CN101022737B - Anti-hypertensive functional food products - Google Patents
Anti-hypertensive functional food products Download PDFInfo
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- CN101022737B CN101022737B CN2005800317561A CN200580031756A CN101022737B CN 101022737 B CN101022737 B CN 101022737B CN 2005800317561 A CN2005800317561 A CN 2005800317561A CN 200580031756 A CN200580031756 A CN 200580031756A CN 101022737 B CN101022737 B CN 101022737B
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Abstract
The present invention provides novel protein hydrolysates with antihypertensive properties, as well as food products and food supplements comprising these.
Description
Technical field
The present invention relates to the egg protein hydrolysate (hydrolysate) of biologically active, the functional food and the food supplement that prepare the method for activated protein hydrolysate and comprise these activated protein hydrolysates of appropriate amount.These functional foods, food supplement and activated protein hydrolysate as functional component all are particularly suitable for bringing high blood pressure down, and can by prophylactically with therapeutic eat.In addition, hydrolysate can be used to treatment or prevent one or more metabolic disease and risk factors of cardiovascular diseases, for example metabolic syndrome with the composition that comprises these hydrolysates.
Background technology
The angiocardiopathy of global range has tremendous influence to overall health status, also affects resident's economic conditions and macroeconomy.In EU member country (European heart alliance), all there are every year 1500000 people to die from angiocardiopathy (CVD).In addition, the CVD life loss that causes of disabling is nearly 1,400,000 years, and surpassing half (more than 700,000 years) all is because apoplexy.Very strong association is arranged between the danger of blood pressure level and apoplexy and coronary heart disease, and the European of significant proportion has (slight) hypertension, special old age group even higher danger is arranged.
Hypertension coexists with a large amount of hazards usually.Angiotensins-I invertase (ACE) inhibitor not only can bring high blood pressure down, and can also forward ground influences many other the aspects in the atherogenicity background.Now verified ACE inhibitor is reducing myocardial infarction and congestive heart failure patient's cardiovascular mortality and the benefit aspect the incidence of disease.Even the slight decline of blood pressure also can influence the death rate and the incidence of disease in forward ground.Recently the dangerous analysis of essence of CVD is shown that diet should be than receiving bigger concern (European heart alliance position paper, 1998) in the past as basic, potential hazards.In this position paper, suggestion need be providing health diet to bring up to the core of CVD prevention action.
ACE goes out the terminal His-Leu of C through cracking from the angiotensin I of non-activity and has generated the Angiotensin II with blood vessel pressurization active (comprising vessel retraction).Known function through inhibition ACE can be treated hypertension.Exist a large amount of chemicalses, for example moexipril, quinapril, enalapril, lisinopril, Perindopril, Ramipril, Trandolapril and benazepil with ACE inhibitor activity in the body.These medicines usually have side effect, and the danger of excessive use is arranged.In addition, they are not suitable for preventative use usually.Therefore, need substituting, natural ACE mortifier, when the not high object of blood pressure (prophylactically) was taken in these mortifiers, they also were harmless, but they can reduce the blood pressure of the object of (slightly) blood pressure rising effectively.Preferably, take in these products as food supplement (for example form such as tablet, wafer) or as functional food (for example beverage, semisolid or solid food form).Use these food supplements or functional food to estimate to cause the number of the trouble CVD among minimizing of the cost of health care aspect (CVD expense 2%) and the EU to reduce 5% regularly.As if the average anosis life-span also can prolong at least 3 years.In addition, compare with chemicals, the production process of these products is that littler and producing cost is also still less to the influence of environment.
A large amount of antihypertensive compositions that is derived from natural origin has been described.EP1228708 has described has the newborn derived protein that reduces hypertensive activity and the purposes of peptide level branch.EP0583074 has also described has active peptide that comprises the Val-Pro-Pro that breast is derived of ACE inhibition and its fermented food.The raw material that WO01/32905 has described through lactic acid bacteria fermentation casein containing protein prepare antihypertensive peptide.US6,514,941 relate to the casein hydrolysate that is rich in the Amid.WO01/85984 has described has the purposes that ACE suppresses the lactalbumin hydrolysate of active and antihypertensive active.EP1094071 relates to the peptide as drug for hypertension that from the flesh of fish, obtains.
The purpose of this invention is to provide and have new composition and the function ingredients that ACE suppresses active and antihypertensive active in vivo.
General Definition
Term " food " referred to herein as the food of the liquid that is suitable for people and/or animal edible, semisolid or solid.Therefore, said food comprises beverage.
" functional food " refers to and comprises one or more active component; The food of one or more egg protein hydrolysate of the present invention particularly; In view of the above; When (slightly) hypertensive object ate these functional foods, said active component had been prevented the generation of hypertension or blood pressure rising in vivo and/or has been reduced blood pressure effectively.
" " refer to the enriching substance that is appropriate to people and/or animal edible, it comprises one or more of the present invention bioactive protolysate as functional component of appropriate amount to food supplement.Enriching substance can be forms such as pill, wafer, powder.
Any member of " object " expression class of mammals includes but not limited to people, inhuman primate, farming animals, domestic animal and experimental animal.
" food-grade " refers to and when being taken in by the human or animal, is considered to harmless component.The food-grade component preferably should have the GRAS state.
Term " comprises " and is interpreted as the existence that is used to explain specified ingredients, step or component, but do not get rid of the existence of one or more other components, step or component.Therefore, the peptide sequence that comprises the X district can comprise other zone, and promptly the X district can be contained in the bigger peptide district.
" effective dose " or " effective dose " refers to the dosage that is enough to cause in vivo treatment or preventive effect.The treatment effective dose is to be enough in vivo (after oral absorption) bring high blood pressure down at least about 0.5mmHg, 1mmHg, 5mmHg, 8mmHg, 10mmHg, 12mmHg, 15mmHg, 20mmHg, 30mmHg, 50mmHg or 100mmHg or more dosage.Importantly; Any measurable reduction of blood pressure has all influenced the final result of the cardiovascular disease incidence rate and the death rate significantly and valuably and (has seen McMahon et al.Lancet1990; 335:765-774 and Murray andLopez1997, Lancet349:1498-1504).Can reduce systole phase and/or diastolic blood pressure simultaneously in this way.
" ACE inhibitor " or " ACE suppress active " referred to herein as protolysate external and/or suppressed the ability of ACE-I (angiotensins-I invertase) in vivo significantly.The IC50 value is considered to have remarkable vitro ACE smaller or equal to the protolysate of 0.5mg/ml and suppresses active and be considered to (possibility) to have in the significant body ACE and suppress activity and (see Sekiya et al.1994, Science45:513-517).IC50 refers to the concentration that has suppressed 50% enzymatic activity.
" ovum " preferably refers to egg at this, although can also can use the ovum of other birds.
" egg protein hydrolysate " as generic term, it refers to whole egg, egg-development stage branch (for example egg white (egg white) or yolk (egg yolk)) or the protolysate (external preparation) of the egg protein of purifying (particularly lipovitellin (lipovitellin), ovomucin (ovomucin), lysozyme (lysozyme), ovalbumin (ovalbumin) and ovotransferrins (ovotransferrin)) basically at this.
" unhydrolysed egg protein " or " indigested egg protein " as generic term, it refers to also not at the external whole egg that is hydrolyzed, egg-development stage branch (for example egg white or yolk) or the egg protein of purifying (particularly lipovitellin, ovomucin, lysozyme, ovalbumin and ovotransferrins) basically at this.
" metabolic syndrome " refers to the multiple clinical disease that is relative to each other; Comprise obesity, insulin resistance and hyperinsulinemia, GI, hypertension and dyslipidemia (high triglyceride and low HDL cholesterol levels); As in Moller and Kaufman (Annual Rev.ofMedicine Vol56, that kind described in 45-62).
" biomarker " refers to the indicant of the diseases related or syndrome (or syndromic single component) of blood pressure.For example, the blood level of people CRP (c reactive protein) (a kind of stress GAP-associated protein GAP) is the biomarker of CVD hazards.The reduction of CRP is the for example indication that reduces of apoplexy and incidence rate of myocardial infarction of angiocardiopathy; The raising of CRP level is the indication of vascular system inflammation, and said inflammation affects blood pressure and CVD is dangerous; The other biological label is the amount of insulin (prompting insulin resistance) that discharges after the glucose load and the amount of the secreted albumen in the urine.
" T-CHOL " refers to LDL-and HDL-cholesterol.
" external digestion simulation " referred to herein as egg protein hydrolysate or for example pepsin, chymotrypsin and tryptic incubation of the enzyme in hydrolysed egg proteins and the object intestines and stomach not, and successively with according to the time physiology course that digests in the simulation body mutually.
" active fragment " of peptide or albumen refers to than full-length proteins or the shorter protein component of protein variant (through the synthetic variant that obtains in enzyme hydrolysis or the body), and it has ACE and suppresses active.
Term " substantially the same ", " substantially the same property " or " similar in essence " or " similitude in essence " expression are when for example with the program GAP that utilizes default parameters or BESTFIT optimization ground two kinds of peptides of comparison or two kinds of nucleotide sequences, and they are shared in the sequence homogeny of other local defined particular percentile at least.GAP utilizes Needleman and the overall alignment algorithm of Wunsch on its whole length, to compare two kinds of sequences, makes the number maximization of coupling and the number of breach minimize.Usually use the GAP default parameters, breach generates compensation=50 (nucleotides)/8 (protein) and breach extends compensation=3 (nucleotides)/2 (protein).For nucleotides, used acquiescence integration matrix is nesgapdna and for protein, acquiescence integration matrix be Blosum62 (Henikoff&Henikoff, 1992, PNAS89,915-919).
Utilize computer program for example GCG Wisconsin software kit (10.3 editions) (from AccelrysInc.; 9685Scranton Road; San Diego, CA92121-3752USA) or common software Emboss for Windows (at present version 2 .7.1-07) can determine the integration of sequence alignment and sequence homogeny percentage.Likewise, for example FASTA, BLAST etc. can determine similitude or homogeny percentage through search database.
In addition, when with indefinite article " " or " a kind of " when quoting element, do not get rid of and have the possibility that surpasses or a kind of element, only if clear from context ground requires to have and only have one or a kind of element here.Indefinite article " one " or " a kind of " so ordinary representation " at least one or a kind of ".
In addition, when quoting nucleotides or amino acid " sequence ", understand that this refers to nucleic acid molecules or protein molecular that physiological molecule promptly has nucleic acid or amino acid sequence.
The detailed description of invention
Be surprised to find, egg protein hydrolysate particularly ovalbumin hydrolysate has significant ACE inhibition activity in vitro detection, and in the mouse body, shows significant antihypertensive active.
Analyze (utilizing the special-purpose Homebrew (proprietary in house software) that albumen is carried out quantitative integration and classification) with silicon chip (in silico) at first and identify the target protein in the egg; It comprises that having ACE suppresses active peptide, and therefore very possible anti-hypertension in vivo.Selection strategy is to calculate the integration that surpasses the reference protein beta-casein that contains known ace inhibitory peptide.According to total quantitative integration and classification; Identify 5 kinds of target proteins; It all comprises ace inhibitory peptide, and when carrying out enzyme hydrolysis, it can give play to antihypertensive active in vivo; And after enzyme hydrolysis randomly enrichment active peptide: lipovitellin (seeing for example SEQ ID NO:1,2,7 and 8), a kind of albumen of in yolk, finding; Ovomucin (seeing for example SEQ ID NO:6 and 9), lysozyme (seeing for example SEQ ID NO:4), ovalbumin (seeing for example SEQ ID NO:8) and ovotransferrins (seeing for example SEQ ID NO:5) (all four kinds all is the albumen of in egg white, finding).
Whether and how can be used to produce hydrolysate in order to analyze egg white and/or livetin, carry out a large amount of experiments as further describing in an embodiment with remarkable ACE inhibition activity.
An embodiment of the invention have provided to be used to generate has the egg protein hydrolysate that ACE suppresses active and antihypertensive active.Method comprises step:
1. liquid solution is provided, the aqueous solution preferably, it comprises one or more target protein;
Randomly with solution be heated to about 90 ℃ about 5 to 20 minutes, preferably 15 minutes so that with albuminous degeneration and/or deactivation (possibly exist) protease inhibitors;
3. the pH value is adjusted to the pH value that the hydrolase that makes institute to use has activity, the preferably optimal pH of used hydrolase;
4. when composition has reached the hydrolysis temperature (the preferably optimum temperature of hydrolysing activity) that is suitable for used hydrolase, add the hydrolase of appropriate amount, preferably add about 2% the hydrolase (w/w) that is equivalent to that total target protein level divides;
5. incubation solution stirred at least about 3 hours simultaneously;
6. randomly add the enzyme (2%w/w) of additional amount and about 2 to 3 hours of incubation solution again, stir simultaneously;
7. randomly use for example heat treatment (for example handling 15 minutes down for 90 ℃) inactivator.
The hydrolysate that obtains with top method also is provided, and the composition that comprises this hydrolysate of appropriate amount, preferably food or food supplement composition.
Solution can be concentrated or freeze-drying (for example lyophilized) and room temperature under stored, be used for further purposes (for example external ACE suppresses to detect), be used for peptide enrichment (as follows) or further peptide purification or be used to produce the food supplement or the food of the hydrolysate that comprises effective dose.
Compare with the activity of all-hydrolytic thing, find that can cause the ACE of soluble fraction to suppress active with centrifugal removal solid constituent sometimes increases by 1.5 to 2 times.Randomly, can separate and use soluble fraction.4, centrifugal about 15 minutes perhaps equivalent conditions can be removed solid fraction under the 500g through for example.Also can be through filtering or other isolation technics removal solid fraction.For other target protein, the removal of solid fraction can cause the ACE of soluble fraction to suppress active reduction, and this is disadvantageous.For these target proteins, solid fraction is preferably remained in or adds to again in the product or be used as active food or food supplement composition (a part).
Understand that some steps in the method for hydrolysis can be modified, and do not change the character of formed hydrolysate.For example, use-case according to appointment 4 to 5 or 5 to 6 hours single enzyme incubation step can replace incubation step 5 and 6.The one skilled in the art can adopt top hydrolysis scheme to be optimized application easily.Preferably in water rather than in buffer solution, be hydrolyzed, particularly for the large-scale production that is used for people and/or animal consumption, because a large amount of salt is unwelcome.For hydrolysis, need to optimize amount, pH value, temperature and the incubation time of used enzyme usually.The instance of suitable hydrolysis scheme is provided in an embodiment.
The protein component of the liquid solution in the step 1 can be selected from one or more following albumen: the holoprotein composition of the purifying basically of the albumen of (fresh) whole egg, separation, the yolk of separation, ovum powder, egg white powder, yolk powder, ovalbumin, lysozyme, ovotransferrins, ovomucin and/or lipovitellin.The instance in suitable target protein source is that obtainable protein composition of commercialization or egg-development stage are divided for example egg white powder of composition.For example can obtain (100% pure protein) lysozyme from Belovo; Can obtain ovotransferrins from Sigma-Aldrich (81.3% pure protein) or from Belovo (89.5% albumen); Can obtain ovomucin from Belovo; Can obtain ovalbumin from Worthington (75.7% albumen) or from Interchema (70.8% albumen), can obtain yolk and can obtain egg white from NIVE (31.6% albumen) from NIVE (79.7% albumen).It is obvious that, can use other commercialization source or can from ovum or egg-development stage branch, be purified into one or more target protein with known purification process.Utilize like recombinant DNA technology known in the art and also can generate albumen for example lipovitellin, lysozyme, ovotransferrins, ovomucin and ovalbumin.For example, utilize chemical synthesis can original position synthetic or can in recombinant host cell, clone and express protein fragments or the full-length proteins of arbitrary SEQ ID NO:1-10 (or its variant).Therefore, can predict through the for example suitable edible animal or even can be with the whole egg, yolk or the egg white that are rich in one or more albumen in lipovitellin, lysozyme, ovotransferrins, ovomucin and the ovum target protein (or the variant of these albumen or its fragment) as raw material of laying eggs through transgenic animals.
" variant " of the target protein of SEQ ID NO:1-10 comprises the similar basically albumen of those albumen with SEQ ID NO:1-10, for example has to comprise the amino acid whose albumen that at least 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence homogeny is arranged with arbitrary sequence of SEQ ID NO:1-10.Can original position synthesize, from natural origin, separate or clone perhaps identifying these variants at silicon chip.
In an embodiment of the invention, be stale as the ovum of dietary protein origin, can store the longer time down or with form of powder room temperature for example, 4 ℃.Find that non-fresh ovum source can not influence to negativity ACE and suppress active.This is good for the hydrolysate generative process, because for example before using, do not need freezing ovum.At room temperature or to store under the temperature between about 4 ℃ and the room temperature that ovum or egg-development stage divided at least 6 weeks and do not lose activity be possible.
Perhaps, can from ovum, extract target protein, subsequently with its purifying.For example, utilize known method can isolate egg white level branch, utilize cation and anion exchange chromatography, gel permeation chromatography, affinity chromatography and additive method known in the art can be purified into special target protein.For example can be purified into ovotransferrins, and can use the heparin affinity chromatography method to separate glycoprotein with the metal chelate chromatography method.Lysozyme concentration in the egg white is than the lysozyme in other sources higher (3-4%).The method that routine is used for the lysozyme purifying is a cation exchange chromatography.Lysozyme is to combine with cation-exchanger in 9 o'clock in the pH value, and this can carry out at stirred-tank reactor or in chromatographic column.After going out adsorption particle with eluting salt, lysozyme is enough pure for food applications.In order to obtain to be applicable to the highly purified lysozyme of medicinal application, need the anion-exchange chromatography under the pH4 to carry out further purifying.The advantage of this purification process is not change raw material (egg white), and the easy large-scale application of this method for food applications, only needs an absorption process and kept biologically active.Therefore, in one embodiment, use the cation exchange chromatography Purification of Lysozyme.For being further purified of arbitrary target protein, can adopt the step of for example film ultrafiltration and diafiltration.
The enzyme that is applicable to hydrolysis can be the protease of arbitrary food-grade or the proteinase mixture in various sources in theory, and said source for example is plant, animal or microorganism for example fungi or bacterium.For example, Newlase F comprises the acid protease that is derived from snow-white head mold (Rhizopus niveus), and Promod184P comprises the protease of pineapple, and Promod258P comprises the protease (papain) of papaya and the protease and the peptase of Eurotium.Pepsin 389P comprises the animal acid protease from the pig gastric mucosa, and Alcalase comprises bacillus licheniformis (Bacillus licheniformi) proteinase mixture, and wherein main enzyme is an endoproteinase.
Find that differential protein-protease combination has discharged biologically active peptide, generated to have the hydrolysate that ACE suppresses activity.Table 1 provides the preferred enzyme that is used to prepare protolysate, and the optimum condition of said enzyme application.
Table 1: be used for the enzyme that hydrolysate generates
| Enzyme | The pH scope | Temperature range (℃) | Supplier | Best pH and temperature |
| Newlase?F | 2-7 | 30-55 | Amano?Enz. | pH3；50℃ |
| Pepsin 389P | 2-5.5 | 40-55 | Biocatalysts | pH3；50℃ |
| Promod258P | 3.5-7.5 | 40-50 | Biocatalysts | pH5.5；45℃ |
| Promod184P | 5-7 | 45-55 | Biocatalysts | pH6；50℃ |
| Flavourzyme | 5-7 | 30-60 | Novozyme | pH7；50℃ |
| Alcalase | 6.5-8.5 | 40-65 | Novozyme | pH8；60℃ |
| PEM | 6-8 | 30-45 | Novozyme | pH8；50℃ |
| PTN6.0S | 6-8 | 30-45 | Novozyme | pH7；45℃ |
| Corolase?PP | 6-10 | 30-55 | AB?Enzymes | pH8.5；45℃ |
| Protex6L | 7-10 | 25-70 | Genencor | pH9.5；60℃ |
In these enzymes of testing, the food-grade albumen enzyme (or proteinase mixture) below finding is specially suitable: Newlase F, Promod258P, Alcalase, PEM (protein cleavage enzymatic mixture), PTN (Pancreatic Trypsin Novo) and Protex6L.Understand can use other interior-with outer-protease (or mixture).The one skilled in the art can determine applicability through preparing described hydrolysate and being used in other local described vitro detection method test ACE inhibition activity.
Have no intention to limit scope of the present invention; But think because the enzyme that the exoproteinase activity discharges quite a high proportion of single amino acids is not too suitable, and the enzyme that discharges quite a high proportion of dipeptides and/or tripeptides (because interior-with outer-proteinase activity or only be endoprotease activity) is only.Enzyme suitably discharges and is less than about 10% free amino acid, preferably is less than about 8%, 7%, 6% or 5%.Preferably, used enzyme discharges quite a high proportion of dipeptides and/or tripeptides, for example surpasses 10%, 15%, 20%, 30%, 40%, 50% or more at least.
Therefore protolysate of the present invention preferably includes the dipeptides and/or the tripeptides of a high proportion of target protein, preferably includes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more.In one embodiment, therefore more long-chain and the peptide of HMW more for example have 4,5,6,7,8 or more a plurality of amino acid whose peptide preferably have only quite little amount (preferably less than 10% total target protein level branch) perhaps not exist.
Therefore the dipeptides of deriving through target protein in one embodiment, and/or the molecular weight distribution of tripeptides (and the distribution in protein fractions of peptide chain length) and/or target peptide can characterize out hydrolysate of the present invention.For example, hydrolysate can comprise at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% the target peptide less than 0.5kD.Embodiment 13 has shown the correlation between molecular weight distribution, hydrolysis degree and the hydrolysate activity of target protein hydrolysate.Preferred molecular weight distribution therefore shown in embodiment 13, and comprise for example about 70:10:20,45:21:34,70:16:14,98:1:1,99:1:0,85:10:5,90:1:9,92:4:4 and 53:17:30 (expression have molecular weight the fragment of 0.5kD: 0.5 and 1.0kD between fragment: the percentage of the fragment of 1kD).Less than the fragment of 0.5kD corresponding to less than 4 to 5 amino acid whose fragments, and the fragment of about 0.5-1.0kD corresponding to 4 to 9 amino acid and the fragment that surpasses 1kD corresponding to surpassing 9 amino acid whose fragments of peptides.Therefore, arbitrary target protein for example comprises that albumen or its variant of the amino acid sequence of SEQ ID NO:1-10 can be hydrolyzed the peptide that generation comprises these continuous amino acid chains.Preferably, the hydrolysis degree of target protein is about 15%, 20% at least, more preferably is 30% or higher at least.Therefore, in one embodiment, the present invention comprises the composition that comprises multiple fragment, and said fragment is selected from one or more of 2 arbitrary in SEQ ID NO:1-10 or its variant continuous amino acids, 3,4,5,6,7,8,9 continuous amino acids.
Therefore the molecular weight distribution of hydrolysate can comprise that 40-70% for example is less than the peptide between 0.5 and the 1kD of the target peptide of 0.5kD and 30-50% etc.For example SDS-PAGE analyzes, HPLC analyzes, MALDI-TOF is (like Kaufmann with methods known in the art; J.Biotechn1995; 41:155-175 and Soeryapranata et al.2002; The mastrix-assisted laser desorption ionization time of flight mass spectrography that the described matrix of J.Food Sci.67:534-538 is auxiliary), HP-GPC is (like Terheggen-Lagro et al.BMC Pediatrics2002; The described high phase gel permeation chromatography of 2:10) and Edman degraded (Siemensma et al.Trends Food Sci Technology1993; The molecular weight distribution, peptide chain length that 4:16-21) can determine hydrolysate distribute and maximum peptide molecular weight, also are shown in embodiment 13.The maximum molecular peptide amount of target protein is preferably less than 10kD, more preferably less than 5kD, more preferably less than 4kD, 3kD, 2kD, 1kD, 0.5kD or less than about 0.3kD.
In a concrete embodiment, the small peptide for example ratio of dipeptides and tripeptides is able to enrichment, and the ACE of formed hydrolysate suppresses activity has in view of the above increased at least 1.5 times, 2 times, preferably increased by 3 times or more.Hydrolysate through comprising the target protein derived peptide filters the enrichment that can realize biologically active peptide through one or more molecular filters (for example ultrafiltration or nanofiltration filter); Preferably used to have molecular weight threshold, more preferably used the molecular filter from 2kD to 5kD as 0.3kD, 0.5kD, 1kD, 2kD, 3kD, 4kD, 5kD molecular filter to 10kD.This enrichment is provided amino acid length be about 2,3,4 and/or 5 weak point, low-molecular-weight peptide and ACE with reinforcement suppress active target protein hydrolysate.Preferably using dimension threshold is 2 to the enrichment of 3kD.For example, after the enrichment small peptide, the ACE that demonstrates the hydrolysate that has increased the hydrolysate that comprises the ovotransferrins peptide and comprised the lysozyme peptide suppresses active (seeing embodiment).The hydrolysate of these enrichments preferably includes antihypertensive active in the body that has improved.In addition, littler amount all is effectively for using every day, and this makes can prepare the more food supplement or the food of small size.
Also can remove the unwanted impurity that possibly exist in the hydrolysate and possibly influence the peptide activity with membrane filtration.
The present invention also relates to produce bioactive protolysate, randomly for example dipeptides and/or tripeptides of enrichment low molecular weight peptide subsequently through combination (mixture) with target protein and/or enzyme.
Perhaps, utilize chemical synthesis process can regenerate similar protolysate product combination thing of the present invention.Particularly, can generate dipeptides and three peptide libraries, form all possible combination of dipeptides and tripeptides in view of the above through combinatorial chemistry.From this combination or dipeptides and three peptide libraries, can generate and have and the mixture of said hydrolyzed thing the active substantially the same activity of blood pressure.
Below the target protein-enzyme combination ACE inhibiting value that generated the 0.5mg/ml hydrolysate be about 50%, 55%, 60%, 65%, 70%, 80% or higher hydrolysate (detailed content is seen embodiment).
Target protein-enzyme
Lysozyme-Alcalase
Lysozyme-Protex6L
Lysozyme-PEM
Lysozyme-PTN
Ovomucin-PEM
Ovomucin-Alcalase
Ovomucin-Protex6L
Ovomucin-PTN
Ovotransferrins-Newlase F
Ovotransferrins-Promod258P
Ovotransferrins-PTN
Ovotransferrins-PEM
Ovotransferrins-Protex6L
Ovotransferrins-Promod258P
Yolk-Alcalase
Yolk-Protex L6
Yolk-PEM
Yolk-Newlase F
Egg white-Alcalase
Egg white-Promod258P
10 kinds the tabulation of activated hydrolysate generated the classification that following ACE suppresses active (vitro detection) (from maximum activity to less activity):
1. ovotransferrins-Newlase F; Ovotransferrins-PTN
2. ovomucin-Alcalase; Ovotransferrins-PEM; Lysozyme-PEM
3. yolk-Protex L6; Ovalbumin-Promod258P; Ovotransferrins-Promod258P; Lysozyme-Alcalase; Lysozyme-Protex6L.
For the commercialization purpose, be suitable for large-scale production for example 500,1000, the production method that surpasses 1000 liters undressed raw material and/or protolysate is the skilled person's in required field the content of routine test.
Therefore provide generate as stated and randomly further enrichment and/or purifying, have an egg protein hydrolysate that significant ACE suppresses antihypertensive active in the active and significant body.The composition of the hydrolysate of the present invention that comprises appropriate amount, particularly food and/or food supplement composition also are provided, as follows.
In another embodiment; Egg protein of unhydrolysed egg protein or partial hydrolysis and uses thereof is provided; And the food supplement and the food that comprise at least a unhydrolysed egg protein of effective dose, at least aly specifically be selected from that whole egg, whole egg are yellow, whole egg is clear, the unhydrolysed egg protein of ovotransferrins, ovomucin, lysozyme and/or ovalbumin.The hydrolysis of the protease in the intestines and stomach of (with the digestion simulation) human body of discovery surprisingly and/or animal target is effective for from target protein, discharging biologically active peptide.Therefore extracorporeal hydrolysis itself is not an essential step just; But it is preferred; So that the variation that acts in the body is minimized, the variation of effect can come from the variation between the object (object possibly have higher digestive ferment amount and/or activity than another object) and/or come from food composition and the variation of structure (having caused the variation of the time of staying in different alimentary canals space) in the said body.
When the egg protein with hydrolysis was used to prepare food supplement or food, preferably the activity of hydrolysate was not modified in vivo, perhaps was at least not modified basically or do not modified by the negativity mode (reducing active).Whether can test hydrolysate by " complete hydrolysis " through external digestion analog detection method, the meaning is not influence with the biologically active that contacts hydrolysate of gastrointestinal enzyme subsequently.In this detects; Hydrolysate is with pepsin, trypsase and chymotrypsin incubation, and the ACE of more initial hydrolysate suppresses the ACE inhibition active (more details is seen embodiment) of active and the hydrolysate after pepsin/trypsase/chymotrypsin is handled.Digestion simulation (with digesting in the body) does not preferably change hydrolysate or comprises that the food supplement of hydrolysate or the ACE of food suppress active.
The food supplement and the food of at least a not hydrolysed egg proteins (or egg protein of partial hydrolysis) of the hydrolysate at least a of the present invention that comprises effective dose and/or effective dose also are provided.Significant ACE suppresses activity and is defined as the ACE activity that 0.5mg/ml protolysate (or still less) can suppress about 50% (or more), for example detects with the vitro detection method.Be used for confirming that it is that known (see for example Matsui et al.1992, Biosc.Biotech.Biochem56 517-518), and describes in an embodiment to some extent that ACE suppresses active vitro detection method.Known ACE inhibitor is used as object of reference, for example captopril rightly.Significant antihypertensive active is defined as the ability that hydrolysate brings high blood pressure down in vivo at this; For example can for example measure in SHR (spontaneous hypertension rat) or the human trial, wherein measure (diastole and/or) systolic blood pressure (SPB) in the regular intervals after oral absorption at experimental animal.The contrast mouse can be for example Wistar mouse or WKY of normotensive mouse.The method that is used to measure the animal model blood pressure is known in this area, and for example remote sensing determination method and cuff method (are seen Van Vliet et al.2000, J Pharmacol ToxicolMethods.44 (2): 361-73).Remote sensing equipment and cuff analysis of blood pressure appearance all are that commercialization is obtainable.
As known in the art, can prepare the food supplement and the food that comprise at least a hydrolysate of the present invention and/or at least a not hydrolysed egg proteins of the present invention.Food supplement for example can be form for example tablet, pill, powder wafer, gel, the capsule etc. of any storage form.The preferably oral absorption of the absorption mode of object.Food can be drink form (for example 100ml bottle, 150ml solution), solid or semi-solid food products for example dessert, dessert, condiment, whole-wheat bread etc.Food is the edible food of rule (preferably every day) preferably, for example staple food (for example bread, noodles, soft drink, dairy products for example cheese, sour milk, fermented dairy product, milk, butter etc.).Therefore can biological active component be added in the raw-food material and perhaps can in the production process of food, biological active component be incorporated in the food.Therefore, comprise any existing food or food supplement product that comprises hydrolysate of the present invention at this.
One preferred embodiment in, food is beverage, preferably based on the beverage of fruit juice or vegetable juice, although also can comprise the beverage based on milk.Can prepare the every day dose volume is 50ml, 100ml, 150ml, 200ml or more beverage.Understand that food supplement or food can also comprise other food-grade component, such as but not limited to flavor enhancement, vitamin, mineral matter, stabilizing agent, emulsifying agent, other biological active component, raw-food material/nutrients for example protein, carbohydrate and/or lipidic component or the like.Arbitrary stage that can be in the normal productive process of food/food supplement adds egg protein hydrolysate or hydrolysed egg proteins not.
Component and the carrier that food/enriching substance also can comprise other non-activities be for example carboxymethyl cellulose (CMC), dolomol, stearic acid, saccharin sodium, talcum powder, magnesium carbonate etc. of glucose, lactose, sucrose, sweet mellow wine, starch, cellulose or cellulose derivative for example.It also can comprise water, electrolyte, essential and nonessential amino acid, trace element, mineral matter, fiber, sweetener, flavor enhancement, colouring agent, emulsifying agent and stabilizing agent (the for example ester class of soybean lecithin, citric acid, monoglyceride or diglyceride), anticorrisive agent, bond, aromatic etc.
The effective dose that needs to add depends on multiple factor for example object (for example human or animal), dosage form (every day, a day are for several times, weekly) and product composition and/or structure.Protolysate or not protein hydrolysate every day effective dose scope be in about 50mg/kg body weight to 100mg/kg, 500mg/kg between the 1000mg/kg body weight or higher.But for the hydrolysate of high activity, about 10mg/kg/ days or amount still less possibly be effective.Utilizing routine test to determine effective dose is within one skilled in the art's limit of power.In one embodiment, provide and comprised 10g, 20g, 30g, 40g or the more food or the food supplement of the hydrolysate of high dose.These compositions are suitable for taking in every day.
For the food supplement of pill or capsule form, can add coating, it can change position and/or time that biologically active peptide discharges in vivo.Slow release formulation is known in this area.
After rule was taken in effective dose (preferably every day), functional food of the present invention and food supplement preferably brought high blood pressure down.According to dosage form and absorption,, will see effect to blood pressure in several weeks for example after 3,4,5,6 or 7 weeks.
These functional foods or food supplement can be marked as to have the effect that brings high blood pressure down and can be suffered from slight elevated blood pressure by diagnosis or hypertensive object is taken in as treatment means.Adult's normal arterial pressure is about 120mmHg (systole phase)/80mmHg (diastole).Raise or high blood pressure can be divided into different horizontal, and for example critical (120-160/90-94), slight (140-160/95-104), moderate (140-180/105-114) and severe (160+/115+).Critical, slight and moderate group that food of the present invention and enriching substance are particularly suitable for treating.They can or be united absorption with chemicals (its dosage can reduce) or other products that brings high blood pressure down by independent absorption.
Perhaps, product can be had object or arbitrary health objects that the danger of blood pressure risings/hypertension takes place and prophylactically taken in.In the human body that has blood pressure to raise, any decline of the blood pressure (mmHg) after the oral absorption composition of the present invention can both significantly advantageously influence M & M.After oral absorption food supplement of the present invention or composition, diastolic blood pressure only reduces 1mmHg (or still less), 5mmHg, 8mmHg, 10mmHg, 12mmHg, 15mmHg, 20mmHg, 30mmHg, 40mmHg or more and all is comprised within the scope of the present invention at this.The present invention comprises that also (additionally or likewise) systolic blood pressure has been lowered like the equal number shown in the above.
In another embodiment; Hydrolysate of the present invention or the composition that comprises one or more hydrolysate of appropriate amount are used to treatment or prevention of metabolic syndrome (perhaps one or more single component of metabolic syndrome), are used to reduce T-CHOL and/or LDL-cholesterol; Be used to reduce blood CRP level, being used for treatment or preventing insulin resistance (is diabetes; Diabetes B or adult-onset diabetes or GI), and/or reduce the total urinary protein level.Therefore, can after rule is taken in the composition that comprises one or more hydrolysate of the present invention, measure biomarker, and can confirm the effect of hydrolysate biomarker.Therefore hydrolysate can be used to one or more biomarker, and has useful health benefit in view of the above.Comprised the method for utilizing hydrolysate regulation and control biomarker of the present invention at this.
Preferably, after rule is taken in hydrolysate, (for example take in after 1 week of appropriate amount, 2 weeks or 3 weeks), total blood cholesterol levels and/or LDL cholesterol levels have reduced 10mg/L, 15mg/L, 25mg/L, 30mg/L or more at least.
Can determine suitable amount with routine test.
Blood CRP level preferably has been lowered 0.3mg/L, 0.5mg/L, 1.0mg/L or more.The urine protein level preferably has been lowered 2%, 5%, 10% or more.
Understand any combination that comprises above content at this.Therefore, take in hydrolysate of the present invention and go for reducing systole phase and diastolic blood pressure, T-CHOL and LDL cholesterol, CRP level etc.
Understand that for example fermentation, chemical synthesis or the expression in genetically modified host cell or host's organism of bacterium or other microorganisms (yeast, other fungies) also can generate the hydrolysate that comprises antihypertensive peptide of the present invention with other method.For example, the dna sequence dna of coding active peptide can operably be connected with suitable promoter (preferably strong promoter) and in plant cell, tissue or organ and/or genetically modified plants, express.Also can design the dna sequence dna of the sequence string that comprises some active peptides of coding that under single promoter, to express.Said peptide can be spaced apart sequence separately, and for example coding can be made can discharge active peptide through enzyme hydrolysis subsequently by the amino acid sequence of protease identification and degraded.
SEQ ID NO1:VIT2_CHICK amino acid sequence
SEQ ID NO2:VIT2_CHICK amino acid sequence
SEQ ID NO3:APV1_CHICK amino acid sequence
SEQ ID NO4:LYC_CHICK amino acid sequence
SEQ ID NO5:TRFE CHICK amino acid sequence
SEQ ID NO6:Q98UI9 amino acid sequence
SEQ ID NO7:VIT1_CHICK amino acid sequence
SEQ ID NO8:OVAL_CHICK amino acid sequence
SEQ ID NO9:Q98UI9 amino acid sequence
SEQ ID NO10:VIT1_CHICK amino acid sequence
For example clear product of the present invention of following non-limiting example and method.
Embodiment
Embodiment 1: the silicon chip analysis
1.1 method
Set up the peptide database that contains the sequence data and the inside of quantitative activity data (IC50 value) for all known ACE peptide for inhibiting.To second database analysis that comprises all chicken/egg proteins this database.In analytic process, removed burst and be not the peptide of total length mature peptide.Calculate integration with program, it has reflected that existing peptide has the possibility that ACE suppresses activity in target protein sequence.
1.2 result
Length (small peptide length is preferential) according to activity prediction and binding peptide has obtained following result.
| Source: EY=yolk EW=egg white | Protein | Weighted integral (the x1000 integration/AA) |
| EY | Vit2 lipovitellin II (VIT2_CHICK; SEQ ID NO:1) | 15.8 |
| EY | Vit2 lipovitellin I (VIT2_CHICK; SEQ ID NO:2) | 9.6 |
| EY | Apovitellenin 1 precursor (APV1_CHICK; SEQ ID NO:3) | 9.5 |
| EW | Lysozyme C precursor (LYC_CHICK; SEQ ID NO:4) | 8.6 |
| EW | The ovotransferrins precursor | 8.3 |
| (TRFE_CHICK；SEQ?ID?NO：5) | ||
| EW | Ovomucin α subunit (starting from 1741) (Q98UI9; SEQ ID NO:6) | 6.0 |
| EY | Vit1 lipovitellin I (VIT1_CHICK; SEQ ID NO:7) | 5.6 |
| EW | Ovalbumin (OVAL_CHICK; SEQ ID NO:8) | 5.3 |
| EW | Ovomucin α subunit (to 1740) (Q98UI9; SEQ ID NO:9) | 5.0 |
| EY | Vit1 lipovitellin II (VIT1_CHICK; SEQ ID NO:10) | 3.8 |
| With reference to albumen | B casein precursor (CASB_BOVIN) | 4.5 |
Have that the target protein of high weighted integral comprises having the peptide that ACE suppresses active (quantity and gross activity), therefore these albumen are the sources likely with hydrolysate of ACE rejection.Target protein the most likely is lipovitellin I and II, lysozyme, ovotransferrins, ovomucin and ovalbumin.
Embodiment 2: prioritization scheme of the present invention
2.1 the scheme of hydrolysed egg proteins
● protein fractions (3%) is dissolved in the water;
● agitating solution;
● with solution 90 ℃ of following incubations 15 minutes (, aggegation not being arranged) to lysozyme;
● be cooled to temperature required after, the pH value is adjusted to required pH value, and adds enzyme (enzyme is 2% with respect to the ratio (w/w) of protein fractions);
● incubation solution 3 hours, stir simultaneously;
● add the enzyme (2%w/w) of additional amount;
● under agitation again incubation solution 2 by 3 hours;
● through 15 minutes inactivators of 90 ℃ of following incubations in water tank;
● with solution freeze-drying and storage at room temperature.
2.2 be used for the scheme of the external digestion simulation of (hydrolysis) sample
● prepare 4% albumen (hydrolysate) sample solution;
● the pH value is set at 2;
● add the 10mg/ml pepsin solution, the ratio of addition is 1/250 (w/w);
● rock down, 37 ℃ of following incubation solution is 2 hours in water tank;
● subsequently, pH is set at 6.5;
● add the solution that contains 10mg/ml trypsase and 10mg/ml chymotrypsin, the ratio of two kinds of enzymes all is 1/250 (w/w);
● rock down, 37 ℃ of following incubation solution is 2.5 hours in water tank;
● through at 15 minutes inactivators of 90 ℃ of following incubations;
● with solution freeze-drying and storage at room temperature.
2.3 be used for determining the active scheme of ACE inhibition of hydrolysate at micro plate
Utilization can go out ACE inhibition activity by external test based on the detection method of AAS.This detection method is to be dependent on through ACE catalysis from the Hip-His-Leu substrate, to discharge hippuric acid.
Used solution:
● contain the 0.1M borate buffer (pH value 8.3) of 300mM NaCl;
● 5mM EDTA.2Na solution;
● contain the 0.5M Bicine of 1M NaCl;
●0.25M?NaOH；
●50mU/ml?ACE；
● 50mM2,4, the 0.1M Na of 6-TNB (TNBS)
2HPO
4Solution;
● contain the borate buffer of the 5mM Hippuryl-L-Histidyl-L-Leucine (Hip-His-Leu) of 1mg/ml lactoferrin;
● albumen (hydrolysate) solution (0.500mg/ml, 0.167mg/ml, 0.055mg/ml, 0.019mg/ml);
● with reference to solution, 0.5mg/ml egg white/Alcalase solution.
ACE suppresses the incubation scheme of detection method miniaturization
EDTA is with ACE sex change (100%ACE inhibition)
In institute is porose, add lactoferrin (native protein) with zero balancing test b icine (buffer solution), adjustment the pH value to 9.1 in porose, i.e. the optimal pH that reacts of TNBS.
The calculating that ACE suppresses
% inhibition=(contrast-blank 2)-(sample-blank 1)/(contrast-blank 2) x100
2.4 with the further hydrolysis (hydrolysis-optimized) of Alcalase to ovomucin
Carry out the scheme that DH measures with the TNBS method
Solution:
● 0.21M sodium phosphate buffer (pH value 8.2)
-0.21M NaH
2PO
4.H2O (25ml solution)
-0.21M Na
2HPO
4.2H2O (500ml solution)
-mixed solution, pH value are 8.2
●0.05％TNBS
-in MQ test tube with the aluminium foil clad, dilution TNBS storage liquid (5% solution), and be stored in the dark place.
● 1%SDS solution
● leucine titer (3mM leucine storage liquid)
-with leucine solution in 1%SDS solution.Press 20x, 10x, 4x, 2x with 1%SDS and dilute storage liquid.
● blank
-dilute not hydrolyzation sample with 1%SDS, make that protein concentration is 0.5mg/ml
● sample
-with the sample of 1%SDS dilution hydrolysis, make that concentration is 0.5mg/ml, the double sample analysis
● 15 μ l sample (leucine titer or sample) suctions are measured in the micro plate; Every kind of twice dilution in the assay plate (Costar, non-binding plate)
● add 45 μ l0.21M sodium phosphate buffers (pH value 8.2)
● add 45 μ l0.05%TNBS.Use the Aluminium Foil Package cover plate.The incubation plate is 60 minutes in 50 ℃ of heaters
● after incubation, add 90 μ l0.1N HCl
● measure the absorbance (micro plate ELIASA) at 340nm place
The calculating of hydrolysis degree (DH)
● make the compensation line of leucine titer.Use this compensation line that the absorbance numerical value of sample is changed into the NH2 equivalent.Mmol NH2-equivalent in the correcting sample proofreaied and correct be the molar equivalent in the hydrolyzation sample not.
● for example: the NH2=0.78mM leucine NH2 that hydrolysis generates, 0.78mM*15 (sample volume) * 10
-6=1.17*10
-5Meq.The protein concentration of sample is 0.5mg/ml, so 15 μ l samples contain 7.5 μ g albumen.h＝1.17*10
-5meq/7.5*10
-6＝1.56meq/g。H tot eiwit (numerical value depends on albumen)=7800meq/kg.％DH＝(1.56/7.8)*100％＝20％。
The result
Experiment 1:
3% ovomucin solution, pH8,60 ℃, Alcalase
| The hydrolysis time total (hour) | Enzyme (%) | DH(％) | ACE suppresses IC50 (mg/ml) |
| 4 | 2％t＝0 | 0 | >2 |
| 5 | 4% (adds 2% after 4 hours) altogether | 5.0 | 0.76 |
| 6 | 4% altogether | 7.7 | 0.66 |
| 7 | 6% (adds 2% after 6 hours) altogether | 12.9 | 0.45 |
| 7.5 | 6% altogether | 15.0 | 0.43 |
Attention: during first hour, do not take place hydrolysis-optimized.
Experiment 2
3% ovomucin solution/Alcalase hydrolysate, pH8,60 ℃, Alcalase
| The hydrolysis time total (hour) | Enzyme (%) | DH | ACE suppresses IC50 (mg/ml) |
| 5 | 4% (2%t=0 added 2% after 3 hours) altogether | 12.0 | 0.54 |
| 8 | 6% (t=5 adds 2%) altogether | 21.7 | 0.39 |
| 10.5 | 8% (adds 2% after 8 hours) altogether | 23.9 | 0.21 |
Experiment 3
3% ovomucin solution/Alcalase hydrolysate (DH23.9, IC
50=0.21mg/ml), pH8,60 ℃, Alcalase
| The hydrolysis time total (hour) | Enzyme (%) | DH | ACE suppresses IC50 (mg/ml) |
| 10.5 | 8% altogether | 21.2 | 0.25 |
| 13.5 | 10% altogether | 22.0 | 0.18 |
| 16.5 | 12% altogether | 21.1 | 0.18 |
Ovomucin/Alcalase the hydrolysate that is used for zooscopy
General introduction to the further hydrolysis of ovomucin/Alcalase hydrolysate
● 6g hydrolysate (ovomucin/Alcalase hydrolysate, 5 hours)
● 60 ℃, pH value 8
●120μl?Alcalase
● after 3 hours, the extra Alcalase of 120 μ l
● after 3 hours, the extra Alcalase of 120 μ l
● after 30 minutes, cessation reaction (90 ℃ are following 15 minutes)
● freeze-drying
Total hydrolysis time (5+6.5)=11.5 hour
ACE is active: IC50=0.23mg/ml
Sample has been used to embodiment 8.2.4.1.
Embodiment 3: initial egg protein hydrolysising experiment (before scheme optimization)
For hydrolysising experiment, 10 kinds of enzyme and 9 kinds of protein samples that commercialization obtains have been used.Following enzyme has been used to hydrolysising experiment (having provided best pH value, temperature and buffer solution in the number of scraping):
● Newlase F (pH3, T50, formic acid buffer solution)
● pepsin 389P (pH3, T50, formic acid buffer solution)
● Promod258P (pH5.5, T45, acetate buffer)
● Promod184P (pH6, T50, Bis-Tris buffer solution)
● Flavourzyme (pH7, T50, phosphate buffer)
● Alcalase (pH8, T60, Tris buffer solution)
● PEM (pH8, T50, Tris buffer solution)
● PTN (pH8, T50, Tris buffer solution)
● Corolase PP (pH8, T50, Tris buffer solution)
● Protex6L (pH8, T60, Tris buffer solution)
The target protein composition of commodity below using (provided in the number of scraping and produced family and explanation):
● lysozyme (Belovo, 100% albumen)
● ovotransferrins (Sigma, Aldrich, 81.3% albumen)
● ovomucin (Belovo, 72.3% albumen)
● ovomucin (Worthington, 75.7% albumen)
● yolk (NIVE, 31.6% albumen)
● egg white 1101 (NIVE, 79.7% albumen)
● egg white 102 (NIVE, 77.2% albumen)
● ovalbumin (Interchema, 70.8% albumen)
● ovotransferrins (Belovo, 89.5% albumen)
Determine albumen percentage according to Kjeldahl (the Kjeldahl coefficient is 6.25).
Under the pH and temperature of the best, in buffer solution, carry out initial hydrolysising experiment.Scheme is:
● protein fractions (100mg albumen) is dissolved in the 3.3ml buffer solution;
● vortex solution;
● when t=0, taking-up sample (300 μ l) also is stored in-20 ℃ of refrigerators;
● with solution incubation 15 minutes (rocking simultaneously) in 90 ℃ of water tanks.Because the formation of agglutinator is arranged, lysozyme is not carried out this step;
● be cooled to temperature required after, add enzyme (1.8mg or 1.8 μ l; Add 2% enzyme with respect to protein fractions);
● after mixing, the vortex test tube;
● when stirring with bar magnet, incubation solution 3 hours, during vortex solution;
● through 15 minutes inactivators (rocking) of 90 ℃ of following incubations in water tank;
● determine the pH value, with the solution freeze-drying and be stored in 4 ℃.
After all target proteins-enzyme combination is initially screened (result does not show), analyze sample the most likely (being the sample of IC50) again less than 0.5mg/ml.Provided ACE inhibition result's (working concentration is the captopril of 6ng/ml) in the table below as the function of protein concentration.
Ovalbumin
Ovotransferrins
Lysozyme
Ovomucin
Yolk
Egg white 1101
Egg white 1102
Demonstrated analysis in the table below to result shown in top.Repeatability that ACE to the combination of target protein-enzyme suppresses active, suppress and outward appearance (this also be to hydrolysate can operated convenience measured value) score.
Have the albumen-enzyme combination that suppresses above 50%ACE during for 0.5mg/ml in concentration:
#: outward appearance; S=solution; The p=particle; The m=emulsus; The a=aggegation; The ts=turbid solution
1:ACE suppresses>60% do ++; Between the 50-60% be+
2: the difference between the highest and minimum 10% be+; Be 0 between the 10-20%; 20% be-
3: dissolubility: solution does ++; Turbid solution is+; Emulsion is+; Particle is 0; Aggegation is 0
Above any target protein in the table (particularly with shown in enzyme or have the combination of enzyme of equal value active) be applicable to that all preparation has bring high blood pressure down active target protein hydrolysate and food supplement/food (these hydrolysates that comprise appropriate amount).
Suppress active for the external ACE after the hydrolysis of albumen in buffer solution, according to integration following albumen-enzyme is made up and carry out classification.For this reason, the integration of each aspect of every kind of target protease combination is added up and obtained below classification:
10 kinds of best target proteins-enzyme combination
| Integration | The source | Enzyme |
| +++++ | Ovotransferrins | NewlaseF |
| Ovotransferrins | PTN | |
| ++++ | Ovomucin | Alcalase |
| Ovotransferrins | PEM | |
| Lysozyme | PEM | |
| +++ | Yolk | Protex6L |
| Ovalbumin | Promod258P | |
| Ovotransferrins | Promod258P | |
| Lysozyme | Alcalase | |
| Lysozyme | Protex6L |
Distribution below the analysis of the albumen that in best group is set up jointly, occurred and enzyme number drawn:
| Protein | # | Enzyme | # |
| Ovotransferrins | 4 | Alcalase | 2 |
| Lysozyme | 3 | Promod258P | 2 |
| Ovomucin | 1 | PEM | 2 |
| Yolk | 1 | Protex6L | 2 |
| Ovalbumin | 1 | PTN | 1 |
| Egg white | 0 | NewlaseF | 1 |
Some enzyme demonstrates it can not discharge biologically active peptide, for example pepsin, Promodl84P, Flavourzyme and CorolasePP effectively from target protein.It should be noted that CorolasePP and Flavourzyme are the enzymes that can generate very many free amino acids (11.5-13.0%), because their exoproteinase (exoprotease) activity.In these hydrolysates, formed free amino acid rather than small peptide, caused the ace inhibitory peptide of low concentration.Be not to be the restriction to scope of the present invention, the enzyme that has proposed to discharge the free amino acid of relatively more measuring than the amount of dipeptides and tripeptides is not too to be fit to.Promod258P also is the mixture of endoproteinase and exoproteinase, and known its can generate considerable free amino acid (9.3%), and it is a kind of gratifying enzyme that is used to generate ACE inhibition hydrolysate.
Embodiment 4: hydrolysis in the water and hydrolysis scheme optimization
Consider and do not hope to use the commercialization of buffer solution to use, in water, be hydrolyzed.If not the situation of clear and definite necessity, the consumption product should not contain additional salt component, so water replaces the optimized buffer liquid condition of every kind of enzyme.In course of reaction, through adding the pH value of NaOH or HCl adjustment solution.
Also further optimized the incubation time of enzyme and target protein.Initial hydrolysis scheme is identical with scheme in the top buffer solution, but after 3 hours, with the enzyme (amount is 2%w/w) of additional amount protein hydrolysate further.Each hour obtained sample.Use ACE to suppress detection method test level branch, the result is shown in the following table.
Demonstrate row as the 0.5mg/ml protein concentration of relevant contrast with shade.If the inhibition percentage of this numerical value is 50% or higher, hydrolysate possibly have antihypertensive active in the body so.
The optimum hydrolysis time of egg white and yolk sample is nearly 5 hours, obtain 0.15 and 0.25mg/ml between the IC50 value.For purifying protein is lysozyme, ovotransferrins and ovomucin; The optimum hydrolysis time is between 4 to 5 hours, and the scope of IC50 value (data computation as from table obtains) is from being lower than 0.1mg/ml (lysozyme and ovotransferrins) to 0.3mg/ml (ovomucin).
Embodiment 5: the external digestion simulated test
5.1
Carry out the external digestion simulated test to study possibly acting on of inherent digestive system with albumen-enzymatic compositions.Transplant activity if endogenous protein lyases negative sense ground has influenced the ACE of the hydrolysate of external optimization, should adopt optimization method to address this problem.Therefore, used following scheme:
● prepare 4% albumen (hydrolysate) sample solution
● the pH value is set at 2
● add the 10mg/dl pepsin solution, the ratio of its amount is 1/250 (w/w)
● rock down, incubation solution is 2 hours in 37 ℃ water tank
● the pH value is set at 6.5
● add respectively and contain the trypsase of 10mg/ml and the solution of chymotrypsin, the ratio of amount is 1/250 (w/w)
● rock down, incubation solution is 2.5 hours in 37 ℃ water tank
● through at 15 minutes inactivators of 90 ℃ of following incubations
● the freeze-drying sample also is stored in 4 ℃
Hydrolyzation sample with 6 little time points is implemented this scheme.Result's (inhibition percentage) is shown in the following table, and the row of the hydrolase of 0.5mg/ml level has shaded background.
As seeing, there is not a kind of ACE inhibition ability of target protein-enzymatic compositions after the external digestion simulation, significant variation to take place from the 0.5mg/ml point.The total result of each time point (comprising all IC50 values) all is shown in the following table.Three different researchs have been summarized: 1. the screening of hydrolysis after 3 hours in buffer solution; 2. to the optimization of hydrolysis in water; 3. the digestion of 6 hours hydrolyzation samples is simulated.
5.2
Carried out another test, with the influence of research external digestion simulation to the hydrolysate of yolk and egg white target protein.
D=external digestion simulation
Y+AIc=yolk hydrolysate-Alcalase
Yolk D=is yellow through (in-vitro simulated) whole egg of digestion
Y+AIc D=yolk hydrolysate-Alcalase digests (in-vitro simulated) afterwards
EW+AIc=egg white hydrolysate-Alcalase
Egg white D=is clear through (in-vitro simulated) whole egg of digestion
EW+AIc D=egg white hydrolysate-Alcalase digests (in-vitro simulated) afterwards
Can see that the ACE that the external digestion simulation can not change hydrolysate suppresses active.
Lyz+AIc=lysozyme hydrolysate-Alcalase
Lyz+PEM=lysozyme hydrolysate-PEM
The external digestion simulation of Lyz D=lysozyme (not by hydrolysis in advance)
Lyz+AIc D=hydrolysate (lysozyme-Alcalase), carry out the external digestion simulation afterwards
OM+AIc=ovomucin hydrolysate-Alcalase
OM+PTN=ovomucin hydrolysate-PTN
The external digestion simulation of OM D=ovomucin (not by hydrolysis in advance)
OM+AIc D=hydrolysate (ovomucin-Alcalase), carry out the external digestion simulation afterwards
OT+NF=ovotransferrins hydrolysate-NewlaseF
OT+Pr258P=ovotransferrins hydrolysate-Promod258P
The external digestion simulation of OT D=ovotransferrins (not by hydrolysis in advance)
OT+AIc D=hydrolysate (ovotransferrins-Promod258P), carry out the external digestion simulation afterwards
Digestion simulation can negative sense ground or the ACE inhibition activity of the formed hydrolysate of forward ground influence.Have only the hydrolysate forward ground of ovotransferrins (OT) to increase ACE inhibition activity.
Generally speaking, external digestion simulation can forward ground or the ACE rejection of negative sense ground change egg protein hydrolysate.The ACE that digestion activity in this description object body can not change hydrolysate suppresses.But this needs to confirm in the body.
5.3
Research to the influence of pepsic two kinds of different incubation times in external digestion
With promptly 30 minutes pepsic two kinds of incubation times and 120 minutes (D30 and D120) repetition external digestion research, this has represented short residence time and long residence time in the stomach respectively.The incubation time of enteron aisle simulation does not change, and promptly still is 150 minutes.
In addition, suppress to have tested initial albumen and protein fractions (not by hydrolysis in advance, also not by external digestion) in the detection method at ACE, active with the possible background of estimating these albumen.
| Yolk | Yolk-D30 | Yolk-D120 | Yolk+Alc | Y+Alc-D30 | Y+Alc-D120 | Concentration (mg/ml) |
| -7.8 | 37.5 | 45.0 | 33.1 | 400 | 44.0 | 0.500 |
| 3.1 | 12.2 | 16.0 | 17.6 | 20.7 | 21.6 | 0.167 |
| 8.3 | 9.0 | 5.9 | 9.5 | 7.3 | 8.1 | 0.056 |
| 10.1 | 1.8 | -5.1 | 13.3 | -1.2 | 2.7 | 0.019 |
| Egg white | EW-D30 | EW-D120 | EW+Alc | EW+Alc-D30 | EW+Alc-D120 | Concentration (mg/ml) |
| 7.9 | 43.6 | 48.9 | 56.8 | 46.7 | 56.9 | 0.500 |
| 4.2 | 17.9 | 20.6 | 28.4 | 25.6 | 27.8 | 0.167 |
| 10.2 | 7.3 | 9.5 | 21.7 | 11.3 | 20.3 | 0.056 |
| 4.6 | 4.0 | -1.0 | 11.8 | 4.6 | 9.9 | 0.019 |
| LysUM | LysD30 | LysD120 | Lys+Alc | Lys+AD30 | Lys+AD120 | Concentration (mg/ml) |
| 2.6 | 71.1 | 78.2 | 78.2 | 79.1 | 80.5 | 0.500 |
| -4.7 | 55.0 | 61.4 | 50.4 | 58.8 | 56.9 | 0.167 |
| 1.3 | 32.1 | 40.4 | 32.6 | 31.3 | 32.4 | 0.056 |
| 0.1 | 16.5 | 17.9 | 24.5 | 14.8 | 16.5 | 0.019 |
| Lys?UM | Lys?D30 | Lys?D120 | Lys+PEM | Lys+PD30 | Lys+PD120 | Concentration (mg/ml) |
| 2.6 | 71.1 | 78.2 | 79.1 | 74.2 | 76.3 | 0.500 |
| -4.7 | 55.0 | 61.4 | 59.0 | 54.7 | 59.3 | 0.167 |
| 1.3 | 32.1 | 40.4 | 38.1 | 32.4 | 39.4 | 0.056 |
| 0.1 | 16.5 | 17.9 | 18.8 | 20.7 | 22.0 | 0.019 |
| OT-UM | OT-D30 | OT-D120 | OT+Pr258P | OT+P-D30 | OT+P-D120 | Concentration (mg/ml) |
| -0.3 | 53.6 | 68.4 | 73.6 | 74.4 | 75.6 | 0.500 |
| 0.7 | 32.7 | 44.5 | 50.7 | 48.3 | 51.3 | 0.167 |
| -4.1 | 12.1 | 12.4 | 28.1 | 304 | 31.0 | 0.056 |
| 0.7 | 7.6 | 7.0 | 15.6 | 10.3 | 12.2 | 0.019 |
| OM-UM | OM-D30 | OM-D120 | OM+Alc | OM+A-D30 | OM+A-D120 | Concentration (mg/ml) |
| 2.1 | 47.1 | 51.8 | 16.0 | 50.0 | 54.3 | 0.500 |
| 2.2 | 21.5 | 26.5 | 7.7 | 23.3 | 30.0 | 0.167 |
| 5.2 | 8.2 | 9.6 | -0.3 | 9.6 | 15.0 | 0.056 |
| -0.4 | 4.0 | 2.1 | 2.5 | 5.6 | 2.3 | 0.019 |
From on can reach a conclusion, promptly initial (unhydrolysed and indigested) albumen (row 1) does not have ACE and suppresses activity.
The ACE of the hydrolysate after the ACE through the hydrolysate of pepsin after 30 minutes suppresses active and handles 120 minutes suppresses do not have too many difference between the activity, and the time of staying in this explanation stomach maybe not can influence the performance of protolysate.
Embodiment 6: the comparison of egg white powder and fresh feed
The purpose of this test is to confirm whether the egg white powder has formed the hydrolysate with inhibition activity different with fresh egg white raw material.The egg white powder of commercialization acquisition and the egg white of prepared fresh are compared, and tested ACE and suppressed active, so that the relatively influence of freshness.
Testing conditions is:
● test number (TN): 3 times
● the EW1101 product that commercialization obtains is a product of removing lysozyme, and for this test, (BeIo vo) carries out enrichment with 3.4% lysozyme
● contrast: without pepsin/chymotrypsin/trypsase digestion (only using the alcalase hydrolysis)
● the pepsin digestion of D30/D120:30 '/120 ', afterwards with α chymotrypsin/trypsase in pH6.5,37 ℃ digestion 2.5 hours down
Determine ACE and suppress active (IC50mg/ml):
The result shows that egg white powder of doing and fresh egg white all have significant ACE and suppress active.
Embodiment 7: the soluble fraction of hydrolysate and the activity of solid fraction
Purpose is to confirm that the soluble fraction of hydrolysate and the ACE that the insolubility level is divided suppress active.So far, with alcalase hydrolysis egg white, lysozyme and ovomucin, and through centrifugal (15 minutes, 4,500xg) the formed hydrolysate of classification.Difference freeze-drying sediment and supernatant level branch.
According to dry weight, divide sediment and supernatant level branch as follows:
| Hydrolysate | Deposition (%) | Supernatant (%) |
| EW/Alcalase | 15.215.9 | 84.884.1 |
| Lysozyme/Alcalase | 1.51.4 | 98.598.6 |
| Ovomucin/Alcalase | 27.224.3 | 72.875.7 |
Determine ACE and suppress active, and its activity with the all-hydrolytic thing of the direct freeze-drying of quilt after hydrolysis is compared.
1: the marker (the egg white 1101/Alcalase that on September 25th, 2003 generated, 0.5mg/ml solution) of in every plate, all measuring the egg white sample
| The all-hydrolytic thing 1 | EW/Alcalase | Lysozyme/Alcalase | Ovomucin/Alcalase |
| IC50(mg/ml) | 0.58 | 0.12 | 0.32 |
1: not centrifugal
In two kinds of situation (being EW1101 and ovomucin), the ACE that has caused supernatant that separates of sediment and supernatant suppresses active increase.But with the difference of whole, not centrifugal hydrolysate less than 2 times.
Shown in condition under, after external digestion simulation, also can from sediment, discharge the main ACE that the insolubility level of ovomucin/alcalase hydrolysate divides and suppress activity:
If possible, the activity that sediment had should be contained in the whole last food.
Embodiment 8: in vivo studies
8.1 first in vivo studies
8.1.1 large-scale first hydrolysate preparation
In order to obtain to be used for the large batch of hydrolysate of in vivo studies, carry out the hydrolysis of 20 to 25 gram scales.Prepare following hydrolysate:
1. lysozyme/Alcalase (Lys+Alc)
2. lysozyme/PEM (Lys+PEM)
3. ovomucin/Alcalase (OM+Alc)
4. ovotransferrins/Promod258P (OT+Pr258P)
5. yolk/Alcalase (Y+Alc)
6. egg white/Alcalase (EW+Alc)
The ACE that has tested these extensive hydrolysates suppresses active, the small-scale hydrolysate of test before its demonstration is similar to very much:
8.1.2 second large-scale experiment
Carry out another large-scale experiment, be used for the hydrolysate of studying in the SHR body with generation.Provided in the table below about various batches of ACE and suppressed active general introduction with sample.The retest sample.
1Reference: the marker (the egg white 1101/Alcalase that on September 25th, 2003 generated, 0.5mg/ml solution) of in every plate, all measuring the egg white sample
Batch: batch
From top table, be clearly shown that the high-caliber repeatability of the IC50 value of particular sample, the operation that the hydrolysis scheme is described, the further processed of hydrolyzation sample and ACE is suppressed to detect all is standardized and accurately.
8.2SHR body build-in test and result
In order to study the effect of hydrolysate, carried out SHR research to blood pressure.Studied the elementary effect of blood pressure and the secondary action that organ injury is taken place.SHR has the mean arterial blood pressure (MAPB) of about 190mmHg, and it exceeds normal arterial pressure far away.
Through the drinking water oral SHR mouse nursing hydrolysate of giving every day.52 mouse of hydrolysate treatment with per kilogram of body weight 1000mg every day.Tested four kinds of hydrolysates:
Hydrolysate A: lysozyme-alcalase
Hydrolysate B: ovotransferrins-newlase F
Hydrolysate C: ovomucin-alcalase
Hydrolysate D: ovalbumin-alcalase
Measure blood pressures with two kinds of methods, promptly the cuff method (measure systolic blood pressure down in anesthesia down or in anesthesia, SBP) and the remote sensing determination method, blood pressure determination value of sensoring acquisition in per 5 minutes through implantation wherein.The contrast mouse is accepted placebo.Radio remote sensing determination techniques makes arterial pressure, heart rate and physical exertion and their rhythm of the heart and them of the unrestricted animal of long-time continuous ground monitoring become possibility to replying of treatment.
8.2.1 cuff method result
The SBP of the anesthesia mouse of feeding through hydrolysate A has been reduced to 183.8mmHg at the 65th day from 201.7, blood pressure drops about 10%.Therefore, this explains that hydrolysate A is effective in vivo at least.For other three kinds of hydrolysates, test also well afoot.For the ACE inhibitory action, the ACE that has also measured in the tissue (non-blood plasma) is active.
8.2.2 remote sensing determination method
Lysozyme-alcalase and ovomucin-alcalase hydrolysate has all demonstrated significant blood pressure reduction effect in vivo.
The remote sensing of clear-headed unrestricted animal is measured
At first, SBP every day of all TM mouse is all along with the time increases to some extent, with desired the same in not treating the SHR mouse.But, 5 whens week behind begin treatment, accept hydrolysate A the TM mouse every day SBP single pattern begin to demonstrate lasting decline.Accept that the decline of SBP of TM mouse of hydrolysate A is the longest to have lasted till 7 weeks behind the begin treatment that it becomes stable afterwards.After 8 weeks of treatment, the SBP of the remote sensing mouse of treating through hydrolysate A continues to have reduced nearly 10%.
8.2.3 the acute treatment effect of lysozyme/alcalase in anesthesia SHR
This research is used to set up blood pressure and the Plasma ACE active effect of lysozyme/alcalase acute oral treatment to the anesthesia mouse.
Method
Rat: SHR mouse (382-440g) is through Harlan, and Horst, The Netherlands be available from Harlan, Oxon, United Kingdom.
Stable breeding: mouse by stable breeding in groups in the center Animal Lab. (UMCG Groningen), and allows that it conformed for 1 week.
Operation: testing the same day, with mouse anesthesia, row arteria carotis is put pipe so that measure blood pressure.In addition, going jugular vein puts pipe so that allow that getting blood carries out that Plasma ACE is active to be detected.
Scheme: when time point t=0, utilize the mode of stomach tube administered through oral nasal feeding to give the mouse study drug-administration.Before the t=0 nasal feeding at once and after t=0.5, t=1, t=2, t=3, t=4, t=5 and t=6 the time blood sampling.All note blood pressure (per 10 minutes mean value) in whole 6 hours research phase.
Research medicine: use lysozyme/alcalase or lysozyme (be 1000mg/kg all, be dissolved in the 1ml drinking water) or application of water only to mouse at random.
The result:
Blood pressure: the research medicine is calculated as the baseline blood pressure percentage change in the single mouse body to the effect of blood pressure.Lysozyme/the alcalase of Orally administered single dosage (hydrolysate A) has caused the significant and temporary transient decline of systolic blood pressure (SBP).The maximum of SBP descends and has reached nearly 35%, and it reached after using lysozyme/alcalase in 1.5-2 hour, and kept blood pressure again and reduced nearly 2 hours.Afterwards, after beginning nearly t=4 hour, SBP began to raise once more, when terminal point t=6 hour of research phase, has reached 20% decline.Using lysozyme (contrast) afterwards, seen closely similar pattern, but the maximum of SBP descends littler (nearly 15%).We have also studied the effect of application of water in these experiments, and after application of water, SBP also descends to some extent, only use lysozyme (promptly reply and be positioned between the two) but maximum descends less than lysozyme/alcalase and slightly is higher than.But the SBP changing pattern that application of water obtained obviously is different from the above two, and is more of short duration specifically, and after 3 hours, SBP has returned to baseline values (promptly 100%) fully in application of water 2.Maybe the better contrast of lysozyme/alcalase than water only with lysozyme.
Plasma ACE is active: the pattern that lysozyme/alcalase (hydrolysate A) and lysozyme (contrast) the Plasma ACE activity pattern afterwards of using is in close proximity in SBP to be seen.Promptly when using t=2 behind lysozyme/alcalase to t=3, it is nearly 30% that the active maximum of Plasma ACE has reduced, and it begins to raise once more afterwards, in the time of t=6 hour, has reached nearly 95% of baseline value.On the contrary, the active largest inhibition of using behind the lysozyme of Plasma ACE is approximately 10%.
Top discovery has clearly confirmed the ability of lysozyme/alcalase to the acute blood pressure reduction of anesthesia SHR mouse; The active synchronous reduction of Plasma ACE explains that this blood pressure reduction effect is because of the inhibition to RAS.Because the number of the mouse that this research institute comprises is quite few, and we hope preferably this effect of checking in clear-headed animal, and we utilize the TM determination method to design the 3rd experimental study (therefore, the TM method can not resemble the very anxiety of clear-headed animal that makes of TC).
8.2.4 the acute treatment effect of lysozyme/alcalase in clear-headed SHR
Carry out this research and treat blood pressure and the active effect of Plasma ACE clear-headed SHR to set up with the acute oral of lysozyme/alcalase.
Method
Rat: SHR mouse (388-515g) is through Harlan, and Horst, The Netherlands be available from Harlan, Oxon, United Kingdom, and be housed in groups the center Animal Lab. (UMCG, Groningen).
Radio remote sensing determination method: the SHR mouse (8) undergo surgery, with the pre ure tra ducer device (TM) of settling remote sensing mensuration and the permanent catheter that is used to get blood.TM is implanted in the arteria coeliaca, and far-end arrives the left side arteria renalis.Permanent catheter is implanted in the jugular vein, and its other end arrives the head of mouse through hypodermic tunnel, and is fixed in wherein.Then, single stable breeding mouse, and allow and recovered for 2 weeks, begin to collect radio remote sensing determination data afterwards, and last till the whole research phase.
Scheme: mouse is assigned with acceptance treatment plan at random then: the lysozyme/alcalase (LHA) of the single dose of three different times of 3 different tests days, lysozyme (L) or water (H2O), during at interval (flushing/contrast) in 2 to 3 days.Lysozyme/alcalase and lysozyme (every kind all is 1000mg/kg) are dissolved in the water, and through stomach tube Orally administered (cumulative volume is nearly 1ml solution); Use 1ml water only for the contrast mouse.All between 9 o'clock to the 10 o'clock morning of testing day, carry out nasal feeding always.
Rat1 Rat2 Rat3 Rat4 Rat5 Rat6 Rat7 Rat8
Day1(22-11-2004)： LHA ?L ?H2O ?LHA ?L ?H2O ?LHA ?L
Day2(25-11-2004)： H2O ?LHA ?L ?L ?H2O ?LHA ?H2O ?LHA
Day3(29-11-2004)： L ?H2O ?LHA ?H2O ?LHA ?L ?L ?H2O
Get blood: on the same day of oral medication, before the nasal feeding nearly 0.5 to 1.0 hours, permanent catheter is connected with extension tube attached, get blood to allow, and handle mouse no longer further.(t=0) at once before nasal feeding and the t=0.5 after it, take out blood sample 1,2,3,4,5 and 6 hour the time.Subsequently, with blood sample at 4 ℃, under the 1600g centrifugal 10 minutes, with its quick-frozen and be stored under-80 ℃ up to detection.
Plasma ACE is active: the ACE with the Hip-His-Leu method is determined in the blood plasma tissue is active, foregoing that kind.Briefly, with plasma sample and ACE substrate Hippury-His-Leu (12.5nM) incubation 10 minutes just in time under 37 ℃.Through adding the conversion of 280mM NaOH termination substrate.After this, the His-Leu that adds 100 μ l phtaldialdehyde marked frees.Respectively excitation wavelength and emission wavelength be 364 with the condition of 486nm under, determine the amount of the His-Leu fluorescence of institute's mark.Comprise control sample, wherein before adding substrate Hippuryl-His-Leu, added NaOH to stop the conversion of substrate.
Analysis to blood pressure data: the relevant time period is generally thought to test the nasal feeding of day and was arrived afterwards 10 hours in preceding 1 hour, promptly from 8 o'clock to 19 o'clock.Every mouse has all obtained these time periods of 3 tests day, utilizes the data computation of all mouse to go out the mean value of certain specific therapy then.Every mouse also obtained before the test day and between the data of identical time period of 6 non-tests (contrast) days, draw the mean value of all mouse then.
The result
Compare with H2O, in 1 hour, just observed LHA and reduced the phenomenon of SBP, and kept this reduction at least 4 hours.Also observed the phenomenon of L reduction SBP, although this has postponed 1 hour than LHA.This possibly be the result to the natural digestion process of L, and the quite high dosage of this requirement, but the LHA of this illustrative less amount is the same with it effective.
8.2.4.1 ovomucin/alcalase is to the acute treatment effect of clear-headed SHR
Method
Rat: the mouse that the remote sensing above using among the 8.2.4 is measured, except natural death during the mouse.In test day, with or without ovomucin/alcalase hydrolysate (1000mg/kg) treatment mouse (n=7), said hydrolysate mixes with 3ml G/W (10%), and (nearly 10 points) are it with flat disc and pass mouse (30 minutes) in the morning; In contrast day, treatment includes only G/W, and between contrast treatment and the active treatment is 2 days at interval.
The analysis of blood pressure data: the relevant time period is generally thought to test the nasal feeding of day and was arrived afterwards 8 hours in preceding 1 hour, promptly from 8 o'clock to 17 o'clock.Every mouse has all obtained these time periods of 2 test days, utilizes the data computation of all mouse to go out the mean value of ovomucin treatment or contrast treatment then.Every mouse also obtained before the test day and between the data of identical time period of 4 non-tests (contrast) days, draw the mean value of all mouse then.
The result
The blood pressure data that a SHR mouse is arranged is extremely low, in further analyzing, has got rid of these data.In test day, most mouse have all eaten the 3ml G/W in 10 minutes.Drink all increasing of G/W with systolic blood pressure; Be this with research in the above in the viewed similar phenomena through stomach tube picked-up back, although the degree (being similar to 25mmHg) that the degree that the SBP after glucose is taken in increases (approximate 10mmHg) increases than the SBP after the oral absorption of stomach tube is lower.To this, when not treating, Japan and China do not observe this effect in contrast.Compare with the contrast treatment that only gives G/W, the active treatment that contains the G/W of ovomucin/alcalase hydrolysate has caused descending faster and more significantly of SBP.Ovomucin/alcalase hydrolysate obtained maximum decline (near 15mmHg) in back 1 hour in absorption, therefore, this with research in the above in clear-headed mouse in the viewed time period closely similar.
Embodiment 9: through membrane filtration enrichment hydrolysate
Whether can realize the enrichment of little peptide and the ACE that this caused are suppressed active increase in order to study, with the ovalbumin (ovotransferrins and lysozyme) of membrane filtration (utilizing the Polyethersulfon milipore filter of bore) two kinds of hydrolysis of classification for 2kD and 10kD.
In the Amicon determination unit, the polyethersulfon milipore filter is used as flat film.Volume is that the 5% protolysate solution of 15ml has been condensed to 5ml (15%), and adds 5ml water.Once more solution concentration is become 5ml (washing step).Repeated washing step 2 time again.Merge the penetrating fluid of inceptive filtering and the penetrating fluid of washing step, and freeze-drying.The penetrating fluid of freeze-drying is stored in 4 ℃, and tests its ACE and suppress active.
Following this test of setting:
-two kinds of hydrolysate: ovotransferrins/promod258 and lysozyme/alcalase
-two kinds of films: a kind of bore is 2kD, and alternative bore is 10kD
-after classification, need not or with the various samples of external digestion simulation process (raw material, penetrating fluid and residue), wherein with the time varying length of pepsin incubation (simulated gastric in hydrolysis), between 0 to 120 minute.
-carry out the pepsin incubation for 2,37 ℃ in the pH value.
-6.5,37 ℃ of pH values down with the incubation of trypsase/α chymotrypsin 2.5 hours.
After the freeze-drying sample, determine IC50 (mg/ml) value of branches at different levels:
Divide relatively with the raw material level, the ACE that the penetrating fluid level is divided suppresses active maximum has increased by 2 times.For ovotransferrins, observed this increase with the 10kD film, and be to use the 2kD film for lysozyme.
This embodiment shows can use membrane filtration enriched biological active peptide, and increases the ACE inhibition activity of hydrolysate.Can through enrichment be reduced to the realization effect essential rule (for example every day) use the amount of the material of consumption, and be integrated into easily in food supplement and the functional food because the minimizing of volume makes.
Embodiment 10: the storage time of ovum and temperature suppress active effect to ACE
Store the ovum research storage time and the Influence of Temperature in 6 weeks with the ovum (isolate egg white and yolk, use alcalase hydrolysis egg white level branch subsequently) of direct processed processing or under 4 ℃ or room temperature.
-with alcalase hydrolysis a) 3 hours or b) 5 hours (after 3 hours, adding extra enzyme)
After hydrolysis, branches at different levels are by freeze-drying, and powder level divides storage at room temperature.The ACE that in identical detection method, determines all each grade branches suppresses active.Make an experiment 2 times.
The storage time of ovum and temperature suppress active effect to ovalbumin ACE:
1: room temperature
The hydrolysis of 5 hours hydrolysis than 3 hours generated to have better and the hydrolysate of the activity of higher repeatability.
Test shows, suppresses the optimum activity of active aspect for the ACE of ovalbumin hydrolysate, and ovum can directly be used, and perhaps can under 4 ℃ or room temperature, store at least 6 weeks.
Embodiment 11: the bitter taste of hydrolysate
In order to estimate bitter taste, analyze hydrolysate by three volunteers with feeling.With the hydrolysate powder dissolution in water (15-20mg/ml) or water and glucose.
| Hydrolysate | Water | Water+0.44% glucose | Water+0.88% glucose |
| Ovotransferrins-newlaseF | Bitter | Bitter | Bitter |
| Ovomucin-alcalase | Middle | ||
| Egg white-alcalase | Hardship/acid | Bitter taste is lighter | Bitter taste is light (identical with 0.44% glucose) |
| Lysozyme-alcalase | Middle |
Two kinds of hydrolysates are bitter, and in addition two kinds do not have bitter taste.Can cover the bitter taste of egg white-alcalase slightly through adding glucose.For a large amount of hydrolysates, in food, need adding flavor enhancement or other taste to cover compound, for example maltodextrin, aspartame, inulin etc.Taste can not influence the desire of the edible hydrolysate of mouse.
Embodiment 12: food supplement and food
Tablet or wafer
The tablet of forming below preparation has (or wafer):
-each tablet contains 250mg target protein hydrolysate (about 60% tablet volume)
-40% volume comprises cornstarch (20%), cellulose (15%) polysaccharide 0.5%), lubricant and polishing material (3%), other (1.5%).
Embodiment 13: the molecular weight distribution of protolysate
(Amersham Biosciences, Uppsala Sweden), measure the molecular weight distribution (MWD) of protolysate with gel permeation chromatography (GPC) to utilize Superdex Peptide PE7.5/300 post.The MW separating ranges that this post has is 100 to 7000.In containing 30% acetonitrile of 0.1%TFA, run post then, condition is with identical with the condition of A&F analyzing proteins hydrolysate.Standard peptide with the known molecular amount is proofreaied and correct post (seeing the following form).Reference material and hydrolysate sample all are dissolved in the standard eluent, and peptide concentration is 5mg/ml.In injection (200 μ l) before, centrifugal sample is removed the insolubility particle.Keep watch on elution mode at the 214nm place.
Standard Mw
L-leucine 131.2
L-arginine 174.2
VVYV 478.5
VVYK 507.6
VVYR 535.6
Insulin chain B-oxidizing ferment 3,496
Press down enzyme peptide 6,500 from the ox lung
From the cromoci of cor bovinum, V-A type 12,327
Lysozyme .HCI 14,300
Do not demonstrate calibration curve.
The MWD of protolysate is represented as the percentage (%) of the peptide level branch with predetermined molecules weight range: less than 500Da, between 500 to 1000Da and greater than 1000Da.This is corresponding with the peptide with following length: less than 4 to 5 amino acid, 4 to 9 amino acid or greater than 9 amino acid.
The result:
| Hydrolysate | DH(％) | IC50(mg/mL) | ％<500Da | ％500-1000Da | ％>1000Da |
| Egg?White/Alcalase(LS250903) | 31.9 | 0.46 | 91.7 | 4.6 | 3.7 |
| Lysozyme/Alcalase(LS250903) | 34.5 | 0.11 | 53.4 | 16.7 | 29.9 |
| Lysozyme/Alcalase(Sup101104) | 34 | 0.15 | 89.4 | 0.9 | 9.7 |
| Lysozyme/PEM(LS250903) | 34 | 0.12 | 99.1 | 0.8 | 0.1 |
| Lysozyme/PEM(LS250903) | 34 | 0.12 | 99.2 | 0.7 | 0.0 |
| Ovomucin/Alcalase(170604) | 12 | 0.54 | 85.1 | 10.0 | 4.9 |
| Ovomucin/Alcalase(281004_1) | 21.7 | 0.39 | 99.1 | 0.9 | 0.0 |
| Ovomucin/Alcalase(281004_2) | 23.9 | 0.21 | 97.7 | 0.8 | 1.6 |
| Ovomucin/Alcalase(5ml5h2BB211004) | 5 | 0.76 | 69.7 | 16.4 | 13.9 |
| Ovomucin/Alcalase(LS250903) | 14.9 | 0.73 | 44.9 | 21.7 | 33.4 |
| Ovotransferrin/Promod258P(LS250903) | 27.3 | 0.15 | 70.2 | 10.7 | 19.1 |
The result shows, between the hydrolysis degree of target protein and IC50 activity, correlation arranged.Particularly, at least about 20%, preferred hydrolysis degree at least about 30% has generated has the hydrolysate that high ACE suppresses activity.
Embodiment 14: research in the human body body that carries out with the ovum peptide of blood pressure that can significantly reduce spontaneous hypertension rat
The composition that is used for the lysozyme hydrolysate of research in the human body body:
Determine the composition of the lysozyme/alcalase hydrolysate of ACE inhibition:
-90% lysozyme
-1.8％alcalase
-5％NaOH
-3.2% water
-lisozima is from Belovo (Schoterweg14,8462TD Rotstergaast): be a kind of less important albumen in the egg white that ion exchange chromatography is purified into, make it reach 99% purity with SDS-PAGE and HPLC chromatography.
-Alcalase Food Grade van Novozymes, Alcalase2.4L: limpid bronzing liquid; Enzyme concentration is the 2.3AU/ liter; Density is approximately 1.18g/ml; Alcalase originates from the selected bacterial strain of bacillus licheniformis; Be a kind of bacterialprotease and endopeptidase.
-NaOH pellets van Merc: in hydrolytic process, the pH value is adjusted to 8 with 33% solution.
For 3% lysozyme (100ml) solution, need approximate 500 μ l1M NaOH solution that the pH value was set at for 8 (the 3g lysozyme needs 500 μ l1M NaOH=0.02g NaOH).After adding enzyme, still need add NaOH is 8 to keep the pH value, therefore in end-product, can have minor N aOH.
The human research
Obtained the final permission of the METc (the Medical Ethics committee) of University Medical Center Groningen (UMCG) to the research of functional food in human body.
Volunteer (n=5) has accepted to contain the lysozyme/alcalase of single dose or the beverage of suitable placebo two different test days.Before absorption, measured blood pressure at once and afterwards the latest by 6 hours.In addition, before absorption at once and the specific time point after absorption get blood, carry out the ACE activity analysis.
Drink formula:
-20g hydrolysate
-40g Karvan Cevitan " red grape fruit "
-60g water
90 ℃ of following Bath Teng methods were sterilized 20 minutes.
Siliker (Ede) microbiologic analysis is OK.
The composition of the red grape fruit of Karvan Cevitan: 60% undyed fruit; The vitamin of 4 kinds of interpolations; 60% concentration of juices agent (red grape fruit, elder berry, cynarrhodion), wherein 54% is red grape fruit; Other component comprises sugar, glucose-fructose juice, food acid (citric acid), spices, vitamin, anticorrisive agent (potassium sorbate).
Control beverage: the same, but do not have hydrolysate.
The result
Test comprises 5 people that accept hydrolysate.After 2 weeks, 3 conducts in 5 contrast.With the mean blood pressure of having examined in 6 hours time periods.Compare with contrast, it is about 5 to 6% that blood pressure on average descends, and demonstrated the obvious effect of hydrolysate.Within 1 hour behind the edible hydrolysate, also observe effect, and after 1.5 hours, reached maximum effect.Decline when the mean blood pressure when 3h and 6h (last time point of being measured) descends with 1.5h is suitable.Reach the peak value reduction in 1 hour that active research is presented at behind the edible hydrolysate to the average blood plasma ACE of hydrolysate, and after 3 hours, returned to base level.
The human research is still underway.
Claims (9)
1. food or food supplement; It comprises the Alcalase hydrolysate of the lysozyme of effective dose; It is characterized in that when people that blood pressure increases takes in said food or food supplement said food or food supplement stop blood pressure to raise or bring high blood pressure down significantly.
2. the enzymatic proteolysis thing that obtains of the Alcalase hydrolysis through lysozyme is characterized in that said hydrolysate has antihypertensive active.
3. claim 1 or 2 food or food supplement or enzymatic proteolysis thing, wherein the ratio of dipeptides and/or tripeptides is at least 10% in the hydrolysate.
4. claim 1 or 2 food or food supplement or enzymatic proteolysis thing, wherein at least 30% hydrolysate is the peptide of molecular weight less than 0.5kD.
5. the protolysate of claim 2 has the food of antihypertensive active or the purposes in the food supplement in preparation.
6. be used to prepare the method for the protolysate with antihypertensive active, it comprises step:
(a) provide and comprise the liquid solution of the lysozyme of purifying basically;
(b) the pH value with said solution is adjusted to the pH value that is suitable for Alcalase; With
(c) in said solution, add Alcalase, and under suitable temperature one suitable period of incubation solution.
7. the method for claim 6 also comprises through the said solution of membrane filtration and obtains the step (d) of the penetrating fluid of the active target peptide of enrichment.
8. pass through the antihypertensive protolysate of the method acquisition of claim 6 or 7.
9. the food or the food supplement that comprise the anti-hypertension protolysate of claim 8.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US58990204P | 2004-07-22 | 2004-07-22 | |
| US60/589,902 | 2004-07-22 | ||
| EP05100544.5 | 2005-01-27 | ||
| EP05100544A EP1685764A1 (en) | 2005-01-27 | 2005-01-27 | Anti-hypertensive functional food products |
| PCT/NL2005/000535 WO2006009448A1 (en) | 2004-07-22 | 2005-07-22 | Anti-hypertensive functional food products |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101022737A CN101022737A (en) | 2007-08-22 |
| CN101022737B true CN101022737B (en) | 2012-08-15 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2005800317561A Active CN101022737B (en) | 2004-07-22 | 2005-07-22 | Anti-hypertensive functional food products |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102076228B (en) * | 2008-04-29 | 2016-08-03 | 帝斯曼知识产权资产管理有限公司 | The tryptophan being combined with peptide and the mixture of the tryptophan being combined with polypeptide |
| CN105637096A (en) * | 2013-10-04 | 2016-06-01 | 英诺威有限公司 | Hydrolysate of animal protein, manufacturing method thereof and use thereof |
| CN107205432A (en) * | 2014-11-11 | 2017-09-26 | 克莱拉食品公司 | Method and composition for generating ovalbumin |
| CN107337710A (en) * | 2017-07-26 | 2017-11-10 | 盐城卫生职业技术学院 | A kind of antihypertensive active peptide The The Pro and application and pharmaceutical composition |
| CN108864279B (en) * | 2018-07-24 | 2021-06-25 | 渤海大学 | ACE inhibitory peptide |
| BR112022000545A2 (en) | 2019-07-11 | 2022-04-05 | Clara Foods Co | Protein compositions and consumer products of these |
| US10927360B1 (en) | 2019-08-07 | 2021-02-23 | Clara Foods Co. | Compositions comprising digestive enzymes |
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