WO2013091301A1 - 利用海参肠自溶制备活性物质的方法 - Google Patents

利用海参肠自溶制备活性物质的方法 Download PDF

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Publication number
WO2013091301A1
WO2013091301A1 PCT/CN2012/071381 CN2012071381W WO2013091301A1 WO 2013091301 A1 WO2013091301 A1 WO 2013091301A1 CN 2012071381 W CN2012071381 W CN 2012071381W WO 2013091301 A1 WO2013091301 A1 WO 2013091301A1
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Prior art keywords
sea cucumber
autolysis
active substance
residue
preparing
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PCT/CN2012/071381
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English (en)
French (fr)
Inventor
朱蓓薇
吴海涛
董秀萍
郑杰
孙黎明
周大勇
李冬梅
杨静峰
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大连工业大学
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Priority to JP2013549709A priority Critical patent/JP2014504160A/ja
Publication of WO2013091301A1 publication Critical patent/WO2013091301A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/50—Molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives

Definitions

  • the invention belongs to the field of deep processing of marine by-products, and relates to the technical field of autolysis, gel column chromatography and vacuum freeze-drying. Background technique
  • Sea cucumber intestine is a by-product of the sea participation process. Its protein content is high, up to 70% (by dry weight), and it is a good raw material for preparing active substances. In China, sea cucumber intestines are often discarded as waste, and the utilization rate is extremely low, resulting in waste of resources and environmental pollution.
  • Sea cucumber intestine has strong autolysis ability. At present, there are no reports on the use of autolysis technology to prepare active substances from sea cucumber intestines.
  • the literature on sea cucumber intestinal autolysis mainly focuses on the separation, purification and enzymatic properties of its endogenous enzymes.
  • Japanese researchers have improved the production method of the traditional Japanese fermented food "Yanxin”.
  • the hydrostatic pressure is not added to the salt to promote the autolysis of sea cucumber, but it does not involve the extraction and separation of the autolyzed active substances from the sea cucumber. Summary of the invention
  • the invention aims to provide a preparation for preparing sea cucumber enteric active substance and its autolyzed residue active material by using autolysis technology, in view of the fact that the sea cucumber intestine in the sea is strong in autolysis, but at the same time it is not fully utilized. method.
  • the invention utilizes the method for preparing the active substance by autolysis of sea cucumber intestine, and takes the sea cucumber intestine as a raw material, and the supernatant is homogenized, autolyzed and centrifuged to obtain the sea cucumber intestinal autolysate; the product can be further concentrated by freezing and concentration to The original volume is 10% ⁇ 20%; it can also be dried by vacuum freeze-drying to a moisture content of 8% or less, so as to obtain crude products of sea cucumber enteric active substances in different product forms.
  • the preferred solution of the autolysis step is as follows: after the slurry is obtained by the slurry, after being irradiated with ultraviolet rays, 1 to 4 times of a buffer solution of citric acid and disodium hydrogen phosphate having a pH of 4.0 to 7.0 is added to perform autolysis.
  • autolysis can take two conditions: when the temperature is 4 ⁇ 10 °C, autolysis is 4 ⁇ 12h, or when the temperature is autolysis at 35 °C ⁇ 60 °C, autolysis is 0.5 ⁇ 4h.
  • the ultraviolet irradiation can be performed by using a 30W ultraviolet lamp, and the autolysis is induced after being irradiated for 5 to 40 minutes at a distance of 0.5 m; when the treatment amount is large, the sea cucumber intestinal slurry can be placed in a stainless steel tank, and the slurry is prepared. Thickness l ⁇ 2cm, place UV lamp at 0.2 ⁇ 0.5m above, control the liquid surface of the slurry The external strength is 60 ⁇ 120 ⁇ /cm 2 , and the autolysis is induced for 5-40 min.
  • the step of taking the supernatant by centrifugation is preferably: centrifugation at 2500 ⁇ 6000r/min for 10 ⁇ 30 minutes, taking the supernatant; or using ultrafiltration to collect components below lOkDa.
  • the present invention obtains a purified active substance by separating and freeze-drying the crude sea cucumber enteric active substance.
  • the separation step is preferably: the crude sea cucumber intestinal active material is applied to a Sephadex G-15 chromatography column, and eluted with deionized water to control a flow rate of 0.3 to 0.5 mL. /min, collects the main components (peak area accounts for 70% to 80% of the total peak area of each component).
  • DPPH diphenyl-tertene free radical
  • the preferred process of the freeze-drying is: drying at a cold trap temperature of -30 to -45 ° C and a vacuum of 50 to 80 Pa.
  • the invention further provides a method for preparing an active substance by autolysis of sea cucumber intestine, mainly for the sea cucumber enteric residue active substance, the purpose of which is to improve the utilization rate of sea cucumber intestine.
  • the specific process is to use sea cucumber as raw material, homogenate and autoly dissolve. Then, the supernatant is separated to obtain the sea cucumber intestinal autolysate, and the remaining residue is used for the following treatment: adding 2 to 4 volumes of deionized water to mix, and heating at 90 to 100 ° C for 5 to 15 minutes. After cooling, a neutral protease of 1500-3000 U/g protein, such as Bacillus subtilis 1398, was enzymatically hydrolyzed at 50 ° C and pH 7.0 for 30-180 min.
  • the enzymatic hydrolysate is heated at 95 ⁇ 100 °C for 5 ⁇ 10min to kill the enzyme. After cooling, centrifuge at 2500 ⁇ 6000r/min for 10 ⁇ 30 min, take the supernatant, or use ultrafiltration to collect lOkDa.
  • the following components are the active ingredients of sea cucumber enteric residue.
  • the product can be further concentrated to 10% to 20% of the original volume by freeze-concentration; or dried by vacuum freeze-drying to a moisture content of 8% or less, thereby obtaining active ingredients of sea cucumber enteric residue in different product forms. Crude.
  • the crude sea cucumber autolysis residue active material was applied to a Sephadex G-25 chromatography column and eluted with deionized water to control the flow rate of 0.2-0.4 mL/min.
  • the main components of 80% to 95% of the total peak area were collected.
  • the purified active material is obtained after lyophilization at a cold trap temperature of -30 to -45 ° C, a vacuum of 50 to 80 Pa, and a moisture content of 8% or less.
  • the above-mentioned autolysis process for preparing an active material by using a residue is preferably: after homogenization, the homogenate is irradiated with ultraviolet rays, and then 1 to 4 volumes of a citric acid and a disodium hydrogen phosphate buffer solution having a pH of 4.0 to 7.0 are added.
  • Autolysis when the amount of treatment is small, the ultraviolet irradiation can be selected from a 30W ultraviolet lamp, and the autolysis is induced after being irradiated for 5 to 40 minutes at a distance of 0.5 m; when the treatment amount is large, the sea cucumber intestinal homogenate can be placed in stainless steel.
  • the thickness of the slurry is l ⁇ 2cm, and the purple is placed at 0.2 ⁇ 0.5m above.
  • the external lamp controls the liquid surface UV intensity of the slurry to be 60 ⁇ 120 ⁇ /cm 2 and maintains the autolysis induction for 5-40 minutes.
  • the processed sea cucumber intestinal active material has certain antioxidant capacity and can be used as a functional base for the development of various functional products, which has great development potential.
  • the autolysis mentioned in the present invention utilizes the endogenous enzyme contained in the sea cucumber intestine to carry out enzymatic hydrolysis, and adopts the autolysis technique to prepare the sea cucumber intestinal active substance, thereby maximally maintaining the active ingredient contained in the sea cucumber intestine and obtaining higher quality.
  • the product At the same time, for the residue after autolysis of sea cucumber intestine, exogenous enzymes can be used to prepare active substances, so that the sea cucumber intestine can be fully utilized.
  • the process parameters mentioned in the preferred process of the present invention are intended to increase the yield and quality of the active material product.
  • the content of TCA soluble oligopeptide in the sea cucumber intestinal autolysate is increased to be self-dissolving. 2 ⁇ 3 times, the peptide yield of the autolyzed residue protein is 10% ⁇ 30% after denaturation treatment and exogenous enzymatic hydrolysis.
  • the invention relates to a sea cucumber enteric active substance and an autolysate residue active substance thereof.
  • the sea cucumber enteric active substance is prepared from sea cucumber intestine by homogenization, autolysis, centrifugation, freeze concentration, gel column separation and freeze-drying; the residue of sea cucumber enteric autolysis can be subjected to denaturing pretreatment, exogenous It is prepared by enzymatic hydrolysis, centrifugation, freeze concentration, gel column separation and freeze drying.
  • the two nutrients are light yellow solid powder, odorless, have the inherent odor of sea cucumber, soluble in water, hygroscopic, have certain anti-oxidation ability in vitro, and can be used as functional base for preparing various functions.
  • Type food is light yellow solid powder, odorless, have the inherent odor of sea cucumber, soluble in water, hygroscopic, have certain anti-oxidation ability in vitro, and can be used as functional base for preparing various functions.
  • Type food
  • the sea cucumber intestinal pulp slurry can be placed in a stainless steel tank, the thickness of the slurry is l ⁇ 2cm, and the ultraviolet lamp is placed at the upper 0.2 ⁇ 0.5m to control the liquid surface ultraviolet intensity of the slurry to be 60 ⁇ 120 W/cm. 2 , for 5 ⁇ 40min for autolysis induction, then add 1 ⁇ 4 volumes of citric acid - disodium hydrogen phosphate buffer solution (pH 4.0 ⁇ 7.0), according to the above method for autolysis.
  • citric acid - disodium hydrogen phosphate buffer solution pH 4.0 ⁇ 7.0
  • the crude sea cucumber intestinal active substance was applied to a Sephadex G-15 gel column and eluted with deionized water to control the flow rate to 0.3 to 0.5 mL/min.
  • the main components peak area accounts for 70% to 80% of the total peak area of each component) are collected while achieving the purpose of desalination.
  • the collected active components are freeze-dried, the cold trap temperature is -30 ⁇ -45 °C, the vacuum degree is 50 ⁇ 80Pa, and the moisture content is less than or equal to 8%. After lyophilization, the sea cucumber enteric autolyzed active substance is obtained. .
  • the residue is taken, and 2 ⁇ 4 times of volume of deionized water is added to the hook, and heated at 90-100 °C for 5-15 minutes to carry out protein denaturation treatment and cooling.
  • the enzymatic hydrolysate is heated at 95 ⁇ 100 °C for 5 ⁇ 10min to kill the enzyme, and after cooling, it is centrifuged at 2500 ⁇ 6000r/min for 10 ⁇ 30 min, and the supernatant is taken, or ultrafiltration is used to collect less than lOkDa.
  • the component is the sea cucumber enteric residue active substance.
  • the product can be further concentrated to 10-20% of the original volume by freeze-concentration; or can be dried by vacuum freeze-drying to a moisture content of 8% or less, thereby obtaining the active substance of the sea cucumber enteric residue in different product forms. mouth
  • the crude sea cucumber autolysis residue active substance was applied to a Sephadex G-25 column and eluted with deionized water.
  • the flow rate was controlled to 0.2-0.4 mL/min, and the main component (peak area)
  • the total peak area of each component is 80% to 95%), and the purpose of desalting is achieved.
  • Example 1 The collected active components are freeze-dried, the temperature of the cold trap is -30 ⁇ -45 °C, the degree of vacuum is 50 ⁇ 80Pa, and the moisture content is less than or equal to 8%. After lyophilization, the autolysis residue of sea cucumber enteric residue is obtained. substance.
  • Example 1 The collected active components are freeze-dried, the temperature of the cold trap is -30 ⁇ -45 °C, the degree of vacuum is 50 ⁇ 80Pa, and the moisture content is less than or equal to 8%. After lyophilization, the autolysis residue of sea cucumber enteric residue is obtained. substance.
  • Example 1 Example 1:
  • Sea cucumber enteric residue add 4 times volume of deionized water and mix, heat at 100 °C for 5 min. After cooling, 1500 U/g protein of Bacillus subtilis 1398 neutral protease was added at 50 ° C, pH 7.0, and the enzyme was hydrolyzed for 180 min. After the enzymatic hydrolysis, the enzymatic hydrolysate was heated at 95 ° C for 10 min to kill the enzyme, centrifuged at 6000 r / min for 10 min, and the supernatant was taken and frozen and concentrated to 10% of the original volume, which is the crude active substance of sea cucumber enteric residue.
  • the concentrate was passed through a Sephadex G-25 column, eluted with deionized water, and the flow rate was controlled at 0.25 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the trap was -30 ° C. Vacuum The degree is 80 Pa, and the moisture content is less than 8%.
  • After lyophilization, the active ingredient of sea cucumber enteric residue is obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.
  • Example 2 Example 2:
  • Sea cucumber enteric residue add 2 times volume deionized water mixed hook, heated at 90 °C for 15min. After cooling, a neutral protease of 2000 U/g protein was added at 50 ° C, pH 7.0, and the solution was digested for 120 min. After the enzymatic hydrolysis, the enzymatic hydrolysate was heated at 100 ° C for 5 min to destroy the enzyme, and 10 kDa was ultrafiltered to collect the components with molecular weight less than 10 kDa, and concentrated to 15% of the original volume, which is the crude active substance of sea cucumber intestinal autolysis residue. The concentrate was passed through a Sephadex G-25 column and eluted with deionized water to control the flow rate to 0.3 mL/min.
  • the main components were collected and freeze-dried.
  • the cold trap temperature was -45 °C
  • the vacuum degree was 50 Pa
  • the moisture content was less than 8%.
  • the active ingredient of sea cucumber enteric residue was obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.
  • Example three Example three:
  • the crude sea cucumber autolysis active substance was separated on a Sephadex G-15 gel column, and the flow rate was controlled at 0.35 mL/min. The main components were collected and freeze-dried. The temperature of the cold trap is adjusted to -45 ° C, the degree of vacuum is 65 Pa, and the moisture content is less than 8%. After lyophilization, the sea cucumber enteric active substance is obtained.
  • Sea cucumber enteric residue add 3 times volume deionized water mixed hook, heat at 95 °C for 10min. After cooling, 3000 U/g protein of Bacillus subtilis 1398 neutral protease was added at 50 ° C, pH 7.0, and enzymatically hydrolyzed for 60 min. After the enzymatic hydrolysis, the enzymatic hydrolysate was heated at 100 ° C for 5 min to kill the enzyme, and lOkDa was ultrafiltered to collect the fraction with molecular weight lower than lOkDa, and frozen and concentrated to 15% of the original volume, which is the crude active substance of sea cucumber enteric residue.
  • the concentrate was passed through a Sephadex G-25 column, eluted with deionized water, and the flow rate was controlled at 0.25 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the trap was -45 °C, vacuum.
  • the degree is 65 Pa, and the moisture content is less than 8%.
  • After lyophilization, the active ingredient of sea cucumber enteric residue is obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.
  • Example four Example four:
  • 500g sea cucumber intestine was washed, chopped and homogenized. After homogenization treatment, ultraviolet (30W, 0.5m) was irradiated for 10 minutes, and 4 times volume of pH 4.0 sodium disodium hydrogen phosphate-citrate buffer solution was added at 60 °C. Dissolved for 0.5h. After the autolysis was completed, centrifuge at 4500 r/min for 15 min, and the supernatant was taken and the residue was used. The supernatant is concentrated by vacuum freezing to 20% of the original volume, which is the crude autologous active substance of sea cucumber. The crude sea cucumber autologous active substance was separated on a Sephadex G-15 gel column, and the flow rate was controlled to 0.4 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the cold trap is adjusted to -35 ° C, the degree of vacuum is 75 Pa, and the moisture content is less than 6%.
  • the sea cucumber enteric active substance is obtained.
  • Sea cucumber enteric residue add 2 times volume of deionized water mixed hook, heat at 100 ° C for 5 min.
  • a neutral protease of 2000 U/g protein was added at 50 ° C, pH 7.0, and the solution was hydrolyzed for 150 min.
  • the enzymatic hydrolysis was heated at 95 ° C for 10 min to kill the enzyme, and centrifuged at 4500 r/min for 15 min.
  • the supernatant was taken and concentrated by vacuum freezing to 20% of the original volume, which was the crude active substance of the sea cucumber enteric residue.
  • the concentrate was passed through a Sephadex G-25 column, eluted with deionized water, and the flow rate was controlled at 0.3 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the trap was -35 °C, vacuum.
  • the degree is 75 Pa, and the moisture content is less than 8%.
  • the active ingredient of sea cucumber enteric residue is obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.
  • Example 5 Example 5:
  • the sea cucumber enteric autolyzed active substance was separated on a Sephadex G-15 gel column, and the flow rate was controlled to 0.5 mL/min, and the main components were collected and freeze-dried.
  • the temperature of the cold trap is adjusted to -40 ° C, the degree of vacuum is 70 Pa, and the moisture content is less than 6%. After lyophilization, the sea cucumber enteric active substance is obtained.
  • Sea cucumber enteric residue add 4 times volume deionized water mixed hook, heated at 90 °C for 10min.
  • a neutral protease of 1500 U/g protein was added at 50 ° C and pH 7.0, and the enzyme was hydrolyzed for 180 min.
  • the enzymatic hydrolysate was heated at 100 °C for 5 min to kill the enzyme, and centrifuged at 5000 r/min for 15 min. The supernatant was taken. The supernatant was vacuum-frozen and concentrated to 15% of the original volume, which is the autolysis residue of sea cucumber. Substance crude.
  • the concentrate was passed through a Sephadex G-25 column, eluted with deionized water, and the flow rate was controlled at 0.4 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the trap was -40 °C.
  • the degree is 70 Pa, and the moisture content is less than 8%.
  • After lyophilization, the active ingredient of sea cucumber enteric residue is obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.
  • Example six Example six:
  • the crude sea cucumber autolysis active substance was separated on a Sephadex G-15 gel column, and the flow rate was controlled to 0.3 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the cold trap is adjusted to -45 ° C, the degree of vacuum is 70 Pa, and the moisture content is less than 6%. After lyophilization, the sea cucumber enteric active substance is obtained.
  • Sea cucumber enteric residue add 2 times volume of deionized water and mix, heat at 100 °C for 5 min. After cooling, a neutral protease of 2500 U/g protein was added at 50 ° C and pH 7.0, and the solution was digested for 120 min. After the enzymatic hydrolysis, the enzymatic hydrolysate was heated at 95 ° C for 10 min to kill the enzyme, and centrifuged at 2500 r / min for 30 min. The supernatant was taken, and the supernatant was frozen and concentrated to 10% of the original volume, which is the sea cucumber enteric residue active substance. Crude.
  • the concentrate was passed through a Sephadex G-25 column, eluted with deionized water, and the flow rate was controlled at 0.3 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the trap was -45 °C, vacuum.
  • the degree is 70 Pa, and the moisture content is less than 8%.
  • After lyophilization, the active ingredient of sea cucumber enteric residue is obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.
  • Example 7 Example 7:
  • sea cucumber intestine was washed, chopped, homogenized, homogenized, irradiated with ultraviolet light (30W, 0.5m) for 5min, and added with 4 times volume of pH 6.0 hydrogen phosphate disodium-citrate buffer solution at 60 °C. Dissolved for 0.5h. After autolysis, centrifuge at 5500 r/min for 10 min, take the supernatant, and use the residue for later use. The supernatant is dried by vacuum and dried to be a crude sea cucumber active substance. The crude sea cucumber intestinal active substance was dissolved in water, separated by a Sephadex G-15 gel column, and the flow rate was controlled to 0.45 mL/min, and the main components were collected and freeze-dried. The temperature of the trap is adjusted to -35 °C, the degree of vacuum is 60Pa, and the moisture content is less than 6%. After lyophilization, the sea cucumber enteric active substance is obtained.
  • Sea cucumber enteric residue add 3 times volume deionized water mixed hook, heated at 90 ° C for 15min. After cooling, a neutral protease of 3000 U/g protein was added at 50 ° C, pH 7.0, and the solution was digested for 120 min. After the enzymatic hydrolysis, the enzymatic hydrolysate was heated at 100 ° C for 5 min to kill the enzyme, and lOkDa was ultrafiltered to collect the fraction with molecular weight lower than lOkDa, and frozen and concentrated to 15% of the original volume, which is the crude active substance of sea cucumber enteric residue.
  • the concentrate was passed through a Sephadex G-25 column, eluted with deionized water, and the flow rate was controlled at 0.25 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the trap was -35 °C, vacuum.
  • the degree is 60 Pa, and the moisture content is less than 8%.
  • After lyophilization, the active ingredient of sea cucumber enteric residue is obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.
  • Example eight Example eight:
  • Sea cucumber enteric residue add 4 times volume deionized water mixed hook, heated at 90 °C for 15min. After cooling, 2000 U/g protein of Bacillus subtilis 1398 neutral protease was added at 50 ° C, pH 7.0, and the enzyme was hydrolyzed for 180 min. After enzymatic hydrolysis, the enzymatic hydrolysate was heated at 95 ° C for 5 min to kill the enzyme, centrifuged at 6000 r / min for 10 min, and the supernatant was taken. The supernatant was vacuum frozen and concentrated to 15% of the original volume, which is the autolysis residue activity of sea cucumber. Substance crude.
  • the concentrate was passed through a Sephadex G-25 column, eluted with deionized water, and the flow rate was controlled at 0.4 mL/min.
  • the main components were collected and freeze-dried.
  • the temperature of the trap was -35 °C, vacuum.
  • the degree is 65 Pa, and the moisture content is less than 8%.
  • After lyophilization, the active ingredient of sea cucumber enteric residue is obtained.
  • the two products are light yellow solid powder, odorless, have the inherent flavor of sea cucumber, soluble in water, hygroscopic, have certain in vitro antioxidant capacity, and can be used as functional base for preparing various functional types. food.

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Abstract

本发明公开了一种利用海参肠自溶制备活性物质的方法，所采用的制备方法是海参肠匀浆后，经紫外线照射诱导，在一定条件下利用内源酶的作用使其自溶，自溶混合液经离心、冷冻浓缩、Sephadex G-15分离、冷冻干燥制成。海参肠自溶活性物质为淡黄色固体粉末，无臭，有海参肠固有的鲜味，易溶于水，有吸湿性，具有一定的体外抗氧化能力，可作为功能基料用于制备多种功能型食品。本发明的突出特点是利用海参肠本身具有的自溶能力制备活性物质，能够最大程度的保持原有的营养成分，同时还减少环境污染。加工处理的工艺合理、简单、成本低，加工产品绿色、无污染。

Description

利用海参肠自溶制备活性物质的方法
技术领域
本发明属于海洋副产物精深加工领域， 同时涉及自溶、 凝胶柱层析和真空 冷冻干燥的技术领域。 背景技术
海参肠是海参加工过程中的副产物， 其蛋白质含量高， 可达 70%左右 （以 干重计）， 是制备活性物质的良好原料。 在中国， 海参肠多被作为废弃物丟弃， 利用率极低， 造成资源浪费及环境污染。
海参肠具有较强的自溶能力。 目前， 有关利用自溶技术从海参肠中制备活 性物质的相关专利未见报道。 关于海参肠自溶的文献报道， 主要集中在对其内 源酶进行分离、 纯化及酶学特性研究。 日本学者对日本传统发酵食品 "盐辛" 的制作方法进行改进， 采用静水压不添加食盐而促进海参肠自溶， 但未涉及海 参肠自溶活性物质的提取及分离制备。 发明内容
针对海参加工中的副产物海参肠自溶能力较强， 但同时并未得到充分利用 的状况， 本发明旨在提供一种利用自溶技术制备海参肠活性物质及其自溶残渣 活性物质的制备方法。
本发明利用海参肠自溶制备活性物质的方法， 以海参肠为原料， 经匀浆、 自溶、 离心取上清液即为海参肠自溶产物； 该产物可进一步采用冷冻浓缩的方 法浓缩至原体积的 10%~20%； 还可以采用真空冷冻干燥的方式干燥至水分含量 小于等于 8%, 从而获得不同产品形式的海参肠活性物质粗品。
其中， 所述自溶工序的优选方案为： 勾浆后所得勾浆液经紫外线照射后， 加入 1 ~ 4倍体积、 pH4.0~7.0的柠檬酸与磷酸氢二钠緩沖溶液， 进行自溶。 根 据实际情况不同自溶可采取两种条件： 温度在 4 ~ 10°C时， 自溶 4 ~ 12h, 或者 温度在 35 °C ~ 60 °C自溶时， 自溶 0.5 ~ 4h。
优选方式下， 处理量较少时， 紫外线照射可以选用 30W紫外灯， 距离 0.5m 照射 5 ~ 40min后进行自溶诱导； 处理量较大时， 可以将海参肠勾浆液放置在不 锈钢槽内， 浆液厚度 l~2cm, 在上方 0.2~0.5m处放置紫外灯， 控制浆液液面紫 外强度为 60~120 μψ/cm2, 保持 5 ~ 40min进行自溶诱导。
此外， 所述离心取上清液的工序， 优选方式为： 2500 ~ 6000r/min离心 10 ~ 30 min, 取上清液； 或采用超滤的方式， 收集 lOkDa以下的组分。
为了获得海参肠活性物质精制品， 本发明在制得海参肠活性物质粗品后， 经分离、 冷冻干燥制得精制活性物质。
其中， 所述分离的工序， 优选方式为： 将所述海参肠活性物质粗品上样于 葡聚糖凝胶 Sephadex G-15层析柱，用去离子水洗脱，控制流速为 0.3~0.5mL/min, 对主要组分（峰面积占各组分总峰面积的 70%~80% )进行收集。 采用二苯代苦 味肼自由基（ DPPH )体系和还原能力测定体系， 已经证明主要组分具有较强的 清除 DPPH自由基及还原能力。
此外， 所述冷冻干燥的优选工序为： 在冷阱温度 -30 ~ -45 °C、真空度为 50 ~ 80Pa条件下进行干燥。
本发明另外提供了一种利用海参肠自溶制备活性物质的方法， 主要针对海 参肠自溶残渣活性物质， 其目的在于提高海参肠的利用率。 具体过程为， 以海 参肠为原料， 经匀浆、 自溶。 而后分离上清液获得海参肠自溶产物， 同时利用 余下残渣作如下处理：加入 2〜4倍体积的去离子水混匀， 90~100°C加热 5~15min。 冷却后于 50°C , pH7.0下， 加入 1500~3000 U/g蛋白的中性蛋白酶， 如枯草芽孢 杆菌 1398 , 酶解 30~180min。 酶解完毕后， 酶解液于 95~100°C加热 5~10min灭 酶， 冷却后于 2500 ~ 6000r/min下离心 10 ~ 30 min, 取上清液， 或采用超滤的方 式， 收集 lOkDa以下的组分， 即为海参肠自溶残渣活性物质。 该产物可进一步 采用冷冻浓缩的方法浓缩至原体积的 10%~20%； 还可以采用真空冷冻干燥的方 式干燥至水分含量小于等于 8% ,从而获得不同产品形式的海参肠自溶残渣活性 物质粗品。
将所述海参肠自溶残渣活性物质粗品上样于葡聚糖凝胶 Sephadex G-25 层 析柱， 用去离子水洗脱， 控制流速为 0.2~0.4mL/min, 对峰面积占各组分总峰面 积的 80%~95%的主要组分进行收集。 最后， 在冷阱温度为 -30 ~ -45 °C , 真空度 为 50 ~ 80Pa, 至水分含量为小于等于 8%为止， 冻干后制得精制活性物质。
上述利用残渣制备活性物质的自溶工序， 优选方案为： 匀浆后所得匀浆液 经紫外线照射后， 加入 1 ~ 4倍体积、 pH4.0~7.0的柠檬酸与磷酸氢二钠緩沖溶 液， 进行自溶。 此外， 优选方式下， 处理量较少时， 紫外线照射可以选用 30W 紫外灯， 距离 0.5m照射 5 ~ 40min后进行自溶诱导； 处理量较大时， 可以将海 参肠匀浆：^置在不锈钢槽内， 浆液厚度为 l~2cm, 在上方 0.2~0.5m处放置紫 外灯，控制浆液液面紫外强度为 60~120 μψ/cm2,保持 5 ~ 40min进行自溶诱导。 本发明突出的优点在于：
1. 提高了海参加工过程中副产物的利用率， 使其所含营养成分和功能因子 得到充分开发， 同时还减少了对环境造成的污染。 加工处理后的海参肠活性物 质具有一定的抗氧化能力， 可作为功能基料用于开发多种功能型产品， 极具开 发潜力。
2. 本发明提及的自溶是利用海参肠自身所含有的内源酶进行酶解， 采用自 溶技术制备海参肠活性物质， 最大限度的保持了海参肠所含有的活性成分， 得 到更优质的产品。 同时， 针对海参肠自溶后的残渣， 可采用外源酶酶解制备活 性物质， 使海参肠得以充分利用。
本发明优选工艺中提及的工艺参数， 旨在提高活性物质产品的得率和质量， 在本发明的工艺参数条件下， 海参肠自溶产物中 TCA可溶性寡肽的含量提高为 自溶前的 2~3 倍， 自溶残渣蛋白质经变性处理、 外源酶酶解后肽得率达到 10%~30%。 具体实施方式
本发明一种海参肠自溶活性物质及其自溶残渣活性物质。 海参肠自溶活性 物质以海参肠为原料， 经匀浆、 自溶、 离心、 冷冻浓缩、 凝胶柱分离及冷冻干 燥制成； 海参肠自溶后的残渣， 可经变性预处理、 外源酶酶解、 离心、 冷冻浓 缩、 凝胶柱分离及冷冻干燥的方式制成。 该两种营养品为淡黄色固体粉末， 无 臭、 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 具有一定的体外抗氧化能力， 可作为功能基料用于制备多种功能型食品。
1. 海参肠自溶活性物质制备具体工艺步骤如下：
( 1 )原料处理
从新鲜海参中取出海参肠、 清洗， 去除泥沙和杂质， 直接匀浆备用或在 -15 °C冷冻备用；
( 2 ) 自溶
利用海参肠所含内源酶的作用， 使其在不同的条件下进行自溶：
海参肠匀浆液经紫外线 （ 30W, 0.5m )照射 5 ~ 40min后， 加入 1 ~ 4倍体 积的柠檬酸-磷酸氢二钠緩沖溶液（ pH4.0~7.0 ), 于 35 °C ~ 60 °C自溶 0.5 ~ 4h或 者于 4°C ~ 10°C自溶 4 ~ 12h。 自溶结束后， 2500 ~ 6000r/min离心 10 ~ 30 min, 取上清液； 或采用超滤的方式， 收集 lOkDa以下的组分。 真空冷冻浓缩后至原 体积的 10% ~ 20%, 或直接冷冻干燥， 即为海参肠自溶活性物质粗品。
处理量较大时，可以将海参肠勾浆：^置在不锈钢槽内，浆液厚度为 l~2cm, 上方 0.2~0.5m处放置紫外灯，控制浆液液面紫外强度为 60~120 W/cm2,保持 5 ~ 40min 进行自溶诱导， 之后加入 1 ~ 4 倍体积的柠檬酸-磷酸氢二钠緩沖溶液 ( pH4.0~7.0 ), 按照上述方法进行自溶。
( 3 )分离
将海参肠活性物质粗品上样于 Sephadex G-15凝胶柱， 用去离子水洗脱， 控 制流速为 0.3~0.5mL/min。 对主要组分（峰面积占各组分总峰面积的 70%~80% ) 进行收集， 同时达到脱盐的目的。
( 4 )干燥
对收集的活性组分进行冷冻干燥， 冷阱温度为 -30 ~ -45 °C , 真空度为 50 ~ 80Pa, 至水分含量为小于等于 8%为止， 冻干后即得到海参肠自溶活性物质。
2. 海参肠自溶残渣活性物质制备具体工艺步骤如下：
( 1 )预处理
获得海参肠自溶产物后，取残渣，加入 2~4倍体积的去离子水混勾， 90-100 °C加热 5~15min, 进行蛋白质变性处理， 冷却。
( 2 )外源酶酶解
在 50°C , pH7.0下， 加入 1500~3000 U/g蛋白的中性蛋白酶（如枯草芽孢杆 菌 1398 ) , 酶解 30~180min。 酶解完毕后， 酶解液于 95~100°C加热 5~10min灭 酶， 冷却后 2500 ~ 6000r/min离心 10 ~ 30 min, 取上清液， 或采用超滤的方式， 收集 lOkDa以下的组分， 即为海参肠自溶残渣活性物质。 该产物可进一步采用 冷冻浓缩的方法浓缩至原体积的 10~20%； 还可以采用真空冷冻干燥的方式干燥 至水分含量小于等于 8%,从而获得不同产品形式的海参肠自溶残渣活性物质粗 口口
( 3 )分离
将海参肠自溶残渣活性物质粗品上样于葡聚糖凝胶 Sephadex G-25层析柱， 用去离子水洗脱， 控制流速为 0.2~0.4mL/min , 对主要组分（峰面积占各组分总 峰面积的 80%~95% )进行收集， 同时达到脱盐的目的。
( 4 )干燥
对收集的活性组分进行冷冻干燥， 冷阱温度为 -30 ~ -45 °C , 真空度为 50 ~ 80Pa,至水分含量为小于等于 8%为止，冻干后即得到海参肠自溶残渣活性物质。 实例一：
将 100g海参肠清洗、 剪碎、 匀浆， 匀浆处理后紫外线（30W, 0.5m )照射 25min, 加入 1.5倍体积的 pH5.0的磷酸氢二钠 -柠檬酸緩沖溶液， 于 40°C自溶 4h。 自溶结束后， 3000r/min离心 25min, 取上清液， 残渣备用。 上清液经真空 冷冻浓缩为原体积的 10%, 即为海参肠自溶活性物质粗品。 海参肠自溶性物质 粗品经 Sephadex G-15凝胶柱分离， 控制流速为 0.4mL/min, 收集主要组分， 进 行冷冻干燥。 调节冷阱温度为 -30°C , 真空度为 80Pa, 至水分含量小于 8%, 冻 干后即得到海参肠自溶活性物质。
海参肠自溶残渣， 加入 4倍体积去离子水混匀， 100 °C加热 5min。 冷却后， 在 50°C , pH7.0下， 加入 1500U/g蛋白的枯草芽孢杆菌 1398中性蛋白酶， 酶解 180min。 酶解完毕后， 酶解液于 95°C加热 lOmin灭酶， 6000r/min离心 lOmin, 取上清液， 冷冻浓缩至原体积的 10%, 即为海参肠自溶残渣活性物质粗品。 浓 缩液经葡聚糖凝胶 Sephadex G-25 层析柱， 用去离子水洗脱， 控制流速为 0.25mL/min, 收集主要组分， 进行冷冻干燥， 冷阱温度为 -30°C , 真空度为 80Pa, 至水分含量为小于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品 为淡黄色固体粉末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 其具 有一定的体外抗氧化能力， 可作为功能基料用于制备多种功能型食品。 实例二：
将 200g海参肠清洗、 剪碎、 匀浆， 匀浆处理后紫外线（30W, 0.5m )照射 40min, 加入 3.5倍体积的 pH6.0的磷酸氢二钠 -柠檬酸緩沖溶液， 于 50°C自溶 3h。 自溶结束后， 4000r/min离心 20min, 取上清液， 残渣备用。 上清液经真空 冷冻浓缩为原体积的 20%, 即为海参肠自溶活性物质粗品。 海参肠自溶活性物 质粗品经 Sephadex G-15凝胶柱分离， 控制流速为 0.5mL/min, 收集主要组分， 进行冷冻干燥。 调节冷阱温度为 -45°C , 真空度为 50Pa, 至水分含含量小于 8%, 冻干后即得到海参肠自溶活性物质。
海参肠自溶残渣， 加入 2倍体积去离子水混勾， 90°C加热 15min。 冷却后， 在 50°C , pH7.0下， 加入 2000U/g蛋白的中性蛋白酶， 酶解 120min。 酶解完毕 后， 酶解液于 100°C加热 5min灭酶， 10kDa超滤， 收集分子量低于 10kDa的组 分， 冷冻浓缩至原体积的 15%, 即为海参肠自溶残渣活性物质粗品。 浓缩液经 葡聚糖凝胶 Sephadex G-25层析柱， 用去离子水洗脱， 控制流速为 0.3mL/min, 收集主要组分， 进行冷冻干燥， 冷阱温度为 -45 °C , 真空度为 50Pa, 至水分含量 为小于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品为淡黄色固 体粉末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 其具有一定的体 外抗氧化能力， 可作为功能基料用于制备多种功能型食品。 实例三：
将 1kg海参肠清洗、剪碎、匀浆，放置在不锈钢槽内，浆液的厚度为 l~2cm, 在上方 0.5m处放置紫外灯， 控制浆液液面紫外强度为 60 μ /c 2, 保持 15min 进行自溶诱导。 然后， 加入 2倍体积的 pH4.0的磷酸氢二钠 -柠檬酸緩沖溶液， 于 45°C自溶 4h。 自溶结束后， 3500r/min离心 20min, 取上清液， 残渣备用。 上 清液经真空冷冻浓缩为原体积的 15%, 即为海参肠自溶活性物质粗品。 海参肠 自溶活性物质粗品经 Sephadex G-15凝胶柱分离，控制流速为 0.35mL/min,收集 主要组分， 进行冷冻干燥。 调节冷阱温度为 -45°C , 真空度为 65Pa, 至水分含量 小于 8%, 冻干后即得到海参肠自溶活性物质。
海参肠自溶残渣， 加入 3倍体积去离子水混勾， 95 °C加热 10min。 冷却后， 在 50°C , pH7.0下， 加入 3000U/g蛋白的枯草芽孢杆菌 1398中性蛋白酶， 酶解 60min。 酶解完毕后， 酶解液于 100°C加热 5min灭酶， lOkDa超滤， 收集分子量 低于 lOkDa的组分， 冷冻浓缩至原体积的 15%, 即为海参肠自溶残渣活性物质 粗品。 浓缩液经葡聚糖凝胶 Sephadex G-25层析柱， 用去离子水洗脱， 控制流速 为 0.25mL/min,收集主要组分，进行冷冻干燥，冷阱温度为 -45 °C ,真空度为 65Pa, 至水分含量为小于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品 为淡黄色固体粉末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 其具 有一定的体外抗氧化能力， 可作为功能基料用于制备多种功能型食品。 实例四：
将 500g海参肠清洗、 剪碎、 匀浆， 匀浆处理后紫外线（30W, 0.5m )照射 lOmin,加入 4倍体积的 pH4.0的磷酸氢二钠 -柠檬酸緩沖溶液，于 60 °C自溶 0.5h。 自溶结束后， 4500r/min离心 15min, 取上清液， 残渣备用。 上清液经真空冷冻 浓缩为原体积的 20%, 即为海参肠自溶活性物质粗品。 海参肠自溶活性物质粗 品经 Sephadex G-15凝胶柱分离， 控制流速为 0.4mL/min, 收集主要组分， 进行 冷冻干燥。 调节冷阱温度为 -35°C , 真空度为 75Pa, 至水分含量小于 6%, 冻干 后即得到海参肠自溶活性物质。 海参肠自溶残渣， 加入 2倍体积去离子水混勾， 100°C加热 5min。 冷却后， 在 50°C , pH7.0下， 加入 2000U/g蛋白的中性蛋白酶， 酶解 150min。 酶解完毕 后， 酶解液于 95°C加热 lOmin灭酶， 4500r/min离心 15min, 取上清液， 经真空 冷冻浓缩为原体积的 20%, 即为海参肠自溶残渣活性物质粗品。 浓缩液经葡聚 糖凝胶 Sephadex G-25层析柱， 用去离子水洗脱， 控制流速为 0.3mL/min, 收集 主要组分， 进行冷冻干燥， 冷阱温度为 -35 °C , 真空度为 75Pa, 至水分含量为小 于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品为淡黄色固体粉 末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 其具有一定的体外抗 氧化能力， 可作为功能基料用于制备多种功能型食品。 实例五：
将 300g海参肠清洗、 剪碎、 匀浆， 匀浆处理后紫外线（30W, 0.5m )照射 30min,加入 1倍体积的 pH5.0的磷酸氢二钠 -柠檬酸緩沖溶液， 于 35°C自溶 6h。 自溶结束后， 5000r/min离心 15min, 取上清液， 残渣备用。 上清液经真空冷冻 浓缩为原体积的 20%, 即为海参肠自溶活性物质粗品。 海参肠自溶活性物质粗 品经 Sephadex G- 15凝胶柱分离， 控制流速为 0. 5mL/min, 收集主要组分， 进行 冷冻干燥。 调节冷阱温度为 -40°C , 真空度为 70Pa, 至水分含量小于 6%, 冻干 后即得到海参肠自溶活性物质。
海参肠自溶残渣， 加入 4倍体积去离子水混勾， 90°C加热 10min。 冷却后， 在 50°C , pH7.0下， 加入 1500U/g蛋白的中性蛋白酶， 酶解 180min。 酶解完毕 后， 酶解液于 100°C加热 5min灭酶， 5000r/min离心 15min, 取上清液， 上清液 经真空冷冻浓缩为原体积的 15%, 即为海参肠自溶残渣活性物质粗品。 浓缩液 经葡聚糖凝胶 Sephadex G-25层析柱，用去离子水洗脱，控制流速为 0.4mL/min, 收集主要组分， 进行冷冻干燥， 冷阱温度为 -40 °C , 真空度为 70Pa, 至水分含量 为小于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品为淡黄色固 体粉末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 其具有一定的体 外抗氧化能力， 可作为功能基料用于制备多种功能型食品。 实例六：
将 1.5kg海参肠清洗、剪碎、 匀浆，放置在不锈钢槽内， 浆液的厚度为 2cm, 在上方 0.3m处放置紫外灯， 控制浆液液面紫外强度在 100 W/cm2, 保持 20min 进行自溶诱导。 然后， 加入 3倍体积的 pH7.0的磷酸氢二钠 -柠檬酸緩沖溶液， 于 50°C自溶 2h。 自溶结束后， 2500r/min离心 30min, 取上清液， 残渣备用。 上 清液经真空冷冻浓缩为原体积的 20%, 即为海参肠自溶活性物质粗品。 海参肠 自溶活性物质粗品经 Sephadex G-15凝胶柱分离， 控制流速为 0.3mL/min, 收集 主要组分， 进行冷冻干燥。 调节冷阱温度为 -45°C , 真空度为 70Pa, 至水分含量 小于 6%, 冻干后即得到海参肠自溶活性物质。
海参肠自溶残渣， 加入 2倍体积去离子水混匀， 100 °C加热 5min。 冷却后， 在 50°C , pH7.0下， 加入 2500U/g蛋白的中性蛋白酶， 酶解 120min。 酶解完毕 后， 酶解液于 95°C加热 lOmin灭酶， 2500r/min离心 30min, 取上清液， 上清液 经冷冻浓缩至原体积的 10%, 即为海参肠自溶残渣活性物质粗品。 浓缩液经葡 聚糖凝胶 Sephadex G-25层析柱， 用去离子水洗脱， 控制流速为 0.3mL/min, 收 集主要组分， 进行冷冻干燥， 冷阱温度为 -45 °C , 真空度为 70Pa, 至水分含量为 小于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品为淡黄色固体 粉末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 其具有一定的体外 抗氧化能力， 可作为功能基料用于制备多种功能型食品。 实例七：
将 800g海参肠清洗、 剪碎、 匀浆， 匀浆处理后紫外线（30W, 0.5m )照射 5min,加入 4倍体积的 pH6.0的磷酸氢二钠 -柠檬酸緩沖溶液，于 60 °C自溶 0.5h。 自溶结束后， 5500r/min离心 lOmin, 取上清液， 残渣备用。 上清液经真空冷冻 干后即为海参肠活性物质粗品。海参肠活性物质粗品用水溶解，经 Sephadex G-15 凝胶柱分离， 控制流速为 0.45mL/min, 收集主要组分， 进行冷冻干燥。 调节冷 阱温度为 -35 °C , 真空度为 60Pa, 至水分含量小于 6%, 冻干后即得到海参肠自 溶活性物质。
海参肠自溶残渣， 加入 3倍体积去离子水混勾， 90°C加热 15min。 冷却后， 在 50°C , pH7.0下， 加入 3000U/g蛋白的中性蛋白酶， 酶解 120min。 酶解完毕 后， 酶解液于 100°C加热 5min灭酶， lOkDa超滤， 收集分子量低于 lOkDa的组 分， 冷冻浓缩至原体积的 15%, 即为海参肠自溶残渣活性物质粗品。 浓缩液经 葡聚糖凝胶 Sephadex G-25层析柱， 用去离子水洗脱， 控制流速为 0.25mL/min, 收集主要组分， 进行冷冻干燥， 冷阱温度为 -35 °C , 真空度为 60Pa, 至水分含量 为小于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品为淡黄色固 体粉末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿性， 其具有一定的体 外抗氧化能力， 可作为功能基料用于制备多种功能型食品。 实例八：
将 2kg海参肠清洗、 剪碎、 匀浆， 放置在不锈钢槽内， 浆液的厚度为 lcm, 在上方 0.5m处放置紫外灯， 使浆液液面紫外强度为 lOO W/cm2, 保持 20min进 行自溶诱导。 然后， 加入 4倍体积的 pH4.0的磷酸氢二钠 -柠檬酸緩沖溶液， 于 55°C自溶 1.5h。 自溶结束后， 6000r/min离心 lOmin, 取上清液， 残渣备用。 上 清液经真空冷冻浓缩后即为海参肠自溶活性物质粗品。然后经 Sephadex G-15凝 胶柱分离， 控制流速为 0.5mL/min, 收集主要组分， 进行冷冻干燥。 调节冷阱温 度为 -35°C , 真空度为 65Pa, 至水分含量小于 6%, 冻干后即得到海参肠自溶活 性物质。
海参肠自溶残渣， 加入 4倍体积去离子水混勾， 90°C加热 15min。 冷却后， 在 50°C , pH7.0下， 加入 2000U/g蛋白的枯草芽孢杆菌 1398中性蛋白酶， 酶解 180min。 酶解完毕后， 酶解液于 95°C加热 5min灭酶， 6000r/min离心 lOmin, 取上清液， 上清液经真空冷冻浓缩为原体积的 15%, 即为海参肠自溶残渣活性 物质粗品。 浓缩液经葡聚糖凝胶 Sephadex G-25层析柱， 用去离子水洗脱， 控制 流速为 0.4mL/min, 收集主要组分， 进行冷冻干燥， 冷阱温度为 -35 °C , 真空度 为 65Pa, 至水分含量为小于 8%为止， 冻干后即得到海参肠自溶残渣活性物质。 两种产品为淡黄色固体粉末， 无臭， 有海参肠固有的鲜味， 易溶于水， 有吸湿 性， 其具有一定的体外抗氧化能力， 可作为功能基料用于制备多种功能型食品。
以上所述， 仅为本发明较佳的具体实施方式， 但本发明的保护范围并不局 限于此， 任何熟悉本技术领域的技术人员在本发明披露的技术范围内， 根据本 发明的技术方案及其发明构思加以等同替换或改变， 都应涵盖在本发明的保护 范围之内。

Claims

1、 一种利用海参肠自溶制备活性物质的方法， 其特征在于， 以海参肠为原 料， 经匀浆、 自溶、 取上清液， 冷冻浓缩至原体积的 10%~20%或真空冷冻干燥 至水分含量小于等于 8%, 制得海参肠活性物质粗品； 再经分离、 冷冻干燥制得 精制活性物质。
2、根据权利要求 1所述利用海参肠自溶制备活性物质的方法，其特征在于， 所述自溶的工序为：
匀浆后所得匀浆液经紫外线照射后， 加入 1 ~ 4倍体积、 pH 4.0~7.0的柠檬 酸与磷酸氢二钠緩沖溶液， 在 35 ~ 60°C下自溶 0.5 ~ 4 h, 或在 4 ~ 10°C时， 自溶 4 ~ 12 h。
3、根据权利要求 2所述利用海参肠自溶制备活性物质的方法， 其特征在于， 所述自溶工序中选用 30瓦紫外灯， 距离 0.5 m照射 5 ~ 40min, 进行自溶诱导； 或者， 将匀浆液放置在不锈钢槽内， 在上方 0.2~0.5m处放置紫外灯， 控制 浆液液面紫外强度为 60~120 μψ/cm2, 保持 5 ~ 40 min进行自溶诱导。
4、根据权利要求 3所述利用海参肠自溶制备活性物质的方法， 其特征在于， 所述取上清液的工序为： 2500 ~ 6000 r/min离心 10 ~ 30分钟， 取上清液； 或者 所述取上清液的工序采用超滤的方式， 收集 lOkDa以下的组分。
5、 根据权利要求 1-4任一所述利用海参肠自溶制备活性物质的方法， 其特 征在于， 所述分离的工序为：
将所述海参肠活性物质粗品上样于葡聚糖凝胶 Sephadex G-15层析柱， 用去 离子水洗脱，控制流速为 0.3~0.5mL/min,对峰面积占各组分总峰面积 70%~80% 的活性组分进行收集。
6、根据权利要求 5所述利用海参肠自溶制备活性物质的方法， 其特征在于， 所述冷冻干燥的工序为： 在冷阱温度 -30 ~ -45°C、 真空度为 50 ~ 80Pa条件下进 行干燥。
7、 一种利用海参肠自溶制备活性物质的方法， 其特征在于， 以海参肠为原 料， 经匀浆、 自溶；
而后取残渣，加入 2~4倍体积的去离子水混匀， 90°C~100 °C加热 5~15 min; 冷却后于 50 °C、 ρΗ7·0下，加入 1500~3000 U/g蛋白的中性蛋白酶，酶解 30-180 min;
酶解液于 95~100 °C加热 5~10 min灭酶， 冷却后于 2500 ~ 6000 r/min下离 心 10 ~ 30 min;
而后取上清液或采用超滤的方式收集 lOkDa以下的组分， 制得海参肠自溶 残渣活性物质。
8、根据权利要求 7所述利用海参肠自溶制备活性物质的方法，其特征在于， 所述自溶的工序为：
匀浆后所得匀浆液经紫外线照射后， 加入 1 ~ 4倍体积、 pH 4.0~7.0的柠檬 酸与磷酸氢二钠緩沖溶液， 在 35 ~ 60°C下自溶 0.5 ~ 4 h, 或在 4°C ~ 10°C时， 自 溶 4 ~ 12 h。
其中， 紫外线照射的工序为： 所述自溶工序中选用 30瓦紫外灯，距离 0.5 m 照射 5 ~ 40 min进行自溶诱导； 或者， 将匀浆液放置在不锈钢槽内， 浆液厚度 1-2 cm, 在上方 0.2~0.5 m处放置紫外灯， 控制浆液液面紫外强度为 60~120 W/cm2 , 保持 5 ~ 40 min进行自溶诱导。
9、根据权利要求 7或 8所述利用海参肠自溶制备活性物质的方法， 其特征 在于， 将制得的海参肠自溶残渣活性物质采用冷冻浓缩的方法浓缩至原体积的 10 %~20 %, 或者采用真空冷冻干燥的方式干燥至水分含量小于等于 8 %, 获得 海参肠自溶残渣活性物质粗品。
10、 根据权利要求 9所述利用海参肠自溶制备活性物质的方法， 其特征在 于，将所述海参肠自溶残渣活性物质粗品上样于葡聚糖凝胶 Sephadex G-25层析 柱， 用去离子水洗脱， 控制流速为 0.2~0.4 mL/min, 对峰面积占各组分总峰面积 的 80%~95%的主要组分进行收集；
最后， 在冷阱温度为 -30 ~ -45 °C , 真空度为 50 ~ 80 Pa条件下进行干燥， 至水分含量小于等于 8 %为止， 冻干后制得精制活性物质。
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