Fermented sea-buckthorn beverage as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fermented food, and particularly relates to a fermented sea-buckthorn beverage as well as a preparation method and application thereof. The fermented hippophae rhamnoides beverage prepared by fermenting the hippophae rhamnoides juice with the volume percentage content of 10%-40% by using the lactobacillus bulgaricus LN6 has obvious capabilities of reducing blood sugar and regulating and controlling blood fat metabolism. Animal experiments show that the fermented sea-buckthorn beverage can improve the symptoms of type II diabetes mellitus, improve the blood sugar level of mice, reduce the serum insulin level, improve the glucagon-like peptide-1 (GLP-1) level and reduce insulin resistance when being used for intragastric administration of type II diabetes mellitus mice. Meanwhile, the fermented hippophae rhamnoides fruit beverage can also recover liver tissue damage and improve abnormal blood lipid metabolism.

Description

Technical Field

[0001] The present invention belongs to the technical field of fermented foods, and particularly relates to a fermented seabuckthorn beverage, a preparation method thereof, and an application thereof. Background Art

[0002] Diabetes is a metabolic disease characterized by hyperglycemia caused by impaired insulin secretion or insensitivity of target organs to it, which imposes a serious burden on individuals, families, and society. Among them, type II diabetes is the most typical and the most prevalent type of diabetes. Type II diabetes, also known as "non-insulin-dependent diabetes", is a complex metabolic disease characterized by hyperglycemia, and its formation is related to multiple factors such as genes, unhealthy diet, and lack of exercise. Drug treatment and dietary intervention are the main means for the treatment and prevention of type II diabetes at present. However, although the existing drugs for treating type II diabetes have a certain effect on maintaining blood glucose in type II diabetes patients, they also have relatively obvious side effects and adverse reactions. For example, biguanide drugs can cause gastrointestinal symptoms such as dry mouth, bitter taste, anorexia, nausea, and vomiting, and diarrhea may also occur. In severe cases, lactic acidosis may be induced. Dietary intervention is a very effective means to control or prevent type II diabetes.

[0003] Seabuckthorn is the fruit of Hippophae rhamnoides L. of the genus Hippophae in the Elaeagnaceae family, and contains various nutritional components such as essential amino acids, organic acids, vitamins, oils, unsaturated fatty acids, and inorganic salts, among which the vitamin content is rich. "Dictionary of Traditional Chinese Medicine" records: "Seabuckthorn fruit has the effects of promoting blood circulation to remove blood stasis, resolving phlegm and widening the chest, strengthening the spleen and stomach, promoting the production of body fluid and quenching thirst, and clearing heat and stopping diarrhea." Although seabuckthorn has many benefits, due to its high water content (about 70%), it is not resistant to storage at room temperature, is easily damaged mechanically and infected by microorganisms, and the taste of seabuckthorn fruit is sour and astringent and is not suitable for fresh consumption. Therefore, it is very necessary to further process seabuckthorn.

[0004] Probiotics refer to active microorganisms beneficial to the host, and have the effects of regulating the host's immunity, improving the intestinal microecological balance, and improving cardiovascular and cerebrovascular diseases. Dairy products represented by yogurt and pickles are currently the fields where lactic acid bacteria are more maturely applied. In recent years, with the development of deep processing technology of fruits and vegetables, lactic acid bacteria-fermented fruit and vegetable juices have gradually become a research hotspot. Although there are related studies on preparing seabuckthorn juice by fermenting probiotics in the prior art, there are few reports on preparing seabuckthorn fermented products by fermenting lactic acid bacteria, especially seabuckthorn fermented products with hypoglycemic effects. Summary of the Invention

[0005] The purpose of the present invention is to provide a fermented seabuckthorn beverage, a preparation method thereof, and an application thereof. The fermented seabuckthorn beverage can effectively play a role in lowering blood sugar and regulating blood lipid metabolism, and can be used as an effective dietary intervention means for diabetes.

[0006] The present invention provides a method for preparing a fermented sea buckthorn beverage, comprising the following steps:

[0007] Sterilize sea buckthorn juice with a volume percentage of 10% to 40% to obtain sterilized sea buckthorn juice;

[0008] Inoculate the sterilized sea buckthorn juice with Lactobacillus caucasicus LN6 bacterial solution and then carry out constant-temperature fermentation to obtain the fermented sea buckthorn beverage.

[0009] Preferably, the sea buckthorn juice comprises sea buckthorn juice with a volume percentage of 10% to 20%.

[0010] Preferably, the concentration of the Lactobacillus caucasicus LN6 bacterial solution is 10 8 ~10 9 CFU / mL.

[0011] Preferably, the inoculation amount of the Lactobacillus caucasicus LN6 bacterial solution is 1% to 5%.

[0012] Preferably, the temperature of the constant-temperature fermentation is 35 to 37 °C, and the time is 20 to 28 h.

[0013] Preferably, the sterilization method is pasteurization, the temperature of the pasteurization is 80 to 85 °C, and the time is 15 to 20 min.

[0014] The present invention also provides a fermented sea buckthorn beverage prepared by using the preparation method described in the above technical solution.

[0015] The present invention also provides the application of the fermented sea buckthorn beverage described in the above technical solution in the preparation of products for lowering blood sugar and / or regulating blood lipid metabolism.

[0016] Preferably, the blood sugar lowering includes one or more of improving diabetic symptoms, improving blood sugar levels, reducing serum insulin levels, increasing glucagon-like peptide-1 (GLP-1) levels, and reducing insulin resistance.

[0017] Preferably, the regulation of blood lipid metabolism includes alleviating liver tissue damage and / or improving abnormal blood lipid metabolism.

[0018] Beneficial effects:

[0019] The present invention provides a method for preparing a fermented seabuckthorn beverage, comprising the following steps: sterilizing seabuckthorn juice with a volume percentage of 10% to 40% to obtain sterilized seabuckthorn juice; inoculating the sterilized seabuckthorn juice with Lactobacillus caucasicus LN6 bacterial liquid and then performing constant-temperature fermentation to obtain the fermented seabuckthorn beverage. The fermented seabuckthorn beverage prepared by fermenting seabuckthorn juice with a volume percentage of 10% to 40% by Lactobacillus caucasicus LN6 has significant blood glucose lowering and blood lipid metabolism regulation capabilities. Through animal experiments, it is found that: gavage of the fermented seabuckthorn beverage to type II diabetic mice can improve the symptoms of type II diabetes, improve the blood glucose level of mice, reduce the serum insulin level and increase the GLP-1 level, and reduce insulin resistance. At the same time, the fermented seabuckthorn beverage can also restore liver tissue damage and improve abnormal blood lipid metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0021] Figure 1 Inhibition rates of pre-fermentation seabuckthorn juice and post-fermentation seabuckthorn beverage on α-glucosidase in Examples 1 to 4;

[0022] Figure 2 Total phenol contents in pre-fermentation seabuckthorn juice and post-fermentation seabuckthorn beverage in Example 1;

[0023] Figure 3 Four-week body weight measurement results of mice in different experimental groups in Test Example 2;

[0024] Figure 4 Four-week fasting blood glucose measurement results of mice in different experimental groups in Test Example 2;

[0025] Figure 5 Fasting blood glucose values at each time point in the oral glucose tolerance test of mice in different experimental groups in Test Example 2;

[0026] Figure 6 Areas under the glucose tolerance curves of mice in different experimental groups in Test Example 2;

[0027] Figure 7 Liver index measurement results of mice in different experimental groups in Test Example 3;

[0028] Figure 8 Kidney index measurement results of mice in different experimental groups in Test Example 3;

[0029] Figure 9 Liver glycogen content measurement results of mice in different experimental groups in Test Example 3;

[0030] Figure 10For the determination results of triglyceride (TG) in the sera of mice in different experimental groups in Test Example 3;

[0031] Figure 11 For the determination results of total cholesterol (TC) in the sera of mice in different experimental groups in Test Example 3;

[0032] Figure 12 For the determination results of low-density lipoprotein cholesterol (LDL-C) in the sera of mice in different experimental groups in Test Example 3;

[0033] Figure 13 For the determination results of high-density lipoprotein cholesterol (HDL-C) in the sera of mice in different experimental groups in Test Example 3;

[0034] Figure 14 For the determination results of serum GLP-1 of mice in different experimental groups in Test Example 3;

[0035] Figure 15 For the determination results of serum insulin of mice in different experimental groups in Test Example 3;

[0036] Figure 16 For the determination results of blood AST of mice in different experimental groups in Test Example 3;

[0037] Figure 17 For the determination results of blood ALT of mice in different experimental groups in Test Example 3;

[0038] Figure 18 For the pathological sections of the liver tissues of mice in Test Example 3 after H&E staining;

[0039] Among them Figures 6 - 17 Different lowercase letters indicate differences between groups (p < 0.05). Specific implementation mode

[0040] The present invention provides a preparation method of a fermented seabuckthorn beverage, comprising the following steps:

[0041] Sterilize seabuckthorn juice with a volume percentage of 10% to 40% to obtain sterilized seabuckthorn juice;

[0042] Inoculate the sterilized seabuckthorn juice with Lactobacillus kefiranofaciens LN6 bacterial liquid and then carry out constant-temperature fermentation to obtain the fermented seabuckthorn beverage.

[0043] As an implementation mode, the present invention mixes seabuckthorn puree with water to obtain seabuckthorn juice with a volume percentage of 10% to 40%. As an implementation mode, the water is purified water. The present invention has no special limitation on the preparation method of the seabuckthorn puree, and the conventional preparation method of seabuckthorn puree in the art can be adopted.

[0044] As an implementation method, after obtaining the sea buckthorn juice with a volume percentage content of 10% - 40%, the present invention adjusts the pH value of the sea buckthorn juice with a volume percentage content of 10% - 40% to 5.0 ± 0.02. As an implementation method, the present invention adjusts the pH value with food-grade sodium bicarbonate; as another implementation method, the concentration of the food-grade sodium bicarbonate can be 1 mol / L. Adjusting the pH value of the sea buckthorn juice with a volume percentage content of 10% - 40% to 5.0 ± 0.02 by the present invention is suitable for the growth of fermentation strains.

[0045] After adjusting the pH value, the present invention sterilizes the sea buckthorn juice with a volume percentage content of 10% - 40% after adjusting the pH value to obtain sterilized sea buckthorn juice. As an implementation method, the sea buckthorn juice is sea buckthorn juice with a volume percentage content of 10% - 20%; as another implementation method, the sea buckthorn juice is sea buckthorn juice with a volume percentage content of 20%. As an implementation method, the sterilization method is pasteurization, and the temperature of the pasteurization is 80 - 85°C; as another implementation method, the temperature of the pasteurization is 80°C. As an implementation method, the time of the pasteurization is 15 - 20 min; as another implementation method, the time of the pasteurization is 15 min.

[0046] After obtaining the sterilized sea buckthorn juice, the present invention inoculates the sterilized sea buckthorn juice with Lactobacillus kefiranofaciens LN6 bacterial liquid and then performs constant-temperature fermentation to obtain the fermented sea buckthorn beverage. The Lactobacillus kefiranofaciens LN6 of the present invention is derived from kefir grains, preserved in the laboratory bacteria bank of the College of Food Science and Technology, Hebei Agricultural University, and publicly available on the website https: / / mp.weixin.qq.com / s / VuhwkJDWehfeK_yAynqa1g, can be obtained through the website https: / / mp.weixin.qq.com / s / VuhwkJDWehfeK_yAynqa1g, and is guaranteed to be distributed to the public within twenty years from the application date. As an implementation method, the concentration of the Lactobacillus kefiranofaciens LN6 bacterial liquid is 10 8 ~10 9 CFU / mL. As an implementation method, the inoculation amount of the Lactobacillus kefiranofaciens LN6 bacterial liquid is 1% - 5%. As an implementation method, the temperature of the constant-temperature fermentation is 35 - 37°C; as another implementation method, the temperature of the constant-temperature fermentation is 36°C. As an implementation method, the time of the constant-temperature fermentation is 20 - 28 h, as another implementation method, the time of the constant-temperature fermentation is 24 h.

[0047] The present invention also provides a fermented sea buckthorn beverage, and the fermented sea buckthorn beverage is prepared by using the preparation method described in the above technical solution.

[0048] The fermented seabuckthorn beverage prepared by the method of the present invention has remarkable ability to reduce blood sugar and regulate blood lipid metabolism.

[0049] Based on the above advantages, the present invention also provides an application of the fermented seabuckthorn beverage described in the above technical solution in the preparation of a product for reducing blood sugar and / or regulating blood lipid metabolism. As an implementation manner, the application is an application in the preparation of a product for reducing blood sugar and regulating blood lipid metabolism. As an implementation manner, the reduction of blood sugar includes one or more of improving diabetic symptoms, improving blood sugar level, reducing serum insulin level, increasing glucagon-like peptide-1 level, and reducing insulin resistance; As an implementation manner, the diabetes is type II diabetes. As an implementation manner, the regulation of blood lipid metabolism includes alleviating liver tissue damage and / or improving abnormal blood lipid metabolism. As an implementation manner, the product can be a food and / or a health product.

[0050] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1

[0052] A preparation method of a seabuckthorn fermented beverage is as follows:

[0053] 1) Raw material treatment: Mix seabuckthorn puree and purified water according to a volume ratio of 1:4 to obtain seabuckthorn juice with a volume percentage of 20%, and the pH value is 2.91 ± 0.02; Use food-grade sodium bicarbonate with a concentration of 1 mol / L to adjust the pH value of the seabuckthorn juice with a seabuckthorn content of 20% to 5.0 ± 0.02.

[0054] 2) Sterilization: Perform pasteurization on the seabuckthorn juice with a volume percentage of 20% prepared in step 1) at 80 °C for 15 min, and quickly cool it to room temperature with flowing cold water to obtain sterilized seabuckthorn juice.

[0055] 3) Preparation of Lactobacillus caucasicus LN6 bacterial liquid: Inoculate Lactobacillus caucasicus LN6 into the sterilized MRS liquid medium at an inoculation amount of 2%, activate it for 2 generations after culturing at 37 °C for 24 h, centrifuge at 5000 r / min for 12 - 15 min, take the precipitate, wash the precipitate 3 times with sterile normal saline, and then adjust the concentration of the bacterial suspension (resuspended with normal saline) to 10 8 CFU / mL to obtain Lactobacillus caucasicus LN6 bacterial liquid for standby.

[0056] 4) Inoculation: Inoculate the sterilized seabuckthorn juice obtained in step 2) with the Lactobacillus caucasicus LN6 bacterial liquid prepared in step 3), and the inoculation amount is 3% of the volume of the sterilized seabuckthorn juice.

[0057] 5) Incubation at a constant temperature: Put the inoculated sea buckthorn juice in step 4) into a biochemical incubator at 36 °C for constant-temperature fermentation for 24 h to obtain a sea buckthorn fermented beverage.

[0058] Example 2

[0059] Use the preparation method of the sea buckthorn fermented beverage in Example 1, the only difference is: replace the sea buckthorn juice with a volume percentage of 20% in step 1) with sea buckthorn juice with a volume percentage of 10%, that is, the volume percentage of sea buckthorn puree in the sea buckthorn juice is 10%.

[0060] Example 3

[0061] Use the preparation method of the sea buckthorn fermented beverage in Example 1, the only difference is: replace the sea buckthorn juice with a volume percentage of 20% in step 1) with sea buckthorn juice with a volume percentage of 30%, that is, the volume percentage of sea buckthorn puree in the sea buckthorn juice is 30%.

[0062] Example 4

[0063] Use the preparation method of the sea buckthorn fermented beverage in Example 1, the only difference is: replace the sea buckthorn juice with a volume percentage of 20% in step 1) with sea buckthorn juice with a volume percentage of 40%, that is, the volume percentage of sea buckthorn puree in the sea buckthorn juice is 40%.

[0064] Test Example 1

[0065] The in vitro hypoglycemic ability of the sea buckthorn fermented beverages prepared in Examples 1 to 4 is as follows:

[0066] Centrifuge the sea buckthorn juice before fermentation and the sea buckthorn fermented beverage prepared by fermentation at 8000 r / min for 12 min respectively, take the supernatant to obtain the supernatant of the sea buckthorn juice before fermentation and the supernatant of the sea buckthorn juice after fermentation respectively.

[0067] 1. Detection of α-glucosidase inhibition rate:

[0068] Prepare an α-glucosidase solution with a concentration of 2 U / mL and a 4-nitrophenyl-β-D-glucopyranoside (PNPG) solution with a concentration of 2.5 mmol / L respectively using a phosphate buffer solution (pH 6.8).

[0069] Add 40 μL of the α-glucosidase solution to the supernatant of the sea buckthorn juice before fermentation and the supernatant of the sea buckthorn juice after fermentation respectively into a 96-well plate, mix well, and incubate at 37 °C for 15 min; then add 20 μL of the PNPG solution respectively, mix well, and incubate at 37 °C for 15 min. Finally, add 150 μL of 0.2 mol / L Na 2 CO 3The solution was used to terminate the reaction. The absorbance at 405 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader and denoted as OD sample. 40 μL of PBS was used instead of seabuckthorn juice as a blank control, and the absorbance at 405 nm measured was denoted as OD blank. PBS was used instead of α-glucosidase as a background group, and the absorbance at 405 nm measured was denoted as OD background. The inhibition rate was calculated according to the formula: inhibition rate = (1 - (OD sample - OD background) / OD blank) × 100.

[0070] The seabuckthorn juices with seabuckthorn contents of 20%, 10%, 30% and 40% in step 1) of Examples 1 to 4 and the corresponding seabuckthorn fermented beverages were respectively measured for the inhibition rate of the seabuckthorn juice supernatant on α-glucosidase before and after fermentation according to the above steps. The results are as Figure 1 shown, where NF represents the seabuckthorn juice supernatant before fermentation and F represents the seabuckthorn juice supernatant after fermentation.

[0071] From Figure 1 it can be concluded that: regardless of whether the volume percentage of seabuckthorn puree in the seabuckthorn juice is 10%, 20%, 30% or 40%, the in vitro hypoglycemic activity of the seabuckthorn beverage after fermentation with Lactobacillus caucasicus LN6 is significantly improved compared with that of the seabuckthorn juice before fermentation. In particular, the in vitro hypoglycemic activity of the seabuckthorn fermented beverage prepared with 10% - 20% seabuckthorn puree by volume is higher.

[0072] 2. The phenolic substances were detected by the Folin-Ciocalteu reagent method: Using gallic acid as the standard product, with the concentration of the gallic acid standard solution as the abscissa and the absorbance value at 760 nm as the ordinate, the standard curve equation was y = 1.916x + 0.052, R 2 = 0.9981. 1 mL of the test solution was taken, 5 mL of Folin-Ciocalteu reagent was added, mixed well, reacted for 5 min, 4 mL of NaCO 3 was added, and the reaction was carried out in the dark at room temperature for 1 h, and the absorbance was measured at 760 nm.

[0073] The method in step 2 was used to measure the content of phenolic substances in the seabuckthorn juice supernatant before and after fermentation in Example 1. The results are as Figure 2 shown, where NF represents the seabuckthorn juice supernatant before fermentation and F represents the seabuckthorn juice supernatant after fermentation.

[0074] From Figure 2 it can be concluded that: compared with the seabuckthorn juice before fermentation, the total phenolic content in the seabuckthorn beverage after fermentation with Lactobacillus caucasicus LN6 is significantly increased.

[0075] Test Example 2

[0076] The use of the seabuckthorn fermented beverage prepared in Example 1 in improving the glucose metabolism of mice is as follows:

[0077] Animal and Feed Preparation: Animal breeding was carried out in the SPF animal experimental center of Hebei Agricultural University. Six-week-old male C57BL / 6J mice with a body weight between (20±2) g were used. All mice were housed in standard polypropylene cages, with free access to food and water. The temperature was (25±2) °C, the humidity was (50±5)%, and a 12h light / 12h dark cycle was maintained. All animal treatment protocols were approved by the Ethics Committee of Hebei Agricultural University, with the approval number 2023193. The production license number for experimental animals is: SCXK (Beijing) 2024-0001. The standard feed GB14924 was purchased from Beijing Speyford Biotechnology Co., Ltd. The high-fat diet XTHF60 was purchased from Jiangsu Xietong Biotechnology Co., Ltd., and the feed production license number is Su Feed License (2019) 01008. The high-fat and high-sugar diet consisted of the following components by mass percentage: 38% standard pellet feed, 28% lard, 5.6% sucrose, 10.8% whole milk powder, 11.5% casein, 2% animal premix, 1.9% microcrystalline cellulose, 1.8% calcium hydrogen phosphate, and 0.4% limestone powder.

[0078] Gavage Sample Preparation: The seabuckthorn juice before fermentation and the fermented seabuckthorn beverage in Example 1 were concentrated in a vacuum rotary evaporator at a temperature of 40 °C and concentrated 2.5 times. The concentrated samples were aliquoted and stored at -20 °C, and thawed before use.

[0079] Establishment of Type 2 Diabetes Mouse Model:

[0080] Type 2 diabetes model mice were induced by a combination of a high-fat and high-sugar diet and streptozotocin (STZ) as follows: After 7 days of adaptive feeding with normal maintenance feed, the mice were randomly divided into a normal group (n = 6) and a disease model group (n = 30). The normal group was fed normal maintenance feed, and the disease model group was fed a high-fat and high-sugar diet (consisting of the following components by mass percentage: 38% standard pellet feed, 28% lard, 5.6% sucrose, 10.8% whole milk powder, 11.5% casein, 2% animal premix, 1.9% microcrystalline cellulose, 1.8% calcium hydrogen phosphate, and 0.4% limestone powder). After 8 weeks of feeding, the mice in the disease model group were fasted but allowed water for 12 h, and then continuously injected intraperitoneally with STZ (first injected with an STZ solution at a concentration of 120 mg / kg, and then injected again with an STZ solution at a concentration of 60 mg / kg. The solvent of the STZ solution was 0.1 mmol / L sodium citrate buffer, pH 4.4, protected from light and ice-bathed, prepared freshly before use and used up within 30 min). The mice in the normal group were injected intraperitoneally with an equal volume of 0.1 mmol / L sodium citrate buffer, and fasted for 2 h after injection. Seven days later, a blood glucose meter was used to collect tail tip blood to measure fasting blood glucose. If the fasting blood glucose was greater than 11.1 mmol / L and accompanied by symptoms such as polydipsia, polyphagia, polyuria, and weight loss, it was considered that the type 2 diabetes mouse model was successfully constructed.

[0081] Grouping of experimental mice: 6 mice in the normal group and 10 mice in each of the other groups;

[0082] Normal group (NG): Normal mice were intragastrically administered normal saline daily;

[0083] Model group (DM): Model mice were intragastrically administered normal saline daily;

[0084] Fermented sea buckthorn juice group (F): Model mice were intragastrically administered concentrated samples of fermented sea buckthorn beverage daily;

[0085] Unfermented sea buckthorn juice group (NF): Model mice were intragastrically administered concentrated samples of unfermented sea buckthorn juice daily;

[0086] The intragastric administration volume was 0.1 mL / 10 g, and intragastric administration was performed once a day. The body weight (BW) and fasting blood glucose (FBG) of the mice in each group were weighed at the same time every week. At the 4th week, the mice were fasted but allowed to drink water for 12 h, and the oral glucose tolerance (OGTT) of the mice was measured.

[0087] Oral glucose tolerance test: After 4 weeks of intragastric administration, the oral glucose tolerance test was performed the next day after fasting but allowing to drink water for 12 h. The mice were intragastrically administered glucose solution (2 g / kg). Starting from the end of intragastric administration, the fasting blood glucose values of each mouse were measured at 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, and 2 h, and the area under the curve (AUC) was calculated.

[0088] AUC (h.mmol / L) = 0.5×(0.25A + 0.5B + 0.75C + D + E + 0.5F), where: A, B, C, D, E, and F are the fasting blood glucose values mmol / L at 0 h, 0.25 h, 0.5 h, 1 h, 1.5 h, and 2.0 h after intragastric administration of glucose solution, respectively.

[0089] Among them, the body weight results of the mice measured every week are as Figure 3 shown, the fasting blood glucose results measured every week are as Figure 4 shown, the fasting blood glucose values at each time point in the oral glucose tolerance test are as Figure 5 shown, and the calculation results of the area under the glucose tolerance curve are as Figure 6 shown.

[0090] It can be concluded from Figure 3 that: compared with the DM group, the body weights of the mice in the F group and the NF group increased after 4 weeks of intragastric administration, but the upward trend in the F group was significantly greater than that in the NF group; it can be concluded from Figure 4 that: compared with DM, the fasting blood glucose values of the mice in the F group and the NF group were significantly decreased after 4 weeks of intragastric administration, and the fasting blood glucose value of the mice in the F group decreased more significantly than that in the NF group. It can be concluded from Figure 5It can be concluded that compared with the DM group, the fasting blood glucose levels of the mice in the F group and the NF group decreased more significantly after oral glucose administration over time, and the mice in the F group were closer to the NC group, indicating that the F group had a stronger ability to lower blood glucose. From Figure 6 It can be concluded that compared with the DM group, the areas under the curves of the F group and the NF group were smaller, and the area under the curve of the F group was relatively smaller. Overall Figures 3 - 6 It can be concluded that sea buckthorn juice has a good regulatory effect on the blood glucose of type 2 diabetic mice, and the ability of the fermented sea buckthorn beverage to regulate blood glucose metabolism is stronger.

[0091] Test Example 3

[0092] 1. Determine the liver index, kidney index, hepatic glycogen content, serum blood lipid level, serum glycated serum protein (GSP) level, alanine aminotransferase (ALT) content, aspartate aminotransferase (AST) content of the mice in each group after the gavage test in Test Example 2, and the results of the H&E stained pathological sections of the mouse liver tissue:

[0093] 1) The liver index is: the ratio of liver weight to body weight. The measurement results are as Figure 7 shown.

[0094] 2) The kidney index is: the ratio of kidney weight to body weight. The measurement results are as Figure 8 shown.

[0095] According to Figures 7 - 8 the results in, it can be concluded that compared with the NG group, the abnormal increase in the liver weight of the mice in the DM group was mainly related to the accumulation of fat. As an important organ for glycolipid metabolism, the accumulation of fat in the liver can induce the occurrence of fatty liver, cause insulin resistance, and exacerbate type 2 diabetes; while compared with the DM group, the liver indices of the F group and the NF group decreased by 25.26% and 17.47% respectively, and the kidney indices decreased by 16.41% and 13.57% respectively. It can be seen that sea buckthorn juice can reduce the liver index and kidney index of type 2 diabetic mice, and the effect of the fermented sea buckthorn beverage is better.

[0096] 3) The method for measuring the hepatic glycogen content is: use the kit of Nanjing Jiancheng Bioengineering Institute and measure it according to the instructions of the kit. The measurement results are as Figure 9 shown.

[0097] Glycogen is an important form of glucose storage in the body. From Figure 9 it can be concluded that the hepatic glycogen of the mice in the NG group was maintained at a relatively high level, while the hepatic glycogen content of the mice in the DM group was significantly reduced. After 4 weeks of intervention with sea buckthorn juice, compared with the DM group, the hepatic glycogen contents of the fermented sea buckthorn juice and the unfermented sea buckthorn juice increased to varying degrees, and the effect of the F group on the hepatic glycogen of the mice was significantly higher than that of the NF group.

[0098] 4) The serum GSP levels of mice in each group were measured. The measurement method was as follows: using the ELISA kit of Jingmei Bioengineering Co., Ltd. for measurement, and the specific measurement steps refer to the kit instruction manual. The results showed that the serum GSP level of mice in the DM group was significantly increased (6.07 mmol / L, p < 0.01); compared with the DM group, after 4 weeks of sea buckthorn juice intervention, the serum GSP levels of mice in each group were decreased to varying degrees (p < 0.05). Specifically, the serum GSP of mice in the F group was decreased by 16.64%, and that of mice in the NF group was decreased by 12.03%.

[0099] 5) The serum lipid levels of mice in each group, namely triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C), were measured. The measurement method was to use the kit of Nanjing Jiancheng Bioengineering Institute and measure according to the kit instruction manual. The results are as Figures 10 - 13 shown.

[0100] It can be Figures 10 - 13 concluded that fermented sea buckthorn juice can significantly improve the abnormal levels of TC and TG in type 2 diabetic mice, and the F group has a better reduction effect than the NF group. Fermented sea buckthorn juice has a good effect on improving lipid metabolism disorders in type 2 diabetic mice. Compared with the DM group, the serum LDL-C level of type 2 diabetic mice in the F group was decreased by 33.11%, and the serum HDL-C level was also significantly increased compared with the DM group.

[0101] 6) The serum insulin (InS), related indexes (insulin secretion index HOMA-β, insulin sensitivity index ISI, and insulin resistance index HOMA-IR), and serum GLP-1 of mice in each group were measured. The measurement method was to use the ELISA kit of Jingmei Bioengineering Co., Ltd. for measurement, and the specific measurement steps refer to the kit instruction manual. The calculation methods of each related index are as follows: Insulin resistance index Homa-IR = (FBG × InS) / 22.5; Insulin secretion index Homa-β = 20 × InS / (FBG - 3.5); Insulin sensitivity index ISI = ln(FBG × InS) -1 .

[0102] . The measurement results are shown in Table 1 and Figures 14 - 15 as follows, where different lowercase letters in Table 1 indicate significant differences between groups (p < 0.05).

[0103] Table 1 Measurement results of serum insulin and related indexes of mice

[0104]

[0105] From Table 1 and Figures 14 - 15It can be concluded that the serum insulin level in the DM group was significantly higher than that in the NG group, showing hyperinsulinemia. Combining with the blood glucose level of the mice, it was judged that the type 2 diabetes model was successfully established. Compared with the DM group, the ISI index of type 2 diabetic mice in the F group and the NF group was significantly increased, and the HOMA-IR index was significantly decreased. The results of the ISI and HOMA-IR indexes showed that fermented sea buckthorn juice could increase the effective utilization of insulin in type 2 diabetic mice, thereby weakening the IR of type 2 diabetic mice.

[0106] 7) Determine the contents of ALT and AST in the blood of mice in each group. The detection method is to use the kit of Nanjing Jiancheng Bioengineering Institute and measure according to the instructions of the kit. The detection results are as Figures 16 - 17 shown.

[0107] Once the liver is damaged, transaminases will enter the blood and the content of blood transaminases will increase. From Figures 16 - 17 the results, it can be concluded that the contents of ALT and AST in the blood of mice in the DM group were significantly higher than those in the NG group. Moreover, after the intervention of sea buckthorn juice, the contents of AST and ALT were significantly decreased, and the decrease in the F group was also more obvious than that in the NF group.

[0108] 8) Prepare pathological sections by H&E staining of the liver tissues of mice in each group. The method is to immerse the fresh liver in 4% paraformaldehyde for 24 hours and observe its effect on the tissue. After paraffin embedding, take tissue sections with a thickness of 5 microns and observe their pathological morphology by double labeling with hematoxylin and eosin. The results are as Figure 18 shown. In Figure 18 , a is the NG group, b is the DM group, c is the F group, and d is the NF group.

[0109] From Figure 18 it can be concluded that the hepatocyte morphology in the NG group was normal, arranged neatly and distributed evenly. The cell morphology in the DM group was abnormal, with oval fat vacuoles of different sizes, and obvious cell necrosis. After the intervention of sea buckthorn juice, the liver fat vacuoles became smaller and the number decreased, the inflammatory reaction was alleviated, the cell arrangement was neater than that in the DM group, the cell necrosis phenomenon decreased, and the fat vacuoles in the F group were smaller than those in the NF group, and the recovery was better. Fermented sea buckthorn juice can effectively relieve liver damage caused by type 2 diabetes.

[0110] It can be concluded from the above examples that the fermented sea buckthorn beverage described in the present invention can effectively play a role in lowering blood glucose and regulating blood lipid metabolism, and can be used as an effective dietary intervention means for diabetes.

[0111] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a fermented seabuckthorn beverage, characterized in that: The steps include: sterilizing seabuckthorn juice with a volume percentage of 10% to 40% to obtain sterilized seabuckthorn juice; The sterilized seabuckthorn juice is inoculated with a bacterial liquid of Lactobacillus caucasus LN6 and then fermented at a constant temperature to obtain the fermented seabuckthorn beverage.

2. The preparation method according to claim 1, characterized in that: The seabuckthorn juice comprises seabuckthorn juice with a volume percentage of 10% to 20%.

3. The preparation method according to claim 1, characterized in that: The concentration of the Lactobacillus caucasus LN6 bacterial solution is 10 8 ~10 9 CFU / mL.

4. The preparation method according to claim 1 or 3, characterized in that: The inoculation amount of the Lactobacillus caucasus LN6 bacterial liquid is 1% to 5%.

5. The preparation method according to claim 1, characterized in that: The constant temperature fermentation temperature is 35-37° C. and the time is 20-28 hours.

6. The preparation method according to claim 1, characterized in that: The sterilization method is pasteurization, the pasteurization temperature is 80-85° C., and the time is 15-20 minutes.

7. A fermented seabuckthorn beverage, characterized in that: The fermented seabuckthorn beverage is prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the fermented seabuckthorn beverage according to claim 7 in the preparation of products for lowering blood sugar and / or regulating blood lipid metabolism.

9. The use according to claim 8, characterized in that: The hypoglycemic effect includes one or more of improving diabetic symptoms, improving blood sugar levels, reducing serum insulin levels, increasing glucagon-like peptide-1 levels, and reducing insulin resistance.

10. The use according to claim 8, characterized in that: The regulating blood lipid metabolism includes alleviating liver tissue damage and / or improving abnormal blood lipid metabolism.

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