product, especially fermented milk, which contains the waxberry extract, also has a significant anti-inflammatory function, and can effectively relieve colitis symptoms and intestinal injury and protect intestinal health.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and relates to a bayberry extract with anti-inflammatory function, a preparation method and application, and a dairy product containing the ingredient, in particular to a composite functional fermented milk. Background Art

[0002] Inflammatory bowel disease (IBD) presents with diarrhea, weight loss, abdominal pain, and bloody stools as its primary clinical manifestations, and its global incidence is significantly increasing. Current clinical treatment strategies primarily include medication, nutritional supplementation, and surgical intervention, but a complete cure remains elusive. Recent studies have demonstrated that dietary intervention plays an important role in regulating the microbiome, metabolome, and intestinal immunity, making it a promising adjunctive therapy for a variety of chronic diseases.

[0003] With rising health awareness, the consumption of functional foods has become a widespread trend. Dairy products, in particular, have garnered significant attention as ideal carriers of nutrients and probiotic microorganisms. Studies have found that functional fermented dairy products can reduce tumor burden and alleviate inflammatory bowel damage in mice with colorectal cancer, promoting beneficial effects on the host. The mechanisms may involve immune regulation and the maintenance of intestinal microbiota homeostasis. Consequently, the addition of functional substances to fermented dairy foods has been proposed as an effective option for improving health benefits.

[0004] Bayberry (Myrica rubra Sieb.et Zucc), a subtropical evergreen tree in the Myricaceae family, is native to China and Southeast Asia. It boasts a rich variety of species and a long history of cultivation dating back two thousand years. Bayberry is not only beloved for its unique flavor, but also attracts significant attention in traditional medicine and modern research for its rich nutritional value and medicinal properties. The folk custom of infusing bayberry into wine is widely recognized for its health benefits, which not only nourishes the stomach and spleen, promotes fluid production and quenches thirst, but also treats diarrhea, improves digestive issues, lowers blood lipids, and eliminates dampness. The traditional medical text "Compendium of Materia Medica" records its benefits as "promoting fluid production, quenching thirst, regulating the five internal organs, and cleansing the stomach and intestines." Modern research indicates that bayberry is rich in bioactive compounds such as phenolic acids, anthocyanins, and flavonols, which may have various biological functions, including improving metabolic disorders and enhancing immune defense. Furthermore, bayberry cultivation and processing have generated significant economic benefits in many regions, becoming an important source of income for farmers. As research deepens, the application potential of bayberry will be further explored, bringing more benefits to human health.

[0005] During the fermentation process of fermented milk, the lactose in the milk is converted into lactic acid by microorganisms, and at the same time a variety of metabolites are produced, such as peptides, organic acids, vitamins and aromatic substances, which give the fermented milk a unique flavor, texture and nutritional value. It has the effects of moisturizing the skin, promoting fluid production and quenching thirst, replenishing the body and stimulating the appetite, moisturizing the intestines and promoting bowel movements, and promoting digestion.

[0006] Adding functional food ingredients to fermented milk imparts additional benefits beyond those of standard fermented milk, such as anti-inflammatory and metabolic improvement. This not only preserves traditional fermentation techniques but also actively responds to modern health needs. However, there is currently a market gap for such products. Summary of the Invention

[0007] The present invention aims to provide a plant extract with anti-inflammatory function, as well as a preparation method and use.

[0008] One purpose of the present invention is to use the above-mentioned plant extracts to prepare dairy products, especially fermented dairy products, to provide a fermented milk with anti-inflammatory function, and to provide a method for preparing the fermented milk.

[0009] The technical solutions of the present invention are as follows:

[0010] A bayberry extract with anti-inflammatory function, selected from at least one of the following or any mixture:

[0011] a. Bayberry pulp ethanol extract;

[0012] b. petroleum ether extract of ethanol extract of bayberry pulp;

[0013] c. ethyl acetate extract of ethanol extract of bayberry pulp;

[0014] d. n-butanol extract of ethanol extract of bayberry pulp;

[0015] e. Aqueous extract of ethanol extract of bayberry pulp.

[0016] The method for preparing the bayberry pulp ethanol extract comprises at least one of the following steps:

[0017] (1) mixing the dried bayberry pulp powder with an ethanol solution, performing ultrasonic extraction, taking the supernatant and drying it to obtain the bayberry ethanol extract;

[0018] (2) suspending the bayberry ethanol extract in distilled water and extracting with at least one organic solvent selected from petroleum ether, ethyl acetate, and n-butanol; collecting each organic solvent extraction section and the aqueous phase to obtain a petroleum ether extract, an ethyl acetate extract, an n-butanol extract, and a water extract of the bayberry pulp ethanol extract.

[0019] In step (1), the solid-liquid ratio of the dried bayberry pulp powder to the ethanol solution is 1:20-50; in a preferred embodiment of the present invention, it is 1:40. The unit of the solid-liquid ratio is g / mL.

[0020] Preferably, the concentration of the ethanol solution is 70%-100% (volume ratio).

[0021] More preferably, the concentration of the ethanol solution is 95% (volume ratio).

[0022] The preferred conditions for ultrasonic extraction are: 50-100 Hz extraction for 3-15 minutes; further, the power of ultrasonic extraction is 20-100W.

[0023] More preferably, the ultrasonic extraction conditions are: 50-100 Hz extraction for 3-15 minutes; further, the ultrasonic extraction power is 20-100W.

[0024] In a preferred embodiment of the present invention, ultrasonic extraction is performed at a power of 20 W and a frequency of 60 Hz for 5-10 minutes.

[0025] In one embodiment of the present invention, the bayberry is the water chestnut bayberry from Xianju County, Taizhou City, Zhejiang Province.

[0026] In step (2), each organic solvent extraction section and the aqueous phase are collected and dried to obtain a petroleum ether extract, an ethyl acetate extract, a n-butanol extract and a water extract of the bayberry pulp ethanol extract.

[0027] Preferably, the number of extractions with each organic solvent is 1-3 times, and the volume ratio of each extraction with the aqueous phase is 1:0.8-1.2; further preferably, the number of extractions with each organic solvent is 3 times, and the volume ratio of each extraction with the aqueous phase is 1:1.

[0028] Preferably, the extraction is performed with n-butanol.

[0029] In one embodiment of the present invention, petroleum ether, ethyl acetate and n-butanol are used for extraction in sequence, and the n-butanol extraction section is dried to obtain the bayberry extract.

[0030] The bayberry extract has an anti-inflammatory effect, can restore weight loss and shortened colon length caused by acute colitis, can alleviate colon inflammation and colon tissue pathological damage caused by acute colitis, and can repair crypt tissue to a certain extent.

[0031] The bayberry extract can be used to prepare drugs, health products or foods for auxiliary anti-inflammatory effects; or, the bayberry extract can be used to prepare drugs, health products or foods for auxiliary treatment or relief of colitis damage, or for preparing drugs, health products or foods for auxiliary healing of ulcerative colitis damage.

[0032] In a preferred embodiment of the present invention, the bayberry extract is used to prepare dairy products, especially fermented milk.

[0033] Another embodiment of the present invention is a dairy product containing the aforementioned bayberry extract. Preferably, the dairy product is fermented milk.

[0034] Preferably, the bayberry extract is the n-butanol extract of bayberry pulp ethanol extract.

[0035] Based on 100 parts of milk as the raw material, the amount of bayberry extract added is 0.1-1.5 parts, preferably 0.25-1 part. The parts mentioned are parts by weight.

[0036] Based on 100 parts of milk as raw material, dairy products also contain the following ingredients:

[0037] 1) 0-7 parts of sweetener, and / or,

[0038] 2) 0-1 part, preferably 0.3-0.5 part, of a stabilizer, and / or

[0039] 3) 0-0.5 parts of leavening agent, preferably 0.1-0.5 parts, more preferably 0.1-0.2 parts.

[0040] The cow's milk is conventional milk in the field, free of antibiotics and bactericides, and has a certain protein and fat content; it includes raw milk, pasteurized milk, ambient temperature milk, or reconstituted milk. The raw milk is conventional raw milk in the field, i.e., the mammary gland secretions of healthy cows, preferably pre-treated raw milk. The raw milk meets the requirements of GB-19301, "National Food Safety Standard - Raw Milk."

[0041] The starter is a direct-injection yogurt starter containing Lactobacillus bulgaricus and Streptococcus thermophilus.

[0042] Preferably, the stabilizer is low-ester pectin, which is a soluble dietary fiber and is more easily absorbed by the human body.

[0043] The sweetener is sugar or a sugar substitute; preferably, the sugar is sucrose.

[0044] The preparation method of the dairy product comprises the following steps:

[0045] a. Mix pasteurized milk with the starter and bayberry extract;

[0046] b. First fermentation at 40-45℃ for 6-10h;

[0047] c. Fermentation at 2-6℃ for 10-20h.

[0048] After the post-fermentation, the milk is broken and aseptically filled, and the fermented milk is refrigerated and stored. The packaging specifications of the fermented milk can be 180-220g / bottle.

[0049] In step a, a sweetener and / or a stabilizer is added to the cow's milk to prepare mixed milk, which is then pasteurized.

[0050] Preferably, the pasteurization method in step a is: heating the raw material to 80-90° C. and maintaining it for 10-20 minutes; more preferably, heating it to 85° C. and maintaining it for 15 minutes.

[0051] Preferably, in step b, the fermentation temperature is 42° C. and the fermentation time is 8 h.

[0052] Preferably, in step c, the post-fermentation temperature is 4° C. and the time is 15 h.

[0053] The obtained functional fermented milk also has significant anti-inflammatory effects, reduces mucosal permeability and alleviates barrier damage, has the effect of improving epithelial barrier function, can effectively relieve the symptoms of colitis and intestinal damage, and has a positive effect on improving colitis, thereby effectively protecting intestinal health.

[0054] Compared with the prior art, the present invention has the following advantages:

[0055] 1. The present invention obtains bayberry extract, which has significant anti-inflammatory effects after animal experiments. It can reduce and repair intestinal mucosal / epithelial barrier damage, reduce intestinal permeability, and is suitable for preventing or assisting in the treatment of inflammatory bowel disease.

[0056] 3. The fermented milk prepared with bayberry extract of the present invention has the characteristics of uniform and delicate tissue morphology, rich nutrition and taste, can reduce the level of pro-inflammatory factors, and has anti-inflammatory function.

[0057] This invention leverages the biological activity and intestinal protective effects of bayberry extract by adding it to fermented milk. The resulting novel functional product retains the advantages of traditional fermented milk while further enhancing its functionality to meet the health needs of diverse populations. Beyond conventional fermented milk, it offers benefits such as anti-inflammatory, improved metabolic disorders, repair of the intestinal barrier and intestinal damage, and protection of intestinal health. This product fills a current gap in this category and provides an important theoretical basis for the adjunctive treatment of ulcerative colitis and the development of functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Evaluation of the effects of different fractions of bayberry extract on body weight and colon length in mice with acute UC, (A) is mouse body weight, (B) is colon length.

[0059] Figure 2Activity evaluation of different fractions of bayberry extract in acute UC mice, (A) inflammatory factors, (B) pathological analysis.

[0060] Figure 3 This study aimed to evaluate the sensory properties of fermented milk with different amounts of bayberry extract (NBA).

[0061] Figure 4 Evaluation of the effects of fermented milk supplemented with bayberry extract on the body weight and colon length of mice with acute UC. (A) is the body weight of mice, and (B) is the colon length.

[0062] Figure 5 The anti-inflammatory effect of fermented milk supplemented with bayberry extract on acute UC mice. (A) is the inflammatory factor, and (B) is the intestinal barrier. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below with reference to the accompanying drawings and specific examples. The specific examples are carried out according to the technical solution of the present invention, and provide detailed implementation methods and operating procedures. However, the scope of protection of the present invention is not limited to the following examples.

[0064] The raw materials used in the embodiments of the present invention are all commercially available products.

[0065] The bayberry is water chestnut bayberry from Xianju County, Taizhou City, Zhejiang Province; the stabilizer is low-ester pectin; and the fermentation agent is a bacterial powder preparation containing thermophilic Streptococcus and Bulgarian Lactobacillus.

[0066] Example 1

[0067] The preparation steps of bayberry extract are as follows:

[0068] (1) The freeze-dried powder of bayberry pulp was mixed with 95% ethanol solution at a solid-liquid ratio of 1:40 (g / mL), and ultrasonic extraction was performed at a power of 30 W and a frequency of 60 kHz. The supernatant was collected and concentrated by rotary evaporation to obtain bayberry ethanol extract (EA).

[0069] (2) The bayberry ethanol extract was suspended in an appropriate amount of distilled water and extracted with equal volumes of petroleum ether, ethyl acetate, and n-butanol in sequence. Each solvent was extracted three times, and the extracts of the same solvent were combined to obtain four segments: petroleum ether layer, ethyl acetate layer, n-butanol layer, and water layer. The obtained segmented materials were concentrated by rotary evaporation to obtain petroleum ether extract (PE), ethyl acetate extract (EAC), n-butanol extract (NBA), and water extract (W), which were stored separately for later use.

[0070] Animal experiments were conducted to study the anti-inflammatory effects of different components of bayberry extract on mice with acute colitis (UC).

[0071] Experimental Animals: Male C57 / BL6J mice (18-20 g, 6 weeks old) were purchased from Spefox Biotechnology Co., Ltd. (Shanghai, China). After adaptive feeding, the mice were randomly divided into 7 groups (n=6): normal group (CK), model group (DSS), ethanol extract group (DSS+EA), petroleum ether group (DSS+PE), ethyl acetate group (DSS+EAC), n-butanol group (DSS+NBA), and water group (DSS+W). Except for the CK group, mice in the other groups were gavage-treated with the corresponding extract for 14 days. On the 15th day, they were anesthetized and sacrificed. Blood, colon contents, and tissues were collected and stored at -80°C for further analysis. The experimental results are as follows:

[0072] Effects of different fractions of bayberry extract on DSS (dextran sulfate sodium salt)-induced UC mice Figure 1 As shown. By evaluating the general symptoms of colitis, including weight changes and colon length, we found that the weight of mice in the DSS-treated group was significantly reduced (p < 0.05), indicating that the UC model was successfully established. However, after treatment with different segmented materials of Bayberry (EA, PE, EAC, NBA and W groups), the weight of mice was significantly restored ( Figure 1 A). In addition, the colon length of mice in the DSS group was significantly shorter than that in the CK group (p<0.0001), while the colon length of the groups treated with Bayberry segmented substances was significantly restored, with the NBA group showing the most significant effect ( Figure 1 B).

[0073] Evaluation of the activity of different fractions of bayberry extract in acute UC mice Figure 2 shown. Figure 2 (A) and (B) are inflammatory factors and pathological analysis, respectively.

[0074] The inflammatory parameters were further analyzed by detecting changes in TNF-α and IL-6 in the serum of mice using pro-inflammatory cytokines. The study found that compared with CK, the levels of TNF-α and IL-6 in mice in the DSS group were significantly increased (p<0.01). These inflammatory mediators are closely related to intestinal mucosal damage, bleeding, and edema. However, intervention in the EA, PE, EAC, NBA, and W groups significantly reduced the levels of these inflammatory factors, among which the effect of the NBA group was the most significant ( Figure 2 A). In addition, colon tissue was histopathologically evaluated ( Figure 2 B), Compared with the CK group, the colon tissue of mice in the DSS group showed significant pathological damage. However, intervention in the EA, PE, EAC, NBA, and W groups significantly alleviated these damages and repaired the crypt structure to a certain extent, indicating that different components of the ethanol extract of bayberry can effectively alleviate the tissue pathological damage of experimental colitis, among which the effect of the NBA group was the most significant.

[0075] Example 2 Preparation of fermented milk

[0076] Raw materials: 100 parts of cow's milk, 6 parts of sweetener, 0.3 parts of stabilizer, 0.15 parts of fermentation agent; and 1 part of bayberry extract NBA of Example 1. The amount of bayberry extract added is 1% based on the cow's milk.

[0077] The preparation steps of fermented milk are as follows:

[0078] (1) 100 parts of milk, 6 parts of sweetener, and 0.3 parts of stabilizer were mixed evenly, stirred, and the mixed milk was pasteurized at 85°C for 15 minutes;

[0079] (2) The material of step (1) was cooled to 42°C, 0.15 parts of the starter culture and 1 part of the NBA of Example 1 were added, the mixture was shaken well, and the mixture was placed in an incubator for constant temperature culture and fermented for 8 hours; then, a post-fermentation was carried out at 4°C for 15 hours;

[0080] (3) The material of step (2) is stirred to break the emulsion and then aseptically filled to obtain the functional fermented milk; the packaging specification can be 180 to 220 g / bottle and refrigerated.

[0081] Example 3 Preparation of fermented milk (2)

[0082] The raw materials and fermentation method are the same as those in Example 2, except that the added amounts of Bayberry extract NBA are 0%, 0.25%, 0.5%, 1.5% and 3% based on milk.

[0083] Sensory evaluation of fermented milk with different NBA addition amounts in Examples 2 and 3 Figure 3 As shown in the figure, the effects of different addition amounts on the sensory perception of fermented milk were analyzed. As can be seen from the figure, when the addition amount of fermented milk was 0%, 0.25% and 0.5%, the overall score was greater than 80, indicating that the fermented milk with these addition amounts was more in line with the taste preferences of consumers; when the addition amount was increased to 1%, the sensory score was 79.9, although the taste was reduced, it was still acceptable; when the addition amount was 1.5% and 3%, the overall score was lower than 80, showing a significant decline in taste and a decrease in consumer preference. Further viable bacteria count determination results showed that the number of lactic acid bacteria in fermented milk with 0%, 0.25%, 0.5% and 1% addition amounts was greater than 1×10 6 CFU / g(mL), in line with national standards.

[0084] The fermented milk can be prepared into various fermented dairy products according to actual conditions. All dairy products prepared using the fermented milk of the present invention fall within the scope of protection of the present invention.

[0085] Example 4 Effect of fermented milk on the anti-inflammatory effect of mice with acute colitis

[0086] The anti-inflammatory effects of fermented milk (0-FD and 1-FD) with 0% and 1% NBA added prepared in Example 2 on mice with acute colitis were studied.

[0087] Experimental Animals: Male C57 / BL6J mice (18-20 g, 6 weeks old) were purchased from Spefox Biotechnology Co., Ltd. (Shanghai, China). After adaptive feeding, the mice were randomly divided into four groups (n=6): a normal group (CK), a model group (DSS), a fermented milk group (DSS+0-FD), and a fermented milk group supplemented with 1% NBA (DSS+1-FD). Mice in the DSS+0-FD and DSS+1-FD groups were gavage-fed with the corresponding fermented milk for 14 days. On the 15th day, they were anesthetized and sacrificed. Blood, colon contents, and tissues were collected and stored at -80°C for further analysis.

[0088] The results are as follows Figure 4 Compared with the CK group, DSS treatment resulted in a significant reduction in the length of the colon in mice (p<0.001). Figure 4 (B) However, after 1-FD intervention, the body weight and colon length of mice were significantly restored, as shown in Figure 4 (A) and (B). Notably, 1-FD intervention significantly prolonged the colon length of mice (p < 0.05), indicating that 1-FD has a positive effect on improving colitis.

[0089] In order to further study the improvement effect of fermented milk with different amounts of NBA on DSS-induced UC, its anti-inflammatory effect on acute UC mice was detected. Figure 5 Changes in TNF-α and IL-6 were detected in the colon tissue of mice. Figure 5 As shown in (A), compared with the CK group, the expression levels of TNF-α and IL-6 in the colon tissue of mice in the DSS-treated group were significantly increased (p<0.05). Excessive secretion of these pro-inflammatory factors can lead to pathological changes such as intestinal mucosal barrier damage, tissue bleeding and edema, thereby aggravating intestinal inflammatory response and functional disorder. However, after 1-FD intervention, the levels of TNF-α and IL-6 in the colon tissue of mice were significantly reduced (p<0.05). In order to further study the effect of 1-FD on parameters related to epithelial barrier function, we analyzed the secretion of IgA and tight junction protein ZO-1, as well as intestinal permeability. The results are shown in Figure 5 (B) shows that 1-FD intervention reduced mucosal permeability and alleviated barrier damage. These results indicate that 1-FD has the effect of improving epithelial barrier function, thereby effectively protecting intestinal health.

[0090] The above embodiments are used to illustrate the present invention and are not used to limit the scope of protection of the present invention. Based on the common sense in this field, the above preferred conditions can be combined arbitrarily.

Claims

1. A bayberry extract with anti-inflammatory function, characterized in that: Selected from at least one of the following or any mixture: a. Bayberry pulp ethanol extract; b. petroleum ether extract of ethanol extract of bayberry pulp; c. ethyl acetate extract of ethanol extract of bayberry pulp; d. n-butanol extract of ethanol extract of bayberry pulp; e. Aqueous extract of ethanol extract of bayberry pulp.

2. The method for preparing the bayberry extract according to claim 1, wherein Includes at least one of the following steps: (1) mixing bayberry pulp powder with ethanol solution, performing ultrasonic extraction, taking the supernatant and drying it to obtain bayberry ethanol extract; or (2) suspending the bayberry ethanol extract in distilled water and extracting with at least one organic solvent selected from petroleum ether, ethyl acetate, and n-butanol; collecting each organic solvent extraction section and the aqueous phase to obtain a petroleum ether extract, an ethyl acetate extract, an n-butanol extract, and a water extract of the bayberry pulp ethanol extract.

3. The preparation method according to claim 2, characterized in that The concentration of the ethanol solution in step (1) is 70%-100%; the ultrasonic extraction conditions are: 50-100 Hz extraction for 3-15 minutes.

4. The preparation method according to claim 2, characterized in that In step (2), n-butanol is used for extraction.

5. Use of the bayberry extract according to claim 1 in the preparation of medicines, health products or foods having the following functions: (1) auxiliary anti-inflammatory; or (2) assist in the treatment or relief of colitis damage; or (3) Assist in the healing of ulcerative colitis lesions.

6. A dairy product, characterized in that Contains the bayberry extract according to claim 1 or 2.

7. The dairy product according to claim 6, characterized in that Based on 100 parts of milk as the raw material, the added amount of bayberry extract is 0.1-1.5 parts.

8. The dairy product according to claim 6 or 7, characterized in that For fermented milk.

9. The method for preparing the dairy product according to any one of claims 6 to 8, characterized in that: The following steps are involved: a. Mix pasteurized milk with the starter and bayberry extract; b. First fermentation at 40-45℃ for 6-10h; c. Fermentation at 2-6℃ for 10-20h.

10. The preparation method according to claim 9, characterized in that In step a, a sweetener and / or a stabilizer is added to the cow's milk to prepare mixed milk, which is then pasteurized.

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