CN1298227C - Effective sterilizing method capable of keeping colostrum immune globulin activity - Google Patents
Effective sterilizing method capable of keeping colostrum immune globulin activity Download PDFInfo
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- CN1298227C CN1298227C CNB2004100993326A CN200410099332A CN1298227C CN 1298227 C CN1298227 C CN 1298227C CN B2004100993326 A CNB2004100993326 A CN B2004100993326A CN 200410099332 A CN200410099332 A CN 200410099332A CN 1298227 C CN1298227 C CN 1298227C
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Abstract
The present invention relates to an effective sterilizing method with the capability of keeping the activity of colostrum immunoglobulin. In the method, colostrum powders of mammals including cattle, sheep, horses, etc., which contain immunoglobulin, pure colostrum immunoglobulin dry powders and physiological saline are first proportionally dissolved; or cold ethanol is added after indissoluble particles in colostrum whey fluid of cattle, sheep, horses, etc. are removed, and the final sterilizing concentration of ethanol ranges from 70 to 75%. The centrifugation removal of supernatant fluid containing ethanol is realized, and precipitates at the bottom are reserved. The precipitates are stirred and dissolved through sterile physiological saline or high-temperature sterilizing emulsions, and the original volume is recovered so that sterile immunoglobulin fluid is prepared. The sterile immunoglobulin fluid can be prepared into liquid milk with immunological activity and can also be dried at a low temperature so as to form sterile dry powders. The method can keep the immunological activity of colostrum and has the advantages of thorough sterilization, low cost and suitability for scale production.
Description
Technical field
The present invention relates to a kind of sterilizing methods that can keep the beestings immune globulin activity.
Background technology
As everyone knows, mammal milk has the immunity of organism regulating action, and immunoglobulin (Ig) wherein (Ig) can directly neutralize a toxin, and suppresses the breeding of pathogen microorganism.A large amount of experimental datas prove that the immunoglobulin (Ig) in a certain animal milk not only is the young baby of self, also can be other different types of animal the passive immunity protection is provided.Various on this basis is that the health products of main active substances arise at the historic moment with the immunoglobulin (Ig).Usually in lactation period, the immunoglobulin content in colostrum stage is the highest.In the colostrum application process, if can solve the biologically active that the sterilization problem can keep colostrum immune globulin again, this has very significance to the health-care effect of giving full play to colostrum immune globulin.
In the animal lactation, exist IgA, IgG and IgM three major types immunoglobulin (Ig), their molecular weight is generally bigger, and the molecular weight of IgA is about 420000, and IgG is about 160000, and IgM about 900000.Its basic structure is identical by two, the bigger heavy chain of relative molecular mass with other two identical, the less light chain of relative molecular mass is formed, these polypeptide chains link together by-S-S-key.In addition, also have intrachain disulfide bond in every chain of immunoglobulin (Ig), be linked into several closed-loop structure, formation stable region and the variable region relevant with the conjugated antigen specificity with every.The specific molecule structures shape that these are complicated the unstability of immunoglobulin (Ig).High temperature and oxidant all can cause the irreversible albuminous degeneration of immunoglobulin (Ig).
Sterilization to dairy products adopts high temperature moment and two kinds of methods of Pasteur usually at present, and the former sterilizes thoroughly, and the immune globulin activity in Ruzhong is lost easily, though sterilization is not thorough but latter's retaining part is active.Other method, as: the miillpore filter that 0.3 μ is following, though also can remove microorganism, it is extremely low that the filter membrane micropore easily stops up production efficiency; The isotopic irradiation sterilization is also very fatal to the oxidative damage of protein.So immune liquid milk popularity rate in the market is very low.
Summary of the invention
The purpose of this invention is to provide a kind of colostrum sterilization method quick, effective, that easily go, not only keep the activity of immunoglobulin (Ig) but also reach thorough sterilization effect, suitable colostrum immune globulin is at the bread and cheese especially application in liquid milk.
Ethanol is a kind of surface disinfection agent commonly used.Experiment according to the inventor finds that under certain conditions, ethanol can thoroughly be killed the microorganism in Ruzhong just, and keeps the biologically active of former Ruzhong immunoglobulin (Ig) substantially.The ethanol of sterilization usefulness belongs to edible or medical grade, and the ethanol residual quantity is extremely low after the sterilization treatment, and is safe in utilization.
Technical scheme of the present invention is:
According to a first aspect of the invention, a kind of effective sterilizing method that can keep the colostrum powder immune globulin activity, its step comprises:
(1) colostrum powder that meets food standard is carried out immunoglobulin content and measures, and get content and be at least 3% colostrum powder as the material powder,
(2) to the material powder got with physiological saline solution, the addition of physiological saline is at least 5 milliliters/gram dry powder, makes colostric fluid,
(3) to described colostric fluid, remove tangible particle and sediment with centrifugal or filter method, make processing whey liquid just,
(4) just whey liquid is according to 95% medical grade or the food grade ethanol of original volume calculating adding precooling to described processing, and the final valid density of the ethanol of sample sterilization treatment is 70～75%, makes milk alcohol liquid sample just,
(5) the first milk alcohol liquid sample that will add behind the ethanol is kept at below 15 ℃, and leaves standstill at least 5 minutes,
(6) with incline ethanol liquid in the first milk alcohol liquid sample of centrifugal process, collect sterilized immunoglobulin deposit thing,
(7) described immunoglobulin deposit thing made with SPSS or sterilization emulsion dissolving contained the immunoglobulin (Ig) sterilization preparation, its pH between 6.0～7.2,
(8) the described immunoglobulin (Ig) sterilization preparation that contains is made sterile dry with cold drying, or be mixed and made into the liquid milk that contains immunoglobulin (Ig) with high-temperature sterilization emulsion desired proportions.
More specifically, the pre-cooled ethanol that adds in the described step (4), its precooling temperature is up to 4 ℃;
The colostrum powder that meets food standard in the described step (1) is bovine colostrum or sheep colostrum powder or horse colostrum powder.
According to a second aspect of the invention, a kind of effective sterilizing method that can keep the colostrum whey immune globulin activity, its step comprises:
(I) get colostrum whey that immunoglobulin content in 7 days branch puerperiums is at least/100 milliliters of 1 grams as colostrum whey material liquid,
(II) colostrum whey material liquid is removed tangible particle and sediment with centrifugal or filter method, makes processing whey liquid just,
(III) just whey liquid is according to 95% medical grade or the food grade ethanol of original volume calculating adding precooling to described processing, and the final valid density of the ethanol of sample treatment is 70～75%, makes milk alcohol liquid sample just,
(IV) will add first milk alcohol liquid sample behind the ethanol, and remain on below 15 ℃, and left standstill at least 5 minutes,
(V) with incline ethanol liquid in the first milk alcohol liquid sample of centrifugal process, collect the sterilized immunoglobulin deposit thing that contains,
(VI) the described immunoglobulin deposit thing that contains is made with SPSS or sterilization emulsion dissolving and is contained the immunoglobulin (Ig) sterilization preparation, its pH between 6.0～7.2,
(VII) the described immunoglobulin (Ig) sterilization preparation that contains is made sterilization dry powder with cold drying, or be mixed and made into the liquid milk that contains immunoglobulin (Ig) with high-temperature sterilization emulsion desired proportions.
More specifically, the pre-cooled ethanol that adds in described step (III) is limited to 4 ℃ on its precooling temperature;
Described in the described step (I) to get the colostrum whey of childbirth in 7 days be colostrum whey or sheep colostrum whey or horse colostrum whey.
According to a third aspect of the present invention, a kind of effective ways that can keep pure colostrum immune globulin powder activity, its step comprises:
(a) to pure colostrum immune globulin powder with physiological saline solution, the addition of physiological saline is at least 5 milliliters/gram dry powder, forms the colostric fluid of dissolving,
(b) colostric fluid that will dissolve is removed tangible particle and sediment with centrifugal or filter method, makes processing whey liquid just,
(c) original volume according to the first whey liquid of processing calculates 95% medical grade or the food grade ethanol that adds precooling, and the final valid density of the ethanol of sample sterilization treatment is 70～75%, makes milk alcohol liquid sample just,
(d) the first milk alcohol liquid sample that will add behind the ethanol keeps below 15 ℃, and leaves standstill at least 5 minutes,
(e) follow, the ethanol liquid at the beginning of inclining with centrifugal process in the milk alcohol liquid sample, collecting has sterilized contains the immunoglobulin deposit thing,
(f) the described immunoglobulin deposit thing that contains is made with SPSS or sterilization emulsion dissolving and is contained the immunoglobulin (Ig) sterilization preparation, its pH between 6.0～7.2,
(g) with cold drying the immunoglobulin (Ig) sterilization preparation is made sterilization dry powder again, or be mixed and made into the liquid milk that contains immunoglobulin (Ig) with high-temperature sterilization emulsion desired proportions.
More specifically, in described step (c), the pre-cooled ethanol of adding is limited to 4 ℃ on its precooling temperature;
At the pure colostrum immune globulin powder described in the described step (a) is pure bovine colostrum immunoglobulin powder or pure horse colostrum immune globulin powder or pure sheep colostrum immune globulin powder.
According to a fourth aspect of the present invention, the sterilization dry powder that above-mentioned first and second and the 3rd described sterilizing methods in aspect of the present invention are made or contain the immunoglobulin (Ig) sterilization preparation can be used as the active immne hardening agent and makes an addition in the food.
Advantage of the present invention is: 1, adopt the ethanol sterilization can effectively kill wherein microorganism to colostric fluid, and former immunoglobulin (Ig) biologically active does not change; 2, disinfectant adopts food grade or medical grade ethanol, has no side effect, and sterilization back sample ethanol residual quantity is low, safe in utilization; 3, the ethanol after this law sterilization, recyclable utilization again reduces production costs; 4, after this method sterilization, the invalid components in first Ruzhong such as salt, monose, aliphatic acid equal size obviously reduce.
Description of drawings
Fig. 1 is the sephadex-G200 column chromatography analysis demonstration figure according to the embodiment of the invention 1 immunoglobulin (Ig) before sterilization treatment;
Fig. 2 is the sephadex-G200 column chromatography analysis demonstration figure according to inventive embodiments 1 immunoglobulin (Ig) after sterilization treatment;
Fig. 3 is the sephadex-G200 column chromatography analysis demonstration figure according to the embodiment of the invention 4 contrast breasts;
Fig. 4 is the sephadex-G200 column chromatography analysis demonstration figure according to the immune milk of the embodiment of the invention 4.
The specific embodiment
Enumerate four embodiment of the present invention below, and described in detail respectively, so that ins and outs of the present invention further are provided.
Get prediction and contain 1.000 kilograms in the colostrum of 80% immunoglobulin (Ig) or sheep colostrum or horse colostrum dry powder, add 15 liters of physiological saline, or every at least gram dry powder adds 5 ml physiological salines and stir that solution filters through 200 mesh filter screens after 15 minutes, and is standby under 10 ℃ of left and right sides conditions of sample.Other is taken at 52 liters of the 95% concentration food grade ethanol of-10 ℃～-20 ℃ of freezer precoolings, and the final concentration of alcohol of solution is slowly joined in the colostric fluid while stirring in 70%～75% scope, leaves standstill after finishing 5～10 minutes, and limit can be 4 ℃ on the pre-cooled ethanol.Centrifugal under gnotobasis, 1000rpm, 5 minutes.The supernatant that inclines adds 50 liters of SPSSs in sediment, stirring and dissolving 20 minutes remains on and left standstill under 15 ℃ 30 minutes or at least 5 minutes.Get the supernatant low temperature drying and get dry powder.All do not detect through hygiene bacterial detection sum, mould, Escherichia coli, pathogenic bacteria.Colostrum dry powder immunoglobulin (Ig) is analyzed through double diffusion immune response and Sephadex-G200 column chromatography before and after the sample sterilization treatment, and demonstration immunoglobulin (Ig) composition and content are not lost, as depicted in figs. 1 and 2.
Embodiment 2
Get 1.56 kilograms of the bovine colostrums of prediction 3%～10% immunoglobulin (Ig), add 13 liters of physiological saline, stir centrifugal 3000rpm after 15 minutes, 10 minutes, abandon sediment.Supernatant is standby under 10 ℃ of left and right sides conditions after 200 mesh filter screens filter.Other is taken at 39 liters of the 95% concentration food grade ethanol of 4 ℃～-20 ℃ of freezer precoolings, slowly joins in the colostrum supernatant while stirring, leaves standstill after finishing 5 minutes.Centrifugal under gnotobasis, 1000rpm, 5 minutes.The supernatant that inclines adds 50 liters of sterile salines in sediment, stirring and dissolving 20 minutes leaves standstill the supernatant of getting these 50 liters of sterilizations at least after 30 minutes 5 minutes and the high-temperature sterilization breast of 3070 liters of coolings and mixes.Automatic subpackaging has 6240 boxes, 500 milliliters in every box.The immunity milk of the bovine colostrum before the sterilization and the back preparation of sterilizing, immunology and the detection of hygiene index are done in placement in the time of 30 days under 7 ℃ of conditions, and the result is as follows:
| Immunoglobulin (Ig) | Total number of bacteria | Escherichia coli | Mould | |
| Colostrum powder | 0.1 gram/gram (dry powder) | 20,000/gram | 40/100 grams | 25/gram |
| Immunity milk | 25 milligrams/500 milliliters | Do not detect | Do not detect | Do not detect |
Embodiment 3
Get 1.56 kilograms of the sheep colostrum powders of prediction 10% or at least 3% immunoglobulin (Ig), sterilizing and be mixed with by the method for embodiment 2 contains sheep colostrum immune globulin liquid milk.This contains sheep colostrum immune globulin liquid milk placed 30 days under 7 ℃ of conditions, still contained immunocompetence, and total number of bacteria, Escherichia coli and mould all do not have and detect.
Embodiment 4
Collect 10 kilograms of the colostrums that cow or mare or ewe divide secretion on the same day in puerperium, low-temperature centrifugation 3000rpm, 30 minutes, degrease.Stir on the limit at ambient temperature, and the limit splashes into 2N HCl, and causing emulsion pH is 4.7.Second day, centrifugal 2000rpm, 10 minutes, abandon sediment, the supernatant of adjusting centrifugal collection with 2N NaOH is pH6.0, and filters through 200 mesh filter screens.Other is taken at 35 liters of the 95% concentration medical grade ethanol of-10～-15 ℃ of freezer precoolings, slowly adds in the colostric fluid while stirring, leaves standstill after finishing 5 minutes.Centrifugal under gnotobasis, 1000rpm, 5 minutes.The supernatant that inclines, the high-temperature sterilization breast that adds 100 liters of coolings in sediment mixes, and stirring and dissolving is after 20 minutes, and automatic subpackaging has 400 boxes, 250 milliliters in every box.Detect average every box through immunology, hygiene and contain 250 milligrams of immunoglobulin (Ig)s; Escherichia coli, total number of bacteria all do not detect.As Fig. 3 and Fig. 4, through the comparative analysis of sephadex-G200 column chromatography: immune milk, be distributed in the 10th and about pipe the immune globulin white peak appears containing; The contrast breast does not then have.
Claims (10)
1, a kind of effective sterilizing method that can keep the colostrum powder immune globulin activity, its step comprises:
(1) colostrum powder that meets food standard is carried out immunoglobulin content and measures, and get content and be at least 3% colostrum powder as the material powder,
(2) to the material powder got with physiological saline solution, the addition of physiological saline is at least 5 milliliters/gram dry powder, makes colostric fluid,
(3) to described colostric fluid, remove tangible particle and sediment with centrifugal or filter method, make processing whey liquid just,
(4) just whey liquid is according to 95% medical grade or the food grade ethanol of original volume calculating adding precooling to described processing, and the final valid density of the ethanol of sample sterilization treatment is 70～75%, makes milk alcohol liquid sample just,
(5) the first milk alcohol liquid sample that will add behind the ethanol is kept at below 15 ℃, and leaves standstill at least 5 minutes,
(6) with incline ethanol liquid in the first milk alcohol liquid sample of centrifugal process, collect sterilized immunoglobulin deposit thing,
(7) described immunoglobulin deposit thing made with SPSS or sterilization emulsion dissolving contained the immunoglobulin (Ig) sterilization preparation, its pH between 6.0～7.2,
(8) the described immunoglobulin (Ig) sterilization preparation that contains is made sterilization dry powder with cold drying, or be mixed and made into the liquid milk that contains immunoglobulin (Ig) with high-temperature sterilization emulsion desired proportions.
2, the effective sterilizing method that can keep the colostrum powder immune globulin activity according to claim 1 is characterized in that, the pre-cooled ethanol that adds in the described step (4), and its precooling temperature is up to 4 ℃.
3, the effective sterilizing method that can keep the colostrum powder immune globulin activity according to claim 1 is characterized in that, the colostrum powder that meets food standard in the described step (1) is bovine colostrum or sheep colostrum powder or horse colostrum powder.
4, a kind of effective sterilizing method that can keep the colostrum whey immune globulin activity, its step comprises:
(I) get colostrum whey that immunoglobulin content in 7 days branch puerperiums is at least/100 milliliters of 1 grams as colostrum whey material liquid,
(II) colostrum whey material liquid is removed tangible particle and sediment with centrifugal or filter method, makes processing whey liquid just,
(III) just whey liquid is according to 95% medical grade or the food grade ethanol of original volume calculating adding precooling to described processing, and the final valid density of the ethanol of sample treatment is 70～75%, makes milk alcohol liquid sample just,
(IV) will add first milk alcohol liquid sample behind the ethanol, and remain on below 15 ℃, and left standstill at least 5 minutes,
(V) with incline ethanol liquid in the first milk alcohol liquid sample of centrifugal process, collect the sterilized immunoglobulin deposit thing that contains,
(VI) the described immunoglobulin deposit thing that contains is made with SPSS or sterilization emulsion dissolving and is contained the immunoglobulin (Ig) sterilization preparation, its pH between 6.0～7.2,
(VII) the described immunoglobulin (Ig) sterilization preparation that contains is made sterilization dry powder with cold drying, or be mixed and made into the liquid milk that contains immunoglobulin (Ig) with high-temperature sterilization emulsion desired proportions.
5, the effective sterilizing method that can keep the colostrum whey immune globulin activity according to claim 4 is characterized in that, the pre-cooled ethanol that adds in described step (III) is limited to 4 ℃ on its precooling temperature.
6, the effective sterilizing method that can keep the colostrum whey immune globulin activity according to claim 4 is characterized in that, described in the described step (I) to get the colostrum whey of childbirth in 7 days be colostrum whey or sheep colostrum whey or horse colostrum whey.
7, a kind of effective sterilizing method that can keep pure colostrum immune globulin powder activity, its step comprises:
(a) to pure colostrum immune globulin powder with physiological saline solution, the addition of physiological saline is at least 5 milliliters/gram dry powder, forms the colostric fluid of dissolving,
(b) the first whey liquid that will dissolve is removed tangible particle and sediment with centrifugal or filter method, makes processing whey liquid just,
(c) original volume according to the first whey liquid of processing calculates 95% medical grade or the food grade ethanol that adds precooling, and the final valid density of the ethanol of sample sterilization treatment is 70～75%, makes milk alcohol liquid sample just,
(d) the first milk alcohol liquid sample that will add behind the ethanol keeps below 15 ℃, and leaves standstill at least 5 minutes,
(e) follow, the ethanol liquid at the beginning of inclining with centrifugal process in the milk alcohol liquid sample, collecting has sterilized contains the immunoglobulin deposit thing,
(f) the described immunoglobulin deposit thing that contains is made with SPSS or sterilization emulsion dissolving and is contained the immunoglobulin (Ig) sterilization preparation, its pH between 6.0～7.2,
(g) with cold drying the immunoglobulin (Ig) sterilization preparation is made sterilization dry powder again, or be mixed and made into the liquid milk that contains immunoglobulin (Ig) with high-temperature sterilization emulsion desired proportions.
8, the effective sterilizing method that can keep pure colostrum immune globulin powder activity according to claim 7 is characterized in that in described step (c), the pre-cooled ethanol of adding is limited to 4 ℃ on its precooling temperature.
9, the effective sterilizing method that can keep pure colostrum immune globulin powder activity according to claim 7, it is characterized in that, be pure bovine colostrum immunoglobulin powder or pure horse colostrum immune globulin powder or pure sheep colostrum immune globulin powder at the pure colostrum immune globulin powder described in the described step (a).
10, a kind of sterilization dry powder of making according to claim 1 or 4 or 7 described methods that can keep colostrum immune globulin activity or the purposes of sterilization preparation is characterized in that, can be used as the active immne hardening agent and make an addition in the food.
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| CNB2004100993326A CN1298227C (en) | 2004-12-30 | 2004-12-30 | Effective sterilizing method capable of keeping colostrum immune globulin activity |
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| CN1298227C true CN1298227C (en) | 2007-02-07 |
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| CN102919365B (en) * | 2012-11-08 | 2014-03-26 | 光明乳业股份有限公司 | Liquid dairy product rich in immune globulin and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6635222B2 (en) * | 1993-07-22 | 2003-10-21 | Clearant, Inc. | Method of sterilizing products |
| CN1541110A (en) * | 2001-06-14 | 2004-10-27 | Methods for sterilizing prepns of monoclonal immunoglobulins |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6635222B2 (en) * | 1993-07-22 | 2003-10-21 | Clearant, Inc. | Method of sterilizing products |
| CN1541110A (en) * | 2001-06-14 | 2004-10-27 | Methods for sterilizing prepns of monoclonal immunoglobulins |
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Assignee: Kexing Biological Tech. Co., Ltd. Shanghai Assignor: Sansheng Biotech Co., Ltd., Shanghai Contract fulfillment period: 2009.8.25 to 2014.8.25 contract change Contract record no.: 2009310000191 Denomination of invention: Effective sterilizing method capable of keeping colostrum immune globulin activity Granted publication date: 20070207 License type: Exclusive license Record date: 2009.9.4 |
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Free format text: EXCLUSIVE LICENSE; TIME LIMIT OF IMPLEMENTING CONTACT: 2009.8.25 TO 2014.8.25; CHANGE OF CONTRACT Name of requester: SHANGHAI KEXING BIOTECHNOLOGY CO., LTD. Effective date: 20090904 |
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