A zephyranthes grandiflora powder enzymatic hydrolysate having an anticoagulant effect

Abstract

This invention discloses an enzymatic hydrolysate of taro flour with anticoagulant properties. The invention uses taro flour as raw material and prepares the taro flour enzymatic hydrolysate through water extraction, alcohol precipitation, resolution, starch removal with amylase, further alcohol precipitation, further resolution, and glycosidic hydrolysis. The enzymes used are galactosidase, fucosidase, and mannosidase. The taro flour enzymatic hydrolysate prepared by this invention can delay blood clotting time and has anticoagulant properties, and can be used as a functional ingredient to make beverages and foods with anticoagulant effects.

Description

Technical Field

[0001] This invention belongs to the food field, specifically relating to an enzymatic hydrolysate of taro flour with anticoagulant properties. Background Technology

[0002] Thrombotic diseases seriously threaten human life and health and are one of the key focuses and hot topics in contemporary medical research. The banana taro, Latin name... Canna edulis Ker Canna indicum is a perennial herbaceous plant belonging to the genus Canna in the family Cannaceae, and is widely cultivated in Yunnan, Sichuan, Guangxi and other regions. Canna indicum starch contains a large amount of starch. Our research found that the enzymatic hydrolysis product of canna indicum starch after starch removal has anticoagulant activity and can be used in food development for the prevention of thrombotic diseases. Summary of the Invention

[0003] The purpose of this invention is to provide an enzymatic hydrolysate of taro starch with anticoagulant properties, prepared by water extraction, alcohol precipitation, resolution, starch removal with amylase, further alcohol precipitation, further resolution, and glycosidic hydrolysis, specifically as follows:

[0004] (1) Water extraction: Extract the banana taro powder with 6-10 times the amount of distilled water 3 times, filter, combine the filtrates from the 3 extractions, and concentrate the filtrate to 10% of the total volume;

[0005] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0006] (3) Resolution: Add distilled water to the precipitate and dissolve it by ultrasonic heating;

[0007] (4) Amylase removal of starch: Take the compound solution and add α-amylase to remove starch;

[0008] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0009] (6) Glycosidic hydrolysis: Distilled water is added to the precipitate, and the mixture is dissolved by ultrasonic heating. Glycosidase is added, and the mixture is hydrolyzed for 24 hours to obtain the banana taro powder hydrolysate.

[0010] The glycosidases are α-galactosidase, β-galactosidase, fucosidase, and mannosidase.

[0011] A beverage with anticoagulant properties, containing enzymatic hydrolysates of taro starch.

[0012] A food product with anticoagulant properties, containing enzymatic hydrolysates of taro starch.

[0013] Advantages of this invention:

[0014] This invention delves into the anticoagulant activity of banana taro powder, which has significant research value and application potential.

[0015] The enzymatic hydrolysate of banana taro flour disclosed in this invention has excellent anticoagulant effects and health benefits. Detailed Implementation

[0016] The present invention will be further described below, but this is not intended to limit the invention in any way. Any modifications made based on the present invention are within the scope of protection of the present invention.

[0017] A banana taro starch enzymatic hydrolysate with anticoagulant properties is prepared by water extraction, alcohol precipitation, resolution, starch removal with amylase, further alcohol precipitation, further resolution, and glycosidic hydrolysis. Specifically:

[0018] (1) Water extraction: Extract the banana taro powder with 6-10 times the amount of distilled water 3 times, filter, combine the filtrates from the 3 extractions, and concentrate the filtrate to 10% of the total volume;

[0019] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0020] (3) Resolution: Add distilled water to the precipitate and dissolve it by ultrasonic heating;

[0021] (4) Amylase removal of starch: Take the compound solution and add α-amylase to remove starch;

[0022] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0023] (6) Glycosidic hydrolysis: Distilled water is added to the precipitate, and the mixture is dissolved by ultrasonic heating. Glycosidase is added, and the mixture is hydrolyzed for 24 hours to obtain the banana taro powder hydrolysate.

[0024] The glycosidases are α-galactosidase, β-galactosidase, fucosidase, and mannosidase.

[0025] A beverage with anticoagulant properties, containing enzymatic hydrolysates of taro starch.

[0026] A food product with anticoagulant properties, containing enzymatic hydrolysates of taro starch.

[0027] Example 1

[0028] The brand of α-amylase is Adamas, CAS number 9000-90-2, and parameter is 2000 U / g.

[0029] The brand of α-galactosidase is Aladdin, CAS number: 9025-35-8, parameter is 2000U / g.

[0030] (1) Water extraction: 1 kg of banana taro powder was extracted three times with 8 times the amount of distilled water, filtered, and the filtrates from the three extractions were combined. The filtrates were then concentrated to 10% of the total volume.

[0031] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0032] (3) Redissolution: Add 1L of distilled water to the precipitate and dissolve it by ultrasonic heating;

[0033] (4) Amylase removal of starch: Take the reconstituted solution and add 1g of α-amylase to remove starch;

[0034] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0035] (6) Glycoside hydrolysis: Add 1L of distilled water to the precipitate, dissolve by ultrasonic heating, add 1g of α-galactosidase, and hydrolyze for 24 hours to obtain the banana taro flour hydrolysate.

[0036] Example 2

[0037] The brand of α-amylase is Adamas, CAS number 9000-90-2, and parameter is 2000 U / g.

[0038] The brand of β-galactosidase is Adamas Life, CAS number: 9031-11-2, parameter is FromKluyveromyces lactis, 5000 NLU / g.

[0039] (1) Water extraction: 1 kg of banana taro powder was extracted three times with 8 times the amount of distilled water, filtered, and the filtrates from the three extractions were combined. The filtrates were then concentrated to 10% of the total volume.

[0040] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0041] (3) Redissolution: Add 1L of distilled water to the precipitate and dissolve it by ultrasonic heating;

[0042] (4) Amylase removal of starch: Take the reconstituted solution and add 1g of α-amylase to remove starch;

[0043] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0044] (6) Glycoside hydrolysis: Add 1L of distilled water to the precipitate, dissolve by ultrasonic heating, add 1g of β-galactosidase, and hydrolyze for 24 hours to obtain the banana taro flour hydrolysate.

[0045] Example 3

[0046] The brand of α-amylase is Adamas, CAS number 9000-90-2, and parameter is 2000 U / g.

[0047] The brand of fucosidase is Adamas Life, CAS number: 9037-65-4, and the parameter is >5U / mL.

[0048] (1) Water extraction: 1g of banana taro powder was extracted three times with 8 times the amount of distilled water, filtered, and the filtrates from the three extractions were combined. The filtrates were then concentrated to 10% of the total volume.

[0049] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0050] (3) Redissolution: Add 1 mL of distilled water to the precipitate and dissolve by ultrasonic heating;

[0051] (4) Amylase removal of starch: Take the reconstituted solution and add 1g of α-amylase to remove starch;

[0052] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0053] (6) Glycoside hydrolysis: Add 1 mL of distilled water to the precipitate, dissolve by ultrasonic heating, add 1 U of fucoidan, and hydrolyze for 24 hours to obtain the banana taro powder hydrolysate.

[0054] Example 4

[0055] The brand of α-amylase is Adamas, CAS number 9000-90-2, and parameter is 2000 U / g.

[0056] The brand of mannosidase is Sigma-Aldrich, CAS number: 9025-42-7, and the parameters are derived from sword bean, ammonium sulfate suspension, ≥15 units / mg protein (biuret).

[0057] (1) Water extraction: 1g of banana taro powder was extracted three times with 8 times the amount of distilled water, filtered, and the filtrates from the three extractions were combined. The filtrates were then concentrated to 10% of the total volume.

[0058] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0059] (3) Redissolution: Add 1 mL of distilled water to the precipitate and dissolve by ultrasonic heating;

[0060] (4) Amylase removal of starch: Take the reconstituted solution and add 1g of α-amylase to remove starch;

[0061] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0062] (6) Glycoside hydrolysis: Add 1 mL of distilled water to the precipitate, dissolve by ultrasonic heating, add 1 mg of mannosidase, and hydrolyze for 24 hours to obtain the banana taro powder hydrolysate.

[0063] Example 5

[0064] The brand of α-amylase is Adamas, CAS number 9000-90-2, and parameter is 2000 U / g.

[0065] The brand of α-galactosidase is Aladdin, CAS number: 9025-35-8, parameter is 2000U / g.

[0066] (1) Water extraction: 1 kg of banana taro powder was extracted three times with 6 times the amount of distilled water, filtered, and the filtrates from the three extractions were combined. The filtrates were then concentrated to 10% of the total volume.

[0067] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0068] (3) Redissolution: Add 1L of distilled water to the precipitate and dissolve it by ultrasonic heating;

[0069] (4) Amylase removal of starch: Take the reconstituted solution and add 1g of α-amylase to remove starch;

[0070] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0071] (6) Glycoside hydrolysis: Add 1L of distilled water to the precipitate, dissolve by ultrasonic heating, add 1g of α-galactosidase, and hydrolyze for 24 hours to obtain the banana taro flour hydrolysate.

[0072] Example 6

[0073] The brand of α-amylase is Adamas, CAS number 9000-90-2, and parameter is 2000 U / g.

[0074] The brand of α-galactosidase is Aladdin, CAS number: 9025-35-8, parameter is 2000U / g.

[0075] (1) Water extraction: 1 kg of banana taro powder was extracted three times with 6 times the amount of distilled water, filtered, and the filtrates from the three extractions were combined. The filtrates were then concentrated to 10% of the total volume.

[0076] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0077] (3) Resolution: Add distilled water to the precipitate and dissolve it by ultrasonic heating;

[0078] (4) Amylase removal of starch: Take the reconstituted solution and add 1g of α-amylase to remove starch;

[0079] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0080] (6) Glycoside hydrolysis: Distilled water was added to the precipitate, and the mixture was dissolved by ultrasonic heating. 1g of α-galactosidase was added, and the mixture was hydrolyzed for 24 hours to obtain the banana taro flour hydrolysate.

[0081] Compare with Example 1

[0082] The brand of α-amylase is Adamas, CAS number 9000-90-2, and parameter is 2000 U / g.

[0083] (1) Water extraction: 1 kg of banana taro powder was extracted three times with 8 times the amount of distilled water, filtered, and the filtrates from the three extractions were combined. The filtrates were then concentrated to 10% of the total volume.

[0084] (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0085] (3) Redissolution: Add 1L of distilled water to the precipitate and dissolve it by ultrasonic heating;

[0086] (4) Amylase removal of starch: Take the reconstituted solution and add 1g of α-amylase to remove starch;

[0087] (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate.

[0088] (6) Glycoside hydrolysis: Add 1L of distilled water to the precipitate and dissolve it by ultrasonic heating to obtain the solution of control example 1.

[0089] Anticoagulant activity test

[0090] The anticoagulant activity was tested by referring to the paper "Chen Yanping, He Juping, Liu Yi, et al. Optimization of ultrasonic-microwave assisted extraction process of Eucommia ulmoides leaf polysaccharide and analysis of its in vitro anticoagulant activity [J]. Food Industry Technology, 2023, 44 (17): 202-211."

[0091] Weigh out 1 mg of α-galactosidase, 1 mg of β-galactosidase, 1 U of fucosidase, and 1 mg of mannosidase, and dissolve them in 1 mL of physiological saline to prepare control solutions. Use physiological saline and heparin sodium solution (2 μg / mL) as negative and positive controls, respectively.

[0092] The enzymatic hydrolysis products of banana taro powder from Examples 1-4 and the control solution were mixed with plasma at a volume ratio of 1:4. Then, the anticoagulant activity was evaluated by measuring three indicators: activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT).

[0093] a. Determination of APTT: Take 100 μL of preheated 37 ℃ mixed plasma to be tested, add 100 μL of APTT, preheat for 3 min, then add 100 μL of preheated calcium chloride solution (0.025 mol / L) and mix well. Start timing with the instrument.

[0094] b. Determination of PT: Take 100 μL of preheated 37 ℃ mixed plasma to be tested, add 200 μL of preheated PT, mix well, and start timing with the instrument.

[0095] c. Determination of TT: Take 100 μL of preheated 37 ℃ mixed plasma to be tested, add 100 μL of preheated TT, mix well, and start timing with the instrument.

[0096] The results are shown in the table below.

[0097] .

[0098] Note: Each sample was measured four times. The data conformed to a normal distribution and were compared pairwise using a t-test. * indicates that the data were statistically significant compared with the saline, control 1 solution, and heparin sodium group. # indicates that the data were statistically significant compared with control 1 solution or saline, but not statistically significant compared with heparin sodium group.

Claims

1. An enzymatic hydrolysate of taro starch with anticoagulant properties, characterized in that, It is prepared by water extraction, alcohol precipitation, resolution, starch removal with amylase, alcohol precipitation again, resolution again, and glycosidic hydrolysis, specifically as follows: (1) Water extraction: Extract the banana taro powder with 6-10 times the amount of distilled water 3 times, filter, combine the filtrates from the 3 extractions, and concentrate the filtrate to 10% of the total volume; (2) Alcohol precipitation: Add 5 times the amount of anhydrous ethanol to the concentrated filtrate while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate. (3) Resolution: Add distilled water to the precipitate and dissolve it by ultrasonic heating; (4) Amylase removal of starch: Take the compound solution and add α-amylase to remove starch; (5) Secondary ethanol precipitation: Add 5 times the amount of anhydrous ethanol while stirring. After the addition is complete, refrigerate and let stand for 24 hours to precipitate. Centrifuge at 4000 r / min for 30 min to separate the precipitate. (6) Glycosidic hydrolysis: Distilled water is added to the precipitate, and the mixture is dissolved by ultrasonic heating. Glycosidase is added, and the mixture is hydrolyzed for 24 hours to obtain the banana taro flour hydrolysate. The glycosidase mentioned therein is α-galactosidase, β-galactosidase, fucosidase, or mannosidase.