CN107868808A - A kind of method for improving bird's nest saliva free acid - Google Patents
A kind of method for improving bird's nest saliva free acid Download PDFInfo
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- CN107868808A CN107868808A CN201711059791.5A CN201711059791A CN107868808A CN 107868808 A CN107868808 A CN 107868808A CN 201711059791 A CN201711059791 A CN 201711059791A CN 107868808 A CN107868808 A CN 107868808A
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
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- C—CHEMISTRY; METALLURGY
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- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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Abstract
The invention discloses a kind of method for improving bird's nest saliva free acid, comprise the following steps：(1) bird's nest is crushed, sieves, obtain bird's nest powder；(2) bird's nest powder is well mixed with distilled water, obtains mixed liquor；(3) pH for adjusting mixed liquor is 78, adds papain, is digested 23 hours in 45 55 DEG C；(4) after the completion of reacting, enzymolysis liquid enzyme deactivation is lived；(5) the enzymolysis liquid cooling after enzyme deactivation is lived, centrifugation, collects supernatant；(6) by supernatant vacuum freeze drying, you can.The method of the present invention for improving bird's nest saliva free acid, is changed into free state from the bonding state of sialoglycoprotein by the sialic acid in bird's nest, gives full play to the nutritive effect of bird's nest, and obtained bird's nest sialic acid has the function that anti-aging and improves memory function.
Description
Technical field
The present invention relates to health product technology field, and in particular to a kind of method for improving bird's nest saliva free acid.
Background technology
Bird's nest is that the saliva of Apodidae (Apedidae) esculent swift secretion or saliva mix bonding with its down and built up
Nest.Main product is in Fujian in the countries in Southeast Asia such as Malaysia, Indonesia, Thailand, Burma and China, Coast of Guangdong Province
Band.Bird's nest one is deep by the country as the high-grade invigorant of medicine-food two-purpose to valuable medicinal and delicious food and in folklore
Outer consumer's likes.
The patent of invention of Application No. 201610261793.1 discloses a kind of processing method of bird's nest, main to include clearly
Wash, sterilize, being soaked, being dehydrated, be quick-frozen, being dried in vacuo several steps, passing through bird's nest made from above-mentioned processing, health, safety, nothing
It is bacterium, additive-free, remain the nutritional ingredient in bird's nest raw material, user can be brewed boiling water, by way of vexed system very short
Time in have high-quality bird's nest.The present invention ideally solves bird's nest all the time and is difficult to, it is difficult to preservation and transport
The problem of, there is high market value.
The patent of invention of Application No. 201410735503.3 discloses a kind of bird's nest complex enzyme hydrolysis processing technology, and it is related to
Bird's nest processing technique field, its processing technology are as follows：Step 1：Pulverized and sieved after bird's nest heated-air drying, add bird's nest dry weight
25 times of amount water, soak 4 hours；Step 2：Under mechanical agitation, temperature control is incubated 60 minutes at 95-99 DEG C；Step 3：
50-60 DEG C is cooled to, adds animal hydrolase 0.3%, neutral proteinase 0.3%, the food flavor enzyme 0.15% of bird's nest dry weight, it is natural
PH value, digest 6 hours；Step 4：Being warming up to 85-90 DEG C again is kept for 20-30 minutes carry out enzyme-deactivating；Step 5：Filter off
Except insoluble impurities, it is concentrated in vacuo or sprays or be freeze-dried；Its processing technology is simple, and yield about 80%, bird's nest characteristic refers to
It is 10.5% to mark sialic acid content, retains bioactivity, total number of bacteria 460cfu/g, meets edible cosmetic product hygienic requirements, water
Dissolubility is good, and flavor has the albumen fragrance of bird's nest, no bitter taste.
Sialic acid is very important a kind of nutriment in bird's nest, its substantially in the form of combination with mucopolysaccharide,
Glycoprotein is connected with the oligomeric sugar chain in sugar ester, is to be bonded first with other cells positioned at the end of cell surface sugar chain
Molecule.But its effect of bird's nest sialic acid existing for reference state form is much not as good as sialic acid existing in the form of free state.
So this invention address that find a kind of method of new raising bird's nest saliva free acid, as much as possible by bird's nest to combine
Sialic acid existing for state form is converted into sialic acid existing in the form of free state.
The content of the invention
The present invention seeks to what is be achieved through the following technical solutions：
One of technical problems to be solved by the invention are to provide a kind of method for improving bird's nest saliva free acid.
The invention discloses a kind of method for improving bird's nest saliva free acid, comprise the following steps：
(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；
(2) by bird's nest powder and distilled water with solid-liquid ratio 1:(20-40) (g/mL) is well mixed, obtains mixed liquor；
(3) pH for adjusting mixed liquor is 7-8, adds papain, the addition of papain is bird's nest powder weight
The 1-4% of amount, 2-3 hours are digested in 45-55 DEG C；
(4) after the completion of reacting, enzymolysis liquid is placed in enzyme deactivation in 100-105 DEG C of water-bath and lived 5-10 minutes；
(5) enzymolysis liquid after enzyme deactivation is lived is cooled to 23-25 DEG C, and 10-15 is centrifuged with 3000-5000 revs/min of rotating speed
Minute, collect supernatant；
(6) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
~-25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09-
0.095MPa, vacuum freeze drying time are 16-20 hours.
The invention discloses a kind of method for improving bird's nest saliva free acid, comprise the following steps：
(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；
(2) by bird's nest powder and distilled water with solid-liquid ratio 1:(20-40) (g/mL) mix, in microwave power 400-500W,
The 50-60 seconds are handled under conditions of microwave frequency 900-2500MHz, obtain mixed liquor；
(3) pH for adjusting mixed liquor is 7-8, adds papain, the addition of papain is bird's nest powder weight
The 1-4% of amount, 1-2 hours are digested in 45-55 DEG C；
(4) after the completion of reacting, enzymolysis liquid is placed in enzyme deactivation in 100-105 DEG C of water-bath and lived 5-10 minutes；
(5) enzymolysis liquid after enzyme deactivation is lived is cooled to 23-25 DEG C, and 10-15 is centrifuged with 3000-5000 revs/min of rotating speed
Minute, collect supernatant；
(6) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
~-25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09-
0.095MPa, vacuum freeze drying time are 16-20 hours.
The invention discloses a kind of method for improving bird's nest saliva free acid, comprise the following steps：
(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；
(2) by acid solution that bird's nest powder and molar concentration are 0.04-0.05mol/L with solid-liquid ratio 1:(20-40)(g/mL)
It is well mixed, obtain mixed liquor；
(3) mixed liquor is extracted into 20-30 minutes in 100-105 DEG C of water-bath, obtains extract solution；
(4) extract solution is cooled to 23-25 DEG C, 10-15 minutes is centrifuged with 3000-5000 revs/min of rotating speed, in collection
Clear liquid；
(5) supernatant is carried out using milipore filter under conditions of molecular cut off 3-5KDa, operating pressure 38-42psi
Ultrafiltration, collect filtrate；
(6) filtrate is concentrated into 1-2 hours under conditions of 50-55 DEG C, vacuum 0.06-0.07MPa, obtains concentrate；
Concentrate is purified using anion exchange resin, loading flow velocity is 0.8-1mL/min, collects refined solution；
(7) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
~-25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09-
0.095MPa, vacuum freeze drying time are 16-20 hours.
The invention discloses a kind of method for improving bird's nest saliva free acid, comprise the following steps：
(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；
(2) preparation molar concentration containing sodium acid carbonate is 1-2mmol/L, dithiothreitol (DTT) molar concentration is 5-6mmol/L, sugar
Molar concentration is 1.0-1.5mmol/L treatment fluid；
(3) bird's nest powder is configured to the 4-5mg/mL aqueous solution, the treatment fluid that it is obtained with step (2) is with volume ratio
1:(1-3) is mixed, and 4-8 hours are stirred with 190-220 revs/min of rotating speed in 60-70 DEG C of water-bath；Gained reaction solution is cooled down
To 23-25 DEG C, vacuum freeze drying obtains solid A；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30~-25
DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09-
0.095MPa, vacuum freeze drying time are 16-20 hours；
(4) by acid solution that solid A and molar concentration are 0.04-0.05mol/L with solid-liquid ratio 1:(20-40) (g/mL) is mixed
Uniformly, mixed liquor is obtained；
(5) mixed liquor is extracted into 20-30 minutes in 100-105 DEG C of water-bath, obtains extract solution；
(6) extract solution is cooled to 23-25 DEG C, 10-15 minutes is centrifuged with 3000-5000 revs/min of rotating speed, in collection
Clear liquid；
(7) supernatant is carried out using milipore filter under conditions of molecular cut off 3-5KDa, operating pressure 38-42psi
Ultrafiltration, collect filtrate；
(8) filtrate is concentrated into 1-2 hours under conditions of 50-55 DEG C, vacuum 0.06-0.07MPa, obtains concentrate；
Concentrate is purified using anion exchange resin, loading flow velocity is 0.8-1mL/min, collects refined solution；
(9) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
~-25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09-
0.095MPa, vacuum freeze drying time are 16-20 hours.
The acid solution can be aqueous sulfuric acid, aqueous citric acid solution, aqueous acetic acid, hydrochloric acid, the chlorogenic acid aqueous solution, apple
One or more mixtures in the tartaric acid aqueous solution, aqueous tartaric acid solution, butanedioic acid aqueous solution.Preferably, the acid solution is
The chlorogenic acid aqueous solution.
The sugar is glucose, one or more of compositions in maltose, trehalose.Preferably, the sugar is Portugal
Grape sugar and trehalose in molar ratio 1:1 mixing.
The method of the present invention for improving bird's nest saliva free acid, the knot by the sialic acid in bird's nest from sialoglycoprotein
Conjunction state is changed into free state, gives full play to the nutritive effect of bird's nest, and obtained bird's nest sialic acid has anti-aging and improvement
The effect of memory function.
Embodiment
Each raw material and introduction of apparatus in embodiment：
Bird's nest, purchased from Zhong Huiyu Industrial Co., Ltd.s of Shenzhen.
Papain, No. CAS：9001-73-4, purchased from this reagent Co., Ltd of Adama, enzyme activity 2000U/mg.
Milipore filter, purchased from Co., Ltd in Mi Libo, production code member U650592, molecular cut off 3KDa.
Citric acid, No. CAS：77-92-9, purchased from Wei Keqi bio tech ltd of Sichuan Province.
Chlorogenic acid, No. CAS：327-97-9, purchased from the Zhangjiajie biological Co., Ltd of complete sincerity.
Dithiothreitol (DTT), No. CAS：3483-12-3, purchased from Suzhou Highfine Biotech Co., Ltd..
Glucose, No. CAS：50-99-7, purchased from Shanghai Mike's woods biochemical technology Co., Ltd.
Trehalose, No. CAS：99-20-7, purchased from lark prestige Science and Technology Ltd..
Anion exchange resin, purchased from Shanghai Kai Ping resins Co., Ltd, model D201.
Embodiment 1
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) by bird's nest powder and distilled water with solid-liquid ratio 1:20 (g/mL) are well mixed, obtain mixed liquor；
(3) mixed liquor is extracted 20 minutes in 102 DEG C, obtains extract solution；
(4) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(5) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 2
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) by bird's nest powder and distilled water with solid-liquid ratio 1:20 (g/mL) are well mixed, obtain mixed liquor；
(3) pH for adjusting mixed liquor is 7, adds papain, the addition of papain is bird's nest powder weight
3%, in 50 DEG C digest 3 hours；
(4) after the completion of reacting, enzymolysis liquid is placed in enzyme deactivation in 100 DEG C of water-baths and lived 5 minutes；
(5) enzymolysis liquid after enzyme deactivation is lived is cooled to 25 DEG C, is centrifuged 15 minutes with 4000 revs/min of rotating speed, in collection
Clear liquid；
(6) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 3
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) by bird's nest powder and distilled water with solid-liquid ratio 1:20 (g/mL) are mixed, in microwave power 400W, microwave frequency
Handled 60 seconds under conditions of 915MHz, obtain mixed liquor；
(3) pH for adjusting mixed liquor is 7, adds papain, the addition of papain is bird's nest powder weight
3%, in 50 DEG C digest 1.5 hours；
(4) after the completion of reacting, enzymolysis liquid is placed in enzyme deactivation in 100 DEG C of water-baths and lived 5 minutes；
(5) enzymolysis liquid after enzyme deactivation is lived is cooled to 25 DEG C, is centrifuged 15 minutes with 4000 revs/min of rotating speed, in collection
Clear liquid；
(6) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 4
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) by sulfuric acid solution that bird's nest powder and molar concentration are 0.05mol/L with solid-liquid ratio 1:20 (g/mL) mixing is equal
It is even, obtain mixed liquor；
(3) mixed liquor is extracted 20 minutes in 100 DEG C of water-baths, obtains extract solution；
(4) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(5) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Found during detection, substantial amounts of sulfuric acid is remained in obtained sample, it is therefore desirable to attempted in extract solution
The sulfuric acid of residual removes.
Embodiment 5
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) by aqueous sulfuric acid that bird's nest powder and molar concentration are 0.05mol/L with solid-liquid ratio 1:20 (g/mL) are mixed
Uniformly, mixed liquor is obtained；
(3) mixed liquor is extracted 20 minutes in 100 DEG C of water-baths, obtains extract solution；
(4) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(5) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3KDa, operating pressure 40psi,
Big molecular impurity is removed, collects filtrate；
(6) filtrate is concentrated 2 hours under conditions of 50 DEG C, vacuum 0.07MPa, obtains concentrate；Concentrate is adopted
Purified with anion exchange resin, remove the sulfuric acid remained in concentrate, loading flow velocity is 1mL/min, collects refined solution；
(7) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 6
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) by aqueous citric acid solution that bird's nest powder and molar concentration are 0.05mol/L with solid-liquid ratio 1:20 (g/mL) are mixed
Close uniformly, obtain mixed liquor；
(3) mixed liquor is extracted 20 minutes in 100 DEG C of water-baths, obtains extract solution；
(4) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(5) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3KDa, operating pressure 40psi,
Big molecular impurity is removed, collects filtrate；
(6) filtrate is concentrated 2 hours under conditions of 50 DEG C, vacuum 0.07MPa, obtains concentrate；Concentrate is adopted
Purified with anion exchange resin, remove the citric acid remained in concentrate, loading flow velocity is 1mL/min, collects purifying
Liquid；
(7) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 7
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) by the chlorogenic acid aqueous solution that bird's nest powder and molar concentration are 0.05mol/L with solid-liquid ratio 1:20 (g/mL) are mixed
Close uniformly, obtain mixed liquor；
(3) mixed liquor is extracted 20 minutes in 100 DEG C of water-baths, obtains extract solution；
(4) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(5) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3KDa, operating pressure 40psi,
Big molecular impurity is removed, collects filtrate；
(6) filtrate is concentrated 2 hours under conditions of 50 DEG C, vacuum 0.07MPa, obtains concentrate；Concentrate is adopted
Purified with anion exchange resin, remove the chlorogenic acid remained in concentrate, loading flow velocity is 1mL/min, collects purifying
Liquid；
(7) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 8
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) preparation sodium acid carbonate molar concentration is 1mmol/L, dithiothreitol (DTT) molar concentration is 5mmol/L, glucose rubs
The treatment fluid that your concentration is 1.2mmol/L；
(3) bird's nest powder is configured to the 4mg/mL aqueous solution, the treatment fluid that it is obtained with step (2) is with volume ratio 1:
1 mixing, is stirred 5 hours in 60 DEG C of water-baths with 190 revs/min of rotating speed；Gained reaction solution is cooled to 25 DEG C, vacuum refrigeration
It is dried to obtain solid A；The process conditions of wherein vacuum freeze drying are：- 30 DEG C of pre-freezing temperature, pre-freeze time are 2 hours, distillation
Temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, and the vacuum freeze drying time is 16 hours；
(4) by the chlorogenic acid aqueous solution that solid A and molar concentration are 0.05mol/L with solid-liquid ratio 1:20 (g/mL) mixing is equal
It is even, obtain mixed liquor；
(5) mixed liquor is extracted 20 minutes in 100 DEG C of water-baths, obtains extract solution；
(6) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(7) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3KDa, operating pressure 40psi,
Big molecular impurity is removed, collects filtrate；
(8) filtrate is concentrated 2 hours under conditions of 50 DEG C, vacuum 0.07MPa, obtains concentrate；Concentrate is adopted
Purified with anion exchange resin, remove the chlorogenic acid remained in concentrate, loading flow velocity is 1mL/min, collects purifying
Liquid；
(9) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 9
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) preparation sodium acid carbonate molar concentration is 1mmol/L, dithiothreitol (DTT) molar concentration is 5mmol/L, trehalose rubs
The treatment fluid that your concentration is 1.2mmol/L；
(3) bird's nest powder is configured to the 4mg/mL aqueous solution, the treatment fluid that it is obtained with step (2) is with volume ratio 1:
1 mixing, is stirred 5 hours in 60 DEG C of water-baths with 190 revs/min of rotating speed；Gained reaction solution is cooled to 25 DEG C, vacuum refrigeration
It is dried to obtain solid A；The process conditions of wherein vacuum freeze drying are：- 30 DEG C of pre-freezing temperature, pre-freeze time are 2 hours, distillation
Temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, and the vacuum freeze drying time is 16 hours；
(4) by the chlorogenic acid aqueous solution that solid A and molar concentration are 0.05mol/L with solid-liquid ratio 1:20 (g/mL) mixing is equal
It is even, obtain mixed liquor；
(5) mixed liquor is extracted 20 minutes in 100 DEG C of water-baths, obtains extract solution；
(6) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(7) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3KDa, operating pressure 40psi,
Big molecular impurity is removed, collects filtrate；
(8) filtrate is concentrated 2 hours under conditions of 50 DEG C, vacuum 0.07MPa, obtains concentrate；Concentrate is adopted
Purified with anion exchange resin, remove the chlorogenic acid remained in concentrate, loading flow velocity is 1mL/min, collects purifying
Liquid；
(9) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Embodiment 10
A kind of method for improving bird's nest saliva free acid, comprises the following steps：
(1) bird's nest is crushed, crosses 35 mesh sieves, obtain bird's nest powder；
(2) preparation sodium acid carbonate molar concentration is 1mmol/L, dithiothreitol (DTT) molar concentration is 5mmol/L, glucose rubs
Your concentration is 0.6mmol/L, the treatment fluid that trehalose molar concentration is 0.6mmol/L；
(3) bird's nest powder is configured to the 4mg/mL aqueous solution, the treatment fluid that it is obtained with step (2) is with volume ratio 1:
1 mixing, is stirred 5 hours in 60 DEG C of water-baths with 190 revs/min of rotating speed；Gained reaction solution is cooled to 25 DEG C, vacuum refrigeration
It is dried to obtain solid A；The process conditions of wherein vacuum freeze drying are：- 30 DEG C of pre-freezing temperature, pre-freeze time are 2 hours, distillation
Temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, and the vacuum freeze drying time is 16 hours；
(4) by the chlorogenic acid aqueous solution that solid A and molar concentration are 0.05mol/L with solid-liquid ratio 1:20 (g/mL) mixing is equal
It is even, obtain mixed liquor；
(5) mixed liquor is extracted 20 minutes in 100 DEG C of water-baths, obtains extract solution；
(6) extract solution is cooled to 25 DEG C, centrifuged 15 minutes with 4000 revs/min of rotating speed, collect supernatant；
(7) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3KDa, operating pressure 40psi,
Big molecular impurity is removed, collects filtrate；
(8) filtrate is concentrated 2 hours under conditions of 50 DEG C, vacuum 0.07MPa, obtains concentrate；Concentrate is adopted
Purified with anion exchange resin, remove the chlorogenic acid remained in concentrate, loading flow velocity is 1mL/min, collects purifying
Liquid；
(9) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30
DEG C, the pre-freeze time is 2 hours, and sublimation temperature is 15 DEG C, and resolution temperature is 35 DEG C, vacuum 0.09MPa, vacuum freeze drying
Time is 16 hours.
Test case 1
The percentage composition of free state sialic acid in embodiment 1-3,5-10 is tested, comprised the following steps：
1st, the content of sialic acid in bird's nest raw material is tested first, comprised the following steps：
(1) preparation of sialic acid sample solution：Bird's nest powder 50.0mg is weighed, adds 40mL 1% phosphoric acid solution in boiling
Hydrolyzed 20 minutes in water-bath, enzymolysis liquid taken out and is cooled to 25 DEG C, then centrifuged 10 minutes with 5000 revs/min of rotating speed,
Supernatant is obtained, waits to derive.
(2) sialic acid standard solutions are prepared：Accurately weighing 0.1000gN- n acetylneuraminic acid ns standard items (is purchased from Beijing rope
Lai Bao Science and Technology Ltd.s), it is configured to the standard items storing solution that concentration is 2mg/mL；Standard items storing solution is taken, respectively will
It is diluted to the standard items of 1,20,40,60,80,100 μ g/mL isoconcentrations and uses liquid, waits to derive.
(3) the determination reference of derivatization conditions and chromatographic condition《The DAD/FLD cascaded H PLC assay methods of sialic acid in bird's nest
Research》(Food Science, Feng Tingyu, 2010,31 (8)：233-236) carry out；Specifically derivatization conditions are：Sample solution 1mL is taken, is added
Enter 20mg/mL o-dihydroxy ammon HCI solution 1mL, 80 DEG C of lucifuge water-baths 40 minutes, after taking out rapid cooling, through 0.45 μm of water
It is filtering with microporous membrane, prepares sample introduction；Chromatographic condition：Chromatographic column：WaterssymmetryC18 posts (4.6mm × 250mm, 5 μ
m)；Mobile phase：1.0% tetrahydrofuran aqueous solution (containing 0.5% phosphoric acid, 0.15% n-butylamine)-acetonitrile (95:5, v/v)；Fluorescence swashs
Send out wavelength 230nm, launch wavelength 425nm；Column temperature：35℃；Flow velocity：1.0mL/min；Sampling volume：20μL.
2nd, the content of free state sialic acid is tested in the sample obtained to embodiment：Derivatization conditions and chromatographic condition are same
Total silicic acid assay in bird's nest raw material.
3rd, free sialic acid percentage composition is calculated, calculation formula is：Free state saliva in the sample that embodiment obtains
The calculation formula of liquid acid percentage composition is as follows：Free state in the sample that free state sialic acid percentage composition (%)=embodiment obtains
Sialic acid gross mass × 100% in sialic acid quality/bird's nest raw material.
Specific test result is shown in Table 1.
Table 1：Free state sialic acid percentage composition test result table
It was found from above-mentioned data, although being extracted in embodiment 1-3 to sialic acid, the form majority of sialic acid is
Occur in the form of reference state, be not translated into the form of free state.Carried in embodiment 4-5 in the form of acid system
Taking, as a result find, the content of sialic acid is converted for the form of free state from the form of reference state, but due to embodiment
Substantial amounts of acid is remained in 4 sample, so embodiment 5 purifies to the extract solution that acidity extraction obtains, removal wherein remains
The material such as most of acid and albumen, reach the effect for improving sialic acid purity.Embodiment 8 is to saliva existing in the form of reference state
Liquid acid it is modified is extracted again, by bird's nest raw material in the form of reference state existing for sialic acid be substantially completely converted into free state
Sialic acid, improve bird's nest in sialic acid recovery rate.
Test case 2
Carried out by effect of the sample that water maze test obtains to embodiment 1-3,5-10 to learning and memory in rats ability
Test, specific method of testing are as follows：
Subjects：Wistar rats, it is purchased from Shanghai Si Laike zooperies Co., Ltd, 21 ages in days, averagely 90, body
Weight is 65-66g.
The raising of subjects：By rat sub-cage rearing, 22 DEG C of raising temperature, relative humidity 60%, daily 12 small time
According to alternately, free water and ingest.Keep the cleaning of rat living environment, regular ventilation.
After rat adaptability is raised 3 days, 9 groups are randomly divided into, sialic acid is supplemented by the way of gavage；During gavage
Sialic acid is configured to the 50mg/L aqueous solution；The dosage of gavage is daily 10mL/kg/d, and the time of gavage is every morning 8：
00-9：00.Continuous gavage 30 days, gavage order follow the order of packet, carry out gavage to rat in sequence successively every time, with
Ensure the unification of every rat oral gavage time.
Water maze is made up of black round pool and security platform.Black circle pool diameter is 180cm, is highly
60cm, four quadrants are divided into according to rectangular co-ordinate.Clear water is injected into pond to predetermined altitude before experiment, water temperature maintains 23 ±
2℃.The a diameter of 10cm of security platform, it is highly 30cm, less than water surface 1cm, is fixed on pond fourth quadrant.During experiment, protect
Hold intensity of illumination and position in room to fix, keep surrounding environment quiet, experimenter keeps constant.
Hide platform and obtain the stage that experiment is study and training.Experiment lasts 5 days, daily training 4 times.Distinguish during training
Rat is put into water by the place of entry fixed at random from 4 quadrants, and each experimental period is 60 seconds, if rat was looked in 60 seconds
To hiding platform, it is allowed to be stood on platform 10 seconds；If rat did not found hiding platform also in 60 seconds, direct it to
Platform is simultaneously stood 20 seconds on platform, and now escape latency is designated as 60 seconds..
After space search is tested for testing rat association searching platform, to the holding energy of original platform space position memory
Power.Removed security platform at the 6th day, arbitrarily select a place of entry rat is put into water, swim 60 seconds, survey in its 60 seconds
The time of target quadrant (quadrant where former security platform) swimming and distance and spanning platform number.
Specific test result is shown in Table 2.
Table 2：Water maze test test result table
From table 2 it can be seen that after the study of 5 days, swimming rate of each group rat in space search experiment is simultaneously
There is no significant difference, after illustrating study and training by early stage, rat is already adapted to pond.This is also eliminated from side
Due to swimming rate difference cause the time that rat moves about in target quadrant again with apart from the different possibility of proportion.Rat
After the bird's nest saliva free acid for supplementing embodiment 5-7, swimming time of the rat in target quadrant and the obvious increase of travelling distance,
Rat spanning platform number increases, and this rat of explanation after sialic acid supplements travelling longer time in target quadrant comes
Find platform, at the same more can locating platform exactly position, it is stronger for the memory capability of original platform position.Implement
Example 8-10 effect is more excellent compared to embodiment 5-7, and this is probably because after treatment, the reference state sialic acid in bird's nest is more
Free state sialic acid is converted into more, and free state sialic acid is more conducive to be absorbed by cerebral tissue.
Preferred embodiment of the invention described in detail above.It should be appreciated that one of ordinary skill in the art without
Creative work can is needed to make many modifications and variations according to the design of the present invention.Therefore, all technologies in the art
Personnel are available by logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Technical scheme, all should be in the protection domain being defined in the patent claims.
Claims (7)
- A kind of 1. method for improving bird's nest saliva free acid, it is characterised in that comprise the following steps：(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；(2) by bird's nest powder and distilled water with solid-liquid ratio 1:(20-40) (g/mL) is well mixed, obtains mixed liquor；(3) pH for adjusting mixed liquor is 7-8, adds papain, the addition of papain is bird's nest powder weight 1-4%, 2-3 hours are digested in 45-55 DEG C；(4) after the completion of reacting, enzymolysis liquid is placed in enzyme deactivation in 100-105 DEG C of water-bath and lived 5-10 minutes；(5) enzymolysis liquid after enzyme deactivation is lived is cooled to 23-25 DEG C, and 10-15 minutes are centrifuged with 3000-5000 revs/min of rotating speed, Collect supernatant；(6) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30~- 25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09- 0.095MPa, vacuum freeze drying time are 16-20 hours.
- A kind of 2. method for improving bird's nest saliva free acid, it is characterised in that comprise the following steps：(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；(2) by bird's nest powder and distilled water with solid-liquid ratio 1:(20-40) (g/mL) is mixed, in microwave power 400-500W, microwave The 50-60 seconds are handled under conditions of frequency 900-2500MHz, obtain mixed liquor；(3) pH for adjusting mixed liquor is 7-8, adds papain, the addition of papain is bird's nest powder weight 1-4%, 1-2 hours are digested in 45-55 DEG C；(4) after the completion of reacting, enzymolysis liquid is placed in enzyme deactivation in 100-105 DEG C of water-bath and lived 5-10 minutes；(5) enzymolysis liquid after enzyme deactivation is lived is cooled to 23-25 DEG C, and 10-15 minutes are centrifuged with 3000-5000 revs/min of rotating speed, Collect supernatant；(6) by supernatant vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30~- 25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09- 0.095MPa, vacuum freeze drying time are 16-20 hours.
- A kind of 3. method for improving bird's nest saliva free acid, it is characterised in that comprise the following steps：(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；(2) by acid solution that bird's nest powder and molar concentration are 0.04-0.05mol/L with solid-liquid ratio 1:(20-40) (g/mL) is mixed Uniformly, mixed liquor is obtained；(3) mixed liquor is extracted into 20-30 minutes in 100-105 DEG C of water-bath, obtains extract solution；(4) extract solution is cooled to 23-25 DEG C, 10-15 minutes is centrifuged with 3000-5000 revs/min of rotating speed, collect supernatant Liquid；(5) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3-5KDa, operating pressure 38-42psi, Collect filtrate；(6) filtrate is concentrated into 1-2 hours under conditions of 50-55 DEG C, vacuum 0.06-0.07MPa, obtains concentrate；Will be dense Contracting liquid is purified using anion exchange resin, and loading flow velocity is 0.8-1mL/min, collects refined solution；(7) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30~- 25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09- 0.095MPa, vacuum freeze drying time are 16-20 hours.
- 4. the method according to claim 3 for improving bird's nest saliva free acid, it is characterised in that comprise the following steps：(1) bird's nest is crushed, crosses 30-40 mesh sieves, obtain bird's nest powder；(2) preparation molar concentration containing sodium acid carbonate is 1-2mmol/L, dithiothreitol (DTT) molar concentration is 5-6mmol/L, sugar mole Concentration is 1.0-1.5mmol/L treatment fluid；(3) bird's nest powder is configured to the 4-5mg/mL aqueous solution, the treatment fluid that it is obtained with step (2) is with volume ratio 1: (1-3) is mixed, and 4-8 hours are stirred with 190-220 revs/min of rotating speed in 60-70 DEG C of water-bath；Gained reaction solution is cooled to 23-25 DEG C, vacuum freeze drying obtains solid A；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30~-25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09- 0.095MPa, vacuum freeze drying time are 16-20 hours；(4) by acid solution that solid A and molar concentration are 0.04-0.05mol/L with solid-liquid ratio 1:(20-40) (g/mL) mixing is equal It is even, obtain mixed liquor；(5) mixed liquor is extracted into 20-30 minutes in 100-105 DEG C of water-bath, obtains extract solution；(6) extract solution is cooled to 23-25 DEG C, 10-15 minutes is centrifuged with 3000-5000 revs/min of rotating speed, collect supernatant Liquid；(7) supernatant is subjected to ultrafiltration using milipore filter under conditions of molecular cut off 3-5KDa, operating pressure 38-42psi, Collect filtrate；(8) filtrate is concentrated into 1-2 hours under conditions of 50-55 DEG C, vacuum 0.06-0.07MPa, obtains concentrate；Will be dense Contracting liquid is purified using anion exchange resin, and loading flow velocity is 0.8-1mL/min, collects refined solution；(9) by refined solution vacuum freeze drying, you can；The process conditions of wherein vacuum freeze drying are：Pre-freezing temperature -30~- 25 DEG C, the pre-freeze time is 2-3 hours, and sublimation temperature is 15-20 DEG C, and resolution temperature is 35-40 DEG C, vacuum 0.09- 0.095MPa, vacuum freeze drying time are 16-20 hours.
- 5. the method for the raising bird's nest saliva free acid according to claim 3 or 4, it is characterised in that the acid solution is sulphur Aqueous acid, aqueous citric acid solution, aqueous acetic acid, hydrochloric acid, the chlorogenic acid aqueous solution, aqueous solution of malic acid, aqueous tartaric acid solution, One or more mixtures in butanedioic acid aqueous solution.
- 6. the method for the raising bird's nest saliva free acid according to claim 3 or 4, it is characterised in that the acid solution is green The ortho acid aqueous solution.
- 7. it is according to claim 4 improve bird's nest saliva free acid method, it is characterised in that it is described sugar for glucose, One or more of compositions in maltose, trehalose.
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