Mulberry leaf fermented extract with slow-release sugar effect, preparation method and application thereof

Abstract

The present invention belongs to the technical field of fermented extract preparation, and specifically relates to a mulberry leaf fermented extract with a slow-release sugar effect, and a preparation method and application thereof. The present invention first adds saccharomyces cerevisiae separated from the surface of mulberry leaves while ensuring that the content of 1-deoxynojirimycin in the mulberry leaf extract remains unchanged, and utilizes the method of microbial fermentation to fully release and retain the nutrition and active ingredients of the mulberry leaves, and ensures the dissolution rate of the active ingredients of the mulberry leaves while also ensuring safety; secondly, the use of organic solvents is reduced by fermentation, and environmental pollution is avoided; again, the bitterness of the mulberry leaf extract is reduced or even avoided, and the fragrance of mulberry leaf tea is improved; finally, the microbial varieties in the method are easy to obtain, the fermentation process is simple, the use cost is low, and the microbial fermentation is efficient and repeatable, whether it is laboratory small-scale preparation or factory large-scale production, it has cost advantages and has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation extract preparation, and particularly relates to a mulberry leaf fermentation extract with a slow-release sugar effect, a preparation method and an application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] As a metabolic disease characterized by high blood sugar, diabetes is mainly manifested by a decline in the function of the pancreas that secretes insulin due to long-term overeating, which leads to insufficient supply of insulin, the transporter of glucose generated by the breakdown of regular staple foods. These glucoses that remain in the blood waiting for insulin to transport them form symptoms of postprandial high blood sugar. Therefore, controlling the decomposition rate of carbohydrates in staple foods can control the symptoms of high blood sugar after meals in diabetics.

[0004] Mulberry belongs to the Moraceae family and the genus Morus. It is a deciduous tree or shrub and is widely distributed in my country. Mulberry leaves, as the main product of mulberry trees, can not only be used as feed for silkworms, but also as traditional Chinese herbal medicines. Mulberry leaves are rich in bioactive substances such as flavonoids, alkaloids, polyphenols, polysaccharides and various amino acids, and have anti-inflammatory, antioxidant, immune-enhancing and lipid-regulating effects. Modern Chinese and Western medicine use mulberry leaves and mulberry leaf biological preparations as drugs to improve diabetes and other difficult and complicated diseases, and believe that their efficacy is extremely wide. There are lungs and moisturizing, cough, heat, phlegm, liver and eyesight, treatment of dizziness, insomnia, elimination of eye fatigue, lowering blood pressure, lowering blood lipids, preventing myocardial infarction, cerebral hemorrhage, lowering blood sugar, and anti-diabetes. In order to maximize the utilization value of the active ingredients in mulberry leaves, the active ingredients in mulberry leaves are currently extracted and purified. Mulberry leaf extracts are rich in 1-deoxynojirimycin (DNJ), which can significantly reduce postprandial blood sugar and insulin sensitivity. Using mulberry leaves in various staple foods can significantly reduce the activity of α-glucosidase and reduce the rate of increase in blood sugar after a meal. However, mulberry leaf extract has a strong bitter taste, which limits the application of mulberry leaf extract. Summary of the invention

[0005] Based on the above-mentioned deficiencies in the prior art, the present invention provides a mulberry leaf fermented extract with a sustained sugar release effect, and a preparation method and application thereof. The present invention separates a strain of brewer's yeast from the surface of mulberry leaves, and utilizes a microbial fermentation method to fully release and retain the nutrients and active ingredients of the mulberry leaves, while effectively reducing or even avoiding the bitterness of the mulberry leaf extract.

[0006] In order to achieve the above technical objectives, the present invention relates to the following technical solutions:

[0007] One aspect of the present invention provides a strain of Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Nice-01, deposited in the China Center for Type Culture Collection (Address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), the deposit date is December 9, 2022, and its biological deposit number is CCTCC NO: M 20221903.

[0008] The second aspect of the present invention provides a microbial agent, which contains at least Saccharomyces cerevisiae Nice-01.

[0009] In a third aspect of the present invention, the above-mentioned Saccharomyces cerevisiae Nice-01 or microbial agent is used in any one or more of the following:

[0010] (a) producing a mulberry leaf fermented extract having a slow-release sugar effect;

[0011] (b) Reduce or eliminate the bitterness of mulberry leaf fermentation products and enhance their fragrance.

[0012] Wherein, the sustained-release sugar comprises 1-deoxynojirimycin.

[0013] The fourth aspect of the present invention provides a mulberry leaf fermentation extract having a slow-release sugar effect, wherein the mulberry leaf fermentation extract is prepared by the following method:

[0014] S1, adding water to the pretreated mulberry leaves, raising the temperature and performing ultrasonic-assisted extraction;

[0015] S2, sterilizing the liquid obtained in S1 and inoculating the above-mentioned brewer's yeast Nice-01 or the above-mentioned microbial agent for fermentation;

[0016] S3. Centrifuge the fermentation product and take the supernatant filtrate.

[0017] The fifth aspect of the present invention provides the use of the above-mentioned mulberry leaf fermentation extract in preparing products.

[0018] The product described is a pharmaceutical product.

[0019] Beneficial technical effects of one or more of the above technical solutions:

[0020] The above technical scheme provides a mulberry leaf fermentation powder with a slow-release sugar effect and a preparation method thereof. While ensuring that the content of 1-deoxynojirimycin (DNJ) in the mulberry leaf extract remains unchanged, firstly, brewer's yeast separated from the surface of the mulberry leaf is added, and the method of microbial fermentation is used to fully release and retain the nutrients and active ingredients of the mulberry leaf, and the dissolution rate of the active ingredients of the mulberry leaf is ensured while ensuring safety; secondly, the use of organic solvents is reduced through fermentation, avoiding environmental pollution; thirdly, the bitterness of the mulberry leaf extract is reduced or even avoided, and the fragrance of the mulberry leaf tea is improved; finally, the microbial species in this method are easy to obtain, the fermentation process is simple, the use cost is low, and the microbial fermentation is efficient and repeatable, whether it is a small-scale laboratory test preparation or a large-scale factory production, it has cost advantages, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 This is the change of postprandial blood sugar in diabetic rats after feeding mulberry leaf fermented powder in the effect verification example of the present invention.

[0023] Figure 2 The changes in the pancreatic organs of diabetic rats before and after feeding mulberry leaf fermented powder in the effect verification example of the present invention; wherein a1 and a2 are mulberry leaf fermented powder feeding groups, and b1 and b2 are diabetic rat groups. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0026] In one embodiment of the present invention, a strain of Saccharomyces cerevisiae ( Saccharomyces cerevisiae) Nice-01, deposited in China Center for Type Culture Collection (Address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), deposited on December 9, 2022, and its biological deposit number is CCTCC NO: M 20221903. The strain was isolated by the inventor from the surface of mulberry leaves, and was confirmed to belong to Saccharomyces cerevisiae after morphological, physiological and biochemical and 18S rDNA sequence analysis.

[0027] In another specific embodiment of the present invention, a microbial agent is provided, which contains at least Saccharomyces cerevisiae Nice-01.

[0028] In another specific embodiment of the present invention, the microbial agent, in addition to the active ingredient, also contains a carrier, which can be a commonly used and biologically inert carrier in the field of microbial preparations.

[0029] The carrier may be a solid carrier or a liquid carrier;

[0030] The solid carrier may be a mineral material, a plant material and / or a polymer compound; the mineral material may be at least one of clay, talc, medical stone, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, bean flour, rice husk flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol;

[0031] The liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0032] The bacterial agent may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule; preferably, powder.

[0033] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0034] In another specific embodiment of the present invention, the above-mentioned Saccharomyces cerevisiae Nice-01 or microbial agent is used in any one or more of the following:

[0035] (a) producing a mulberry leaf fermented extract having a slow-release sugar effect;

[0036] (b) Reduce or eliminate the bitterness of mulberry leaf fermentation products and enhance their fragrance.

[0037] Wherein, the sustained-release sugar comprises 1-deoxynojirimycin.

[0038] In another specific embodiment of the present invention, a mulberry leaf fermentation extract with a slow-release sugar effect is provided, and the mulberry leaf fermentation extract is prepared by the following method:

[0039] S1, adding water to the pretreated mulberry leaves, raising the temperature and performing ultrasonic-assisted extraction;

[0040] S2, sterilizing the liquid obtained in S1 and inoculating the above-mentioned brewer's yeast Nice-01 or the above-mentioned microbial agent for fermentation;

[0041] S3. Centrifuge the fermentation product and take the supernatant filtrate.

[0042] Wherein, in the step S1, the mulberry leaves are preferably mulberry leaves with a high DNJ content, and can generally be selected from mulberry leaves picked in Guangdong and Guangxi regions in October each year;

[0043] The pretreatment method includes: drying (such as drying) and crushing (such as ultrafine crushing) the harvested mulberry leaves, so as to facilitate subsequent extraction and fermentation.

[0044] The volume ratio of mulberry leaves to water is 1:10-20, the heating temperature is controlled to be 80-100°C, and in the ultrasonic-assisted extraction, the ultrasonic condition is controlled to be 70-90 kHz; the processing time is controlled to be 60-200 min.

[0045] In the step S2, the sterilization may be performed by any known sterilization treatment. In a specific embodiment of the present invention, the sterilization may be performed by boiling sterilization. Specifically, the feed liquid is heated to 100-101° C. for boiling sterilization for 1-10 min.

[0046] The specific method of the fermentation treatment is: fermentation at 21-40° C. for 4-10 days; more specifically, aerobic fermentation is adopted, and the rotation speed is controlled at 100-300 rpm.

[0047] Among them, the inoculation amount of brewer's yeast Nice-01 is controlled at 1-3%.

[0048] In step S3, the specific conditions for the centrifugal treatment are: 4-30° C., 1000-8000 rpm.

[0049] At this time, the mulberry leaf fermentation extract is liquid. For the convenience of storage, transportation and use, the filtrate can be spray-dried to obtain a solid powder, namely, mulberry leaf fermentation powder.

[0050] In another specific embodiment of the present invention, there is provided application of the above-mentioned mulberry leaf fermentation extract in preparing products.

[0051] The product described is a pharmaceutical product.

[0052] The drug can be administered in a unit dosage form, liquid dosage form, solid dosage form. The liquid dosage form can be a true solution, colloid, microparticle dosage form, emulsion dosage form, suspension dosage form, etc. Other dosage forms include tablets, capsules, dripping pills, pills, powders, emulsions, granules, suppositories, inclusion compounds, landfills, etc.

[0053] The medicine of the present invention may also contain a commonly used carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, alumina, aluminum stearate, lecithin, glycerol, sorbitol, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, water, salt or electrolyte, etc. The content of the carrier in the medicine may be 1% by weight to 98% by weight, usually accounting for about 80% by weight.

[0054] The present invention is further explained by the following examples, but it does not constitute a limitation of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The mulberry leaves used in each embodiment are all mulberry leaves from the same mulberry garden in Liuzhou, Guangxi, picked in October 2021.

[0055] Example 1

[0056] A mulberry leaf fermented powder with a slow-release sugar effect, and a preparation method thereof, comprising the following steps:

[0057] (1) picking mulberry leaves, drying them and then ultra-fine grinding them;

[0058] (2) Add 10 times the volume of water, heat to 100 °C, and perform 90 kHz ultrasonic-assisted extraction for 60 min;

[0059] (3) After the extraction is completed, the liquid is transferred to a fermentation tank and heated to 100 °C for boiling sterilization for 6 min;

[0060] (4) Inoculate with 2% brewer's yeast and ferment at 180 rpm and 30 °C for 1 day;

[0061] (5) After fermentation, centrifuge at 20°C and 1000 rpm to obtain the supernatant filtrate;

[0062] (6) The filtrate is further spray-dried to obtain mulberry leaf fermented powder.

[0063] Example 2

[0064] A mulberry leaf fermented powder with a slow-release sugar effect, and a preparation method thereof, comprising the following steps:

[0065] (1) picking mulberry leaves, drying them and then ultra-fine grinding them;

[0066] (2) Add 10 times the volume of water, heat to 90 °C, and perform 80 kHz ultrasonic-assisted extraction for 110 min;

[0067] (3) After the extraction is completed, the liquid is transferred to a fermentation tank and heated to 101°C for boiling sterilization for 1 min;

[0068] (4) Inoculate with 4% brewer's yeast and ferment at 200 rpm and 28 °C for 7 days;

[0069] (5) After fermentation, centrifuge at 21 °C and 4000 rpm to obtain the supernatant filtrate;

[0070] (6) The filtrate is further spray-dried to obtain mulberry leaf fermented powder.

[0071] Example 3

[0072] A mulberry leaf fermented powder with a slow-release sugar effect, and a preparation method thereof, comprising the following steps:

[0073] (1) picking mulberry leaves, drying them and then ultra-fine grinding them;

[0074] (2) Add 10 times the volume of water, heat to 80 °C, and perform 100 kHz ultrasonic-assisted extraction for 190 min;

[0075] (3) After the extraction is completed, the liquid is transferred to a fermentation tank and heated to 91°C for boiling sterilization for 10 min;

[0076] (4) Inoculate with 3% brewer's yeast and ferment at 300 rpm and 31 °C for 6 days;

[0077] (5) After fermentation, centrifuge at 28 °C and 6000 rpm to obtain the supernatant filtrate;

[0078] (6) The filtrate is further spray-dried to obtain mulberry leaf fermented powder.

[0079] Example 4

[0080] A mulberry leaf fermented powder with a slow-release sugar effect, and a preparation method thereof, comprising the following steps:

[0081] (1) The harvested mulberry leaves are dried and then ultra-finely crushed;

[0082] (2) Add 10 times the volume of water, heat to 80 °C, and perform 100 kHz ultrasonic-assisted extraction for 190 min;

[0083] (3) After the extraction is completed, the liquid is transferred to a fermentation tank and heated to 91°C for boiling sterilization for 10 min;

[0084] (4) Inoculate 3% of purchased industrial brewer's yeast and ferment at 300 rpm and 31 °C for 6 days;

[0085] (5) After fermentation, centrifuge at 28 °C and 6000 rpm to obtain the supernatant filtrate;

[0086] (6) The filtrate is further spray-dried to obtain mulberry leaf fermented powder.

[0087] Effect verification example

[0088] 1. The detection of DNJ content in mulberry leaf fermented powder was carried out using the mulberry leaf extract on the market as a control and the national standard GB / T40642-2021, as shown in Table 1.

[0089] Table 1 DNJ content in mulberry leaf extract and mulberry leaf fermented powder

[0090]

[0091] From the test data in Table 1, it can be seen that the content of DNJ in mulberry leaf fermentation powder before and after fermentation with the microorganisms isolated by us is equivalent to that of mulberry leaf extract sold on the market, and remains basically unchanged.

[0092] 2. Bitterness Detection

[0093] Weigh 3.0 g of mulberry leaf fermented powder and mulberry leaf extract, add them into 300 mL of pure water, taste the taste, and record the bitterness range value, as shown in Table 2.

[0094] Table 2 Bitterness range of mulberry leaf extract and mulberry leaf fermented powder

[0095]

[0096] Comprehensive taste evaluation of the bitterness of mulberry leaf fermented powder before and after fermentation shows that, while keeping the DNJ content basically unchanged, the bitterness of the mulberry leaf powder fermented by the microorganisms we isolated is significantly reduced compared with the mulberry leaf extract at the same concentration.

[0097] III. Application of mulberry leaf fermented powder in Example 3 in blood sugar regulation

[0098] (1) Establishment of type 2 diabetes mellitus (T2DM) rat model: Several healthy male Wistar rats with uniform body weight of 200±20 g were selected. Fasting plasma glucose (FPG) was tested before the experiment. Those with normal fasting plasma glucose were selected for the experiment. The rats were numbered and adaptively fed for 1 week. After the adaptive feeding, they were fed a high-sugar and high-fat diet. After the high-sugar and high-fat diet was finished, a small dose of streptozotocin was injected intraperitoneally once according to the rat's body weight in the fasting state. FPG was tested. A successful T2DM model was considered when fasting plasma glucose ≥7.0 mmol / L.

[0099] (2) Feeding diabetic rats with mulberry leaf fermented powder: The model rats were divided into groups for feeding, including a diabetic control group (0.2 mL / kg / d of normal saline per meal), an acarbose group (10 mg / kg / d of acarbose), and a mulberry leaf fermented powder group (200 mg / kg / d of mulberry leaf fermented powder). The feeding time for each group was 8:00 a.m. and 20:00 p.m. Every 3 days, blood was collected from the rats’ tail vertebrae 1 hour after the end of feeding in the morning for blood glucose measurement. After about 4 weeks of feeding, the rats were euthanized, the chest cavity was opened, the heart was punctured for blood collection, and the pancreas and other major organs were removed. They were quickly frozen with liquid nitrogen and quickly transferred to a -80°C refrigerator for storage. The sections were sliced ​​and stained with HE to observe pathological changes.

[0100] (3) Effect of mulberry leaf fermented powder on blood sugar regulation: Through experiments, it can be seen that Figure 1 and Figure 2 As shown, the postprandial blood sugar of rats fed with mulberry leaf fermented powder rose slowly, and the blood sugar control effect was better, which was similar to the blood sugar control effect of the diabetes drug acarbose. After feeding, it was found that the pancreatic acinar cells of rats in the mulberry leaf fermented powder group had normal morphology and clear boundaries, while the rats in the diabetic control group showed a small amount of capillary dilation (black arrows) and a small amount of inflammatory cell infiltration (white arrows).

[0101] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A strain of Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Nice-01, deposited in China Center for Type Culture Collection, the deposit date is December 9, 2022, and its biological deposit number is CCTCC NO: M 20221903.

2. A microbial agent, characterized in that: The method comprises at least the cerevisiae yeast Nice-01 according to claim 1.

3. The microbial agent according to claim 2, characterized in that In addition to the active ingredients, the microbial agent also contains a carrier.

4. Use of the cerevisiae yeast Nice-01 according to claim 1 or the microbial agent according to claim 2 in any one or more of the following: (a) producing a mulberry leaf fermented extract containing 1-deoxynojirimycin; (b) reducing or eliminating the bitter taste in the fermented mulberry leaf extract and improving its fragrance.

5. A mulberry leaf fermentation extract containing 1-deoxynojirimycin comprising the cerevisiae Nice-01 of claim 1, characterized in that: The mulberry leaf fermented extract is prepared by the following method: S1, adding water to the pretreated mulberry leaves, raising the temperature and performing ultrasonic-assisted extraction; S2, sterilizing the liquid obtained in S1 and inoculating the above-mentioned brewer's yeast Nice-01 for fermentation; S3. Centrifuge the fermentation product and take the supernatant filtrate.

6. The mulberry leaf fermented extract according to claim 5, characterized in that In the step S1, the mulberry leaves are selected from mulberry leaves picked in Guangdong and Guangxi regions in October each year; The pretreatment method comprises: drying and crushing the picked mulberry leaves.

7. The mulberry leaf fermented extract according to claim 5, characterized in that In the step S1, the volume ratio of the mulberry leaves to water is 1:10-20, the heating temperature is controlled to be 80-100°C, and in the ultrasonic-assisted extraction, the ultrasonic condition is controlled to be 70-90 kHz; the processing time is controlled to be 60-200 min.

8. The mulberry leaf fermented extract according to claim 5, characterized in that In step S2, the sterilization is performed by boiling sterilization; specifically, the feed liquid is heated to 100-101° C. for boiling sterilization for 1-10 min; The specific method of the fermentation treatment is: fermentation at 21-40°C for 4-10 days; Among them, the inoculation amount of brewer's yeast Nice-01 is controlled at 1-3%.

9. The mulberry leaf fermented extract according to claim 8, characterized in that Aerobic fermentation was adopted, and the rotation speed was controlled at 100-300 rpm.

10. The mulberry leaf fermented extract according to claim 5, characterized in that In step S3, the specific conditions for the centrifugal treatment are: 4-30° C., 1000-8000 rpm.

11. The mulberry leaf fermented extract according to claim 5, characterized in that The supernatant filtrate is spray-dried to obtain a solid powder, namely, mulberry leaf fermented powder.

12. Use of the mulberry leaf fermented extract according to any one of claims 5 to 11 in the preparation of medicines for treating type 2 diabetes.

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