CN109295146B - Enzymatic abalone extract and preparation method thereof - Google Patents
Enzymatic abalone extract and preparation method thereof Download PDFInfo
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Abstract
According to the invention, the abalone is subjected to finish machining treatment by means of bioengineering, and macromolecular substances such as protein in the abalone are degraded into active micromolecules to the maximum extent by the combination of autolytic enzymolysis, active strain fermentation, pulse ultrasonic treatment and enzymolysis processes, so that the utilization degree and the nutritional value of nutritional ingredients of the abalone are improved, the obtained abalone enzyme method extract has good antioxidant activity and anti-fatigue effect, can be used as a processing raw material of health-care food and medicines with corresponding effects, and the additional value and the industrial value of abalone processing are greatly improved.
Description
The technical field is as follows:
the invention belongs to the field of bioengineering, relates to enzyme engineering and fermentation engineering technology, and particularly relates to an abalone enzyme method extract and a preparation method thereof.
Background art:
abalone is a mollusk, belonging to the class of gastropoda, gilles, order of primitive gastropoda, family of abalone, genus of abalone, and is a rare marine product. Abalone is wide in geographical distribution, is mainly carried by Australia, Japan, China, New Zealand, America, Europe and the middle east, is a traditional high-grade nourishing food, is praised as 'soft gold' on a dining table, can be used as both medicine and food, and is recorded in famous medical records, so that the abalone is sweet and salty in nature and warm in nature, and has the functions of nourishing liver and kidney, and replenishing vital essence and improving eyesight; recorded in Ben Cao gang mu, abalone is neutral in nature, sweet and salty in taste, and has the functions of improving eyesight, tonifying deficiency, clearing heat, nourishing yin, nourishing blood, benefiting stomach, and nourishing liver and kidney.
With the application of the abalone artificial culture technology, the Fujian province and the Guangdong province become the largest abalone culture bases in China, wherein the Shanshan City of the Guangdong province forms one of the largest abalone culture bases in China, and 900 t abalones are produced annually and show a continuous rising trend; the sea water in part of the Fujian province is pure, the seaweed is rich, the abalone culture medium is very suitable for the growth of the abalones, the yield of the abalones accounts for about three quarters of the national yield, meanwhile, most of Fujian abalone varieties are hybrid abalones, the individuals are large, the abalones are suitable for the growth environment of southern sea areas, the culture time is short, the survival rate is high, the quality and the quantity of the abalones are high, the Fujian abalones are provided with a vigorous development opportunity, and the Zhang Zhou abalones and the Lianjiang abalones become famous brands in the abalone industry in China.
However, at present, the processing and utilization of abalones are still in the primary stage, the processing of the existing abalones mainly focuses on the dry and instant abalones, and the development of products of the abalone mainly focuses on the improvement of the individual size of the abalones, the rehydration technology of dry products, the flavor of the instant abalones and the like. At present, few researches on processing, nutrient components and physiological activity of abalone are reported, and few literature reports mention that abalone is processed into abalone juice or is processed into an enzymolysis extract by an enzymolysis technology.
The invention content is as follows:
based on the development condition of the existing abalone production and processing technology, the invention aims to provide an abalone enzyme method extract and a preparation method thereof.
In order to solve the technical problems, the invention adopts the following technical scheme:
an enzymatic extract of abalone, which is characterized by being prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, and soaking the abalone meat in an ozone water solution for disinfection treatment;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture, and placing the mixture in a colloid mill for grinding to obtain abalone pretreatment slurry;
step three, autolysis hydrolysis: irradiating the abalone pretreatment slurry prepared in the step two by using ultraviolet rays, continuously stirring in the process, and performing autolysis hydrolysis;
step four, fermentation treatment: adding isomaltooligosaccharide and glucose into the autolyzed hydrolysate prepared in the third step, inoculating saccharomyces cerevisiae and lactobacillus casei, and performing fermentation treatment to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5-8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 40-42 ℃, and carrying out enzymolysis treatment for 3.5-4 hours to obtain an enzymolysis solution, wherein the weight parts of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme are 3 (1-1.5) to (1-2) to (2-3), and the addition amount of the complex enzyme is 0.5-0.7%;
and seventhly, inactivating enzyme and concentrating to obtain the abalone enzyme method extract.
Specifically, the invention relates to the following specific technical scheme:
an enzymatic extract of abalone, which is characterized by being prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 2-3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2-4, placing the mixture in a colloid mill for grinding, sieving the mixture by a 30-50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the step two at the temperature of 2-4 ℃, irradiating by ultraviolet rays with the wavelength of 200-280nm, continuously stirring in the process, performing autolytic hydrolysis for 3-4 hours, and performing autolytic hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain an autolytic hydrolysate;
step four, fermentation treatment: adding isomaltooligosaccharide and glucose into the autolyzed hydrolysate prepared in the third step, wherein the mass ratio of the autolyzed hydrolysate to the isomaltooligosaccharide to the glucose is 100 (0.5-1) to (2-3), inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at the temperature of 28-32 ℃ for 20-24 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 10-12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5-8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 40-42 ℃, and carrying out enzymolysis treatment for 3.5-4 hours to obtain an enzymolysis solution, wherein the weight parts of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme are 3 (1-1.5) to (1-2) to (2-3), and the addition amount of the complex enzyme is 0.5-0.7%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the original volume, namely the abalone enzyme method extract.
The invention also relates to a preparation method of the abalone enzymatic extract, which is characterized by comprising the following steps:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 2-3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2-4, placing the mixture in a colloid mill for grinding, sieving the mixture by a 30-50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the step two at the temperature of 2-4 ℃, irradiating by ultraviolet rays with the wavelength of 200-280nm, continuously stirring in the process, performing autolytic hydrolysis for 3-4 hours, and performing autolytic hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain an autolytic hydrolysate;
step four, fermentation treatment: adding isomaltooligosaccharide and glucose into the autolyzed hydrolysate prepared in the third step, wherein the mass ratio of the autolyzed hydrolysate to the isomaltooligosaccharide to the glucose is 100 (0.5-1) to (2-3), inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at the temperature of 28-32 ℃ for 20-24 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 10-12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5-8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 40-42 ℃, and carrying out enzymolysis treatment for 3.5-4 hours to obtain an enzymolysis solution, wherein the weight parts of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme are 3 (1-1.5) to (1-2) to (2-3), and the addition amount of the complex enzyme is 0.5-0.7%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the original volume, namely the abalone enzyme method extract.
Preferably, the mass ratio of the autolyzed hydrolysate, the isomaltooligosaccharide and the glucose in the fourth step is 100:1:2 or 100:0.5: 3.
Preferably, in the sixth step, the weight part ratio of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the compound enzyme is 3: 1.5:2:3 or 3:1:2: 2. Wherein the alkaline protease, papain, neutral protease and complex flavor protease are all commercially available food processing enzymes, such as the related enzymes from Novistin.
Based on the technical scheme, the invention has the following advantages and beneficial effects:
firstly, the abalone is subjected to finish machining treatment by adopting a bioengineering method, macromolecular substances such as protein in the abalone are degraded into active micromolecules to the maximum extent by the combination of autolytic enzymolysis, active strain fermentation, pulse ultrasonic treatment and enzymolysis technology, the utilization degree and the nutritional value of nutritional ingredients of the abalone are improved, the obtained abalone enzyme method extract has good antioxidant activity and anti-fatigue effect, can be used as a processing raw material of health-care food and medicine with corresponding effects, and the added value and the industrial value of abalone processing are greatly improved. In addition, aiming at the current situation of abalone overproduction, the method provided by the invention can effectively remove the abalone production stock, and avoids abalone price reduction caused by abalone yield increase.
Secondly, the invention initiatively adopts a combined treatment process of autolytic hydrolysis, active strain fermentation, pulse ultrasonic treatment and enzymolysis, wherein the autolytic hydrolysis utilizes rich enzymes contained in abalone, after the grinding treatment, abalone tissues and cells are destroyed, hydrolytic enzymes such as protease in the cells are released into tissue homogenate, the enzymes contained in the autolytic hydrolysis can carry out enzymolysis on histoprotein, polysaccharide molecules and the like of the abalone to release active ingredients, in the autolytic hydrolysis process, ultraviolet irradiation with specific wavelength is adopted, the autolytic enzymolysis can be excited, meanwhile, pollution bacteria in the surrounding environment such as air and the like can be killed, the pollution is avoided, the autolytic hydrolysis can also provide good nutrition for the next active strain fermentation, and the active strain can be proliferated and fermented very quickly after inoculation; secondly, in the active strain fermentation process, the method is carried out in a mode of combining saccharomyces cerevisiae and lactobacillus casei, isomaltooligosaccharide and glucose are added into an autolyzed hydrolysate, the specific proportion of the isomaltooligosaccharide and the glucose ensures that the two bacteria can be relatively and uniformly proliferated, particularly, the isomaltooligosaccharide can only be utilized by the lactobacillus casei, the isomaltooligosaccharide can promote the proliferation of the lactobacillus casei, and then macromolecular nutrient components for degrading the abalones are micromolecular active substances which are utilized by the saccharomyces cerevisiae and promote the proliferation of the saccharomyces cerevisiae; the invention creatively utilizes the pulse ultrasonic treatment, and obtains the optimal pulse ultrasonic treatment mode by adjusting the pulse ultrasonic frequency, the power, the treatment time and the pulse frequency, the pulse ultrasonic treatment can further break the tissue cells of the abalone and fully release macromolecular substances such as protein, meanwhile, the pulse ultrasonic treatment can break and proliferate active strains, namely lactobacillus casei and saccharomyces cerevisiae, and release tissue enzymes in the cells of the lactobacillus casei and the saccharomyces cerevisiae for the next enzymolysis, and meanwhile, the broken yeast cells have good delicate flavor, and the good flavor of the product is fully ensured; finally, the abalone enzymolysis extract is obtained through enzymolysis processing, the inventor determines that the complex enzyme consists of alkaline protease, papain, neutral protease and compound flavor protease through a large number of tests, wherein the proportion of various enzymes is synergistic with the result of pulse ultrasonic treatment, namely, enzymes in the complex enzyme and enzymes released after active strains are crushed jointly act, the degradation of abalone macromolecular substances is effectively promoted, the full hydrolysis of the macromolecular substances, namely protein and polysaccharide molecules in the abalone is ensured, and the abalone enzymolysis extract with good flavor and high activity is finally prepared.
The specific implementation mode is as follows:
example 1: an enzymatic extract of abalone, which is characterized by being prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2, placing the mixture in a colloid mill for grinding, sieving the mixture with a 50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the second step at the temperature of 4 ℃, irradiating by ultraviolet rays with the wavelength of 220nm, continuously stirring in the process, carrying out autolysis hydrolysis for 4 hours, and carrying out autolysis hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain autolysis hydrolysate;
step four, fermentation treatment: adding isomaltose hypgather and glucose into the autolyzed hydrolysate obtained in the third step, wherein the mass ratio of the isomaltose hypgather to the glucose is 100:1:2, inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at 30 ℃ for 22 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 42 ℃, and carrying out enzymolysis treatment for 3.5 hours to obtain an enzymolysis solution, wherein the weight part ratio of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme is 3: 1.5:2:3, and the addition amount of the complex enzyme is 0.6%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone enzyme method extract.
Example 2: an enzymatic extract of abalone, which is characterized by being prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 2 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:4, placing the mixture in a colloid mill for grinding, sieving the mixture by a 30-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the second step at the temperature of 2 ℃, irradiating by ultraviolet rays with the wavelength of 280nm, continuously stirring in the process, carrying out autolysis hydrolysis for 4 hours, and carrying out autolysis hydrolysis on macromolecules such as protein of the abalone through enzymes contained in the abalone to obtain autolysis hydrolysate;
step four, fermentation treatment: adding isomaltose hypgather and glucose into the autolyzed hydrolysate obtained in the third step, wherein the mass ratio of the isomaltose hypgather to the glucose is 100:0.5:3, inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at 28 ℃ for 24 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavourzyme, heating to 40 ℃, and carrying out enzymolysis treatment for 4 hours to obtain an enzymolysis liquid, wherein the weight ratio of the alkaline protease, the papain, the neutral protease and the compound flavourzyme in the complex enzyme is 3:1:2:2, and the addition amount of the complex enzyme is 0.7%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone enzyme method extract.
Example 3: a preparation method of an abalone enzymatic extract is characterized by comprising the following steps:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 2 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2-4, placing the mixture in a colloid mill for grinding, sieving the mixture with a 40-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the second step at the temperature of 3 ℃, irradiating by ultraviolet rays with the wavelength of 250nm, continuously stirring in the process, carrying out autolysis hydrolysis for 3.5 hours, and carrying out autolysis hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain autolysis hydrolysate;
step four, fermentation treatment: adding isomaltose hypgather and glucose into the autolyzed hydrolysate obtained in the third step, wherein the mass ratio of the isomaltose hypgather to the glucose is 100:1:2, inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at 30 ℃ for 22 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 11 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.8, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 42 ℃, and carrying out enzymolysis treatment for 3.5 hours to obtain an enzymolysis solution, wherein the weight part ratio of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme is 3: 1.5:2:3, and the addition amount of the complex enzyme is 0.5%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone enzyme method extract.
Example 4: in order to respectively verify the influence of autolytic hydrolysis, pulse ultrasonic treatment and compound enzyme composition on the enzymatic extract of the abalone, the test process of the invention is specially provided with the following three groups of control examples:
comparative example a (to verify the effect of autolytic hydrolysis on enzymatic extracts):
an abalone extract A, which is prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2, placing the mixture in a colloid mill for grinding, sieving the mixture with a 50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, fermentation treatment: adding isomaltose hypgather and glucose into the pretreated serous fluid prepared in the step two, wherein the mass ratio of the autolyzed hydrolysate to the isomaltose hypgather to the glucose is 100:1:2, inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at the temperature of 30 ℃ for 22 hours to obtain a fermentation product;
step four, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step three, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step five, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fourth step to 8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 42 ℃, and carrying out enzymolysis treatment for 3.5 hours to obtain an enzymolysis solution, wherein the weight ratio of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme is 3: 1.5:2:3, and the addition amount of the complex enzyme is 0.6%;
step six, enzyme deactivation and concentration: heating the enzymolysis liquid prepared in the step five to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone extract A.
Comparative example B (to verify the effect of pulsed sonication on enzymatic extracts):
an abalone extract B, which is prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2, placing the mixture in a colloid mill for grinding, sieving the mixture with a 50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the second step at the temperature of 4 ℃, irradiating by ultraviolet rays with the wavelength of 220nm, continuously stirring in the process, carrying out autolysis hydrolysis for 4 hours, and carrying out autolysis hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain autolysis hydrolysate;
step four, fermentation treatment: adding isomaltose hypgather and glucose into the autolyzed hydrolysate obtained in the third step, wherein the mass ratio of the isomaltose hypgather to the glucose is 100:1:2, inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at 30 ℃ for 22 hours to obtain a fermentation product;
step five, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fourth step to 8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 42 ℃, and carrying out enzymolysis treatment for 3.5 hours to obtain an enzymolysis solution, wherein the weight ratio of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme is 3: 1.5:2:3, and the addition amount of the complex enzyme is 0.6%;
step six, enzyme deactivation and concentration: heating the enzymolysis liquid prepared in the step five to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone extract B.
Comparative example C (to verify the effect of complex enzyme composition on enzymatic extracts):
an abalone extract C, which is prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2, placing the mixture in a colloid mill for grinding, sieving the mixture with a 50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the second step at the temperature of 4 ℃, irradiating by ultraviolet rays with the wavelength of 220nm, continuously stirring in the process, carrying out autolysis hydrolysis for 4 hours, and carrying out autolysis hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain autolysis hydrolysate;
step four, fermentation treatment: adding isomaltose hypgather and glucose into the autolyzed hydrolysate obtained in the third step, wherein the mass ratio of the isomaltose hypgather to the glucose is 100:1:2, inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at 30 ℃ for 22 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavourzyme, heating to 42 ℃, and carrying out enzymolysis treatment for 3.5 hours to obtain an enzymolysis solution, wherein the weight ratio of the alkaline protease to the papain in the complex enzyme is 3:2, and the addition amount of the complex enzyme is 0.5%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone enzyme method extract.
Example 5: safety test
Acute toxicity test of mice: the products of the embodiments 1-3 of the invention are partially used for replacing the feed of mice to feed, 2-3mL of the abalone enzyme method extract is fed every day, the abalone is continuously fed for 48 days, no death and abnormal toxic reaction are found during the test period, the test mice are dissected after the test is finished, and the liver, the kidney, the spleen, the stomach, the heart, the lung and the intestines are observed, and are not abnormally changed. The products of examples 1-3 were of non-toxic grade according to the acute toxicity criteria.
Chronic toxicity test: the products of the embodiments 1-3 of the invention are partially used for replacing the feed of mice to feed, 4-6mL of the abalone enzyme method extract is fed every day, and the results of the continuous feeding for 220 days show that: the mice fed with the products of the invention examples 1-3 have smooth skin and hair, normal behavior and activity, normal weight increase, normal drinking and eating, fluctuation of hematology and blood biochemical indexes in a normal range, fluctuation of marrow and urine conventional indexes in a normal range, no abnormality of tissue organs and no toxicological reaction on the whole.
As can be seen from the above experiments, the product of the invention is safe and nontoxic.
Example 6: sensory evaluation
Sensory evaluation was performed on the enzymatic abalone extracts of examples 1, 2 and 3 of the present invention and the abalone extracts a to C prepared in comparative examples a to C, with a full score of 10 and a 6-score of pass, and the sensory evaluation was performed on the flavor by 10 professional sensory evaluators and 50 randomly invited general consumers, respectively, and the specific results (average score) are shown in table 1 below:
TABLE 1 flavor sensory score results
| Group of | Example 1 | Example 2 | Example 3 | Comparison A | Comparison B | Comparison C |
| Professional rater scoring | 9.51 | 9.33 | 9.42 | 8.31 | 7.89 | 6.47 |
| General consumer rating | 9.36 | 9.22 | 9.25 | 6.89 | 6.56 | 5.28 |
Wherein the products of examples 1-3 all had good flavor and a relatively good umami taste; whereas the product of comparative example A had a slightly poorer flavour than the products of examples 1-3, comparative examples B and C had a certain bitterness, in particular a stronger bitterness after taste than comparative example C.
Example 7: animal testing
140 Kunming mice are taken, half of the mice are female and male, the mice are randomly divided into 7 groups, and 10 males and 10 females in each group. Wherein group 1 is a blank control group, and is fed with conventional mouse feed; groups 2-4 are experimental groups, the abalone enzymatic extract of examples 1-3 of the present invention was mixed with conventional mouse feed at a ratio of 1:4 and then fed to mice, groups 5-7 were comparative experimental groups, the abalone extract a-C of comparative experimental examples a-C was mixed with conventional mouse feed at a ratio of 1:4 and then fed to mice, and seven groups were fed for 15 days with the same feeding conditions except for different feed compositions. Then, various tests were performed:
(1) and (3) testing the activity level of the antioxidant enzyme:
on the 10 th day of continuous feeding, a small amount of whole blood is respectively taken through a rat tail vein, the activity of glutathione peroxidase (GSH-Px) in the whole blood is determined, and the activity range of the GSH-Px of mice in a group 1 (blank control group) is 25 +/-4U/mL of the whole blood through detection; the viability of GSH-Px fed mice with enzymatic extracts of abalone of groups 2-4 (i.e., examples 1-3) ranged from 38 ± 5U/mL whole blood; GSH-Px activity of the paua extracts of controls 5-7 (i.e., comparative examples a-C) fed mice with GSH-Px ranged from 28 ± 3U/mL whole blood (comparative example a), 30 ± 4U/mL whole blood (comparative example B), 29 ± 2U/mL whole blood (comparative example C). Therefore, the abalone enzymolysis extract disclosed by the embodiments 1-3 can improve the antioxidant level of mice, and compared with a blank control group and comparative test examples A-C, the abalone enzymolysis extract has unexpected technical effects, and the antioxidant level of the abalone enzymolysis extract is improved by more than 30%.
(2) And (3) animal experiments for relieving physical fatigue: after 15 days of continuous feeding, a weight-bearing swimming experiment is carried out, after 120min of the last feeding, the mice are placed in a swimming tank, the water depth is 40cm, the length and the width of the water tank are 0.75m x 0.5m respectively, the wall of the water tank is smooth, the water temperature is 25 +/-0.5 ℃, and 5 percent of weight of lead skin is loaded on the root parts of the rat tail.
The time from the beginning of swimming to death of the mice was the swimming time of the mice, and the results are shown in the following table 2:
TABLE 2 functional animal experiment results for physical fatigue alleviation
| Group of | Swimming time/min |
| Group 1 (blank control) | 55.8±10.1 |
| Group 2 (example 1) | 232.5±15.7 |
| Group 3 (example 2) | 220.6±12.2 |
| Group 4 (example 3) | 210.5±11.3 |
| Group 5 (comparative example A) | 150.3±11.2 |
| Group 6 (comparative example B) | 138.9±9.7 |
| Group 7 (comparative example C) | 147.5±10.5 |
From the test results, compared with a blank control group fed by a common feed, the products of the embodiments 1 to 3 can greatly improve the time for the mice to carry heavy swim, and the time for the mice fed by the products to carry heavy swim is 3 to 4 times that of the mice fed by the common feed, which shows that the products of the invention have the obvious function of relieving physical fatigue; although comparative examples A-C also increased the duration of heavy swimming, the anti-fatigue effect was less than that of the products of examples 1-3 of the present invention, and it was due to the synergistic effect of the process steps of the present invention that the products of the present invention achieved an unexpected anti-fatigue effect compared to comparative examples A-C.
Claims (6)
1. The preparation method of the enzymatic extract of the abalone is characterized by comprising the following steps of:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, and soaking the abalone meat in an ozone water solution for disinfection treatment;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture, and placing the mixture in a colloid mill for grinding to obtain abalone pretreatment slurry;
step three, autolysis hydrolysis: irradiating the abalone pretreatment slurry prepared in the step two by using ultraviolet rays, continuously stirring in the process, and performing autolysis hydrolysis;
step four, fermentation treatment: adding isomaltooligosaccharide and glucose into the autolyzed hydrolysate prepared in the third step, inoculating saccharomyces cerevisiae and lactobacillus casei, and performing fermentation treatment to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5-8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 40-42 ℃, and carrying out enzymolysis treatment for 3.5-4 hours to obtain an enzymolysis solution, wherein the weight parts of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme are 3 (1-1.5) to (1-2) to (2-3), and the addition amount of the complex enzyme is 0.5-0.7%;
and seventhly, inactivating enzyme and concentrating to obtain the abalone enzyme method extract.
2. A process for the preparation of an enzymatic extract of abalone according to claim 1, characterised in that it comprises the following steps:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 2-3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2-4, placing the mixture in a colloid mill for grinding, sieving the mixture by a 30-50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the step two at the temperature of 2-4 ℃, irradiating by ultraviolet rays with the wavelength of 200-280nm, continuously stirring in the process, performing autolytic hydrolysis for 3-4 hours, and performing autolytic hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain an autolytic hydrolysate;
step four, fermentation treatment: adding isomaltooligosaccharide and glucose into the autolyzed hydrolysate prepared in the third step, wherein the mass ratio of the autolyzed hydrolysate to the isomaltooligosaccharide to the glucose is 100 (0.5-1) to (2-3), inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at the temperature of 28-32 ℃ for 20-24 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 10-12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5-8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 40-42 ℃, and carrying out enzymolysis treatment for 3.5-4 hours to obtain an enzymolysis solution, wherein the weight parts of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme are 3 (1-1.5) to (1-2) to (2-3), and the addition amount of the complex enzyme is 0.5-0.7%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone enzyme method extract.
3. An enzymatic extract of abalone, which is characterized by being prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, and soaking the abalone meat in an ozone water solution for disinfection treatment;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture, and placing the mixture in a colloid mill for grinding to obtain abalone pretreatment slurry;
step three, autolysis hydrolysis: irradiating the abalone pretreatment slurry prepared in the step two by using ultraviolet rays, continuously stirring in the process, and performing autolysis hydrolysis;
step four, fermentation treatment: adding isomaltooligosaccharide and glucose into the autolyzed hydrolysate prepared in the third step, inoculating saccharomyces cerevisiae and lactobacillus casei, and performing fermentation treatment to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5-8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 40-42 ℃, and carrying out enzymolysis treatment for 3.5-4 hours to obtain an enzymolysis solution, wherein the weight parts of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme are 3 (1-1.5) to (1-2) to (2-3), and the addition amount of the complex enzyme is 0.5-0.7%;
and seventhly, inactivating enzyme and concentrating to obtain the abalone enzyme method extract.
4. An enzymatic extract of abalone, which is characterized by being prepared by the following method:
step one, pretreatment: taking fresh abalone, removing shells and viscera, taking abalone meat, soaking the abalone meat in an ozone water solution for disinfection treatment, wherein the treatment time is 20min, the treatment temperature is 5 ℃, and the ozone concentration in the ozone water solution is 2-3 ppm;
step two, preparing slurry: mixing the abalone meat pretreated in the step one with an ice water mixture according to a mass ratio of 1:2-4, placing the mixture in a colloid mill for grinding, sieving the mixture by a 30-50-mesh sieve, and uniformly stirring to prepare abalone pretreatment slurry;
step three, autolysis hydrolysis: placing the abalone pretreatment slurry prepared in the step two at the temperature of 2-4 ℃, irradiating by ultraviolet rays with the wavelength of 200-280nm, continuously stirring in the process, performing autolytic hydrolysis for 3-4 hours, and performing autolytic hydrolysis on macromolecules such as protein of the abalone by enzymes contained in the abalone to obtain an autolytic hydrolysate;
step four, fermentation treatment: adding isomaltooligosaccharide and glucose into the autolyzed hydrolysate prepared in the third step, wherein the mass ratio of the autolyzed hydrolysate to the isomaltooligosaccharide to the glucose is 100 (0.5-1) to (2-3), inoculating saccharomyces cerevisiae and lactobacillus casei, and fermenting at the temperature of 28-32 ℃ for 20-24 hours to obtain a fermentation product;
step five, pulse ultrasonic pretreatment: performing pulse ultrasonic pretreatment on the fermentation product obtained in the step four, wherein the ultrasonic frequency is 20kHz, the power is 200W, the ultrasonic time is 10-12 minutes, and the pulse ultrasonic working gap time ratio is 2s/2s to obtain a pulse ultrasonic pretreatment product;
step six, enzymolysis: adjusting the pH value of the pulse ultrasonic pretreatment product prepared in the fifth step to 7.5-8.2, adding a complex enzyme consisting of alkaline protease, papain, neutral protease and compound flavor protease, heating to 40-42 ℃, and carrying out enzymolysis treatment for 3.5-4 hours to obtain an enzymolysis solution, wherein the weight parts of the alkaline protease, the papain, the neutral protease and the compound flavor protease in the complex enzyme are 3 (1-1.5) to (1-2) to (2-3), and the addition amount of the complex enzyme is 0.5-0.7%;
step seven, inactivating enzyme and concentrating: heating the enzymolysis liquid prepared in the sixth step to 80 ℃ in a water bath, inactivating the enzyme for 10 minutes to inactivate the complex enzyme, filtering, and concentrating to 1/3 of the volume of the enzymolysis liquid, namely the abalone enzyme method extract.
5. An abalone enzymatic extract according to claim 3 or 4, characterised in that the mass ratio of autolysed hydrolysate, isomaltooligosaccharide and glucose in step four is 100:1:2 or 100:0.5: 3.
6. The enzymatic extract of abalone according to claim 3 or 4, wherein in step six, the weight ratio of alkaline protease, papain, neutral protease and flavourzyme in the complex enzyme is 3: 1.5:2:3 or 3:1:2: 2.
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