KR102607676B1 - Probiotics composition for companion animals comprising aerotolerant Bifidobacterium strain as effective component and production method thereof - Google Patents
Probiotics composition for companion animals comprising aerotolerant Bifidobacterium strain as effective component and production method thereof Download PDFInfo
- Publication number
- KR102607676B1 KR102607676B1 KR1020200167269A KR20200167269A KR102607676B1 KR 102607676 B1 KR102607676 B1 KR 102607676B1 KR 1020200167269 A KR1020200167269 A KR 1020200167269A KR 20200167269 A KR20200167269 A KR 20200167269A KR 102607676 B1 KR102607676 B1 KR 102607676B1
- Authority
- KR
- South Korea
- Prior art keywords
- bifidobacterium longum
- freeze
- cfu
- companion animals
- strain
- Prior art date
Links
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 239000006041 probiotic Substances 0.000 title abstract description 34
- 235000018291 probiotics Nutrition 0.000 title abstract description 34
- 241001465754 Metazoa Species 0.000 title abstract description 28
- 239000000203 mixture Substances 0.000 title abstract description 18
- 241000186000 Bifidobacterium Species 0.000 title abstract description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 51
- 241001608472 Bifidobacterium longum Species 0.000 claims description 42
- 229940009291 bifidobacterium longum Drugs 0.000 claims description 42
- 239000000843 powder Substances 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 17
- 230000001965 increasing effect Effects 0.000 claims description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 13
- 238000004108 freeze drying Methods 0.000 claims description 9
- 235000020183 skimmed milk Nutrition 0.000 claims 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 description 7
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 239000002577 cryoprotective agent Substances 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 claims description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 6
- 239000004223 monosodium glutamate Substances 0.000 claims description 6
- 239000010690 paraffinic oil Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 239000013028 medium composition Substances 0.000 claims description 4
- 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 claims description 3
- 229930195725 Mannitol Natural products 0.000 claims description 3
- 239000000594 mannitol Substances 0.000 claims description 3
- 235000010355 mannitol Nutrition 0.000 claims description 3
- 239000008176 lyophilized powder Substances 0.000 claims 1
- 230000000529 probiotic effect Effects 0.000 abstract description 25
- 241000894006 Bacteria Species 0.000 abstract description 23
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 18
- 239000004310 lactic acid Substances 0.000 abstract description 9
- 235000014655 lactic acid Nutrition 0.000 abstract description 9
- 239000004480 active ingredient Substances 0.000 abstract description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 8
- 239000001301 oxygen Substances 0.000 abstract description 8
- 229910052760 oxygen Inorganic materials 0.000 abstract description 8
- 241000588724 Escherichia coli Species 0.000 abstract description 4
- 208000004232 Enteritis Diseases 0.000 abstract description 3
- 244000005709 gut microbiome Species 0.000 abstract description 3
- 210000000936 intestine Anatomy 0.000 abstract description 2
- 235000015872 dietary supplement Nutrition 0.000 abstract 1
- 239000002609 medium Substances 0.000 description 26
- 239000000047 product Substances 0.000 description 19
- 210000003608 fece Anatomy 0.000 description 16
- 241000282472 Canis lupus familiaris Species 0.000 description 15
- 238000000034 method Methods 0.000 description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 10
- 230000000968 intestinal effect Effects 0.000 description 9
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 238000003860 storage Methods 0.000 description 7
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 6
- 239000005662 Paraffin oil Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000036541 health Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 5
- 238000012937 correction Methods 0.000 description 5
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 5
- 239000004201 L-cysteine Substances 0.000 description 4
- 235000013878 L-cysteine Nutrition 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002550 fecal effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 240000006024 Lactobacillus plantarum Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 3
- 235000015140 cultured milk Nutrition 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 2
- 244000199866 Lactobacillus casei Species 0.000 description 2
- 235000013958 Lactobacillus casei Nutrition 0.000 description 2
- 241000191998 Pediococcus acidilactici Species 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000037406 food intake Effects 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229940017800 lactobacillus casei Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 201000004384 Alopecia Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 206010054272 Helicobacter gastritis Diseases 0.000 description 1
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 1
- 239000006154 MacConkey agar Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 206010033307 Overweight Diseases 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 208000012873 acute gastroenteritis Diseases 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 235000008452 baby food Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 235000014048 cultured milk product Nutrition 0.000 description 1
- 230000013872 defecation Effects 0.000 description 1
- 230000000741 diarrhetic effect Effects 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 208000024963 hair loss Diseases 0.000 description 1
- 230000003676 hair loss Effects 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- RSAZYXZUJROYKR-UHFFFAOYSA-N indophenol Chemical compound C1=CC(O)=CC=C1N=C1C=CC(=O)C=C1 RSAZYXZUJROYKR-UHFFFAOYSA-N 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 229940072205 lactobacillus plantarum Drugs 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000011120 smear test Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
본 발명은 내산소성 비피도박테리움 균주를 유효성분으로 포함하는 반려동물용 생균제 조성물 및 이의 제조방법에 관한 것으로, 본 발명에 따른 반려동물용 생균제는 반려동물의 장내 유산균과 비피더스 균의 수는 증가시키고, 대장균의 수는 감소시켜 장내 미생물총을 개선할 수 있으므로, 장염 또는 배변활동이 불편한 반려동물을 위한 건강보조사료로 유용하게 활용될 수 있을 것이다.The present invention relates to a probiotic composition for companion animals containing an oxygen-resistant Bifidobacterium strain as an active ingredient and a method for producing the same. The probiotic for companion animals according to the present invention increases the number of lactic acid bacteria and bifidobacteria in the intestines of companion animals. Since it can improve the intestinal microflora by reducing the number of E. coli, it can be useful as a health supplement for pets with enteritis or uncomfortable bowel movements.
Description
본 발명은 내산소성 비피도박테리움 균주를 유효성분으로 포함하는 반려동물용 생균제 조성물 및 이의 제조방법에 관한 것이다.The present invention relates to a probiotic composition for companion animals containing an oxygen-resistant Bifidobacterium strain as an active ingredient and a method for producing the same.
반려동물산업에 있어서, 2011년 7월부터 항생제 금지조치에 따른 대체품목인 생균제 시장의 규모가 17여 개의 보조사료 중 44%를 차지하고 있다. 종래 반려동물을 위한 보조사료는 털이나 뼈, 관절 등 주요 기능성 제품의 판매가 주를 이뤘다면 최근에는 유산균, 스트레스 완화, 눈 건강 등 기능적이고 특화된 제품들에 대한 수요가 증가하고 있는 추세이다. 국내시장의 경우, 고가의 유기농 제품과 프리미엄 제품을 포함하여 수입제품이 시장의 대부분을 차지하고 있으며, 반려동물 사료수입량이 지속적으로 증가하면서 2001년 17,169톤에서 2011년 36,308톤 규모로 성장하였으며, 2012년에는 전체 반려동물 사료시장의 약 58%가 수입제품으로 확인되었다. 수입제품을 찾는 소비자들의 수요가 증가하면서 해외 유명제품을 수입해서 판매하는 구매대행 업체가 늘어나고 있으나, 현지 가격과의 차이가 상당한 것으로 확인되었다. 이에 따라 수입제품에 버금가는 국내제품을 개발하고 사양실험, 급여효과, 사례 등을 철저히 분석하는 한편 가격경쟁력을 갖추어 향후 증가할 것으로 예상되는 시장상황에 유기적으로 대응할 필요가 있는 상황이다.In the companion animal industry, the market size of probiotics, which are replacement items following the ban on antibiotics since July 2011, accounts for 44% of 17 supplementary feeds. Previously, supplementary feed for pets was mainly sold on major functional products such as fur, bones, and joints, but recently, demand for functional and specialized products such as lactic acid bacteria, stress relief, and eye health has been increasing. In the domestic market, imported products, including expensive organic products and premium products, account for most of the market, and the volume of pet food imports continues to increase, growing from 17,169 tons in 2001 to 36,308 tons in 2011 and 2012. Approximately 58% of the total pet food market was confirmed to be imported products. As consumer demand for imported products increases, the number of purchasing agencies that import and sell famous overseas products is increasing, but the difference with local prices has been confirmed to be significant. Accordingly, there is a need to develop domestic products comparable to imported products, thoroughly analyze specification experiments, salary effects, and cases, while maintaining price competitiveness and organically responding to market conditions that are expected to increase in the future.
반려동물 시장의 급격한 성장에 따라 건강 및 위생문제도 심각하게 대두되고 있다. 특히 피부염, 급성 위장염, IBD(Inflammatory bowel disease), 알레르기, 탈모, 스트레스, 비만과 과체중, 비뇨생식기 감염, 헬리코박터 위염, 기생충 감염 등이 주된 질병으로 밝혀졌다, 치료뿐 아니라 예방차원 목적에서도 생균제의 관심이 매우 높아지고 있다.With the rapid growth of the companion animal market, health and hygiene issues are becoming more serious. In particular, dermatitis, acute gastroenteritis, IBD (Inflammatory bowel disease), allergies, hair loss, stress, obesity and overweight, genitourinary infections, Helicobacter gastritis, and parasitic infections were found to be the main diseases. Probiotics are of interest not only for treatment but also for preventive purposes. This is getting very high.
비피더스(bifidus)는 모유 영양아의 분변에서 최초로 분리되었으며, 유아 및 성인에 있어서도 비피더스 변화가 스트레스, 알레르기 또는 암 등과 밀접하게 관련되어 있음이 밝혀짐에 따라 건강 기여효과가 인정되고 있으며, 발효유, 유아식, 건강식품, 정장제 등에 널리 활용되고 있다. 비피더스는 혐기성균으로 혐기조건을 필요로 하기때문에 산업적으로 이용하는데 제한적이며, 발효유 내 비피더스 균수 문제가 제기되고 있다. 이는 비피더스가 원료유에서 잘 자라지 않기 때문에 발효유 제조 후 혐기적으로 배양한 비피더스를 첨가하여 유통하게 되는데, 유통과정에서 비피더스 생육조건인 혐기조건 형성이 어려워 지속적으로 사멸하는 문제점이 발생하기 때문이다. 비피더스의 다양한 생리활성을 식품산업에 이용하는 추세이나, 편성혐기성균(偏性嫌氣性菌)이라는 점과 현재 국내에서 발효유 제품에 사용하고 있는 유산균은 외국인에서 유래된 균주로서 한국인에게서 우수한 생리활성을 나타내지 못한다는 점도 지적되고 있다. 비피더스의 건강증진작용으로는 감염에 대한 저항성, 항암, 항종양, 혈중 콜레스테롤 함량 저하 및 정장효과 등의 다양한 생리활성이 보고되어 있다.Bifidus was first isolated from the feces of breastfed infants, and its contribution to health has been recognized as changes in bifidus have been found to be closely related to stress, allergies, or cancer in infants and adults, and it is being used in fermented milk and baby food. It is widely used in health foods, bowel preparations, etc. Bifidus is an anaerobic bacterium and requires anaerobic conditions, so its industrial use is limited, and problems are being raised about the number of bifidus bacteria in fermented milk. This is because bifidus does not grow well in raw milk, so after manufacturing fermented milk, anaerobically cultured bifidus is added and distributed. This is because it is difficult to create anaerobic conditions, which are the growth conditions for bifidus, during the distribution process, causing the problem of continuous death. There is a trend to use the various physiological activities of Bifidobacterium in the food industry, but the fact that it is an organized anaerobic bacterium and the lactic acid bacteria currently used in fermented milk products in Korea are strains originating from foreign countries, showing excellent physiological activity in Koreans. It is also pointed out that it cannot be shown. Various physiological activities such as resistance to infection, anti-cancer, anti-tumor, lowering blood cholesterol content, and intestinal regulation effect have been reported as health-promoting effects of bifidus.
한편, 한국등록특허 제2003822호에는 '반려동물의 모질을 개선하는 효능을 갖는 유산균과 이를 포함하는 생균제 조성물을 유효성분으로 함유하는 모질 개선을 위한 동물 사료용 첨가제 및 동물 사료 조성물'이 개시되어 있고, 한국등록특허 제0568036호에는 '장기능 활성화 및 면역기능 강화작용을 하는 비피토박테리움 균주 및 이를 함유하는 생균제제'가 개시되어 있으나, 본 발명의 내산소성 비피도박테리움 균주를 유효성분으로 포함하는 반려동물용 생균제 조성물 및 이의 제조방법에 대해서는 기재된 바가 없다.Meanwhile, Korean Patent No. 2003822 discloses 'An additive for animal feed and an animal feed composition for improving coat quality containing lactic acid bacteria with the effect of improving the coat quality of companion animals and a probiotic composition containing the same as active ingredients.' Korean Patent No. 0568036 discloses 'Bifitobacterium strains that activate intestinal function and enhance immune function and probiotics containing the same', but include the oxygen-resistant Bifidobacterium strain of the present invention as an active ingredient. There is no description of the probiotic composition for companion animals and its manufacturing method.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 반려동물용 사료첨가제로 사용되는 것을 특징으로 하는 프로바이오틱스 조성물로서 열 또는 동결건조 공정으로 제조가 가능한 조성물을 개발하고자, 국립축산과학원으로부터 내산소성을 가지는 비피도박테리움 롱검(Bifidobacterium longum) 균주(기탁번호 KACC91563P)를 기술이전받아 상기 균주의 대량생산을 위한 최적화된 산업용 배지 및 배양조건을 개발하였고, 분말형태의 생균제를 생산하기 위해 최적 냉동건조 조건을 확립하여 반려동물용 생균제 제품을 개발하였다. 또한, 상기 개발한 반려동물용 생균제 제품을 이용하여 반려견에 대한 사양실험을 수행한 결과, 본 발명에서 개발한 생균제 제품을 섭취한 반려견의 장내환경 및 분변지수가 생균제 섭취 전에 비해 개선되는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was derived from the above-mentioned needs, and the present inventors sought to develop a probiotic composition characterized by being used as a feed additive for companion animals that can be manufactured by a heat or freeze-drying process. We received technology transfer of the plastic Bifidobacterium longum strain (accession number KACC91563P), developed optimized industrial media and culture conditions for mass production of the strain, and optimized freezing to produce powdered probiotics. A probiotic product for companion animals was developed by establishing drying conditions. In addition, as a result of conducting a feeding experiment on dogs using the probiotic product for companion animals developed above, it was confirmed that the intestinal environment and fecal index of dogs that consumed the probiotic product developed in the present invention were improved compared to before ingestion of the probiotic. , completed the present invention.
상기 과제를 해결하기 위해, 본 발명은 기탁번호가 KACC91563P인 비피도박테리움 롱검(Bifidobacterium longum) 균주를 배양배지에 접종하여 배양하는 단계; 및 상기 배양된 균주 배양액의 냉동건조물에 냉동보호제를 첨가한 후 동결건조하는 단계;를 포함하는, 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말의 제조방법을 제공한다.In order to solve the above problem, the present invention includes the steps of inoculating a Bifidobacterium longum strain with accession number KACC91563P into a culture medium and culturing it; and adding a cryoprotectant to the freeze-dried product of the cultured strain and then freeze-drying it. A method for producing a freeze-dried powder with increased room temperature stability of Bifidobacterium longum is provided.
또한, 본 발명은 상기 제조방법에 의해 제조된 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말을 제공한다.In addition, the present invention provides a freeze-dried powder of Bifidobacterium longum prepared by the above production method with increased room temperature stability.
또한, 본 발명은 상기 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말을 유효성분으로 포함하는 장내 환경 및 분변지수 개선을 위한 반려동물용 생균제 조성물을 제공한다.In addition, the present invention provides a probiotic composition for companion animals for improving the intestinal environment and fecal index, which contains as an active ingredient a freeze-dried powder of Bifidobacterium longum with increased room temperature stability.
본 발명에 따른 반려동물용 생균제는 반려동물의 장내 유산균과 비피더스 균의 수는 증가시키고, 대장균의 수는 감소시켜 장내 미생물총을 개선할 수 있으므로, 장염 또는 배변활동이 불편한 반려동물을 위한 건강보조사료로 유용하게 활용될 수 있을 것이다.The probiotic for pets according to the present invention can improve the intestinal microflora by increasing the number of lactic acid bacteria and bifidobacteria in the intestines of pets and reducing the number of E. coli, thereby providing health support for pets with enteritis or uncomfortable bowel movements. It could be useful as feed.
도 1은 본 발명에 따른 비피도박테리움 롱검(Bifidobacterium longum) 균주(기탁번호 KACC91563P)를 함유하는 생균제의 시제품 사진이다.
도 2는 본 발명에 따른 비피도박테리움 롱검 균주를 함유하는 생균제 섭취에 따른 반려견 분변의 황화수소(H2S) 및 암모니아(NH4) 변화를 보여준다.Figure 1 is a photograph of a prototype of a probiotic containing the Bifidobacterium longum strain (accession number KACC91563P) according to the present invention.
Figure 2 shows changes in hydrogen sulfide (H 2 S) and ammonia (NH 4 ) in dog feces according to ingestion of probiotics containing the Bifidobacterium longum strain according to the present invention.
본 발명의 목적을 달성하기 위하여, 본 발명은 기탁번호가 KACC91563P인 비피도박테리움 롱검(Bifidobacterium longum) 균주를 배양배지에 접종하여 배양하는 단계; 및 상기 배양된 균주 배양액의 냉동건조물에 냉동보호제를 첨가한 후 동결건조하는 단계;를 포함하는, 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말의 제조방법을 제공한다.In order to achieve the object of the present invention, the present invention includes the steps of inoculating a Bifidobacterium longum strain with accession number KACC91563P into a culture medium and culturing it; and adding a cryoprotectant to the freeze-dried product of the cultured strain and then freeze-drying it. A method for producing a freeze-dried powder with increased room temperature stability of Bifidobacterium longum is provided.
본 발명에 있어서, 상기 기탁번호가 KACC91563P인 비피도박테리움 롱검(Bifidobacterium longum) 균주는 내산소성(aerotolerant)을 가지며, 단독으로 커드의 형성이 가능한 비피도박테리움 롱검 6-T-4-55(Bifidobacterium longum 6-T-4-55) 균주이다 (한국등록특허 제1250463호).In the present invention, the Bifidobacterium longum strain with the accession number KACC91563P is aerotolerant and independently capable of forming curds, Bifidobacterium longum 6-T-4-55 ( Bifidobacterium longum 6-T-4-55) strain (Korean Patent No. 1250463).
본 발명의 일 구현 예에 따른 제조방법에 있어서, 상기 배양배지는 바람직하게는, 배지 조성물의 단위부피 L 당 옥수수침지액(corn steep liquor) 20~40 ㎖, 효모 추출물 1~3 g, 글루코스 1~3 g, 탈지유 분말 0.1~2 g, 수산화나트륨 0.1~2 g, 염화나트륨 3~7 g, L-시스테인(cysteine) 0.3~0.7 g 및 파라핀유(paraffinic oil) 30~50 ㎖을 포함하는 것일 수 있고, 더욱 바람직하게는 배지 조성물의 단위부피 L 당 옥수수침지액 25~35 ㎖, 효모 추출물 1.5~2.5 g, 글루코스 1.5~2.5 g, 탈지유 분말 0.5~1.5 g, 수산화나트륨 0.5~1.5 g, 염화나트륨 4~6 g, L-시스테인 0.4~0.6 g 및 파라핀유 35~45 ㎖을 포함하는 것일 수 있으며, 더 더욱 바람직하게는 배지 조성물의 단위부피 L 당 옥수수침지액 30 ㎖, 효모 추출물 2 g, 글루코스 2 g, 탈지유 분말 1 g, 수산화나트륨 1 g, 염화나트륨 5 g, L-시스테인 0.5 g 및 파라핀유 40 ㎖을 포함하는 것일 수 있으나, 이에 제한되지 않는다.In the production method according to one embodiment of the present invention, the culture medium preferably contains 20 to 40 ml of corn steep liquor, 1 to 3 g of yeast extract, and 1 glucose per unit volume L of the medium composition. ~3 g, may contain 0.1~2 g of skim milk powder, 0.1~2 g of sodium hydroxide, 3~7 g of sodium chloride, 0.3~0.7 g of L-cysteine, and 30~50 ml of paraffinic oil. and, more preferably, per unit volume L of the medium composition, 25 to 35 ml of corn steep liquid, 1.5 to 2.5 g of yeast extract, 1.5 to 2.5 g of glucose, 0.5 to 1.5 g of skim milk powder, 0.5 to 1.5 g of sodium hydroxide, and sodium chloride 4. It may contain ~6 g, 0.4~0.6 g of L-cysteine, and 35~45 ml of paraffin oil, and more preferably, 30 ml of corn steep liquid per unit volume L of the medium composition, 2 g of yeast extract, and 2 g of glucose. g, 1 g of skim milk powder, 1 g of sodium hydroxide, 5 g of sodium chloride, 0.5 g of L-cysteine, and 40 ml of paraffin oil, but is not limited thereto.
본 발명에 따른 기탁번호가 KACC91563P인 비피도박테리움 롱검 균주는 동일한 배양배지에서 종균배양(seed culture)한 후, 본 배양배지의 0.6~1.0 중량%(v/v)로 접종하여 배양하였다. 상기 배양은 32~37℃의 온도에서 22~50시간 동안 수행되는 것일 수 있으나, 이에 제한되지 않는다.The Bifidobacterium longum strain with the accession number KACC91563P according to the present invention was seed cultured in the same culture medium and then inoculated with 0.6 to 1.0% by weight (v/v) of the main culture medium. The culturing may be performed at a temperature of 32 to 37°C for 22 to 50 hours, but is not limited thereto.
또한, 본 발명에 따른 제조방법에 있어서, 상기 냉동보호제는 배양된 균주 배양액의 냉동건조물을 동결건조하기 전 단계에서 첨가하는 것으로, 바람직하게는 배양배지의 0.1~1.0 중량%(w/v)로 첨가되는 것일 수 있고, 더욱 바람직하게는 배양배지의 0.4~0.7 중량%(w/v)로 첨가되는 것일 수 있으며, 더 더욱 바람직하게는 배양배지의 0.56 중량%(w/v)로 첨가되는 것일 수 있으나, 이에 제한되지 않는다.In addition, in the production method according to the present invention, the cryoprotectant is added in the step before freeze-drying the freeze-dried product of the cultured strain culture medium, preferably at 0.1 to 1.0% by weight (w/v) of the culture medium. It may be added, more preferably at 0.4 to 0.7 weight% (w/v) of the culture medium, and even more preferably at 0.56 weight% (w/v) of the culture medium. may, but is not limited to this.
또한, 상기 냉동보호제는 탈지분유, MSG (monosodium glutamate) 및 만니톨이 동일 중량으로 혼합된 것일 수 있으나, 이에 제한되지 않는다.Additionally, the cryoprotectant may be a mixture of skim milk powder, MSG (monosodium glutamate), and mannitol in equal weight, but is not limited thereto.
본 발명에 따른 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말의 제조방법은, 전술한 배양배지 및 냉동보호제의 사용을 통해 동결건조분말 내 비피도박테리움 롱검의 생균수가 26~30℃의 온도에서 102~106일 동안 5.0 x 109 내지 1.0 x 1010 cfu/g을 유지될 수 있도록 하여, 열에 약한 비피더스의 보존성을 현저하게 증가시킨 것이 특징이다.The method for producing a freeze-dried powder with increased room temperature stability of Bifidobacterium longum according to the present invention allows the viable cell count of Bifidobacterium longum in the freeze-dried powder to be increased to 26-30°C through the use of the above-mentioned culture medium and cryoprotectant. It is characterized by significantly increasing the preservation of bifidus , which is vulnerable to heat, by maintaining 5.0
본 발명은 또한, 상기 제조방법에 의해 제조된 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말을 제공한다.The present invention also provides a freeze-dried powder of Bifidobacterium longum prepared by the above production method with increased room temperature stability.
본 발명에 따른 동결건조분말은 26~30℃의 온도에서 102~106일 동안 비피도박테리움 롱검의 생균수가 5.0 x 109 내지 1.0 x 1010 cfu/g으로 유지될 수 있고, 상기 온도뿐만 아니라 3~5℃의 온도에서도 동일 기간 유사한 수준으로 생균수를 유지하는 것이 특징이다.The freeze-dried powder according to the present invention can maintain the viable cell count of Bifidobacterium longum at 5.0 x 10 9 to 1.0 x 10 10 cfu/g for 102 to 106 days at a temperature of 26 to 30 ° C. It is characterized by maintaining the number of viable bacteria at a similar level for the same period even at a temperature of 3~5℃.
본 발명은 또한, 본 발명에 따른 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말을 유효성분으로 포함하는, 장내 환경 및 분변지수 개선을 위한 반려동물용 생균제 조성물을 제공한다.The present invention also provides a probiotic composition for companion animals for improving the intestinal environment and fecal index, comprising as an active ingredient the freeze-dried powder of Bifidobacterium longum with increased room temperature stability according to the present invention.
본 명세서에서 용어 '생균제'는 장내 미생물의 균형을 개선하여 숙주동물에게 유익한 작용을 하는 물질을 의미하는 것으로, 프로바이오틱스(probiotics)로도 불리운다. 본 발명의 반려동물용 생균제 조성물은 유효성분으로 비피도박테리움 롱검(Bifidobacterium longum) KACC91563P 균주, 이의 배양액, 상기 배양액의 농축액 또는 그의 건조물을 포함할 수 있고, 당업계에 공지된 방법에 따라 다양한 제형과 방법으로 제조 및 투여될 수 있다.As used herein, the term 'probiotic' refers to a substance that has a beneficial effect on the host animal by improving the balance of intestinal microorganisms, and is also called probiotics. The probiotic composition for companion animals of the present invention may include the Bifidobacterium longum KACC91563P strain, its culture, a concentrate of the culture, or a dried product thereof as an active ingredient, and can be formulated into various formulations according to methods known in the art. It can be manufactured and administered by the following methods.
예를 들어, 약제학적 분야에서 통상적으로 사용되는 담체와 혼합하여 산제(powder), 액제(liquids and solutions), 정제(tablet), 캡슐(capsule), 시럽(syrup), 현탁제(suspension) 또는 과립제(granule) 등의 형태로 제조될 수 있고, 상기 담체로는 예를 들어, 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소 및 향료 등일 수 있으나, 이에 제한되지 않는다. 또한, 투여 용량은 체내에서의 활성성분의 흡수도, 불활성률, 배설속도, 피투여자의 연령, 성별, 축종, 상태 및 질병의 중증 정도 등에 따라 적절히 선택할 수 있다.For example, it can be mixed with carriers commonly used in the pharmaceutical field to form powders, liquids and solutions, tablets, capsules, syrups, suspensions or granules. It may be manufactured in the form of (granule), etc., and the carrier may be, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, and a flavoring agent, but is limited thereto. It doesn't work. In addition, the administered dose can be appropriately selected depending on the absorption of the active ingredient in the body, the inactivation rate, the excretion rate, the age, gender, species, condition, and severity of the disease of the recipient.
본 발명의 상기 생균제 조성물은 비피도박테리움 롱검 KACC91563P 균주 외에, 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 플랜타룸(L. plantarum), 및 페디오코커스 애시도락티시(Pediococcus acidilactici)를 추가로 포함할 수 있으며, 면역증강물질 및 피부알러지 억제물질 또한 추가로 포함할 수 있으나, 이에 제한되지 않는다.The probiotic composition of the present invention includes, in addition to the Bifidobacterium longum KACC91563P strain, Lactobacillus casei , Lactobacillus plantarum ( L. plantarum ), and Pediococcus acidilactici . It may include, and may additionally include, but is not limited to, immune enhancing substances and skin allergy suppressing substances.
본 발명에 따른 반려동물용 생균제 조성물에 있어서, 상기 반려동물은 이에 한정되지 않으나, 반려견 또는 반려묘일 수 있다.In the probiotic composition for companion animals according to the present invention, the companion animal is not limited thereto, but may be a dog or a cat.
본 발명의 상기 반려동물용 생균제 조성물은 반려동물의 장내 유산균 수 및 배변지수를 증가시키며, 장내 대장균 수 및 분변 내 황화수소 및 암모니아를 저감시키는 효과가 있어, 장염 또는 배변활동이 불편한 반려동물을 위한 건강보조사료로 이용될 수 있다.The probiotic composition for companion animals of the present invention increases the number of intestinal lactic acid bacteria and defecation index of companion animals, and has the effect of reducing the number of intestinal coliforms and hydrogen sulfide and ammonia in feces, providing health for companion animals with enteritis or uncomfortable bowel movements. It can be used as supplementary feed.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
실시예 1. 비피도박테리움 롱검(Example 1. Bifidobacterium longum ( Bifidobacterium longumBifidobacterium longum ) 균주(기탁번호: KACC91563P)의 대량 생산을 위한 최적화 산업용 배지 및 조건 개발) Development of optimized industrial media and conditions for mass production of strains (Accession Number: KACC91563P)
1-1. 산소차단 유무에 따른 액상산업배지에서의 비피더스 생장 확인1-1. Confirmation of Bifidus growth in liquid industrial medium with and without oxygen blocking
비피도박테리움 롱검 균주를 유산균 전용배지인 MRS(대조구)와 선바이오에서 조합한 LAB Industrial broth, CSL Industrial broth 또는 SMP Industrial broth에 각각 0.5%씩 접종한 후 32℃에서 배양하였다. 각각의 배지는 121℃에서 15분간 멸균처리한 후 사용하였다. 또한, 산소차단을 목적으로 파라핀 오일을 첨가한 그룹과 무첨가그룹으로 각각 나누어 비피도박테리움 롱검 균주의 생장을 분석하였다.The Bifidobacterium longum strain was inoculated at 0.5% each into MRS (control), a dedicated lactic acid bacteria medium, and LAB Industrial broth, CSL Industrial broth, or SMP Industrial broth prepared by Sunbio, and then cultured at 32°C. Each medium was sterilized at 121°C for 15 minutes before use. In addition, the growth of Bifidobacterium longum strains was analyzed by dividing them into a group with and without paraffin oil added for the purpose of blocking oxygen.
그 결과, 대부분의 배지에서 2일 배양 시 최대수치인 2.0x109 cfu/㎖까지 생장하였으며 이후 감소하는 추세를 보여 1-2일 배양이 최적배양시간임을 알 수 있었다(표 2). 또한, 파라핀오일 첨가구와 무첨가구에서의 비피도박테리움 롱검의 생장에 큰 차이를 보이지 않아 비피도박테리움 롱검 KACC91563P 균주가 내산소성이 있음을 확인할 수 있었다.As a result, in most media, the growth reached the maximum value of 2.0x109 cfu/ml when cultured for 2 days, and then showed a decreasing trend, showing that 1-2 days of culture was the optimal culture time (Table 2). In addition, there was no significant difference in the growth of Bifidobacterium longum in the group with and without paraffin oil, confirming that the Bifidobacterium longum KACC91563P strain was oxygen resistant.
첨가paraffin oil
adding
무첨가paraffin oil
No additives
1-2. pH 조절과 온도에 따른 비피더스 생장 확인1-2. Verification of bifidus growth according to pH control and temperature
CSL 산업배지와 LAB 산업배지에서 pH 및 온도에 따른 비피도박테리움 롱검의 생장 상태를 알아보기 위하여 32℃와 37℃, 염화나트륨(NaOH) 보정과 무보정 그룹 등 4개 처리구로 나누어 실험하였다. 스타터 균주는 각 배양액 1L 기준 5㎖씩 접종하였다. pH 조절은 배양 16시간 경과 후에 pH가 5이하로 떨어진 처리구에 1M NaOH를 추가하여 pH를 6.0 이상으로 조절하여 배양하였다.To determine the growth status of Bifidobacterium longum according to pH and temperature in CSL industrial medium and LAB industrial medium, experiments were conducted in four treatment groups, including 32°C and 37°C, sodium chloride (NaOH) correction and no correction groups. The starter strain was inoculated at 5 ml per 1 L of each culture medium. For pH control, 1M NaOH was added to the treatment group where the pH dropped below 5 after 16 hours of incubation, and the pH was adjusted to above 6.0.
그 결과, 하기 표 3 및 표 4에서와 같이 32℃와 37℃ 배양에 따른 차이는 볼 수 없었으나, 단지 배양 2일째 pH 무보정 CSL 산업배지가 1.3x109 cfu/㎖로 균수가 가장 높은 것을 확인할 수 있었다. 또한, 전체적으로 CSL 산업배지가 LAB 산업배지보다 균수가 높은 것을 알 수 있었다. 또한, NaOH 보정과 무보정에 따른 차이는 온도와 마찬가지로 볼 수 없었으며, 단지 배양 2일째부터 pH 조절 즉, NaOH 보정 시 균수 감소가 확인되었다.As a result, as shown in Tables 3 and 4 below, no difference was observed between cultures at 32°C and 37°C, but on the second day of culture, the pH-free CSL industrial medium had the highest bacterial count at 1.3x109 cfu/ml. I was able to confirm. In addition, overall, it was found that the CSL industrial badge had a higher bacterial count than the LAB industrial badge. In addition, the difference between NaOH correction and no correction was not seen as with temperature, and a decrease in the number of bacteria was confirmed when pH was adjusted, that is, NaOH correction, only from the second day of culture.
실시예 2. 비피도박테리움 롱검(Example 2. Bifidobacterium longum ( Bifidobacterium longumBifidobacterium longum ) 균주(기탁번호: KACC91563P)의 분말 생균제품 대량생산을 위한 최적 냉동건조 조건 개발) Development of optimal freeze-drying conditions for mass production of powdered live bacterial products of the strain (Accession number: KACC91563P)
SMP 산업배지와 CSL 산업배지는 표 1에 개시된 조성으로 1톤을 제조하여 90℃에서 1시간 열처리 후 32℃로 냉각시켰다. 그 후 비피도박테리움 롱검의 종균 배양액을 8L (0.8%) 접종하고, CO2 가스를 30분간 투입하였다. 배양 24시간 후, 균주 배양액을 원심분리하였고, 냉동건조기(EYELA Co., Japan ; condencer capacity: 300kg)를 사용하여 (0.05 torr, -40℃ 이하)에서 48시간 냉동건조하였으며, 탈지분유, MSG (monosodium glutamate) 및 만니톨이 동일 중량으로 혼합된 냉동보호제 5.6 kg을 첨가한 후 동결건조하였다.One ton of SMP industrial medium and CSL industrial medium were prepared with the compositions shown in Table 1, heat treated at 90°C for 1 hour, and then cooled to 32°C. Afterwards, 8L (0.8%) of the seed culture of Bifidobacterium longum was inoculated, and CO 2 gas was introduced for 30 minutes. After 24 hours of incubation, the strain culture was centrifuged and freeze-dried for 48 hours at (0.05 torr, -40°C or lower) using a freeze dryer (EYELA Co., Japan; condenser capacity: 300kg). Skimmed milk powder, MSG ( 5.6 kg of cryoprotectant mixed with equal weights of monosodium glutamate) and mannitol were added and then freeze-dried.
= 3.9x1015cfu/㎖1,000kg x 3.9x10 9 cfu/ml
= 3.9x10 15 cfu/ml
= 2.0x1015cfu/g72kg x 2.7x10 10 cfu/g
= 2.0x10 15 cfu/g
= 2.6x1014cfu/㎖928kg x 2.8x10 7 cfu/ml
= 2.6x10 14 cfu/ml
= 8.8x1013cfu/g8kg x 1.1x10 10 cfu/g
= 8.8x10 13 cfu/g
= 2.8x1015cfu/㎖1,000kg x 2.8x10cfu/㎖ 9
= 2.8x10 15 cfu/ml
= 5.6x1014cfu/g180kg x 3.1x10 10 cfu/g
= 5.6x10 14 cfu/g
= 2.46x1014cfu/㎖820kg x 3.0x10 8 cfu/ml
= 2.46x10 14 cfu/ml
= 1.1x1013cfu/g37.4kg x 2.9x10 8 cfu/g
= 1.1x10 13 cfu/g
CSL 배지 1 ton을 이용한 비피도박테리움 롱검의 배양액의 총균수는 3.9x1015 cfu/㎖이었으며, 최종 동결건조(FD) 분말 8kg에 함유된 균수는 8.8x1013 cfu/g으로, FD 수율은 8.8/390 = 2.2564%로 확인되었다. SMP 배지 1 ton을 이용한 비피도박테리움 롱검의 배양액의 총균수는 2.8x1015 cfu/㎖이며, 최종 FD 분말 37.4kg에 함유된 균수는 1.1x1013 cfu/g으로, FD 수율이 1.1/280 = 0.39%로 나타났다. CSL 배지와 SMP 배지의 FD 분말 수율을 비교하면 88:11로, CSL 배지가 SMP 배지보다 8배 정도 높아 CSL 배지가 비피도박테리움 롱검의 FD 공정에 적합한 배지임을 확인하였다.The total number of bacteria in the culture medium of Bifidobacterium longum using 1 ton of CSL medium was 3.9x10 15 cfu/ml, the number of bacteria contained in 8kg of final freeze-dried (FD) powder was 8.8x10 13 cfu/g, and the FD yield was 8.8. /390 = 2.2564%. The total number of bacteria in the culture medium of Bifidobacterium longum using 1 ton of SMP medium is 2.8x10 15 cfu/ml, and the number of bacteria contained in 37.4kg of final FD powder is 1.1x10 13 cfu/g, FD yield = 1.1/280 It was found to be 0.39%. Comparing the FD powder yield of CSL medium and SMP medium, it was 88:11, and CSL medium was about 8 times higher than SMP medium, confirming that CSL medium was a suitable medium for the FD process of Bifidobacterium longum.
SMP 산업배지를 사용하여 배양에서부터 동결건조과정을 거친 FD 분말을 4℃(냉장)와 28℃(실온)에서 각각 보관하면서 저장성을 조사하였다. 그 결과, 4℃ 냉장보관의 경우 초기 3.5x108 cfu/g에서 시작하여 약 4달(113일) 경과 후에 2.0x107 cfu/g으로 94.3%의 균 사멸을 보였다. 또한, 28℃ 실온보관의 경우도 초기 3.5x108 cfu/g에서 약 1달만에 3.2x105 cfu/g, 4달 후에는 1.0x104 cfu/g으로 대부분 사멸하여, SMP 산업배지가 온도에 상당히 약한 모습을 보였다.The storability of FD powder, which had undergone a freeze-drying process from cultivation using SMP industrial medium, was stored at 4°C (refrigerated) and 28°C (room temperature), respectively. As a result, in the case of refrigerated storage at 4℃, it started at 3.5x10 8 cfu/g initially and reached 2.0x10 7 cfu/g after about 4 months (113 days), showing 94.3% of bacterial death. In addition, in the case of storage at room temperature at 28℃, most of the death occurred from the initial 3.5x10 8 cfu/g to 3.2x10 5 cfu/g in about 1 month, and 1.0x10 4 cfu/g after 4 months, so SMP industrial media is considerably sensitive to temperature. He appeared weak.
(냉장보관)(Keep refrigerated)
(실온보관)(Stored at room temperature)
반면, CSL 산업배지를 사용하여 배양에서부터 동결건조과정을 거친 FD 분말은 4℃ 냉장보관의 경우 초기 1.1x1010 cfu/g에서 한달 후에 7.7x109 cfu/g로 70%의 생존을 보였으며, 약 4달(104일) 경과 후에 7.3x109 cfu/g으로 66%의 생존율을 보여주었다. 또한, 28℃ 실온보관의 경우 초기 1.1x1010 cfu/g에서 약 1달만에 8.3x109 cfu/g으로 냉장보관보다 오히려 생균수가 높았으며, 4달 후(104일)에 7.0x109 cfu/g으로 63%의 생존율을 보여 냉장보관과 큰 차이를 볼 수 없었다. 상기 결과를 통해 CSL 산업배지로 제조한 비피더스 제품의 경우 실온 유통 가능성을 높일 수 있을 것으로 판단되었다.On the other hand, FD powder, which went through the process from culture to freeze-drying using CSL industrial medium, showed 70% survival from 1.1x10 10 cfu/g initially to 7.7x10 9 cfu/g after a month when refrigerated at 4°C. After 4 months (104 days), the survival rate was 66% at 7.3x10 9 cfu/g. In addition, in the case of room temperature storage at 28℃, the number of viable bacteria was higher than that of refrigerated storage, from 1.1x10 10 cfu/g initially to 8.3x10 9 cfu/g in about 1 month, and 7.0x10 9 cfu/g after 4 months (104 days). The survival rate was 63%, showing no significant difference from refrigerated storage. Based on the above results, it was determined that the possibility of room temperature distribution could be increased for Bifidus products manufactured with CSL industrial medium.
(냉장보관)(Keep refrigerated)
(실온보관)(Stored at room temperature)
실시예 3. 반려견을 대상으로 한 사양실험Example 3. Feeding experiment on dogs
본 발명자는 CSL 산업배지를 이용하여 내산소성을 가지는 비피도박테리움 롱검 균주(기탁번호: KACC91563P)를 배양하고 이를 동결건조하여 반려동물을 위한 비피더스 생균제를 제조하였다. 상기 반려동물을 위한 생균제는 비피도박테리움 롱검 KACC91563P 균주(1.0x108 cfu/g 이상) 외, 락토바실러스 카제이(Lactobacillus casei, 1.0x107 cfu/g 이상), 락토바실러스 플랜타룸(L. plantarum, 1.0x107 cfu/g 이상), 페디오코커스 애시도락티시(Pediococcus acidilactici, 1.0x107 cfu/g 이상)를 포함하도록 제조하였다.The present inventor cultivated an oxygen-resistant Bifidobacterium longum strain (accession number: KACC91563P) using CSL industrial medium and freeze-dried it to prepare Bifidobacterium probiotics for companion animals. The probiotics for companion animals include Bifidobacterium longum KACC91563P strain (1.0x10 8 cfu/g or more), Lactobacillus casei (1.0x10 7 cfu/g or more), and L. plantarum . , 1.0x10 7 cfu/g or more), and Pediococcus acidilactici (1.0x10 7 cfu/g or more).
상기 제조된 생균제를 이용하여 반려견 11마리를 이용하여 반려견 사양시험을 실시하였다. 사양시험은 반려견 장내 미생물 개선효과 확인을 위하여 분변내 유산균, 대장균 수를 도말검사를 통하여 확인하였으며, 반려견 환경 개선효과 확인을 위하여 Bristol stool chart를 통한 설사지수 검사, 발색법을 통한 분변의 암모니아 측정, 키트를 통한 분변의 황화수소를 측정하였다. 본 사양시험에 참여하는 시험동물은 보호자를 통한 자원으로 모집되었으며, 총 11마리의 반려견이 모집되었고, 반려견의 종과 나이, 성별, 중성화 등의 기본 정보는 하기 표 8과 같다.A dog feeding test was conducted using 11 dogs using the probiotic prepared above. In the feeding test, the number of lactic acid bacteria and E. coli in the feces was confirmed through a smear test to confirm the effect of improving the dog's intestinal microflora. To confirm the effect of improving the dog's environment, the diarrhea index test was performed using a Bristol stool chart, ammonia in feces was measured using a coloring method, and Hydrogen sulfide in feces was measured using a kit. The test animals participating in this specification test were recruited as volunteers through their guardians, and a total of 11 dogs were recruited. Basic information such as the breed, age, gender, and neutering of the dogs is shown in Table 8 below.
시료의 섭취는 본 발명의 생균제와 사료를 함께 섞어 급이하는 방식으로 진행되었으며, 하루 2회 섭취하도록 보호자에게 설명하였다. 반려견의 분변은 시험 시작 후 0, 42, 85일째에 사진촬영 후 분석에 사용되었다. 분변의 미생물 수는 멸균된 생리식염수 10 ㎖에 분변 1g을 섞은 후 충분히 현탁시키고, 이를 십진희석법으로 희석한 후, Macconkey agar, MRS agar 및 Bifidobacterium selective agar에 도말하여 37℃에서 혐기 혹은 일반 배양을 실시한 후 생성된 콜로니를 계측기를 이용하여 측정한 뒤 균수를 계산하였다. 분변 내 황화수소의 측정을 실험실에 도착한 분변이 담겨있는 튜브의 입구에서 약 lcm 높이의 위치에서 황화수소 측정 검치관을 장착한 Gastec GV-100S를 이용하여 측정하였다. 분변 내 암모니아의 측정은 발색법인 인도페놀법을 이용하여 측정하였다. 멸균된 생리식염수에 현탁된 분변의 상등액을 동 식염수를 이용하여 2배 희석한 뒤, 시료 2.5 ㎖를 페놀 용액 0.1 ㎖, Nitroprusside sodium 용액 0.1 ㎖, 산화용액 0.25 ㎖를 넣고 실온에서 1시간 반응시켰다. 반응이 완료된 시료는 분광광도계를 이용하여 흡광도를 측정하였다. 시료 섭취에 따른 반려견 분변의 설사지수의 확인은 Bristol stool chart를 바탕으로 측정하였다.The intake of the sample was carried out by mixing the probiotic of the present invention and feed and feeding it, and the guardian was instructed to consume it twice a day. The dog's feces were photographed and used for analysis at 0, 42, and 85 days after the start of the test. To determine the number of microorganisms in feces, mix 1 g of feces in 10 ml of sterilized saline solution, suspend it sufficiently, dilute it using the decimal dilution method, spread it on Macconkey agar, MRS agar, and Bifidobacterium selective agar, and perform anaerobic or general culture at 37°C. The resulting colonies were measured using a measuring instrument and the number of bacteria was calculated. Hydrogen sulfide in feces was measured using a Gastec GV-100S equipped with a hydrogen sulfide measuring tube at a height of approximately lcm from the entrance of the tube containing the feces that arrived at the laboratory. Ammonia in feces was measured using the indophenol method, a colorimetric method. The supernatant of feces suspended in sterilized saline solution was diluted two-fold using the same saline solution, and then 0.1 ml of phenol solution, 0.1 ml of nitroprusside sodium solution, and 0.25 ml of oxidation solution were added to 2.5 ml of sample and reacted at room temperature for 1 hour. The absorbance of the sample for which the reaction was completed was measured using a spectrophotometer. The diarrheal index of dog feces according to sample intake was determined based on the Bristol stool chart.
그 결과, 본 발명의 생균제를 섭취한 반려견의 분변 내 대장균 수 변화는 하기 표 9와 같이 생균제의 섭취 기간이 길어질수록 감소하는 것을 알 수 있었으며, 유산균 및 비피더스 균은 표 10 및 표 11에 개시된 것과 같이 생균제의 섭취 기간이 길어질수록 증가되는 것을 알 수 있었다.As a result, it was found that the change in the number of E. coli in the feces of dogs that consumed the probiotic of the present invention decreased as the period of intake of the probiotic increased, as shown in Table 9 below, and the lactic acid bacteria and bifidobacteria were as shown in Tables 10 and 11. Likewise, it was found that the longer the period of probiotic intake, the greater the increase.
또한, 상기와 같은 장내 미생물의 변화에 따라 반려견의 분변에서 황화수소 및 암모니아의 생성 또한 저감되는 것을 확인할 수 있었다(도 2). 상기의 결과를 통해 비피도박테리움 롱검(Bifidobacterium longum) 균주(기탁번호:KACC91563P)를 함유하는 본 발명의 생균제는 반려동물의 장내 환경 개선 및 분변지수 개선의 효과가 있어, 반려동물을 위한 건강보조사료로 사용될 수 있음을 확인할 수 있었다.In addition, it was confirmed that the production of hydrogen sulfide and ammonia in the dog's feces was also reduced according to the above changes in intestinal microorganisms (Figure 2). Based on the above results, the probiotic of the present invention containing Bifidobacterium longum strain (Accession number: KACC91563P) is effective in improving the intestinal environment and fecal index of companion animals, providing health support for companion animals. It was confirmed that it could be used as feed.
Claims (8)
상기 배양된 균주 배양액의 냉동건조물에 탈지분유, MSG (monosodium glutamate) 및 만니톨이 동일 중량으로 혼합된 냉동보호제를 배양배지의 0.1~1.0 중량%(w/v)로 첨가한 후 동결건조하는 단계;를 포함하는, 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말의 제조방법으로서,
상기 배양배지는 배지 조성물의 단위부피 L 당 옥수수침지액(corn steep liquor) 20~40 ㎖, 효모 추출물 1~3 g, 글루코스 1~3 g, 탈지유 분말 0.1~2 g, 수산화나트륨 0.1~2 g, 염화나트륨 3~7 g, L-시스테인(cysteine) 0.3~0.7 g 및 파라핀유(paraffinic oil) 30~50 ㎖을 포함하는 것을 특징으로 하는 비피도박테리움 롱검의 실온 안정성이 증가된 동결건조분말의 제조방법.Inoculating a Bifidobacterium longum strain with accession number KACC91563P into a culture medium and culturing it; and
Adding a cryoprotectant mixed with equal weights of skim milk powder, MSG (monosodium glutamate) and mannitol to the freeze-dried product of the cultured strain at 0.1 to 1.0% by weight (w/v) of the culture medium and then freeze-drying; A method for producing a freeze-dried powder with increased room temperature stability of Bifidobacterium longum, comprising:
The culture medium contains 20-40 ml of corn steep liquor, 1-3 g of yeast extract, 1-3 g of glucose, 0.1-2 g of skim milk powder, and 0.1-2 g of sodium hydroxide per unit volume L of the medium composition. , lyophilized powder with increased room temperature stability of Bifidobacterium longum, characterized in that it contains 3 to 7 g of sodium chloride, 0.3 to 0.7 g of L-cysteine, and 30 to 50 ml of paraffinic oil. Manufacturing method.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020190159635 | 2019-12-04 | ||
| KR20190159635 | 2019-12-04 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20210070220A KR20210070220A (en) | 2021-06-14 |
| KR102607676B1 true KR102607676B1 (en) | 2023-11-29 |
Family
ID=76417654
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020200167269A KR102607676B1 (en) | 2019-12-04 | 2020-12-03 | Probiotics composition for companion animals comprising aerotolerant Bifidobacterium strain as effective component and production method thereof |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR102607676B1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004097222A (en) | 2002-09-06 | 2004-04-02 | Alfa Wassermann Spa | Bacillus bifidus and preparation containing the same |
| US20070280967A1 (en) | 2003-12-03 | 2007-12-06 | Tim Lee | Cryo-protective agents for microorganisms |
| KR101604633B1 (en) * | 2015-08-28 | 2016-03-18 | 주식회사 락토메이슨 | Medium composition for culturing lactic acid bacteria and producing method of powder of lactic acid bacteria using the same |
| JP2016105705A (en) * | 2009-04-30 | 2016-06-16 | イントレクソン・アクトバイオテイクス・エヌブイＩｎｔｒｅｘｏｎ Ａｃｔｏｂｉｏｔｉｃｓ ＮＶ | Cryoprotectants for freeze drying of lactic acid bacteria |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR0155223B1 (en) * | 1995-10-25 | 1998-10-15 | 이은선 | Culture medium for lactic acid bacteria with corn steep liquor and molasses |
| KR101008568B1 (en) * | 2008-11-11 | 2011-01-19 | 대한민국 | Novel Aquitalea sp. 5YN1-3 strain for effective denitrification |
| KR101250463B1 (en) * | 2010-10-12 | 2013-04-15 | 대한민국 | Oxygen tolerant Bifidobacterium longum from Korean neonate fecal samples and probiotic compositions produced by the same |
| KR101793498B1 (en) * | 2015-08-27 | 2017-11-03 | 선 바이오 (주) | Method for producing multi-functional probiotics for feed additive having antibacterial activity and enzyme activity through solid state fermentation using lactic acid bacteria, Bacillus sp. and yeast strain and multi-functional probiotics for feed additive thereof |
| KR102027029B1 (en) * | 2017-05-25 | 2019-09-30 | (주)엠앤씨생명과학 | Natural liposome comprising lactic acid bacteria, process for the preparation thereof and food or pharmaceutical composition comprising the same |
| KR20200004179A (en) * | 2018-07-03 | 2020-01-13 | 고혁진 | Aronia fermented beverage with antioxidant function and method for manufacturing the same |
-
2020
- 2020-12-03 KR KR1020200167269A patent/KR102607676B1/en active IP Right Grant
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004097222A (en) | 2002-09-06 | 2004-04-02 | Alfa Wassermann Spa | Bacillus bifidus and preparation containing the same |
| US20070280967A1 (en) | 2003-12-03 | 2007-12-06 | Tim Lee | Cryo-protective agents for microorganisms |
| JP2016105705A (en) * | 2009-04-30 | 2016-06-16 | イントレクソン・アクトバイオテイクス・エヌブイＩｎｔｒｅｘｏｎ Ａｃｔｏｂｉｏｔｉｃｓ ＮＶ | Cryoprotectants for freeze drying of lactic acid bacteria |
| US20170151292A1 (en) | 2009-04-30 | 2017-06-01 | Intrexon Actobiotics Nv | Cryoprotectants for freeze drying of lactic acid bacteria |
| KR101604633B1 (en) * | 2015-08-28 | 2016-03-18 | 주식회사 락토메이슨 | Medium composition for culturing lactic acid bacteria and producing method of powder of lactic acid bacteria using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20210070220A (en) | 2021-06-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106282072B (en) | Compound lactobacillus microecological preparation and preparation method and application thereof | |
| CN101603021B (en) | Metabolically active micro organisms and methods for their production | |
| CN109749957B (en) | Preparation and application of lactobacillus gasseri preparation with aquatic pathogenic bacteria antagonistic property | |
| CN112011481B (en) | Lactobacillus reuteri for preventing and treating bacterial diarrhea of livestock and poultry and application thereof | |
| CN107312732B (en) | Probiotic feed additive | |
| CN106578064B (en) | Donkey-hide gelatin and lactic acid bacteria beverage and preparation method thereof | |
| CN102783566B (en) | Feed premix with intestine prompting function for aquatic livestock and preparation method for feed premix | |
| CN113549574B (en) | Bacillus coagulans and application thereof | |
| CN113122467B (en) | Lactobacillus paracasei and composition thereof | |
| CN102517227B (en) | Enterococcus faecalis and applications and feed additive and leavening agent thereof | |
| CN110577907B (en) | Bifidobacterium animalis and application thereof | |
| CN114085789B (en) | Pediococcus pentosaceus MA.WTPQJ01 and application thereof | |
| CN113088468B (en) | Lactobacillus casei Ma. GLRGJ1 and application thereof | |
| EP2166083B1 (en) | Novel Lactobacillus paracasei subsp. paracasei SG96, a bacteriostatic composition containing the same and use thereof | |
| KR20200042165A (en) | Novel lactobacillus salivarius and feed additives for aquacultural fish and crustacea comprising the same | |
| CN106617095B (en) | Donkey-hide gelatin probiotic oral liquid and preparation method thereof | |
| KR102003822B1 (en) | Lactic acid bacteria improving hair quality, additives and composition for animal feed for improving hair quality comprising thereof as effective component | |
| CN112226389A (en) | Planting culture method of intestinal probiotic groups of Sanhuang young chickens and application of intestinal probiotic groups | |
| KR102607676B1 (en) | Probiotics composition for companion animals comprising aerotolerant Bifidobacterium strain as effective component and production method thereof | |
| KR100240687B1 (en) | Lactobacillus acidophilus ky 2104 and its uses | |
| JP2019517810A (en) | Bacillus licheniformis NY1505 strain producing a large amount of α-glucosidase inhibitor | |
| CN109897800A (en) | The strong enterococcus A8-1 of one plant of selenium-rich and its application | |
| CN114456969A (en) | Probiotic preparation for losing weight and reducing blood sugar as well as preparation method and application thereof | |
| CN108690820B (en) | High ampicillin resistance lactobacillus plantarum and breeding method and application thereof | |
| CN107189968B (en) | Enterococcus faecalis and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| E902 | Notification of reason for refusal | ||
| AMND | Amendment | ||
| E601 | Decision to refuse application | ||
| AMND | Amendment | ||
| X701 | Decision to grant (after re-examination) |