CN105248833A - Method for extracting shrimp protein nutrients used for enhancing immunity and relieving fatigue - Google Patents
Method for extracting shrimp protein nutrients used for enhancing immunity and relieving fatigue Download PDFInfo
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Abstract
The invention relates to the technical field of shrimp protein extraction, in particular to a method for extracting shrimp protein nutrients used for enhancing immunity and relieving fatigue. The method includes the following steps that 1, brine shrimps are dried through hot air; 2, after being dried, the brine shrimps are smashed and screened; 3, after the brine shrimps are smashed and screened, water is added to soak the brine shrimps; 4, a compound enzyme is added to perform limited enzymatic hydrolysis to prepare enzymatic hydrolysate; 5, the prepared enzymatic hydrolysate is concentrated; 6, 5% of active carbon is added to perform decoloration and deodorization, and then the active carbon is filtered out; 7, finally, spray drying is performed to obtain the nutrients. The compound enzyme added in the step 4 is a papain, alkaline protease and flavourzyme compound, and the proportion of papain to alkaline protease to flavourzyme in the compound enzyme added in the step 4 is 1.5-2.5:1:1.5-2.0. As the limited enzymatic hydrolysis technology is utilized for controlling the enzymatic hydrolysis degree, enzymatic hydrolysis is not thorough, the nutrients contain abundant amino acid, polypeptide and protein which can be easily absorbed and utilized by the human body and are in a proper proportion, and the total protein extraction ratio reaches 76.3%.
Description
Technical field
The present invention relates to shrimp protein extractive technique field, be specifically related to the shrimp protein nutrient method of a kind of extraction for develop immunitypty and fatigue-relieving.
Background technology
Modern is along with the enhancing of operating pressure, and the body immunity of people is low, and sense of fatigue becomes clear day by day, and increasing people is in sub-health state.Therefore the pharmaceutical methods research for the effect such as develop immunitypty, fatigue-relieving is quite a few.
" research of shrimp processing byproduct enzymolysis process " that " food engineering " as Chinese document was published in June, 2012, by the efficiency of more different hydrolases shrimp processing byproducts, determines that alkali protease is hydrolysis shrimp processing byproduct enzyme.Investigated solid-liquid ratio, time, temperature, initial ph value, enzyme addition to the impact of protein extracting ratio, determined that optimum enzymolysis process condition is: enzyme addition be shrimp med quality 0.8%, temperature 60 C, solid-liquid ratio 4g:100mL, initial pH9.0, time 2.0h, protein extracting ratio be 65.3%.In addition, in " research of hydrolyzing technology of shrimp offal by proteinase " that " food additives " were published in December, 2005, this test take degree of hydrolysis as index, have studied the ability of 5 kinds of protease hydrolyzed shrimp processing fents, and determining Flavourzyme and Alcalase is best complex enzyme.Utilize Response Surface Method, obtain the peak enzymolysis-ability parameter of complex enzyme: initial ph value 6.74, hydrolysis temperature 48.3 DEG C, concentration of substrate 7.3%.Under this optimal conditions, repeating test 3 times, average degree of hydrolysis reaches 23.16%.Although all carried out some researchs, not further to the research of nutrient in develop immunitypty and effect of relieving physical fatigue in above-mentioned two class technology to protease hydrolyzed shrimp.
Summary of the invention
For the technical problem existed in above-mentioned prior art, the present invention aims to provide the shrimp protein nutrient method of a kind of extraction for develop immunitypty and fatigue-relieving.
For realizing this technical purpose, the solution of the present invention is: take salted shrimp gravy as raw material, the proportioning of papain, alkali protease and flavor protease is utilized to combine, by controlled enzymatic hydrolysis condition, enzymolysis time and enzyme addition and ratio, carry out limited enzymolysis, then through decolouring, deodorization process passable to aromatic flavour, without fishy smell, nutrient without bitter taste, odorless.
Preparation method of the present invention is specific as follows:
(1) salted shrimp gravy is passed through heated-air drying;
(2) crushed after being dried, through screening;
(3) soak after crushing and screening;
(4) complex enzyme limited enzymolysis is added, obtained enzymolysis liquid;
(5) concentrated obtained enzymolysis liquid;
(6) add the active carbon of 5%, carry out decoloration and deodorization process, afterwards elimination active carbon;
(7) last, obtain nutrient after spray drying treatment.
Further, the interpolation complex enzyme in described step (4) is the combination of papain, alkali protease and flavor protease;
Further, the interpolation compound enzyme amount in described step (4) is 0.5%-1.0% (with shrimp podwer Mass Calculation);
Further, the ratio papain of the interpolation complex enzyme in described step (4): alkali protease: flavor protease=1.5-2.5:1:1.5-2.0;
Further, the hydrolysis temperature in described step (4) 50 DEG C-55 DEG C, PH is 7.0-7.5, and enzymolysis time is 5h-7h;
Further, limited enzymolysis in described step (4) is extracted from salted shrimp gravy by protein, recovery rate is controlled in the level being greater than 75%, enzymatic hydrolyzation (amino acid/gross protein) is controlled at 35%-40% simultaneously, make total protein content high, simultaneously containing the composite nutrient being easy to the amino acid of assimilation ratio, polypeptide, protein;
Further, in described step (2), screening order number is 40;
Further, the heated-air drying temperature in described step (1) is 85 DEG C;
Further, the salted shrimp gravy quality in described step (1) is 50KG.
Beneficial effect:
1, the present invention uses limited enzymolysis technology, controlled enzymatic hydrolysis degree, thus complete thorough enzymolysis, containing abundant amino acid, polypeptide, the albumen that are easy to absorption of human body and utilize proper ratio, and gross protein extraction rate reached 76.3%.
2, compared with existing product, abundant raw material source, cheap, the nutrient nutrition of waste is more comprehensively abundant, and is easy to absorption of human body; The papain selected, alkali protease, flavor protease reasonable mixture ratio, protein extracting ratio is higher, reaches 76.3%.
3, this product natural health, do not add anticorrisive agent, steady quality, best in quality, in evaluating by function of health food, the animal model of develop immunitypty and link physical fatigue carries out Efficacy experiments, prove that the absorption rate of this product is apparently higher than unprocessed salted shrimp gravy and routine protein powder product, and through animal efficacy validation, there is the function of develop immunitypty and alleviating physical fatigue simultaneously.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further details.
Example one:
1,50kg salted shrimp gravy used 85 DEG C of heated-air dryings, pulverize 40 mesh sieves, enzyme concentration (with shrimp podwer Mass Calculation) 0.5%, add the ratio papain of complex enzyme: alkali protease: flavor protease=1.5:1:1.5, hydrolysis temperature 50 DEG C, pH is 7.0, and enzymolysis time is 5h.After measured, after enzymolysis, extraction rate of protein reaches 75.2%, and degree of hydrolysis is 35.5%, add the active carbon of 5%, carry out decoloration and deodorization process, afterwards elimination active carbon, carry out spraying dry and obtain nutrient, obtain oligopeptide content (10000d)=21.2g/100g.
2, product Animals fed experimental: after basic for feeding rats material adaptability is raised a week, is in the male SD rat 50 in growth period, is divided into 5 groups at random: 1. casein group; 2. without nitrogen group; 3. non-enzymolysis group (feed that feeding shrimp podwer is configured to); 4. product group (feed that this products configuration of feeding becomes); 5. albumen powder product group (feed that feeding commercial protein powder is configured to).
In order to ensure that every mouse is identical at the Feed consumption of duration of test, namely the intake of nitrogen is identical, and every rat is put into a nitrogen metabolism cage by this experiment respectively, adopts and raises the method for feeding, freely drink water.Ambient parameter: temperature (20 ± 1) DEG C, relative humidity (40 ± 5) %.
9:00 weighed, fed intake every day, and recorded the body weight of rat and the addition of feed.First 15 days that test, 1. 2. organize equal feeding casein diet, from the 16th day, 2. group starts to change and feeds without nitrogen feed, from 17 days, collects ight soil and the urine of 2. rat every day, measure nitrogen content, after the discharge rate of nitrogen is constant, 1. 3. 4. 5. rat carry out seven days metabolisms.Carry out nitrogen metabolism experimental session, collecting ight soil and the urine of rat early morning every day, utilize kjeldahl determination to measure nitrogen content.After experiment terminates, all Rat Fast 12h, after leg arterial blood extracting, dislocate lethal, get liver organization to be measured.
3, nutrient nutrient value evaluation index calculating method and result:
The total intake of nitrogen in absorbed nitrogen=food-(fecal nitrogen-Faecal metabolic nitrogen)
Nitrogen=absorbed nitrogen-(urinary nitrogen-urine endogenous nitrogen)=food nitrogen-(total nitrogen output-total endogenous nitrogen) is stayed in storage
Table 1-1: rat nitrogen balance and evaluation of nutrition index
Nitrogen balance can reflect the situation of body protein metabolism.At growth and development stage, the speed of protein anabolism exceedes catabolic speed, so the animal being in growth and development stage is usually expressed as positive nitrogen equilibrium.Experimental result shows, four groups of rats all belong to positive nitrogen equilibrium state, illustrates that all feeds of preparation all can meet the needs of rat growthing development.
The excretion of non-enzymolysis group ight soil and urine is apparently higher than product group and albumen powder group, and wherein higher than two protease hydrolyzed group 2-3 doubly, urinary nitrogen is but starkly lower than other groups to fecal nitrogen content.Prove the natural macromolecular amount protein that salted shrimp gravy self contains, the degree digested and assimilated in vivo is general, and the macromolecular weight protein major part taken in body is not all absorbed by body, but directly along with refuse is discharged in body through urine and ight soil.Wherein, the storage of product group stays nitrogen the highest, and illustrate that the protein anabolism in this group rat body is the most vigorous, the net utilization of protein is also the highest.
In order to reach when not increasing food volume, meet the object of the needs of body, people evaluate the nutritive effect of protein with regard to needing.The nutritive value of food protein depends primarily on its protein content and the degree that be absorbed and used of protein in body.The indexs such as protein true digestibility, net utilization, biological value are protein nutritive value evaluation indexes the most frequently used in nutrition.
The rat protein matter true digestibility of product group and net protein utilization all higher than non-enzymolysis group and albumen powder group, this illustrate small molecular weight titanium class contained in the enzymolysis liquid utilizing compound protease enzymolysis salted shrimp gravy to obtain comparatively in salted shrimp gravy body natural macromolecular amount protein be more easy to body and absorb.
Also can be found out by contrast, product group comparatively market albumen powder group also has obvious advantage in indices.
4, develop immunitypty functional evaluation
4.1 dosage and grouping: select inbred mouse 80, body weight is 20g ± 1g, experiment is divided into control group and basic, normal, high dosage group totally four groups by 5 times (0.25g/kgBW), 10 times (0.5g/kgBW) and 30 times (1.5g/kgBW) of human body recommended amounts every day (in the body weight 70kg that is grown up, recommended amounts is 0.05g/kgBW).Control group feeding white granulated sugar, gives 30 days time.
4.2 mouse delayed allergy experiments (sufficient sole of the foot thickness increases method)
The 4th day immune animal before experiment terminates, by 2% (v/v) sheep red blood cell (SRBC) lumbar injection 0.2mL sensitized animal, left back sufficient sole of the foot portion thickness is measured after 5 days, then in the every mouse of this place's hypodermic injection 20% (v/v) sheep red blood cell (SRBC) 20 μ L/), inject and measure left back sufficient sole of the foot portion thickness three times in latter 24 hours, obtain mean value.
The mouse spleen lymphocyte conversion test (mtt assay) of 4.3ConA induction
Lymphproliferation response: add in 24 well culture plates by a cell suspension point holes, every hole 1mL, a hole 75 μ LConA liquid (being equivalent to 7.5 μ g/mL), 5%CO2 in contrast, is put in another hole, cultivates 72h for 37 DEG C.Cultivation terminates first 4 hours, and every hole sucks supernatant 0.7mL, adds 0.7mL serum-free RPMI1640 nutrient solution, adds MTT (5mg/mL) 50 μ L/ hole simultaneously, continues to cultivate 4h.After cultivation terminates, every hole adds 1mL isopropyl alcohol, and piping and druming mixing, makes purple crystal dissolve, and divides and is added in 96 well culture plates, makes the Duplicate Samples in 3 holes, measures OD value with 570nm wavelength.
4.4 experimental result
Table 1-2 is on the impact (n=10, x+s) of mouse thymus, spleen weight
Each dosage group internal organs/body weight ratio compares with control group, difference there are no significant meaning (seeing the above table).Show that product has no significant effect the spleen weight of mouse and thymic weight.
Table 1-3 is on the impact (x+s) of mouse delayed allergy
Mouse delayed allergy test (the sufficient sole of the foot thickens method) of sheep red blood cell (SRBC) induction as can be seen from the above table, wherein, the high dose group foot sole of the foot thickens value and compares with control group, and difference has significant.
Table 1-4 agent is on the impact (x+s) of mouse spleen lymphocyte conversion reaction
From upper table, to the mouse spleen lymphocyte conversion reaction of ConA induction, its high dose group optical density difference is higher than control group, and difference has significant.Show that the Splenic vein hemodynamics result of the test of product is for positive, given the test agent has the effect of enhancing cellular immune function.
Judge according to develop immunitypty functional evaluation standard in " health food inspection and assessment technical specification " (2003 editions), this nutrient has develop immunitypty function.
5, alleviating physical fatigue functional evaluation
5.1 dosage and grouping: carry out with develop immunitypty functional evaluation simultaneously.Select inbred mouse 80, body weight is 20g ± 1g, experiment is divided into control group and basic, normal, high dosage group totally four groups by 5 times (0.25g/kgBW), 10 times (0.5g/kgBW) and 30 times (1.5g/kgBW) of human body recommended amounts every day (in the body weight 70kg that is grown up, recommended amounts is 0.05g/kgBW).Control group feeding white granulated sugar, gives 30 days time.
5.2 swimmings with a load attached to the body experiments: after last gavage 30min, often group gets 10, and its root of the tail portion load 5% weight sheet lead is placed on depth of water 30cm, the swimming trunk went swimming of water temperature (25.0 ± 1.0) DEG C.Record is the mice burden swimming time from beginning to the time that the whole submerged 8s of head can not emerge of swimming.
5.3 oxygen deficit tolerance experiments: after last gavage 1h, often group is got 10 and put into the 250mL port grinding bottle (1 every bottle) filling 5g soda lime, bottleneck, with after vaseline sealing, records the time of mouse death because of anoxic.
5.4 experimental result
The measurement result (n=10) of table 1-5 mice burden swimming time and Hypoxia under normal pressure
As seen from the above table, after this nutrient of mouse stomach, walking weight load and Hypoxia under normal pressure comparatively control group all have significant difference, and product has effect for alleviating physical fatigue.
Judge according to alleviating physical fatigue functional evaluation standard in " health food inspection and assessment technical specification " (2003 editions), this nutrient has alleviating physical fatigue function.
Illustrate through above-mentioned zoopery, this product comparatively process before salted shrimp gravy and soyabean protein powder, have and better absorb value.There is the function of develop immunitypty and alleviating physical fatigue simultaneously.
Example two
1,50kg salted shrimp gravy used 85 DEG C of heated-air dryings, pulverize 40 mesh sieves, enzyme concentration (with shrimp podwer Mass Calculation) 0.75%, add the ratio papain of complex enzyme: alkali protease: flavor protease=1.5:1:2.0, hydrolysis temperature 52 DEG C, pH is 7.5, and enzymolysis time is 6h.
After measured, after enzymolysis, extraction rate of protein reaches 76.0%, and degree of hydrolysis is 36.8%, add the active carbon of 5%, carry out decoloration and deodorization process, afterwards elimination active carbon, carry out spraying dry and obtain nutrient, obtain oligopeptide content (10000d)=22.1g/100g.
2, product Animals fed experimental: after basic for feeding rats material adaptability is raised a week, is in the male SD rat 50 in growth period, is divided into 5 groups at random: 1. casein group; 2. without nitrogen group; 3. non-enzymolysis group (feed that feeding shrimp podwer is configured to); 4. product group (feed that this products configuration of feeding becomes); 5. albumen powder product group (feed that feeding commercial protein powder is configured to).
In order to ensure that every mouse is identical at the Feed consumption of duration of test, namely the intake of nitrogen is identical, and every rat is put into a nitrogen metabolism cage by this experiment respectively, adopts and raises the method for feeding, freely drink water.Ambient parameter: temperature (20 ± 1) DEG C, relative humidity (40 ± 5) %.
9:00 weighed, fed intake every day, and recorded the body weight of rat and the addition of feed.First 15 days that test, 1. 2. organize equal feeding casein diet, from the 16th day, 2. group starts to change and feeds without nitrogen feed, from 17 days, collects ight soil and the urine of 2. rat every day, measure nitrogen content, after the discharge rate of nitrogen is constant, 1. 3. 4. 5. rat carry out seven days metabolisms.Carry out nitrogen metabolism experimental session, collecting ight soil and the urine of rat early morning every day, utilize kjeldahl determination to measure nitrogen content.After experiment terminates, all Rat Fast 12h, after leg arterial blood extracting, dislocate lethal, get liver organization to be measured.
3, nutrient nutrient value evaluation index calculating method and result:
The total intake of nitrogen in absorbed nitrogen=food-(fecal nitrogen-Faecal metabolic nitrogen)
Nitrogen=absorbed nitrogen-(urinary nitrogen-urine endogenous nitrogen)=food nitrogen-(total nitrogen output-total endogenous nitrogen) is stayed in storage
Table 2-1 rat nitrogen balance and evaluation of nutrition index
Nitrogen balance can reflect the situation of body protein metabolism.At growth and development stage, the speed of protein anabolism exceedes catabolic speed, so the animal being in growth and development stage is usually expressed as positive nitrogen equilibrium.Experimental result shows, four groups of rats all belong to positive nitrogen equilibrium state, illustrates that all feeds of preparation all can meet the needs of rat growthing development.
The excretion of non-enzymolysis group ight soil and urine is apparently higher than product group and albumen powder group, and wherein higher than two protease hydrolyzed group 2-3 doubly, urinary nitrogen is but starkly lower than other groups to fecal nitrogen content.Prove the natural macromolecular amount protein that salted shrimp gravy self contains, the degree digested and assimilated in vivo is general, and the macromolecular weight protein major part taken in body is not all absorbed by body, but directly along with refuse is discharged in body through urine and ight soil.Wherein, the storage of product group stays nitrogen the highest, and illustrate that the protein anabolism in this group rat body is the most vigorous, the net utilization of protein is also the highest.
In order to reach when not increasing food volume, meet the object of the needs of body, people evaluate the nutritive effect of protein with regard to needing.The nutritive value of food protein depends primarily on its protein content and the degree that be absorbed and used of protein in body.The indexs such as protein true digestibility, net utilization, biological value are protein nutritive value evaluation indexes the most frequently used in nutrition.
The rat protein matter true digestibility of product group and net protein utilization all higher than non-enzymolysis group and albumen powder group, this illustrate small molecular weight titanium class contained in the enzymolysis liquid utilizing compound protease enzymolysis salted shrimp gravy to obtain comparatively in salted shrimp gravy body natural macromolecular amount protein be more easy to body and absorb.
Also can be found out by contrast, product group comparatively market albumen powder group also has obvious advantage in indices.
4, develop immunitypty functional evaluation
4.1 dosage and grouping: select inbred mouse 80, body weight is 20g ± 1g, experiment is divided into control group and basic, normal, high dosage group totally four groups by 5 times (0.25g/kgBW), 10 times (0.5g/kgBW) and 30 times (1.5g/kgBW) of human body recommended amounts every day (in the body weight 70kg that is grown up, recommended amounts is 0.05g/kgBW).Control group feeding white granulated sugar, gives 30 days time.
4.2 mouse delayed allergy experiments (sufficient sole of the foot thickness increases method)
The 4th day immune animal before experiment terminates, by 2% (v/v) sheep red blood cell (SRBC) lumbar injection 0.2mL sensitized animal, left back sufficient sole of the foot portion thickness is measured after 5 days, then in the every mouse of this place's hypodermic injection 20% (v/v) sheep red blood cell (SRBC) 20 μ L/), inject and measure left back sufficient sole of the foot portion thickness three times in latter 24 hours, obtain mean value.
The mouse spleen lymphocyte conversion test (mtt assay) of 4.3ConA induction
Lymphproliferation response: add in 24 well culture plates by a cell suspension point holes, every hole 1mL, a hole 75 μ LConA liquid (being equivalent to 7.5 μ g/mL), 5%CO2 in contrast, is put in another hole, cultivates 72h for 37 DEG C.Cultivation terminates first 4 hours, and every hole sucks supernatant 0.7mL, adds 0.7mL serum-free RPMI1640 nutrient solution, adds MTT (5mg/mL) 50 μ L/ hole simultaneously, continues to cultivate 4h.After cultivation terminates, every hole adds 1mL isopropyl alcohol, and piping and druming mixing, makes purple crystal dissolve, and divides and is added in 96 well culture plates, makes the Duplicate Samples in 3 holes, measures OD value with 570nm wavelength.
4.4 experimental result
Table 2-2 is on the impact (n=10, x+s) of mouse thymus, spleen weight
Each dosage group internal organs/body weight ratio compares with control group, difference there are no significant meaning (seeing the above table).Show that product has no significant effect the spleen weight of mouse and thymic weight.
Table 2-3 is on the impact (x+s) of mouse delayed allergy
Mouse delayed allergy test (the sufficient sole of the foot thickens method) of sheep red blood cell (SRBC) induction as can be seen from the above table, wherein, the high dose group foot sole of the foot thickens value and compares with control group, and difference has significant.
Table 2-4 agent is on the impact (x+s) of mouse spleen lymphocyte conversion reaction
From upper table, to the mouse spleen lymphocyte conversion reaction of ConA induction, its high dose group optical density difference is higher than control group, and difference has significant.Show that the Splenic vein hemodynamics result of the test of product is for positive, given the test agent has the effect of enhancing cellular immune function.
Judge according to develop immunitypty functional evaluation standard in " health food inspection and assessment technical specification " (2003 editions), this nutrient has develop immunitypty function.
5, alleviating physical fatigue functional evaluation
5.1 dosage and grouping: carry out with develop immunitypty functional evaluation simultaneously.Select inbred mouse 80, body weight is 20g ± 1g, experiment is divided into control group and basic, normal, high dosage group totally four groups by 5 times (0.25g/kgBW), 10 times (0.5g/kgBW) and 30 times (1.5g/kgBW) of human body recommended amounts every day (in the body weight 70kg that is grown up, recommended amounts is 0.05g/kgBW).Control group feeding white granulated sugar, gives 30 days time.
5.2 swimmings with a load attached to the body experiments: after last gavage 30min, often group gets 10, and its root of the tail portion load 5% weight sheet lead is placed on depth of water 30cm, the swimming trunk went swimming of water temperature (25.0 ± 1.0) DEG C.Record is the mice burden swimming time from beginning to the time that the whole submerged 8s of head can not emerge of swimming.
5.3 oxygen deficit tolerance experiments: after last gavage 1h, often group is got 10 and put into the 250mL port grinding bottle (1 every bottle) filling 5g soda lime, bottleneck, with after vaseline sealing, records the time of mouse death because of anoxic.
5.4 experimental result
The measurement result (n=10) of table 2-5 mice burden swimming time and Hypoxia under normal pressure
As seen from the above table, after this nutrient of mouse stomach, walking weight load and Hypoxia under normal pressure comparatively control group all have significant difference, and product has effect for alleviating physical fatigue.
Judge according to alleviating physical fatigue functional evaluation standard in " health food inspection and assessment technical specification " (2003 editions), this nutrient has alleviating physical fatigue function.
Illustrate through above-mentioned zoopery, this product comparatively process before salted shrimp gravy and soyabean protein powder, have and better absorb value.There is the function of develop immunitypty and alleviating physical fatigue simultaneously.
Example three:
1,50kg salted shrimp gravy used 85 DEG C of heated-air dryings, pulverize 40 mesh sieves, enzyme concentration (with shrimp podwer Mass Calculation) 1.0%, adds the ratio papain of enzyme: alkali protease: flavor protease=2.0:1:2.0, hydrolysis temperature 55 DEG C, pH is 7.5, and enzymolysis time is 6h.
After measured, after enzymolysis, extraction rate of protein reaches 76.0%, and degree of hydrolysis is 38.8%, adds the active carbon of 5%, carries out decoloration and deodorization process, afterwards elimination active carbon, carries out spraying dry and obtains nutrient.Oligopeptide content (10000d)=23.0g/100g.2, product Animals fed experimental: after basic for feeding rats material adaptability is raised a week, is in the male SD rat 50 in growth period, is divided into 5 groups at random: 1. casein group; 2. without nitrogen group; 3. non-enzymolysis group (feed that feeding shrimp podwer is configured to); 4. product group (feed that this products configuration of feeding becomes); 5. albumen powder product group (feed that feeding commercial protein powder is configured to).
In order to ensure that every mouse is identical at the Feed consumption of duration of test, namely the intake of nitrogen is identical, and every rat is put into a nitrogen metabolism cage by this experiment respectively, adopts and raises the method for feeding, freely drink water.Ambient parameter: temperature (20 ± 1) DEG C, relative humidity (40 ± 5) %.
9:00 weighed, fed intake every day, and recorded the body weight of rat and the addition of feed.First 15 days that test, 1. 2. organize equal feeding casein diet, from the 16th day, 2. group starts to change and feeds without nitrogen feed, from 17 days, collects ight soil and the urine of 2. rat every day, measure nitrogen content, after the discharge rate of nitrogen is constant, 1. 3. 4. 5. rat carry out seven days metabolisms.Carry out nitrogen metabolism experimental session, collecting ight soil and the urine of rat early morning every day, utilize kjeldahl determination to measure nitrogen content.After experiment terminates, all Rat Fast 12h, after leg arterial blood extracting, dislocate lethal, get liver organization to be measured.
3, nutrient nutrient value evaluation index calculating method and result:
The total intake of nitrogen in absorbed nitrogen=food-(fecal nitrogen-Faecal metabolic nitrogen)
Nitrogen=absorbed nitrogen-(urinary nitrogen-urine endogenous nitrogen)=food nitrogen-(total nitrogen output-total endogenous nitrogen) is stayed in storage
Table 3-1 rat nitrogen balance and evaluation of nutrition index
Nitrogen balance can reflect the situation of body protein metabolism.At growth and development stage, the speed of protein anabolism exceedes catabolic speed, so the animal being in growth and development stage is usually expressed as positive nitrogen equilibrium.Experimental result shows, four groups of rats all belong to positive nitrogen equilibrium state, illustrates that all feeds of preparation all can meet the needs of rat growthing development.
The excretion of non-enzymolysis group ight soil and urine is apparently higher than product group and albumen powder group, and wherein higher than two protease hydrolyzed group 2-3 doubly, urinary nitrogen is but starkly lower than other groups to fecal nitrogen content.Prove the natural macromolecular amount protein that salted shrimp gravy self contains, the degree digested and assimilated in vivo is general, and the macromolecular weight protein major part taken in body is not all absorbed by body, but directly along with refuse is discharged in body through urine and ight soil.Wherein, the storage of product group stays nitrogen the highest, and illustrate that the protein anabolism in this group rat body is the most vigorous, the net utilization of protein is also the highest.
In order to reach when not increasing food volume, meet the object of the needs of body, people evaluate the nutritive effect of protein with regard to needing.The nutritive value of food protein depends primarily on its protein content and the degree that be absorbed and used of protein in body.The indexs such as protein true digestibility, net utilization, biological value are protein nutritive value evaluation indexes the most frequently used in nutrition.
The rat protein matter true digestibility of product group and net protein utilization all higher than non-enzymolysis group and albumen powder group, this illustrate small molecular weight titanium class contained in the enzymolysis liquid utilizing compound protease enzymolysis salted shrimp gravy to obtain comparatively in salted shrimp gravy body natural macromolecular amount protein be more easy to body and absorb.
Also can be found out by contrast, product group comparatively market albumen powder group also has obvious advantage in indices.
4, develop immunitypty functional evaluation
4.1 dosage and grouping: select inbred mouse 80, body weight is 20g ± 1g, experiment is divided into control group and basic, normal, high dosage group totally four groups by 5 times (0.25g/kgBW), 10 times (0.5g/kgBW) and 30 times (1.5g/kgBW) of human body recommended amounts every day (in the body weight 70kg that is grown up, recommended amounts is 0.05g/kgBW).Control group feeding white granulated sugar, gives 30 days time.
4.2 mouse delayed allergy experiments (sufficient sole of the foot thickness increases method)
The 4th day immune animal before experiment terminates, by 2% (v/v) sheep red blood cell (SRBC) lumbar injection 0.2mL sensitized animal, left back sufficient sole of the foot portion thickness is measured after 5 days, then in the every mouse of this place's hypodermic injection 20% (v/v) sheep red blood cell (SRBC) 20 μ L/), inject and measure left back sufficient sole of the foot portion thickness three times in latter 24 hours, obtain mean value.
The mouse spleen lymphocyte conversion test (mtt assay) of 4.3ConA induction
Lymphproliferation response: add in 24 well culture plates by a cell suspension point holes, every hole 1mL, a hole 75 μ LConA liquid (being equivalent to 7.5 μ g/mL), 5%CO2 in contrast, is put in another hole, cultivates 72h for 37 DEG C.Cultivation terminates first 4 hours, and every hole sucks supernatant 0.7mL, adds 0.7mL serum-free RPMI1640 nutrient solution, adds MTT (5mg/mL) 50 μ L/ hole simultaneously, continues to cultivate 4h.After cultivation terminates, every hole adds 1mL isopropyl alcohol, and piping and druming mixing, makes purple crystal dissolve, and divides and is added in 96 well culture plates, makes the Duplicate Samples in 3 holes, measures OD value with 570nm wavelength.
4.4 experimental result
Table 3-2 is on the impact (n=10, x+s) of mouse thymus, spleen weight
Each dosage group internal organs/body weight ratio compares with control group, difference there are no significant meaning (seeing the above table).Show that product has no significant effect the spleen weight of mouse and thymic weight.
Table 3-3 is on the impact (x+s) of mouse delayed allergy
Mouse delayed allergy test (the sufficient sole of the foot thickens method) of sheep red blood cell (SRBC) induction as can be seen from the above table, wherein, the high dose group foot sole of the foot thickens value and compares with control group, and difference has significant.
Table 3-4 agent is on the impact (x+s) of mouse spleen lymphocyte conversion reaction
From upper table, to the mouse spleen lymphocyte conversion reaction of ConA induction, its high dose group optical density difference is higher than control group, and difference has significant.Show that the Splenic vein hemodynamics result of the test of product is for positive, given the test agent has the effect of enhancing cellular immune function.
Judge according to develop immunitypty functional evaluation standard in " health food inspection and assessment technical specification " (2003 editions), this nutrient has develop immunitypty function.
5, alleviating physical fatigue functional evaluation
5.1 dosage and grouping: carry out with develop immunitypty functional evaluation simultaneously.Select inbred mouse 80, body weight is 20g ± 1g, experiment is divided into control group and basic, normal, high dosage group totally four groups by 5 times (0.25g/kgBW), 10 times (0.5g/kgBW) and 30 times (1.5g/kgBW) of human body recommended amounts every day (in the body weight 70kg that is grown up, recommended amounts is 0.05g/kgBW).Control group feeding white granulated sugar, gives 30 days time.
5.2 swimmings with a load attached to the body experiments: after last gavage 30min, often group gets 10, and its root of the tail portion load 5% weight sheet lead is placed on depth of water 30cm, the swimming trunk went swimming of water temperature (25.0 ± 1.0) DEG C.Record is the mice burden swimming time from beginning to the time that the whole submerged 8s of head can not emerge of swimming.
5.3 oxygen deficit tolerance experiments: after last gavage 1h, often group is got 10 and put into the 250mL port grinding bottle (1 every bottle) filling 5g soda lime, bottleneck, with after vaseline sealing, records the time of mouse death because of anoxic.
5.4 experimental result
The measurement result (n=10) of table 3-5 mice burden swimming time and Hypoxia under normal pressure
As seen from the above table, after this nutrient of mouse stomach, walking weight load and Hypoxia under normal pressure comparatively control group all have significant difference, and product has effect for alleviating physical fatigue.
Judge according to alleviating physical fatigue functional evaluation standard in " health food inspection and assessment technical specification " (2003 editions), this nutrient has alleviating physical fatigue function.
Illustrate through above-mentioned zoopery, this product comparatively process before salted shrimp gravy and soyabean protein powder, have and better absorb value.There is the function of develop immunitypty and alleviating physical fatigue simultaneously.
Claims (2)
1. extract the shrimp protein nutrient method being used for develop immunitypty and fatigue-relieving, it is characterized in that, the method comprises the steps:
(1) salted shrimp gravy is passed through heated-air drying;
(2) crushed after being dried, through screening process;
(3), after crushing and screening, soak;
(4) complex enzyme limited enzymolysis is added, obtained enzymolysis liquid;
(5) concentrated obtained enzymolysis liquid;
(6) add the active carbon of 5%, carry out decoloration and deodorization process, afterwards elimination active carbon;
(7) last, obtain nutrient after spray drying treatment;
Heated-air drying temperature in described step (1) is 85 DEG C;
In described step (2), the order number of screening is 40;
Interpolation complex enzyme in described step (4) is the combination of papain, alkali protease and flavor protease;
The ratio papain of the interpolation complex enzyme in described step (4): alkali protease: flavor protease=1.5-2.5:1:1.5-2.0;
Interpolation compound enzyme amount in described step (4) is 0.5%-1.0% (with shrimp podwer Mass Calculation);
Hydrolysis temperature in described step (4) 50 DEG C-55 DEG C, PH is 7.0-7.5, and enzymolysis time is 5h-7h;
Limited enzymolysis in described step (4) is extracted from salted shrimp gravy by protein, recovery rate is controlled in the level being greater than 75%, enzymatic hydrolyzation (amino acid/gross protein) is controlled at 35%-40% simultaneously, make total protein content high, simultaneously containing the composite nutrient being easy to the amino acid of assimilation ratio, polypeptide, protein.
2. a kind of shrimp protein nutrient method extracted for develop immunitypty and fatigue-relieving as claimed in claim 1, it is characterized in that, the salted shrimp gravy quality in described step (1) is 50KG.
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| CN109221972A (en) * | 2018-09-03 | 2019-01-18 | 东山县太元水产有限公司 | A kind of sea shrimp nutrient extacting method |
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