CN101292639A - Method for improving lactoferrin content in cow's milk - Google Patents
Method for improving lactoferrin content in cow's milk Download PDFInfo
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- CN101292639A CN101292639A CNA2007100977026A CN200710097702A CN101292639A CN 101292639 A CN101292639 A CN 101292639A CN A2007100977026 A CNA2007100977026 A CN A2007100977026A CN 200710097702 A CN200710097702 A CN 200710097702A CN 101292639 A CN101292639 A CN 101292639A
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Abstract
The invention relates to a method for improving the content of lactoferrin (Lf) in cowmilk. By injecting ARD (Antigen Releasing Device) to the position of lymph node in the hip of cows, the method makes a deep research on the Immunogenicity and stability of ARD (Antigen Releasing Device) and ISCOMs (immune stimulating complexes) and has emphasis on checking immunoglobulin in cowmilk; after the ARD is implanted, the content of IgG is increased by 56 percent, which provides an experimentation basis for producing immune milk containing high-level specific antibody. The method of the invention is widely applied to the fields of nutrition food and health food.
Description
Lactoferrin (Lactoferrin is called for short Lf) is a kind of main lactalbumin, and molecular weight is about 80,000, approximately be made of 700 amino acid residues, lactoferrin takes on a red color because of its crystal, claims again " red eggs are white ", about 1 grams per liter of content in the colostrum, the about 0.02-0.35 grams per liter of the normal milk content of ox.Has different physiological roles: as the breeding of the growth of the transmission that promotes iron and absorption, antibacterial activity, antivirus action, immunization, promotion enterocyte and Bifidobacterium, natural, the generation of adjusting bone marrow cell etc.
The present invention is by improving the immunogenicity and the stability of antigen releasing agent ARD (Antigen Releasing Device) and immunostimulating complex (ISCOMs), the sample content of expanding test animal, launch deep research, research ISCOMs vaccine is to the rule that influences of milk cow cellular immunity, lactoferrin in the emphasis check cow's milk is for the immune milk production of contained high levels specific antibody provides test basis.
1, materials and methods
1.1 sample: antigen releasing agent ARD (Antigen Releasing Device)
The antigen releasing agent ARD (Antigen Releasing Device) that selects for use Australian Agri-BIOTECH company to produce, immunostimulating complex (ISCOMs) is encapsulated with the polyactide of variable concentrations, and the 0d after heeling-in, 14d and 28d discharge the immune response generation immunoglobulin that immunostimulating complex causes milk cow respectively.Heeling-in rifle and ARD design diagram are seen accompanying drawing 1.
1.2 animal:
Reach the good fortune dairy farm in Daxing District, Beijing deep blue and select the lactation adult healthy holstein cow in early stage, carry out mastitis with the recessive mastitis diagnosticum and detect the sick ox of rejecting recessive mastitis, and according to its milk yield, age, parity, lactation stage select 40 cow heads and the grouping, 20 of immune group (numbering No.1～20), 20 of control groups (numbering No.21～40).The milk cow situation of participating in the experiment sees Table 1.On November 16th, 2,006 20 oxen of immune group are carried out heeling-in ARD, the heeling-in site is at milk ox Kua endolymph knot place, 3 kinds of different AR D of every ox heeling-in, and experimental period is 4 months.
Table 1 test milk cow situation
Test cattle breeding management is undertaken by the normal feeding and management program of cattle farm, milking cow feed formula in the employing, and TMR feeds, and smart slightly than being 45: 55, grain is formed and trophic level sees Table 2.The milk cow free choice feeding, the record feed intake is fed 3 times, and feeding time is respectively 7:00,14:00 and 20:00.Milk 3 times, the time is 7:00,14:00 and 20:00, adopts Delaval plshy bone open milking parlor to carry out mechanical milking, when milking " secondary dipping, a time paper handkerchief is dried ".Observe the milk cow health situation, especially note it being whether inflammation of the wound in the 7d after the heeling-in.
1.3 test cattle breeding management and sample collecting:
Test cattle breeding management is undertaken by the normal feeding and management program of cattle farm, milking cow feed formula in the employing, and TMR feeds, and smart slightly than being 45: 55, grain is formed and trophic level sees Table 2.The milk cow free choice feeding, the record feed intake is fed 3 times, and feeding time is respectively 7:00,14:00 and 20:00.Milk 3 times, the time is 7:00,14:00 and 20:00, adopts Delaval plshy bone open milking parlor to carry out mechanical milking, when milking " secondary dipping, a time paper handkerchief is dried ".Observe the milk cow health situation, especially note it being whether inflammation of the wound in the 7d after the heeling-in.
Table 2 test grain is formed and trophic level
The milk yield of every ox of per two days records of 0d～10d is measured every ox rectal temperature in the set time of every day, gathers the newborn sample of every ox; Gather the newborn sample of every ox in per 3 days during 21d～60d, write down feed intake, milk yield and body temperature weekly.Gather newborn sample 3 every day when adopting newborn sample, mixes altogether take a sample 100mL, 4 ℃ preservations at 4: 3: 3 in the morning, noon and afternoon ratio; 50mL detects milk somatic cell sum and conventional ingredients such as butterfat, milk protein in Beijing Milk Cow Center; 50mL milk sample is used for sepg whey.
Gather the colostrum sample that deep blue reaches other milk cows of good fortune cattle farm simultaneously, every every 24h of childbirth milk cow gathers the milk sample 1 time, gathers more than the 200mL at every turn, and continuous acquisition 7d is in order to analyze relative immunity whey and colostrum whey.Gather anti-freezing and short blood coagulation when collecting newborn sample.
1.4 experimental technique
1.4.1 mastitis and somatic number detect
Get 50mL breast sample and measure somatic number and butterfat percnetage (Fat), milk protein (Pro), lactose (Lac), total solid (TS), 5 conventional ingredients of non-fat solid content (SNF) with milk composition analyzer (model: Fossomatic 5000, Milkoscan 4000).
1.4.2 indirect ELISA method is measured the Ig of the anti-Lipase of specificity
Method with reference to Guan doctor Tay, catch specific antibody in the sample to be checked with Lipase Antigent coated elisa plate, the diluted sample multiple is 1: 200, adopts the anti-goat IgG of AP enzyme mark donkey anti-as two, the p-NPP colour developing, total Ig of the anti-Lipase of mensuration specificity.The negative contrast of FCS (hyclone), the positive contrast of 2 positive wheys that provide with Australian Agri-BIOTECH company (ox number be respectively 168Q and 460X).
1.4.3 sandwich ELISA method is measured Ig and lactoferrin absolute content
Select for use the IgG/IgA/IgM/Lf quantification kit of Bethyl to measure its absolute content, adopt capture antibody, standard to set up calibration curve, set up regression equation and detect its content with reference to albumen, HRP labelled antibody etc.
1.4.4 SDS-PAGE electrophoresis and quantitative analysis method
Make 5% and concentrate glue and 12% separation gel, carry out the SDS-PAGE of colostrum, normal breast and immune milk whey, voltage is 80V (concentrating glue) and 120V (separation gel), and electrophoresis time is about 1.5h, coomassie brilliant blue staining.Volumes Quick Guide (quantitative analysis) with Bio-Rad gel imaging system Quantity One software carries out relative quantitative assay to the Ig band, obtains the relative concentration of different I g.
1.5 data are handled
With Excel edit data, carry out statistical analysis with SAS V8.
2, result
2.1 ARD is to the influence of milk somatic cell number
Somatic number is not only an important indicator weighing cow breast health in the milk, also is the important indicator of raw material milk quality evaluation simultaneously, and it is all influential to aspects such as dairy products nutritive value, processing and storage characteristic and dairy food-like flavours.Somatic cell comprises polytype cell such as neutrophil, macrophage, lymphocyte, acidophic cell and various galactophore epithelial cells etc.In healthy cow mammary gland, most of somatic cell is macrophage and lymphocyte, and these cells come autoblood, and after mammary gland is infected or damages, these cells will be impregnated into this position, thereby reaches the effect that external microbe infects of resisting.The Ruzhong somatic number of healthy cow is generally about 200,000/mL, just milking cow and the more satisfactory milk cow Ruzhong somatic number of feeding and management may be lower than 100,000/mL, if the Ruzhong somatic number is higher than 500,000/mL, then cow breast is infected probably or wound arranged.Impaired breast tissue shows as the reduction of complex functionality on the one hand, causes milk production to reduce; Show as blood breast barrier quilt destruction to a certain degree on the other hand, not only the synthetic precursor of influence breast enters mammary gland, and other material enters milk in the increase blood, thereby causes milk composition to change, and further newborn nutritive value and other characteristics is produced relatively wide influence.
Somatic number can be up to millions of even several ten million in the individual milk of the milk cow of mastitis owing to suffer from, and other normal milk cow somatic number is below 200,000, therefore the somatic cell numerical value of mastitis individuality can cause tremendous influence to statistics, studies ARD and has removed the fall ill data of ox of mastitis when somatic number influence in the milk.
Heeling-in ARD can cause certain damage to cow breast, and whether heeling-in can increase somatic number is the problem that we relatively worry.Milk somatic cell is counted measurement result and is seen Table 3 in experimental period, experimental group and control group result difference be remarkable (p＞0.05) not, but experimental group milk cow No.8 and No.12 somatic number obviously rise in heeling-in operation back and (see somatic number situation of change in accompanying drawing 2 milk, accompanying drawing 3 No.8 and No.12 milk cow somatic number situation of change), but the original level of very fast recovery.
Table 3 milk somatic cell is counted measurement result
2.2 ARD heeling-in operation is to the influence of the mastitis for milk cows incidence of disease
(4 months) have 8 ox morbidities in experimental period, and wherein experimental group is 2,6 of control groups; Have 6 oxen mastitis takes place, wherein experimental group is 2,4 of control groups, and details sees Table 4.
Table 4 milk cow incidence in experimental period
The outstanding feature of mastitis is that the somatic cell in the milk increases, and the proterties quality of breast takes place unusual.The sick bovine somatic cells number of mastitis obviously increases as can be seen from Table 4, up to millions of even several ten million.
Mastitis is that breast tissue is subjected to physics, chemistry, microorganism and stimulates a kind of inflammatory that is taken place to change, and when milk cow was subjected to bad feeding and management or resistance and descends, pathogenic microorganism was by milk, and blood and lymph liquid are invaded breast tissue and caused.The cause of disease that causes mastitis mainly is to be caused by multiple unspecific pathogenic microorganism, bacterium, Mycoplasma, fungi, virus etc. are arranged, be subjected to weather, feeding and management, influence of various factors such as the mode of milking, lactation amount, lactation stage and nipple form, different breasts district, heredity.Can not cause milk cow generation mastitis from the incidence of disease heeling-in operation of mastitis.
2.3 specific antibody content rule
2.3.1 specific antibody content rule in the serum
Discharge ISCOMs in special time after the ARD heeling-in, enter lymph node or enter spleen, thereby humoral immunoresponse(HI) and cellullar immunologic response take place through blood through lymph circulation.The ISCOMs that enters lymph node is caught by antigen presenting cell APC, handles forming antigenic peptides-MHC II molecular complex through processing, and submission is given the CD4+TH cell.The TH cell is divided into the CD4+TH2 cell by identification, activation, propagation to antigen, produces cell factor and acts on the B cell, impels the B cell activation.The B cell of activation can be expressed the various kinds of cell factor acceptor, and the classification conversion takes place under the different cytokines effect, and proliferation and differentiation is the thick liquid cell of the different classes of Ig of energy synthesis secretion, synthetic justacrine antibody, performance specific immunity effect.In this process, part B cell differentiation is a memory B cell, and keeping has long-term memory to specific antigen, and when replying once more, proliferation and differentiation is a thick liquid cell rapidly, and synthetic more multispecific antibody enlarges immunological effect.The antibody that the ARD inducing cows produces through lymph liquid and blood transport to whole body, in breast tissue is optionally transported IgG into milk by the acinus epithelium.Therefore, the antibody Changing Pattern should be consistent in cow's milk and the blood, and the Ruzhong antibody concentration is influenced by the blood antibody concentration.
The anti-Lipase TPPA of specificity the results are shown in accompanying drawing 4 in the serum, behind the heeling-in ARD, specific antibody level all obviously raises in 20 cow's serums of immune group, and compares than first phase test (2 ox immuning failures in 5 oxen), illustrate that this time to test the ARD production effect better, immunogenicity is better.10d can detect high-level antibody after the heeling-in, lasts till that always 40d just begins slow decline.
2.3.2 specific antibody content rule in the whey
9d just can detect the antibody of the anti-Lipase of specificity in the whey after the heeling-in, and its antibody content changes sees accompanying drawing 5, and it is replied, and rule is consistent with the release of 3 kinds of ARD, and 17～40d level is higher, and after this specific antibody level descends always.As seen, the antibody high level can be kept more than 20 day, and the duration is not very desirable.After this antibody content descend very fast, control group and just difference is not remarkable behind the 75d.
2.3.3 just Ruzhong specific antibody Changing Pattern and and immune milk between comparison
Detect in the colostrum whey specific antibody Ig content and with Australian immune milk positive control 168Q, 460X, this time test immune milk aggregate sample (17d～35d) compare (seeing that accompanying drawing 6 different whey specific antibody content relatively), as seen, Australian 2 immune milk sample specific antibody content can reach the level of early stage colostrum, and the immune milk of this test can reach the level of 2d～3d colostrum.
2.3.4 whey specific antibody growth and decline law-analysing
Though second phase test effect obviously is better than first phase test, has obtained containing the more immune milk of antibody with high specificity, each cow head of labor immune group reply rule, can find that there is some difference between effect, release time and the milk cow individuality of 3 kinds of ARD.
2.3.5 the ideal model of specific antibody growth and decline rule in the whey
The universal law of humoral immunoresponse(HI) is: primary response is long latent period, antibody could occur in about 1w～2w serum after the antigenic stimulus, is mainly IgM, and antibody content is low, the IgG weak point of holding time in vivo; Reply the weak point in latent period once more, be generally 1d～2d, mainly produce IgG, antibody concentration height, the length of holding time.What accompanying drawing 7 was represented is the ideal model of specific antibody growth and decline rule in the whey.3 kinds of ARD successively stimulate, and it replys the rule variation obviously, can detect specific antibody about 10d after the heeling-in, and the weak point of holding time descends rapidly; ARD2 discharges during 14d, causes secondary immune response, and antibody rises rapidly, slowly descends again; ARD3 discharges during 30d, causes immune response the 3rd time, and antibody content should rise to higher level, longer duration, and decrease speed is slow.
2.4 the comparison of IgG/IgA/IgM/Lf content in colostrum, normal breast and the immune whey
IgG, IgA, IgM and Lf absolute content measurement result see Table 5 in colostrum, normal breast and the immune whey, and the activated protein content is fairly obvious in the Bovine Colostrum, descend rapidly along with the increase of childbirth age in days, and the milk cow interindividual variation is very big.Lf has improved 56% after the ARD heeling-in.
IgG, IgA, IgM and Lf (unit: mg/mL) in table 5 colostrum, normal breast and the immune whey
2.5 colostrum, normal breast and immune milk albumin SDS-PAGE analyze
Carry out colostrum, normal breast and immune milk albumin SDS-PAGE electrophoresis, the results are shown in accompanying drawing 8.
The coloring degree of protein band is deposited certain difference, the content that these protein ingredients are described there are differences, with Volumes QuickGuide the Ig band is carried out relative quantitative assay, if the normal newborn Ig of this test, Lf content are 1, can obtain the relative concentration of other wheys IgG and Lf, the IgG of sandwich ELISA method and SDS-PAGE relative quantitative assay, IgA, IgM and Lf Changing Pattern are similar, analyze Lf Changing Pattern shown in the Lf Changing Pattern as accompanying drawing 9 SDS-PAGE relative quantitative assay Lf variation and accompanying drawing 10 sandwich ELISA methods.
3.5.3 immunostimulation is to the influence of Lf content
Analyze Lf content (seeing accompanying drawing 11) after the milk cow immunity such as No.8, No.11, No.12, immunity back Lf can raise, and with release, the whey specific antibody Changing Pattern of ARD certain correlation is arranged.Influence of various factors such as Lf content is subjected to parity, the stage of giving milk, health status.
3. discuss
3.1 the heeling-in operation does not have harmful effect to the milk cow health situation, does not cause mastitis for milk cows, cow feeding amount, milk yield, milk composition do not have variation significantly.This has tentatively illustrated the safety of Nai Niu Kua endolymph knot heeling-in ARD.
3.2 the immune milk specific antibody of this test can reach the level of later stage colostrum.IgG, IgA, IgM and Lf absolute content change fairly obviously in colostrum, normal breast and the immune whey, descend rapidly along with the increase of childbirth age in days, and the milk cow interindividual variation is bigger;
Lf has improved 56% after the ARD heeling-in.
3.3 immune milk is novel functional food, has vast market prospect, by improving immunogenicity and the stability of ARD and ISCOMs, the sample content of expanding test animal, launch more deep research work, be expected to produce the immune milk of contained high levels specific antibody.
Claims (2)
1, the method for lactoferrin (Lf) content in the raising cow's milk of the present invention, by method at the Nai Niu Kua endolymph knot injections of antigens releasing agent ARD of place (Antigen Releasing Device), the deep research antigen releasing agent ARD (AntigenReleasing Device) and the immunogenicity and the stability of immunostimulating complex (ISCOMs).
2, according to the method for claim 1, IgG, IgA, IgM and Lf absolute content change fairly obviously in colostrum, normal breast and the immune whey, and the content of Lf has improved 56% after the ARD heeling-in.
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| CN102869788A (en) * | 2010-01-27 | 2013-01-09 | 维亚拉克什亚生物科学(新西兰)有限公司 | Marker assisted selection of a mammalian subject for desired phenotype |
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| CN102869788A (en) * | 2010-01-27 | 2013-01-09 | 维亚拉克什亚生物科学(新西兰)有限公司 | Marker assisted selection of a mammalian subject for desired phenotype |
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Open date: 20081029 |