KR100429495B1 - Manufacturing method of Dual-coated Lactic acid bacteria powder using protein and polysaccharlde - Google Patents
Manufacturing method of Dual-coated Lactic acid bacteria powder using protein and polysaccharlde Download PDFInfo
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- KR100429495B1 KR100429495B1 KR10-2001-0010397A KR20010010397A KR100429495B1 KR 100429495 B1 KR100429495 B1 KR 100429495B1 KR 20010010397 A KR20010010397 A KR 20010010397A KR 100429495 B1 KR100429495 B1 KR 100429495B1
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- lactic acid
- protein
- acid bacteria
- aqueous solution
- coating
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 136
- 239000004310 lactic acid Substances 0.000 title claims abstract description 68
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 68
- 241000894006 Bacteria Species 0.000 title claims abstract description 64
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 52
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 52
- 239000000843 powder Substances 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 17
- 230000009977 dual effect Effects 0.000 title 1
- 238000000576 coating method Methods 0.000 claims abstract description 39
- 239000011248 coating agent Substances 0.000 claims abstract description 38
- 150000004676 glycans Chemical class 0.000 claims abstract description 36
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 36
- 239000005017 polysaccharide Substances 0.000 claims abstract description 36
- 239000007864 aqueous solution Substances 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 26
- 238000000855 fermentation Methods 0.000 claims abstract description 24
- 230000004151 fermentation Effects 0.000 claims abstract description 20
- 239000000243 solution Substances 0.000 claims abstract description 20
- 239000002577 cryoprotective agent Substances 0.000 claims abstract description 12
- 235000020183 skimmed milk Nutrition 0.000 claims abstract description 12
- 238000004108 freeze drying Methods 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 8
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 8
- 239000000284 extract Substances 0.000 claims abstract description 8
- 239000008103 glucose Substances 0.000 claims abstract description 8
- 235000013372 meat Nutrition 0.000 claims abstract description 8
- 239000002994 raw material Substances 0.000 claims abstract description 8
- 239000012138 yeast extract Substances 0.000 claims abstract description 8
- 244000068988 Glycine max Species 0.000 claims abstract description 7
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 7
- 108091005804 Peptidases Proteins 0.000 claims abstract description 7
- 230000002797 proteolythic effect Effects 0.000 claims abstract description 4
- 230000001954 sterilising effect Effects 0.000 claims abstract description 4
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 4
- 239000001913 cellulose Substances 0.000 claims description 17
- 229920002678 cellulose Polymers 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229940099596 manganese sulfate Drugs 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- AIHDCSAXVMAMJH-GFBKWZILSA-N levan Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(CO[C@@H]2[C@H]([C@H](O)[C@@](O)(CO)O2)O)O1 AIHDCSAXVMAMJH-GFBKWZILSA-N 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 claims description 4
- MKSMEGFVJFHTAL-UHFFFAOYSA-L dipotassium;diformate Chemical compound [K+].[K+].[O-]C=O.[O-]C=O MKSMEGFVJFHTAL-UHFFFAOYSA-L 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims 1
- 239000012460 protein solution Substances 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 15
- 241000186660 Lactobacillus Species 0.000 abstract description 11
- 229940039696 lactobacillus Drugs 0.000 abstract description 11
- 102000035195 Peptidases Human genes 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 238000000265 homogenisation Methods 0.000 abstract description 2
- 239000013589 supplement Substances 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 39
- 210000004027 cell Anatomy 0.000 description 23
- 239000002253 acid Substances 0.000 description 13
- 210000000941 bile Anatomy 0.000 description 12
- 230000004083 survival effect Effects 0.000 description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 238000003756 stirring Methods 0.000 description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 5
- 229930195725 Mannitol Natural products 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- 230000008014 freezing Effects 0.000 description 5
- 239000000594 mannitol Substances 0.000 description 5
- 235000010355 mannitol Nutrition 0.000 description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000005913 Maltodextrin Substances 0.000 description 4
- 229920002774 Maltodextrin Polymers 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 4
- 229940035034 maltodextrin Drugs 0.000 description 4
- 235000004252 protein component Nutrition 0.000 description 4
- 239000008199 coating composition Substances 0.000 description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000004051 gastric juice Anatomy 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 235000020130 leben Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 108010043393 protease N Proteins 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 241001608472 Bifidobacterium longum Species 0.000 description 2
- 240000001046 Lactobacillus acidophilus Species 0.000 description 2
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 2
- 241000194020 Streptococcus thermophilus Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229940009291 bifidobacterium longum Drugs 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011814 protection agent Substances 0.000 description 2
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000002702 enteric coating Substances 0.000 description 1
- 238000009505 enteric coating Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000008570 general process Effects 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 230000008991 intestinal motility Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 229940066779 peptones Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- 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
Abstract
본 발명은 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법에 관한 것이다. 본 이중코팅 유산균 원말의 제조방법은 1%-10%농도의 대두분리단백과 탈지분유를 소정의 혼합비로 혼합하여 1%-10%수용액으로 만든 후 유산균주에 따라서 대단백질 중량비 0.01%-1%범위의 단백분해 효소용액을 가하여 효소처리하는 단백질효소분해단계와, 상기 효소처리액에 중량비 1%-5%범위의 포도당, 0.1%-1.5%범위의 효모추출물, 0.1%-1.5%범위의 육엑기스, 0.01%-0.1%범위의 이온성분을 첨가하여 용해시키고 발효관에서 스팀살균 후 유산균을 배양하는 유산균 발효단계와, 상기 발효된 발효액을 고속원심분리기에 의해 분리 농축하여 균체를 분리하고 코팅하는 1차 단백질코팅단계와, 상기 회수된 균체에 대비하여 중량비 1%-10%의 동결보호제 성분을 35%-45% 수용액으로 제조하고, 회수균체에 대비하여 1%-10%의 다당류 성분을 1%-10%수용액으로 제조하여 균체, 동결보호제 수용액, 다당류 수용액을 혼합균질화 하여 코팅하고 동결건조하는 2차코팅단계를 포함한다. 이와 같이, 본 발명은 수용액상에서의 균질화 및 동결건조공정만으로 이중코팅된 유산균체를 획득할 수 있어 별도의 설비없이 제조공정이 유지되므로 공정의 융통성 및 호환성이 큰 장점이 있다.The present invention relates to a method for preparing double-coated lactic acid bacteria raw materials using proteins and polysaccharides. This double-coated lactobacillus powder is prepared by mixing 1% -10% soybean isolate protein and skim milk powder in a predetermined mixing ratio to make 1% -10% aqueous solution, and then, according to the lactic acid bacteria, large protein weight ratio 0.01% -1%. A proteolytic step of enzymatic treatment by adding a proteolytic enzyme solution in the range, and glucose in the range of 1% -5% by weight, yeast extract in the range of 0.1% -1.5%, meat in the range of 0.1% -1.5% Lactobacillus fermentation step of dissolving extract by adding ionic component in the range of 0.01% -0.1% and cultivating lactic acid bacteria after steam sterilization in fermentation tube, separating and coating the fermented fermentation broth by high-speed centrifuge The first protein coating step, and prepared by the 35% -45% aqueous solution of a 1% -10% cryoprotectant component by weight relative to the recovered cells, 1% -10% of the polysaccharide component compared to the recovered cells 1 Cells, frozen supplement prepared with% -10% aqueous solution By mixing the homogenized first aqueous solution, the polysaccharide solution includes a second coating step of coating and freeze-dried. As such, the present invention can obtain the double-coated lactic acid cells only by the homogenization and freeze-drying process in an aqueous solution, so the manufacturing process is maintained without a separate facility, and thus the flexibility and compatibility of the process are great.
Description
본 발명은 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법에 관한 것으로써 좀더 상세하게 설명하면 유산균에 단백질과 다당류를 이중코팅하는 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법에 관한 것이다.The present invention relates to a method for producing double-coated lactic acid bacteria using protein and polysaccharide, and more particularly, to a method for preparing double-coated lactic acid bacteria using protein and polysaccharide for double-coating protein and polysaccharide in lactic acid bacteria.
일반적으로 유산균은 장에 정착하여 장관운동 활성화, 유해균 억제, 비타민 및 면역증강 물질 촉진 등 다양한 생리활성 효과를 발휘하는데, 인체의 구조상 사람이 유산균 섭취시 장까지 도달하기 까지에는 위를 거치게 되어있어 위에서 분비되는 위산으로 인해 유산균이 사멸하여 유산균 본래의 생리활성 기능을 발휘하지 못하게 되는 경우가 많다.In general, lactic acid bacteria settle in the intestine and exert various physiological effects such as activation of intestinal motility, suppression of harmful bacteria, promotion of vitamins and immune enhancing substances. Due to the secreted stomach acid, lactic acid bacteria are often killed, so that lactic acid bacteria cannot exert their original biologically active functions.
특히, 종래의 비코팅 유산균 원말의 제조공정은 도 1에서와 같이 유산균 발효, 균체 회수, 동결건조의 과정으로 이루어지는데, 이러한 비코팅 유산균 분말은 공기, 수분, 온도조건에 민감하게 반응하므로 저장 및 유통안정성과 2차 제품 제조시의 가공안정성을 확보하기에 어려움이 있다. 또한 사람 및 동물의 섭취시에는 위산 및 담즙산과의 직접적인 접촉에 의해 급격히 사멸하므로 장내정착성 및 유산균 고유의 효능, 효과를 보장할 수 없는 문제점이 있다.In particular, the manufacturing process of the conventional uncoated lactic acid bacteria raw material is made of a process of fermentation, cell recovery, lyophilization, such as lactic acid bacteria, these uncoated lactobacillus powders are sensitive to air, moisture, temperature conditions, storage and Difficulties in securing distribution stability and processing stability in secondary product manufacturing. In addition, when ingested in humans and animals is rapidly killed by direct contact with gastric acid and bile acid, there is a problem that can not guarantee intestinal fixation and intrinsic efficacy, effect.
이러한 문제점을 해결하기 위해 유산균에 코팅을 하여 사용하게 되었는데, 유산균 코팅 기술로는 캡슐제를 이용한 장용코팅제와 젤라틴, 당류, 껌류 등을 이용한 마이크로캡슐화(Microencapsulation)공정 등이 있는데 유산균체의 회수공정 이후에 코팅제를 투입하는 별도의 코팅공정에 의해 실시되어 진다.In order to solve this problem, the lactic acid bacteria were coated and used. The lactic acid bacteria coating technology includes an enteric coating agent using a capsule and a microencapsulation process using gelatin, sugars, and gums. The coating is carried out by a separate coating process.
종래 유산균 코팅공정은 건조 완료된 유산균 분말에 코팅제를 첨가하여 실시되는 경우가 많으며, 일반적인 공정은 도 1과 같다. 유산균 배양(M1,M2)은 일반적으로 펩톤류, 육엑기스, 효모추출물, 포도당 및 무기이온이 조합된 발효배지를 이용하여 혐기적 발효장치 내에서 이루어진다. 이들 배지 성분은 주로 유산균이 증식하는 데에 이용되는 수용성 성분만으로 구성되어 진다. 유산균체의 회수(M3)는 원심분리 또는 한외여과기를 이용하여 이루어지며 균체와 배양 여액과의 분리 및 균체의 농축만을 목적으로 하고 있다. 급속동결 및 동결건조 과정(M4)에서 유산균의 급격한 사멸이 발생하므로 당류, 아미노산등을 조합한 유산균 동결보호제(M5)를 첨가하여 건조분말화(M6) 시킨다. 이후 유산균 분말에 수용액상의 코팅제 조성물이 가해지고 교반 및 혼합 후 동결 건조되어 진다. 또는 매우 미세한 구형의 비드(Bead) 형성능이 있는 코팅제 조성물이 가해지고 노즐분사에 의해 마이크로캡슐화 공정이 이루어 진다.Conventional lactic acid bacteria coating process is often carried out by adding a coating agent to the dried lactic acid bacteria powder, the general process is as shown in FIG. Lactic acid bacteria culture (M1, M2) is generally carried out in an anaerobic fermentation apparatus using a fermentation medium in combination with peptones, meat extract, yeast extract, glucose and inorganic ions. These media components consist mainly of the water-soluble components used for propagation of lactic acid bacteria. Recovery of the lactic acid cells (M3) is made by centrifugation or ultrafiltration, and is intended only for separation of the cells from the culture filtrate and concentration of the cells. Rapid freezing and freeze-drying process (M4) occurs because the rapid killing of lactic acid bacteria, lactic acid bacteria freeze protection agent (M5) in combination with sugars, amino acids, etc. is added to dry powder (M6). Thereafter, the coating composition of the aqueous phase is added to the lactic acid bacteria powder, and the mixture is freeze-dried after stirring and mixing. Alternatively, a coating composition having a very fine spherical bead formation ability is added and a microencapsulation process is performed by nozzle spraying.
이러한, 종래 유산균 코팅공정은 유산균체의 회수 및 건조분말화 공정 이후에 코팅제의 혼합교반 또는 마이크로캡슐화 공정이 이루어지므로 고가의 코팅제 및 공정추가에 따르는 비용부담이 발생하며 기타 미생물의 혼입이 우려되므로 무균조작에 어려움이 발생할 수 있다. 또한, 액상코팅 후 동결건조과정에서는 우수한 생존률 및 안정성을 확보하기 위해서 동결보호제 및 안정제의 사용이 필요하므로 재료 또는 공정의 충돌이나 중복이 발생하는 문제점이 있다.In the conventional lactic acid bacteria coating process, the mixing and stirring or microencapsulation of the coating agent is carried out after the recovery and drying powder of the lactic acid bacteria, so that the cost burden due to the expensive coating agent and the addition of the process is generated, and other microorganisms are mixed. Difficulty in operation may occur. In addition, the freeze-drying process after the liquid coating requires the use of a cryoprotectant and a stabilizer in order to secure excellent survival rate and stability, so there is a problem in that a collision or duplication of materials or processes occurs.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출한 것으로써, 공기, 수분과의 직접적인 반응이 억제되며 내열성, 내산성, 내담즙성이 강화되어 생균 안정성 및 가공 안정성이 증강된 유산균 원말의 제조 및 이와 관련된 유산균 코팅공정을 단백질 소재를 이용하여 저렴하고 신속한 제조방법에 의해 실시하는 단백질코팅 유산균에 추가적으로 잔탐검(Xantan gum), 셀룰로즈(Cellulose), 리벤(Levan)과 같은 다당류를 균주에 따라서 단독, 또는 혼합 사용하여 2중으로 코팅하는 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법을 제공하는데 그 목적이 있다.The present invention has been made in order to solve the above problems, the direct reaction with air, moisture is suppressed, and heat resistance, acid resistance, bile resistance is enhanced, the production of lactic acid bacteria powder with enhanced viable stability and processing stability and this In addition to protein-coated lactobacillus which performs the related lactobacillus coating process by using protein material inexpensively and quickly, polysaccharides such as Xantan gum, cellulose, and Leben may be used alone, or It is an object of the present invention to provide a method for preparing double-coated lactic acid bacteria by using a double-coated protein and a polysaccharide mixed.
도 1은 종래 코팅 유산균의 제조방법에 따른 공정도이고,1 is a process chart according to the method for preparing a conventional coating lactic acid bacteria,
도 2는 본 발명인 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법에 따른 공정도이고,Figure 2 is a process chart according to the production method of the original double-coated lactic acid bacteria using the present invention proteins and polysaccharides,
도 3은 본 발명인 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법에 따른 실시 예를 나타내는 공정도이다.Figure 3 is a process chart showing an embodiment according to the production method of double-coated lactic acid bacteria using the present invention protein and polysaccharide.
상기 목적은, 본 발명에 따라, 1%-10%농도의 대두분리단백과 탈지분유를 소정의 혼합비로 혼합하여 1%-10%수용액으로 만든 유산균주에 따라서 대단백질 중량비 0.01%-1%범위의 단백분해 효소용액을 가하여 효소처리하는 단백질효소분해단계와, 상기 효소처리액에 중량비 1%-5%범위의 포도당, 0.1%-1.5%범위의 효모추출물, 0.1%-1.5%범위의 육엑기스, 0.01%-0.1%범위의 이온성분을 첨가하여 용해시키고 발효관에서 스팀살균 후 유산균을 배양하는 유산균 발효단계와, 상기 발효된 발효액을 고속원심분리기에 의해 분리 농축하여 균체를 분리하고 동시에 발효액에 존재하는 단백질 침적물 내에 균체를 코팅하는 1차 단백질코팅단계와, 상기 회수된 균체에 대비하여 중량비 1%-10%의 동결보호제 성분을 35%-45% 수용액으로 제조하고, 회수균체에 대비하여 중량비 1%-10%의 다당류 성분을 1%-10% 수용액으로 제조하여 균체, 동결보호제 수용액, 다당류 수용액으로 혼합 균질화하여 코팅하고 동결건조하는 2차코팅단계를 포함하는 것을 특징으로 하는 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법에 의해 달성된다.The above object, according to the present invention, soybean isolate protein of 1% -10% concentration and skim milk powder mixed in a predetermined mixing ratio according to the lactic acid strain made of 1% -10% aqueous solution, large protein weight ratio 0.01% -1% range The proteolytic step of enzymatic treatment by adding the proteolytic enzyme solution of the enzyme, and the glucose treatment in the weight ratio of 1% -5%, yeast extract in the range of 0.1% -1.5%, the meat extract in the range of 0.1% -1.5% , Lactobacillus fermentation step of dissolving by adding ionic components in the range of 0.01% -0.1% and cultivating lactic acid bacteria after steam sterilization in the fermentation tube, and separating and concentrating the fermented fermentation broth by high-speed centrifuge A first protein coating step of coating the cells in the protein deposits present, and prepared by the 35% -45% aqueous solution of a 1% -10% cryoprotectant component by weight relative to the recovered cells, the weight ratio compared to the recovered cells 1% -10% polysaccharides Preparation of double-coated lactic acid bacteria using a protein and a polysaccharide, characterized in that it comprises a secondary coating step of preparing a component in a 1% -10% aqueous solution, mixing and homogenizing the mixture, a homogenizer solution, a polysaccharide solution, and lyophilization. Is achieved by the method.
여기서, 상기 이온성분으로 암모니움 시트레이트, 소디움 아세테이트, 디포타시움 포스테이트, 마그네슘 설페이트, 망간 설페이트, 소디움 클로라이드의 이온성분을 사용하는 것이 바람직하다.Here, it is preferable to use an ionic component of ammonium citrate, sodium acetate, dipotassium formate, magnesium sulfate, manganese sulfate, sodium chloride as the ionic component.
또한, 상기 다당류 수용액으로 잔탐검(Xantan gum), 셀룰로즈(Cellulose), 리벤(Levan)을 사용하는 것이 바람직하다.In addition, it is preferable to use Xantan gum, cellulose (Cellulose), Liben (Levan) as the polysaccharide aqueous solution.
그리고, 상기 1차 단백질코팅단계 이후 동결건조하는 단계를 더 포함하는 것이 바람직하다.And, it is preferable to further include a step of lyophilization after the first protein coating step.
이하, 첨부한 도면인 도 2와 도3을 참고하여 본 발명인 단백질 및 다당류를이용한 이중코팅 유산균 원말의 제조방법을 설명하면 다음과 같다. 도 2는 균체회수 후 단백질 및 다당류를 사용하여 이중코팅 후 동결건조하여 제조하는 이중코팅 원말의 제조공정도이고, 도 3은 단백질 코팅하여 제조된 유산균 원말에 대하여 다당류 수용액을 사용하여 2차 코팅한 후 동결건조하여 제조하는 이중코팅유산균 원말의 제조공정도이다.Hereinafter, with reference to the accompanying drawings, Figures 2 and 3 will be described the method of producing the original double-coated lactic acid bacteria using the protein and the polysaccharide of the present invention. Figure 2 is a manufacturing process of the double-coated raw material prepared by lyophilization after the double coating using the protein and polysaccharides after the cell recovery, Figure 3 is a secondary coating using a polysaccharide aqueous solution for the lactic acid bacteria prepared by protein coating It is a manufacturing process chart of the double-coated lactic acid bacteria raw material prepared by lyophilization.
본 발명인 단백질 및 다당류를 이용한 이중코팅 유산균 원말의 제조방법은 발효배지 안에 포함된 단백질 코팅제에 의해 1차 코팅하고 다당류수용액을 사용하여 다시 코팅하는 이중코팅방법으로 단백질효소분해단계와 유산균발효단계와 1차 단백질코팅단계와 2차 코팅단계로 이루어진다.The present invention is a method of producing a double-coated lactic acid bacteria using protein and polysaccharides is a double coating method of the first coating with a protein coating agent contained in the fermentation medium and recoated using a polysaccharide aqueous solution and a lactic acid bacteria fermentation step and 1 It consists of a secondary protein coating step and a secondary coating step.
상기 단백질효소분해단계(S1)에서는 발효배지로 사용되는 탈지분유와 대두분리단백을 각각 단독으로 또는 혼합하여 1%-10%수용액으로 만든 후 유산균주에 따라서 대단백질 중량비 0.01%-1%범위의 단백분해 효소용액을 가하여 효소처리하게 된다. 여기서, 탈지분유(Skim Milk)와 대두분리단백(Isolated Soy Protein)의 효소처리 가수분해물은 유산균 증식에 이용되는 수용성 또는 저분자량의 펩타이드 성분과 유산균체의 포집 및 코팅에 사용되어지는 반수용성 또는 고분자량의 펩타이드 성분으로 구별된다.In the proteolytic digestion step (S1), the skim milk powder and the soybean separation protein used as fermentation medium, respectively, alone or mixed to make 1% -10% aqueous solution, and then, according to the lactic acid strain, the protein ratio of 0.01% -1% Proteolytic enzyme solution is added to enzymatic treatment. Here, the enzyme-treated hydrolysates of skim milk and isolated soy protein are semi-water soluble or high-molecular weight peptides used for the growth of lactic acid bacteria and semi-water-soluble or high-molecular weights used for the collection and coating of lactic acid bacteria. Molecular weight peptide components.
상기 유산균발효단계(S2)에서는 효소처리액에 중량비 1%-5%범위의 포도당, 0.1%-1.5%범위의 효모추출물, 0.1%-1.5%범위의 육엑기스, 0.01%-0.1%범위의 암모니움 시트레이트, 소디움 아세테이트, 디포타시움 포스테이트, 마그네슘 설페이트, 망간 설페이트, 소디움 클로라이드 등의 이온성분을 첨가하여 용해시키고 혐기적발효관에서 스팀 살균 후 유산균을 배양한다.The lactic acid bacteria fermentation step (S2) in the enzyme treatment solution in the weight ratio of 1% -5% glucose, 0.1% -1.5% range yeast extract, 0.1% -1.5% range meat extract, 0.01% -0.1% range ammonia Ionic components such as um citrate, sodium acetate, dipotassium formate, magnesium sulfate, manganese sulfate and sodium chloride are added to dissolve and culture the lactic acid bacteria after steam sterilization in an anaerobic fermentation tube.
상기 1차 단백질코팅단계(S3)에서는 발효된 발효액을 15,000RPM이상의 고속원심분리기에 의해 분리 및 농축한다. 이때 발효액 중의 잔존 단백질 성분이 균체와 함께 침적되면서 균체를 포집 및 포괄하는 코팅기작이 발생한다. 여기서, 가수분해물 조성은 유산균종의 생리특성에 따라 단백질의 농도 및 효소처리율을 가감함으로써 고농도의 유산균 증식효과를 얻을 수 있고, 급속동결시에는 균체를 포괄하여 아이스크리스탈의 형성으로부터 균체를 방어하므로 동결보호제로서의 효과도 동시에 지니게 된다.In the first protein coating step (S3), the fermented fermentation broth is separated and concentrated by a high-speed centrifuge of 15,000 RPM or more. At this time, as the remaining protein components in the fermentation broth are deposited with the cells, a coating mechanism for collecting and enclosing the cells occurs. Here, the hydrolyzate composition can obtain a high concentration of lactic acid bacteria growth effect by reducing the protein concentration and enzyme treatment rate according to the physiological characteristics of the lactic acid species, and during rapid freezing, it protects the cells from the formation of ice crystals by freezing them, thus freezing them. It also has the effect as a protective agent.
상기 2차 코팅단계(S4)에서는 회수된 균체에 대비하여 동결보호제로써 중량비 1%-10%의 트리할로즈, 말토덱스트린, 만니톨, 탈지분유 성분을 35%-45% 수용액으로 조성하고, 회수균체 대비 1%-10%의 잔탐검(Xantan gum), 셀룰로즈(Cellulose), 리벤(Levan)을 각각 단독으로 또는 혼합으로 사용하여 1%-10% 수용액으로 제조하여 가압살균한 후에 회수균체, 동결보호제 수용액과 혼합하고 교반기에서 교반하여 균질화시킨 후 동결건조한다. 또는 단백질코팅 후 동결건조된 단백질 코팅 유산균 원말에 대비하여 중량비 1%-10%의 트리할로즈, 말토덱스트린, 만니톨, 탈지분유 수용액을 35%-45%의 중량비의 농도로 조성하고 유산균 원말에 대비하여 1%-10%의 잔탐검(Xantan gum), 셀룰로즈(Cellulose), 리벤(Levan)을 각각 단독으로 또는 혼합하여 1%-10% 수용액으로 제조하여, 가압살균 후에 단백질코팅된 유산균 원말과 혼합하여 교반기에서 교반하여 균질화시킨 후 동결건조(S3-1)하여 2차 코팅(S4)한다. 다당류 수용액에 균질화된 단백질코팅 유산균은 동결건조 후 다당류가 보유한 강력한 접착력에 의해 균체사이의 결합이 발생하며 매우 치밀한 구조를 지닌 균괴를 형성하고 원말의 분쇄공정을 거쳐 40-120의 일정한 입도를 갖게 된다.In the second coating step (S4) to prepare the recovered cells in a 35% -45% aqueous solution of trihalose, maltodextrin, mannitol, skim milk powder components of 1% -10% by weight as a cryoprotectant 1% -10% Xantan gum, cellulose (Cellulose) and Leben (Levan) alone or in combination to prepare a 1% -10% aqueous solution and then autoclaved and recovered bacteria, cryoprotectant It is mixed with an aqueous solution, stirred in a stirrer, homogenized, and lyophilized. Alternatively, prepare protein-coated lactobacillus powder, lyophilized, and then prepare a solution of trihalose, maltodextrin, mannitol, and skim milk powder in a weight ratio of 35% -45% in preparation for the lyophilized protein-coated lactobacillus. 1% -10% Xantan gum, cellulose (Cellulose) and Liben (Levan) alone or mixed to prepare a 1% -10% aqueous solution, after the autoclaving and mixed with the protein-coated lactic acid bacteria powder After homogenizing by stirring in a stirrer and lyophilized (S3-1) and secondary coating (S4). Protein-coated lactobacillus homogenized in polysaccharide solution is lyophilized and the binding between cells is caused by strong adhesive force of polysaccharide, and it forms a very compact structure and has a uniform particle size of 40-120 through the original grinding process. .
그림 1. 이중 코팅 유산균 원말의 형상도Figure 1.Schematic diagram of the original double-coated lactobacillus
여기서, 상기 2차 코팅제로 사용되는 다당류의 특성을 살펴보면 특유의 점착성에 의해 각 균체를 결합하여 균괴를 형성시키며 친수성을 지니므로 1차 코팅제인 단백질과의 결합은 물론 기타 수용성의 당류, 아미노산 성분인 동결보호제 및 유산균 안정제와의 병용사용이 가능한 장점이 있다. 특히, 다당류재료는 수용액 상태일 때 산성의 조건에서는 용해도가 극히 낮고 중성 이상의 pH에서는 쉽게 해리, 용출되므로 산성조건에서의 유산균의 안정성 및 장내정착성이 매우 우수해질 수 있다.Here, looking at the properties of the polysaccharide used as the secondary coating agent by combining the respective cells by the unique adhesiveness to form a mass and has a hydrophilic property, as well as other water-soluble sugars, amino acid components of the primary coating agent binding There is an advantage that can be used in combination with cryoprotectant and lactic acid bacteria stabilizer. In particular, the polysaccharide material is very low solubility in acidic conditions in the aqueous solution state and easily dissociated and eluted at a pH higher than neutral so that the stability and intestinal fixation of lactic acid bacteria in acidic conditions can be very excellent.
이러한 2중 코팅방법을 사용하여 제조된 제품에 따른 몇 가지 실시 예를 들면 아래와 같다. 첫째, 단백질 코팅된 Lactobacillus acidophilus CBT-LH원말의 생산은 탈지분유 4Kg, 대두분리단백 2kg을 물 100Kg에 현탁시킨 후 저속 교반기, 온도조절장치, pH조절장치가 장착된 효소처리조에서 55℃ 온도, 초기 pH7.0의 조건에서 단백분해효소제(Amano사 제품, Protease-N) 1.02g을 100ml의 물에 용해시켜서 첨가하고 pH가 6.0으로 감소할 때까지 가수분해하였다. 이 가수분해 용액에 포도당 5Kg, 육엑기스 1Kg, 효모추출물 0.5Kg, 디포타슘 포스페이트 50g, 암모니움 시트레이트 50g, 소디움 아세테이트 50g, 마그네슘 설페이트 10g, 망간 설페이트 10g을 첨가하여 용해시키고 200L 용량의 혐기적 발효관에 이송하여 121℃에서 15분간 살균하고 종균 2L를 접종하여 암모니아로 pH를 6.0으로 유지하면서 12시간 발효하였다. 발효액을 60L/Hr의 유속으로 연속식 원심분리기로 이송시키면서 균체와 잔존 단백성분을 침적시키고 회수하였다. 트리할로즈 1Kg, 만니톨 1Kg, 말토덱스트린 1Kg으로 조성된 동결보호제 수용액 10L를 가압살균하여 제조하고, 10g 잔탐검(Xantan gum), 10g 셀룰로즈(Cellulose), 10g 리벤(Levan)을 용해시킨 다당류 수용액 3L를 가압살균하여 회수균체, 동결보호제 수용액, 다당류 수용액을 혼합한 후 교반기에서 5,000RPM으로 교반하여 균질화시키고 -55℃의 냉동고에서 급속동결한 후 0℃에서 40℃사이의 조작조건에서 동결건조 하였다. 원심분리 침적과정에서 반 수용성의 잔존 단백성분은 균체를 포괄하고 수용성 펩타이드 성분은 균체의 세포벽에 침착하여 단백질코팅을 형성하였으며 다당류 성분은 균체 사이의 결합에 의해 매우 치밀한 구조를 지닌 균괴를 형성하였다. 잔탐검(Xantan gum), 셀룰로즈(Cellulose), 리벤(Levan)의 혼합 조성물에 의한 이중코팅 유산균은 비코팅 유산균에 비하여 우수한 가속시험 안정성, 내산성, 내담즙성을 나타내었다. 이에 따른 시험결과는 아래(표1)와 같다.Some examples of products manufactured using such a double coating method are as follows. First, the production of protein-coated Lactobacillus acidophilus CBT-LH powder was suspended in 4 kg of skim milk powder and 2 kg of soybean isolate in 100 kg of water, followed by 55 ° C in an enzyme treatment tank equipped with a slow stirrer, temperature controller, and pH controller. At the initial pH of 7.0, 1.02 g of protease (Amano, Protease-N) was added in 100 ml of water and hydrolyzed until the pH was reduced to 6.0. To this hydrolysis solution, 5 Kg of glucose, 1 Kg of meat extract, 0.5 Kg of yeast extract, 50 g of dipotassium phosphate, 50 g of ammonium citrate, 50 g of sodium acetate, 10 g of magnesium sulfate and 10 g of manganese sulfate were dissolved and dissolved in 200 L anaerobic fermentation. Transfer to the tube and sterilized for 15 minutes at 121 ℃, inoculated with 2L spawn and fermented for 12 hours while maintaining the pH to 6.0 with ammonia. The fermentation broth was transferred to a continuous centrifuge at a flow rate of 60 L / Hr, and the cells and remaining protein components were deposited and recovered. 10L cryoprotectant solution composed of 1Kg of trihalose, 1Kg of mannitol, and 1Kg of maltodextrin was prepared by autoclaving, and 3L of polysaccharide solution in which 10g Xantan gum, 10g Cellulose, and 10g Liben were dissolved. After autoclaving, the recovered cells, an aqueous solution of a cryoprotectant and an aqueous polysaccharide solution were mixed, homogenized by stirring at 5,000 RPM in a stirrer, and rapidly frozen in a freezer at -55 ° C., and lyophilized at an operating condition between 0 ° C. and 40 ° C. During centrifugal deposition, the semi-soluble residual protein contained the cells and the water-soluble peptide was deposited on the cell walls of the cells to form protein coatings. The polysaccharide component formed a very compact structure by binding between the cells. Double-coated lactic acid bacteria by the mixed composition of Xantan gum, cellulose (Cellulose) and Leben (Levan) showed excellent accelerated test stability, acid resistance, and bile resistance compared to the uncoated lactic acid bacteria. The test results are as follows (Table 1).
표1. 단백질 코팅된 Lactobacillus acidophilus CBT-LH의 내산성, 내담즙성, 가속시험 결과.Table 1. Acid resistance, bile resistance and accelerated test results of protein coated Lactobacillus acidophilus CBT-LH.
둘째, 단백질 코팅된 Streptococcus thermophilus CBT-ST 원말의 생산은 탈지분유 6Kg을 물 100Kg에 현탁시킨 후 저속 교반기, 온도조절장치, pH조절장치가 장착된 효소처리조에서 55℃ 온도, 초기 pH7.0의 조건에서 단백분해효소제(Amano사 제품, Protease-N) 6g을 100ml의 물에 용해시켜서 첨가하고 pH가 6.2로 감소할 때까지 가수분해하였다. 이 가수분해 용액에 포도당 4Kg, 육엑기스 0.5Kg, 효모추출물 0.5Kg, 디포타슘 포스페이트 50g, 암모니움 시트레이트 50g, 소디움 아세테이트 50g, 마그네슘 설페이트 20g, 망간 설페이트 5g을 첨가하여 용해시키고 200L 용량의 혐기적 발효관에 이송하여 121℃에서 15분간 살균하고 종균 2L를 접종하여 암모니아로 pH를 6.0으로 유지하면서 12시간 발효하였다. 발효액을 60L/Hr의 유속으로 연속식 원심분리기에 이송시키면서 균체와 잔존 단백성분을 침적시키고 회수하였다. 트리할로즈 0.5Kg, 만니톨 0.5Kg, 말토텍스트린 0.5Kg, 탈지분유 1.5Kg으로 조성하고 가압살균하여 제조한 동결보호제 수용액 10L와 잔탐검(Xantan gum)15g, 셀룰로즈(Cellulose) 15g을 가하여 용해시키고 가압살균하여 제조한 다당류 수용액 3L를 균체침적물과 혼합하여 교반기에서 5,000RPM으로 교반하여 균질화시키고 -55℃의 냉동고에서 급속동결한 후 0℃에서 40℃사이의 온도에서 동결건조하였다. 탈지분유에 의한 단백질 코팅 및 잔탐검(Xantan gum), 셀룰로즈(Cellulose)혼합조성에 의한 이중코팅 유산균은 비코팅 유산균에 비하여 우수한 가속시험 안정성, 내산성, 내담즙성을 나타내었다.Second, the production of protein-coated Streptococcus thermophilus CBT-ST powder was prepared by suspending 6Kg of skim milk powder in 100Kg of water and then applying a low temperature stirrer, temperature controller, and pH controller to 55 ℃ and initial pH 7.0. Under the conditions, 6 g of protease (Amano, Protease-N) was dissolved in 100 ml of water and added and hydrolyzed until the pH decreased to 6.2. To this hydrolysis solution, glucose 4Kg, meat extract 0.5Kg, yeast extract 0.5Kg, dipotassium phosphate 50g, ammonium citrate 50g, sodium acetate 50g, magnesium sulfate 20g, manganese sulfate 5g were dissolved and dissolved in a 200L anaerobic volume. Transfer to the fermentation tube, sterilized for 15 minutes at 121 ℃, seedling 2L inoculated and fermented for 12 hours while maintaining the pH to 6.0 with ammonia. The fermentation broth was transferred to a continuous centrifuge at a flow rate of 60 L / Hr, and the cells and remaining protein components were deposited and recovered. It is made up of 0.5Kg of trihalose, 0.5Kg of mannitol, 0.5Kg of maltotextile, 1.5Kg of skim milk powder, and dissolved by adding 10L of an aqueous solution of cryoprotectant prepared by autoclaving, 15g of Xantan gum, and 15g of Cellulose. 3L of the polysaccharide aqueous solution prepared by autoclaving was mixed with the cell suspension, homogenized by stirring at 5,000 RPM in a stirrer, and rapidly frozen in a freezer at -55 ° C, and lyophilized at a temperature between 0 ° C and 40 ° C. Protein coated by skim milk powder, double coated lactic acid bacteria by Xantan gum, Cellulose mixture composition showed excellent accelerated test stability, acid resistance, and bile resistance compared to uncoated lactic acid bacteria.
표2. 단백질 코팅된 Streptococcus thermophilus CBT-ST의 내산성, 내담즙성, 가속시험 결과.Table 2. Acid, Bile and Accelerated Test Results of Protein Coated Streptococcus thermophilus CBT-ST.
세째, 단백질 코팅된 Bifidobacterium longum CBT-BG원말의 생산은 대두분리단백 2Kg을 물 100Kg에 현탁시킨 후 저속 교반기, 온도조절장치, pH조절장치가 장착된 효소처리조에서 55℃온도, 초기 pH7.0의 조건에서 단백분해효소제(Amano사 제품, protease-N) 5.4g을 100ml의 물에 용해시켜서 첨가하고 pH가 6.2로 감소할 때까지 가수분해하였다. 이 가수분해 용액에 포도당 4Kg, 육엑기스 0.5Kg, 효모추출물 0.5Kg, 카제인 펩톤 1Kg, 디포타슘 포스페이트 50g, 포타슘 포스페이트 50g, 마그네슘 설페이트 10g, 소디움 클로라이드 1g, 망간 설페이트 10g을 첨가하여 용해시키고 200L용량의 혐기적 발효관에 이송하여 121℃에서 15분간 살균하고 종균 2L를 접종하여 암모니아로 pH를 6.0으로 유지하면서 15시간 발효하였다. 발효액을 60L/Hr의 유속으로 연속식 원심분리기에 이송시키면서 균체와 잔존 단백성분을 침적시키고 회수하여 미리 살균한 트리할로즈 0.5Kg, 만니톨 0.5Kg, 말토덱스트린 0.5Kg, 탈지분유 1.5Kg으로 조성된 동결보호제 수용액 10L를 가하여 교반기에서 5,000RPM으로 교반하여 균질화시키고 -55℃의 냉동고에서 급속동결한 후 0℃에서 40℃사이의 온도에서 동결건조하였다. 유산균 동결건조 분말 100g과 물 3L에 15g의 잔탐검(Xantan gum)과 15g의 셀룰로즈(Cellulose)를 가하여 용해시키고 가압살균한 다당류 코팅 조성물을 혼합한 후 교반기에서 5,000RPM으로 교반하여 균질화시키고 -55℃의 냉동고에서 급속동결한 후 0℃에서 40℃사이의 온도에서 동결건조하였다. 대두분리단백에 의한 단백질 코팅 및 잔탐검(Xantan gum), 셀룰로즈(Cellulose)의 혼합조성에 의한 이중코팅 유산균은 비코팅 유산균에 비하여 우수한 가속시험 안정성, 내산성, 내담즙성을 나타내었다.Third, the production of protein-coated Bifidobacterium longum CBT-BG powder was suspended in 2 kg of soybean isolate protein in 100 kg of water, followed by 55 ° C temperature and initial pH 7.0 in an enzyme treatment tank equipped with a slow stirrer, temperature controller, and pH controller. Under the conditions of 5.4 g of protease (Amano, protease-N) was dissolved in 100 ml of water and added and hydrolyzed until the pH decreased to 6.2. To this hydrolysis solution, glucose 4Kg, meat extract 0.5Kg, yeast extract 0.5Kg, casein peptone 1Kg, dipotassium phosphate 50g, potassium phosphate 50g, magnesium sulfate 10g, sodium chloride 1g, 10g manganese sulfate were added to dissolve and 200L capacity. Transfer to an anaerobic fermentation tube was sterilized for 15 minutes at 121 ℃, seedling 2L inoculated and fermented for 15 hours while maintaining the pH to 6.0 with ammonia. The fermentation broth was transferred to a continuous centrifuge at a flow rate of 60 L / Hr, and the cells and residual protein components were deposited and recovered to sterilize trihalose 0.5Kg, mannitol 0.5Kg, maltodextrin 0.5Kg, and skim milk powder 1.5Kg. 10 L of an aqueous solution of a cryoprotectant was added thereto, homogenized by stirring at 5,000 RPM in a stirrer, and freeze-dried in a freezer at -55 ° C, and lyophilized at a temperature between 0 ° C and 40 ° C. 15 g of Xantan gum and 15 g of cellulose were added to 100 g of lactic acid bacteria lyophilized powder and 3 L of water, and the autoclaved polysaccharide coating composition was mixed and homogenized by stirring at 5,000 RPM in a stirrer. After freezing rapidly in the freezer of lyophilized at a temperature between 0 ℃ to 40 ℃. Double coated lactic acid bacteria by protein coating by soybean isolate, Xantan gum, and cellulose (Cellulose) showed superior accelerated test stability, acid resistance, and bile resistance compared to uncoated lactic acid bacteria.
표3. 단백질 코팅된 Bifidobacterium longum CBT-BG의 내산성, 내담즙성, 가속시험 결과.Table 3. Acid, Bile and Accelerated Test Results of Protein Coated Bifidobacterium longum CBT-BG.
이와 같이 본 발명으로 제조된 이중코팅 유산균 원말은 비코팅 유산균 및 1차 코팅 유산균에 비교하여 매우 우수한 저장 안정성과 내산성, 내담즙성, 내열성을 갖는다.Thus, the double-coated lactic acid bacteria prepared by the present invention has a very excellent storage stability and acid resistance, bile resistance, heat resistance compared to the uncoated lactic acid bacteria and the primary coating lactic acid bacteria.
본 발명은 균주에 따라서 여러 종류의 동결보호제 및 안정제 등과 혼합하여 사용이 가능하고 수용액상에서의 균질화 및 동결건조공정만으로 이중코팅된 유산균체를 획득할 수 있으므로 별도의 설비사용이 없이 제조공정이 유지될 수 있으며 건조 전의 회수 균체 또는 건조 균체에 모두 적용 할 수 있어 공정의 융통성 및 호환성이 크며, 균체의 생존율은 50%-90%로 유산균 원말의 회수율이 특히 개선되어질 수 있어 생산성이 향상되며, 이 공정에 의해 생산되는 이중코팅 유산균 원말은 내열성, 내산성, 내담즙성, 저장안정성이 크게 개선되고, 인체에 투여시에는 장내정착성이 매우 우수하여 유산균 고유의 생리활성 기능의 발휘가 탁월하므로 사용자의 사용상 효율성을 극대화한다.The present invention can be used by mixing with various kinds of freeze protection agent and stabilizer according to the strain and can obtain the double-coated lactobacillus only by homogenization and freeze-drying process in aqueous solution, so that the manufacturing process can be maintained without the use of additional equipment. It can be applied to both recovered and dried cells before drying, so the flexibility and compatibility of the process is great, and the survival rate of the cells is 50% -90%, so the recovery rate of the lactic acid bacteria can be especially improved, which improves productivity. The double-coated lactic acid bacteria produced by the raw material is greatly improved in heat resistance, acid resistance, bile resistance, and storage stability. Maximize your efficiency.
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| WO2006122965A1 (en) | 2005-05-18 | 2006-11-23 | Dsm Ip Assets B.V. | Compositions for enteral application of microorganisms |
| KR101000364B1 (en) | 2010-04-26 | 2010-12-13 | 고려대학교 산학협력단 | Double-coating methods for enhancing viability |
| CN104611256A (en) * | 2014-12-17 | 2015-05-13 | 光明乳业股份有限公司 | Microbial freeze-dried protective agent and preparation method and application thereof |
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| KR100493354B1 (en) * | 2002-08-26 | 2005-06-07 | 한국생명공학연구원 | Powder of lactic acid bacteria coated with levan and alginate bead having improved survival ability and method of preparing the same |
| US20040247580A1 (en) | 2003-06-06 | 2004-12-09 | Myung-Jun Chung | Process for preparing double-coated lactic acid bacteria powder using protein and polysaccharide and product by the same |
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| KR101355003B1 (en) | 2010-11-02 | 2014-01-24 | 정명준 | Lactic acid bacteria having multi coating layers and preparing method thereof |
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