Mulberry leaf fermentation extract as well as preparation method and application thereof

Abstract

The invention discloses a mulberry leaf fermentation extract as well as a preparation method and application thereof, and belongs to the technical field of fermentation extract preparation. The preparation method of the mulberry leaf fermentation extract comprises the following steps: S1, mincing dry mulberry leaves, putting the minced mulberry leaves into an ethanol water solution, carrying out ultrasonic extraction, and distilling; adding distilled water to a constant volume, adjusting pH, and sterilizing at high temperature to obtain mulberry leaf feed liquid; s2, firstly inoculating fungi into the mulberry leaf feed liquid, performing liquid fermentation treatment for 2-3 days, then inoculating lactobacillus plantarum and pediococcus pentosaceus, performing liquid fermentation treatment for 4-5 days to obtain a fermentation product, centrifuging the fermentation product, taking supernatant filtrate, and drying to obtain the mulberry leaf fermentation extract. Comprising the following active components: 1-deoxynojirimycin (DNJ) and flavone, the content of 1-deoxynojirimycin is greater than 1.2%, and the content of flavone is greater than 1%; the bitter taste is greatly reduced, and the product can be applied to hypoglycemic food.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation extract preparation, and specifically relates to a mulberry leaf fermentation extract and a preparation method and application thereof. Background Art

[0002] 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 in food and other fields.

[0003] The patent application with publication number CN 107836707 A discloses a preparation method and application of a healthy food base material with the function of improving glucose and lipid metabolism disorders, comprising the following steps: picking and pretreatment of mulberry leaves; preparation of fresh mulberry leaf puree; preparation of mulberry leaf acid polysaccharides; preparation of bitter buckwheat extract; preparation of oat extract; preparation of composite puree; sterilization and fermentation; drying, packaging and sterilization. This application retains most of the active ingredients, but the flavor can be further improved. Therefore, it is extremely necessary to use microbial fermentation to promote the degradation of macromolecular substances in mulberry leaves, promote the transformation and generation of volatile components, ensure that the effective ingredients in mulberry leaves are not lost, and reduce the green smell of mulberry leaves. Summary of the invention

[0004] The purpose of the present invention is to provide a mulberry leaf fermentation extract and a preparation method and application thereof, so as to increase the content of active ingredients in the mulberry leaf fermentation extract.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A method for preparing a mulberry leaf fermented extract comprises the following steps:

[0007] S1. Crush the dried mulberry leaves, place them in an ethanol aqueous solution for ultrasonic extraction, and distill them; add distilled water to make up the volume, adjust the pH, and sterilize them at high temperature to obtain a mulberry leaf liquid;

[0008] S2. The mulberry leaf liquid is first inoculated with fungi, and then inoculated with Lactobacillus plantarum and Pediococcus pentosaceus after liquid fermentation for 2 to 3 days. After liquid fermentation for 4 to 5 days, a fermentation product is obtained. The fermentation product is centrifuged to obtain the supernatant filtrate, and the mulberry leaf fermentation extract is obtained after drying.

[0009] Furthermore, the concentration of the ethanol aqueous solution is 65% to 75%; the usage ratio of the mulberry leaves and the ethanol aqueous solution is 8 to 10 g: 100 to 150 mL; the ultrasonic extraction time is 0.5 to 1 h; and the distillation method is rotary evaporation at 80°C.

[0010] Furthermore, the pH is adjusted to 5-6; and the high temperature sterilization condition is sterilization at 110-120° C. for 20-30 min.

[0011] Furthermore, the fungus is one of Aspergillus oryzae and Mucor.

[0012] Furthermore, the inoculation amount of Aspergillus oryzae is 6% to 8% of the mass of dry mulberry leaves, and the inoculation amount of Lactobacillus plantarum and Pediococcus pentosaceus is 8% to 10% of the mass of dry mulberry leaves; the inoculation concentration of Aspergillus oryzae is 1*10 6 ~1*10 7 cfu / mL, the inoculation concentration of Lactobacillus plantarum and Pediococcus pentosaceus was 1*10 7 ~5*10 7 cfu / mL.

[0013] Furthermore, the fermentation treatment conditions are 30-40°C and 150-200r / min.

[0014] Furthermore, the mulberry leaf fermentation extract comprises an active ingredient 1-deoxynojirimycin (DNJ), and the content of 1-deoxynojirimycin is greater than 1.2%.

[0015] Furthermore, the mulberry leaf fermentation extract also includes flavonoids, and the flavonoid content is greater than 1%.

[0016] Furthermore, the mulberry leaf fermented extract is used in the preparation of blood sugar lowering food.

[0017] It should be noted that the term "food" used in the present invention has a broad meaning, and generally refers to any item that can be eaten by humans. Therefore, the food mentioned not only includes common food suitable for all people, but also covers special food, wherein special food includes but is not limited to food with specific health functions and nutritional formula food designed for special medical needs.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention adopts a method for preparing a mulberry leaf fermentation extract, which reduces the green smell of the mulberry leaf fermentation extract while ensuring that the main active ingredients of the mulberry leaf fermentation extract are not lost. The Aspergillus oryzae secretes proteases, cellulases, etc., which are beneficial to the production of soluble substances and reduce the crude fiber content; Lactobacillus plantarum has the ability to deglycosylate bound phenolic substances to increase the content of free phenolic substances. The hydrolase secreted by Pediococcus pentosaceus decomposes the plant cell wall, hydrolyzes complex phenolic compounds into simpler forms, and releases soluble conjugated or insoluble bound phenolic and flavonoid compounds.

[0020] (2) The fungi used in the present invention can produce a variety of enzymes, such as proteases and amylases, which help to decompose complex organic substances and provide more easily digestible substrates for Lactobacillus plantarum and Pediococcus pentosaceus, thereby promoting their growth and metabolic activities. The multiple inoculations also reduce the competition between fungi and bacteria. In addition, the co-inoculation of Lactobacillus plantarum and Pediococcus pentosaceus increases the diversity of the bacterial community, which helps to improve the quality of the mulberry leaf fermented extract.

[0021] (3) The ultrasonic extraction method of ethanol-water solution adopted in the present invention can better extract the active ingredients in mulberry leaves compared with the traditional water decoction. At the same time, the removal temperature of ethanol is lower than that of water, which reduces the loss of effective active ingredients. On the other hand, the liquid fermentation adopted has sufficient material exchange, high fermentation uniformity, and easy real-time monitoring and control of fermentation conditions, which is conducive to the full contact and absorption of nutrients by microorganisms, so that the microorganisms can grow and ferment rapidly. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] This embodiment provides a mulberry leaf fermentation extract, which is prepared by the following steps:

[0025] S1. Crush 10g of dry mulberry leaves, place them in 150mL of 75% ethanol aqueous solution for ultrasonic extraction for 1h, and remove ethanol by rotary evaporation at 80℃; add distilled water to make the volume 120mL, adjust the pH to 6, and sterilize at 115℃ for 20min to obtain mulberry leaf liquid;

[0026] S2, inoculate mulberry leaf liquid with 6% concentration of 5*106 cfu / mL Aspergillus oryzae, treated at 35°C and 150 r / min, and inoculated with 8% and 1*10 7 cfu / mL of Lactobacillus plantarum and Pediococcus pentosaceus were treated at 35°C and 150r / min for 4 days to obtain a fermentation product. The fermentation product was centrifuged to obtain the supernatant filtrate, which was dried to obtain the mulberry leaf fermentation extract.

[0027] The mulberry leaf fermented extract is used in foods for lowering blood sugar.

[0028] Example 2

[0029] The present embodiment is different from the embodiment 1 in that the "75% ethanol aqueous solution" in S1 is changed to "65% ethanol aqueous solution".

[0030] The remaining raw materials and preparation process remain the same as in Example 1.

[0031] Example 3

[0032] The present embodiment is different from the embodiment 1 in that the “ultrasonic extraction for 1 h” in S1 is changed to “ultrasonic extraction for 0.5 h”.

[0033] The remaining raw materials and preparation process remain the same as in Example 1.

[0034] Example 4

[0035] The present embodiment is different from the embodiment 1 in that "adjusting the pH to 6" in S1 is changed to "adjusting the pH to 5".

[0036] The remaining raw materials and preparation process remain the same as in Example 1.

[0037] Example 5

[0038] The difference between this embodiment and embodiment 1 is that the "first inoculation of 6% with a concentration of 5*10 6 cfu / mL Aspergillus oryzae, inoculated with 8% concentration of 1*10 7 cfu / mL Lactobacillus plantarum and Pediococcus pentosaceus" was changed to "First inoculate 8% concentration of 5*10 6 cfu / mL Aspergillus oryzae, and 10% of the concentration was 1*10 7 cfu / mL Lactobacillus plantarum and Pediococcus pentosaceus”.

[0039] The remaining raw materials and preparation process remain the same as in Example 1.

[0040] Example 6

[0041] The present embodiment is different from the embodiment 1 in that "Aspergillus oryzae" in S2 is changed to "Mucor".

[0042] The remaining raw materials and preparation process remain the same as in Example 1.

[0043] Example 7

[0044] Compared with Example 1, this embodiment differs in that the post-inoculation treatment time in S2 is prolonged, and the specific implementation steps are as follows:

[0045] S2, inoculate mulberry leaf liquid with 6% concentration of 5*10 6 cfu / mL Aspergillus oryzae, treated at 35°C and 150 r / min, and inoculated with 8% and 1*10 7 cfu / mL of Lactobacillus plantarum and Pediococcus pentosaceus were treated at 35°C and 150r / min for 5 days in liquid fermentation to obtain a fermentation product. The fermentation product was centrifuged to obtain the supernatant filtrate, which was dried to obtain the mulberry leaf fermentation extract.

[0046] The remaining raw materials and preparation process remain the same as in Example 1.

[0047] Comparative Example 1

[0048] Compared with Example 1, this comparative example is different in that the extraction is carried out by water decoction. The specific implementation method is as follows:

[0049] S1. Crush 10g of dry mulberry leaves, place in 150mL of water and boil for 1h, adjust the pH to 6, and sterilize at 115℃ for 20min to obtain mulberry leaf liquid;

[0050] S2, inoculate mulberry leaf liquid with 6% concentration of 5*10 6 cfu / mL Aspergillus oryzae, treated at 35°C and 150 r / min, 2 to 3 days later, 8% of the concentration was inoculated at 1*10 7 cfu / mL of Lactobacillus plantarum and Pediococcus pentosaceus, treated at 35°C and 150r / min, and subjected to liquid fermentation treatment for 4-5 days to obtain a fermentation product. The fermentation product was centrifuged to obtain the supernatant filtrate, and mulberry leaf fermentation extract was obtained after drying.

[0051] The mulberry leaf fermented extract is used in foods for lowering blood sugar.

[0052] The remaining raw materials and preparation process remain the same as in Example 1.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 1 is that Aspergillus oryzae is not added in S2.

[0055] The remaining raw materials and preparation process remain the same as in Example 1.

[0056] Comparative Example 3

[0057] The difference between this comparative example and Example 1 is that Lactobacillus plantarum is not added to S2.

[0058] The remaining raw materials and preparation process remain the same as in Example 1.

[0059] Comparative Example 4

[0060] The difference between this comparative example and Example 1 is that Pediococcus pentosaceus is not added to S2.

[0061] The remaining raw materials and preparation process remain the same as in Example 1.

[0062] Comparative Example 5

[0063] The difference between this comparative example and Example 1 is that Lactobacillus plantarum and Pediococcus pentosaceus are not added to S2.

[0064] The remaining raw materials and preparation process remain the same as in Example 1.

[0065] Comparative Example 6

[0066] The difference between this comparative example and Example 1 is that in S2, "Pediococcus pentosaceus" is replaced by "Lactobacillus fermentum".

[0067] The remaining raw materials and preparation process remain the same as in Example 1.

[0068] Comparative Example 7

[0069] Compared with Example 1, this comparative example is different in that solid-state fermentation is used for extraction. The specific implementation method is as follows:

[0070] S1. Crush 10 g of dry mulberry leaves and sterilize at 115°C for 20 min to obtain mulberry leaf crushed material;

[0071] S2, mulberry leaf fragments were first inoculated with 6% concentration of 5*10 6 cfu / mL Aspergillus oryzae, treated at 35°C and 150 r / min, and inoculated with 8% and 1*10 7 cfu / mL Lactobacillus plantarum and Pediococcus pentosaceus, the bacterial liquid was diluted to 20mL and inoculated by spraying, treated at 35°C and 150r / min, and subjected to solid-state fermentation for 4d to obtain a fermentation product, which was placed in 150mL of 75% ethanol aqueous solution for ultrasonic extraction for 1h, and the ethanol was removed by rotary evaporation at 80°C. After drying, the mulberry leaf fermentation extract was obtained.

[0072] The mulberry leaf fermented extract is used in foods for lowering blood sugar.

[0073] The remaining raw materials and preparation process remain the same as in Example 1.

[0074] Comparative Example 8

[0075] This comparative example is a commercially available mulberry leaf extract, specification: mulberry leaf DNJ 1% / 1kg (Shanxi Shengdayue Biotechnology Co., Ltd.).

[0076] The performance tests were performed on Examples 1 to 7 and Comparative Examples 1 to 8.

[0077] According to GB / T40642-2021, the 1-deoxynojirimycin content of the mulberry leaf fermented extracts obtained in various embodiments and comparative examples of the present application was detected; the total phenol content and flavonoid content of the mulberry leaf fermented extracts obtained in various embodiments and comparative examples of the present application were detected by high performance liquid chromatography; 1.0 g of the mulberry leaf fermented extract prepared by the present invention and 1.0 g of the commercially available mulberry leaf extract were weighed, added to 100 mL of pure water, tasted the taste, and recorded the bitterness sensory value (excellent, good, good, poor, poor).

[0078] The results are shown in Table 1:

[0079] Table 1

[0080]

[0081]

[0082] As can be seen from Table 1, compared with Comparative Example 8, Example 1 has an increased 1-deoxynojirimycin content of 24%, an increased flavonoid content of 18%, and a significantly reduced bitterness; compared with Example 1, Examples 2 to 4 and Example 6 differ only in that the preparation conditions and raw materials are adjusted within a reasonable range, and the final results are not much different from Example 1; Compared with Example 1, Examples 5 and Example 7 show a decrease in the content of the active ingredient. From the comparison between Example 5 and Example 7, it can be seen that the higher the bacterial concentration or the longer the fermentation time, the more effective ingredients will be decomposed, thereby resulting in a decrease in the content.

[0083] Compared with Example 1, the organic components in the mulberry leaf liquid obtained by ethanol extraction are better than those extracted by water decoction, and the changes in the content of 1-deoxynojirimycin and flavonoids in the results also prove this point; compared with Example 1, the synergistic effect of fermentation between fungi and bacteria in Comparative Example 2 to Comparative Example 6 can lead to an increase in the content of effective ingredients in the mulberry leaf fermentation extract. The reason is that the various enzymes produced by the fungi help to decompose complex organic matter and provide more easily digestible substrates for Lactobacillus plantarum and Pediococcus pentosaceus, thereby promoting their growth and metabolic activities; in addition, the co-inoculation of Lactobacillus plantarum and Pediococcus pentosaceus can increase the diversity of the bacterial community and increase the content of active substances; compared with Example 1, the solid-state fermentation method has sufficient material exchange in the liquid fermentation, and the fermentation conditions are easy to monitor and control in real time, which is conducive to the full contact and absorption of nutrients by microorganisms, so that the microorganisms can grow and ferment rapidly, thereby improving the quality of the fermentation product.

[0084] In order to explore the application of 1-deoxynojirimycin content in blood glucose regulation, a diabetic mouse model was established. After one week of adaptive feeding, the mice were fasted for 24 hours, and alloxan saline solution (50 mg / kg) was injected into the tail vein. After 72 hours, the same dose was used for reinforcement. After one week, blood glucose was measured by fasting for 4-5 hours through the tail vein. The diabetic mouse model was defined as a blood glucose concentration ≥11.1 mmol / L. 35 diabetic mice were obtained and divided equally into 7 groups.

[0085] The mice were given oral administration based on body weight, wherein Group A was given the fermented extract prepared in Example 1 at a dosage of 0.040 g / kg / d; Group B was given the fermented extract prepared in Example 1 at a dosage of 0.035 g / kg / d; Group C was given the fermented extract prepared in Comparative Example 1 at a dosage of 0.040 g / kg / d; Group D was given the fermented extract prepared in Comparative Example 5 at a dosage of 0.040 g / kg / d; Group E was given the fermented extract prepared in Comparative Example 7 at a dosage of 0.040 g / kg / d; Group F was given the fermented extract prepared in Comparative Example 8 at a dosage of 0.040 g / kg / d; Group G was used as a blank control group and was given an equal volume of normal saline by oral administration, and blood glucose was measured by taking the tail vertebrae blood of the mice 1 hour after the feeding.

[0086] The results are shown in Table 2:

[0087] Table 2

[0088]

[0089]

[0090] It can be seen from Table 2 that the mulberry leaf fermented extract prepared in Example 1 has a good hypoglycemic effect, and the hypoglycemic effect is better as the concentration of the hypoglycemic component (1-deoxynojirimycin) increases; in summary, the present invention provides a mulberry leaf fermented extract and a preparation method and application thereof, and the prepared mulberry leaf fermented extract has high 1-deoxynojirimycin content and flavonoid content, and the bitter taste is greatly reduced, and animal model experiments have also proved that it has a good hypoglycemic effect.

[0091] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0092] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a mulberry leaf fermentation extract, characterized in that: The following steps are involved: S1. Crush the dried mulberry leaves, place them in an ethanol aqueous solution for ultrasonic extraction, and distill them; add distilled water to make up the volume, adjust the pH, and sterilize them at high temperature to obtain a mulberry leaf liquid; S2. The mulberry leaf liquid is first inoculated with fungi, and then inoculated with Lactobacillus plantarum and Pediococcus pentosaceus after 2-3 days of liquid fermentation. After liquid fermentation for 4-5 days, a fermentation product is obtained. The fermentation product is centrifuged to obtain the supernatant filtrate, and the mulberry leaf fermentation extract is obtained after drying.

2. The method for preparing a mulberry leaf fermented extract according to claim 1, characterized in that: The concentration of the ethanol aqueous solution is 65% to 75%; the usage ratio of the mulberry leaf to the ethanol aqueous solution is 8 to 10 g: 100 to 150 mL; the ultrasonic extraction time is 0.5 to 1 h; and the distillation method is rotary evaporation at 80° C.

3. The method for preparing a mulberry leaf fermented extract according to claim 1, characterized in that: The pH is adjusted to 5-6; the high temperature sterilization condition is sterilization at 110-120°C for 20-30 minutes.

4. The method for preparing a mulberry leaf fermented extract according to claim 1, characterized in that: The fungus is one of Aspergillus oryzae and Mucor.

5. The method for preparing a mulberry leaf fermented extract according to claim 1, characterized in that: The inoculation amount of the fungus is 6% to 8% of the mass of the dry mulberry leaves, and the inoculation amount of Lactobacillus plantarum and Pediococcus pentosaceus is 8% to 10% of the mass of the dry mulberry leaves; the inoculation concentration of the fungus is 1*10 6 ~1*10 7 cfu / mL, the inoculation concentration of Lactobacillus plantarum and Pediococcus pentosaceus was 1*10 7 ~5*10 7 cfu / mL.

6. The method for preparing a mulberry leaf fermented extract according to claim 1, characterized in that: The fermentation treatment conditions are 30-40° C. and 150-200 r / min.

7. A mulberry leaf fermented extract, characterized in that: The mulberry leaf fermentation extract prepared by the preparation method according to any one of claims 1 to 6 comprises the active ingredient 1-deoxynojirimycin, and the content of the 1-deoxynojirimycin is greater than 1.2%.

8. The mulberry leaf fermented extract according to claim 7, characterized in that: The mulberry leaf fermented extract also includes flavonoids, and the flavonoid content is greater than 1%.

9. Use of the mulberry leaf fermented extract according to any one of claims 7 to 8 in preparing blood sugar-lowering food.

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