Preparation process of astringency-removed mulberry leaf powder

Abstract

The application discloses a mulberry leaf astringency removing treatment process and super-micro powder thereof. The process mainly uses a fermented coix as an astringency removing agent to remove the astringency of frost mulberry leaves in combination with three steaming and hot air drying methods. The application solves the problems of astringency and green smell of the mulberry leaves by using a relatively simple process, retains the unique taste and fragrance of the mulberry leaves, and adds unique alcohol and ester fragrance, so that the taste and smell of the mulberry leaves are rich in layers. The prepared astringency removed mulberry leaf super-micro powder has high retention rate of main nutritional components, has obvious inhibition effect on alpha-glucosidase and pancreatic lipase, and can be used as a food base to develop health food for reducing blood sugar and blood fat.

Description

Technical Field

[0001] This invention relates to a process for removing astringency from mulberry leaves, specifically involving the removal of astringency from mulberry leaves and the preparation of ultrafine powder, belonging to the field of mulberry product processing technology. Technical Background

[0002] Mulberry leaves are sweet and bitter in taste, and cold in nature. They enter the lung and liver meridians, and have the effects of dispersing wind-heat, clearing the lungs and moistening dryness, and clearing the liver and improving eyesight. They are best harvested after the first frost. Modern medical research shows that mulberry leaves are rich in various bioactive substances, such as flavonoids, polyphenols, polysaccharides, alkaloids, phytosterols, and γ-aminobutyric acid (GABA), which have health benefits such as lowering blood sugar, lowering blood pressure, lowering blood lipids, and antiviral effects. Mulberry leaves have been a traditional medicinal and edible plant since ancient times. With the modern consumer's pursuit of healthy foods, mulberry leaf products are receiving increasing attention, and mulberry leaves have become an important raw material for natural health foods.

[0003] Processing mulberry leaves into mulberry leaf powder allows them to be used as a food additive in various mulberry leaf products. However, certain substances in mulberry leaves, such as tannins and organic acids, can impart unpleasant tastes to these products, including astringency and a strong grassy aroma. This is especially true for frost-covered mulberry leaves, making them unsuitable for consumer consumption.

[0004] In existing technology, patent CN 1081901C discloses a method for removing astringency from mulberry leaf health products, which involves steaming and air-drying repeatedly 9-10 times to produce mulberry leaf tea. This method involves excessive steaming, causing the mulberry leaves to turn yellow and damaging their nutritional components, as well as their unique aroma and flavor. Patent 201710562991.6 discloses a deodorizing and decolorizing process for mulberry leaves, which involves adding a deastringent agent during the pile fermentation and deastringency removal process, and a deodorizing agent during the steaming and deodorizing process. After rinsing, filtration, secondary steaming, and roasting with flavor enhancers, the bitterness and off-flavors of the mulberry tea are removed. This method is cumbersome; the added deodorizing agent needs to be removed through rinsing, which can easily lead to the loss of mulberry leaf nutrients, and the addition of flavor enhancers alters the unique aroma and flavor of the mulberry leaves. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a process for deastringing mulberry leaves using coix seed fermentation as a deastringent agent combined with steam deastringency. This method solves the problem of poor taste of mulberry leaves with a relatively simple procedure, and the deastringent mulberry leaf ultrafine powder prepared has a high retention rate of major nutrients, and can be used as a food base to produce mulberry leaf health foods.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] A process for removing astringency from mulberry leaves includes the following steps:

[0008] (1) After the frost, pick fresh mulberry leaves that are free from disease, pests and pollution, wash them with clean water and dry them, cut off the leaf stalks, cut the leaves into strips, steam them to kill the green and spread them out to dry.

[0009] (2) Spray the coix seed fermentation material evenly at 50-95% of the weight of mulberry leaf strips, knead and stir evenly, spread it in a sealed container, let it stand at 20-40℃ for 4-12 hours, and dry it with hot air at 60-65℃ for 110-150 minutes.

[0010] (3) Spread dried mulberry leaf strips on a steaming tray to a thickness of 2-4 cm, and steam for 5-10 minutes after the steam rises. Repeat step (2).

[0011] (4) Repeat step (3) and continue to heat to 90~95℃ for hot air drying for 10~20 minutes until the moisture content is below 5%.

[0012] In step (1), the steaming of mulberry leaves involves spreading mulberry leaf strips 2-4 cm thick on a steaming tray and steaming for 3-5 minutes after the steam rises.

[0013] The coix seed ferment in step (2) is prepared using the following steps:

[0014] A. Activate the yeast strain;

[0015] B. Prepare a coix seed fermentation medium by weight, containing 5-30 parts coix seed powder, 10-20 parts whey powder, 5-15 parts sucrose, and make up the remainder with water. After high temperature and high pressure sterilization, inoculate with 3-6 parts yeast liquid, control the temperature at 18-30℃, and ferment in a shaker at 150-200 rpm for 1-3 days.

[0016] C. After fermentation, freeze-centrifuge to collect the supernatant of the fermentation broth, and freeze-dry it;

[0017] D. The freeze-dried powder is prepared into a water-soluble substance with a mass fraction of 20-50% to obtain the coix seed fermentation product.

[0018] The yeast strain used in step A of the preparation of the coix seed fermentation product is a mixture of Saccharomyces p. and Kluyveromyces sp., preferably brewer's yeast, brewer's yeast, baker's yeast, as well as Kluyveromyces lactis and Kluyveromyces marx, with a mixing ratio of 1:1 to 4:1 (based on colony count).

[0019] The live yeast inoculation amount in step B of the preparation of the coix seed ferment is 1.21 × 10⁻⁶. 8 ~2.39×10 10 CFU / mL.

[0020] The average particle size of the deastringent mulberry leaf ultrafine powder prepared according to the method of the present invention is less than 20 μm.

[0021] The de-astringent mulberry leaf ultrafine powder prepared according to the method of the present invention can be used as a food base for health foods that help lower blood sugar and lipids.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The mulberry leaf de-astringency treatment process of the present invention has a simple operation process, and the prepared mulberry leaf ultrafine powder has obvious de-astringency and aroma enhancement. The coix seed fermentation product can effectively combine with the tannins in mulberry leaves, greatly reducing the astringency of mulberry leaves and preserving the unique taste and fragrance of mulberry leaves; moreover, the interaction between the fermentation product, organic acids, alcohols and other components in mulberry leaves adds a unique ethanol and ester aroma to the mulberry leaf ultrafine powder, resulting in a good taste and strong layering.

[0024] (2) The mulberry leaf ultrafine powder prepared by the deastringency treatment in this invention has a high retention rate of nutrients and a significant inhibitory effect on α-glucosidase and pancreatic lipase. It is suitable as a food base for developing health foods that help lower blood sugar and lipids. Attached Figure Description

[0025] Figure 1 Comparative graphs of the inhibitory effects of hot air drying treatment, Example 1, and comparative mulberry leaf ultrafine powder on α-glucosidase.

[0026] Figure 2 Comparative graphs of the inhibitory effects of hot air drying treatment, Example 1, and comparative mulberry leaf ultrafine powder prepared on pancreatic lipase. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments.

[0028] Example 1:

[0029] 1. Preparation of Coix Seed Fermentation

[0030] (1) Single colonies of yeast strains Saccharomyces cerevisiae DC-2 and Kluyveromyces marxianus Y-3 were inoculated into YPD liquid medium and cultured at 30℃ and 180rpm for 2 days to activate the strains.

[0031] (2) The two activated yeast strains were transferred to YPD liquid medium at an inoculation rate of 2% and cultured at 30℃ and 180 rpm for 1 day. The colony count of S. cerevisiae DC-2 was 2.52 × 10⁻⁶. 8 CFU / mL, K. marxianus Y-3 is 1.13×10 8 CFU / mL, the two were mixed at a colony count ratio of 1:1 to form a seed culture, with a viable count of 1.56 × 10⁻⁶. 8CFU / mL.

[0032] (3) Prepare a coix seed fermentation medium by weight, containing 20 parts coix seed powder, 10 parts whey powder, 8 parts sucrose, and the remainder water. Sterilize under high temperature and high pressure, inoculate with 3 parts of compound seed liquid, control the temperature at 30℃, and ferment at 200 rpm for 2 days.

[0033] (3) After fermentation, freeze-centrifuge to obtain the supernatant of the fermentation broth, and freeze-dry it;

[0034] (4) Prepare a 50% water-soluble product from the freeze-dried powder to obtain the coix seed fermentation product.

[0035] 2. De-astringency treatment of mulberry leaves

[0036] (1) After the frost, pick fresh mulberry leaves that are free from disease, pests and pollution, wash them with clean water and dry them, cut off the leaf stalks, cut the leaves into strips, steam them to kill the green and spread them out to dry.

[0037] (2) Spray 50% coix seed fermentation evenly at 95% of the weight of mulberry leaf strips, knead and stir evenly, spread in a sealed container, let stand at 28℃ for 12 hours, and dry with hot air at 65℃ for 120 minutes;

[0038] (3) Spread dried mulberry leaf strips on a steaming tray to a thickness of 2-4 cm, and steam for 6 minutes after the steam rises. Repeat step (2).

[0039] (4) Repeat step (3) and continue to heat to 95°C for hot air drying for 20 minutes until the moisture content is below 5%.

[0040] 3. The processed dried mulberry leaves are pulverized to 80 mesh using a high-speed pulverizer, and then processed into deastringent mulberry leaf ultrafine powder using an airflow ultrafine pulverizer, with an average particle size of less than 20 μm.

[0041] Example 2:

[0042] 1. Preparation of Coix Seed Fermentation

[0043] (1) Single colonies of yeast strains Saccharomyces cerevisiae DC-2 and Kluyveromyces lactis Y-1 were inoculated into YPD liquid medium and cultured at 30℃ and 180rpm for 2 days to activate the strains.

[0044] (2) The two activated yeast strains were transferred to YPD liquid medium at an inoculation rate of 5% and cultured at 30℃ and 180 rpm for 1 day. The colony count of S. cerevisiae DC-2 was 8.9 × 10⁻⁶. 9 CFU / mL, K. lactis Y-1 was 3.55 × 10⁻⁶. 9CFU / mL, the two were mixed at a colony count ratio of 4:1 to form a seed culture, with a viable count of 6.84 × 10⁻⁶. 9 CFU / mL.

[0045] (3) Prepare a coix seed fermentation medium by weight, containing 30 parts coix seed powder, 20 parts whey powder, 5 parts sucrose, and the remainder water. Sterilize under high temperature and high pressure, inoculate with 6 parts of compound seed liquid, control the temperature at 30℃, and ferment at 180 rpm for 3 days.

[0046] (3) After fermentation, freeze-centrifuge to obtain the supernatant of the fermentation broth, and freeze-dry it;

[0047] (4) Prepare a 30% water-soluble product from the freeze-dried powder to obtain the coix seed fermentation product.

[0048] 2. De-astringency treatment of mulberry leaves

[0049] (1) After the frost, pick fresh mulberry leaves that are free from disease, pests and pollution, wash them with clean water and dry them, cut off the leaf stalks, cut the leaves into strips, steam them to kill the green and spread them out to dry.

[0050] (2) Spray 30% coix seed fermentation evenly at 95% of the weight of mulberry leaf strips, knead and stir evenly, spread in a sealed container, let stand at 37℃ for 5 hours, and dry with hot air at 60℃ for 150 minutes.

[0051] (3) Spread dried mulberry leaf strips on a steaming tray to a thickness of 2-4 cm, and steam for 8 minutes after the steam rises. Repeat step (2).

[0052] (4) Repeat step (3) and continue to heat to 90°C and dry with hot air for 10 minutes until the moisture content is below 5%.

[0053] 3. The processed dried mulberry leaves are pulverized to 80 mesh using a high-speed pulverizer, and then processed into deastringent mulberry leaf ultrafine powder using an airflow ultrafine pulverizer, with an average particle size of less than 20 μm.

[0054] Comparative example:

[0055] The coix seed fermentation product in Example 1 was replaced with water, and the mulberry leaves were subjected to the same deastringency treatment as in Example 1, as a comparative example.

[0056] 1. The tannin content in the mulberry leaf ultrafine powder of the examples and comparative examples was determined according to the determination of tannin content (General Chapter 2202) in the Chinese Pharmacopoeia (2015 edition). The test results are shown in Table 1. The sensory differences between the ultrafine powders are shown in Table 2. It can be seen that compared with hot air drying, the tannin content in Example 1 was reduced by 84.61%, which is 2.45 times the reduction in tannin content in the comparative example. From the sensory indicators, the mulberry leaf ultrafine powder of Example 1 is significantly less astringent and more fragrant than the comparative example, with a better taste and richer layers of flavor.

[0057] Table 1. Comparison of tannin content in mulberry leaf ultrafine powder between Example 1 and the comparative example.

[0058] Steaming times Comparative example (mg / g) Tannin reduction rate (%) Example 1 (mg / g) Tannin reduction rate (%) first 4.23±0.09 20.87 2.33±0.08 56.39 The second 3.74±0.12 30.03 1.40±0.08 73.83 The third 3.5±0.12 34.52 0.82±0.04 84.61

[0059] Note: The tannin content of the ultrafine powder prepared from mulberry leaves after hot air drying (65℃ hot air drying for 120 minutes, 95℃ hot air drying for 20 minutes) is 5.35±0.09 mg / g.

[0060] Table 2 Sensory comparison between Mulberry Leaf Ultrafine Powder in Example 1 and Comparative Mulberry Leaf Ultrafine Powder

[0061] Sensory content Comparative Example 1 Example 1 color yellow-green Green, slightly yellowish The green odor of mulberry leaves Lighter none aroma Mulberry leaf aroma Mulberry leaf aroma, slightly sweet, mellow. taste Slightly sweet, with a mild astringency Sweet, not astringent, with a slightly sour aftertaste.

[0062] 2. Crude polysaccharides were extracted from mulberry leaf ultrafine powder using a water extraction and alcohol precipitation method. 2 g of mulberry leaf ultrafine powder was extracted at a material-to-liquid ratio of 1:30 in a 90℃ water bath with stirring for 3 hours. The mixture was then filtered, and the filtrate was retained. The residue was extracted once more using the above method. The two filtrates were combined, and 80% ethanol was added for overnight precipitation. The precipitate was collected by centrifugation and freeze-dried to obtain crude polysaccharides. The crude polysaccharide content of the mulberry leaf ultrafine powder in the examples and comparative examples was determined using the sulfuric acid-phenol method, as shown in Table 3. It can be seen that the polysaccharide content prepared from mulberry leaves using the process described in Example 1 and the comparative example increased by 44.09% and 21.73% respectively compared to the hot air drying treatment. The increase in polysaccharide content in Example 1 was 2.02 times that in the comparative example.

[0063] Table 3 Comparison of polysaccharide content in mulberry leaf ultrafine powder of Example 1 and Comparative Example 1

[0064] Steaming times Comparative example (mg / g) Polysaccharide increase rate (%) Example 1 (mg / g) Polysaccharide increase rate (%) first 88.57±0.89 13.11 101.60±1.31 29.75 The second 93.21±1.71 19.04 108.47±1.07 38.53 The third 95.32±1.83 21.73 112.82±1.52 44.09

[0065] Note: The polysaccharide content of the ultrafine powder prepared from mulberry leaves after hot air drying (65℃ hot air drying for 120 minutes, 95℃ hot air drying for 20 minutes) was 78.30±0.88 mg / g.

[0066] 3. DNJ was extracted from mulberry leaf ultrafine powder using water extraction. 0.5 g of mulberry leaf ultrafine powder was taken and extracted in a water bath at 80 ℃ with stirring for 2 hours at a material-to-liquid ratio of 1:70. The mixture was then filtered, and the residue was extracted again with 15 mL of purified water. The two filtrates were combined and diluted to 50 mL with purified water to obtain the sample extract.

[0067] Derivatization of DNJ: Take 300 μL of DNJ extract (or standard solution) into a 10.0 mL centrifuge tube, add 300 μL of 0.4 mol / L potassium borate buffer solution (pH 8.5), then add 300 μL of 5 mmol / L FMOC-Cl (dissolved in acetonitrile), mix well, and incubate at 25 ℃ for 20 min. Add 300 μL of 1 mol / L glycine to neutralize the remaining FMOC-Cl to terminate the reaction. Dilute with 300 μL of 1% acetic acid solution and 1200 μL of ultrapure water, centrifuge at 6000 r / min for 10 min, and then filter through a 0.45 μm microporous membrane filter. Collect the filtrate for later use.

[0068] High-performance liquid chromatography (HPLC) conditions: Column: C18 column (150 mm × 4.6 mm, 5 μm); Mobile phase: acetonitrile-0.1% acetic acid (50:50, V / V); Flow rate: 1.0 mL / min; Column temperature: 25 ℃; Injection volume: 10 μL; UV detector, detection wavelength: 254 nm.

[0069] The DNJ content of mulberry leaf ultrafine powder in Example 1 and the comparative example is shown in Table 4. It can be seen that the DNJ extraction amount of mulberry leaves processed according to the process of Example 1 and the comparative example increased by 22.32% and 2.72% respectively compared with hot air drying, and the increase in DNJ in Example 1 was 8.21 times that in the comparative example.

[0070] Table 4 Comparison of DNJ content in mulberry leaf ultrafine powder of Example 1 and Comparative Example 1

[0071] Steaming times Comparative example (mg / g) DNJ increase rate (%) Example 1 (mg / g) DNJ increase rate (%) first 2.45±0.06 5.06 2.58±0.10 11.02 The second 2.44±0.02 4.58 2.67±0.04 14.59 The third 2.39±0.08 2.72 2.85±0.08 22.32

[0072] Note: The DNJ content of the ultrafine powder prepared from mulberry leaves after hot air drying (65℃ hot air drying for 120 minutes, 95℃ hot air drying for 20 minutes) was 2.33±0.06 mg / g.

[0073] 4. Preparation of mulberry leaf ultrafine powder ethanol extract and determination of its inhibitory effect on α-glucosidase. Take 10 g of mulberry leaf ultrafine powder, add 150 mL of 75% ethanol and extract for 24 hours; filter, recover the ethanol from the filtrate under reduced pressure below 60℃ to obtain a thick extract, place it in a vacuum drying oven at 60℃ and dry under reduced pressure for 24 hours to obtain the sample.

[0074] According to Table 5, take a certain amount of potassium phosphate buffer, enzyme solution, substrate, and test solution into each well of a 96-well plate. After incubating at 37°C for 30 min, add sodium carbonate solution as a stop agent, and measure the absorbance at 405 nm. The α-glucosidase inhibition rate is calculated using the following formula:

[0075]

[0076] Among them, AE A is the absorbance value without a sample; EB A represents the absorbance without sample or enzyme solution. I A is the absorbance value when the inhibitor is added. IB The absorbance value is the value when the inhibitor is added but the enzyme solution is not added.

[0077] Table 5. Assay system for α-glucosidase activity

[0078] Conditions (μL) Sample group Sample blank control group negative control group negative blank control group Enzyme solution (dissolved in buffer) 100 0 100 0 buffer solution 0 100 0 100 Substrate 30 30 30 30 sample 10 10 0 0 distilled water 0 0 10 10

[0079] Appendix Figure 1 The results showed that the half-maximal inhibitory rates (ICP-C) of the hot-air dried mulberry leaf ultrafine powder, Example 1, and the comparative mulberry leaf ultrafine powder against α-glucosidase were 1.82 mg / mL, 1.23 mg / mL, and 0.86 mg / mL, respectively. It can be seen that the de-astringent mulberry leaf ultrafine powder of Example 1 exhibited the best inhibitory effect on α-glucosidase and can be applied to the development of hypoglycemic health foods.

[0080] 5. Prepare mulberry leaf ultrafine powder alcohol extract and determine its inhibitory effect on pancreatic lipase.

[0081] The preparation of the ethanol extract was the same as before. According to Table 1, take a certain amount of Tris-HCl (pH 7.4) buffer, enzyme solution, substrate, and test solution into a 96-well plate, incubate at 37℃ for 20 min, and immediately measure the absorbance at 405 nm. The pancreatic lipase inhibition rate was calculated using the following formula:

[0082]

[0083] Among them, A E A is the absorbance value without a sample; I The absorbance value obtained when the inhibitor is added; A IB The absorbance is the value when the inhibitor is added but the substrate and enzyme solution are not added.

[0084] Table 6 Pancreatic Lipase Activity Assay System

[0085] Conditions (μL) Sample group Sample blank control group negative control group Tris-HCl buffer 140 140 140 enzyme solution 20 0 20 Substrate 20 0 20 sample 20 20 0 distilled water 0 40 20

[0086] Appendix Figure 2 The results showed that the half-maximal inhibitory rates (ICP-C) of the hot-air dried mulberry leaf ultrafine powder, Example 1, and the comparative mulberry leaf ultrafine powder against pancreatic lipase were 2.12 mg / mL, 1.84 mg / mL, and 1.29 mg / mL, respectively. It can be seen that the de-astringent mulberry leaf ultrafine powder of Example 1 exhibited the best inhibitory effect on pancreatic lipase and can be applied to the development of lipid-lowering health foods.

Claims

1. A process for removing astringency from mulberry leaves, characterized in that, Includes the following steps: (1) After the frost, pick fresh mulberry leaves that are free from disease, pests and pollution, wash them with clean water and dry them, cut off the leaf stalks, cut the leaves into strips, steam them to kill the green and spread them out to dry. (2) Spray the coix seed fermentation material evenly at 50-95% of the weight of mulberry leaf strips, knead and stir evenly, spread it in a sealed container, let it stand at 20-40℃ for 4-12 hours, and dry it with hot air at 60-65℃ for 110-150 minutes. (3) Spread dried mulberry leaf strips 2-4 cm thick on a steaming tray and steam for 5-10 minutes after the steam rises; (4) Repeat steps (2) and (3) three times, raise the temperature to 90~95℃ and dry with hot air for 10~20 minutes until the moisture content is below 5%; The aforementioned coix seed fermentation product is prepared using the following steps: (1) Activate yeast strains; (2) Prepare a coix seed fermentation culture medium by weight, containing 5-30 parts coix seed powder, 10-20 parts whey powder, 5-15 parts sucrose, and the remainder water. After high temperature and high pressure sterilization, inoculate 3-6 parts yeast liquid, control the temperature at 18-30℃, and ferment at 150-200 rpm for 1-3 days. (3) After fermentation, freeze-centrifuge to obtain the supernatant of the fermentation broth, and freeze-dry it; (4) Prepare a water-soluble product with a mass fraction of 20-50% from the freeze-dried powder to obtain the coix seed fermentation product; The yeast strain used in the coix seed fermentation product is a mixture of yeast strains and Kluyveromyces strains, with a ratio of 1:1 to 4:1 based on the number of colonies.

2. The mulberry leaf astringency removal process according to claim 1, characterized in that, The yeast strains include brewer's yeast, brewer's yeast, baker's yeast, as well as Kluyveromyces lactis and Kluyveromyces mascarpium.

3. The mulberry leaf astringency removal process according to claim 1, characterized in that, The steam blanching process involves spreading mulberry leaf strips 2-4 cm thick on a steaming tray and steaming for 3-5 minutes after the steam rises.

4. The mulberry leaf astringency removal process according to claim 1, characterized in that, The live yeast culture inoculation rate was 1.21 × 10⁻⁶. 8 ~2.39×10 10 CFU / mL.

5. The de-astringent mulberry leaf ultrafine powder prepared by the process according to any one of claims 1-4, characterized in that, The average particle size of the ultrafine powder is below 20 μm.

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