Preparation method and application of grape pomace freeze-dried powder with high hypoglycemic activity

Abstract

The invention relates to a preparation method of grape pomace freeze-dried powder with high hypoglycemic activity, which comprises the following steps: raw material cleaning, ethanol soaking extraction, low-temperature centrifugation, supernate treatment, residue treatment, mixing, fermentation and freeze drying. According to the present invention, with methods of extraction of enriched functional active substances, exogenous addition of amino acids and the like, the inhibition of the product on alpha-amylase and alpha-glucosidase is improved, the blood sugar reducing effect is enhanced, and the resource utilization of the grape skin residue and other agricultural wastes is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of functional foods, and in particular to a preparation method and application of grape pomace freeze-dried powder with high blood sugar-lowering activity. Background Art

[0002] With the continuous improvement of living standards, people have a deeper understanding of the demand for the intake of active substances in food nutrition functions, and the demand for food quality and function is also increasing. Among them, foods with hypoglycemic activity are increasingly attracting close attention from consumers due to their unique advantages in health care.

[0003] In the process of grape by-product processing, such as winemaking and NFC (non-concentrated reduced) juice production, a large amount of grape pomace will be produced. At present, the development and utilization of these pomace is relatively low, resulting in a large amount of grape pomace resources being wasted. Grape pomace is rich in a variety of nutrients, such as anthocyanins, polyphenols, flavonoids, resveratrol, dietary fiber and saponins. These functional active substances not only have good hypoglycemic activity, but also can inhibit the activity of α-glucosidase and α-amylase, thus showing great potential in the prevention and treatment of diabetes.

[0004] In addition, the primary product of the Maillard reaction, Amadori compounds, also exhibit strong hypoglycemic activity. Studies have shown that fructose-glutamate can inhibit the activity of α-amylase, while fructose-arginine can inhibit the activity of pancreatic amylase and glucosidase, further confirming its role in lowering blood sugar. At the same time, the products of probiotic fermentation also have high α-amylase and α-glucosidase inhibition, providing a scientific basis for the development of new hypoglycemic foods.

[0005] Diabetes is a chronic metabolic disorder caused by multiple causes, characterized by hyperglycemia. Its etiology involves genetics, environment, immune inflammation and other aspects. Insulin resistance leads to increased inflammatory stress in the body, and the interaction between intestinal flora and environmental and genetic factors further leads to the occurrence of diabetes. At present, the treatment methods for diabetes are mostly oral hypoglycemic drugs, such as sulfonylureas, biguanides, or injections of insulin secretagogues, insulin analogs and insulin inhibitors. Although these drugs are effective in treating diabetes, most hypoglycemic drugs cause certain damage to the liver, kidneys and gastrointestinal tract. Therefore, there is an urgent need for a safe treatment for diabetes with no side effects or mild side effects.

[0006] In this context, selecting health foods with hypoglycemic effects from natural foods through technical optimization and probiotic fermentation has become an important direction for the current development of diabetes treatment. This not only helps to realize the resource utilization of agricultural waste such as grape skin residues, but also provides consumers with safe and effective hypoglycemic food options, which has important social and economic value. Summary of the invention

[0007] The present invention aims to provide a preparation method and application of grape pomace freeze-dried powder with high hypoglycemic activity. The method makes full use of the rich functional active substances in grape pomace and realizes the effective development and utilization of grape pomace resources through a series of scientific and reasonable process steps.

[0008] To achieve the purpose of the present invention, the present invention provides a preparation method and application of grape pomace freeze-dried powder with high hypoglycemic activity, the method comprising the following steps:

[0009] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0010] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0011] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0012] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0013] (5) Residue treatment: exogenously add glutamate and glucose, dissolve them in 200 mL of deionized water, add them to the extract residue, mix them by ultrasonic at 45 °C for 30 min, then place the mixed solution in a round-bottom flask and perform rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid;

[0014] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0015] (7) fermentation step: adding fermentation bacteria to the grape skin residue liquid obtained in step (6) for low-temperature mixed fermentation, and fermenting in a shake flask at 25° C. for 2 days to obtain a mixed fermentation liquid;

[0016] (8) Freeze-drying step: freeze-drying the mixed fermentation liquid obtained in step (7) to finally obtain freeze-dried grape pomace powder with high hypoglycemic activity.

[0017] Furthermore, in step (2), the concentration of the ethanol solution is 75%.

[0018] Furthermore, in the step (5), the amount of glutamic acid added is 1 mmol, and the amount of glucose added is 5 mmol.

[0019] Furthermore, in step (7), the fermentation bacteria are Lactobacillus plantarum and Lactobacillus acidophilus.

[0020] Furthermore, in step (8), the freeze-drying time is 72 hours.

[0021] The method for preparing a freeze-dried grape pomace powder with high hypoglycemic activity provided by the present invention has the following advantages:

[0022] 1. Extraction and enrichment of functional active substances: The present invention effectively enriches the anthocyanins, resveratrol and other blood sugar-lowering active ingredients in grape pomace through scientific extraction technology, significantly improves the functionality of the product, and promotes the resource utilization of agricultural waste such as grape pomace.

[0023] 2. Enhancement of enzyme inhibition: The present invention forms the Amadori compound (Fru-Glu) by exogenously adding amino acids and glucose and combining it with rotary evaporation, which greatly improves the product's inhibition of α-amylase and α-glucosidase and enhances the blood sugar lowering effect.

[0024] 3. Probiotic fermentation optimization: The present invention introduces probiotics for low-temperature fermentation, which not only improves the taste and flavor of the product, but also further enhances the inhibitory effect of α-amylase and α-glucosidase, making the product have extremely high blood sugar-lowering activity.

[0025] 4. Low temperature treatment and retention of active ingredients: The present invention is always kept at a relatively low temperature during the entire preparation process, and is finally freeze-dried to retain the functional ingredients with hypoglycemic activity to a great extent, ensuring the long-term stability and effectiveness of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of Amadori compound (Fru-Glu) concentration on α-amylase inhibition rate is shown;

[0027] Figure 2 The effects of anthocyanin and acarbose concentrations on α-amylase inhibition rates were shown;

[0028] Figure 3 The effects of resveratrol and acarbose concentrations on α-glucosidase inhibition rates were shown;

[0029] Figure 4 The effects of Lactobacillus plantarum, Lactobacillus acidophilus, and co-culture of Lactobacillus plantarum / Lactobacillus acidophilus on the inhibition rate of α-glucosidase are shown. DETAILED DESCRIPTION

[0030] Example 1

[0031] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0032] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0033] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0034] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0035] (5) Residue treatment: 1 m mol glutamic acid and 1 m mol glucose were added exogenously, dissolved in 200 mL of deionized water, added to the extract residue, and ultrasonically mixed at 45 °C for 30 min. The mixed solution was then placed in a round-bottom flask and subjected to rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid;

[0036] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0037] (7) Freeze-drying step: freeze-drying the grape pomace liquid obtained in step (6) to finally obtain grape pomace freeze-dried powder with high hypoglycemic activity.

[0038] Example 2

[0039] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0040] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0041] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0042] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0043] (5) Residue treatment: 1 m mol glutamic acid and 3 m mol glucose were added exogenously, dissolved in 200 mL of deionized water, added to the extract residue, and ultrasonically mixed at 45 °C for 30 min. The mixed solution was then placed in a round-bottom flask and subjected to rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid;

[0044] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0045] (7) Freeze-drying step: freeze-drying the grape pomace liquid obtained in step (6) to finally obtain grape pomace freeze-dried powder with high hypoglycemic activity.

[0046] Example 3

[0047] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0048] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0049] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0050] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0051] (5) Residue treatment: 1 m mol glutamic acid and 5 m mol glucose were added exogenously, dissolved in 200 mL of deionized water, added to the extract residue, and ultrasonically mixed at 45 °C for 30 min. The mixed solution was then placed in a round-bottom flask and subjected to rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid;

[0052] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0053] (7) Freeze-drying step: freeze-drying the grape pomace liquid obtained in step (6) to finally obtain grape pomace freeze-dried powder with high hypoglycemic activity.

[0054] Example 4

[0055] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0056] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0057] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0058] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0059] (5) Residue treatment: 1 m mol glutamic acid and 7 m mol glucose were added exogenously, dissolved in 200 mL of deionized water, added to the extract residue, and ultrasonically mixed at 45 °C for 30 min. The mixed solution was then placed in a round-bottom flask and subjected to rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid.

[0060] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0061] (7) Freeze-drying step: freeze-drying the grape pomace liquid obtained in step (6) to finally obtain grape pomace freeze-dried powder with high hypoglycemic activity.

[0062] Example 5

[0063] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0064] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0065] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0066] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0067] (5) Residue treatment: 1 m mol glutamic acid and 5 m mol glucose were added exogenously, dissolved in 200 mL of deionized water, added to the extract residue, and ultrasonically mixed at 45 °C for 30 min. The mixed solution was then placed in a round-bottom flask and subjected to rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid;

[0068] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0069] (7) fermentation step: adding 10, 20, and 40 uL of Lactobacillus plantarum to the grape skin residue liquid obtained in step (6), respectively, and fermenting in a shake flask at 25° C. for 2 days to obtain a mixed fermentation liquid;

[0070] (8) Freeze-drying step: freeze-drying the mixed fermentation liquid obtained in step (7) to finally obtain freeze-dried grape pomace powder with high hypoglycemic activity.

[0071] Example 6

[0072] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0073] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0074] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0075] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0076] (5) Residue treatment: 1 m mol glutamic acid and 5 m mol glucose were added exogenously, dissolved in 200 mL of deionized water, added to the extract residue, and ultrasonically mixed at 45 °C for 30 min. The mixed solution was then placed in a round-bottom flask and subjected to rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid;

[0077] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0078] (7) fermentation step: adding 10, 20, and 40 uL of Lactobacillus acidophilus to the grape pomace liquid obtained in step (6), respectively, and fermenting in a shake flask at 25° C. for 2 days to obtain a mixed fermentation liquid;

[0079] (8) Freeze-drying step: freeze-drying the mixed fermentation liquid obtained in step (7) to finally obtain freeze-dried grape pomace powder with high hypoglycemic activity.

[0080] Example 7

[0081] (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue;

[0082] (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances;

[0083] (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue;

[0084] (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities;

[0085] (5) Residue treatment: 1 m mol glutamic acid and 5 m mol glucose were added exogenously, dissolved in 200 mL of deionized water, added to the extract residue, and ultrasonically mixed at 45 °C for 30 min. The mixed solution was then placed in a round-bottom flask and subjected to rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid;

[0086] (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity;

[0087] (7) fermentation step: adding 10, 20, and 40 uL of Lactobacillus plantarum and Lactobacillus acidophilus to the grape skin residue liquid obtained in step (6) for mixed fermentation, and shaking flask fermentation at 25° C. for 2 days to obtain a mixed fermentation liquid;

[0088] (8) Freeze-drying step: freeze-drying the mixed fermentation liquid obtained in step (7) to finally obtain freeze-dried grape pomace powder with high hypoglycemic activity.

[0089] Analysis of the above embodiments:

[0090] Table 1 Effect of different Amadori compound contents on α-amylase

[0091]

[0092] As shown in Table 1, the different ratios of glutamic acid and glucose added have a significant effect on the α-amylase inhibition rate; when the glutamic acid and glucose added are 1m mol and 5m mol respectively, the inhibition effect on the α-amylase inhibition rate is the best. The reason for this phenomenon is that amino acids and glucose generate Amadori compounds under vacuum rotary evaporation conditions, which have a good inhibitory effect on α-amylase. The results are shown in Figure 1 In the examples, the exogenously added glucose will also combine with other amino acids in the grape pomace to form different Amadori compounds, which significantly increases the content of the Amadori compounds and promotes the effect of lowering blood sugar.

[0093] Depend on Figure 4 It can be seen that, with Example 3 as a control, fermentation was performed using Lactobacillus plantarum and Lactobacillus acidophilus respectively. It was found that the fermentation broths in Examples 5, 6, and 7 had significant inhibitory effects on α-glucosidase, among which the mixed fermentation in Example 7 had the best inhibitory effect and had a higher hypoglycemic activity.

Claims

1. A method for preparing freeze-dried grape pomace powder with high hypoglycemic activity, comprising the following steps: (1) Raw material cleaning: The grape skin residue is first cleaned with tap water and then re-washed with deionized water to obtain clean grape skin residue; (2) Ethanol immersion extraction: The cleaned grape pomace is completely immersed in an ethanol solution, subjected to ultrasonic extraction at 20°C for 30 minutes, and then placed in a refrigerator at 4°C for 24 hours to extract anthocyanins, tannins, flavonoids, polyphenols, resveratrol and other substances; (3) Low temperature centrifugation: centrifuge the refrigerated extracted solution at 8000 rpm for 15 min at 4°C to separate the supernatant and the extract residue; (4) Supernatant treatment: The supernatant was subjected to low-temperature rotary evaporation at 40°C and 0.05 MPa for 3 hours to remove ethanol, and then filtered through a 0.22 nm filter membrane to obtain a composite extract with multiple functional activities; (5) Residue treatment: exogenously add glutamate and glucose, dissolve them in 200 mL of deionized water, add them to the extract residue, mix them by ultrasonic at 45 °C for 30 min, then place the mixed solution in a round-bottom flask and perform rotary evaporation at 95 °C and 0.01 MPa to obtain a vacuum rotary evaporation liquid; (6) mixing: mixing the composite extract obtained in step (4) with the vacuum rotary evaporation liquid obtained in step (5), and ultrasonicating at 37° C. for 30 minutes to obtain a grape skin residue liquid having hypoglycemic activity; (7) fermentation step: adding fermentation bacteria to the grape skin residue liquid obtained in step (6) for low-temperature mixed fermentation, and fermenting in a shake flask at 25° C. for 2 days to obtain a mixed fermentation liquid; (8) Freeze-drying step: freeze-drying the mixed fermentation liquid obtained in step (7) to finally obtain freeze-dried grape pomace powder with high hypoglycemic activity.

2. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the ethanol solution is 75%.

3. The preparation method according to claim 1, characterized in that: In step (5), the amount of glutamate added is 1 mmol, and the amount of glucose added is 5 mmol.

4. The preparation method according to claim 1, characterized in that: In step (7), the fermentation bacteria are Lactobacillus plantarum and Lactobacillus acidophilus.

5. The preparation method according to claim 1, characterized in that: In step (8), the freeze-drying time is 72 hours.

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