CN104651435A - Method for preparing antihypertensive peptides by fermenting marine organisms through probiotics - Google Patents
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Abstract
The invention relates to a method for preparing antihypertensive peptides by fermenting marine organisms through probiotics. The method comprises the following steps: with marine organisms, such as sea cucumber, jellyfish, acaudina molpadioidea or sleeve-fish, rich in collagen, as raw materials, desalting, cleaning, carrying out pulping and grinding treatment, and heating for killing microorganisms; cooling, adding protease, carrying out liquefaction treatment, carrying out enzyme deactivation, adding glucose, and then putting into a fermentation tank; sterilizing, inoculating a probiotic leavening agent, fermenting, and filtering out residues; adding a cryoprotectant to fermented filtrate, carrying out freeze-drying to directly obtain a composite antihypertensive biological preparation containing active probiotics and bioactive peptides; and in addition, further carrying out tubular membrane purification, ultrafiltration membrane intercept classification and nanofiltration membrane concentration and desalting on the fermented filtrate, and carrying out spray drying or freeze-drying to obtain antihypertensive peptide products with relatively high purity. The marine organisms rich in collagen are adopted as the raw materials; the technical scheme of combining protease liquefaction with probiotic fermentation is adopted. Compared with a traditional enzymatic preparation technology, the method has the advantages that the preparation efficiency of high-activity antihypertensive oligopeptide is improved and enhanced; and the preparation technology is reasonable and effective, and is high in operability and suitable for industrialized production.
Description
Technical field
The invention belongs to biological technical field.A kind of specifically method utilizing probiotics fermention marine organisms to prepare blood pressure lowering peptide.
Background technology
Hypertension is kind of a common chronic cardiovascular disease, and latent period is long, and complication is many, needs the development of long-term pharmacological agent ability symptom management.At present, although the Altace Ramipril of synthetic is evident in efficacy, in the process of clinical application, multiple side effect is created.Therefore, be present in the blood pressure-lowering functional factor in natural animal-plant resource, at home and abroad obtain and pay close attention to widely.Wherein, blood pressure lowering peptide has become a focus in the research of current hypertension prevention and control, have safe and effective, the feature such as have no side effect.
At present, the preparation method of blood pressure lowering peptide mainly contains: extracting directly, enzyme or chemical hydrolysis, enzyme or chemosynthesis.Enzymatic hydrolysis mild condition, high specificity, does not produce deleterious effect, can adopt single enzyme or prozyme, and can be a step or multistep hydrolysis, be the method be most widely used at present.At present, zymyhydrolyzed protein is prepared the technical bottleneck of blood pressure lowering peptide and is: the preparation efficiency of this kind of effective functional component of high reactivity blood pressure lowering peptide is lower.Research in a large number based on blood pressure lowering peptide structure activity relationship shows, high reactivity blood pressure lowering peptide mainly molecular weight is less than the oligopeptide of 1kDa, usually containing proline(Pro) and oxyproline residue in its aminoacid sequence.Therefore, breaking through the key point preparing the hypotensive oligopeptide technical bottleneck of high reactivity is: (1) selects to be rich in the quality protein of high reactivity blood pressure lowering peptide characteristic sequence; (2) directionally hydrolyzing albumen is selected to produce the specific enzymes of high reactivity blood pressure lowering peptide; (3) the high high efficiency technical scheme of proteolysis degree is designed.
Containing the proline(Pro) up to 15-25% and oxyproline residue in the aminoacid sequence of collagen protein, is the potential quality protein producing high reactivity blood pressure lowering peptide.China marine site is vast, abounds with the marine organisms of being rich in collagen protein, as sea cucumber, jellyfish, Haiti melon, squid etc., for the industrialization preparation of high reactivity blood pressure lowering peptide provides abundant natural resources.
Probiotic bacterium is a class living microorganism that can produce one or more beneficial effects to human body.Wherein, the probiotic bacterium such as plant lactobacillus, lactobacterium casei, lactobacterium helveticus, can secrete specific extracellular protease, and the albumen in directionally hydrolyzing matrix produces high reactivity blood pressure lowering peptide, thus produces hypotensive activity to human body; In addition, the specific meta-bolites of this kind of probiotic bacterium, as peptidoglycan, also has significant hypotensive activity.Therefore, the probiotic bacterium with hypotensive activity becomes another focus in the research of current hypertension prevention and control.This kind of probiotic bacterium is that directionally hydrolyzing albumen is prepared high reactivity blood pressure lowering peptide and provided potential specificity extracellular enzyme.
At present, utilize marine organisms to prepare technological line that blood pressure lowering peptide mainly adopts enzymatic hydrolysis; In addition, utilizing probiotics fermention to prepare blood pressure lowering peptide mainly adopts milk-protein as protolysate.The preparation efficiency how strengthening the hypotensive oligopeptide of high reactivity is a problem demanding prompt solution, and wherein, the selection of albumen and enzyme and the design of technical scheme are gordian techniquies.At present, the marine organisms that yet there are no to be rich in collagen protein are raw material, adopt the technical scheme that proteolytic enzyme liquefies and probiotics fermention combines to report to the research preparing blood pressure lowering peptide.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiencies in the prior art, there is provided a kind of with the marine organisms of being rich in collagen protein for raw material, proteolytic enzyme liquefaction and probiotics fermention is adopted to combine, rational technology is effective, preparation efficiency is high, and the probiotics fermention marine organisms that utilize being applicable to suitability for industrialized production prepare the method for blood pressure lowering peptide.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of method utilizing probiotics fermention marine organisms to prepare blood pressure lowering peptide, it is with sea cucumber, jellyfish, the marine organisms that Haiti melon or squid etc. are rich in collagen protein are raw material, carry out pulling an oar and milled processed after desalination and cleaning, heating is gone out except microorganism, add proteolytic enzyme after cooling and carry out liquefaction processing, go out and add glucose after enzyme and obtain Preliminary fermentation liquid, then fermentor tank is put into, inoculate probiotic bacterium liquid starter after sterilizing to ferment, elimination residue after fermentation, cryoprotectant is added in ferment filtrate, the hypotensive biotechnological formulation of compound containing active probiotic and bioactive peptide is directly obtained through lyophilize, in addition, ferment filtrate also after further tubular membrane removal of impurities, ultra-filtration membrane retain classification, nanofiltration membrane concentrating and desalinating, can obtain the blood pressure lowering peptide goods of higher degree by spraying dry or lyophilize.
Concrete technology step of the present invention is:
A. the choosing and pre-treatment of raw material: choosing marine organisms sea cucumber, jellyfish, Haiti melon or the squid of being rich in collagen protein is raw material, clear water soaks and removes salinity, pull out after cleaning and drain away the water, hollander is used to carry out making beating process, adopt colloidal mill to carry out grinding thinning processing after adding the pure water of 2 ~ 30 times of volumes, obtain soup compound;
B. proteolytic enzyme liquefaction: the soup compound obtained by step a is heated to core temperature and reaches 95 ~ 100 DEG C, keep 10min, go out except microorganism, be cooled to 35 ~ 55 DEG C, regulate pH to 2 ~ 9 with NaOH and HCl, add proteolytic enzyme and carry out liquefaction processing, its consumption is 0.5 ~ 10 ‰ of marine organisms weight, after enzymolysis 1 ~ 3h, liquefier is heated to core temperature and reaches 95 ~ 100 DEG C, keep 10min, go out and dezymotize work, must go out enzyme liquefaction liquid;
C. the preparation of Preliminary fermentation liquid: what obtain in step b goes out in enzyme liquefaction liquid, and percentage ratio 0.5 ~ 3% adds glucose or sucrose by weight, regulates pH to 6.5 ~ 7.5 with NaOH and HCl, obtains Preliminary fermentation liquid;
D. probiotics fermention: the Preliminary fermentation liquid obtained by step c is put into the sky fermentor tank that disappears and carried out reality and disappear, the real condition disappeared of empty slake is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access probiotic bacterium liquid starter, this liquid starter be by prebiotic bacterial classification by weight percentage ratio 0.01 ~ 1% access 37 DEG C of constant-temperature tables sealing cultivations in the Preliminary fermentation liquid of sterilizing and form for 2 ~ 4 days, inoculum size is 0.1 ~ 10% of Preliminary fermentation liquid percent by volume, this inoculation mixed solution ferments 2 ~ 5 days at the condition lower seal of 37 DEG C and 100 ~ 300rpm mixing speed, fermented liquid elimination residue, obtain ferment filtrate,
E. the preparation of blood pressure lowering peptide goods: add the cryoprotectants such as 2 ~ 10% trehaloses, 1 ~ 5% maltodextrin, 0.5 ~ 2% glycerine in the ferment filtrate that steps d obtains,-80 ~-20 DEG C of pre-freeze 2 ~ 6h, then lyophilize 24 ~ 48h under the condition of-50 ~-40 DEG C of condenser temperatures and < 150Pa vacuum tightness, directly obtains the composite blood pressure lowering peptide biotechnological formulation containing active probiotic and bioactive peptide; Or, the ferment filtrate employing aperture that steps d obtains is that the tubular membrane of 0.1 ~ 0.5 μm removes macromole, then molecular weight cut-off is adopted to be that the ultra-filtration membrane of 3kDa retains classification, trapped fluid adopts nanofiltration membrane concentrating and desalinating, then spraying dry under the condition of 175 ~ 185 DEG C of inlet temperature, 75 ~ 85 DEG C of air outlet temperatures and 0.35 ~ 0.45MPa spray pressure, or lyophilize 24 ~ 48h under the condition of-50 ~-40 DEG C of condenser temperatures and < 150Pa vacuum tightness, obtains blood pressure lowering peptide goods.
Described proteolytic enzyme is one or more in papoid, flavor protease, Sumizyme MP, bromeline, neutral protease and trypsinase, and its consumption is 0.5 ~ 10 ‰ of marine organisms weight.
Described probiotic bacterium liquid starter be by prebiotic bacterial classification by weight percentage ratio 0.01 ~ 1% access 37 DEG C of constant-temperature tables sealing cultivations in the Preliminary fermentation liquid of sterilizing and form for 2 ~ 4 days, inoculum size is 0.1 ~ 10% of Preliminary fermentation liquid percent by volume, and prebiotic bacterial classification is one or more in plant lactobacillus and lactobacterium casei, lactobacterium helveticus, bifidus bacillus, Lactobacterium acidophilum, lactobacillus bulgaricus and thermophilus streptococcus.
What the present invention was scientific and reasonable chooses the marine organisms such as sea cucumber, jellyfish, Haiti melon or squid is raw material, and its collagen protein be rich in is the quality protein being rich in high reactivity blood pressure lowering peptide characteristic sequence; Plant lactobacilluss used etc. have the probiotic bacterium of hypotensive activity, can secrete the specificity extracellular protease that directionally hydrolyzing albumen produces high reactivity blood pressure lowering peptide; Adopt the process of proteolytic enzyme primary liquefaction, improve the efficiency that probiotic bacterium subsequent fermentation prepares blood pressure lowering peptide.After fermentation, cryoprotectant can be added in fermented liquid, the hypotensive biotechnological formulation of compound is directly obtained through lyophilize, said preparation is rich in other functional factor that the probiotic bacterium metabolism such as the hypotensive oligopeptide of high reactivity, active probiotic, blood pressure lowering peptide glycan produce, and said preparation also retains other functional factor naturally occurring in marine organisms; In addition, after fermented liquid also can adopt membrane filtration purifying further, obtained the blood pressure lowering peptide goods of higher degree by spraying dry or lyophilize.Present invention achieves the efficiently and directionally preparation of the hypotensive oligopeptide of high reactivity, started a new operational path, the hypotensive biotechnological formulation of compound and the high purity blood pressure lowering peptide goods of gained have good application prospect and economic results in society.
Embodiment
Below in conjunction with embodiment, the present invention will be further described.
A kind of method utilizing probiotics fermention marine organisms to prepare blood pressure lowering peptide, it is with sea cucumber, jellyfish, the marine organisms that Haiti melon or squid etc. are rich in collagen protein are raw material, carry out pulling an oar and milled processed after desalination and cleaning, heating is gone out except microorganism, add proteolytic enzyme after cooling and carry out liquefaction processing, go out and add glucose after enzyme and obtain Preliminary fermentation liquid, then fermentor tank is put into, inoculate probiotic bacterium liquid starter after sterilizing to ferment, elimination residue after fermentation, cryoprotectant is added in ferment filtrate, the hypotensive biotechnological formulation of compound containing active probiotic and bioactive peptide is directly obtained through lyophilize, in addition, ferment filtrate also after further tubular membrane removal of impurities, ultra-filtration membrane retain classification, nanofiltration membrane concentrating and desalinating, can obtain the blood pressure lowering peptide goods of higher degree by spraying dry or lyophilize.
Embodiment 1: a kind of method utilizing probiotics fermention sea cucumber to prepare blood pressure lowering peptide, its concrete technology step is as follows:
A. the choosing and pre-treatment of raw material: choose the fresh sea cucumber of 100kg, put into boiling water after removing intestines and boil 20min, pulling out drains away the water removes ginseng mouth, then adopt cannery retort 0.1MPa, 121 DEG C, 30min carries out sofening treatment, the sea cucumber of sofening treatment is 7kg, then adopts clear water to soak and removes salinity, pull out after cleaning and drain away the water, use hollander to carry out making beating process, adopt colloidal mill to carry out grinding thinning processing after adding the pure water of 200L, obtain soup compound;
B. proteolytic enzyme liquefaction: the soup compound of gained is heated to core temperature and reaches 100 DEG C, keep 10min, go out except microorganism, be cooled to 50 DEG C, regulate pH to 5.2 with HCl, add after 40g papoid carries out liquefaction processing 2h, liquefier is heated to core temperature and reaches 100 DEG C, keep 10min, go out and dezymotize work, must go out enzyme liquefaction liquid;
C. the preparation of Preliminary fermentation liquid: going out in enzyme liquefaction liquid at gained, adds 3kg glucose, regulates pH to 7.0, obtain Preliminary fermentation liquid with NaOH;
D. probiotics fermention: Preliminary fermentation liquid is put into the sky fermentor tank that disappears and carry out reality and disappear, the real condition disappeared of empty slake is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access 2L probiotic bacterium liquid starter, this liquid starter be by plant lactobacillus, lactobacillus bulgaricus and thermophilus streptococcus all by weight percentage ratio 1% to access in the Preliminary fermentation liquid of sterilizing 37 DEG C of constant-temperature tables sealing cultivations and form for 3 days, this inoculation mixed solution ferments 4 days at the condition lower seal of 37 DEG C and 200rpm mixing speed, fermented liquid elimination residue, obtains ferment filtrate;
E. blood pressure lowering peptide is rough: in the ferment filtrate of gained, add the cryoprotectants such as 6% trehalose, 3% maltodextrin, 1% glycerine;-50 DEG C of pre-freeze 3h; then lyophilize 48h under the condition of-50 DEG C of condenser temperatures and 100Pa vacuum tightness; be ground into powder, directly obtain the composite blood pressure lowering peptide biotechnological formulation containing active probiotic and bioactive peptide.
The hypotensive biotechnological formulation of this compound is rich in other functional factor that the probiotic bacterium metabolism such as the hypotensive oligopeptide of high reactivity, active probiotic, blood pressure lowering peptide glycan produce, said preparation also retains other functional factor such as naturally occurring functional polysaccharide and saponin(e in sea cucumber, and function, nutrition, mouthfeel and local flavor are all good.
Embodiment 2: a kind of method utilizing probiotics fermention sea cucumber to prepare blood pressure lowering peptide, its concrete technology step is as follows:
A. the choosing and pre-treatment of raw material: choose 5kg dry Stichopus japonicus, put into cold water to soak 1 day, pull out and put into boiling water and boil 20min, pull out rear employing cannery retort 0.1MPa, 121 DEG C, 30min carries out sofening treatment, then adopt clear water to soak and remove salinity, pull out after cleaning and drain away the water, use hollander to carry out making beating process, adopt colloidal mill to carry out grinding thinning processing after adding the pure water of 100L, obtain soup compound;
B. proteolytic enzyme liquefaction: the soup compound of gained is heated to core temperature and reaches 95 DEG C, keep 10min, go out except microorganism, be cooled to 55 DEG C, add after 50g flavor protease carries out liquefaction processing 1h, liquefier is heated to core temperature and reaches 95 DEG C, keep 10min, go out and dezymotize work, must go out enzyme liquefaction liquid;
C. the preparation of Preliminary fermentation liquid: going out in enzyme liquefaction liquid at gained, adds 1kg glucose, regulates pH to 7.0, obtains Preliminary fermentation liquid;
D. probiotics fermention: Preliminary fermentation liquid is put into the sky fermentor tank that disappears and carry out reality and disappear, the real condition disappeared of empty slake is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access 1L probiotic bacterium liquid starter, this liquid starter be by plant lactobacillus by weight percentage ratio 1% to access in the Preliminary fermentation liquid of sterilizing 37 DEG C of constant-temperature tables sealing cultivations and form for 4 days, this inoculation mixed solution ferments 4 days at the condition lower seal of 37 DEG C and 150rpm mixing speed, and fermented liquid elimination residue, obtains ferment filtrate;
E. the purifying of blood pressure lowering peptide: the tubular membrane being 0.2 μm by the ferment filtrate of gained employing aperture removes macromole, then molecular weight cut-off is adopted to be that the ultra-filtration membrane of 3kDa retains classification, trapped fluid adopts nanofiltration membrane concentrating and desalinating, then spraying dry under the condition of 180 DEG C of inlet temperature, 80 DEG C of air outlet temperatures and 0.4MPa spray pressure, obtains the blood pressure lowering peptide goods of higher degree.
Compare traditional proteinase hydrolization method, the yield of high purity blood pressure lowering peptide goods prepared by present method improves more than 30%, goods middle-molecular-weihydroxyethyl be less than 1kDa, containing the hypotensive oligopeptide of high reactivity of proline(Pro) or oxyproline residue, as Glu-Pro-Gly-Pro-Thr and Thr-Cys-Ser-Pro etc., ratio improve more than 35%.
Embodiment 3: a kind of method utilizing probiotics fermention jellyfish to prepare blood pressure lowering peptide, its concrete technology step is as follows:
A. the choosing and pre-treatment of raw material: choose the fresh jellyfish of 100kg, clear water soaks and removes salinity, pulls out and drain away the water after cleaning, and uses hollander to carry out making beating and processes, adopt colloidal mill to carry out grinding thinning processing, obtain soup compound after adding the pure water of 200L;
B. proteolytic enzyme liquefaction: the soup compound of gained is heated to core temperature and reaches 100 DEG C, keep 10min, go out except microorganism, be cooled to 50 DEG C, add after 50g flavor protease carries out liquefaction processing 2h, liquefier is heated to core temperature and reaches 100 DEG C, keep 10min, go out and dezymotize work, must go out enzyme liquefaction liquid;
C. the preparation of Preliminary fermentation liquid: going out in enzyme liquefaction liquid at gained, adds 2kg glucose, regulates pH to 7.0, obtains Preliminary fermentation liquid;
D. probiotics fermention: Preliminary fermentation liquid is put into the sky fermentor tank that disappears and carry out reality and disappear, the real condition disappeared of empty slake is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access 3L probiotic bacterium liquid starter, this liquid starter be by plant lactobacillus by weight percentage ratio 1% to access in the Preliminary fermentation liquid of sterilizing 37 DEG C of constant-temperature tables sealing cultivations and form for 4 days, this inoculation mixed solution ferments 3 days at the condition lower seal of 37 DEG C and 200rpm mixing speed, and fermented liquid elimination residue, obtains ferment filtrate;
E. the purifying of blood pressure lowering peptide: the tubular membrane being 0.2 μm by the ferment filtrate of gained employing aperture removes macromole, then molecular weight cut-off is adopted to be that the ultra-filtration membrane of 3kDa retains classification, trapped fluid adopts nanofiltration membrane concentrating and desalinating, then spraying dry under the condition of 185 DEG C of inlet temperature, 80 DEG C of air outlet temperatures and 0.4MPa spray pressure, obtains the blood pressure lowering peptide goods of higher degree.
Embodiment 4: a kind of method utilizing probiotics fermention Haiti melon to prepare blood pressure lowering peptide, its concrete technology step is as follows:
A. the choosing and pre-treatment of raw material: choose 100kg fresh Haiti melon, put into boiling water after removing intestines and boil 20min, pull out and drain away the water, then adopt cannery retort 0.1MPa, 121 DEG C, 30min carries out sofening treatment, Haiti melon of sofening treatment is 7kg, then adopts clear water to soak and removes salinity, pull out after cleaning and drain away the water, use hollander to carry out making beating process, adopt colloidal mill to carry out grinding thinning processing after adding the pure water of 200L, obtain soup compound;
B. proteolytic enzyme liquefaction: the soup compound of gained is heated to core temperature and reaches 100 DEG C, keep 10min, go out except microorganism, be cooled to 50 DEG C, regulate pH to 5.0 with HCl, add after 45g papoid carries out liquefaction processing 2h, liquefier is heated to core temperature and reaches 100 DEG C, keep 10min, go out and dezymotize work, must go out enzyme liquefaction liquid;
C. the preparation of Preliminary fermentation liquid: going out in enzyme liquefaction liquid at gained, adds 2.5kg glucose, regulates pH to 7.0, obtain Preliminary fermentation liquid with NaOH;
D. probiotics fermention: Preliminary fermentation liquid is put into the sky fermentor tank that disappears and carry out reality and disappear, the real condition disappeared of empty slake is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access 2L probiotic bacterium liquid starter, this liquid starter be by plant lactobacillus, lactobacillus bulgaricus and thermophilus streptococcus all by weight percentage ratio 1% to access in the Preliminary fermentation liquid of sterilizing 37 DEG C of constant-temperature tables sealing cultivations and form for 4 days, this inoculation mixed solution ferments 4 days at the condition lower seal of 37 DEG C and 200rpm mixing speed, fermented liquid elimination residue, obtains ferment filtrate;
E. Antihypertensive Peptides is rough: in the ferment filtrate of gained, add the cryoprotectants such as 5% trehalose, 3% maltodextrin, 1% glycerine;-50 DEG C of pre-freeze 3h; then lyophilize 48h under the condition of-50 DEG C of condenser temperatures and 100Pa vacuum tightness; be ground into powder, directly obtain the hypotensive biotechnological formulation of compound containing active probiotic and bioactive peptide.
The hypotensive biotechnological formulation of this compound is rich in other functional factor that the probiotic bacterium metabolism such as the hypotensive oligopeptide of high reactivity, active probiotic, blood pressure lowering peptide glycan produce, said preparation also retains other functional factor such as naturally occurring functional polysaccharide and saponin(e in the melon of Haiti, and function, nutrition, mouthfeel and local flavor are all good.
Embodiment 5: a kind of method utilizing probiotics fermention Haiti melon to prepare blood pressure lowering peptide, its concrete technology step is as follows:
A. the choosing and pre-treatment of raw material: choose 5kg and do Haiti melon, put into cold water to soak 1 day, pull out and put into boiling water and boil 20min, pull out rear employing cannery retort 0.1MPa, 121 DEG C, 30min carries out sofening treatment, then adopt clear water to soak and remove salinity, pull out after cleaning and drain away the water, use hollander to carry out making beating process, adopt colloidal mill to carry out grinding thinning processing after adding the pure water of 100L, obtain soup compound;
B. proteolytic enzyme liquefaction: the soup compound of gained is heated to core temperature and reaches 95 DEG C, keep 10min, go out except microorganism, be cooled to 55 DEG C, add after 50g flavor protease carries out liquefaction processing 1h, liquefier is heated to core temperature and reaches 95 DEG C, keep 10min, go out and dezymotize work, must go out enzyme liquefaction liquid;
C. the preparation of Preliminary fermentation liquid: going out in enzyme liquefaction liquid at gained, adds 1.5kg glucose, regulates pH to 7.0, obtains Preliminary fermentation liquid;
D. probiotics fermention: Preliminary fermentation liquid is put into the sky fermentor tank that disappears and carry out reality and disappear, the real condition disappeared of empty slake is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access 1L probiotic bacterium liquid starter, this liquid starter be by plant lactobacillus by weight percentage ratio 1% to access in the Preliminary fermentation liquid of sterilizing 37 DEG C of constant-temperature tables sealing cultivations and form for 4 days, this inoculation mixed solution ferments 4 days at the condition lower seal of 37 DEG C and 200rpm mixing speed, and fermented liquid elimination residue, obtains ferment filtrate;
E. the purifying of Antihypertensive Peptides: the tubular membrane being 0.2um by the ferment filtrate of gained employing aperture removes macromole, then molecular weight cut-off is adopted to be that the ultra-filtration membrane of 3kDa retains classification, trapped fluid adopts nanofiltration membrane concentrating and desalinating, then spraying dry under the condition of 180 DEG C of inlet temperature, 80 DEG C of air outlet temperatures and 0.4MPa spray pressure, obtains the blood pressure lowering peptide goods of higher degree.
Compare traditional proteinase hydrolization method, the yield of high purity blood pressure lowering peptide goods prepared by present method improves more than 28%, goods middle-molecular-weihydroxyethyl be less than 1kDa, improve more than 35% containing the ratio of the hypotensive oligopeptide of high reactivity of proline(Pro) or oxyproline residue.
Claims (3)
1. utilize probiotics fermention marine organisms to prepare a method for blood pressure lowering peptide, it is characterized in that, comprise following processing step:
A. the choosing and pre-treatment of raw material: choosing marine organisms sea cucumber, jellyfish, Haiti melon or the squid of being rich in collagen protein is raw material, clear water soaks and removes salinity, pull out after cleaning and drain away the water, hollander is used to carry out making beating process, adopt colloidal mill to carry out grinding thinning processing after adding the pure water of 2 ~ 30 times of volumes, obtain soup compound;
B. proteolytic enzyme liquefaction: the soup compound obtained by step a is heated to core temperature and reaches 95 ~ 100 DEG C, keep 10min, go out except microorganism, be cooled to 35 ~ 55 DEG C, regulate pH to 2 ~ 9 with NaOH and HCl, add proteolytic enzyme and carry out liquefaction processing, its consumption is 0.5 ~ 10 ‰ of marine organisms weight, after enzymolysis 1 ~ 3h, liquefier is heated to core temperature and reaches 95 ~ 100 DEG C, keep 10min, go out and dezymotize work, must go out enzyme liquefaction liquid;
C. the preparation of Preliminary fermentation liquid: what obtain in step b goes out in enzyme liquefaction liquid, and percentage ratio 0.5 ~ 3% adds glucose or sucrose by weight, regulates pH to 6.5 ~ 7.5 with NaOH and HCl, obtains Preliminary fermentation liquid;
D. probiotics fermention: the Preliminary fermentation liquid obtained by step c is put into the sky fermentor tank that disappears and carried out reality and disappear, the real condition disappeared of empty slake is 121 DEG C, 20min, treat tank temperature drop to 37 DEG C, access probiotic bacterium liquid starter, this liquid starter be by prebiotic bacterial classification by weight percentage ratio 0.01 ~ 1% access 37 DEG C of constant-temperature tables sealing cultivations in the Preliminary fermentation liquid of sterilizing and form for 2 ~ 4 days, inoculum size is 0.1 ~ 10% of Preliminary fermentation liquid percent by volume, this inoculation mixed solution ferments 2 ~ 5 days at the condition lower seal of 37 DEG C and 100 ~ 300rpm mixing speed, fermented liquid elimination residue, obtain ferment filtrate,
E. the preparation of blood pressure lowering peptide goods: add the cryoprotectants such as 2 ~ 10% trehaloses, 1 ~ 5% maltodextrin, 0.5 ~ 2% glycerine in the ferment filtrate that steps d obtains,-80 ~-20 DEG C of pre-freeze 2 ~ 6h, then lyophilize 24 ~ 48h under the condition of-50 ~-40 DEG C of condenser temperatures and < 150Pa vacuum tightness, directly obtains the composite blood pressure lowering peptide biotechnological formulation containing active probiotic and bioactive peptide; Or, the ferment filtrate employing aperture that steps d obtains is that the tubular membrane of 0.1 ~ 0.5 μm removes macromole, then molecular weight cut-off is adopted to be that the ultra-filtration membrane of 3kDa retains classification, trapped fluid adopts nanofiltration membrane concentrating and desalinating, then spraying dry under the condition of 175 ~ 185 DEG C of inlet temperature, 75 ~ 85 DEG C of air outlet temperatures and 0.35 ~ 0.45MPa spray pressure, or lyophilize 24 ~ 48h under the condition of-50 ~-40 DEG C of condenser temperatures and < 150Pa vacuum tightness, obtains blood pressure lowering peptide goods.
2. the method utilizing probiotics fermention marine organisms to prepare blood pressure lowering peptide according to claim 1, it is characterized in that, the proteolytic enzyme described in step b is one or more in papoid, flavor protease, Sumizyme MP, bromeline, neutral protease and trypsinase.
3. the method utilizing probiotics fermention marine organisms to prepare blood pressure lowering peptide according to claim 1, it is characterized in that, the prebiotic bacterial classification described in steps d is one or more in plant lactobacillus and lactobacterium casei, lactobacterium helveticus, bifidus bacillus, Lactobacterium acidophilum, lactobacillus bulgaricus and thermophilus streptococcus.
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| CN110269249A (en) * | 2019-07-19 | 2019-09-24 | 蓬莱深奥生物科技研究所 | A kind of sea cucumber intestine ovum ferment and preparation method thereof |
| CN110269250A (en) * | 2019-07-19 | 2019-09-24 | 蓬莱深奥生物科技研究所 | A kind of wall of sea cucumber Stichopus japonicus ferment and preparation method thereof |
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| CN114712400A (en) * | 2022-04-13 | 2022-07-08 | 济南瑞隆安生物技术有限公司 | Probiotic fermented clam powder composition capable of reducing blood sugar and blood pressure and preparation method thereof |
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| CN105385748A (en) * | 2015-11-20 | 2016-03-09 | 青岛贝宝海洋科技有限公司 | Preparation method for fermentative production of marine peptides streptozotocin by using sea cucumber leftovers |
| CN106480147A (en) * | 2016-11-01 | 2017-03-08 | 福建农林大学 | A kind of method that red koji fermentation sea-tangle hydrolyzate prepares blood pressure lowering peptide |
| CN107868810B (en) * | 2017-11-13 | 2021-04-09 | 中国科学院兰州化学物理研究所 | Sea cucumber active peptide with hypoglycemic activity |
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| CN110269250A (en) * | 2019-07-19 | 2019-09-24 | 蓬莱深奥生物科技研究所 | A kind of wall of sea cucumber Stichopus japonicus ferment and preparation method thereof |
| CN110269249A (en) * | 2019-07-19 | 2019-09-24 | 蓬莱深奥生物科技研究所 | A kind of sea cucumber intestine ovum ferment and preparation method thereof |
| CN110403151A (en) * | 2019-08-12 | 2019-11-05 | 浙江海洋大学 | A kind of preparation method of flavor tuna nutrients biological product |
| CN110403153A (en) * | 2019-08-28 | 2019-11-05 | 浙江海洋大学 | A kind of preparation method of flavor squid nutrients biological product |
| CN111165750A (en) * | 2019-09-16 | 2020-05-19 | 烟台东宇海珍品有限公司 | Method for preparing sea cucumber pollen by fermentation technology |
| CN110894230A (en) * | 2019-12-30 | 2020-03-20 | 温州科技职业学院 | Preparation method of acaudina molpadioides biological active peptide |
| CN110973342A (en) * | 2020-03-05 | 2020-04-10 | 中国科学院烟台海岸带研究所 | Apostichopus japonicus oligopeptide and preparation method and application thereof |
| CN114712400A (en) * | 2022-04-13 | 2022-07-08 | 济南瑞隆安生物技术有限公司 | Probiotic fermented clam powder composition capable of reducing blood sugar and blood pressure and preparation method thereof |
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