CN101978841A - Protein allergy-resisting and nutritious infant formula milk powder - Google Patents
Protein allergy-resisting and nutritious infant formula milk powder Download PDFInfo
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Abstract
The invention relates to protein allergy-resisting and nutritious infant formula milk powder. The milk powder is prepared from the following raw materials in part by ton weight: 1,000 parts of fresh milk (115 parts of dried substance), 500 parts of demineralized whey powder, 235 parts of refined vegetable oil, 45 parts of lactose, 50 parts of whey small peptide with the molecular weight of less than 10,000 daltons, 20 parts of fructooligosaccharide, 6 parts of arachidonic acid (ARA), 5 parts of docosahexaenoic acid (DHA), 0.42 part of nucleotide, 0.45 part of taurine, 0.42 part of choline, 0.06 part of L-carnitine, 0.012 part of beta-carotene, 2.5 parts of complex vitamin, 3.5 parts of amino acid common trace element chelate and 1.5 parts of selenium-enriched yeast powder. The formula milk powder has the advantages that: the formula milk powder contains the whey small peptide with the molecular weight of less than 10,000 daltons, has the effects of regulating immunity and resisting protein allergy, has the characteristics of high water solubility, high absorptivity, no stimulus on stomach and intestine and the like, and can meet the requirements of human bodies on the supplement of trace elements and amino acid.
Description
Technical field
The invention belongs to technical field of processing dairy products, especially a kind of anti-albumen allergy and nutrition babies ' formula milk powder.
Background technology
Cow's milk is the nutraceutical of high-quality, is again simultaneously more easily to cause one of irritated food.Especially concerning being the infant of main food with cow's milk, lactoprotein allergy has seriously influenced the absorption of infant to lactoprotein.Research is in recent years found protein again after the degraded of alimentary canal enzymatic, mainly is the form absorption with small peptide in the human small intestine, and easier, faster than the absorption of complete free amino acid.Some little peptide can not only provide people's bulk-growth, grow required nutriment, and has antianaphylaxis, reduces cholesterol, brings high blood pressure down, and regulates immunity, promotes biological function such as growth.
Normal trace element is used in baby formula milk powder as nutrition fortifier and has been experienced first generation inorganic salts and the simple organic compound of the second generation.And the third generations such as the normal microelement chelate of amino acids, the normal trace-element complex of polysaccharide, the normal micro-thing of biological concentration are often micro-, the nutrition, digestion and the metabolic effects that the infant are had highly significant, new step has been pushed in development of third generation trace element and production, be called as the third generation product that baby formula milk powder adds trace element.With preceding two generations product comparison, obvious advantages is arranged.
At present domestic patent in the dairy produce field mainly lays particular emphasis on aspect the compatibility of milk powder and the appearance design of packaging bag, and patent is less.Patent of invention CN1439276 at being born to 6 months baby's specialized designs, can provide the nutrition of general equilibrium according to the characteristics of breast milk nutritional labeling for baby.Remove in the invention and added baby grow up necessary multivitamin and trace element, also added L-carnitine, taurine, beta carotene, nucleotides, lactoferrin etc., make its nutritional labeling more reasonable.Wherein composite trace element comprises a kind of and several in ferrous lactate, zinc lactate, copper sulphate, Potassiumiodate, calcium carbonate, the manganese sulfate.One kind of patent of invention CN1107656 with fresh milk, desalted whey powder, purified soyabean oil, make a gift of raw materials such as sugar, maltodextrin, cream, B B-complex, compound dimension secondary element, isomerizing lactose, inositol and carnitine and make milk powder simulating breast milk for infants.Product lays particular emphasis on nutrient formulation, and the nutritional labeling that the baby needs is complete, easily digests and assimilates, and infantile health is grown up.The prescription of a patent of invention CN1300549 lactoferrin baby milk, its manufacture craft is at first that raw milk is newborn only, preheating then, homogeneous, sterilization, concentrating, dusting promptly becomes semi-finished product, adds lactoferrin and mixes and be finished product, and invention lays particular emphasis on manufacture craft.Patent of invention CN1459235 invention utilizes biotechnology that alpha-casein in the cow's milk and beta lactoglobulin are decomposed with compound protease, makes to be difficult to digest and the protein-modified of sensitization arranged, and has eliminated its influence.Invention has successfully solved fresh milk because of the bad difficult problem of the product special flavour that enzymolysis brought, and is the innovation kind in the dairy products.
In sum, the patented invention content at infant's goods aspect aspect the background technology mainly concentrates on prescription and the nutrition aspect, mainly is nutrition arrangement on claim, and does not all relate to anti-albumen allergy and the normal trace element of the third generation etc.
Summary of the invention
The present invention will solve the shortcoming of above-mentioned prior art, a kind of anti-albumen allergy and nutrition babies ' formula milk powder are provided, can reduce the prescription of infant's albumen allergy and the compound that has changed normal trace element, utilize the normal micro-huge legendary turtle compound of amino acids to replace second generation trace element often, and determined rational collocation, the nutrition of highly significant is arranged, by adding the Se-enriched yeast powder, digestion and metabolic effects strengthen, and are particularly suitable for preemie, 0～12 month infant etc.
The invention solves difficult problems such as the infant's albumen allergy and the first generation, the absorption of the normal trace element of the second generation, for the simulation breast milk composition provides a kind of new method.
The present invention solves the technical scheme that its technical problem adopts: this anti-albumen allergy and nutrition babies ' formula milk powder are to be made by the raw material of following ton weight portion: fresh milk 1000 (material 115 of giving money as a gift); desalted whey powder 500; refining vegetable oil 235; lactose 45; molecular weight is less than 10000 dalton's whey small peptides 50; FOS 20; arachidonic acid (ARA) 6; DHA (DHA) 5; nucleotides 0.42; taurine 0.45; choline 0.42; L-carnitine 0.06; beta carotene 0.012; B B-complex 2.5; the normal microelement chelate of amino acids (compound normal trace element) 3.5; Se-enriched yeast powder 1.5.
The normal microelement chelate of described amino acids is meant one or several in calcium glycine or calcium aspartate or Methionine calcium salt or Threonine calcium salt or the calcium lysinate.
The normal microelement chelate of described amino acids is meant one or several in glycine zine or zinc methionine or aspartic acid zinc or threonine zinc or the lysine-zn.
The normal microelement chelate of described amino acids is meant one or several in cupric glycinate or aspartic acid copper or copper methionine or copper-threonine or the Copper lysinate.
Se content 1000～2500mg/kg in the described Se-enriched yeast powder.
The effect that the present invention is useful is:
Compare with the conventional baby formula milk, the present invention contains molecular weight less than 10000 dalton's whey small peptides, has the effect of immunity regulated and anti-albumen allergy;
Compare with the conventional baby formula milk, the present invention contains one or several in calcium glycine or calcium aspartate or Methionine calcium salt or Threonine calcium salt or the calcium lysinate, contain in cupric glycinate or aspartic acid copper or copper methionine or copper-threonine or the Copper lysinate one or several, has good water solubility, the absorptivity height, characteristics such as stimulating gastrointestinal can not satisfy human body simultaneously to mending trace element and mending amino acid whose needs;
Compare with the conventional baby formula milk, the present invention contains the Se-enriched yeast powder, is rich in immune polysaccharide, manna oligosacchride and chitin, can be selectively by beneficial bacteria in the enteron aisle as the energy, promote its procreation, thereby there is also harmful bacterium suppress or kill salmonella etc., has chemical prebiotic effect, promotes trace element to digest and assimilate, improve its biology utilization rate, improve the stomach environment, immune response stimulating improves infant immunity;
Comprehensively discuss, compare with background technology, the present invention utilizes molecular weight to replace second generation trace element often less than 10000 dalton's whey small peptides, the normal micro-huge legendary turtle compound of amino acids and the normal micro-yeast class material of biological concentration, and determined rational collocation, nutrition, digestion and the metabolic effects of highly significant arranged.
The specific embodiment
The invention will be further described below in conjunction with embodiment:
Embodiment 1: this anti-albumen allergy and nutrition babies ' formula milk powder, and be to be made by the raw material of following ton weight portion: fresh milk 1000 (material 115 of giving money as a gift), desalted whey powder 500, refining vegetable oil 235, lactose 45, molecular weight are less than 10000 dalton's whey small peptides 50, FOS 20, arachidonic acid (ARA) 6, DHA (DHA) 5, nucleotides 0.42, taurine 0.45, choline 0.42, L-carnitine 0.06, beta carotene 0.012, B B-complex 2.5, the normal microelement chelate 3.5 of amino acids, Se-enriched yeast powder 1.5; Se content 1000～2500mg/kg in the described Se-enriched yeast powder contains the Se-enriched yeast powder in the milk powder, have the enhancing digestive function.The normal microelement chelate of described amino acids is meant one or several in calcium glycine or calcium aspartate or Methionine calcium salt or Threonine calcium salt or the calcium lysinate.
Contain molecular weight in the formula milk less than 10000 dalton's whey small peptides, have the effect of immunity regulated and anti-albumen allergy; Molecular weight is less than the preparation method of 10000 dalton's whey small peptides: PURE WHEY----Jia enzyme hydrolysis----evaporation and concentration----spray-drying-----whey small peptides powder (protein 80%, degree of hydrolysis 15-20%, lactose≤5%, fat≤7%).
Embodiment 2: difference from Example 1 is: the normal microelement chelate of described amino acids is meant one or several in glycine zine or zinc methionine or aspartic acid zinc or threonine zinc or the lysine-zn.
Embodiment 3: difference from Example 1 is: the normal microelement chelate of described amino acids is meant one or several in cupric glycinate or aspartic acid copper or copper methionine or copper-threonine or the Copper lysinate.
Immunoregulation effect and the preliminary experiment of antiallergy effect: the ICR mouse with 18-22g carries out carbon particle clearance test and serum hemolysin mensuration, carry out the lymphproliferation response test and NK (NKT) cytoactive is tested with the BALB/c mouse of 18-22g, carry out passive cutaneous anaphylaxis test (PCA) with the SD rat of 180-220g.Observe the influence of whey small peptides to mouse carbon granules phagocytic index, mice serum HD50 value, observe whey small peptides to the SI of mouse lymphocyte breeder reaction and the influence of NK cytoactive, observe whey small peptides the locus coeruleus area of models of passive skin irritability rat, the influence of locus coeruleus optical density (OD) value.
1. test principle and purpose
Observe the influence of whey small peptides, the non-specific immune function effect of whey small peptides to mouse is described animal body immunologic function and passive cutaneous anaphylaxis.The research whey small peptides is to the influence of carbon particle clearance function in the mouse body, and within the specific limits, the clearance rate of carbon granule and its dosage are the finger functional relation, and the speed of promptly engulfing is directly proportional with the blood concentration of carbon, and is inversely proportional to the carbon granules amount of having engulfed.Behind the SRBC immune animal, produce anti-SRBC antibody (hemolysin), utilize the degree of its aggegation SRBC to detect the level of hemolysin, obtain whey small peptides SRBC is caused the influence that the mouse hemolytic antibody generates, analyze the effect that suppresses the specific humoral immunity function.The research whey small peptides is to the influence of mice spleen lymphocytes proliferation, blast transformation after stimulating, T lymphocyte receptor ConA takes place, living cells particularly proliferative cell is decomposed into blue purple crystal by the mitochondria hydrolase with MTT (a kind of flaxen azoles nitrogen salt) and develops the color, and its OD value can reflect that the propagation situation of cell reflects the specific cellular immunity function to mouse.The research whey small peptides is to the influence of NK cells in mice activity, and the endochylema of living cells contains lactic dehydrogenase (LDH).Under the normal condition, the obstructed permeate through cell membranes of LDH, after cell was subjected to killing and wounding of NK cell, LDH was discharged into the extracellular.LDH can make the lithium lactate dehydrogenation, and then make NADH (NAD) be reduced into NADH, the latter is again through hydrogen carrier azophenlyene dimethyl ester sulfate (PMS) reduction p-Iodonitrotetrazolium violet (INT), and INT accepts H+ and is reduced into and praises compounds by the aubergine first moon.On ELIASA, use the 490nm colorimetric estimation.The research whey small peptides is to the influence of rat passive cutaneous anaphylaxis of the same race (PCA), passive cutaneous anaphylaxis test is that the serum (containing abundant IgE antibody) that will be tried the thing sensitized animal is given the intact animal intracutaneous injection, the Fc end of IgE and the special receptors bind on the mast cell surface of skin, form the compound of IgE, make mast cell sensitization.When antigen is attacked, the Fab of IgE end combines on antigen and the mast cell surface, cause the change of IgE molecular structure, cause mast cell degranulation, discharged sensitive media such as histamine, slow reacting substance etc., the permeability of local skin blood vessel is increased, make the concurrently injected Evans blue dyestuff of intravenous injection antigen ooze out painted at this skin place.Dye scope or degree according to local skin indigo plant, the size that the decidable vasopermeability changes is judged the degree of cutaneous anaphylaxis then.
This test with distilled water in contrast, research infant nutrition prescription emulsifiable powder, the clear little peptide of various dose whey to the influence of carbon particle clearance function in the mouse body, to SRBC cause influence that the mouse hemolytic antibody generates, to the influence of mice spleen lymphocytes proliferation, to the influence of NK cells in mice activity, to the influence of rat passive cutaneous anaphylaxis of the same race (PCA) etc., in the hope of obtaining infant nutrition prescription emulsifiable powder, whey small peptides in the effect of regulating immunity, anti-albumen allergy etc.
2. be subjected to the reagent thing
2.1 whey small peptides, white powder, degree of hydrolysis 15-20%, below the molecular weight 10000D, make date received through enzymolysis, embedding, spray-drying etc.: 20091207, the unit of providing: Scientific and Technological Institutes Of Zhejiang is biological makes by oneself with Chemical Engineering institute, storage procedures: lucifuge, drying, normal temperature (below 25 ℃) are preserved, compound method: face with preceding under aseptic condition with dissolved in distilled water to desired concn;
2.2 infant nutrition prescription emulsifiable powder: white powder, specification: the 385g/ jar, the free from lactose cane sugar type, storage procedures: lucifuge, drying, normal temperature (below 25 ℃) are preserved, compound method: face with preceding under aseptic condition with dissolved in distilled water to desired concn.
3. experimental animal
3.1 rat: SD rat, SPF level, body weight 180-220g, west, Shanghai pul-Bi Kai animal used as test Co., Ltd [production licence: SCXK (Shanghai) 2008-0016];
3.2 mouse: closed colony ICR mouse, inbred strais BALB/c, SPF level, body weight 18-22g, west, Shanghai pul-Bi Kai animal used as test Co., Ltd [production licence: SCXK (Shanghai) 2008-0016];
3.3 cavy: regular grade, body weight 250-300g, male and female dual-purpose, city, Huzhou west wind ocean special economic animal plant [SCXK (Zhejiang) 2008-0037].
4. experiment condition
The zoopery condition: the large and small mouse raising experiment of SPF level barrier system chamber, regular grade cavy receptacle, temperature: 22 ± 1 ℃, humidity: 50-70%, illumination: 150-200Lx, light and shade replaced in 12 hours, noise＜50dB.
Drinking-water: the tap water filter sterilization places autoclaved drinking-water bottle freely to drink.
Feed: Co
60The aseptic large and small mouse full nutrition pellet of irradiation.
Feeding patterns: rat, mouse, cavy free choice feeding.
5. dosage group (dosage setting and consigner require formulate)
5.1 mouse is used dosage:
Normal control group: distilled water 20ml/kg;
Model control group: distilled water 20ml/kg;
Whey small peptides high dose group: whey small peptides 0.4g/kg (20mg/ml);
Whey small peptides low dose group: whey small peptides 0.2g/kg (10mg/ml);
Infant nutrition prescription emulsifiable powder+10% whey small peptides: 4.0g/kg contains the prescription emulsifiable powder of 10% whey small peptides;
Infant nutrition prescription emulsifiable powder+5% whey small peptides: 4.0g/kg contains the prescription emulsifiable powder of 5% whey small peptides;
Infant nutrition prescription emulsifiable powder group: infant nutrition prescription emulsifiable powder 4.0g/kg.
5.2 rat is used dosage:
Normal control group: solvent 10ml/kg;
Model control group: solvent 10ml/kg;
Whey small peptides high dose group: whey small peptides 0.2g/kg;
Whey small peptides low dose group: whey small peptides 0.1mg/kg;
Infant nutrition prescription emulsifiable powder+10% whey small peptides: 2.0g/kg contains the formula milk of 10% whey small peptides;
Infant nutrition prescription emulsifiable powder+5% whey small peptides: 2.0g/kg contains the formula milk of 5% whey small peptides;
Infant nutrition prescription emulsifiable powder group: infant nutrition prescription emulsifiable powder 2.0g/kg.
5.3 method of administration and capacity
Give a mouthful filling stomach, mouse 20ml/kg (0.2ml/10g), rat 10ml/kg (1ml/100g).
Key instrument and reagent material
6.1 key instrument
The Micro-automatic biochemistry analyzer, Dutch Merck Vitalab Micro;
HH-4 type digital control constant temperature water-bath, Co., Ltd of Gome;
DL-5-B type centrifuge, Anting Scientific Instrument Factory, Shanghai;
Disposable autocontrol micropipet, Jiaojiang City medical apparatus instrument factory;
Hai'an booth science and technology instrument company on the TG-16 type high speed freezing centrifuge;
725 types-86 ℃ low temperature refrigerator, U.S. FORMA;
AG204-electronic analytical balance: Switzerland METTLER;
Baking oven: Gome;
HH-4 type digital control constant temperature water-bath: Gome;
128C-340 type ELIASA: Austrian CliniBio;
CB-150 type CO
2Incubator: German Binder company;
The hot vapour sterilizer of the portable pressure of YXQ-SG41-280 electric heating: Shanghai Medical Nuclear Instrument Factory;
ZEISS AXIOVERT 200 inverted microscopes: German ZEISS.
6.2 main agents and material
India ink, the long-range Science and Technology Ltd. in Chinese and Western, Beijing, 080321, get certain prepared Chinese ink, 1000rpm/min, centrifugal 10min is with 3 times of (V/V) dilutions of physiological saline;
Sodium carbonate: go up marine rainbow photoinitiator chemical factory, the AR level, the 500g/ bottle, 051109, be made into 0.1% sodium carbonate liquor during use;
Preserve liquid (Alsever): glucose 2.05g, sodium chloride 0.42g; Natrium citricum 0.8g, distilled water 100mL: standby after the aseptic filtration;
Sheep red blood cell (SRBC) suspension (SRBC): under aseptic condition, get blood from healthy adult sheep external jugular vein, put blood jog 10min in the conical flask that bead is arranged and defibrinate to remove, add the preservation liquid of 2 times of amounts, it is standby to put 4 ℃ of refrigerators.Face the time spent with physiological saline washing 3 times, get packed red cells, be diluted to desired concn again through the centrifugal 10min of 2000rpm.
Guinea pig heart blood sampling separation of serum, frozen standby; Hemoglobin conversion fluid (cyanmethemoglobin method): Ningbo Ci Cheng biochemical reagents factory, lot number: 031114, this reagent is 100 times of concentrates, contains sodium acid carbonate 10%, high-potassium ferricyanide 2%, potassium cyanide 0.5%;
Natrium citricum: AR level, Shanghai reagent one factory, lot number: examination-2000-07-01, specification: 500g;
Canavalin (ConA): sigma company, 20071001;
Lipopolysaccharides (LPS): sigma company, 024K4067;
RPMI1640 nutrient solution (containing 10% calf serum): get RPMI1640 culture medium dry powder 10.4g (specification is 1L), add the dissolving of 1000mL ultra-pure water, add the 2.1g sodium acid carbonate again, 4.77g HEPES (HEPES), dissolving back mixing, 0.22 the degerming of μ m membrane filtration, 4 ℃ of preservations.Measure hyclone 10mL, add RPMI1640 nutrient solution 90mL, 4 ℃ of preservations through deactivation in 56 ℃, 30 minutes.
Hank ' s liquid: get sodium chloride 80g, sodium hydrogen phosphate (Na
2HPO
412H3O) 0.6g, potassium chloride 4.0g, potassium dihydrogen phosphate 0.6g, magnesium sulfate (MgSO
47H
2O) 2.0g, glucose 10g, anhydrous calcium chloride 1.4g, adding water to 1000mL is stoste.Get stoste 100mL during preparation, add water 896mL, add 0.5% phenol red 4mL, mixing is through 121 ℃, 15 minutes autoclavings, 4 ℃ of preservations.Before facing usefulness, add aseptic 7% sodium bicarbonate solution and transfer pH7.2;
Tetrazolium bromide (MTT) solution: sigma company, 27196EJ takes by weighing 0.5gMTT and is dissolved in 100mL phosphate buffer (PBS), and making concentration is 5mg/mL, 0.22 μ m membrane filtration degerming, 4 ℃ keep in Dark Place;
Dimethyl sulfoxide (DMSO): the large biological Co., Ltd in sea, Wuxi, lot number: 20060607, every bottle of 500mL;
The K562 cell; Lithium lactate; Tris-HCl buffer solution (pH8.2), 1%NP40 or the 2.5%Triton of nitro tetrazolium chloride (INT), azophenlyene dimethyl ester sulfate (PMS), NAD, 0.2mol/L.
The ivens orchid, 10g/ bottle, Fluka import, 82-11-02.
Adsorbed Diphtheria Tetanus Pertussis Combined Vaccine, 2ml/ prop up (4 human dosage), Shanghai Vaccine and Serum Institute, and 2008120101,2-8 ℃ keeps in Dark Place.
Oralbumin, white powder, the 10g/ bottle, 9006-59-1, the sigma product, day Bioisystech Co., Ltd's import packing of Hangzhou sky, airtight, put 4 ℃ of storages.
7. experimental technique
7.1 whey small peptides is to the influence of carbon particle clearance function in the mouse body
Get body weight and be 70 of the male ICR mouses of 18-22g, be divided into 7 groups at random by body weight, be normal control group, model control group, whey small peptides high dose group, whey small peptides low dose group, infant nutrition prescription emulsifiable powder+10% whey small peptides group, infant nutrition prescription emulsifiable powder+5% whey small peptides group and infant nutrition prescription emulsifiable powder control group, every group 10, each group gives the relative medicine and the solvent of respective concentration respectively, once a day, successive administration is 30 days.Behind the last administration 24h, remove normal control group tail vein injection saline, all the other each group injection india inks, after the title mouse body weight, mousetail 75% alcohol wipe is injected the india ink of 4 times of dilutions from the tail vein, calculates by every 10g body weight 0.1mL.Treat that prepared Chinese ink injects timing immediately.Inject behind the prepared Chinese ink 2,10min, get blood 10uL from the angular vein clump respectively, existing side by side is about to it and is added to 1mL 0.1%Na
2CO
3In the solution.With 722 spectrophotometers at 600nm wavelength place's photometry density value (OD), with Na
2CO
3Solution is made blank.Mouse is put to death, get liver and spleen, blot the organ surface blood stains, weigh with filter paper.
(the following OD that uses respectively
2And OD
10The optical density of representing 2min and 10min institute blood sampling), be calculated as follows and clean up index K value.
The K value after body weight and liver spleen heavily convert, phagocytic index α value:
7.2 whey small peptides causes the influence that the mouse hemolytic antibody generates to SRBC
7.2.1SRBC the sheep jugular vein is got blood, the sterilization conical flask of sheep blood being put into bead shakes towards a direction, with defiber, puts into 4 ℃ of refrigerators and preserves standbyly, can preserve for 2 weeks.
7.2.2 the preparation complement is gathered guinea pig blood, isolates serum (pooled serums of 8 cavys), SRBC joins in the 5mL GPS with the 1mL hematocrit, puts 4 ℃ of refrigerator 30min, constantly vibration, centrifuging and taking supernatant, packing ,-70 ℃ of preservations.Time spent dilutes by 1: 8 with SA liquid.
7.2.3 50 of the male ICR mouses that body weight is 18-22g are got in the grouping administration, be divided into 7 groups at random by body weight, be normal control group, model control group, whey small peptides high dose group, whey small peptides low dose group, infant nutrition prescription emulsifiable powder+10% whey small peptides group, infant nutrition prescription emulsifiable powder+5% whey small peptides group and infant nutrition prescription emulsifiable powder control group, every group 10, each group gives the relative medicine and the solvent of respective concentration respectively, once a day, successive administration immunity after 30 days.
7.2.4 separating, immune animal and serum gets sheep blood, with physiological saline washing 3 times, centrifugal at every turn (2000r/min) 10min.Hematocrit SRBC is made into the cell suspension of 20% (v/v) with physiological saline, and every mouse lumbar injection 0.2mL carries out immunity.Behind the 4d, extract eyeball and get blood in centrifuge tube, place about 1h, serum is fully separated out, the centrifugal 10min of 3000r/min collects serum.
7.2.5 getting serum, hemolytic reaction dilutes 500 times with the SA buffer solution.Serum 1mL after the dilution is put in vitro, add 10% (v/v) SRBC 0.5mL successively, complement 1mL (diluting by 1: 8) with SA liquid.Other establishes the not control tube of increase serum (replacing with SA liquid).After putting in 37 ℃ of waters bath with thermostatic control insulation 15-30min, the ice bath cessation reaction.The centrifugal 10min of 2000r/min.Get supernatant 1mL, add Dou Shi reagent 3mL, get 10% (v/v) SRBC 0.25mL simultaneously and add Dou Shi reagent to 4mL, fully mixing is placed 10min, sentences control tube in 540nm and makes blank, measures respectively and respectively manages OD value, calculation sample pipe HD50 value HC
50
7.3 whey small peptides is to the influence of mice spleen lymphocytes proliferation
Get body weight and be 60 of the male BALB/c mouses of 18-22g, be divided into 6 groups at random by body weight, be blank group, whey small peptides high dose group, whey small peptides low dose group, infant nutrition prescription emulsifiable powder+10% whey small peptides group, infant nutrition prescription emulsifiable powder+5% whey small peptides group and infant nutrition prescription emulsifiable powder control group, every group 10, each group gives the relative medicine or the solvent of respective concentration respectively, once a day, successive administration is 30 days.Carry out T, bone-marrow-derived lymphocyte propagation SI mensuration, mouse is plucked the eyeball bloodletting and causes death, and the aseptic spleen of getting is smashed to pieces in culture medium, with syringe pump piping and druming, cell is uniformly dispersed, and filters, and is centrifugal, removes supernatant, nutrient solution washing 2 times.Check the percentage of living cells: splenocyte is made (5～10) * 10
6Individual/mL, other take a morsel splenocyte suspension and equivalent 0.5% trypan blue mixing left standstill the percentage of the dead cell that counting nuclear is colored, the cell number of checkmating deduction several minutes from total cell number.Fabric swatch: the every hole of 96 orifice plates adds cell 200 μ l, and each sample repeats 4 holes, sets up blank simultaneously.Sample well adds ConA2.5 μ g/mL or LPS5.0 μ g/mL, and control wells does not add, 37 ℃ of 5%CO
2Cultivate 72h in the incubator.Add MTT10 μ L, continue to cultivate 4h, abandon supernatant, add dimethyl sulfoxide (DMSO) 150 μ L, fully vibration is surveyed the OD value in 570nm.Calculate SI: SI=the add average OD value of the average OD value ÷ control wells of ConA or LPS cell hole.
7.4 whey small peptides is to the influence of NK cells in mice activity
Get body weight and be 60 of the male BALB/c mouses of 18-22g, be divided into 6 groups at random by body weight, be blank group, whey small peptides high dose group, whey small peptides low dose group, infant nutrition prescription emulsifiable powder+10% whey small peptides group, infant nutrition prescription emulsifiable powder+5% whey small peptides group and infant nutrition prescription emulsifiable powder group, every group 10, each group gives the relative medicine or the solvent of respective concentration respectively, once a day, successive administration is 30 days.Carrying out the NK cytoactive detects.
The NK cytoactive detects: get target cell (K562 cell 1*10
5Cell/mL) and effector cell (splenocyte 1*10
7Cell/mL) each 100uL (imitating target than 100: 1) adds in 96 well culture plates; Target cell nature release aperture adds target cell and each 100uL of nutrient solution, and the maximum release aperture of target cell adds target cell and each 100uL of 1%NP40; Above-mentioned every three multiple holes of all establishing, in 37 ℃, 5%CO2 incubator, cultivate 4h, then with 96 well culture plates with the centrifugal 5min of 1500r/min, draw at the bottom of the supernatant 100uL horizontalization in 96 well culture plates in every hole, add LDH matrix liquid 100uL simultaneously, reaction 3min, every hole adds the HCl 30uL of 1mol/L, measures OD value (OD) at ELIASA 490nm place.
Be calculated as follows the NK cytoactive (NK cell activity, %)=(OD
T-(OD
S-OD
E))/OD
T* 100%, OD
T: the blank OD value of target cell, OD
S: sample OD value, OD
E: the blank OD value of effector cell.
7.5 whey small peptides is to the influence of rat passive cutaneous anaphylaxis of the same race (PCA)
Principle: passive cutaneous anaphylaxis test is that the serum (containing abundant IgE antibody) that will be tried the thing sensitized animal is given the intact animal intracutaneous injection, the Fc end of IgE and the special receptors bind on the mast cell surface of skin, and the compound of formation IgE makes mast cell sensitization.When antigen is attacked, the Fab of IgE end combines on antigen and the mast cell surface, cause the change of IgE molecular structure, cause mast cell degranulation, discharged sensitive media such as histamine, slow reacting substance etc., the permeability of local skin blood vessel is increased, make the concurrently injected Evans blue dyestuff of intravenous injection antigen ooze out painted at this skin place.Dye scope or degree according to local skin indigo plant, the size that the decidable vasopermeability changes.
7.5.1 preparation antiserum:
The SD rat, 8, to every rat paw injection egg protein (20mg/mL), each sufficient sole of the foot injection 0.1mL, 0.4mL, lumbar injection Adsorbed Diphtheria Tetanus Pertussis Combined Vaccine 0.5mL simultaneously altogether.The next day sensitization once, totally three times, after 10 days, from abdominal aortic blood, centrifugal (3000rpm 10min), makes antiserum in last sensitization.Preserve in two weeks standby for-20 ℃.7.5.2 administration and passive sensitization of skin:
Get 70 of SD rats, be divided into 7 groups at random, normal control group, model control group, whey small peptides high dose group, whey small peptides low dose group, infant nutrition prescription emulsifiable powder+10% whey small peptides group, infant nutrition prescription emulsifiable powder+5% whey small peptides group and infant nutrition prescription emulsifiable powder control group, 10 every group.Each group gives the relative medicine or the solvent of respective concentration respectively, once a day, and successive administration 30d, administration 28d, back depilation, 2 points of every side-draw, every some interval 1.5-2.0cm, the antiserum of every rat back intracutaneous injection dilution in 1: 5,1: 10, each dilution factor injection 0.1mL.
7.5.3 antigen is attacked:
Carry out antigen behind the injection 48h and attack, each organizes rat intravenous injection egg protein 20mg/kg, and egg protein is made into 2mg/mL with 0.5% azovan blue solution.Every 100g body weight injection azovan blue solution 1mL.Put to death rat behind the 30min, the upset skin of back behind the measurement locus coeruleus diameter, is laid the locus coeruleus skin graft respectively with the card punch of diameter 8mm, shred, add acetone physiological saline (7: 3) mixed liquor 5mL, soak, in 37 ℃ of thermostat water baths, place and spend the night, centrifugal (3000rpm, 10min), get supernatant, spectrophotometer 610nm place measures absorbance (OD) value.
8. observation index
8.1 carbon granules phagocytic index.
8.2 serum HD50 value (HC
50).
8.3 the SI of lymphproliferation response (SI).
8.4NK cytoactive.
8.5 locus coeruleus area, locus coeruleus OD value.
9. statistical analysis
Carry out statistical analysis with SPSS13.0 software, all data are with mean ± standard deviation
T test evaluation result of the test is used in expression, measurement data.
10. result
10.1 whey small peptides is to the influence of carbon particle clearance function in the mouse body
Show by table 1, with the normal control group relatively, phagocytic index obviously raise (P＜0.01) behind the model control group mouse mainline india ink; Compare with model control group, give 0.2gkg
-1And 0.4gkg
-1Behind the whey small peptides, mouse carbon granules phagocytic index has trend of rising (P＞0.05), gives 4.0gkg
-1The infant nutrition prescription emulsifiable powder, contain the infant nutrition prescription emulsifiable powder of 5% or 10% whey small peptides after, mouse carbon granules phagocytic index all obviously raise (P＜0.05).
Table 1 whey small peptides is to the influence of carbon particle clearance function in the mouse body
Annotate: compare Δ P＜0.05, Δ Δ P＜0.01 with the normal control group; Compare with model control group,
*P＜0.05,
*P＜0.01; Compare aP＜0.05, aaP＜0.01 with infant nutrition prescription emulsifiable powder group.
10.2 whey small peptides causes the mouse hemolytic antibody to SRBC and generates influence
Show by table 2, with the normal control group relatively, HD50 value obviously raise (P＜0.01) after the model control group mouse SRBC sensitization; Compare with model control group, give 0.4gkg
-1Behind the whey small peptides, mouse HD50 value obviously reduces (P＜0.05), the 4.0gkg that gives
-1Behind the infant nutrition prescription emulsifiable powder, mouse HD50 value obviously raise (P＜0.01); Compare with infant nutrition prescription emulsifiable powder group, give 4.0gkg
-1After containing the infant nutrition prescription emulsifiable powder of 5% or 10% whey small peptides, mouse HD50 value obviously reduces (P＜0.05, P＜0.01).
Table 2 whey small peptides causes the mouse hemolytic antibody to SRBC and generates influence
Annotate: compare Δ P＜0.05, Δ Δ P＜0.01 with the normal control group; Compare with model control group,
*P＜0.05,
*P＜0.01; Compare aP＜0.05, aaP＜0.01 with infant nutrition prescription emulsifiable powder group.
10.3 whey small peptides is to mice spleen lymphocytes proliferation reaction influence
Show by table 3, compare, give 0.4gkg with the blank group
-1Whey small peptides and 4.0gkg
-1After containing the infant nutrition prescription emulsifiable powder of 10% whey small peptides, mouse T lymphocyte SI value obviously reduces (P＜0.05), and the no significant change of mouse bone-marrow-derived lymphocyte SI value; Compare with infant nutrition prescription emulsifiable powder group, give 4.0gkg
-1After containing the infant nutrition prescription emulsifiable powder of 10% whey small peptides, mouse T lymphocyte SI value obviously reduces (P＜0.05), and the no significant change of mouse bone-marrow-derived lymphocyte SI value.
Table 3 whey small peptides is to mice spleen lymphocytes proliferation reaction influence
Annotate: compare with the blank group,
*P＜0.05,
*P＜0.01; Compare aP＜0.05, aaP＜0.01 with infant nutrition prescription emulsifiable powder group.
10.4 whey small peptides is to the influence of NK cells in mice activity
Show by table 4, compare, give 0.2gkg with the blank group
-1And 0.4gkg
-1Behind the whey small peptides, the active no significant change (P＞0.05) of NK cells in mice gives 4.0gkg
-1The infant nutrition prescription emulsifiable powder, contain the infant nutrition prescription emulsifiable powder of 5% or 10% whey small peptides after, NK cells in mice activity all obviously raise (P＜0.01).
Table 4 whey small peptides is to the influence of NK cells in mice activity
Annotate: compare Δ P＜0.05, Δ Δ P＜0.01 with the normal control group; Compare with model control group,
*P＜0.05,
*P＜0.01; Compare aP＜0.05, aaP＜0.01 with infant nutrition prescription emulsifiable powder group.
10.5 whey small peptides is to the influence of rat passive cutaneous anaphylaxis of the same race (PCA)
Show by table 5, with the normal control group relatively, locus coeruleus OD value and area significantly raise (P＜0.01) after the model control group rat egg protein sensitization; Compare with model control group, give 0.2gkg
-1, 0.4gkg
-1Whey small peptides and 4.0gkg
-1After containing 10% whey small peptides infant nutrition prescription emulsifiable powder, rat locus coeruleus OD value and area significantly reduce (P＜0.01); Compare with infant nutrition prescription emulsifiable powder group, give 4.0gkg
-1After containing the infant nutrition prescription emulsifiable powder of 10% whey small peptides, mouse locus coeruleus OD value and area significantly reduce (P＜0.01), give 4.0gkg
-1After containing the infant nutrition prescription emulsifiable powder of 5% whey small peptides, mouse locus coeruleus OD value and locus coeruleus area (1: 5) significantly reduce (P＜0.05).
Table 5 whey small peptides is to the influence of rat passive cutaneous anaphylaxis of the same race (PCA)
Annotate: compare Δ P＜0.05, Δ Δ P＜0.01 with the normal control group; Compare with model control group,
*P＜0.05,
*P＜0.01; Compare aP＜0.05, aaP＜0.01 with infant nutrition prescription emulsifiable powder group.
Conclusion: above result of the test shows that whey small peptides has the adjusting immunization, can strengthen the function of mouse monokaryon macrophage system slightly, has the effect of the non-specific immune function of slight enhancing mouse; Whey small peptides can suppress the generation of the hemolytic antibody of SRBC immune mouse, suppresses the specific humoral immunity function of mouse; And can suppress the breeder reaction of mouse lymphocyte, suppress the specific cellular immunity function of mouse; Can suppress the effect of models of passive skin irritability of rats reaction.The infant nutrition prescription emulsifiable powder has certain humidification and the non-specific immune function of mouse is had humidification the NK cellular immune function of mouse.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (5)
1. anti-albumen allergy and nutrition babies ' formula milk powder is characterized in that: be to be made by the raw material of following ton weight portion: fresh milk 1000 (material 115 of giving money as a gift); desalted whey powder 500; refining vegetable oil 235; lactose 45; molecular weight is less than 10000 dalton's whey small peptides 50; FOS 20; arachidonic acid (ARA) 6; DHA (DHA) 5; nucleotides 0.42; taurine 0.45; choline 0.42; L-carnitine 0.06; beta carotene 0.012; B B-complex 2.5; the normal microelement chelate 3.5 of amino acids; Se-enriched yeast powder 1.5.
2. anti-albumen allergy according to claim 1 and nutrition babies ' formula milk powder is characterized in that: the normal microelement chelate of described amino acids is meant one or several in calcium glycine or calcium aspartate or Methionine calcium salt or Threonine calcium salt or the calcium lysinate.
3. anti-albumen allergy according to claim 1 and nutrition babies ' formula milk powder is characterized in that: the normal microelement chelate of described amino acids is meant one or several in glycine zine or zinc methionine or aspartic acid zinc or threonine zinc or the lysine-zn.
4. anti-albumen allergy according to claim 1 and nutrition babies ' formula milk powder is characterized in that: the normal microelement chelate of described amino acids is meant one or several in cupric glycinate or aspartic acid copper or copper methionine or copper-threonine or the Copper lysinate.
5. anti-albumen allergy according to claim 1 and nutrition babies ' formula milk powder is characterized in that: Se content 1000～2500mg/kg in the described Se-enriched yeast powder.
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