CN108576218A - A kind of method that Roy formula lactobacillus protease prepares the breast active beverage of sheep containing Antihypertensive Peptides - Google Patents
A kind of method that Roy formula lactobacillus protease prepares the breast active beverage of sheep containing Antihypertensive Peptides Download PDFInfo
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- CN108576218A CN108576218A CN201710963846.9A CN201710963846A CN108576218A CN 108576218 A CN108576218 A CN 108576218A CN 201710963846 A CN201710963846 A CN 201710963846A CN 108576218 A CN108576218 A CN 108576218A
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- 241000186660 Lactobacillus Species 0.000 title claims abstract description 70
- 229940039696 lactobacillus Drugs 0.000 title claims abstract description 61
- 210000000481 breast Anatomy 0.000 title claims abstract description 35
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- 235000013361 beverage Nutrition 0.000 title claims abstract description 29
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 24
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 20
- 230000003276 anti-hypertensive effect Effects 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 12
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 11
- 239000004365 Protease Substances 0.000 title claims abstract description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 claims abstract description 38
- 108090000790 Enzymes Proteins 0.000 claims abstract description 38
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 210000002421 cell wall Anatomy 0.000 claims abstract description 28
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 24
- 150000004676 glycans Chemical class 0.000 claims abstract description 20
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 20
- 239000005017 polysaccharide Substances 0.000 claims abstract description 20
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- 239000000843 powder Substances 0.000 claims abstract description 18
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- 230000001954 sterilising effect Effects 0.000 claims abstract description 7
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- 238000005119 centrifugation Methods 0.000 claims description 11
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- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 238000011534 incubation Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
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- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
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- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
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- 235000013351 cheese Nutrition 0.000 description 1
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- 239000002934 diuretic Substances 0.000 description 1
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- GBXSMTUPTTWBMN-XIRDDKMYSA-N enalapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)CC1=CC=CC=C1 GBXSMTUPTTWBMN-XIRDDKMYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
- A23C9/1216—Other enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
Abstract
A kind of method that Roy formula lactobacillus protease prepares the breast active beverage of sheep containing Antihypertensive Peptides, will be added phosphate buffer in Roy formula lactobacillus bacterium mud, centrifuge supernatant be protein in cell wall enzyme enzyme solution, freeze-drying obtains Roy formula lactobacillus protein in cell wall enzyme bacterium powder；Roy formula lactobacillus protein in cell wall enzyme bacterium powder and CaCl is added after to degreasing in fresh goat milk2Then enzymolysis adds beta cyclodextrin, hovenia polysaccharides, xylo-oligosaccharide, antierythrite, xylitol, L malic acid, citric acid to obtain the sheep breast active beverage containing Antihypertensive Peptides after homogeneous, sterilization after sodium citrate is uniformly mixed again.The present invention is using sheep breast as raw material, GRAS microorganisms --- the protein in cell wall enzyme of Roy formula lactobacillus is biocatalyst, sheep protein of milk is hydrolyzed into the oligopeptides with ACE inhibitory activity, the Antihypertensive Peptides sheep breast active beverage of preparation can be used as aided blood pressure-lowering, hypoglycemic and anti-caries food, have multiple healthcare function.
Description
Technical field
The present invention relates to a kind of preparation method of functional goat milk beverage, more particularly to a kind of Roy formula lactobacillus protease
The method for preparing the breast active beverage of sheep containing Antihypertensive Peptides.
Background technology
Shaanxi Province is the traditional milch goat main producing region in China, and Guanzhong area is the maximum milch goat breeding production base in the whole nation
Ground.2015 end of the year the whole province milch goat livestock on hands more than 1,800,000, more than 500,000 ton of sheep milk production, the goat milk powder market share up to 95% or more,
Milch goat quantity, sheep milk production and goat milk powder yield are national first, but the less varieties of domestic sheep milk product, predominantly goat milk
Powder and liquid goat milk, wherein most goat milk powder is as raw material of industry exportation abroad, and low, added value that there are the technological element of a product is not
The problems such as high and homogeneity is serious, external sheep milk product type is more, and main includes fermentation sheep milk product (such as cheese, degreasing buttermilk
Or acidophilus goat milk), frozen product (such as ice cream or freezing acidophilus goat milk), liquid milk, butter, functional goat milk piece.Therefore, it develops
Sheep breast new product is necessary.
Hypertension is the most common chronic disease in the world, is the largest one of epidemic disease and cardiovascular and cerebrovascular diseases are most important
Risk factor.Common antihypertensive drugs includes calcium channel blocker, angiotensin converting enzyme inhibitor (ACEI), angiotensins
Receptor blocker (ARB), six class of diuretics, α receptor blocker and beta-blocker, wherein Angiotensin-Converting inhibit
Agent (ACEI) has good target-organ protection and cardiovascular endpoints event prevention effect, and antihypertensive effect for hypertensive patient
It is clear, it has no adverse effects to glycolipid metabolism, is to be depressured one kind with fastest developing speed in class drug now.Common ACEI class drug packets
Captopril, enalapril, benazepil etc. are included, but the ACEI of these synthesis can cause many adverse reactions, most commonly
Dry persistent coughs.Press down with ACE studies have shown that natural ace inhibitory peptide is one kind that protein is decomposed through enzyme and generated
Active polypeptides matter is made, usually contains 3-10 amino acid, molecular weight, can be quickly through digestion between 300-10000
Mucous membrane enters blood circulation, with the potent combinations of ACE to inhibit its activity, plays the role of decompression, therefore be called Antihypertensive Peptides.Portion
The protein in cell wall enzyme energy lactoalbumin hydrolysate of probiotics is divided to release peptides, some of which small peptide has bioactivity, such as drops blood
Pressure, but there is presently no the goat milk beverages of blood pressure lowering.
Honey raisin tree is called papaw, scientific name trifoliate orange Dulcis, and polysaccharide has antidiabetic function, but hovenia polysaccharides prepare presence at present
The low problem of purity, major impurity is monosaccharide and disaccharide, therefore it is that prepare high-purity honey raisin tree more to remove mono-and di-saccharides and retain polysaccharide
The critical issue of sugar.
Invention content
Present invention solves the problem in that a kind of Roy formula lactobacillus protease preparation with blood pressure reduction effect of offer contains
The method of Antihypertensive Peptides sheep breast active beverage.
In order to achieve the above objectives, the technical solution adopted by the present invention is：
1) prepared by Roy formula lactobacillus enriched medium：
By glucose 15-25g, yeast extract 15-25g, manganese sulfate 0.02-0.05g, Tween 80 0.5-1.0mL, synanthrin
1.5-2.5g, casein peptone 4-6g, disodium hydrogen phosphate 4-6g, leucine 15-20mg are added in pure water, are settled to 1L, adjust
PH value is saved to 5.5-7.0, sterilize to obtain Roy formula lactobacillus enriched medium at 121 DEG C；
2) culture of Roy formula lactobacillus and microorganism collection：
Roy formula lactobacillus is accessed by the inoculum concentration of 2%-5% in Roy formula lactobacillus enriched medium, 35-41 DEG C of perseverance
Temperature culture 18-24h, is then centrifuged for outwelling supernatant, collects bacterial sediment, sieve is centrifuged to obtain after being washed later with sterile saline
Her formula lactobacillus bacterium mud；
3) prepared by Roy formula lactobacillus protein in cell wall enzyme：
By the phosphate buffer for adding 20-30mL in every g Roys formula lactobacillus bacterium mud, centrifuged after 35-45 DEG C of heat preservation 1-2h
The enzyme solution that supernatant is protein in cell wall enzyme is obtained, small-molecule substance is removed using 1-3KDa ultrafiltration, is then added in retaining liquid
CaCl2, control CaCl2A concentration of 8-15mM, finally by freeze-drying obtain Roy formula lactobacillus protein in cell wall enzyme bacterium powder；
4) preparation of sheep breast active beverage：
Fresh goat milk is adjusted after degreasing to the Roy formula that 1-3% is added to 6.0-8.0 thereto by mass volume ratio for pH value
The CaCl of lactobacillus protein in cell wall enzyme bacterium powder and 0.01-0.04%2, 2-3h is digested at 35-45 DEG C, then presses quality volume again
Than add 0.5-1.0% beta-cyclodextrin, the hovenia polysaccharides of 0.2-0.4%, the xylo-oligosaccharide of 0.3-0.5%, 4-6% red moss
The L MALIC ACID of sugar alcohol, the xylitol of 3-5%, 0.1-0.3%, the citric acid of 0.10-0.25%, the lemon of 0.10-0.25%
Sour sodium obtains the sheep breast active beverage containing Antihypertensive Peptides, ACE inhibitory activity 75- after being uniformly mixed after homogeneous, sterilization
88%.
The centrifugation of the step 2) is to centrifuge 15-25min at 4-10 DEG C, 6000-8000r/min.
The pH=7.0 of the phosphate buffer of the step 3), lithium chloride containing 180-210g, 0.2- in every liter of phosphate buffer
0.6g lysozymes and 3000-5000U mutanolysin.
The centrifugation of the step 3) is to centrifuge 20-30min at 4-10 DEG C, 4000-6000r/min.
The homogeneous of the step 4) is that temperature is 15-30 DEG C.
The hovenia polysaccharides preparation process of the step 4) is：It takes fresh honey raisin tree to be beaten after cleaning, is then added 20
The water of times honey raisin tree weight, stirs evenly, 2h is kept the temperature under the conditions of 90 DEG C, then filters and obtains hovenia polysaccharides extracting solution, is cooled to
By the Paula enlightening yeast activated liquid of mass volume ratio access 2% after 38 DEG C, 37 DEG C of constant temperature incubations remove for 24 hours monosaccharide in honey raisin tree and
Paula enlightening yeast thalline is collected by centrifugation in disaccharide, supernatant is concentrated into 1/20 with Rotary Evaporators, then press 1:3 volume ratio adds
Enter absolute ethyl alcohol, is then filtered with Buchner funnel and obtain honey raisin tree Thick many candies, then chloroform and positive fourth is added in honey raisin tree Thick many candies rehydration
Chloroform and n-butanol is mutually added in alcohol removing protein, then water intaking again, obtains hovenia polysaccharides by freeze-drying after de- albumen three times.
The present invention is using sheep breast as raw material, GRAS microorganisms --- and the protein in cell wall enzyme of Roy formula lactobacillus is living things catalysis
Sheep protein of milk is hydrolyzed into the oligopeptides with ACE inhibitory activity by agent, and consumer's intake can be added with aided blood pressure-lowering
Hovenia polysaccharides both had the function of stabilizer and reduce blood glucose, the functionality of sheep breast active beverage can be increased；That adds is oligomeric
Xylose is Super Life-benefit Material-X ylo, and it is work(that can promote the proliferation of beneficial bacteria of intestinal tract and the absorption of nutriment, antierythrite and xylitol
Can property sugar alcohol, have the characteristics that low sugariness, it is low in calories, without saprodontia, prepared by present invention Antihypertensive Peptides sheep small to blood sugar influence
Newborn active beverage can be used as aided blood pressure-lowering, hypoglycemic and anti-caries food, have multiple healthcare function.
Specific implementation mode
It is to be described in further details in conjunction with specific embodiments to the present invention below.
Embodiment 1：
1) prepared by Roy formula lactobacillus enriched medium：
By glucose 15g, yeast extract 18g, manganese sulfate 0.03g, Tween 80 0.6mL, synanthrin 2g, casein peptone 4.5g,
Disodium hydrogen phosphate 5g, leucine 18mg are added in pure water, are settled to 1L, adjust pH value to 6.0, sterilize at 121 DEG C
Roy formula lactobacillus enriched medium；
2) culture of Roy formula lactobacillus and microorganism collection：
Roy formula lactobacillus is accessed by 3% inoculum concentration in Roy formula lactobacillus enriched medium, 37 DEG C of constant temperature incubations
22h is then centrifuged for outwelling supernatant, collects bacterial sediment, Roy formula breast bar is centrifuged to obtain after being washed later with sterile saline
Bacterium bacterium mud；
The centrifugation is to centrifuge 20min at 8 DEG C, 7000r/min；
3) prepared by Roy formula lactobacillus protein in cell wall enzyme：
By the phosphate buffer of addition 20mL, pH=7.0 in every g Roys formula lactobacillus bacterium mud (in every liter of phosphate buffer
Lithium chloride containing 200g, 0.5g lysozymes and 4000U mutanolysin), after 40 DEG C keep the temperature 1.5h, centrifuged at 4 DEG C, 5000r/min
25min obtains the enzyme solution that supernatant is protein in cell wall enzyme, small-molecule substance is removed using 1-3KDa ultrafiltration, then in retaining liquid
Add CaCl2, control CaCl2A concentration of 10mM, finally by freeze-drying obtain Roy formula lactobacillus protein in cell wall enzyme bacterium
Powder；
4) preparation of sheep breast active beverage：
Fresh goat milk is adjusted after degreasing pH value to 6.0 be added thereto by mass volume ratio 2.5% Roy formula breast bar
Mycetocyte wall-held protein enzyme bacterium powder and 0.03% CaCl2, 2h is digested at 45 DEG C, then presses mass volume ratio addition 0.8% again
Beta-cyclodextrin, 0.3% hovenia polysaccharides, 0.4% xylo-oligosaccharide, 4.5% antierythrite, 3% xylitol, 0.1%
L MALIC ACID, 0.2% citric acid, 0.15% sodium citrate obtain after 15 DEG C of homogeneous, sterilizations containing drop after being uniformly mixed
Press the sheep breast active beverage of peptide, ACE inhibitory activity 80%.
Embodiment 2：
1) prepared by Roy formula lactobacillus enriched medium：
By glucose 20g, yeast extract 15g, manganese sulfate 0.05g, Tween 80 0.8mL, synanthrin 1.5g, casein peptone 5g,
Disodium hydrogen phosphate 4g, leucine 20mg are added in pure water, are settled to 1L, adjust pH value to 5.5, sterilize at 121 DEG C
Roy formula lactobacillus enriched medium；
2) culture of Roy formula lactobacillus and microorganism collection：
Roy formula lactobacillus is accessed by 5% inoculum concentration in Roy formula lactobacillus enriched medium, 35 DEG C of constant temperature incubations
For 24 hours, it is then centrifuged for outwelling supernatant, collects bacterial sediment, Roy formula breast bar is centrifuged to obtain after being washed later with sterile saline
Bacterium bacterium mud；
The centrifugation is to centrifuge 25min at 4 DEG C, 6000r/min；
3) prepared by Roy formula lactobacillus protein in cell wall enzyme：
By the phosphate buffer of addition 23mL, pH=7.0 in every g Roys formula lactobacillus bacterium mud (in every liter of phosphate buffer
Lithium chloride containing 180g, 0.4g lysozymes and 3500U mutanolysin), after 35 DEG C keep the temperature 2h, centrifuged at 6 DEG C, 4000r/min
30min obtains the enzyme solution that supernatant is protein in cell wall enzyme, small-molecule substance is removed using 1-3KDa ultrafiltration, then in retaining liquid
Add CaCl2, control CaCl2A concentration of 8mM, finally by freeze-drying obtain Roy formula lactobacillus protein in cell wall enzyme bacterium powder；
4) preparation of sheep breast active beverage：
Fresh goat milk is adjusted after degreasing pH value to 8.0 be added thereto by mass volume ratio 1% Roy formula lactobacillus
Protein in cell wall enzyme bacterium powder and 0.01% CaCl2, 3h is digested at 38 DEG C, then presses the β-of mass volume ratio addition 0.5% again
Cyclodextrin, 0.25% hovenia polysaccharides, 0.5% xylo-oligosaccharide, 6% antierythrite, 4% xylitol, 0.2% L- apples
Tartaric acid, 0.10% citric acid, 0.20% sodium citrate obtain containing Antihypertensive Peptides after 20 DEG C of homogeneous, sterilizations after being uniformly mixed
Sheep breast active beverage, ACE inhibitory activity 75%.
Embodiment 3：
1) prepared by Roy formula lactobacillus enriched medium：
By glucose 18g, yeast extract 25g, manganese sulfate 0.02g, Tween 80 0.5mL, synanthrin 2.3g, casein peptone 6g,
Disodium hydrogen phosphate 6g, leucine 15mg are added in pure water, are settled to 1L, adjust pH value to 6.5, sterilize at 121 DEG C
Roy formula lactobacillus enriched medium；
2) culture of Roy formula lactobacillus and microorganism collection：
Roy formula lactobacillus is accessed by 2% inoculum concentration in Roy formula lactobacillus enriched medium, 40 DEG C of constant temperature incubations
20h is then centrifuged for outwelling supernatant, collects bacterial sediment, Roy formula breast bar is centrifuged to obtain after being washed later with sterile saline
Bacterium bacterium mud；
The centrifugation is to centrifuge 15min at 10 DEG C, 8000r/min；
3) prepared by Roy formula lactobacillus protein in cell wall enzyme：
By the phosphate buffer of addition 27mL, pH=7.0 in every g Roys formula lactobacillus bacterium mud (in every liter of phosphate buffer
Lithium chloride containing 210g, 0.2g lysozymes and 5000U mutanolysin), after 45 DEG C keep the temperature 1h, centrifuged at 8 DEG C, 6000r/min
20min obtains the enzyme solution that supernatant is protein in cell wall enzyme, small-molecule substance is removed using 1-3KDa ultrafiltration, then in retaining liquid
Add CaCl2, control CaCl2A concentration of 12mM, finally by freeze-drying obtain Roy formula lactobacillus protein in cell wall enzyme bacterium
Powder；
4) preparation of sheep breast active beverage：
Fresh goat milk is adjusted after degreasing pH value to 7.0 be added thereto by mass volume ratio 3% Roy formula lactobacillus
Protein in cell wall enzyme bacterium powder and 0.04% CaCl2, 2.5h is digested at 35 DEG C, then presses mass volume ratio addition 1.0% again
Beta-cyclodextrin, 0.4% hovenia polysaccharides, 0.3% xylo-oligosaccharide, 5% antierythrite, 5% xylitol, 0.3% L-
Malic acid, 0.15% citric acid, 0.25% sodium citrate obtain after 25 DEG C of homogeneous, sterilizations containing decompression after being uniformly mixed
The sheep breast active beverage of peptide, ACE inhibitory activity 83%.
Embodiment 4：
1) prepared by Roy formula lactobacillus enriched medium：
By glucose 25g, yeast extract 20g, manganese sulfate 0.04g, Tween 80 1.0mL, synanthrin 2.5g, casein peptone 4g,
Disodium hydrogen phosphate 5.5g, leucine 17mg are added in pure water, are settled to 1L, adjust pH value to 7.0, sterilize at 121 DEG C
Obtain Roy formula lactobacillus enriched medium；
2) culture of Roy formula lactobacillus and microorganism collection：
Roy formula lactobacillus is accessed by 4% inoculum concentration in Roy formula lactobacillus enriched medium, 41 DEG C of constant temperature incubations
18h is then centrifuged for outwelling supernatant, collects bacterial sediment, Roy formula breast bar is centrifuged to obtain after being washed later with sterile saline
Bacterium bacterium mud；
The centrifugation is to centrifuge 18min at 6 DEG C, 6000r/min；
3) prepared by Roy formula lactobacillus protein in cell wall enzyme：
By the phosphate buffer of addition 30mL, pH=7.0 in every g Roys formula lactobacillus bacterium mud (in every liter of phosphate buffer
Lithium chloride containing 190g, 0.6g lysozymes and 3000U mutanolysin), after 38 DEG C keep the temperature 2h, centrifuged at 10 DEG C, 4500r/min
28min obtains the enzyme solution that supernatant is protein in cell wall enzyme, small-molecule substance is removed using 1-3KDa ultrafiltration, then in retaining liquid
Add CaCl2, control CaCl2A concentration of 15mM, finally by freeze-drying obtain Roy formula lactobacillus protein in cell wall enzyme bacterium
Powder；
4) preparation of sheep breast active beverage：
Fresh goat milk is adjusted after degreasing pH value to 6.5 be added thereto by mass volume ratio 2% Roy formula lactobacillus
Protein in cell wall enzyme bacterium powder and 0.02% CaCl2, 2h is digested at 42 DEG C, then presses the β-of mass volume ratio addition 0.6% again
Cyclodextrin, 0.2% hovenia polysaccharides, 0.45% xylo-oligosaccharide, 4% antierythrite, 5% xylitol, 0.25% L-
Malic acid, 0.25% citric acid, 0.10% sodium citrate obtain after 30 DEG C of homogeneous, sterilizations containing decompression after being uniformly mixed
The sheep breast active beverage of peptide, ACE inhibitory activity 88%.
Hovenia polysaccharides in above example are prepared using following methods：Fresh honey raisin tree is taken to be beaten after cleaning, so
The water of 20 times of honey raisin tree weight is added afterwards, stirs evenly, 2h is kept the temperature under the conditions of 90 DEG C, then filters and obtains hovenia polysaccharides extraction
Liquid is cooled to after 38 DEG C and removes honey raisin tree for 24 hours by the Paula enlightening yeast activated liquid of mass volume ratio access 2%, 37 DEG C of constant temperature incubations
In monosaccharide and disaccharide, Paula enlightening yeast thalline is collected by centrifugation, supernatant is concentrated into 1/20 with Rotary Evaporators, then press 1:3
Volume ratio be added absolute ethyl alcohol, then with Buchner funnel filter obtain honey raisin tree Thick many candies, then by honey raisin tree Thick many candies rehydration be added
Chloroform and n-butanol is mutually added in chloroform and n-butanol removing protein, then water intaking again, is obtained by freeze-drying after taking off albumen three times
Obtain hovenia polysaccharides.
Claims (6)
1. a kind of method that Roy formula lactobacillus protease prepares the breast active beverage of sheep containing Antihypertensive Peptides, it is characterised in that：
1) prepared by Roy formula lactobacillus enriched medium：
By glucose 15-25g, yeast extract 15-25g, manganese sulfate 0.02-0.05g, Tween 80 0.5-1.0mL, synanthrin 1.5-
2.5g, casein peptone 4-6g, disodium hydrogen phosphate 4-6g, leucine 15-20mg are added in pure water, are settled to 1L, adjust pH
Value is to 5.5-7.0, and sterilize to obtain Roy formula lactobacillus enriched medium at 121 DEG C；
2) culture of Roy formula lactobacillus and microorganism collection：
By Roy formula lactobacillus by the inoculum concentration access Roy formula lactobacillus enriched medium of 2%-5%, 35-41 DEG C of constant temperature is trained
18-24h is supported, is then centrifuged for outwelling supernatant, collects bacterial sediment, Roy formula is centrifuged to obtain after being washed later with sterile saline
Lactobacillus bacterium mud；
3) prepared by Roy formula lactobacillus protein in cell wall enzyme：
By the phosphate buffer for adding 20-30mL in every g Roys formula lactobacillus bacterium mud, centrifuged after 35-45 DEG C of heat preservation 1-2h
Clear liquid is the enzyme solution of protein in cell wall enzyme, removes small-molecule substance using 1-3KDa ultrafiltration, is then added in retaining liquid
CaCl2, control CaCl2A concentration of 8-15mM, finally by freeze-drying obtain Roy formula lactobacillus protein in cell wall enzyme bacterium powder；
4) preparation of sheep breast active beverage：
Fresh goat milk is adjusted after degreasing to the Roy formula breast bar that 1-3% is added to 6.0-8.0 thereto by mass volume ratio for pH value
The CaCl of mycetocyte wall-held protein enzyme bacterium powder and 0.01-0.04%2, 2-3h is digested at 35-45 DEG C, is then added again by mass volume ratio
Add the beta-cyclodextrin of 0.5-1.0%, the hovenia polysaccharides of 0.2-0.4%, the xylo-oligosaccharide of 0.3-0.5%, 4-6% erythrose
The L MALIC ACID of alcohol, the xylitol of 3-5%, 0.1-0.3%, the citric acid of 0.10-0.25%, the citric acid of 0.10-0.25%
Sodium obtains the sheep breast active beverage containing Antihypertensive Peptides, ACE inhibitory activity 75-88% after being uniformly mixed after homogeneous, sterilization.
2. the method that Roy formula lactobacillus protease according to claim 1 prepares the breast active beverage of sheep containing Antihypertensive Peptides,
It is characterized in that：The centrifugation of the step 2) is to centrifuge 15-25min at 4-10 DEG C, 6000-8000r/min.
3. the method that Roy formula lactobacillus protease according to claim 1 prepares the breast active beverage of sheep containing Antihypertensive Peptides,
It is characterized in that：The pH=7.0 of the phosphate buffer of the step 3), lithium chloride containing 180-210g in every liter of phosphate buffer,
0.2-0.6g lysozymes and 3000-5000U mutanolysin.
4. the method that Roy formula lactobacillus protease according to claim 1 prepares the breast active beverage of sheep containing Antihypertensive Peptides,
It is characterized in that：The centrifugation of the step 3) is to centrifuge 20-30min at 4-10 DEG C, 4000-6000r/min.
5. the method that Roy formula lactobacillus protease according to claim 1 prepares the breast active beverage of sheep containing Antihypertensive Peptides,
It is characterized in that：The homogeneous of the step 4) is that temperature is 15-30 DEG C.
6. the method that Roy formula lactobacillus protease according to claim 1 prepares the breast active beverage of sheep containing Antihypertensive Peptides,
It is characterized in that：The hovenia polysaccharides preparation process of the step 4) is：It takes fresh honey raisin tree to be beaten after cleaning, is then added 20
The water of times honey raisin tree weight, stirs evenly, 2h is kept the temperature under the conditions of 90 DEG C, then filters and obtains hovenia polysaccharides extracting solution, is cooled to
By the Paula enlightening yeast activated liquid of mass volume ratio access 2% after 38 DEG C, 37 DEG C of constant temperature incubations remove for 24 hours monosaccharide in honey raisin tree and
Paula enlightening yeast thalline is collected by centrifugation in disaccharide, supernatant is concentrated into 1/20 with Rotary Evaporators, then press 1:3 volume ratio adds
Enter absolute ethyl alcohol, is then filtered with Buchner funnel and obtain honey raisin tree Thick many candies, then chloroform and positive fourth is added in honey raisin tree Thick many candies rehydration
Chloroform and n-butanol is mutually added in alcohol removing protein, then water intaking again, obtains hovenia polysaccharides by freeze-drying after de- albumen three times.
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| CN116606784A (en) * | 2023-07-18 | 2023-08-18 | 华南农业大学 | Application of novel Lactobacillus reuteri anti-freezing protective agent in vacuum freeze drying process |
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