JP3436581B2 - Method for producing cells with high lactose-degrading enzyme activity - Google Patents
Method for producing cells with high lactose-degrading enzyme activityInfo
- Publication number
- JP3436581B2 JP3436581B2 JP07424494A JP7424494A JP3436581B2 JP 3436581 B2 JP3436581 B2 JP 3436581B2 JP 07424494 A JP07424494 A JP 07424494A JP 7424494 A JP7424494 A JP 7424494A JP 3436581 B2 JP3436581 B2 JP 3436581B2
- Authority
- JP
- Japan
- Prior art keywords
- acidophilus
- lactose
- degrading enzyme
- enzyme activity
- bile acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 230000000694 effects Effects 0.000 title claims description 38
- 102000004190 Enzymes Human genes 0.000 title claims description 35
- 108090000790 Enzymes Proteins 0.000 title claims description 35
- 238000004519 manufacturing process Methods 0.000 title claims description 7
- 239000003613 bile acid Substances 0.000 claims description 25
- 240000001046 Lactobacillus acidophilus Species 0.000 claims description 24
- 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 claims description 22
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 21
- 229940039695 lactobacillus acidophilus Drugs 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 14
- 239000004094 surface-active agent Substances 0.000 claims description 14
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims description 8
- 229960003964 deoxycholic acid Drugs 0.000 claims description 8
- KXGVEGMKQFWNSR-UHFFFAOYSA-N deoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 KXGVEGMKQFWNSR-UHFFFAOYSA-N 0.000 claims description 8
- 241000186660 Lactobacillus Species 0.000 claims description 5
- 229940039696 lactobacillus Drugs 0.000 claims description 5
- 241000478814 Acidops Species 0.000 claims 1
- 241000894006 Bacteria Species 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 24
- 239000002609 medium Substances 0.000 description 20
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 12
- 201000010538 Lactose Intolerance Diseases 0.000 description 10
- 238000012258 culturing Methods 0.000 description 10
- 235000015063 acidophilus milk Nutrition 0.000 description 8
- 230000001965 increasing effect Effects 0.000 description 8
- 230000001580 bacterial effect Effects 0.000 description 7
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 6
- 239000004310 lactic acid Substances 0.000 description 6
- 235000014655 lactic acid Nutrition 0.000 description 6
- 239000008101 lactose Substances 0.000 description 6
- 210000000941 bile Anatomy 0.000 description 5
- 235000013365 dairy product Nutrition 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 241000283690 Bos taurus Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 210000001035 gastrointestinal tract Anatomy 0.000 description 3
- 239000006872 mrs medium Substances 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 102220201851 rs143406017 Human genes 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 235000013618 yogurt Nutrition 0.000 description 2
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 241000225382 Chironomus acidophilus Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 208000037656 Respiratory Sounds Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000194024 Streptococcus salivarius Species 0.000 description 1
- 206010047924 Wheezing Diseases 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000001967 plate count agar Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 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 description 1
- 239000012137 tryptone Substances 0.000 description 1
Landscapes
- Dairy Products (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、ラクトバチルス・アシ
ドフィルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐ
ｈｉｌｕｓ）の菌体を、界面活性剤の胆汁酸及び／又は
デオキシコール酸を添加した培地で培養して、乳糖分解
酵素活性の高い菌体を製造する方法に関する。本発明の
方法で得られたラクトバチルス・アシドフィルス（Ｌａ
ｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ）の
菌体は、乳糖分解酵素の活性が高いので、アシドフィル
スミルクを製造する際の乳酸菌スターターとして特に有
用である。The present invention relates to a Lactobacillus acidophilus (Lactobacillus acidop
hilus ), and the surfactant bile acid and / or
The present invention relates to a method for producing cells having high lactose-degrading enzyme activity by culturing in a medium containing deoxycholic acid . Lactobacillus acidophilus ( La obtained by the method of the present invention)
The bacterium of Ctobacillus acidophilus ) is particularly useful as a lactic acid bacterium starter when producing Acidophilus milk because of its high activity of lactose-degrading enzyme.
【０００２】[0002]
【従来の技術】乳糖不耐症は、牛乳などに含まれている
乳糖を摂取した際に、下痢、腹痛、腹鳴などが起こる症
状で、この症状は、消化されない二糖類が腸管内の浸透
圧を高めて多量の水を保持し、細菌による発酵が起こっ
て乳酸が産生されることによるものであり、便は酸臭を
呈する。また、この症状が長く続くと発育が阻害される
ことになる。このような乳糖不耐症は、小腸粘膜の刷子
縁膜に局在する乳糖分解酵素が先天的に欠損しているか
あるいは低下すると発症する。また、黒人や黄色人種の
多くは乳児期に正常な乳糖分解酵素の活性を示すが、加
齢と共に乳糖分解酵素活性が低下し、病的に酵素活性が
低くなると乳糖不耐症の症状となって現れる。BACKGROUND OF THE INVENTION Lactose intolerance is a symptom of diarrhea, abdominal pain, wheezing, etc. when ingesting lactose contained in milk and the like. This symptom is infiltration of indigestible disaccharides into the intestinal tract. This is because the pressure is increased to retain a large amount of water, fermentation by bacteria occurs and lactic acid is produced, and stool exhibits an acid odor. Moreover, if this symptom continues for a long time, the growth will be impaired. Such lactose intolerance occurs when the lactose-degrading enzyme localized in the brush border membrane of the small intestinal mucosa is congenitally deficient or reduced. Also, many blacks and yellows show normal lactose-degrading enzyme activity in infancy, but lactose-degrading enzyme activity decreases with aging, and when the enzyme activity becomes pathologically low, lactose intolerance symptoms occur. Appears.
【０００３】一方、この乳糖不耐症を改善するには、ラ
クトバチルス・デルブルッキー・サブスピーシズ・ブル
ガリクス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕ
ｅｃｋｉｉ ｓｕｂｓｐ．ｂｕｌｇａｒｉｃｕｓ、以
下、ブルガリア菌と略記する）やストレプトコッカス・
サリバリウス・サブスピーシズ・サーモフィルス（Ｓｔ
ｒｅｐｔｏｃｏｃｃｕｓ ｓａｌｉｖａｒｉｕｓ ｓｕ
ｂｓｐ．ｔｈｅｒｍｏｐｈｉｌｕｓ、以下、サーモフィ
ルス菌と略記する）を乳酸菌スターターとして用いたヨ
ーグルトを摂取することが有効であることが知られてい
る。そして、アシドフィルスミルクのような非発酵型の
乳製品を摂取することも乳糖不耐症を改善するのに有効
であることも知られているが、ブルガリア菌やサーモフ
ィルス菌の乳糖分解酵素活性に比べ、ラクトバチルス・
アシドフィルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉ
ｄｏｐｈｉｌｕｓ、以下、アシドフィルス菌と略記す
る）の乳糖分解酵素活性は低いので、アシドフィルスミ
ルクの乳糖不耐症改善効果は、ブルガリア菌やサーモフ
ィルス菌を用いたヨーグルトより弱いとされている。し
かし、アシドフィルス菌は、乳糖分解酵素の活性は弱い
ものの糞便中から高い頻度で分離されることから、腸管
定着性がある乳酸菌とされている。また、アシドフィル
ス菌には、胆汁酸に対する耐性が強いものが多く、ブル
ガリア菌に比べて乳糖不耐症改善効果が弱いのは、この
胆汁酸耐性によるものであるという報告もある。したが
って、アシドフィルス菌の腸管内における乳糖分解酵素
活性の発現を期待する場合には、ブルガリア菌が示す程
度の弱い胆汁酸耐性を有する株を選択する必要があると
いわれている。ところが、本来アシドフィルス菌は胆汁
酸耐性が強いという性質を有するので、このような胆汁
酸耐性が強いアシドフィルス菌の乳糖分解酵素活性を高
める方法が求められていた。On the other hand, in order to improve the lactose intolerance, Lactobacillus delbrux delbrucus
eckii subsp. bulgaricus , hereinafter abbreviated as "Bulgarian fungus") and Streptococcus
Salivarius Subspecies Thermophilus ( St
reptococcus salivarius su
bsp. It is known that it is effective to ingest yogurt using thermophilus (hereinafter abbreviated as thermophilus ) as a lactic acid bacterium starter. It is also known that ingesting a non-fermenting dairy product such as Acidophilus milk is also effective in improving lactose intolerance, but it does not affect the lactose-degrading enzyme activity of Bulgaria or Thermophilus. By comparison, Lactobacillus
Acidophilus ( Lactobacillus aci
dofilus (hereinafter, abbreviated as Acidophilus bacterium) has a low lactose-degrading enzyme activity, and thus the effect of Lactose intolerance improvement of Acidophilus milk is said to be weaker than that of yogurt using Bulgaria bacterium or Thermophilus bacterium. However, Lactobacillus acidophilus is a lactic acid bacterium that is capable of colonizing the intestinal tract because it is frequently isolated from faeces although the activity of lactose-degrading enzyme is weak. In addition, it is reported that many Acidophilus bacteria are highly resistant to bile acids, and that the effect of improving lactose intolerance is weaker than that of Bulgarian bacteria due to this bile acid resistance. Therefore, when it is expected that lactose-degrading enzyme activity in the intestinal tract of Lactobacillus acidophilus is expected, it is said that it is necessary to select a strain having a weak bile acid resistance as shown by Bulgaria. However, since Acidophilus bacteria originally have a property of being highly resistant to bile acids, there has been a demand for a method of enhancing the lactose-degrading enzyme activity of such Acidophilus bacteria having strong bile acid resistance.
【０００４】通常の培養方法で得られたアシドフィルス
菌の菌体を胆汁酸などの界面活性剤で処理することによ
り、その菌体の乳糖分解酵素活性が高まることは知られ
ているが、アシドフィルス菌の培養にむしろマイナス要
因となる胆汁酸などの界面活性剤を培地に添加し、菌体
の乳糖分解酵素活性が高めることは行われていなかっ
た。なお、集菌した菌体を界面活性剤で処理する方法
は、処理後、再度、菌体を洗浄するという操作が必要で
あり、煩雑であるという問題があった。It is known that the Lactose-degrading enzyme activity of B. acidophilus is increased by treating the B. acidophilus cells obtained by a usual culture method with a surfactant such as bile acid. It has not been carried out to increase the lactose-degrading enzyme activity of the bacterial cells by adding a surfactant such as bile acid, which is a negative factor to the culture, to the medium. The method of treating the collected bacterial cells with a surfactant requires a procedure of washing the bacterial cells again after the treatment, which is a problem in that it is complicated.
【０００５】[0005]
【発明が解決しようとする課題】本発明者らは、アシド
フィルスミルクの製造などに用いるアシドフィルス菌の
乳糖不耐症改善効果の発現について、種々検討を行った
ところ、胆汁酸などの界面活性剤を添加した培地でもア
シドフィルス菌を培養することができることが判明し、
しかも、その菌体の乳糖分解酵素活性が飛躍的に高まる
ことを見出し、本発明をなすに至った。したがって、本
発明は、乳糖分解酵素の活性が高められたアシドフィル
ス菌の菌体を製造する方法を提供することを課題とす
る。DISCLOSURE OF THE INVENTION The present inventors have conducted various studies on the expression of the effect of improving Lactose intolerance of Acidophilus bacteria used for the production of Acidophilus milk, etc., and found that surfactants such as bile acids were used. It was found that the Acidophilus can be cultivated in the added medium,
Moreover, they have found that the lactose-degrading enzyme activity of the bacterial cells is dramatically increased, and have completed the present invention. Therefore, an object of the present invention is to provide a method for producing a bacterium of Lactobacillus acidophilus in which the activity of lactose-degrading enzyme is enhanced.
【０００６】本発明の方法で製造したアシドフィルス菌
の菌体は、乳糖分解酵素の活性が高いので、この菌体を
乳酸菌スターターとして用いて製造したアシドフィルス
ミルクなどの乳製品は、乳糖不耐症の改善に有用であ
る。[0006] Since the Lactobacillus acidophilus cells produced by the method of the present invention have a high activity of lactose-degrading enzyme, dairy products such as Acidophilus milk produced by using these cells as a lactic acid bacterium starter are lactose intolerant. It is useful for improvement.
【０００７】[0007]
【課題を解決するための手段】本発明では、菌体の乳糖
分解酵素活性を高めるために、胆汁酸やデオキシコール
酸などの界面活性剤を添加した培地でアシドフィルス菌
を培養する。In the present invention, in order to enhance the lactose-degrading enzyme activity of bacterial cells, bile acid or deoxychol is used.
C. acidophilus is cultured in a medium to which a surfactant such as acid is added.
【０００８】本発明で用いるアシドフィルス菌は、胆汁
酸存在下でも十分生育が可能であるという特性を有する
菌株を用いる。なお、胆汁酸耐性の高い菌株とは、０．
５％濃度となるように胆汁酸を添加した培地にアシドフ
ィルス菌を１％接種して３７℃で６時間培養したときの
生菌数が、１×１０7 ｃｆｕ／ｍｌ以上となる菌株を言
う。また、本発明は乳糖不耐症改善効果をアシドフィル
ス菌に賦与することを目的とするものであるので、乳糖
分解酵素活性が高い菌株であることがより好ましい。As the Acidophilus used in the present invention, a strain having a characteristic that it can grow sufficiently even in the presence of bile acid is used. The strain having high bile acid resistance is 0.
It refers to a strain in which the viable cell count is 1 × 10 7 cfu / ml or more when 1% of Acidophilus is inoculated into a medium to which bile acid has been added so as to have a concentration of 5% and cultured at 37 ° C. for 6 hours. Further, since the present invention is intended to impart the lactose intolerance-improving effect to Lactobacillus acidophilus, a strain having a high lactose-degrading enzyme activity is more preferable.
【０００９】一方、本発明で用いる界面活性剤として
は、市販の胆汁酸塩やウシ胆汁酸粉末（Ｏｘ ｂｉｌ
ｅ）、デオキシコール酸などを例示することができる
が、特に限定されるものではない。さらに、アシドフィ
ルス菌を培養する培地は、通常、アシドフィルス菌の培
養に用いる培地であって、ＭＲＳ培地やチーズホエーに
０．５％酵母エキス及び１％トリプトンを添加した培地
などを例示することができるが、いずれにしても、アシ
ドフィルス菌の生育が良好であって、かつ乳糖または適
当な乳糖分解酵素誘導剤を含む培地であれば良い。On the other hand, as the surfactant used in the present invention, commercially available bile salt or bovine bile acid powder (Ox bil) is used.
Examples thereof include e) and deoxycholic acid, but are not particularly limited. Further, the medium for culturing Acidophilus is usually a medium used for culturing Acidophilus, and examples thereof include MRS medium and cheese whey to which 0.5% yeast extract and 1% tryptone are added. However, in any case, any medium can be used as long as it has good growth of Acidophilus and contains lactose or a suitable lactose-degrading enzyme inducer.
【００１０】次に、本発明におけるアシドフィルス菌の
培養について説明する。前培養したアシドフィルス菌を
上記したような培地に胆汁酸やデオキシコール酸などの
界面活性剤を添加した培地に接種し、通常、乳酸菌を培
養する際に用いることのできる装置で培養する。なお、
アシドフィルス菌の接種量、培養時間、培養温度などの
培養条件、あるいは添加する胆汁酸やデオキシコール酸
などの界面活性剤については、アシドフィルス菌の生育
が良好であれば特に制限されるものではない。そして、
培養したアシドフィルス菌の菌体については、遠心分離
などの処理によって集菌し、必要に応じて、リン酸生理
緩衝液などの適当な緩衝液あるいは生理食塩水で洗浄し
て、乳糖分解酵素活性が高められた菌体とすることがで
きる。Next, the culture of the acidophilus in the present invention will be described. The pre-cultured Acidophilus bacterium is inoculated into a medium prepared by adding a surfactant such as bile acid or deoxycholic acid to the above-mentioned medium, and usually cultivated in an apparatus that can be used for culturing lactic acid bacteria. In addition,
Culture conditions such as inoculum size of Acidophilus, culture time, culture temperature, or added bile acid and deoxycholic acid
The surfactants such as are not particularly limited as long as the Acidophilus bacterium grows well. And
The cultured cells of Acidophilus are collected by a treatment such as centrifugation, and if necessary, washed with an appropriate buffer solution such as a physiological phosphate buffer solution or physiological saline to reduce lactose-degrading enzyme activity. It can be an elevated microbial cell.
【００１１】このように、胆汁酸やデオキシコール酸な
どの界面活性剤を添加した培地でアシドフィルス菌を培
養することにより、菌体の乳糖分解酵素活性を高めるこ
とができる。また、アシドフィルス菌などの菌体の乳糖
分解酵素活性を高めるための方法として従来知られてい
た方法、すなわち、通常の方法で培養した菌体を回収し
た後、その菌体を界面活性剤で処理する方法よりも工程
が短縮できる。さらに、本発明の方法で得られたアシド
フィルス菌の菌体は、乳糖分解酵素の活性が高まってい
るので、アシドフィルスミルクなどの乳製品を製造する
際に有効に利用することができる。以下、実施例を示
し、本発明をさらに詳しく説明する。As described above, by culturing Acidophilus bacteria in a medium containing a surfactant such as bile acid or deoxycholic acid , the lactose-degrading enzyme activity of the cells can be enhanced. In addition, a method that has been conventionally known as a method for increasing the lactose-degrading enzyme activity of bacteria such as Acidophilus, that is, after collecting the cultured bacteria by a usual method, treating the bacteria with a surfactant. The process can be shortened as compared with the method. Further, since the Lactobacillus acidophilus cells obtained by the method of the present invention have increased activity of lactose-degrading enzyme, they can be effectively used in the production of dairy products such as Acidophilus milk. Hereinafter, the present invention will be described in more detail with reference to examples.
【００１２】[0012]
【実施例１】胆汁酸耐性の高いアシドフィルス菌の菌株
として、ラクトバチルス・アシドフィルス（Ｌａｃｔｏ
ｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ）ＳＢＴ２
０６８株（ＦＥＲＭ Ｐ−１４１６１）を用いた。０．
５％濃度となるようウシ胆汁酸を添加した乳糖含有ＭＲ
Ｓ培地に、前培養した上記のアシドフィルス菌を１％接
種し、３７℃で１６時間培養した。なお、培養は１ｌ容
のフラスコに８００ｍｌの培地を添加して行った。ま
た、培養に用いた乳糖含有ＭＲＳ培地は、ペプトン１０
ｇ、肉エキス５ｇ、酵母エキス５ｇ、ラクトース２０
ｇ、リン酸水素二カリウム２ｇ、クエン酸アンモニウム
２ｇ、酢酸ナトリウム５ｇ、硫酸マグネシウム・７水和
物０．２ｇ及び硫酸マンガン・４水和物０．０５ｇを蒸
留水１ｌに溶解し、０．５Ｎ水酸化ナトリウムでｐＨを
６．２に調整したものを用いた。また、対照として、ラ
クトバチルス・アシドフィルス（Ｌａｃｔｏｂａｃｉｌ
ｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ）ＳＢＴ２０６２株
（ＦＥＲＭ Ｐ−１０７３０）についても同様の方法で
培養し、それぞれの菌株の培養後の生菌数をＢＣＰ（Ｂ
ｒｏｍｃｒｅｓｏｌ Ｐｕｒｐｌｅ）加プレートカウン
トアガー上で測定した。その結果を表１に示す。Example 1 As a strain of Acidophilus highly resistant to bile acid, Lactobacillus acidophilus ( Lacto)
bacillus acidophilus ) SBT2
Strain 068 (FERM P-14161) was used. 0.
Lactose-containing MR supplemented with bovine bile acid to a concentration of 5%
The S culture medium was inoculated with 1% of the above-mentioned pre-cultured Acidophilus bacterium and cultured at 37 ° C. for 16 hours. The culture was performed by adding 800 ml of medium to a 1-liter flask. Also, the lactose-containing MRS medium used for the culture was peptone 10
g, meat extract 5g, yeast extract 5g, lactose 20
g, dipotassium hydrogen phosphate 2 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate heptahydrate 0.2 g and manganese sulfate tetrahydrate 0.05 g were dissolved in distilled water 1 l to give 0.5 N The one whose pH was adjusted to 6.2 with sodium hydroxide was used. In addition, as a control, Lactobacillus acidophilus (Lactobacil
L. acidophilus ) SBT2062 strain (FERM P-10730) was also cultured in the same manner, and the viable cell count of each strain after culturing was determined to be BCP (B
romcresol Purple) plate count agar. The results are shown in Table 1.
【００１３】[0013]
【表１】 ──────────────────────────────────── 生菌数（ｃｆｕ／ｍｌ） 菌 株 ──────────────────────── 胆汁酸添加培地 胆汁酸無添加培地 ──────────────────────────────────── ＳＢＴ２０６２株 ８．０×１０8 ８．５×１０8 ＳＢＴ２０６８株 １．２×１０9 １．５×１０9 ────────────────────────────────────[Table 1] ──────────────────────────────────── Viable cell count (cfu / ml) Strain ──────────────────────── Bile acid-containing medium Bile acid-free medium ───────────────── ─────────────────── SBT2062 strain 8.0 × 10 8 8.5 × 10 8 SBT2068 strain 1.2 × 10 9 1.5 × 10 9 ─── ──────────────────────────────────
【００１４】次に、上記のようにして培養して得られた
菌体をリン酸緩衝生理食塩水で３回洗浄した後、乳糖分
解酵素の活性を測定した。なお、乳糖分解酵素活性の測
定は〔Ｂ．Ｆｏｏｄ Ｍｉｃｒｏｂｉｏｌ．，２，２３
〜２９（１９８５）〕などの文献に記載のＯＮＰＧ−ガ
ラクトピラノシドを用いる方法に従って行った。また、
乳糖分解酵素活性は、菌体湿重量１０ｍｇ当たりの活性
単位（Ｕ）で表した。その結果を表２に示す。Next, the cells obtained by culturing as described above were washed 3 times with phosphate buffered saline and the activity of lactose-degrading enzyme was measured. The lactose-degrading enzyme activity is measured by [B. Food Microbiol. , 2 , 23
~ 29 (1985)] and the like using ONPG-galactopyranoside. Also,
The lactose-degrading enzyme activity was expressed as an activity unit (U) per 10 mg of wet cell weight. The results are shown in Table 2.
【００１５】[0015]
【表２】 ──────────────────────────────────── 乳糖分解酵素活性（Ｕ） 菌 株 ──────────────────────── 胆汁酸添加培地 胆汁酸無添加培地 ──────────────────────────────────── ＳＢＴ２０６２株 ０．５８ ０．１５ ＳＢＴ２０６８株 ６．７ ０．１３ ────────────────────────────────────[Table 2] ──────────────────────────────────── Lactose-degrading enzyme activity (U) Strain ──────────────────────── Bile acid-free medium Bile acid-free medium ──────────────────────────────────── SBT2062 strain 0.58 0.15 SBT2068 strain 6.7 0.13 ────────────────────────────────────
【００１６】このように、胆汁酸添加培地で培養したア
シドフィルス菌の乳糖分解酵素活性は、胆汁酸無添加培
地で培養したアシドフィルス菌に比べて上昇しているこ
とが判る。As described above, it is understood that the lactose-degrading enzyme activity of the Acidophilus bacterium cultured in the bile acid-added medium is higher than that of the Acidophilus bacterium cultured in the bile acid-free medium.
【００１７】[0017]
【実施例２】界面活性剤として、デオキシコール酸を培
地に０．０５％添加した以外は実施例１と同様に培養し
たアシドフィルス菌の培養後の生菌数と乳糖分解酵素活
性を表３に示す。[Example 2] Table 3 shows the viable cell count and lactose-degrading enzyme activity after culturing of Acidophilus that was cultured in the same manner as in Example 1 except that 0.05% of deoxycholic acid was added to the medium as a surfactant. Show.
【００１８】[0018]
【表３】 ──────────────────────────────────── 菌 株 生菌数（ｃｆｕ／ｍｌ） 乳糖分解酵素活性（Ｕ） ──────────────────────────────────── ＳＢＴ２０６２株 ２．５×１０8 ０．３３ ＳＢＴ２０６８株 １．４×１０8 ３．５ ────────────────────────────────────[Table 3] ──────────────────────────────────── bacteria strain viable cell count (cfu / ml) Lactose-degrading enzyme activity (U) ──────────────────────────────────── SBT2062 strain 2.5 × 10 8 0.33 SBT2068 strain 1.4 × 10 8 3.5 ──────────────────────────────────── ─
【００１９】[0019]
【参考例１】ウシ胆汁酸を０．５ ％添加した乳糖含有
ＭＲＳ培地５ｌを調製した。この培地にラクトバチルス
・アシドフィルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃ
ｉｄｏｐｈｉｌｕｓ）ＳＢＴ２０６８（ＦＥＲＭ Ｐ−
１４１６１）の前培養物５０ｍｌを添加し、３７℃で１
６時間培養した。培養後、８０００ｒｐｍで１０分間培
養液を遠心分離し、アシドフィルス菌の菌体を集菌し
た。そして、この菌体を生理食塩水で２回洗浄した後、
殺菌済の生乳５ｌに添加してよく攪拌し、菌体を懸濁し
てアシドフィルスミルクを製造した。Reference Example 1 5 L of lactose-containing MRS medium supplemented with 0.5% bovine bile acid was prepared. Lactobacillus acidophilus ( Lactobacillus ac
idophilus ) SBT2068 (FERM P-
14161) pre-cultured 50 ml and added at 37 ° C for 1
Cultured for 6 hours. After culturing, the culture solution was centrifuged at 8000 rpm for 10 minutes to collect the bacteria of Acidophilus. Then, after washing the cells twice with physiological saline,
It was added to 5 liters of sterilized raw milk and stirred well to suspend the bacterial cells to produce Acidophilus milk.
【００２０】[0020]
【発明の効果】本発明のように、胆汁酸やデオキシコー
ル酸などの界面活性剤を添加した培地でアシドフィルス
菌を培養することにより、菌体の乳糖分解酵素活性を高
めることができる。また、アシドフィルス菌などの菌体
の乳糖分解酵素活性を高めるための方法として従来知ら
れていた方法よりも工程が短縮できる。さらに、得られ
たアシドフィルス菌の菌体は、乳糖分解酵素の活性が高
まっているので、アシドフィルスミルクなどの乳製品を
製造する際に有効に利用することができる。As in the present invention, bile acid and deoxycholic acid
By culturing Acidophilus bacteria in a medium to which a surfactant such as acid is added, the lactose-degrading enzyme activity of the cells can be increased. In addition, the steps can be shortened as compared with the conventionally known method for increasing the lactose-degrading enzyme activity of bacterial cells such as Acidophilus. Further, since the obtained Lactobacillus acidophilus cells have increased activity of lactose-degrading enzyme, they can be effectively used in the production of dairy products such as Acidophilus milk.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ Ｃ１２Ｒ 1:23） Ｃ１２Ｒ 1:23 (56)参考文献 特開 平５−292947（ＪＰ，Ａ) Ｎｅｗ Ｆｏｏｄ Ｉｎｄｕｓｔｒ ｙ，1991，Ｖｏｌ．33，Ｎｏ．12，ｐ. 39−47 (58)調査した分野(Int.Cl.7，ＤＢ名) C12N 1/20 C12N 1/38 ＢＩＯＳＩＳ／ＷＰＩ（ＤＩＡＬＯＧ) ＰｕｂＭｅｄ─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. 7 Identification code FI C12R 1:23) C12R 1:23 (56) Reference JP-A-5-292947 (JP, A) New Food Industry, 1991, Vol. 33, No. 12, p. 39-47 (58) Fields surveyed (Int.Cl. 7 , DB name) C12N 1/20 C12N 1/38 BIOSIS / WPI (DIALOG) PubMed
Claims (3)
ｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ）の菌
体を、界面活性剤の胆汁酸及び／又はデオキシコール酸
を添加した培地で培養した後、回収することを特徴とす
る乳糖分解酵素活性の高い菌体の製造法。1. Lactobacillus acidophilus ( Lac
Tobacillus acidophilus ) cells are cultivated in a medium to which a surfactant bile acid and / or deoxycholic acid is added, and then the cells are recovered, which is a method for producing cells having a high lactose-degrading enzyme activity. Law.
ｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ）が、
胆汁酸耐性の高い菌株である請求項１記載の製造法。2. Lactobacillus acidophilus ( Lac
tobacillus acidophilus )
The method according to claim 1, which is a strain having high bile acid resistance.
フィルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐ
ｈｉｌｕｓ）の菌株が、ラクトバチルス・アシドフィル
ス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌ
ｕｓ）ＳＢＴ２０６８株（ＦＥＲＭ Ｐ−１４１６１）
である請求項２記載の製造法。3. A high bile acid resistance Lactobacillus acidophilus (Lactobacillus acidop
strains of hilus) is, Lactobacillus acidophilus (Lactobacillus acidophil
us ) SBT2068 strain (FERM P-14161)
The method according to claim 2, wherein
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP07424494A JP3436581B2 (en) | 1994-03-18 | 1994-03-18 | Method for producing cells with high lactose-degrading enzyme activity |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP07424494A JP3436581B2 (en) | 1994-03-18 | 1994-03-18 | Method for producing cells with high lactose-degrading enzyme activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH07255467A JPH07255467A (en) | 1995-10-09 |
| JP3436581B2 true JP3436581B2 (en) | 2003-08-11 |
Family
ID=13541563
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| GB9814056D0 (en) | 1998-06-29 | 1998-08-26 | Imp Biotechnology | Cheese ripening process |
| JP2002193817A (en) | 2000-12-28 | 2002-07-10 | Calpis Co Ltd | Drug for controlling intestinal functions |
| CN101479382B (en) * | 2006-06-26 | 2012-07-04 | 财团法人粮食研究会 | Lactic acid bacterium for amelioration of lactose intolerance |
| EP3244903B1 (en) * | 2015-01-14 | 2023-06-07 | Infant Bacterial Therapeutics AB | Preparation comprising lactobacillus reuteri and citrate for medical use |
-
1994
- 1994-03-18 JP JP07424494A patent/JP3436581B2/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| Ｎｅｗ Ｆｏｏｄ Ｉｎｄｕｓｔｒｙ，１９９１，Ｖｏｌ．３３，Ｎｏ．１２，ｐ．３９−４７ |
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