CN113637609A - 一种降低乳蛋白抗原性的植物乳杆菌 - Google Patents
一种降低乳蛋白抗原性的植物乳杆菌 Download PDFInfo
- Publication number
- CN113637609A CN113637609A CN202110944351.8A CN202110944351A CN113637609A CN 113637609 A CN113637609 A CN 113637609A CN 202110944351 A CN202110944351 A CN 202110944351A CN 113637609 A CN113637609 A CN 113637609A
- Authority
- CN
- China
- Prior art keywords
- milk
- lactobacillus plantarum
- protein
- milk protein
- strains
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 102000014171 Milk Proteins Human genes 0.000 title claims abstract description 40
- 108010011756 Milk Proteins Proteins 0.000 title claims abstract description 40
- 235000021239 milk protein Nutrition 0.000 title claims abstract description 40
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 20
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 20
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 20
- 102000011632 Caseins Human genes 0.000 claims abstract description 14
- 108010076119 Caseins Proteins 0.000 claims abstract description 14
- 235000020247 cow milk Nutrition 0.000 claims abstract description 10
- 102000008192 Lactoglobulins Human genes 0.000 claims abstract description 9
- 108010060630 Lactoglobulins Proteins 0.000 claims abstract description 9
- 235000021249 α-casein Nutrition 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims description 3
- 235000013336 milk Nutrition 0.000 abstract description 14
- 239000008267 milk Substances 0.000 abstract description 14
- 210000004080 milk Anatomy 0.000 abstract description 14
- 235000013365 dairy product Nutrition 0.000 abstract description 11
- 235000021001 fermented dairy product Nutrition 0.000 abstract description 2
- 238000012545 processing Methods 0.000 abstract description 2
- 235000013601 eggs Nutrition 0.000 abstract 1
- 235000020183 skimmed milk Nutrition 0.000 description 31
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 20
- 238000000034 method Methods 0.000 description 17
- 238000012216 screening Methods 0.000 description 16
- 241000894006 Bacteria Species 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 13
- 208000009793 Milk Hypersensitivity Diseases 0.000 description 11
- 201000010859 Milk allergy Diseases 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 10
- 235000014655 lactic acid Nutrition 0.000 description 10
- 239000004310 lactic acid Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 238000000855 fermentation Methods 0.000 description 9
- 230000004151 fermentation Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000000427 antigen Substances 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 230000007062 hydrolysis Effects 0.000 description 7
- 238000006460 hydrolysis reaction Methods 0.000 description 7
- 230000008520 organization Effects 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000013566 allergen Substances 0.000 description 6
- 235000015140 cultured milk Nutrition 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 6
- 206010020751 Hypersensitivity Diseases 0.000 description 5
- 208000026935 allergic disease Diseases 0.000 description 5
- 230000007815 allergy Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 239000012460 protein solution Substances 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 108090000942 Lactalbumin Proteins 0.000 description 3
- 102000004407 Lactalbumin Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000006041 probiotic Substances 0.000 description 3
- 230000000529 probiotic effect Effects 0.000 description 3
- 235000018291 probiotics Nutrition 0.000 description 3
- 230000017854 proteolysis Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 235000019640 taste Nutrition 0.000 description 3
- 235000021241 α-lactalbumin Nutrition 0.000 description 3
- 235000021247 β-casein Nutrition 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 101710089384 Extracellular protease Proteins 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000005903 acid hydrolysis reaction Methods 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000000172 allergic effect Effects 0.000 description 2
- 230000027645 antigenic variation Effects 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 2
- 235000021240 caseins Nutrition 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 235000013350 formula milk Nutrition 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000003118 sandwich ELISA Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 235000021119 whey protein Nutrition 0.000 description 2
- 206010002199 Anaphylactic shock Diseases 0.000 description 1
- 108050008461 Beta-lactoglobulin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101000798100 Bos taurus Lactotransferrin Proteins 0.000 description 1
- 102100037840 Dehydrogenase/reductase SDR family member 2, mitochondrial Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016946 Food allergy Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000010445 Lactoferrin Human genes 0.000 description 1
- 108010063045 Lactoferrin Proteins 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101710188053 Protein D Proteins 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 101710132893 Resolvase Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000002009 allergenic effect Effects 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019658 bitter taste Nutrition 0.000 description 1
- 229910021538 borax Inorganic materials 0.000 description 1
- 229940072440 bovine lactoferrin Drugs 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 201000005356 cow milk allergy Diseases 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 235000014048 cultured milk product Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- UQGFMSUEHSUPRD-UHFFFAOYSA-N disodium;3,7-dioxido-2,4,6,8,9-pentaoxa-1,3,5,7-tetraborabicyclo[3.3.1]nonane Chemical compound [Na+].[Na+].O1B([O-])OB2OB([O-])OB1O2 UQGFMSUEHSUPRD-UHFFFAOYSA-N 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
- 235000021242 lactoferrin Nutrition 0.000 description 1
- 229940078795 lactoferrin Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229940054441 o-phthalaldehyde Drugs 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 239000004328 sodium tetraborate Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
- 235000021246 κ-casein Nutrition 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Dairy Products (AREA)
Abstract
本发明属于发酵乳制品加工技术领域,具体涉及一株降低牛乳中乳蛋抗原性的植物乳杆菌及其应用。该植物乳杆菌7‑2于2020年11月30日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO.M20200816。植物乳杆菌7‑2能够降低α‑酪蛋白和β‑乳球蛋白抗原性,从而降低乳制品中乳蛋白致敏性。
Description
技术领域
本发明属于发酵乳制品加工技术领域,具体涉及一株降低牛乳中乳蛋白抗原性的植物乳杆菌及其应用。
背景技术
乳制品中含有人体必需的氨基酸、丰富的钙、磷是作为人类蛋白质的重要来源。现在,乳制品是人类不可缺少的营养制品,尤其对于婴幼儿来说,牛乳是除了母乳之外的重要营养物质。然而,流行病学的研究结果显示,乳制品也是引起过敏的食物之一,其中乳蛋白是主要过敏原,且在学前儿童中最为普遍,发生率约为1%~17.5%(Fiocchi A等,WorldAllergy Organization(WAO)Diagnosis and Rationale for Action against Cow'sMilk Allergy(DRACMA) Guidelines[J].World Allergy Organization Journal,2010,3(4):57-161.),大约有2%-3%的婴儿在 1岁时食用乳制品时会发生乳蛋白过敏现象(Saarinen等,Clinical course and prognosis of cow's milk allergy are dependedon milk-specific Ig-E status[J].Journal of Allergy and Clinical Immunology,2005,116(4):869-875.),涉及皮肤、胃肠道、呼吸系统等,导致婴幼儿出现荨麻疹,哮喘,鼻炎和过敏性休克等严重不良临床反应。
避免食用牛乳及其制品是可靠的解决牛乳过敏方案,但婴幼儿不食用乳蛋白会导致营养不良和生长缓慢。目前,虽然临床上用不同配方蛋白水解物替代牛乳,也能够减少牛乳过敏的几率,但水解蛋白婴儿配方粉的苦味和婴儿生长缓慢等营养等问题仍未解决,所以解决乳制品中过敏源问题迫在眉睫。现阶段,对原料乳进行热加工、酶处理(水解、交联等)、高压、微波、超声、糖基化反应(Aguiar S S J等,Control of milk allergenicity[J].Journal of Dairy& Veterinary Sciences,2017,2(5).),但这些处理方式虽然能够降低乳蛋白的过敏原,但是都存在不可避免的缺陷,例如,在破坏现有过敏表位的同时产生新的表位、蛋白变性、影响营养价值等。相比较而言,其中蛋白酶水解乳蛋白降敏效果较好,但是会产生苦味肽,影响口感和风味,发酵乳品可以控制口感和风味,又能有效降低致敏性,可以突破其他降敏方法的局限性。目前,发酵乳已经由营养阶段进入功能阶段,但大多数发酵剂菌种多为国外垄断,我国益生乳酸菌目前仍处在开发阶段,而且多集中于调节肠道和增强免疫等方面(于洁,张和平. 益生菌发酵乳的研究及产业化进展[J].中国食品学报,2020,20(10):1-7.),所以筛选、开发新型益生乳酸菌是有必要的。
鲜牛乳中蛋白质的含量约为3-4g/100mL,其中至少含有25种不同的蛋白质,所有这些蛋白质都可能作为抗原。乳蛋白分为酪蛋白和乳清蛋白两大类,分别为酪蛋白(αs1-酪蛋白 (αs1-CN)、αs2-酪蛋白(αs2-CN)、β-酪蛋白(β-CN)和κ-酪蛋白(κ-CN))和乳清蛋白(主要包括β-乳球蛋白(β-LG)、α-乳清蛋白(α-LA)、牛乳铁蛋白(LF)、牛血清白蛋白(BSA) 和牛免疫球蛋白(Ig)等)。目前,能够引起婴幼儿主要的过敏现象的蛋白α-CN、β-CN、β-LG 和α-LA。
乳酸菌(Lactic Acid Bacteria,LAB)是指是一类能利用可发酵碳水化合物产生大量乳酸的细菌的统称。乳酸菌可以促进蛋白质、单糖、钙、镁等营养物质的吸收,具有调节肠道菌群和提高免疫等功能,已经广泛应用于食品、酶制剂等方面中。乳酸菌发酵是有效降低乳蛋白致敏性的方式之一。经乳酸菌发酵的乳制品不但保证了乳中的营养成,而且具有口感好、易吸收、调节免疫和安全可靠的优势。目前,降低牛乳中过敏原仍是一个尚待解决的问题,所以筛选一株能够降低牛乳中过敏原性的乳酸菌是极其必要的。
发明内容
本发明要解决的技术问题是提供了一株降解乳蛋白过敏原性的菌株7-2,命名为植物乳杆菌(Lactobacillus plantarum)7-2,于2020年11月30日保藏于中国典型培养物保藏中心,保藏编号为CCTCC No:M 2020806,保藏地址为:中国武汉,武汉大学。
所述植物乳杆菌7-2能够降低牛乳过敏原α-酪蛋白和β-乳球蛋白的致敏性。
本发明还提供了一种应用所述植物乳杆菌降低乳蛋白抗原性的条件:培养环境为11%的脱脂牛乳(蛋白质含量约为3.5-3.6g/100g),接种量为2%,37℃下培养18h。
本发明所述的植物乳杆菌(Lactobacillus plantarum)7-2的筛选方法包括以下步骤:
(1)收集新疆北疆地区的不同农家乳制品在脱脂乳培养基中培养,挑选菌落周围有蛋白水解圈的革兰氏阳性菌株在MRS培养基上培养得到备用菌株;备用菌株接种于MRS液体培养基培养,培养后注入打孔的脱脂乳培养基中,筛选出可能产胞外蛋白酶的菌株;
(2)步骤(1)中筛选的菌株接种于脱脂乳中,测定不同菌株发酵的乳样品的游离氨基酸含量,筛选出氨基酸水解能力强的菌株;
(3)步骤(2)筛选出的菌株接种于脱脂乳中,发酵至凝乳,测定可溶性蛋白含量和乳蛋白水解情况;
(4)步骤(2)筛选出的菌株分别接种于不同乳蛋白溶液中,通过双抗夹心ELISA法进行抗原含量分析;
(5)综合步骤(3)和步骤(4)中可溶性蛋白含量低、乳蛋白水解能力强、降低α-酪蛋白和β-乳球蛋白抗原含量,最终筛选出所需菌株。
上述筛选方法中,进一步地,步骤(4)中乳蛋白溶液分别是α-乳白蛋白,β-乳球蛋白,α-酪蛋白和β-酪蛋白,α-乳白蛋白浓度为1.6mg/mL,β-乳球蛋白浓度为4.0mg/mL,α-酪蛋白浓度为15.36mg/mL,β-酪蛋白浓度为12.48mg/mL。
上述筛选方法中,进一步地,步骤(2)和步骤(3)的脱脂乳为乳蛋白含量为3.5-3.6g/100g 的脱脂乳。
上述筛选方法中,进一步地,步骤(1)在脱脂乳培养基37℃恒温培养48-72h,所述脱脂乳培养基为:乳蛋白含量(0.70-0.72)g/100g的脱脂乳液和3.0%(w/v)琼脂溶液等体积混合。
上述筛选方法中,进一步地,步骤(1)中,在打孔的脱脂乳培养基中37℃孵育24h。
上述筛选方法中,进一步地,步骤(1)中,在MRS培养基上培养条件为37℃下培养18h,菌株接种量2%。
与现有技术相比,本发明的有益效果:本发明方法筛选出的植物乳杆菌(Lactobacillus plantarum)7-2,能够降低α-酪蛋白和β-乳球蛋白抗原性,从而降低乳制品中乳蛋白致敏性。
附图说明
图1是本发明植物乳杆菌7-2的菌落形态图;
图2是本发明植物乳杆菌7-2的显微镜图(100×10);
图3为部分菌株水解乳蛋白的情况(左图:脱脂乳固体培养基,右图:部分菌株对于乳蛋白的水解圈)
图4为55株乳酸菌发酵11%脱脂乳24h后,发酵乳样品中游离氨基酸的含量(仅显示水解能力较强的10株菌的水解能力)。
图5为脱脂乳不同菌株发酵至凝乳时主要可溶性乳蛋白图谱(由上至下分别为:经菌株M8-7、7-2和6-1发酵的脱脂乳以及未经发酵的脱脂乳)。
图6为脱脂乳经不同菌株发酵至凝乳时主要乳蛋白水解情况(M:marker;M8-7:经菌株M8-7发酵后脱脂乳;7-2:经菌株7-2发酵的脱脂乳;6-1:经菌株6-1发酵的脱脂乳;K:未发酵脱脂乳)
图7为牛乳中不同乳蛋白经三株菌发酵后抗原性的变化情况(同种蛋白,经不同菌株发酵抗原含量变化差异性由不同小写字母表示,p<0.05)。
图8为植物乳杆菌7-2的16S rDNA序列进化树。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
游离氨基酸测定方法:采用邻苯二甲醛(OPA)法。(参考文献:Church F C,ProterD H, et al.Spectrophotometric assay using o-phthalaldyhyde for determinationof proteolysis in milk and isolated milk protein[J].Journal of Dairy Science,1983,66(6):1219-1227.)
可溶性蛋白测定方法:采用反向高效液相色谱(RP-HPLC)法。(参考文献:王浩,张志国,常彦忠,段相林,赵述强,张楠,石振华,WANGHao,ZHANGZhi-guo,CHANGYan-zhong,DUANXiang-lin,ZHAOShu-qiang,ZHANGNan,SHIZhen-hua.RP-HPLC法对乳制品中主要牛奶蛋白的分离及定量测定[J].食品科学,2009,30(24):376-380.)
乳蛋白水解情况的分析:采用SDS-PAGE电泳。(参考文献:郭尧君.蛋白质电泳实验技术(第2版)[M].科学出版社,2005.)
实施例1产胞外蛋白酶菌株的筛选
(1)样品:新疆北疆地区的不同农家乳制品;
(2)配制生理盐水(0.85%NaCl),并于121℃下灭菌20min;
(3)配制脱脂乳固体培养基(乳蛋白含量0.36g/100g):配制0.72g/100g的脱脂乳,105℃灭菌15min;3.0%(w/v)琼脂溶于水中,121℃灭菌20min;趁热等体积混合上述两种溶液,制备平板。
(4)配制MRS液体和固体培养基,121℃灭菌20min;
(5)取步骤(1)中不同样品各1mL,与步骤(2)制备的生理盐水按照1:9混合,震荡混匀;
(6)对步骤(5)获得重悬液,采用10倍梯度稀释法依次稀释,根据样品活菌数的差异,稀释到合适的梯度。
(7)取步骤(6)的稀释液100μL,加入步骤(3)制备的脱脂乳固体培养基平板中,用无菌涂布棒涂布均匀,置于37℃恒温培养箱倒置培养48-72h。
(8)挑取菌落周围有蛋白水解圈的所有革兰氏阳性菌株,在步骤(4)所述MRS固体培养基上进行反复划线纯化培养,获得备用菌株;
(9)将步骤(8)获得的菌株以2%(v/v)接种量接种于步骤(4)所述的MRS液体培养基中,37℃培养18h;
(10)使用无菌牛津杯对步骤(3)制备的脱脂乳培养基进行打孔,并取100μL步骤(9)所述菌株培养液注入孔中,37℃孵育24h;筛选出55株具有明显水解圈的菌株进行后续的筛选实验。
实施例2高效水解乳蛋白菌株的筛选
(1)配制乳蛋白含量约3.6g/100g的脱脂乳,并于105℃下灭菌15min。
(2)将实施例1中的筛选出的55株乳酸菌以2%接种量(v/v),接种于步骤(1)中的脱脂乳中,于37℃下发酵24h。
(3)测定步骤(2)获得的不同菌株发酵的乳样品的游离氨基酸含量。游离氨基酸含量测定:采用邻苯二甲醛(OPA)法测定样品中游离氨基酸的含量。取5mL样品与1mL的蒸馏水和10mL三氯乙酸混匀,3000rpm下离心5min。取上清液150μL加入3mL OPA试剂(50mL 0.1mM四硼酸钠,5mL 20%十二烷基硫酸钠,80mg邻苯二甲醛,2mL甲醇,200μLβ-巯基乙醇,200mL蒸馏水),25℃下,静置时反应2min,测量其在340nm处吸光度。对照组不加入发酵样品。
标准酪氨酸含量曲线方程:Y=1.2162x+0.0262(R2=0.9916)。
(4)根据步骤(3)的结果,筛选出氨基酸水解能力强的菌株,分别是7-2,6-1和M8-7,用于后续筛选实验。经三株菌发酵的脱脂乳中,测的氨基酸含量分别为0.295mg/mL、0.272 mg/mL和0.255mg/mL,即最优的是7-2。
实施例3可溶性蛋白含量的测定
(1)配制乳蛋白含量约3.6g/100g的脱脂乳,并于105℃下灭菌15min。
(2)将实施例2筛选出的三株菌均以2%接种量(v/v),接种于步骤(1)中的脱脂乳中,于37℃下发酵至凝乳。
(3)通过反相高效液相色谱法分析步骤(2)获得发酵乳中的水溶性蛋白。取400μL发酵乳样品加入到1600μL处理液(6mol/L盐酸胍溶液,内含0.1mmM EDTA-Na2,pH 6.0)中,充分混匀至没有明显泡沫出现。室温孵育90min。上机前用0.45μm尼龙滤膜过滤。
其色谱条件如下:
色谱柱:ZORBAX 300SB-C18,
柱温:35℃,
流速:1mL/min,
进样量:20μL,
检测波长:214nm,
洗脱时间:40min,
流动相组成:A的组成10%乙腈+90%去离子水+0.1%TFA,
B的组成90%乙腈+10%去离子水+0.1%TFA,
流动相B梯度(变化率)如下:从28.5%到35.5%洗脱10min(0.70%B/min),从35.5%到 37%洗脱6min(0.25%B/min),从37.0%到37.0%洗脱6min(0%B/min),从37.0%到38.0%洗脱2min(0.5%B/min),从38.0%到38.0%洗脱2min(0%B/min),从38.0%到47.0%洗脱15 min(0.6%B/min)。
(4)对步骤(3)获得的图谱进行分析,结果表明,经三株菌发酵的脱脂乳中,可溶性乳蛋白含量明显降低,且菌株7-2发酵样品中的含量整体要少于另外两组。
实施例4乳蛋白水解情况的分析
(1)同实施例3(1)。
(2)同实施例3(2)。
(3)通过SDS-PAGE分析步骤(2)获得发酵乳中乳蛋白的水解情况。
(4)对步骤(3)获得的SDS-PAGE电泳条带进行分析,结果表明,经三株菌发酵的脱脂乳中,乳蛋白受到不同程度的水解,其中菌株7-2水解的能力较强。
实施例5乳蛋白抗原变化的分析
(1)配制不同乳蛋白溶液,α-LA(1.6mg/mL),β-LG(4.0mg/mL),α-CN(15.36mg/mL),β-CN(12.48mg/mL),并于105℃下灭菌15min。
(2)将实施例2筛选出的三株菌均以2%接种量(v/v),接种于步骤(1)中的乳蛋白溶液中,于37℃下发酵18h。
(3)将步骤(1)制备的乳蛋白和步骤(2)所述乳蛋白发酵产物进行稀释,稀释倍数如下:α-LA及其发酵产物稀释1×104倍,β-LG及其发酵产物稀释2×105倍,α-CN及其发酵产物稀释1×108倍,β-CN及其发酵产物稀释1×105倍。
(4)通过双抗夹心ELISA法(试剂盒)对步骤(3)获得的不同乳蛋白及其发酵产物稀释液进行抗原含量分析。结果显示,三株菌均对乳蛋白的抗原含量有影响,但菌株7-2能够有效地降低主要过敏原α-酪蛋白和β-乳球蛋白的抗原含量。
综合实施例3、4、5的实验分析,最终确定7-2为所需菌株,经16S rDNA分析,结果确定为植物乳杆菌,命名为植物乳杆菌(Lactobacillus plantarum)7-2。
对于任何熟悉本领域的技术人员而言,在不脱离本发明技术方案范围情况下,都可利用上述揭示的技术内容对本发明技术方案作出许多可能的变动和修饰,或修改为等同变化的等效实施例。因此,凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化及修饰,均应仍属于本发明技术方案保护的范围内。
Claims (2)
1.一株降低乳蛋白抗原性的植物乳杆菌(Lactobacillusplantarum)7-2,其保藏编号为:CCTCCNo.M20208067-2。
2.权利要求1所述的植物乳杆菌在降低牛乳中α-酪蛋白和β-乳球蛋白抗原性的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110944351.8A CN113637609A (zh) | 2021-08-17 | 2021-08-17 | 一种降低乳蛋白抗原性的植物乳杆菌 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110944351.8A CN113637609A (zh) | 2021-08-17 | 2021-08-17 | 一种降低乳蛋白抗原性的植物乳杆菌 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113637609A true CN113637609A (zh) | 2021-11-12 |
Family
ID=78422444
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110944351.8A Pending CN113637609A (zh) | 2021-08-17 | 2021-08-17 | 一种降低乳蛋白抗原性的植物乳杆菌 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113637609A (zh) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012014971A1 (ja) * | 2010-07-30 | 2012-02-02 | 株式会社明治 | メタボリックシンドローム改善効果を有する乳酸菌 |
US20140037792A1 (en) * | 2011-02-25 | 2014-02-06 | Bio-Competence Centre of Healthy Dairy Products | Isolated microorganism strains Lactobacillus plantarum MCC1 DSM 23881 and Lactobacillus gasseri MCC2 DSM 23882 and their use |
WO2015013932A1 (en) * | 2013-07-31 | 2015-02-05 | Meiji Co., Ltd. | Nutritional composition for inhibiting growth of tumor |
CN109837219A (zh) * | 2017-11-24 | 2019-06-04 | 江南大学 | 一种水解牛乳过敏源β-乳球蛋白蛋白酶的分离纯化及应用 |
-
2021
- 2021-08-17 CN CN202110944351.8A patent/CN113637609A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012014971A1 (ja) * | 2010-07-30 | 2012-02-02 | 株式会社明治 | メタボリックシンドローム改善効果を有する乳酸菌 |
US20140037792A1 (en) * | 2011-02-25 | 2014-02-06 | Bio-Competence Centre of Healthy Dairy Products | Isolated microorganism strains Lactobacillus plantarum MCC1 DSM 23881 and Lactobacillus gasseri MCC2 DSM 23882 and their use |
CN103649304A (zh) * | 2011-02-25 | 2014-03-19 | 保健乳制品生物免疫中心 | 分离的微生物菌株植物乳杆菌(lactobacillus plantarum)mcc1 dsm 23881和格氏乳杆菌(lactobacillus gasseri)mcc2 dsm 23882及其用途 |
WO2015013932A1 (en) * | 2013-07-31 | 2015-02-05 | Meiji Co., Ltd. | Nutritional composition for inhibiting growth of tumor |
CN109837219A (zh) * | 2017-11-24 | 2019-06-04 | 江南大学 | 一种水解牛乳过敏源β-乳球蛋白蛋白酶的分离纯化及应用 |
Non-Patent Citations (2)
Title |
---|
许倩: "不同加工处理对牛乳蛋白抗原性及过敏原性的影响", 《中国优秀博士学位论文全文数据库(电子期刊)》 * |
许倩等: "乳酸杆菌粗酶液对浓缩乳蛋白抗原性及过敏原性的影响", 《中国科技论文》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111631261A (zh) | 一种基于降低牛乳致敏性的液体乳制备方法 | |
CN109517763B (zh) | 瑞士乳杆菌h11及其应用 | |
CN109287749B (zh) | 一种富含活性植物乳杆菌的双蛋白发酵乳及其制备方法 | |
CN112626155B (zh) | 一种豌豆肽的制备方法 | |
Kinariwala et al. | Exploring the potentiality of lactobacillus cultures on the production of milk-derived bioactive peptides with antidiabetic activity | |
CN100502699C (zh) | 蛋乳生物多肽果蔬汁乳酒饮料的制备方法 | |
CN105230783A (zh) | 一种鱼骨发酵乳及其制备方法 | |
CN111903761A (zh) | 一种具有抗氧化功能酸奶及其制备方法 | |
NZ236295A (en) | Immunostimulant containing n-acetyl muramylpeptides derived from cell walls of lactic acid bacteria using lysozyme | |
CN105558091B (zh) | 一种澄清透明的乳清发酵液的制备方法 | |
CN102781261A (zh) | 通过柠檬酸钠的添加、加热处理及乳酸发酵来使鸡蛋蛋白质的抗原性降低的乳酸发酵鸡蛋及其制造方法 | |
CN112075529A (zh) | 一种酶解乳清蛋白粉制备抗氧化肽粉的方法及在奶茶粉中的应用 | |
CN106455601B (zh) | 酸奶的生产 | |
CN113637609A (zh) | 一种降低乳蛋白抗原性的植物乳杆菌 | |
CN106722382A (zh) | 一种利用微生物降低鱼类过敏原活性的方法 | |
KR101498226B1 (ko) | 유청과 표고버섯 자실체 추출물의 혼합 발효를 통한 기능성 음료의 제조방법 | |
KR101381090B1 (ko) | 고초균과 젖산균을 이용한 이단발효를 통한 gaba와 펩타이드가 증진된 발효유 제조방법 | |
CN105483201B (zh) | 乳清抗氧化肽的发酵制备方法 | |
CN109837219A (zh) | 一种水解牛乳过敏源β-乳球蛋白蛋白酶的分离纯化及应用 | |
KR20080068702A (ko) | 셀리악병에 의한 영향을 받는 사람을 위한 “무글루텐”블루바인 유제품 | |
CN109984287B (zh) | 小龙虾过敏原性消减的食品加工方法 | |
Hidayat et al. | Molecular identification 16S rRNA gene of active proteolytic lactic acid bacteria (LAB) isolated from kelengkeng (Dimocarpus longan) fruit | |
Wang et al. | Directed‐Vat‐Set starter producing ACE‐inhibitory peptides: Optimization and evaluation of stability | |
CN113068762A (zh) | 一种乳清蛋白的制备方法 | |
CN110226666A (zh) | 利用超声预处理和马克思克鲁维酵母发酵联用降低乳清蛋白致敏性的方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211112 |
|
RJ01 | Rejection of invention patent application after publication |