阅读说明：本技术 一种桑葚发酵产物及其制备方法和应用 (Mulberry fermentation product and preparation method and application thereof ) 是由 李发荣 党媛婕 魏倩倩 周蓉存 万娟 高婧茹 张宝善 韩松林 塔及古丽·牙库甫 于 2021-07-28 设计创作，主要内容包括：本发明提供了一种桑葚发酵产物,属于生物发酵技术领域。本发明以桑葚为原料,利用植物乳杆菌CICC24202(LactobacillusplantarumCICC24202)和短乳杆菌YM1301(LactobacillusbrerisYM1301)这两种益生菌混合悬液对桑葚匀浆进行发酵处理,得到桑葚发酵产物。本发明得到的桑葚发酵产物对2型糖尿病具有明显改善作用,能够有效降低血糖,增加葡萄糖耐受能力,提高胰岛素敏感性,改善胰岛素抵抗,降低2型糖尿病小鼠的脂肪蓄积,一定程度上改善肾功能,缓解或治疗2型糖尿病并发症,并且安全性高、生产容易、质量稳定性高。(The invention provides a mulberry fermentation product, and belongs to the technical field of biological fermentation. The mulberry fermentation method takes mulberry as a raw material, and utilizes two probiotic mixed suspensions of lactobacillus plantarum CICC24202(Lactobacillus plantarum CICC24202) and lactobacillus brevis YM1301(Lactobacillus brevis YM1301) to ferment mulberry homogenate to obtain a mulberry fermentation product. The mulberry fermentation product obtained by the invention has an obvious improvement effect on type 2diabetes, can effectively reduce blood sugar, increase glucose tolerance, improve insulin sensitivity, improve insulin resistance, reduce lipopexia of type 2diabetes mice, improve renal function to a certain extent, relieve or treat type 2diabetes complications, and has high safety, easy production and high quality stability.)

1. A mulberry fermentation product is characterized by being obtained by fermenting mulberry raw pulp with mixed probiotic suspension, wherein the mixed probiotic suspension is obtained by mixing Lactobacillus brevis YM1301 and Lactobacillus plantarum CICC24202 according to a ratio of 1: 0.1-10.

2. The mulberry fermented product according to claim 1, wherein the raw pulp of mulberry is prepared from mulberry powder and water in a weight ratio of 1g: 4-6 mL.

3. The mulberry fermentation product of claim 1, wherein the mulberry homogenate has a pH of 5.5 to 6.5.

4. The preparation method of the mulberry fermentation product according to any one of claims 1 to 3, comprising the steps of: homogenizing and sterilizing the mulberry, cooling to room temperature, inoculating the mixed probiotic suspension, and fermenting and culturing.

5. The preparation method of claim 4, wherein the inoculation amount of the mixed probiotic suspension is 8-12% of the volume of the mulberry puree.

6. The method of claim 4, wherein the fermentation conditions are: culturing at the constant temperature of 36-38 ℃ for 96-120 h.

7. The preparation method of claim 4, wherein the fermented mulberry raw juice is quickly frozen in a low-temperature environment, and then is freeze-dried and pulverized to obtain the fermented mulberry freeze-dried powder.

8. Use of the mulberry fermentation product according to any one of claims 1 to 3 in the preparation of food and health care products.

9. Use of a mulberry fermentation product according to any one of claims 1 to 3 in the preparation of a medicament for the alleviation or treatment of type 2 diabetes.

10. Use of a mulberry fermentation product according to any one of claims 1 to 3 in the preparation of a medicament for alleviating or treating complications of type 2diabetes, wherein the complications include dyslipidemia, obesity, diabetic nephropathy.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a mulberry fermented product, and a preparation method and application thereof.

Background

Diabetes Mellitus (DM) is a chronic disease in which disturbances of carbohydrate and lipid metabolism in the body are caused by absolute or relative insufficiency of insulin in the body, or by a decrease in the sensitivity of peripheral tissues to insulin. With the escalating increase of economic and people's living standards, diabetes has become the third largest non-infectious disease that endangers human health.

DM is classified into Type 1Diabetes Mellitus (T1 DM), Type 2Diabetes Mellitus (T2 DM), Gestational Diabetes Mellitus (GDM) and other specific types of Diabetes according to its different etiologies and mechanisms. T1DM is characterized in that under the influence of various factors, the autoimmune system is abnormal, islet beta cell specific antibodies are generated, and islet beta cells are permanently damaged, so that absolute insulin deficiency is caused; t2DM is the normal level of insulin secretion in vivo, but the decrease of insulin sensitivity of the body produces insulin resistance effect, which results in the decrease of insulin production level, insufficient insulin secretion and failure to effectively reduce blood sugar level of the body, which is the main cause of the onset of type 2diabetes, and about 90% of patients with diabetes are T2DM patients. Complications such as diabetic nephropathy and diabetic ophthalmopathy are also induced by long-term hyperglycemia or insulin resistance, and the diabetes-related complications are major factors causing the quality of life of diabetic patients to be reduced.

The main approaches for treating type 2diabetes are diet therapy, drug therapy, etc., and the central idea of treatment is to reduce sugar intake and achieve the purpose of treatment by improving the sensitivity of peripheral organs to insulin through various methods. However, the existing common treatment methods have certain disadvantages, and patients who take oral medicines for clinically treating diabetes mellitus are easy to cause adverse reactions such as hypoglycemia, lactic acidosis, gastrointestinal discomfort and the like after taking the oral medicines for a long time, so that the development of high-efficiency and low-toxicity antidiabetic medicines is still a hot point of research in the field of medicine.

Mulberry (Mori Fructus) originally recorded in Tang. New repair materia Medica is mature fruit of Moraceae plant Mulberry, has effects of nourishing yin and supplementing blood, and treating yin deficiency and little body fluid, and is a traditional medicinal and edible medicinal material variety in China. The 2015 pharmacopoeia records that the main functions of the traditional Chinese medicine composition are nourishing yin, enriching blood, promoting the production of body fluid and moistening dryness. Can be used for treating liver and kidney yin deficiency, vertigo, tinnitus, palpitation, insomnia, thirst due to body fluid consumption, and internal heat. The mulberry requires the growth environment, so that the mulberry has the advantages of nature and no pollution, and is called as folk cherry tomato and Chinese cherry tomato. The mulberry contains abundant nutritive value and contains various active ingredients such as various trace elements, anthocyanin, alkaloid, saccharide and the like. The components such as flavone, polyphenol, polysaccharide and the like contained in the mulberry are found to have good blood sugar reducing effect and are main components in a plurality of blood sugar reducing traditional Chinese medicine formulas, but the mulberry fruit has high content of micromolecular sugar, and is not suitable for being taken by diabetes patients for a long time. Therefore, how to improve the pharmacological value of the mulberry, which is convenient for the diabetics to take and has better blood sugar reducing effect is a technical problem to be solved in the field.

Disclosure of Invention

In view of the above, the invention aims to provide a mulberry fermentation product, which can effectively reduce the content of small molecular sugar in mulberries, has an obvious improvement effect on type 2diabetes, can obviously reduce blood sugar, improve sugar tolerance, improve insulin resistance, reduce lipopexia, and improve kidney function.

The invention provides a mulberry fermentation product, which is obtained by fermenting mulberry raw pulp with mixed probiotic suspension, wherein the mixed probiotic suspension is obtained by mixing lactobacillus brevis YM1301 and lactobacillus plantarum CICC24202 according to the ratio of 1: 0.1-10.

Preferably, the mulberry raw pulp is prepared from mulberry powder and water according to the weight ratio of 1g: 4-6 mL.

Preferably, the pH value of the mulberry homogenate is 5.5-6.5.

The invention also provides a preparation method of the mulberry fermented product, which comprises the following steps: sterilizing the primary pulp of the mulberry, cooling to room temperature, inoculating the mixed probiotic suspension, and fermenting and culturing.

Preferably, the inoculation amount of the mixed probiotic suspension is 8-12% of the volume of the mulberry raw pulp.

Preferably, the fermentation conditions are: culturing at the constant temperature of 36-38 ℃ for 96-120 h.

Preferably, the fermented mulberry raw juice is quickly frozen in a low-temperature environment, and then is freeze-dried and crushed to obtain the fermented mulberry freeze-dried powder.

The invention also provides application of the mulberry fermentation product in preparation of foods and health-care products.

The invention also provides application of the mulberry fermentation product in preparation of medicines for relieving or treating type 2 diabetes.

The invention also provides application of the mulberry fermentation product in preparation of medicines for relieving or treating type 2diabetes complications, wherein the complications comprise dyslipidemia, obesity and diabetic nephropathy.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the mulberry is fermented by using the mixed probiotic suspension obtained by mixing lactobacillus brevis YM1301 and lactobacillus plantarum CICC24202, small molecular saccharides such as sucrose, fructose and the like in the mulberry can be removed, the small molecular saccharide content of the mulberry is reduced, the mulberry is convenient for diabetics to take, and the obtained mulberry fermentation product is moderate in sweetness and sourness, has obvious mulberry fragrance and special fermentation flavor, and is good in taste.

The mulberry fermentation product obtained by the invention has an obvious improvement effect on type 2diabetes, can obviously reduce blood sugar, improve sugar tolerance, improve insulin resistance and relieve lipopexia and renal function, and has important significance in preparing medicines for relieving or treating type 2diabetes and related complications thereof.

Drawings

FIG. 1 shows changes in acidity of primary pulp of Mori fructus during fermentation;

FIG. 2 is a graph showing the pH value of primary pulp of Mori fructus during fermentation;

FIG. 3 is a graph showing the change in glucose content in a mulberry fermented product during fermentation;

FIG. 4 is a graph showing the variation of reducing sugar content in a mulberry fermented product during fermentation;

FIG. 5 shows the change of the sensory flavor of the mulberry fermented product during the fermentation process;

FIG. 6 shows the effect of each experiment on body weight, food intake and water intake of type 2 diabetic mice;

FIG. 7 is a graph showing the effect of various experiments on fasting plasma glucose in type 2 diabetic mice;

FIG. 8 is the effect of each experiment on OGTT and AUC of type 2 diabetic mice;

FIG. 9 is the effect of each experiment on ITT, AUC of type 2 diabetic mice;

FIG. 10 shows the effect of various experiments on FINS and HOMA-IR in type 2 diabetic mice;

FIG. 11 is a graph of the effect of various groups of experiments on GHb in type 2 diabetic mice;

FIG. 12 is a graph showing the effect of various groups of experiments on hepatic glycogen and muscle glycogen in type 2 diabetic mice;

FIG. 13 is a graph of the effect of various groups of experiments on GLP-1 in type 2 diabetic mice;

FIG. 14 is a graph of the effect of various groups of experiments on the levels of TG, T-CHO, HDL-C and LDL-C in type 2 diabetic mice;

FIG. 15 is a graph of the effect of various groups of experiments on BUN in type 2 diabetic mice;

FIG. 16 shows the effect of each experiment on Scr in type 2 diabetic mice.

Detailed Description

The invention provides a mulberry fermentation product, which is obtained by fermenting mulberry raw pulp with mixed probiotic suspension, wherein the mixed probiotic suspension is obtained by mixing lactobacillus brevis YM1301 and lactobacillus plantarum CICC24202 according to the ratio of 1: 0.1-10.

Lactobacillus brevis (Lactobacillus brevis) is selected from milk, kefir, cheese, sauerkraut, etc., and has thick and long rod shape with two blunt ends, and can generate various products such as organic acid, amino acid, polysaccharide, etc. during growth and propagation. The lactobacillus brevis YM1301 detected by the invention has better aminobutyric acid production capacity and is very suitable for serving as a fermentation strain for functional health products.

Lactobacillus plantarum (Lactobacillus plantarum) is a common fermentation strain, has high viable count, can produce a large amount of acid, and can relieve lactose intolerance. The lactobacillus plantarum CICC24202 is a strain in lactobacillus plantarum, is aerobic, is a tiny circular white colony, is convex and has regular edges; the cells are rod-shaped, short chain, non-spore and gram-positive. The lactobacillus plantarum CICC24202 has good heat resistance and acid resistance, and is a strain suitable for plant materials as a culture medium.

The Lactobacillus plantarum CICC24202(Lactobacillus plantarum CICC24202) is suitable for fermenting plant-derived materials and has good acid resistance; the Lactobacillus brevis YM1301(Lactobacillus brevis YM1301) can produce high-yield aminobutyric acid through screening verification, small molecular saccharides in the mulberry can be effectively removed by fermenting the mulberry with the two strains, and meanwhile, high-activity probiotics contained in a fermentation product can bring certain health effect, so that the mulberry and the probiotics generate synergistic effect.

According to the invention, lactobacillus brevis YM1301 and lactobacillus plantarum CICC24202 are selected as probiotics to ferment the mulberry, the mulberry fermented by mixing the two strains has a synergistic effect, the content of small molecular sugar in the mulberry can be obviously reduced, and meanwhile, due to the metabolic activity of the two strains, the content of gamma-aminobutyric acid and polysaccharide in a product is improved, the GLP-1 secretion level of an organism can be effectively improved, so that the effect of effectively relieving diabetes is achieved. The specific sources of Lactobacillus brevis YM1301 and Lactobacillus plantarum CICC24202 are not limited in the present invention.

In the invention, the mixed probiotic suspension is prepared by mixing lactobacillus brevis YM1301 and lactobacillus plantarum CICC24202 according to the ratio of 1: 0.1-10, preferably 1: 0.5-2, and more preferably 1: 1. According to the invention, the mulberry is fermented by the mixed probiotic suspension liquid in the proportion, the obtained two strains have moderate metabolite content, and a synergistic effect is generated between the two strains and pharmacological active substances contained in the mulberry, so that the blood sugar reduction effect of the mulberry fermented product is further improved.

The mulberry homogenate is prepared by mixing mulberry powder and water according to the weight ratio of 1g: 4-6 mL, and the material-liquid ratio is preferably 1g:5 mL. As an optional embodiment, the mulberry pulp is prepared by washing purchased mulberry with clear water, naturally drying and crushing the mulberry, and preparing fermented mulberry pulp with distilled water according to the proportion of 1g to 5mL (m/V). The mulberry homogenate can also be obtained by directly grinding fresh mulberries without adding a solution, and can be directly used for subsequent fermentation. The specific sources of the mulberries and the cleaning, airing and crushing modes are not limited.

The pH value of the mulberry raw pulp is adjusted to 5.5-6.5, and preferably 6.0. The pH value of the mulberry homogenate prepared from the mulberry powder or fresh mulberries is slightly lower. According to the invention, alkaline substances are added to adjust the pH value of the mulberry homogenate, and the pH value of the mulberry raw pulp is controlled to ensure that the mulberry raw pulp is the most suitable pH value for mixed probiotic suspension fermentation, so that the fermentation effect of the mulberry can be effectively improved, and the blood sugar reduction effect of the fermentation product is ensured. The present invention is not limited to a specific pH adjustment method.

The obtained mulberry raw pulp is sterilized, and as an optional implementation mode, the mulberry raw pulp is sterilized at 121 ℃ for 20-25 min. The present invention is not limited to a specific sterilization method. And cooling the sterilized mulberry primary pulp to room temperature for later use.

In the invention, in a sterile environment, the probiotic suspension is inoculated into the primary pulp of the mulberry for fermentationAnd (5) culturing. The inoculation amount of the probiotic suspension is 8-12% of the volume of the mulberry raw pulp, preferably 9-11%, and more preferably 10%. The invention discovers that the inoculation amount can effectively degrade micromolecule sugar in the mulberry, and the obtained mulberry fermentation product has high content of viable probiotics, namely 2.28 multiplied by 10⁵Above CFU/g, the mulberry fruit extract and the hypoglycemic active ingredient in the mulberry fermentation product have a synergistic effect, and the alleviation effect of the fermentation product on type 2diabetes is increased.

The fermentation culture condition is constant temperature culture at 36-38 ℃ for 96-120h, and preferably constant temperature culture at 37 ℃ for 110-115 h. The invention discovers that the acidity value of the mulberry fermentation product obtained in the fermentation time is higher, meanwhile, the contents of glucose and fructose after fermentation for 96 hours are reduced and tend to be stable, the mulberry fermentation product can be consumed and decomposed to generate acidic substances, and the flavor and the taste of the obtained fermentation product are better.

According to the invention, the mulberry fermented product obtained by fermentation can be quickly frozen in a low-temperature environment, and then freeze-dried and crushed to obtain the fermented mulberry freeze-dried powder. As an optional implementation mode, the mulberry raw juice is frozen into blocks at the temperature of between 20 ℃ below zero and 25 ℃ below zero, and then the blocks are freeze-dried in a freeze-drying machine at the temperature of between 80 ℃ below zero, and are crushed after freeze-drying, so that the mulberry freeze-dried powder is obtained. The specific preparation method of the mulberry freeze-dried powder is not limited.

The mulberry raw juice or mulberry freeze-dried powder obtained by the invention can be used for preparing food or health products. The mulberry raw pulp or mulberry freeze-dried powder obtained by the invention has obvious mulberry fragrance and special mulberry fermentation flavor, is moderate in sour and sweet taste and pure in taste, and can be used for preparing foods with mulberry flavor, such as mulberry flavored tablet candies, mulberry flavored drinks and the like. The mulberry raw pulp or mulberry freeze-dried powder obtained by the invention contains a large amount of mulberry polysaccharide, gamma-aminobutyric acid, probiotics and the like, and can be used for preparing health products for assisting diabetes treatment and food or health products for regulating gastrointestinal flora.

The mulberry raw juice or mulberry freeze-dried powder obtained by the invention can be used for preparing medicines for relieving or treating type 2 diabetes. The content of glucose and reducing sugar in the obtained mulberry fermented product is obviously reduced, but the content of total polysaccharide is higher than that of unfermented mulberries, and the content of gamma-aminobutyric acid and probiotics in the fermented product is increased, so that the GLP-1 (pancreatic hyperglycemia peptide-like factor) secretion level of an organism can be effectively improved, intestinal microorganisms are regulated, blood sugar is reduced, sugar tolerance is improved, insulin resistance is improved, and the effect of effectively relieving diabetes is achieved.

The mulberry raw juice or mulberry freeze-dried powder obtained by the invention can be used for preparing medicines for relieving or treating type 2diabetes complications, wherein the complications comprise dyslipidemia, obesity and diabetic nephropathy.

The mulberry fermented product obtained by the invention can obviously reduce the levels of TG (triglyceride), T-CHO (total cholesterol), LDL-C (low density lipoprotein) and HDL-C (high density lipoprotein) in the serum of a mouse fed with high fat for a long time, and can obviously reduce the epididymal peripheral fat mass (EAM) and epididymal peripheral fat index (EAMI) of a diabetic mouse. The mulberry fermentation product is shown to improve dyslipidemia and reduce fat accumulation while reducing blood sugar of the type 2diabetes patients, and has certain effect on losing weight and reducing fat of the type 2diabetes patients.

The mulberry fermentation product obtained by the invention can obviously reduce the kidney weight and kidney index of a diabetic mouse, and reduce the serum content levels of Blood Urea Nitrogen (BUN) and serum creatinine (Scr) of the diabetic mouse. Meanwhile, probiotics contained in the mulberry fermentation product has positive regulation effect on the renal function of the diabetic mouse. The mulberry fermentation product can improve the renal function of the type 2diabetes patients to a certain extent.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the embodiment of the invention, the mulberries are purchased from Turpan city of Uygur autonomous region in Xinjiang; lactobacillus plantarum CICC24202(Lactobacillus plantarum CICC24202, Lp for short) purchased from China center for culture Collection of industrial microorganisms; lactobacillus brevis YM1301(Lactobacillus brevis YM1301, abbreviated as Lb) is stored in the institute of microbiology, Yunnan province.

Example 1

The embodiment provides a preparation method of a mulberry fermentation product, which comprises the following steps:

(1) preparation of zymophyte liquid

Sterilizing MRS solid culture medium at 121 deg.C for 25min, placing the slant, cooling to room temperature, streaking in a superclean bench, respectively inoculating Lp and Lb bacteria liquid, culturing at 37 deg.C for 36h in an incubator, and repeatedly activating for 2-3 times.

Pouring 100mL of MRS liquid culture medium into a 250mL conical flask, sterilizing at 121 ℃ for 25min, cooling the liquid culture medium to room temperature, selecting a single colony growing well in a solid culture medium, and inoculating the single colony into the liquid culture medium for culture. And (3) selecting the bacterial liquid which grows to the logarithmic phase according to the growth period of the bacterial liquid, centrifuging to obtain a precipitate, and adding sterile normal saline with the same volume to obtain a bacterial suspension.

(2) Pretreatment of fermented mulberries

The mulberry is cleaned by clear water, dried and crushed, and distilled water is added into mulberry powder according to the proportion of 1g to 5mL (m/V) to prepare fermented mulberry primary pulp. Controlling pH value of the primary pulp of Mori fructus at 6.0 + -0.5, and sterilizing at 121 deg.C for 25 min.

(3) Fermentation inoculation strain suspension

Cooling the sterilized fermented mulberry primary pulp to room temperature, inoculating 10% strain suspension (Lb: Lp ═ 1: 1) in a sterile environment, uniformly mixing, and fermenting in an incubator at 37 ℃ for 120 h.

(4) Preparation of lyophilized powder

And (3) quickly freezing the fermented mulberry pulp at-20 ℃, placing the frozen mulberry pulp in a freeze dryer at-80 ℃ for freeze drying, and crushing to obtain the fermented mulberry freeze-dried powder.

Example 2

This example differs from example 1 in that: and (3) adding distilled water into the mulberry powder according to the proportion of 1g to 6mL (m/V) in pretreatment of the fermented mulberry in the step (2) to prepare fermented mulberry raw pulp. Controlling pH value of the primary pulp of Mori fructus at 6.0 + -0.5, and sterilizing at 121 deg.C for 25 min.

Example 3

This example differs from example 1 in that: inoculating 10% of strain suspension (Lb: Lp ═ 1: 2) into the strain suspension obtained in the step (3) through fermentation, uniformly mixing, and fermenting in an incubator at 37 ℃ for 120 hours.

Example 4

This example differs from example 1 in that: inoculating 12% of strain suspension (Lb: Lp ═ 1: 1) into the strain suspension obtained in the step (3) through fermentation, uniformly mixing, and fermenting in an incubator at 37 ℃ for 120 hours.

Example 5

This example measured the acidity of the mulberry fermented by the probiotic of example 1.

And (4) respectively taking the primary mulberry pulp for 0h, 24h, 48h, 72h, 96h and 120h in the fermentation process in the step (3), diluting the primary mulberry pulp by 10 times to a 100mL volumetric flask, adding water to dilute the primary mulberry pulp to a scale mark, uniformly mixing the primary mulberry pulp at room temperature, and standing for 30 min. Distilled water was used as a control. Precisely sucking 10mL of the solution to be detected into a beaker, and immersing the composite electrode into a proper position of the solution to be detected. Titrating with 0.1mol/L sodium hydroxide solution, taking the titration endpoint when the pH meter shows 8.3 +/-0.1, and recording the volume V of consumed alkali liquor₁. The total acid, expressed as mass of acetic acid per 100mL of sample, was calculated as follows:

C_{total acid}＝(C×(V₁-V₂)×K)×100/(V×M/100)。

In the formula: c_{Total acid}-total acid content in the sample (g/100 mL in acetic acid); c-concentration of sodium hydroxide solution (mol/L); v₁-titration of the volume of spent lye (mL); v₂-control liquid consumption lye volume (mL); v-volume (mL) of liquid to be measured is sucked in titration; m-sample mass or volume (g or mL); k — the converted acid coefficient, expressed in acetic acid, K ═ 0.060.

The acidity value can reflect the effective acidity of the probiotic fermented mulberry sample, and is an important index for reflecting the acidity and fermentation degree of the sample. The results of the acidity values of the raw mulberry pulp measured in this example are shown in fig. 1. As can be seen from FIG. 1, the acidity value increased with the increase of the fermentation time, but the increase of the acidity value was almost stabilized after 96 hours of fermentation. Therefore, 96-120h is the better fermentation time.

Example 6

This example measured the pH during fermentation of mulberries by the probiotic bacteria of example 1.

And (3) respectively taking the primary mulberry pulp of 0h, 24h, 48h, 72h, 96h and 120h in the fermentation process in the step (3), and measuring the pH value of the primary mulberry pulp by using a pH meter, wherein the result is shown in figure 2. The pH value can reflect the effective acidity in the probiotic fermented mulberry sample, wherein the pH value represents H in the sample⁺The activity of (2) is an important index for reflecting the acidity and fermentation degree of the sample. As can be seen from fig. 2, the pH of the raw mulberry pulp tends to decrease with the increase of the fermentation time, but the pH decreases steadily after 96 hours of fermentation. Therefore, 96h-120h is the better fermentation time.

Example 7

In the embodiment, the glucose content in the process of fermenting the mulberry by the probiotics in the embodiment 1 is measured.

And (3) respectively taking the primary mulberry pulp in the fermentation process of the step (3) in the example 1 for 0h, 24h, 48h, 72h, 96h and 120h, preparing the freeze-dried mulberry powder according to the step (4) in the example 1, and measuring the glucose content in the freeze-dried mulberry powder with different time gradients. Measuring by using a glucose oxidase-peroxidase method and a spectrophotometer:

(1) preparing a solution to be detected: and preparing the mulberry sample fermented in different time periods into a solution with the concentration of 50mg/mL by using distilled water, and centrifuging to obtain a supernatant.

(2) Preparing a working solution: reagent 1 (phenol) and reagent 2 (disodium hydrogen phosphate, potassium dihydrogen phosphate, 4-aminoantipyrine, POD, NaN3, GOD) were homogeneously mixed in equal volumes.

(3) Blank tubes, calibration tubes and sample tubes were set, and 1mL of working solution and 10mL of the corresponding solution were added to each tube. Mixing well, and water bathing at 37 deg.C for 15 min. The absorbance values (A) of the calibration tube and the various tubes were determined at a wavelength of 505nm, with blank zeroing.

(4) Calculating the formula: the glucose concentration in the sample was calculated as "glucose concentration C (mg/mL)" [ sample tube absorbance (a)/calibration tube absorbance (a) × calibration solution concentration (5.550) ] × 180/1000, and the glucose content in the sample W (%) ═ glucose concentration (C)/sample concentration (C) × 100%.

The results of the glucose content measurement are shown in FIG. 3. As can be seen from fig. 3, in the fermentation process, the glucose content in the mulberry fermented product tends to decrease with the increase of the fermentation time, but the glucose content decreases steadily after 96 hours of fermentation. Therefore, 96h-120h is the better fermentation time.

Example 8

This example measured the reducing sugar content of the mulberry fermented by the probiotic of example 1.

And (3) respectively taking the primary mulberry pulp in the fermentation process of the step (3) in the example 1 for 0h, 24h, 48h, 72h, 96h and 120h, preparing the freeze-dried mulberry powder according to the step (4) in the example 1, and measuring the content of reducing sugar in the freeze-dried mulberry powder with different time gradients. Measured by the dinitrosalicylic acid method (DNS method) at a wavelength of 520 nm:

(1) preparation of reducing sugar Standard Curve

Accurately weighing 30mg of dry glucose standard substance, and preparing the standard substance with the concentration of 0.30mg/mL by using distilled water; the standard was diluted to 0mg/mL, 0.05mg/mL, 0.15mg/mL, 0.2mg/mL, 0.25mg/mL, and 0.3mg/mL of the standard solution, 1.0mL of each solution was pipetted into 6 tubes with 10mL plugs, 2.0mL of the LDNS solution was added to each group, followed by shaking and mixing, cooling in a water bath at 90 ℃ for 5min, cooling at room temperature for 20min, and then measuring the absorbance at a wavelength of 520 nm. And taking the detected absorbance A as an abscissa axis and the concentration C (mg/mL) of the standard solution as an ordinate axis to obtain a glucose standard curve: the standard curve of glucose is Y-0.0805X-0.0305, R²＝0.9989。

(2) Determination of sample reducing sugar

Precisely weighing 300mg of dried mulberry powder and fermented mulberry powder into 20mL test tubes with stoppers, respectively adding 10.0mL of distilled water, and diluting the obtained supernatant to 0.3mg/mL to obtain a sample solution for measuring the content of reducing sugar. Accurately sucking 1.0mL of sample solution to be detected into 3 10mL test tubes with plugs, adding 2.0mL of LDNS solution into each group, fully oscillating and uniformly mixing, carrying out water bath at 90 ℃ for 5min, cooling at room temperature for 20min, and then measuring the light absorption value at the wavelength of 520 nm.

And calculating the reducing sugar content of different samples according to a standard curve, wherein the reducing sugar content of the sample W (%) ═ concentration of reducing sugar in the sample (C)/concentration of reducing sugar in the sample (C) multiplied by 100%.

The results of the reducing sugar assay are shown in FIG. 4. During the fermentation process, the lactobacillus plantarum and the lactobacillus brevis consume and decompose glucose, fructose and other monosaccharides to generate acidic substances. As can be seen from fig. 4, in the fermentation process, the content of reducing sugar in the mulberry fermented product tends to decrease with the increase of the fermentation time, but the content of reducing sugar slightly increases during 120h of fermentation, so 96h-115h is a better fermentation time.

Example 9

This example was conducted to evaluate the sensory properties of the product of example 1 during fermentation of mulberry with probiotic bacteria.

The mulberry raw juice of 0h, 24h, 48h, 72h, 96h and 120h in the fermentation process in the step (3) in the example 1 is respectively taken to prepare the mulberry freeze-dried powder according to the step (4) in the example 1, 10 classmates (without special taste preference) which accord with sensory evaluation are selected to form an evaluation group, and the sensory evaluation of the mulberry freeze-dried powder with different time gradients is carried out according to the standards listed in the table 1 (the full score is 10).

TABLE 1 sensory evaluation criteria for probiotic fermented mulberries

The evaluation results were collected and subjected to homogenization treatment, and the results are shown in fig. 5. The sensory evaluation of the mulberry fermentation product is mainly considered from two factors of flavor and taste, wherein the acidity is an important index in the investigation and evaluation of the taste. As can be seen from fig. 5, the sensory score tends to increase first and then decrease slightly with the increase of the fermentation time, the probiotic fermentation flavor and the acidity of the mulberry gradually increase and the sweetness gradually decreases with the increase of the fermentation time, and when the fermentation time exceeds 96h, the fermentation flavor and the sweetness of the mulberry probiotic are not obviously changed, and the acidity slightly increases, so that 72-96h is a more reasonable fermentation time.

Example 10

The polysaccharide contents of the mulberry fermented product and the mulberry obtained in example 1 were measured, respectively.

The polysaccharide content is measured by adopting a phenol-sulfuric acid method:

(1) drawing a glucose standard curve

Accurately weighing 10mg of dry glucose standard, and preparing into standard with concentration of 0.10mg/mL with distilled waterPreparing a standard product; diluting the standard substance to 0mg/mL, 0.02mg/mL, 0.04mg/mL, 0.06mg/mL, 0.08mg/mL, 0.1mg/mL of standard substance solution, sucking 1.0mL of the solution into 6 tubes with plugs, adding 1.0mL of 5% phenol solution into each group, slowly adding 5mL of concentrated sulfuric acid, shaking thoroughly, mixing, cooling in 90 deg.C water bath for 20min, cooling at room temperature for 30min, and measuring the absorbance at 490 nm. And taking the detected absorbance A as an abscissa axis and the concentration C (mg/mL) of the standard solution as an ordinate axis to obtain a glucose standard curve: Y0.2014X-0.122, R²＝0.9913。

(2) Mulberry and mulberry fermented product polysaccharide content determination

Weighing 1.0g of dried mulberry fermentation product powder, adding 20 times of distilled water to dissolve in a 50mL conical flask, heating and extracting for 2h in a boiling water bath, repeatedly extracting for 3 times, combining concentrated extracting solutions, adding 4 times of 95% ethanol in volume to filtrate, standing overnight, discarding supernatant, repeatedly precipitating the lower layer part with 95% ethanol, centrifuging, and freeze-drying the precipitate to obtain mulberry polysaccharide (MPP) and mulberry fermentation product polysaccharide (FMPP).

50mg of MPP and FMPP are precisely weighed and are prepared into 0.1mg/mL MPP and FMPP solution to be tested by using distilled water. Sucking 1.0mL of sample solution to be detected into 10mL test tubes with plugs, adding 1.0mL of 5% phenol solution into each test tube, slowly adding 5.0mL of concentrated sulfuric acid, fully oscillating and uniformly mixing, heating in a boiling water bath for 20min, cooling at room temperature for 30min, and measuring the light absorption value at 490 nmnm. The polysaccharide concentration (mg/mL) in the sample was calculated from the standard curve, and the polysaccharide content W (%) in the sample was defined as polysaccharide concentration (C) in the sample/sample concentration (C) × 100%.

The absorbance of the sample was substituted into the standard curve to calculate the polysaccharide content, and the results are shown in Table 2.

TABLE 2 polysaccharide content in Mulberry and Mulberry fermentations: (n＝3)

Note: the letters a and b indicate that the difference has significance by t test (p <0.01)

As can be seen from table 2, the polysaccharide content in the mulberry is 35.09%, the polysaccharide content in the mulberry fermented product after probiotic fermentation is 63.09%, and the difference between the two sets of data is very significant (p < 0.01). The polysaccharide content in the mulberry subjected to fermentation treatment is increased, probably because small molecular saccharides in the mulberry are consumed by probiotics, the polysaccharide content is relatively increased, and the polysaccharide is also probably produced in the fermentation process.

Example 11

The anthocyanin contents of the mulberry fermented product and the mulberry obtained in example 1 were measured, respectively.

Determining the content of anthocyanin by adopting a pH differential method:

weighing 100mg of a sample to be detected, fully dissolving the mulberry and the mulberry fermentation product by using distilled water to obtain 10mg/mL of solution to be detected, sucking 1mL of the solution to be detected, dividing the solution into 3 parts, and respectively fixing the volume to 10mL by using buffer solutions with pH1.0 and pH4.5. Equilibrium was reached after 80min and absorbance was measured at 510nm and 700nm, respectively. Substituting the measured absorbance value into the following formula to obtain anthocyanin content (A)_510nmph1.0-A_700nmph1.0)-(A_510nmph4.5-A_700nmph4.5) The results are shown in Table 3.

TABLE 3 anthocyanin content in Mulberry and Mulberry fermented products: (n＝3)

Note: the letter A, B indicates that the difference between the two is of great significance by t test (p <0.01)

As can be seen from table 3, the mulberry contains 36.74% of anthocyanin, the anthocyanin content in the mulberry fermented product obtained by fermentation treatment is 25.04%, and the two groups of data have very significant differences, which indicates that the anthocyanin content of the mulberry is reduced after fermentation treatment.

Example 12

In this example, the blood glucose-lowering effect of the mulberry fermented product obtained in example 1 was examined.

(1) Molding: 50 male Kunming mice with the weight of 18g +/-2 g are bred in a clean animal room, the indoor constant temperature is kept at 24 +/-1 ℃, the relative humidity is 50-70%, the illumination is carried out for 12 hours, standard experiment feed and free drinking water are adopted, and the experiment is started after the mice are bred for one week adaptively. Mice were randomized into 2 groups: normal control group (n.control) and model building group, normal control group (10) was given with normal feed, model building group (40) was given with high fat feed.

At week 5, a T2DM mouse model was induced by high-fat diet feeding combined with multiple small dose of STZ (70mg/kg) by intraperitoneal injection (STZ dissolved in 0.1mol/L citric acid-sodium citrate buffer, pH4.4, prepared as a 1% strength solution, ready for use). The normal control group was injected with a corresponding volume of citric acid-sodium citrate buffer.

And determining fasting blood glucose by a glucometer 72h after each STZ injection, wherein the mice with the fasting blood glucose of more than or equal to 11.1mmol/L and the fasting blood glucose of more than or equal to 11.1mmol/L after 3 weeks consider the T2DM model mice with successful model building.

(2) Control experiment: mice successfully modeled were randomly divided into 4 groups: model group (Model), positive medicine group (P.control), mulberry group (MP) and mulberry ferment group (FMPH). Beginning intragastric administration of each group of mice in 9 weeks, and administering common feed, drinking water and normal saline 0.2ml per day to normal control group; the model group is fed with high-fat feed and normal diet drinking water, and is intragastrically filled with 0.2ml of normal saline every day; administering high-fat feed and drinking water with normal diet to the positive medicine group, intragastrically irrigating 150mg/kg metformin hydrochloride every day, and preparing a suspension with physiological saline; feeding high-fat feed and drinking water in normal diet to the mulberry group, and preparing 1.0g/kg of mulberry powder into a suspension by using physiological saline every day; and (3) feeding high-fat feed and drinking water by a normal diet in the mulberry fermentation group, and preparing 1.0g/kg of mulberry fermentation product freeze-dried powder into suspension by using physiological saline every day. The weight change and water intake of the mice were recorded regularly during the test period.

(3) Detection of mouse living body related index

Weight and water intake of the mice: during the experiment, the body weight and water intake of the mice were measured and recorded at fixed times, as shown in fig. 6.

As can be seen from fig. 6, the weight of the n.control group mice before administration was slightly higher than that of the four groups of the other models, and the data was very significantly different (p <0.01), and after 4 weeks of administration treatment, the weight of the model group mice still decreased, the weight average of the four groups of the other mice increased slightly, and during the treatment period, it was clearly seen that the food intake and water intake of the model group mice were slightly larger than those of the other groups, and the behavior was daily less active, the response was slower, the crouched dorsum was crouched, the hair was slightly sweated, and the skin was soft; and the symptoms of mice in other administration groups are all reduced, the mice are more active, the hair is smooth, and no obvious skin and hair sweating phenomenon exists.

Measurement of fasting blood glucose: the mice were fasted for 12 hours before and after 4 weeks of administration, and their tail venous blood fasting blood glucose values were measured by a glucometer, and the results are shown in fig. 7.

As can be seen from FIG. 7, the blood glucose levels before administration, except for the N.control group, reached 11.0mmol/L in all four groups, and after 4 weeks of treatment, fasting plasma glucose was increased in the Model group mice compared to before treatment, while fasting plasma glucose was significantly decreased in the other groups. Compared with the rest four groups after four weeks of administration treatment, the fasting blood glucose value has very significant difference (p is less than 0.01), wherein the two groups of MP and FMPH have obvious blood glucose reducing effect compared with the model group, and the fasting blood glucose value data difference before and after treatment of the P.control group and the FMPH group is relatively large and has very significant data difference (p is less than 0.01) by comparing the fasting blood glucose value change before and after treatment of each administration group.

Oral glucose tolerance test: the mice were subjected to an Oral Glucose Tolerance Test (OGTT) 1 week prior to sacrifice. Before the test, the patient is fasted for 12 hours without water, and the glucose is perfused at the dose of 2 g/kg. Blood glucose sampling points are fasting, 30min, 60min and 120min after the glucose load, a glucometer measures the blood glucose value of tail vein blood, the blood glucose value at each time point is marked as G0, G30, G60 and G120, a glucose tolerance curve graph is drawn according to the detection result, and the area under the curve (AUC) is calculated. The calculation formula is as follows: AUC (mmol/L) ═ 1/2 × G0+ G30+ G60+1/2 × G120)/2. The results are shown in FIG. 8.

As can be seen from FIG. 8, Model mice did not quickly recover their blood glucose levels to their initial levels after glucose administration, and the impaired glucose tolerance was relieved to varying degrees in the remaining four groups after treatment. Wherein, the area under FMPH glucose curve (AUC) is greatly different from the Model group and the MP group (p is less than 0.01), which indicates that the mulberry leavening with high dosage has better treatment effect.

Fourthly, mouse insulin tolerance test: the mice were subjected to an Insulin Tolerance Test (ITT) 3 days before sacrifice. Fasting for 2h before the test, measuring the blood glucose value of the tail vein blood at 0min, immediately injecting insulin at 0.75IU/kg (ultra-short-acting) in the abdominal cavity, measuring the blood glucose value of the tail vein blood at 30min, 60min and 120min after injection respectively, marking the blood glucose value of each time point as G0, G30, G60 and G120, drawing an insulin tolerance curve graph according to the detection result, and calculating the area under the curve (AUC). The calculation formula is as follows: AUC (mmol/L) ═ 1/2 × G0+ G30+ G60+1/2 × G120)/2. The results are shown in FIG. 9.

As can be seen from FIG. 9, the blood sugar of the model group mice decreased slowly when insulin was injected compared with the other groups of mice, and the insulin resistance appeared significantly, and the blood sugar of the other four groups decreased slowly after 30min after insulin injection to 120 min. The blood glucose values of the FMPH group and the MP group are significantly different, and the area under the FMPH insulin curve (AUC) and the area under the model group and the mulberry group are significantly different, so that the high-dose mulberry fermentation product has a good treatment effect. In addition, the blood sugar value of the MP group is increased most obviously, but the treatment effect of the high-dose fermented mulberry is better than that of the mulberry comprehensively.

(4) Detection of mouse related biochemical indexes

Detecting serum Insulin (INS) level and insulin resistance index (HOMA-IR) of the mouse: after fasting for 12h, measuring fasting blood glucose of the mice, taking blood from orbital venous plexus, centrifuging to take serum, subpackaging and storing at-20 ℃ for later use; insulin content mouse Insulin (INS) ELISA kit was used, following kit instructions. Calculating the insulin resistance index: HOMA-IR is Fasting Plasma Glucose (FPG) x Fasting Insulin (FINS)/22.5, and the results are shown in fig. 10.

As can be seen from fig. 10, the FINS level and the HOMA-IR level were significantly increased in the Model group mice compared to the n.control group mice and the data were very different, and both the FINS level and the HOMA-IR level were decreased to different degrees in the remaining three treatments. The FINS level and the HOMA-IR level of the FMPH group and the MP group and the FINS level and the HOMA-IR level of the Model group have significant differences, and the FINS level and the HOMA-IR level of the FMPH group and the MP group have significant differences, so that the effect of relieving insulin resistance of type 2diabetes mellitus by using the high-dose mulberry fermentation product is better. The results are combined to show that the mulberry fermentation product can improve insulin sensitivity and insulin resistance, and has better effect than unfermented mulberry fermentation product.

Measurement of mouse serum glycated hemoglobin (GHb): the mouse GHb ELISA test kit was used, the kit instructions were followed and the results are shown in FIG. 11.

As can be seen from FIG. 11, Model group mice showed significantly higher GHb than other group mice, and all four groups showed different reductions in GHb than the Model group. The content of GHb in the N.control group and the Model group is very different, and the content of GHb in the FMPH group and the MP group is different, which shows that the effect of the high-dose mulberry fermentation product on long-term blood sugar control is better.

Testing fasting liver glycogen and muscle glycogen of mice: the results are shown in FIG. 12 following the protocol of the glycogen assay kit.

As can be seen from fig. 12, liver glycogen was significantly increased in the Model group mice compared to the n.control group mice, and liver glycogen was decreased in all of the other three administered treatments compared to the Model group to different extents, since glycogen stored in the liver was not utilized by decomposition or since glucose was converted into liver glycogen after fasting in the Model group mice, thereby stimulating insulin secretion, whereas liver glycogen increase in the Model group mice may be associated with an increase in FINS or insulin sensitivity. The fasting muscle glycogen content of the mice has no obvious difference between the N.control group and the Model group.

Measuring mouse glucagon-like peptide 1 (GLP-1): the results are shown in FIG. 13 using a mouse glucagon-like peptide 1(GLP-1) ELISA test kit, according to the kit instructions.

As can be seen from FIG. 13, the GLP-1 level of the Model group mice was significantly reduced and the data was very significantly different from that of the N.control group mice, and the GLP-1 level of the other three drug treatments was increased to different extents from that of the Model group; the GLP-1 levels of the MP group and the FMPH group and the GLP-1 levels of the Model group are remarkably or extremely remarkably different, and the fact that the mulberry fermentation product can effectively promote cells to secrete GLP-1 is shown.

Example 13

In this example, the blood lipid effect of the mulberry fermentation product obtained in example 1 on T2DM mice was tested.

(1) The serum total cholesterol (T-CHO), total Triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) content of each group of experimental mice in example 12 were measured according to the kit instructions, and the results are shown in FIG. 14.

As can be seen from FIG. 14, the blood lipid levels (TG, T-CHO, LDL-C and HDL-C) of the Model group were all higher than those of the N.control group, indicating that the mice were caused by lipid metabolism disorder due to long-term high-fat feeding, the serum levels of T-CHO, TG, LDL-C and HDL-C of the Model group were all significantly increased and significantly different compared with the N.control group, and the levels of T-CHO, TG, LDL-C and HDL-C of the remaining three administered treatments were all decreased to different degrees compared with the Model group; among them, the P.control, MP and FMPH groups all had very significant differences in T-CHO, TG, LDL-C and HDL-C levels compared to the Model group. Compared with the FMPH group, the MP group and the FMPH group have extremely obvious or obvious difference in T-CHO and TG levels, which indicates that both MP and FMPH can reduce the levels of TG, T-CHO, LDL-C and HDL-C in serum, and the MP group and the FMPH group have higher HDL-C levels, which indicates that FMPH has better blood fat reducing effect.

(2) Determination of epididymal fat weight and epididymal fat index

Mice were dissected and epididymal fat weight (EAM) isolated and weighed. The epididymal fat index (EAMI) was calculated according to the formula: epididymal fat index (epididymal fat weight/body weight) × 100%. The results are shown in Table 4.

TABLE 4 Effect of MP and FMPH on EAM and EAMI in type 2 diabetic mice: (n＝10)

Note: p <0.05, p <0.01 for comparison to model groups; # p <0.05, # p <0.01, indicating comparison with the normal control group.

As can be seen from Table 4, the MP group and FMPH group reduced fat accumulation in the periphery of epididymis, and the EAM and EAMI of the three-dose treatment groups were all decreased to different degrees compared to the Model group; among them, the EAM and EAMI of mice were significantly different in both the MP group and the FMPH group compared to the Model group (p < 0.05). The mulberry fermentation product is shown to reduce the blood sugar of the type 2diabetes patients and reduce the fat accumulation, and has certain effect on losing weight and reducing fat.

Example 14

This example was conducted to test the effect of the mulberry fermentation product obtained in example 1 on the kidney function of T2DM mice.

The common complication of diabetes is diabetic nephropathy, and the change of renal function is mainly reflected in the filtration of related components in the urine generation process, wherein the blood urea nitrogen level and the serum creatinine level represent the change of the renal filtration function, and the blood urea nitrogen level and the serum creatinine level are increased in the renal pathological changes. In this experiment, compared with the model group, the blood urea nitrogen level and the serum creatinine level given to the mulberry fermented product group were decreased in Ming county, wherein the decrease in blood urea nitrogen level was more significant.

(1) Mouse Blood Urea Nitrogen (BUN) assay: the results are shown in FIG. 15 using a mouse Urea Nitrogen (BUN) ELISA test kit, according to the kit instructions.

As can be seen from fig. 15, the BUN levels were significantly increased in the Model group mice compared to the n.control group mice and the data were very poor (p <0.01), with all three remaining treatments administered being reduced to different extents compared to the Model group; among them, BUN levels were very significantly different (p <0.01) for the p.control, MP, and FMPH groups versus the Model group; the FMPH group showed lower BUN expression levels than the MP group, and the data were very significantly different (p < 0.01). The BUN value of FMPH group is 2.20mmol/L at the lowest, and the BUN content of Model group is 4.61mmol/L, which shows that the mulberry fermentation product can obviously reduce the BUN of the mouse.

(2) Mouse serum creatinine (Scr) assay: the mouse urea nitrogen (Scr) ELISA test kit was used, according to the kit instructions, and the results are shown in figure 16.

As can be seen from fig. 16, Model group mice showed significantly higher Scr levels and very poor data (p <0.01) compared to n.control group mice, and Scr levels were decreased in both p.control group and FMPH group compared to Model group; the levels of the p.control group, and FMPH group and the Model group Scr were very significantly different (p < 0.01); the expression level of Scr in the MP group is slightly reduced compared with that in the Model group, but the difference is not statistically significant (p > 0.05); the FMH group MP group had a reduced level of Scr expression compared to the control group and had a very significant difference (p < 0.01). The content of Scr in a Model group is 19.88ng/mL, and the content of Scr in an FMPH group is 17.81ng/mL, which indicates that the mulberry fermentation product can obviously reduce the Scr of the mice.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.