CA1274482A - Biodeodorizer and the process thereof - Google Patents
Biodeodorizer and the process thereofInfo
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- CA1274482A CA1274482A CA000486901A CA486901A CA1274482A CA 1274482 A CA1274482 A CA 1274482A CA 000486901 A CA000486901 A CA 000486901A CA 486901 A CA486901 A CA 486901A CA 1274482 A CA1274482 A CA 1274482A
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- lactobacillus
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- odoriferous compounds
- ability
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Abstract
ABSTRACT OF THE DISCLOSURE
A biodeodorizer and a process to prepare the biodeodorizer are described, the biodeodorizer comprising (a) at least one strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus clearans and a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus, and (b) Streptococcus faecalis having the ability to produce antibiotics.
A biodeodorizer and a process to prepare the biodeodorizer are described, the biodeodorizer comprising (a) at least one strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus clearans and a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus, and (b) Streptococcus faecalis having the ability to produce antibiotics.
Description
~2744~3~
BIODEODORIZE~ AND THE PROCES5 THEREOF
FIELD OF THE INVENTION
. . . _ . _ The present invention relates to a biodeodorizer and the process thereof and, more ~articularly, ~o a biodeodorizer comprising a mixture of a certain Lactoba~illus . . _ . . _ strains and Streptococcus faecalis.
. .
BACKGROUND OF THE INVENTION
It is already known to use various microorganisms for deodorizing decomposing matter and the like, or for ~ 10 reducing or removing offensive odors arising from decomposing - matter or the like. Some bacterial have been used in vivo and some bacteria have been used in the environment by dispersion.
For instance, Japanese Patent Publication No. 7297/83 and British Patent 1,584,693 teach that bacteria which are used in the environment by dispersion have deodorizing function. Further, microorganisms having deodorizing function in vivo were already developed by the inventors concerned. ~For instance, see U.S. Patent No. 4,345,032, British Patent No. 1,584,694, etc.) ~nong these deodorant microorganisms for use in vivo Lactobacillus deodorans, Lactobacillus clearans, Lactobacillus sulfurica, Lactobacillus nitrosus, etc. are mentioned as preferred.
1;~7448~
These deodor~nt microorganisms, however, have the disadvantage that, when they were administered to man and anlmals, the deodorant efrects attained were not always the same but varied depending on the individual or on the animal species and on the conditions of administration, even when the same strains were administered in the same amount. Therefore, it has been desired to make further improvements so that a definite or fixed effect could be attained by administration of these microorganisms to man and animals, irrespectively of the individual or animal species.
Under these circumstances, various investigations have now been made to heighten the deodorant effect of Lactobacillus deodorans, Lactobacillus clearans, Lactobacillus sulfurica and Lactobacillus nitrosus and overcome the defects . _ as described above, and the present invention has been accomplished.
SUMMARY OF THE INVENTION
. . ~_ .
Thus~ an object of the present invention is to provide a novel biodeodorizer and a process thereof.
Another object of the present invention is to provide a novel biodeodorizer exhibitin~, on application to man and animals, a fixed deodorant effect irrespectively of the individual and animal species and the conditions of administration, and a process thereof.
~2~4482 A further object of the present invention is to provid~ a biodeodorizer exhibiting, on administration to man and animals, an excellent and durable deodorant efrect irrespectively of the individual and animaL species and the conditions.of the administration, and a process thereof.
Another object of the present-i-nvention-is to provide a biodeodorizer exhibiting, on administration to man and animals, a deodorant effect which appears within a short time after administration and is durable for a long tLme after the stoppage of the administration, irrespectively of the individual and animal species and the conditions of the administration, and a process thereof.
It has now been found after earnest investigations that the above objects are attained by using the above-mentioned microorganisms in combination with an othercertain microorganism, rather than by improving the micro-organisms per se.
The objects as described above of the present invention have been attained by using (a) at least one Lactobacillus strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus clearans and a mixed culture or Lactobacillus sulfurica and Lactobacillus .
nitrosus, all belonging to deodorant Lactobacillus, in combination with (b) Streptococcus faecalis having the abilit~ to produce antibiotics.
lZ7~482 Thus, the present invention provides a biodeod~rizer comprising (a) at least one strain selected from the group consisting of Lactobacillus deodorans, ~actobaci-llus.slearans.
and a mixed strain of Lactobacillus sulrurica and Lactobacillus . .
nitrosus, and (b) Streptococcus faecalis having the ability to produce antibiotics~
The present invention provides a process to prepare a biodeodorizer comprising mixing la~ at least one strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus clearans and a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus, and (b) Streptococcus faecalis having the ability to produce ~ .
antibiotics.
The present invention further provides a biodeodorizer comprising a combination of ta) at least one strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus -clearans and a mixed strain of .
Lactobacillus-sulfurica and Lactobacillus nitrosus, (b) Streptococcus faecalis having the ability to produce an antibiotics, and further (c) a Bifidobacterium strain and/or a Bacillus strain.
The present invention provides a process to prepare a biodeodorizer comprising mixing ~a) at least one strain selected rrom the group consisting of Lactobacillus deodorans, Lactobacillus clearans, and a mixed strain of 1~74~82 Lactobacillus sulfurica and Lactobacillus nitrosus, , . . .. _ .
(b) Streptcoccus faecalis having the ability to produce antibiotics and (c) a Bifidobacterium strain and/or a -Bacillus strain, c S DETAILED DESCRIPTION OF THE INVENTION
- The characteristics of the Lactobacillus deodorans used in the present invention, and a method of isolating it, are described in Japanese Patent Publication No. 49193/82, Lactobacillus deodorans has the following proper~ies:
(l) a) Odoriferous compounds containing sulfur atoms (such as Na2S 9H2~) and odoriferous compounds containing nitrogen atoms (such as NH3) are essential substances for growth of Lactobacillus deodorans.
For example, even when Lactobacillus deodorans cannot grow in Stephanson-Whetham (S-W) medium only, . it becomes capable of growing in the presance of such odoriferous compounds containing sulfur atoms and/or such odoriferous compounds containing -nitrogen atoms.
b) When Lactobacillus deodorans is cultured in a medium used for the culture according to the present invention, for example, Stephanson-t~hetham medium to which Casamino acid and vitamins are added, growth is promoted or accelerated by adding odoriferous compounds containing sulfur atoms, ~7~48Z
odoriferous compounds containing nitrogen atoms and/or odoriferous compounds containing carbon atoms to the medium during the logarithmic growth stage.
c) When such odoriferous compounds containing a sul~ur atom, odoriferous compounds containing a nitrogen atom and/or odoriferous compounds containing a carbon atom are present in the culture medium, Lactobacillus deodorans continues to grow until these compounds are consumed or assimilated .completelyO
BIODEODORIZE~ AND THE PROCES5 THEREOF
FIELD OF THE INVENTION
. . . _ . _ The present invention relates to a biodeodorizer and the process thereof and, more ~articularly, ~o a biodeodorizer comprising a mixture of a certain Lactoba~illus . . _ . . _ strains and Streptococcus faecalis.
. .
BACKGROUND OF THE INVENTION
It is already known to use various microorganisms for deodorizing decomposing matter and the like, or for ~ 10 reducing or removing offensive odors arising from decomposing - matter or the like. Some bacterial have been used in vivo and some bacteria have been used in the environment by dispersion.
For instance, Japanese Patent Publication No. 7297/83 and British Patent 1,584,693 teach that bacteria which are used in the environment by dispersion have deodorizing function. Further, microorganisms having deodorizing function in vivo were already developed by the inventors concerned. ~For instance, see U.S. Patent No. 4,345,032, British Patent No. 1,584,694, etc.) ~nong these deodorant microorganisms for use in vivo Lactobacillus deodorans, Lactobacillus clearans, Lactobacillus sulfurica, Lactobacillus nitrosus, etc. are mentioned as preferred.
1;~7448~
These deodor~nt microorganisms, however, have the disadvantage that, when they were administered to man and anlmals, the deodorant efrects attained were not always the same but varied depending on the individual or on the animal species and on the conditions of administration, even when the same strains were administered in the same amount. Therefore, it has been desired to make further improvements so that a definite or fixed effect could be attained by administration of these microorganisms to man and animals, irrespectively of the individual or animal species.
Under these circumstances, various investigations have now been made to heighten the deodorant effect of Lactobacillus deodorans, Lactobacillus clearans, Lactobacillus sulfurica and Lactobacillus nitrosus and overcome the defects . _ as described above, and the present invention has been accomplished.
SUMMARY OF THE INVENTION
. . ~_ .
Thus~ an object of the present invention is to provide a novel biodeodorizer and a process thereof.
Another object of the present invention is to provide a novel biodeodorizer exhibitin~, on application to man and animals, a fixed deodorant effect irrespectively of the individual and animal species and the conditions of administration, and a process thereof.
~2~4482 A further object of the present invention is to provid~ a biodeodorizer exhibiting, on administration to man and animals, an excellent and durable deodorant efrect irrespectively of the individual and animaL species and the conditions.of the administration, and a process thereof.
Another object of the present-i-nvention-is to provide a biodeodorizer exhibiting, on administration to man and animals, a deodorant effect which appears within a short time after administration and is durable for a long tLme after the stoppage of the administration, irrespectively of the individual and animal species and the conditions of the administration, and a process thereof.
It has now been found after earnest investigations that the above objects are attained by using the above-mentioned microorganisms in combination with an othercertain microorganism, rather than by improving the micro-organisms per se.
The objects as described above of the present invention have been attained by using (a) at least one Lactobacillus strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus clearans and a mixed culture or Lactobacillus sulfurica and Lactobacillus .
nitrosus, all belonging to deodorant Lactobacillus, in combination with (b) Streptococcus faecalis having the abilit~ to produce antibiotics.
lZ7~482 Thus, the present invention provides a biodeod~rizer comprising (a) at least one strain selected from the group consisting of Lactobacillus deodorans, ~actobaci-llus.slearans.
and a mixed strain of Lactobacillus sulrurica and Lactobacillus . .
nitrosus, and (b) Streptococcus faecalis having the ability to produce antibiotics~
The present invention provides a process to prepare a biodeodorizer comprising mixing la~ at least one strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus clearans and a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus, and (b) Streptococcus faecalis having the ability to produce ~ .
antibiotics.
The present invention further provides a biodeodorizer comprising a combination of ta) at least one strain selected from the group consisting of Lactobacillus deodorans, Lactobacillus -clearans and a mixed strain of .
Lactobacillus-sulfurica and Lactobacillus nitrosus, (b) Streptococcus faecalis having the ability to produce an antibiotics, and further (c) a Bifidobacterium strain and/or a Bacillus strain.
The present invention provides a process to prepare a biodeodorizer comprising mixing ~a) at least one strain selected rrom the group consisting of Lactobacillus deodorans, Lactobacillus clearans, and a mixed strain of 1~74~82 Lactobacillus sulfurica and Lactobacillus nitrosus, , . . .. _ .
(b) Streptcoccus faecalis having the ability to produce antibiotics and (c) a Bifidobacterium strain and/or a -Bacillus strain, c S DETAILED DESCRIPTION OF THE INVENTION
- The characteristics of the Lactobacillus deodorans used in the present invention, and a method of isolating it, are described in Japanese Patent Publication No. 49193/82, Lactobacillus deodorans has the following proper~ies:
(l) a) Odoriferous compounds containing sulfur atoms (such as Na2S 9H2~) and odoriferous compounds containing nitrogen atoms (such as NH3) are essential substances for growth of Lactobacillus deodorans.
For example, even when Lactobacillus deodorans cannot grow in Stephanson-Whetham (S-W) medium only, . it becomes capable of growing in the presance of such odoriferous compounds containing sulfur atoms and/or such odoriferous compounds containing -nitrogen atoms.
b) When Lactobacillus deodorans is cultured in a medium used for the culture according to the present invention, for example, Stephanson-t~hetham medium to which Casamino acid and vitamins are added, growth is promoted or accelerated by adding odoriferous compounds containing sulfur atoms, ~7~48Z
odoriferous compounds containing nitrogen atoms and/or odoriferous compounds containing carbon atoms to the medium during the logarithmic growth stage.
c) When such odoriferous compounds containing a sul~ur atom, odoriferous compounds containing a nitrogen atom and/or odoriferous compounds containing a carbon atom are present in the culture medium, Lactobacillus deodorans continues to grow until these compounds are consumed or assimilated .completelyO
(2) Lactobacillus deodorans.has low auxotroph-y~,and can grow in Stephanson-Whetham medium consisting of inorganic salts and.sugar or in such Stephanson-Whetham medium to which either Casamino acid or vitamins are added.
(~) Lactobacillus deodorans has a high assimilating power for decomposing matters and a strons intestinal stationary ability. Namely, it has a strong cleaning power in vivo.
(4) Lactobacillus deodorans is a non-pathogenic strain.
(5) To maintain the strong deodorant power o~ Lactobacillus deodorans, it is desirable to incorporate amino acids - containing sulfur atoms and/or odoriferous compounds containing sulfur atoms, nitrogen atoms or carbon atoms to the nutrient medium.
(6). For the storage of the cell body, it is desirable to place those substances as mentioned a~ove and/or glycine, ~27~4~3Z
glutamic acid, lysine, alanine, phenylalanine, alginine, aspartic acid, etc. around the cell body of Lactobacillus deodorans.
Any strains of Lactobacillus deodorans which are prepared according to an isolating method of the above Publication or which have the above described characteristics may be used in the present invention.
Especially preferred strains of Lactobacillus deodorans which can be used in the present invention are those strains deposited at the Agency of Industrial Science &
Technology, Fermentation Research Institute ~Japan) under Domestic Registration FERM-P Nos. 1946, 2742, 2779, 2780, 2781 and 2782. FERM-P Nos. 1946 and 2779 are deposlted also at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14450 and 14451.
The characteristics of Lact.obacillus clearans used in the present invention, and a method of isolating it, are described in U.S. Patent No. 4,579,734. (The term "OPI" as used herein refers to a "published unexamined Japanese Patent.
Application".) Lactobacillus clearans has the following characteristics.
(1) a) Odoriferous compounds containing sulfur atoms such as Na2S.9H20), odoriferous compounds containing nitrogen atoms (such as NH3) and/or odoriferous .....
~74~1~Z
compounds containing carbon atoms (such as a lower fatty acid) are not essential for the growth of Lactobaclllus clearans. A medium in which Lactobacillus clearans does not grow cannot be changed to one in which Lactobacillus clearans can grow~ even by addition of the above-mentioned substances.
b) Promotion or acceleration of gro~th does not occur by adding odoriferous compounds containing sulfur atoms, odoriferous compounds containing nitrogen atoms and/or odoriferous compounds containing carbon atoms to the culture medium for Lactobacillus clearans .
at the logarithmic growth stage.
c) Lactobaci-llus clearans utilizes or assimlates both odoriferous compounds containing sulfur atoms and odoriferous compounds containing nitrogen atoms to some degree. (Many strains utilize about 30 ~ 90~ thereof.) However,-Lactobacillus clearans cannot utilize these materials completely.
That is to say, they remain unutilized. (It is noted that Lactobacillus deodorans assimilates the compounds completely.) ~2) The auxotrophy of Lactobacillus clearans is low.
It can grow in Stephanson-Whetham medium by merely adding thereto ~oth Casamino acid and vitamins.
(3~ The multiplying power of Lactobacillus clearans is within the range of 0.9 ~ l.l times in meat extract bouillon, as compared with Eschericria Coli.
12749,~z Lactobacillus clearans utilizes decompos'ng matter, _ although not completely, and so it has a cleaning power in vivo to some degree for about 2 weeks at a maximum, generally for 3 to 6 days, after administration.
(4) Lactobacillus cle~rans corresponds to the Genus . .
Lactobacillus in all propertles~ However, it is quite .
different from previously known strains in respect of the ability of utilizing or assimilating odoriferous compounds containing sulfur atcms (such as ha2S 9~2O) and odori-ferous compounds containing nitrogen atoms (such as NX3), as described above. (Previously known strains except ~or Lactobacillus deodorans do not utilize these types of compound.i Its auxotrophy is low.
(5) The intestinal stationary ability of Lactobacillus clearans is lower than that of Lactobacillus deodorans, .
although it is stronger than that of previously known Lactobacillus strains.) (6) Lactobacillus clearans used in the present invention .
is a non-pathogenic strain.
0 (7) To maintain the strong deodorant power of Lac-tobacillus clearans used in the present invention, it is desirable to incorporate amino acids containing sulfur atoms and/or odoriferous compounds containing sulfur atoms, nitro~en atoms and/or carbon atoms in the nutrient medium.
_ g _ 127~41~2 (8) For the storage of cell body, it is desirable to place those substances as mentioned above for Lactobacillus deodorans around the cell body of Lactobacillus cIearans.
Any strains of Lactobacillus clearans which are prepared according to the above isolating method or which have the above described characteristics may be used in the present invention.
Especially preferred strains of Lactobacillus clearans which can be used in the present invention are those deposited at the Agency of Industrial Science &
Technology, Fermentation Research Institute (Japan) under Domestic fi.egistration FERM-P Nos. 6587,6588,6589 and 6590. Among these strains, strains of FERM-P Nos.
6587 and 6590 are deposited also at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14253 and 1425~.
The characeristics of Lactobacillus sulfurica and Lactobacillus nitrosus used in the present invention, and a method of isolating each of them, are described in ~.S. Patent No. 4,579,734.
Lactobacillus sulfurica used in the present invention utilizes only odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus utilizes only lZ744~3Z
odori~erous compounds containing nitrogen atoms, although the utilization is not complete but r~ns at about 90~ or less in each case~ However, except for this property, they have t~e same properties as Lactobacillus clearans. Therefore, on carrying out deodorization ln vivo with Lactobac~llus sulfuri~~a and Lactobacillus nitrosus, a satisfactory effect cannot be expected unless the two are used in combination.
Any strains of Lactobacillus sulfurica and Lactobacillus nitrosus which are prepared according to the isolating method as described above or which have the above characteristics may be used in the present invention.
Especially preferred strains of Lactobacillus sulfurica are those deposited at the Agency of Industrial Science & Technology, Fermentation Research Institute (Japan) under Domestic Registration FERM-P Nos. 7383 and 7384, which are also deposited at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14255 and 14256, respectively, and especially preferred strains of Lactobacillus nitrosus are those deposited at Fermentation Research Institute the Agency of Industrial Science and Technology (Japan) under Domestic Registration FERM-P Nos. 7385 and 7386, which are also deposited at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14257 and 14258, respectively.
1~74~8Z
The differences in the four types of Lactcbacil1u6 mentioned above from previously known Lactoba _llus.
(1) Previously known Lactobac~llus strains do not require odoriferous compounds containing sulfur atoms and odoriferou~
compounds containing nitrogen atoms as essential substances for their growth. Even when these odoriferous compounds are added to a culture medium in which known Lactobacillus strains cannot grow, the strains do not begin to grow.
Further, even when these compounds are added at the logarithmic growth stage of the known strains, promotion or acceleration of growth does not occur. Previously known Lacto~acillus strains do not at all consume these compounds, and so the compounds remain ultimately in the amount originally added~
(22 The previously known Lactobacillus strains are separated in respect of their auxotrophy into a group of strains which can grow in Stephanson-Whetham medium by adding Caramino acid and vitamins thereto and a group of strains which cannot grow in Stephanson-Whetham medium unless other ingredients are furhter added thereto in addition to Casamino acid and vitamins. (3) The previously known Lactobacillus strains are inferior in multiplying power in usual medium to Escherichia coli, etc. (for example, 0.5 times that of Escherichia coli)~ Their intestinal stationary ability is at a degree of 1 or 2 days.
The cleaning power against even odriferous compounds ~Z7~3Z
containing carbon atoms is unknown.
The four kinds of Lactobacillus strains all have the following prope-ties of Lactobac_llus:
That is, they are Gram-positive, anaerobic or micro aerophilic, non-spore forming rods and, depending on the strains, may be spherical rod-like, curved rod-like, coryne-like or thread-like~ They are non-motile, negative to catalase and do not reduce nitrates. They do not decmpose gelatin and do not produce indole or hydrogen sulfide. Some of them are bipolar-stained.
Their ability to decompose protein and fat is very weak, if any. They show better growth under anaerobic'or microaexobic, conditions rather than under aerobic conditions, having strong ability to decompose sugars, and are acid-fast. When used for glucose fermentation, they produce lactic acid in an yield of more than 50%.
Their growth is promoted by adding acetic acid.
They are not pathogenic to animals and plants.
Lac_obacillus clearans acts very mildly even when it is orally administered in a large amount and reduces offensively odoriferous substances in feces, without an uncomfortable feeling, while the administration continues. However, when administration is stopped, the strain is excreted through intestinal tract to lose its deodorant ~2744~Z
n vivo within a short time.
Lactobacillus deodorans has a deodorant power of about several tens times or more, compared with Lactobacillus clea~ans, and morover it deodorizes completely, that is, it degrades offensively odoriferous substances completely.
For example, human feces were deodorized completely within 48 hours by administration of a tenth part (by weight) of a culture solution of Lactobacil-lus deodorans.
The LactobacillUs deodorans - used in the present invention is obtained by cultivation of Lactobacillus lactis which multiplies and is capable or not capable of producin~
antibiotics in either one of the minimal nutrient mediums shown in the following table, in a medium not affecting the properties of these strains, and the subsequent isolation.
Classifi-cation Minimal Nutrien~ Medium . . . _ . _ (a) Glucose or starch, inorganic salts and water (b) An odoriferous compound containing sulfur, nitrogen or carbon, which is present in feces, is added to ta3 (c~ Vitamins are added individually or in combination, to (a) (d) Amino acids are added individually or in combi-nation, to (a) (e) Specific vitamins are added individually or in combination, to ta) ~Z~4~82 (f) Tryptophane is added to (a) (g) ~mino acids containing sulfur are added individually or in combination, to (2) It is preferable to endow resistance against antibacterial agent, if necessary. The Lactobacillus lactis so obtained can be processed into preparations according to conventional- methods The above-mentioned medium not affecting the properties of Lactobacillus lactis is each of the minimal nutrient mediums as stated in the present invention, a combination of them, or an a~ar medium similar thereto. As the case may be, usual agar medium may be used intermediately.
Further, Lactobacillus clearans is isolated using the medium which is higher in nutrient than that for Lacto-acillus deodorans~ Por example, the medium for isolation of Lactobacillus clearans comprises meat extract, peptone, Na2S-9H2O, sodium butyrate, glucose and agar. As a preferable medium for Lactobacillus nitrosus, it- comprises meat extract, peptone, NH3, sodium butyrate, glucose and agar. As a prefer-able medium for Lactobacillus sulfurica, it comprises meatextract, peptone, Na2S-9K2O, sodium butyrate, glucose, CaCO3, and agar.
Of colonies thus appeared, colonies which margin is transparency are screened and subjected to gas generation ~LZ7448Z
testing and non-gas generation cells are retained. Sub-sequently, cells thus obtained were subjected to hemolysis testing to obtain non-hemolytic cells and then cultured in the LBS medium. After confirmation tha-t the cells belong to Lactobacillus, degree of decrease of Na2S.9H20 and NH3, and accelerated growth for the I.actobacillus strains are examined and classified to Lactobacillus deodorans, Lacto-bacillus clearans, Lactobacillus sulfurica and Lactobacillus nitrosus. As to each Lactobacillus, ones which have higher u value are collected.
The Lactobacillus lactis used in the present invention was isolated, for example, in the following manner:
In a medium (A) [Stephanson Wetham medium S-W] +
vitamin + agar (wherein [S-W3 consists c,f KH2P04 1 g, ~gSO4.7H20 0.7 g, NaC 1 g, (NH4)2HPO4 4 g, FeSO4.7H20 0.03 g and glucose 5 g) or (B) [S-W] + C'asamino acid +
agar, colony was formed by anaerobic culture of feces.
Then, the colony was cultivated in LBS medium [a selective medium. for Lactobacillus lactis, consisting of trypticase 10 g, yeast extract 5 g, meat extract 10 g, KH2PO4 6 g, ammonium hydrogen citrate 2 g, glucose 20 g, Tween 80* 1 g sodium acetate 40 g, solution B 15 ml and glacial acetic acid (99.5/O) 3.7 ml, which requires no pH-adjustment and no sterilization, the solution B
*trade name of KAO Atlas Company for Polyoxyethylene sorbitan monooleate ~; ' ~Z74~32 bein~ composed of FeSO~ 7H2O 0~5 g, MnSO4 nH~O 2.47 g, NaCQ 0.5 g, MgSO~-7H2o 10 g and water 250 ml] and bacterial cells srown on the medium were collected. The cells were cultivated in a normal agar medium, and among the strains which grew only those s~rains which agreed accurately to the definition of Lactobacillus lactis were retained.
_ Then, the strains were classified according to variation in auxotrophy; by means of the following numeral nutrient mediums:
(a). glucose or starch, inorganic salts and water;
(b) An odoriferous compound containing sulfur, nitrogen or carbon, which is limited to one contained in feces, is added to (a);
(c) Vitamins are added individually or in comhination, to (~);
(d~ Amino acids are added individually or in combination, to (a);
(e) Specific vitamins are added individually or in combi-nation, to (a);
(f) Tryptophane is added to (a);
(g) Amino acids containing sulfur are added ~ndividually or in combination, to (a):
1~7~4~32 In the above described medium, vitamin includes vitamin A, vitamin Bl, vitamin B2r vitamin B6~ vitamin B12, nicotinamide, calcium panthothenate, vitamin C, vitamin ~12' etc., specific vitamin includes vitamins other than the above described vitamins, such as folic acid, biotin, etcO
Amino acid includes ones other than specific amino acids and sulfur-containin~ amino acids, such as glycine, glutamic acid,lysine, alanine, phenylalanine, alginine, aspartic acid, etc., and specific amino acid includeS
tryptophan~ -Sulfur-containing amino acid includes cystine, cysteine, methionine, taurine, etc.
- : Streptococcus faecalis used in the present invention is one which is usually added bo feed stuffs for domestic animals. However, it must be a strain capable of produclng an antibiotic, because the antibiotic produced suppresses the growth and multiplic~tion of intestinal saprophytes and assists indirectly the multiplication of the Lactobacillus used in the present invention.
For the isolation of such St-reptococcus faecalis, feces of man or domestic animals are used as the source for isolation and they are cultivated at about 34 ~ 39C
under aerobic conditions in a selective medium for Stroptococcus faecalis, such as SF medium consisting of ~274482 peptone 20 g, glucose 5 g, K2H~O4 4 g, KH2PO4 1^5 g, NaN3 0.5 g, NaC~ S g, BCP tBn~cresol Purple) 0.032 g, agar 15 g and water 1 and ha~ing a pH of 6.9 ~ 7.2. The Streptococcus faecalis obtained is subjected to hemolysis testing to retain non-hemolytic ones, from which these having the ability toproduce antibiotics in further retained. The strain thus retained is inoculated to a medium consisting of skimmed milk and sucroseO From the resulting bacterial cells, those having good odor and taste are selected and used in the present invention.
A representative testing procedure to determine whether the strain is one prod~cing antibiotics comprises coating Stre~tcoccus faecalis on an agar me,dium containing ; sugar and CaC03 in an amount of 10 mm of diameter, 15 culturing at 37C for 48 to 72 hours, streaking a disease germ on the mdium in a radiated state from the center of the cultured Streptoccus faecalis and continue to culture , .
for 24 to 48 hours to examine that the Streptoccus faecalis produces antibiotics.
The antibiotics formed by Streptococcus faecalis are aminoglucoside having lower heat-resistance, and 4~5 ~inds of antibiotics having a narrow spectrum of antimicrobial activity are formed from one strain. These 4 ~ 5 ~inds lZ744~32 of antibiotics having a narrow spectrum of antimicrobial activity exhibit corporately the s~me property as a single antibiotic having a very wide spectrum of antimicrobial activity.
As regards the toxicity, Streptococcus faecalis is rather non-toxic, as compared with activity and metabolism of other intestinal bacteria, amounting upto lQ0 billions~
As for the non-hemolytic Streptococcus faecalis strain producing antibiotics, it is considered that thus strain rather assists the activity of Lactobacillus and Bifidobacterium and suppresses the activity of pathogenic bacteria~
Especially preferred strains of Streptococcus faecalis are those deposited at the Institute of Fermen-tation~ Osaka, Japan under International Registration IFO Nos. 14452 and 14453.
As described a~ove, at least one of Lactobacillus deodorans, Lactobacillus cl-earans is a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus is used . .
in combination with the a~ove-mentioned Streptococcus faecalis according to t~e present invention. The ratio of the combination of Lactobacillus to Streptococcus faecalis .
is 150 ~ 0.01 : l by weight, preferably 100 ~ 0.01 : l, 1274~4~z more preferably 30 ~ 0O03 : 1 by weight. Above all, a ratio of about 10Ol, which is the ratio of intestinal flora, is usually employed. For direct application to the mucosa of the skin, Lactobacillus is used in an amount less than that of Streptococcus, that is, in a ratio of Lactobacillus to StreDtococcus of preferably 1 ~ 0.03 by weight. By t~e way, in order to attain the same effect as Lactobacillus clearans by using Lactobac~llus sulfurica and Lactobacillus nitrosus, it is sufficient to use a _ mixture-of Lactobacillus sulfurica and Lactobacillus nitrosus, each present in the same amount as Lactobacillus c~earans.
For preparing dry ~acterial preparations of such Lactobac lus and Streptococcus faecalis, to which , Bifidobacterium and/or Bacillus is further'added if desired, it is sufficient to mix them together, preferably, in a ratio of 5 ~ 15 parts by weight of-Lactobacil-lus, 10 ~ 100 parts by weight of ~ifidob-acterium and 0.05 ~ 0.15 parts by weight of 8ac _lus to 1 part by weight of Streptococcus faecalisO
. _ _ On using the above four Lactobacillus strains and Streptococcus faecalis in combination, according to the present invention, Bifidobacterium, Bacillus or a mixture of Bif~dobacterium and Bacillus may also be used in combination therewith. Bifidobacterium is effective for maintainin~ the deodorant effect still after the administration _ 21 -~2~448~
is stopped, and Bacillus is effective for heightening the ability of Lactobacillus and Streptococcus in di.gesting offensively odoriferous substances for deodorization.
The Bifidobacterium used in the present invention may be either a Bifidobacterium strain which is usually used for the production of yogurt or that used for feed stuffs or food. In the present invention, strains deposited at ATCC were used.
As the acillus used in the present invention, Bacillus coagulans strains which are excellent as seed __ bacteria for yogurt, ~acillus subtilis strains which are excellent as bacteria for producing fermented soy~eans, or the like are prefera~ly used. Othér Baci-llus strains used for feed stuffs or food may also be employed.
~ The Bifidobacterium is used preferably in an . .
amount of 1 ~ 10 times ~y weight of the Lactobacillus used in the present in~ention, and the ~aclllus Ln an amount of 1 ~ 0.Ql times by wei~ht of the Lactobacillus used in the present invention.
2a It has been found that the ratio of individual strains obtained after cultivation of the strains used in the present invention corresponds to the ratio of individual strains added originally to the culture medium.
However, for the cultivation of several strains including Bifidobacterium, it is desirable to select a medium in which the Bifidobacterium grows well, as the medium for cultivating them simultaneously~ so that the Bifidobacterium having a lower growth rate may begin multiplication sooner than otner kinds of strains.
The deodorizers of the present invention are prepared mainly in the form of a live bacterial preparation, for example, by the following processes:
One process is ~1) a process wherein the moisture content of the wet cells of the Lactobacillus used in the present invention and Streptococcus faecalis collected by centrifugation is reduced (prefera~ly by wei~ht), and the cells are mixed with excipients and dried in vacuum (preferably to a moisture content of about 2~ ~y weight).
Another process is (2) a process wherein wet cells of the Lactobacillus used in the present invention and Streptococcus faecalis collected by centrifugation are _ dispersed in a solution of excipients, and the resulting dispersion is freeze-dried and if necessary further mixed with dry excipients. Further, another process is (3) a process wherein preparations containing a single strain or a plurality of strains prepared by the above process (1) or ~2) are mixed to obtain a preparation containing the proper amounts of individual strains.
Suitable excipients, include starch, lactose, sucrose, skimmed milk, dextrin, gelatin, calcium carbonate .
4~2 and an amino acid containing sulfur, used individually or as a mixture of two or more thereof. It is preferred to use an excipient in an amount of 1 ~ 50 parts by weight per 1 part by weight oE wet cells. ~hen it is desired to use BiCidobacterium and/or Bacillus together with the above . _ . _ . .. . _ wet cells, the cells or Bifidobacterium and/or Bacillus are admixed so as to form an emulsion which gives a dry bacteria preparation ~aving t~e effective ratio of the individual stra;ns. It is preferred to use 5 ~ 100 parts by weight of the Lactobacillus used in the present invention -and 0.05 ~ 0.15 parts ~y weight of Bifidobacterium and/or .
Bacillus per 1 part by wei~ht of-Streptococcus-faecalis.
Suitable solutions of dispersions of excipient used in the above process (2) include solutions or dispersions of lactose, sucrose, skimmed milk, starch and the like.
In-the above processes ll) to (3), at least one compound selected from the group of a sulfur-containing amino acid such as cystine, cysteine, methionine, tauline, etc., a specific amino acid, such as glutamic-acid-sodium glutamat~
glycine, alanine, histidine, tyrosine, etc., or odoriferous compounds containing nitrogen atoms, sulfur atoms or carbon atoms are preferably contained in an amount of 0.1 to 10 weight percent. The sulfur containing amino acid, the spe-cific amino acid or the odoriferous compound are effective in preventing lowering of deodorizing ability of the deodorizer 1~44B2 which is caused during culture or storage. In view of the effect~ sulfur~containing amino acid is the most preferable.
In the specific amino acids, it's effect becomes lower according to the ordex o~ glutamic acid, sodium glutamate, glycine, alanine; histidine and tyrosine.
The deodorizer of the present invention can ~e used for the prepara~ion of a suppository and surgical absorbent cotton. Then it is preferable to convert the Lactobacillus and Streptococcus faecalis ~eforehand into resistant strains against disinfectants to be used.
The suppos;tory can be prepared by mixing the Lactobacillus and Streptococcus faecalis with isotonic sodium chloride :
solution in a ratio desired, for example, in a rat~o of lQ:l, letting the mixture stand for a suitable period, for example, for 5 minutes to 2 hours, and absorbing well the cotton with the mixture. For the preparation of surgical cotton absorbent, both strains are dried seperately and adhered to cotton successively, or both strains are mixed with isotonic sodium chloride solution and the cotton is dipped in the xesulting mixture and dried. Of course, the methods of preparing the suppository or surgical absorbent cotton are not limited to those methods described above.
That is, it is preferahle that the deodorizer is under dried condition to restore its ahility.
1;~74482 The deodorizers of the present invention can be used as valuable additives for feed stuffs and the like.
For addition to feed stuffs, the Lactobacillus and Streptococcus faecalis are cultivated at the same time in one medium in a desired ratio, for example, in a ratio of 10:1, and the culture solution i5 ~ after centrifugation, dried and used as additive for feed stuffs, or the Lactobacillus and Streptococcus faecalis are cultivated separately to prepare dried cells, and t~e two kinds of 1~ dried cells are mixed together in a desired ratio, for examplç, in a ratio of 10:1, and used as an additive for feed stuffs. Since there is a difference in heat resistance between the Lactobacillus and Streptococcus faecalis ., .. _ .. . . _ (the latter being more resistant to heat, than the former), it is rather economical to employ the latter method for ad~ition in producing the preparation.
Generally speaking, when the stationary ability into mucous membranes is taken into consideration, whether an aerobic strain is used or an anaerobic strain is used is determined by whether the mucous membrane to which the strain is applied exists under aerobic conditions or under anaerobic conditions. Although Lactobacillus and Streptococcus are representatives of bacteria having good affinity for mucous membranes, Streptococcus preferring aerobic conditions is suitable for application to mucous 12744~2 membrane of the mouth and Lactobacillus capable of being active even under anaerobic conditions is suitable for application to mucous m~brane of the vaginal region.
However, when contaminant bacteria such as saprogens and pathogens were present in the vaginal region and the region related to the vagina ~especially the vulva or pudenda)., it was difficult in many cases to attain the desired effect by the use of Lactobacillus only.
Although a process for reducin~ these contaminant bacteria beforehand by means of a disinfectant and using Lacto~acillus resistant to the disinfectant was undertaken, it was still difficult to obtain the effect as desired, because the effect of the disinfectant did not endure for a long time.
The deodorizer of the present invention acts effectively particularly at such regions. Because of the effect of antibiotics-formed by Streptococcus faecalis and of the high affinity to mucous membrane under aerobic conditions of Streptococcus faecalis, the Lactobacillus can exhibit its inherent properties effectively. Thus, the deodorizers of the present invention are valuable as --suppositories and furt~er as medicines for external use.
It is preferable to administer a disinfectant beforehand and then apply the suppository or the medicines for external use of the present invention, to which resistance against the disinfectant is provided.
1~74q~8Z
Thus, the deodorizers of the present invention exhibit an excellent effect, not only in intestinum but also in other mucosae especially at the pubic region.
Hereinafter, a method of isolating the Streptococcus used in the present invention is e~plained in detail.
However, the invention is not to be construed as being limited to such method.
Isolation Method for Streptococcus faecalis:
.
(1) SF medium (cons,isting of peptone 20 g, glucose 5 g, K~HPO4 4 g, KH2PO4 1.5 g, NaN3 0-5 g~ NaCQ 5 g~
BCP 0O032 g, agar 15 g and water 1 Q and having a p~ of 6.9 ~ 7.2), w~ich is a selective medium for Str~ptococcus faecalis, was employed as a culture ' medium. Feces of man and animals, were suspended in 15 ' the SF medium and cultivated at 37,C under aerobic conditions for 48 hours.
(2) Those cells which were recognized as Streptococcus faecalis by classification, were subjected to hemolysis testing and non-hemolytic cells were retained.
(~) Lactobacillus deodorans has a high assimilating power for decomposing matters and a strons intestinal stationary ability. Namely, it has a strong cleaning power in vivo.
(4) Lactobacillus deodorans is a non-pathogenic strain.
(5) To maintain the strong deodorant power o~ Lactobacillus deodorans, it is desirable to incorporate amino acids - containing sulfur atoms and/or odoriferous compounds containing sulfur atoms, nitrogen atoms or carbon atoms to the nutrient medium.
(6). For the storage of the cell body, it is desirable to place those substances as mentioned a~ove and/or glycine, ~27~4~3Z
glutamic acid, lysine, alanine, phenylalanine, alginine, aspartic acid, etc. around the cell body of Lactobacillus deodorans.
Any strains of Lactobacillus deodorans which are prepared according to an isolating method of the above Publication or which have the above described characteristics may be used in the present invention.
Especially preferred strains of Lactobacillus deodorans which can be used in the present invention are those strains deposited at the Agency of Industrial Science &
Technology, Fermentation Research Institute ~Japan) under Domestic Registration FERM-P Nos. 1946, 2742, 2779, 2780, 2781 and 2782. FERM-P Nos. 1946 and 2779 are deposlted also at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14450 and 14451.
The characteristics of Lact.obacillus clearans used in the present invention, and a method of isolating it, are described in U.S. Patent No. 4,579,734. (The term "OPI" as used herein refers to a "published unexamined Japanese Patent.
Application".) Lactobacillus clearans has the following characteristics.
(1) a) Odoriferous compounds containing sulfur atoms such as Na2S.9H20), odoriferous compounds containing nitrogen atoms (such as NH3) and/or odoriferous .....
~74~1~Z
compounds containing carbon atoms (such as a lower fatty acid) are not essential for the growth of Lactobaclllus clearans. A medium in which Lactobacillus clearans does not grow cannot be changed to one in which Lactobacillus clearans can grow~ even by addition of the above-mentioned substances.
b) Promotion or acceleration of gro~th does not occur by adding odoriferous compounds containing sulfur atoms, odoriferous compounds containing nitrogen atoms and/or odoriferous compounds containing carbon atoms to the culture medium for Lactobacillus clearans .
at the logarithmic growth stage.
c) Lactobaci-llus clearans utilizes or assimlates both odoriferous compounds containing sulfur atoms and odoriferous compounds containing nitrogen atoms to some degree. (Many strains utilize about 30 ~ 90~ thereof.) However,-Lactobacillus clearans cannot utilize these materials completely.
That is to say, they remain unutilized. (It is noted that Lactobacillus deodorans assimilates the compounds completely.) ~2) The auxotrophy of Lactobacillus clearans is low.
It can grow in Stephanson-Whetham medium by merely adding thereto ~oth Casamino acid and vitamins.
(3~ The multiplying power of Lactobacillus clearans is within the range of 0.9 ~ l.l times in meat extract bouillon, as compared with Eschericria Coli.
12749,~z Lactobacillus clearans utilizes decompos'ng matter, _ although not completely, and so it has a cleaning power in vivo to some degree for about 2 weeks at a maximum, generally for 3 to 6 days, after administration.
(4) Lactobacillus cle~rans corresponds to the Genus . .
Lactobacillus in all propertles~ However, it is quite .
different from previously known strains in respect of the ability of utilizing or assimilating odoriferous compounds containing sulfur atcms (such as ha2S 9~2O) and odori-ferous compounds containing nitrogen atoms (such as NX3), as described above. (Previously known strains except ~or Lactobacillus deodorans do not utilize these types of compound.i Its auxotrophy is low.
(5) The intestinal stationary ability of Lactobacillus clearans is lower than that of Lactobacillus deodorans, .
although it is stronger than that of previously known Lactobacillus strains.) (6) Lactobacillus clearans used in the present invention .
is a non-pathogenic strain.
0 (7) To maintain the strong deodorant power of Lac-tobacillus clearans used in the present invention, it is desirable to incorporate amino acids containing sulfur atoms and/or odoriferous compounds containing sulfur atoms, nitro~en atoms and/or carbon atoms in the nutrient medium.
_ g _ 127~41~2 (8) For the storage of cell body, it is desirable to place those substances as mentioned above for Lactobacillus deodorans around the cell body of Lactobacillus cIearans.
Any strains of Lactobacillus clearans which are prepared according to the above isolating method or which have the above described characteristics may be used in the present invention.
Especially preferred strains of Lactobacillus clearans which can be used in the present invention are those deposited at the Agency of Industrial Science &
Technology, Fermentation Research Institute (Japan) under Domestic fi.egistration FERM-P Nos. 6587,6588,6589 and 6590. Among these strains, strains of FERM-P Nos.
6587 and 6590 are deposited also at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14253 and 1425~.
The characeristics of Lactobacillus sulfurica and Lactobacillus nitrosus used in the present invention, and a method of isolating each of them, are described in ~.S. Patent No. 4,579,734.
Lactobacillus sulfurica used in the present invention utilizes only odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus utilizes only lZ744~3Z
odori~erous compounds containing nitrogen atoms, although the utilization is not complete but r~ns at about 90~ or less in each case~ However, except for this property, they have t~e same properties as Lactobacillus clearans. Therefore, on carrying out deodorization ln vivo with Lactobac~llus sulfuri~~a and Lactobacillus nitrosus, a satisfactory effect cannot be expected unless the two are used in combination.
Any strains of Lactobacillus sulfurica and Lactobacillus nitrosus which are prepared according to the isolating method as described above or which have the above characteristics may be used in the present invention.
Especially preferred strains of Lactobacillus sulfurica are those deposited at the Agency of Industrial Science & Technology, Fermentation Research Institute (Japan) under Domestic Registration FERM-P Nos. 7383 and 7384, which are also deposited at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14255 and 14256, respectively, and especially preferred strains of Lactobacillus nitrosus are those deposited at Fermentation Research Institute the Agency of Industrial Science and Technology (Japan) under Domestic Registration FERM-P Nos. 7385 and 7386, which are also deposited at the Institute of Fermentation, Osaka, Japan under International Registration IFO Nos. 14257 and 14258, respectively.
1~74~8Z
The differences in the four types of Lactcbacil1u6 mentioned above from previously known Lactoba _llus.
(1) Previously known Lactobac~llus strains do not require odoriferous compounds containing sulfur atoms and odoriferou~
compounds containing nitrogen atoms as essential substances for their growth. Even when these odoriferous compounds are added to a culture medium in which known Lactobacillus strains cannot grow, the strains do not begin to grow.
Further, even when these compounds are added at the logarithmic growth stage of the known strains, promotion or acceleration of growth does not occur. Previously known Lacto~acillus strains do not at all consume these compounds, and so the compounds remain ultimately in the amount originally added~
(22 The previously known Lactobacillus strains are separated in respect of their auxotrophy into a group of strains which can grow in Stephanson-Whetham medium by adding Caramino acid and vitamins thereto and a group of strains which cannot grow in Stephanson-Whetham medium unless other ingredients are furhter added thereto in addition to Casamino acid and vitamins. (3) The previously known Lactobacillus strains are inferior in multiplying power in usual medium to Escherichia coli, etc. (for example, 0.5 times that of Escherichia coli)~ Their intestinal stationary ability is at a degree of 1 or 2 days.
The cleaning power against even odriferous compounds ~Z7~3Z
containing carbon atoms is unknown.
The four kinds of Lactobacillus strains all have the following prope-ties of Lactobac_llus:
That is, they are Gram-positive, anaerobic or micro aerophilic, non-spore forming rods and, depending on the strains, may be spherical rod-like, curved rod-like, coryne-like or thread-like~ They are non-motile, negative to catalase and do not reduce nitrates. They do not decmpose gelatin and do not produce indole or hydrogen sulfide. Some of them are bipolar-stained.
Their ability to decompose protein and fat is very weak, if any. They show better growth under anaerobic'or microaexobic, conditions rather than under aerobic conditions, having strong ability to decompose sugars, and are acid-fast. When used for glucose fermentation, they produce lactic acid in an yield of more than 50%.
Their growth is promoted by adding acetic acid.
They are not pathogenic to animals and plants.
Lac_obacillus clearans acts very mildly even when it is orally administered in a large amount and reduces offensively odoriferous substances in feces, without an uncomfortable feeling, while the administration continues. However, when administration is stopped, the strain is excreted through intestinal tract to lose its deodorant ~2744~Z
n vivo within a short time.
Lactobacillus deodorans has a deodorant power of about several tens times or more, compared with Lactobacillus clea~ans, and morover it deodorizes completely, that is, it degrades offensively odoriferous substances completely.
For example, human feces were deodorized completely within 48 hours by administration of a tenth part (by weight) of a culture solution of Lactobacil-lus deodorans.
The LactobacillUs deodorans - used in the present invention is obtained by cultivation of Lactobacillus lactis which multiplies and is capable or not capable of producin~
antibiotics in either one of the minimal nutrient mediums shown in the following table, in a medium not affecting the properties of these strains, and the subsequent isolation.
Classifi-cation Minimal Nutrien~ Medium . . . _ . _ (a) Glucose or starch, inorganic salts and water (b) An odoriferous compound containing sulfur, nitrogen or carbon, which is present in feces, is added to ta3 (c~ Vitamins are added individually or in combination, to (a) (d) Amino acids are added individually or in combi-nation, to (a) (e) Specific vitamins are added individually or in combination, to ta) ~Z~4~82 (f) Tryptophane is added to (a) (g) ~mino acids containing sulfur are added individually or in combination, to (2) It is preferable to endow resistance against antibacterial agent, if necessary. The Lactobacillus lactis so obtained can be processed into preparations according to conventional- methods The above-mentioned medium not affecting the properties of Lactobacillus lactis is each of the minimal nutrient mediums as stated in the present invention, a combination of them, or an a~ar medium similar thereto. As the case may be, usual agar medium may be used intermediately.
Further, Lactobacillus clearans is isolated using the medium which is higher in nutrient than that for Lacto-acillus deodorans~ Por example, the medium for isolation of Lactobacillus clearans comprises meat extract, peptone, Na2S-9H2O, sodium butyrate, glucose and agar. As a preferable medium for Lactobacillus nitrosus, it- comprises meat extract, peptone, NH3, sodium butyrate, glucose and agar. As a prefer-able medium for Lactobacillus sulfurica, it comprises meatextract, peptone, Na2S-9K2O, sodium butyrate, glucose, CaCO3, and agar.
Of colonies thus appeared, colonies which margin is transparency are screened and subjected to gas generation ~LZ7448Z
testing and non-gas generation cells are retained. Sub-sequently, cells thus obtained were subjected to hemolysis testing to obtain non-hemolytic cells and then cultured in the LBS medium. After confirmation tha-t the cells belong to Lactobacillus, degree of decrease of Na2S.9H20 and NH3, and accelerated growth for the I.actobacillus strains are examined and classified to Lactobacillus deodorans, Lacto-bacillus clearans, Lactobacillus sulfurica and Lactobacillus nitrosus. As to each Lactobacillus, ones which have higher u value are collected.
The Lactobacillus lactis used in the present invention was isolated, for example, in the following manner:
In a medium (A) [Stephanson Wetham medium S-W] +
vitamin + agar (wherein [S-W3 consists c,f KH2P04 1 g, ~gSO4.7H20 0.7 g, NaC 1 g, (NH4)2HPO4 4 g, FeSO4.7H20 0.03 g and glucose 5 g) or (B) [S-W] + C'asamino acid +
agar, colony was formed by anaerobic culture of feces.
Then, the colony was cultivated in LBS medium [a selective medium. for Lactobacillus lactis, consisting of trypticase 10 g, yeast extract 5 g, meat extract 10 g, KH2PO4 6 g, ammonium hydrogen citrate 2 g, glucose 20 g, Tween 80* 1 g sodium acetate 40 g, solution B 15 ml and glacial acetic acid (99.5/O) 3.7 ml, which requires no pH-adjustment and no sterilization, the solution B
*trade name of KAO Atlas Company for Polyoxyethylene sorbitan monooleate ~; ' ~Z74~32 bein~ composed of FeSO~ 7H2O 0~5 g, MnSO4 nH~O 2.47 g, NaCQ 0.5 g, MgSO~-7H2o 10 g and water 250 ml] and bacterial cells srown on the medium were collected. The cells were cultivated in a normal agar medium, and among the strains which grew only those s~rains which agreed accurately to the definition of Lactobacillus lactis were retained.
_ Then, the strains were classified according to variation in auxotrophy; by means of the following numeral nutrient mediums:
(a). glucose or starch, inorganic salts and water;
(b) An odoriferous compound containing sulfur, nitrogen or carbon, which is limited to one contained in feces, is added to (a);
(c) Vitamins are added individually or in comhination, to (~);
(d~ Amino acids are added individually or in combination, to (a);
(e) Specific vitamins are added individually or in combi-nation, to (a);
(f) Tryptophane is added to (a);
(g) Amino acids containing sulfur are added ~ndividually or in combination, to (a):
1~7~4~32 In the above described medium, vitamin includes vitamin A, vitamin Bl, vitamin B2r vitamin B6~ vitamin B12, nicotinamide, calcium panthothenate, vitamin C, vitamin ~12' etc., specific vitamin includes vitamins other than the above described vitamins, such as folic acid, biotin, etcO
Amino acid includes ones other than specific amino acids and sulfur-containin~ amino acids, such as glycine, glutamic acid,lysine, alanine, phenylalanine, alginine, aspartic acid, etc., and specific amino acid includeS
tryptophan~ -Sulfur-containing amino acid includes cystine, cysteine, methionine, taurine, etc.
- : Streptococcus faecalis used in the present invention is one which is usually added bo feed stuffs for domestic animals. However, it must be a strain capable of produclng an antibiotic, because the antibiotic produced suppresses the growth and multiplic~tion of intestinal saprophytes and assists indirectly the multiplication of the Lactobacillus used in the present invention.
For the isolation of such St-reptococcus faecalis, feces of man or domestic animals are used as the source for isolation and they are cultivated at about 34 ~ 39C
under aerobic conditions in a selective medium for Stroptococcus faecalis, such as SF medium consisting of ~274482 peptone 20 g, glucose 5 g, K2H~O4 4 g, KH2PO4 1^5 g, NaN3 0.5 g, NaC~ S g, BCP tBn~cresol Purple) 0.032 g, agar 15 g and water 1 and ha~ing a pH of 6.9 ~ 7.2. The Streptococcus faecalis obtained is subjected to hemolysis testing to retain non-hemolytic ones, from which these having the ability toproduce antibiotics in further retained. The strain thus retained is inoculated to a medium consisting of skimmed milk and sucroseO From the resulting bacterial cells, those having good odor and taste are selected and used in the present invention.
A representative testing procedure to determine whether the strain is one prod~cing antibiotics comprises coating Stre~tcoccus faecalis on an agar me,dium containing ; sugar and CaC03 in an amount of 10 mm of diameter, 15 culturing at 37C for 48 to 72 hours, streaking a disease germ on the mdium in a radiated state from the center of the cultured Streptoccus faecalis and continue to culture , .
for 24 to 48 hours to examine that the Streptoccus faecalis produces antibiotics.
The antibiotics formed by Streptococcus faecalis are aminoglucoside having lower heat-resistance, and 4~5 ~inds of antibiotics having a narrow spectrum of antimicrobial activity are formed from one strain. These 4 ~ 5 ~inds lZ744~32 of antibiotics having a narrow spectrum of antimicrobial activity exhibit corporately the s~me property as a single antibiotic having a very wide spectrum of antimicrobial activity.
As regards the toxicity, Streptococcus faecalis is rather non-toxic, as compared with activity and metabolism of other intestinal bacteria, amounting upto lQ0 billions~
As for the non-hemolytic Streptococcus faecalis strain producing antibiotics, it is considered that thus strain rather assists the activity of Lactobacillus and Bifidobacterium and suppresses the activity of pathogenic bacteria~
Especially preferred strains of Streptococcus faecalis are those deposited at the Institute of Fermen-tation~ Osaka, Japan under International Registration IFO Nos. 14452 and 14453.
As described a~ove, at least one of Lactobacillus deodorans, Lactobacillus cl-earans is a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus is used . .
in combination with the a~ove-mentioned Streptococcus faecalis according to t~e present invention. The ratio of the combination of Lactobacillus to Streptococcus faecalis .
is 150 ~ 0.01 : l by weight, preferably 100 ~ 0.01 : l, 1274~4~z more preferably 30 ~ 0O03 : 1 by weight. Above all, a ratio of about 10Ol, which is the ratio of intestinal flora, is usually employed. For direct application to the mucosa of the skin, Lactobacillus is used in an amount less than that of Streptococcus, that is, in a ratio of Lactobacillus to StreDtococcus of preferably 1 ~ 0.03 by weight. By t~e way, in order to attain the same effect as Lactobacillus clearans by using Lactobac~llus sulfurica and Lactobacillus nitrosus, it is sufficient to use a _ mixture-of Lactobacillus sulfurica and Lactobacillus nitrosus, each present in the same amount as Lactobacillus c~earans.
For preparing dry ~acterial preparations of such Lactobac lus and Streptococcus faecalis, to which , Bifidobacterium and/or Bacillus is further'added if desired, it is sufficient to mix them together, preferably, in a ratio of 5 ~ 15 parts by weight of-Lactobacil-lus, 10 ~ 100 parts by weight of ~ifidob-acterium and 0.05 ~ 0.15 parts by weight of 8ac _lus to 1 part by weight of Streptococcus faecalisO
. _ _ On using the above four Lactobacillus strains and Streptococcus faecalis in combination, according to the present invention, Bifidobacterium, Bacillus or a mixture of Bif~dobacterium and Bacillus may also be used in combination therewith. Bifidobacterium is effective for maintainin~ the deodorant effect still after the administration _ 21 -~2~448~
is stopped, and Bacillus is effective for heightening the ability of Lactobacillus and Streptococcus in di.gesting offensively odoriferous substances for deodorization.
The Bifidobacterium used in the present invention may be either a Bifidobacterium strain which is usually used for the production of yogurt or that used for feed stuffs or food. In the present invention, strains deposited at ATCC were used.
As the acillus used in the present invention, Bacillus coagulans strains which are excellent as seed __ bacteria for yogurt, ~acillus subtilis strains which are excellent as bacteria for producing fermented soy~eans, or the like are prefera~ly used. Othér Baci-llus strains used for feed stuffs or food may also be employed.
~ The Bifidobacterium is used preferably in an . .
amount of 1 ~ 10 times ~y weight of the Lactobacillus used in the present in~ention, and the ~aclllus Ln an amount of 1 ~ 0.Ql times by wei~ht of the Lactobacillus used in the present invention.
2a It has been found that the ratio of individual strains obtained after cultivation of the strains used in the present invention corresponds to the ratio of individual strains added originally to the culture medium.
However, for the cultivation of several strains including Bifidobacterium, it is desirable to select a medium in which the Bifidobacterium grows well, as the medium for cultivating them simultaneously~ so that the Bifidobacterium having a lower growth rate may begin multiplication sooner than otner kinds of strains.
The deodorizers of the present invention are prepared mainly in the form of a live bacterial preparation, for example, by the following processes:
One process is ~1) a process wherein the moisture content of the wet cells of the Lactobacillus used in the present invention and Streptococcus faecalis collected by centrifugation is reduced (prefera~ly by wei~ht), and the cells are mixed with excipients and dried in vacuum (preferably to a moisture content of about 2~ ~y weight).
Another process is (2) a process wherein wet cells of the Lactobacillus used in the present invention and Streptococcus faecalis collected by centrifugation are _ dispersed in a solution of excipients, and the resulting dispersion is freeze-dried and if necessary further mixed with dry excipients. Further, another process is (3) a process wherein preparations containing a single strain or a plurality of strains prepared by the above process (1) or ~2) are mixed to obtain a preparation containing the proper amounts of individual strains.
Suitable excipients, include starch, lactose, sucrose, skimmed milk, dextrin, gelatin, calcium carbonate .
4~2 and an amino acid containing sulfur, used individually or as a mixture of two or more thereof. It is preferred to use an excipient in an amount of 1 ~ 50 parts by weight per 1 part by weight oE wet cells. ~hen it is desired to use BiCidobacterium and/or Bacillus together with the above . _ . _ . .. . _ wet cells, the cells or Bifidobacterium and/or Bacillus are admixed so as to form an emulsion which gives a dry bacteria preparation ~aving t~e effective ratio of the individual stra;ns. It is preferred to use 5 ~ 100 parts by weight of the Lactobacillus used in the present invention -and 0.05 ~ 0.15 parts ~y weight of Bifidobacterium and/or .
Bacillus per 1 part by wei~ht of-Streptococcus-faecalis.
Suitable solutions of dispersions of excipient used in the above process (2) include solutions or dispersions of lactose, sucrose, skimmed milk, starch and the like.
In-the above processes ll) to (3), at least one compound selected from the group of a sulfur-containing amino acid such as cystine, cysteine, methionine, tauline, etc., a specific amino acid, such as glutamic-acid-sodium glutamat~
glycine, alanine, histidine, tyrosine, etc., or odoriferous compounds containing nitrogen atoms, sulfur atoms or carbon atoms are preferably contained in an amount of 0.1 to 10 weight percent. The sulfur containing amino acid, the spe-cific amino acid or the odoriferous compound are effective in preventing lowering of deodorizing ability of the deodorizer 1~44B2 which is caused during culture or storage. In view of the effect~ sulfur~containing amino acid is the most preferable.
In the specific amino acids, it's effect becomes lower according to the ordex o~ glutamic acid, sodium glutamate, glycine, alanine; histidine and tyrosine.
The deodorizer of the present invention can ~e used for the prepara~ion of a suppository and surgical absorbent cotton. Then it is preferable to convert the Lactobacillus and Streptococcus faecalis ~eforehand into resistant strains against disinfectants to be used.
The suppos;tory can be prepared by mixing the Lactobacillus and Streptococcus faecalis with isotonic sodium chloride :
solution in a ratio desired, for example, in a rat~o of lQ:l, letting the mixture stand for a suitable period, for example, for 5 minutes to 2 hours, and absorbing well the cotton with the mixture. For the preparation of surgical cotton absorbent, both strains are dried seperately and adhered to cotton successively, or both strains are mixed with isotonic sodium chloride solution and the cotton is dipped in the xesulting mixture and dried. Of course, the methods of preparing the suppository or surgical absorbent cotton are not limited to those methods described above.
That is, it is preferahle that the deodorizer is under dried condition to restore its ahility.
1;~74482 The deodorizers of the present invention can be used as valuable additives for feed stuffs and the like.
For addition to feed stuffs, the Lactobacillus and Streptococcus faecalis are cultivated at the same time in one medium in a desired ratio, for example, in a ratio of 10:1, and the culture solution i5 ~ after centrifugation, dried and used as additive for feed stuffs, or the Lactobacillus and Streptococcus faecalis are cultivated separately to prepare dried cells, and t~e two kinds of 1~ dried cells are mixed together in a desired ratio, for examplç, in a ratio of 10:1, and used as an additive for feed stuffs. Since there is a difference in heat resistance between the Lactobacillus and Streptococcus faecalis ., .. _ .. . . _ (the latter being more resistant to heat, than the former), it is rather economical to employ the latter method for ad~ition in producing the preparation.
Generally speaking, when the stationary ability into mucous membranes is taken into consideration, whether an aerobic strain is used or an anaerobic strain is used is determined by whether the mucous membrane to which the strain is applied exists under aerobic conditions or under anaerobic conditions. Although Lactobacillus and Streptococcus are representatives of bacteria having good affinity for mucous membranes, Streptococcus preferring aerobic conditions is suitable for application to mucous 12744~2 membrane of the mouth and Lactobacillus capable of being active even under anaerobic conditions is suitable for application to mucous m~brane of the vaginal region.
However, when contaminant bacteria such as saprogens and pathogens were present in the vaginal region and the region related to the vagina ~especially the vulva or pudenda)., it was difficult in many cases to attain the desired effect by the use of Lactobacillus only.
Although a process for reducin~ these contaminant bacteria beforehand by means of a disinfectant and using Lacto~acillus resistant to the disinfectant was undertaken, it was still difficult to obtain the effect as desired, because the effect of the disinfectant did not endure for a long time.
The deodorizer of the present invention acts effectively particularly at such regions. Because of the effect of antibiotics-formed by Streptococcus faecalis and of the high affinity to mucous membrane under aerobic conditions of Streptococcus faecalis, the Lactobacillus can exhibit its inherent properties effectively. Thus, the deodorizers of the present invention are valuable as --suppositories and furt~er as medicines for external use.
It is preferable to administer a disinfectant beforehand and then apply the suppository or the medicines for external use of the present invention, to which resistance against the disinfectant is provided.
1~74q~8Z
Thus, the deodorizers of the present invention exhibit an excellent effect, not only in intestinum but also in other mucosae especially at the pubic region.
Hereinafter, a method of isolating the Streptococcus used in the present invention is e~plained in detail.
However, the invention is not to be construed as being limited to such method.
Isolation Method for Streptococcus faecalis:
.
(1) SF medium (cons,isting of peptone 20 g, glucose 5 g, K~HPO4 4 g, KH2PO4 1.5 g, NaN3 0-5 g~ NaCQ 5 g~
BCP 0O032 g, agar 15 g and water 1 Q and having a p~ of 6.9 ~ 7.2), w~ich is a selective medium for Str~ptococcus faecalis, was employed as a culture ' medium. Feces of man and animals, were suspended in 15 ' the SF medium and cultivated at 37,C under aerobic conditions for 48 hours.
(2) Those cells which were recognized as Streptococcus faecalis by classification, were subjected to hemolysis testing and non-hemolytic cells were retained.
(3) The a~ility to produce antibiotics was tested and those having the a~ility of producing anti~iotics were retained.
(4) Then, the strain retained was inoculated to a yogurt medium (consisting of skimmed milk 100 g and sucrose 100 g) and cultivated at 37C for 48 hours to prepare yogurt. The yogurt was subjec'ed to odor and taste 12744~2 testing, and the cells having good odor and taste were retained.
The following example is exemplary of the collect-ion of cells.
Collection of Cells from Culture Solution:
.
In the casPs of Lactobacillus-clearans, Lactobacillus deodorans, Lactobacillus sulfurica and Lactobacillus nitrosus used in the present invention,-Streptococcus ~fae'calis and Bacillus, each strain was inoculated solely to the above-mentioned MRS-lactose medium, i.e. a medium consisting of meat extract 10 g, peptone lQ g, yeast extract 5 g, lactose 20 g, Tween80 lml, XH2P04 2 g, sodium acetate 5 g, ammonium citrate 2 g, MgSO4-7H2O 0.2 g and MnSQ4-4H2O. 0.05 g, cultivation was effected at 37C for 72 ~ours, and the cells were collected using a refrigerated centrifuge.
In the case of Bifidobacterium, the strain was inoc'ulated to the MRS-lactose medium to which 1 mg of vitamin B2, 8 mg of calcium pantothenate and 0.1 mg of biotin were added, cultivation was effected at 37C for 96 hours, and the cells are collected using a refrigerated centrifuge.
The following examples are given to illustrate the present invention in greater detail. Vnless aderwise indicated herein, all parts, percents, ratios and the like are by weight.
~Z~44~2 EXAMPLE
The degree of deodorization of Na2S~9H20 and NH3 using a mixture of Lactobacillus strain and Stre~tococcus _ faecalis was examined~
The following Lactobacillus strains and Streptococcus faecalis were inoculated to the medium cmprising meat extract 5 g, peptone 5 g, glucose 1 g, CaC03 1 g and feces 1 g, which contains Na2S 9H20 0.5 g or NH3 0.5 g, and cultivated to examine the degree of decrease of Na2S~9H20 and NH3. The results are shown in the following Table 1.
~L2744~3Z
.
o o o ~ U~ o o o o o o o o CO ~ ~1 ~ ~ _I
,, , ~ ~-, Ql o O CO O U~ IJ'I Ul O O OO O O O O O
U~
~ ~ I o o O o O O O O O O Ul U~ O U~
_., t~i I O O ~") O o O ~ t`~ r N ~ CO t~l Z ~ ~ ~ ~ er ~r n u~ ~1 EQ~ l ~ .
o o o o oo o o o o o o o o o ~ ~ ~ c~ a~
o ~1 u~
o~ 1 o u~ ~ o o u7 o o o o o ~ o o P . ~`1 N ~ _I 11'1 U'l ~1 t~
o U~
c~l ~r O o o o o u~ o o oo ~ 1~ o In ~ ~ ~ o o u~ I O O co ~r ~ ~ ~o Z ~ ,-~ ~ ~ u~u~ ~r ~ ~1 tn _I
.
q~
.,,~ ~ U~
U7 U) ~a ~ r ~ a~ o :~L'~ O _I N
.R ~ CO a~ U7:1 Q 5-1 :000 h ~ ~ r CO '~
O ~ O ~ ~ ~ O O~
1~ D W ~ 41 1` r ~0~
z u~ a f ~27~8~
For comparison, the same ~rocedures were repeated exce?t that the following Lactobacillus were solel~ used instead of the above mixture or Lactobacillus and Streytcoccus faecalis. The results are shown in S the follo~ing Table 2.
~Z7~a~82 .
~ o oo ~, o o U~ o o o o o o o Ql I_ Ul O O U~
Q
o ~1 o 1n Ln ' 1n O o ' O O O
m ¦ O U~ O OO ~ ) O u~ O UO) Z ~ 0 ~ I ~ er ~r u~ u~ N _I N N C`J ~1 C2~ ~
.4o ~ I ~ Ooo , o 1~')Ir~ o o o o o o o col o o o u~ ~n o . o o o o o o o o O ~ o u~ u~ ~ ,1 ~; ~ O ~ ~ O O r~ ~
a~
Cl~
00 0 ~0 ~0 U~ O O O O O O O O
~O ~D ~ . ~D 1~ r-- 1~ t~ -1 N N N N N
_~
1274~8Z
As is apparent from the Tables, the combinated use of Lactobacillus and Stre~tcoccus faecalis results in an increase of assimilating ability by 10 to 20~ as compared with the sole use of Lactob cillus.
S Lactobacillus deodorans is stronger in assimilating ability than Lactobacillus clearans. The former consumes Na2S-9H20 and ~H3 completely in 72 hours, and while, the latter can not consume them completely to leave over, Lactobacillus nitrosus and Lactobacillus sulfurica also can not-consume them completely.
The strains used in the experiments are shown in Table -3 below:
~Z7448~:
Table 3 Strains used in Experiments ~e~osition . ~
Fermentation Institute for Research Institute Fer~entation, Osaka ATCC
Domestic International International Registration RegistrationRegistration Kind PERM-P FFRM-BP IFO
No. 6587 No. 14253 Lactobacillus No. 6588 clearans No. 6589 No. 6590 No. 14254 .
No. 1946 No. 14450 No. 2742 ~actobacillus No. 2779 No. 14451 deodorans No. 2780 -No. 2781 No. 2782 . ~
Lactobacillus No. 7385 No. 14257 nitrosus No. 7386 No. 14258 Lactobacillus No. 7383 No. 14255 sulfurica No. 7384 No. 14256 ~ ~ No. 20~2 ~ 1445 ( ~ strain) No. 7382 - No. 14453 Streptococcus ( ~ strain) faecalis No.6569~ ~ strain) No.14506t ~ strain) No.11146( ~ strain~
Bifidobacterium No.11863( ~ strain) .. . _ .. _ .. , . , .,, ., ., ., _, ~ . ., .. , . .. _ _ .. _ . _ .. ~ .. _ . _ . _ _ _ . _ _ . _, _ . _ _ Bacillus No.2930 (A strain) No.3335 tB strain) (Note) Deposition numbers given on the same line show the identical strain.
The abbreviated name used in this specification is given in the parentheses after the Registration number.
The ~ strain and ~ strain of Streptococcus faecalis produce antibiotics.
~2744~
The ~ strain and ~ strain of Streptococcus faecalis do not produce antibiotics. The A s.rain of the Bacillus genus i~ a seed cell for yogurt (Bacillus coagulans). The ~ strain of the Bacillus genus is a seed cell for fermented soybeans (Bacillus subtilis).
The follcwing e~p~in~nts were effected using IFO
Nos. 14253 and 14450, as Lactobacillus clearans and .
Lactobaclllus deodorans, respectively.
To l g of fresh human feces was added 9 ml of isotonic sodium chloride solution. To (l) one mixture 50 obtained l ml of a cultured cell solution of Lactobacillus clearans was added, and to (2) another mixture l ml of a cultured cell solution of Lactobacillus clearans plus 0.5 ml of a cultured cell solution of ~ strain were added, and then the mixtures were allowed to stand at 37C
for 48 hours.
By judsement of odor, it was noted that the de~ree of deodorization was higher in the group of experiments of the case of mixture (2) and the amount of feces not deodorized was reduced by half as compared with the case of mixture (l), although the feces was not deodorized in two instances of ten instances of the latter case tested.
By oral administration of Lactobacillus clearans ~2'~4~32 only and of Lactobacillus clearans plus ~ strain, it was recognized as the degree of deodorization was examined with respect to the feces excreted, that the deodorant effect was higher and instances of lack of deodorization were less, in the cases wherein Lac_obacillus clearans plus ~ strain were administered.
These experiments (in vivo) show that Lactobacillus clearans as well as Lactobacillus clearans plus @~ strain are effective in intestines where feces (containing saprogens, most of which are anaerobic, amounting upto l.5 x lOll cellstg of faecalis) are presentO
Although a considerable effect was noticed by contact of either of Lactobacillus clearans and Lactobacillus . clearans plus @~ strain, the combination of Lactobacillus clearans plus ~ strain was more effective, as expected, compared with Lactobacillus clearans only.
Here, the effect means that a high degree of deodorization was attained and instances of lack of deodorization were reduced.
Although the above experiments were those relating to Lactobacillus clearans, the same comparative experiments -as these experiments were effected with respect to Lactobacillus deodorans and to Lactobacillus sulfurica plus Lactobacillus nitro~us, using IFO Nos. 14255, 14256, 14453. That is, comparative experiments between Lactobacillus deodorans and Lactobac llus deodorans plus ~;~74482, ~9 strain and those between Lactobaclllus sulfurica plus Lactobacillus nitrosus and Lactobacillus sulfurlca plus Lactobacillus nitrosus plus ~ strain were effected.
The same tendency as in Lactobacillus clearans
The following example is exemplary of the collect-ion of cells.
Collection of Cells from Culture Solution:
.
In the casPs of Lactobacillus-clearans, Lactobacillus deodorans, Lactobacillus sulfurica and Lactobacillus nitrosus used in the present invention,-Streptococcus ~fae'calis and Bacillus, each strain was inoculated solely to the above-mentioned MRS-lactose medium, i.e. a medium consisting of meat extract 10 g, peptone lQ g, yeast extract 5 g, lactose 20 g, Tween80 lml, XH2P04 2 g, sodium acetate 5 g, ammonium citrate 2 g, MgSO4-7H2O 0.2 g and MnSQ4-4H2O. 0.05 g, cultivation was effected at 37C for 72 ~ours, and the cells were collected using a refrigerated centrifuge.
In the case of Bifidobacterium, the strain was inoc'ulated to the MRS-lactose medium to which 1 mg of vitamin B2, 8 mg of calcium pantothenate and 0.1 mg of biotin were added, cultivation was effected at 37C for 96 hours, and the cells are collected using a refrigerated centrifuge.
The following examples are given to illustrate the present invention in greater detail. Vnless aderwise indicated herein, all parts, percents, ratios and the like are by weight.
~Z~44~2 EXAMPLE
The degree of deodorization of Na2S~9H20 and NH3 using a mixture of Lactobacillus strain and Stre~tococcus _ faecalis was examined~
The following Lactobacillus strains and Streptococcus faecalis were inoculated to the medium cmprising meat extract 5 g, peptone 5 g, glucose 1 g, CaC03 1 g and feces 1 g, which contains Na2S 9H20 0.5 g or NH3 0.5 g, and cultivated to examine the degree of decrease of Na2S~9H20 and NH3. The results are shown in the following Table 1.
~L2744~3Z
.
o o o ~ U~ o o o o o o o o CO ~ ~1 ~ ~ _I
,, , ~ ~-, Ql o O CO O U~ IJ'I Ul O O OO O O O O O
U~
~ ~ I o o O o O O O O O O Ul U~ O U~
_., t~i I O O ~") O o O ~ t`~ r N ~ CO t~l Z ~ ~ ~ ~ er ~r n u~ ~1 EQ~ l ~ .
o o o o oo o o o o o o o o o ~ ~ ~ c~ a~
o ~1 u~
o~ 1 o u~ ~ o o u7 o o o o o ~ o o P . ~`1 N ~ _I 11'1 U'l ~1 t~
o U~
c~l ~r O o o o o u~ o o oo ~ 1~ o In ~ ~ ~ o o u~ I O O co ~r ~ ~ ~o Z ~ ,-~ ~ ~ u~u~ ~r ~ ~1 tn _I
.
q~
.,,~ ~ U~
U7 U) ~a ~ r ~ a~ o :~L'~ O _I N
.R ~ CO a~ U7:1 Q 5-1 :000 h ~ ~ r CO '~
O ~ O ~ ~ ~ O O~
1~ D W ~ 41 1` r ~0~
z u~ a f ~27~8~
For comparison, the same ~rocedures were repeated exce?t that the following Lactobacillus were solel~ used instead of the above mixture or Lactobacillus and Streytcoccus faecalis. The results are shown in S the follo~ing Table 2.
~Z7~a~82 .
~ o oo ~, o o U~ o o o o o o o Ql I_ Ul O O U~
Q
o ~1 o 1n Ln ' 1n O o ' O O O
m ¦ O U~ O OO ~ ) O u~ O UO) Z ~ 0 ~ I ~ er ~r u~ u~ N _I N N C`J ~1 C2~ ~
.4o ~ I ~ Ooo , o 1~')Ir~ o o o o o o o col o o o u~ ~n o . o o o o o o o o O ~ o u~ u~ ~ ,1 ~; ~ O ~ ~ O O r~ ~
a~
Cl~
00 0 ~0 ~0 U~ O O O O O O O O
~O ~D ~ . ~D 1~ r-- 1~ t~ -1 N N N N N
_~
1274~8Z
As is apparent from the Tables, the combinated use of Lactobacillus and Stre~tcoccus faecalis results in an increase of assimilating ability by 10 to 20~ as compared with the sole use of Lactob cillus.
S Lactobacillus deodorans is stronger in assimilating ability than Lactobacillus clearans. The former consumes Na2S-9H20 and ~H3 completely in 72 hours, and while, the latter can not consume them completely to leave over, Lactobacillus nitrosus and Lactobacillus sulfurica also can not-consume them completely.
The strains used in the experiments are shown in Table -3 below:
~Z7448~:
Table 3 Strains used in Experiments ~e~osition . ~
Fermentation Institute for Research Institute Fer~entation, Osaka ATCC
Domestic International International Registration RegistrationRegistration Kind PERM-P FFRM-BP IFO
No. 6587 No. 14253 Lactobacillus No. 6588 clearans No. 6589 No. 6590 No. 14254 .
No. 1946 No. 14450 No. 2742 ~actobacillus No. 2779 No. 14451 deodorans No. 2780 -No. 2781 No. 2782 . ~
Lactobacillus No. 7385 No. 14257 nitrosus No. 7386 No. 14258 Lactobacillus No. 7383 No. 14255 sulfurica No. 7384 No. 14256 ~ ~ No. 20~2 ~ 1445 ( ~ strain) No. 7382 - No. 14453 Streptococcus ( ~ strain) faecalis No.6569~ ~ strain) No.14506t ~ strain) No.11146( ~ strain~
Bifidobacterium No.11863( ~ strain) .. . _ .. _ .. , . , .,, ., ., ., _, ~ . ., .. , . .. _ _ .. _ . _ .. ~ .. _ . _ . _ _ _ . _ _ . _, _ . _ _ Bacillus No.2930 (A strain) No.3335 tB strain) (Note) Deposition numbers given on the same line show the identical strain.
The abbreviated name used in this specification is given in the parentheses after the Registration number.
The ~ strain and ~ strain of Streptococcus faecalis produce antibiotics.
~2744~
The ~ strain and ~ strain of Streptococcus faecalis do not produce antibiotics. The A s.rain of the Bacillus genus i~ a seed cell for yogurt (Bacillus coagulans). The ~ strain of the Bacillus genus is a seed cell for fermented soybeans (Bacillus subtilis).
The follcwing e~p~in~nts were effected using IFO
Nos. 14253 and 14450, as Lactobacillus clearans and .
Lactobaclllus deodorans, respectively.
To l g of fresh human feces was added 9 ml of isotonic sodium chloride solution. To (l) one mixture 50 obtained l ml of a cultured cell solution of Lactobacillus clearans was added, and to (2) another mixture l ml of a cultured cell solution of Lactobacillus clearans plus 0.5 ml of a cultured cell solution of ~ strain were added, and then the mixtures were allowed to stand at 37C
for 48 hours.
By judsement of odor, it was noted that the de~ree of deodorization was higher in the group of experiments of the case of mixture (2) and the amount of feces not deodorized was reduced by half as compared with the case of mixture (l), although the feces was not deodorized in two instances of ten instances of the latter case tested.
By oral administration of Lactobacillus clearans ~2'~4~32 only and of Lactobacillus clearans plus ~ strain, it was recognized as the degree of deodorization was examined with respect to the feces excreted, that the deodorant effect was higher and instances of lack of deodorization were less, in the cases wherein Lac_obacillus clearans plus ~ strain were administered.
These experiments (in vivo) show that Lactobacillus clearans as well as Lactobacillus clearans plus @~ strain are effective in intestines where feces (containing saprogens, most of which are anaerobic, amounting upto l.5 x lOll cellstg of faecalis) are presentO
Although a considerable effect was noticed by contact of either of Lactobacillus clearans and Lactobacillus . clearans plus @~ strain, the combination of Lactobacillus clearans plus ~ strain was more effective, as expected, compared with Lactobacillus clearans only.
Here, the effect means that a high degree of deodorization was attained and instances of lack of deodorization were reduced.
Although the above experiments were those relating to Lactobacillus clearans, the same comparative experiments -as these experiments were effected with respect to Lactobacillus deodorans and to Lactobacillus sulfurica plus Lactobacillus nitro~us, using IFO Nos. 14255, 14256, 14453. That is, comparative experiments between Lactobacillus deodorans and Lactobac llus deodorans plus ~;~74482, ~9 strain and those between Lactobaclllus sulfurica plus Lactobacillus nitrosus and Lactobacillus sulfurlca plus Lactobacillus nitrosus plus ~ strain were effected.
The same tendency as in Lactobacillus clearans
5 was observed in Lactobacillus sulfurica plus Lactobacillus nitrosus.
The effect of ~ strain was larger and more significant in the case of Lactobacillus deodorans.
That is, the use of ~ strain, a Streptococcus faecalis lO strain producing antibiotics, showed a very significant effect against offensive odor not only by oral administration but also by application to the.female pubic region. ~
~he results obtained are shown in Example 6.
r EXAMPLE 3 (Preparation of biodeodorizer?
15 Preparation of Yoqurt: -(l) Preparation of yogurt by ferment,ation of Lactobacillus clearans and Streptococcus faecalis:
A medium for seed cells (containing 30 g of skimmed milk and 3.g o-f calcium carbonate and having a pH
20 of 7.0) was divided into two and strilized. Then, the above strains were inoculated to each medium, respectively, and cultivated at 37C for 24 hours to obtain seed cells of Lactobacillus clearans and seed cells of Streptococcus faecalis. (The number of cells was 1.5 x 109 cells/ml for 25 Lactobacillus clearans and 2 x lO9 cells/ml for StrePtococcus - 3~ -faecalis.) On the other hand, 200 ml of a medium for yogurt consisting of skimmed milk 100 g, sucrose 100 g and agar 2 g, was sterilized.
To the medium for yogurt so obtained, 10 ml of the seed cells of Lactobacillus clearans and 0.8 ml of .
the seed cells of Streptococcus faecalis were added.
Then, cultivation was effected at 37C for 24 hours to prepare yogurt~
- The yogurt prepared contained 2~5 x 109 cells~ml of Lactobacillus clearans and 2.5 x 108 cells/ml of Streptococcus faecalis. The ratio of Lactobacillus clearans to Streptococcus faecalis was 10:1 (by weight~
:
- This yogurt was administered to man and animals.
(2) Preparation of yogurt by fermentation of Lactobacillus clearans, Streptococcus faecalis and Bifidobacterium:
.. .. _ Seed cells of Lactobacillus clearans and Streptococcus f~ecalis were obtained by preparing a medium for seed cells, inoculating each strain~to the medium and cultivating at 37C for 24 hours. Separately, Bifiobacterium was inoculated to a medium for seed cells consisting of skimmed milk 30 g cysteine 1 g, Vitamin B2 1 mg,calcium pantothenate 8 mg and biotin 0.1 mg, and cultivated at 37C for 72 hours.
On the other hand, 200 ml of a medium for yogurt consisting of skimmed milk 100 g, sucrose 100 g, agar 2 g lZ 7a~4~2 and cystein 1 g, was sterilized and subjeeted to sterile filtration. Then, 1 mg of vitamin ~2~ 8 mg of calcium pantothenate and 0.1 mg of biotin were added to the medium.
To the medium thus obtained, 10 ml of the seed cells of Bifidobacterium was added, and cultivation was effected at 37C for 36 hours.
Then, 10 ml of the seed cells of Lactobacillus clearans and 0.8 ml of the seed cells of Strep ococcus faecalis were further added simultaneously to the culture medium, and cultivation was further continued at 37C for 36 hours to prepare yogurt.
The yogurt thus prepared contained 2 x 109 cells/ml of Lactbacillus clearans, 2 x 108 cells/ml of Stre~tococcus faecalis and 3 x 109 cells/ml of Bifidobacterium. The ratio of Lactobacilius clearans: Streptococcus faecalis:
Bifidobacterium - 10 : 1 : 15 (by weight).
EXAMPLE 4 (PreDaration of biodeodorizer) Preparation of Wet Cell Preparations :
To MRS-lactose medium consisting of meat extract 10 g, peptone 10 g, yeast extract 5 g, lactose 20 g, Iween 80 1 ml, KH2Po4 2 g, sodium acetate 5 g, ammonium citrate 2 g, MgSO27H2O 0.2 g and MnSO4 4H20 0.05 g, Lactobacillus clearans, Streptococcusu faecalis and ~acillus were inoculated separately, and pure cultivation of each strain was effected at 37C for 72 hours. After completion ~Z~744~2 of the cultivation, cells were collected by centrifugation.
IThe yield was l g for Lactobacilus clearans, 1.2 g for StreDtococcus faecalis and 1 g for Bacillus, per lO00 ml.) In the czse of Bifidobacterium, cysteine l g, ~itamin B~ l mg, calciu~. pantothenate 8 mg and biotin 0.1 mg were added to MRS-lactose medium and the cultivation was effected at 37C
for 96 hours. After completion of cultivation, cells were collected by centrifugation. ~The yield was 0.5 g per lO00 ml.
(a) Preparations containing Lactobacillus clearans and Streptococcus faecalis:
By mixing lO g of the above collected cells of Lactobacillus clearans with l g of those of Streptococcus faecalisr a preparation was formed. The preparation was admixed with a feed stuff. (For chickens, 0.5 g of-the preparation was added to l kg of the feed stuff, and for pigs, 0.8 g of the preparation was added to l kg of the feed stuff.) (b) Preparations containing Lactobacillus clearans, Streptococcus faecalis and Bacillus:
By mixing 10 g of the above collected cells of Lactobacillus clearans, l g of those of Streptococcus faecalis and lO0 g of those of Bifidobacterium together, a preparation was formed. The preparation was admixed wi,h a feed stuff (for example, in an amount of 0.6 g per l kg of a feed stuff for chickens, and in an amount of 1 g per 1 kg of a feed stuff for pigs).
(c) Preparations containing Lactobacillus clearans, Stre~tococcus faecalis~ Bifidobacterium and Bacillus:
- By mixing 10 g of the above collected cells of Lacto~acillus clearans, 1 g of those of StxePtococcus faecalis, 100 g of those of Bifidobacterium and 0.5 g of those of Bacillus together~ a preparation was formed.
The preparation was added to a feed stuff (for example, in an amount of 0O6 g per 1 Kg of a feed stufr for chickens and in an amount of 1 g per 1 kg of a feed stuff for pits).
Pre~aration of Dry Cell PreParations:
(1) Semi-dry cell preparations:
To the above-described MRS~lactose medium and the above-described medium for Bifidobacterium, Lactobacillus clearans, Stre~tococcus faecalis and Bifidobacterium were inoculated individually, and pure cultivation of each strain was effected. To 10 g of each of the wet cells collected by centrifugation 300 g of starch dried by heating to have a moisture content of 0.2% and 1 g of cystine were ad~ed and mixed to form a wet preparation having finally a moisture content of 2~ or less.
(a) Preparations containing Lactobacillus clearans and StreDtococcus faecalis:
.
By mixing 10 g of the Lactobacillus clearans thus dried and coated, with 1 g of the StreDtococcus lZ'~448Z
faecalis thus dried and coated, a semi-dry preparation was formed~ The preparation was put into a vial, sealed under nitrogen gas and stored in a cold and dark place.
(b) Preparations containing Lactobacillus clearans, Streptococcus faecalis and Bifidobacterium:
By mixing 10 g of the Lactobacillus clearans thus dried and coated, 1 g of the Stre~tococcus faecalis dried and coated and 100 g of the Bifidobacterium dried and coated, together, a semi-dry preparation was formed.
The preparation was put into a vial, sealed under nitrogen gas and stored in a cold and dark place.
(2) Dry cell preparations:
.
To the above-described MRS-lactose medium and the above-described medium for Bifidobacterium, Lactobacillus clearans, Streptococcus faecalis and Bifidobacterium were individually inoculated, and pure cultivation of each strain was effected~ Ten g of each of the wet cells collected by centrifugation was dispersed in 100 ml of a solution containing 1% of lactose and 0.1% of cystine, and the solution was then freeze-dried.
(a) Preparations containing Lactobacillus clearans and strePtococcus faecalis:
By mixing 10 g of the live cell preparation thus obtained of Lactobacillus clearans with 1 g of the live cell preparation thus obtained of Stre~tococcus faecalis, a dry - 4~ -cell preparation was formedO The preparation was filled in a capsule and storedO
(b) Preparatlons_cQntaini~g Lactabacillus.clearans~
Streptococcus faecalis and Biid~bacteLium:
S By m.ixing lO g.Qf the Lactohacillus_clear~s obtained above, 1 g of the StrePtococcus faecalis obtained above and 100 g of the Bifidobacterium obtained abover together, a dry cell preparation was formed. The preparation was processed into tablets and stored in a vial.
Procedures:
(1) Mixed culture of Lactobacillus clearans, Lactobacillus deodorans or an equally mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus and/or Bacillus genus was effected using a medium consisting of skimmed milk 100 g, sucrose 100 g and agar 2 g. First, the medium was sterilized at 100C for 30 minutes, ~nter-mittently for 3 days. Then, the above strains were inoculated to the medium simultaneously in a desired cell number ratio, and culture was effected at 37C
for 24 hours to prepare a product.
(2) Mixed culture of the strains used in ~1) above with Bifidobacterium was effected in the above-mentioned medium to which cystine 1 g, vitamin B2 1 mg, calcium pantothenate 8 mg and biotin 0.1 mg were added under - 4~ -1~744~Z
sterile conditions, Bifidobacterium was cultivated previouqly at 37C for 36 hours~ and then the other strains were inoculated simultaneously and the cultiuatio~ ~as co~tinued further-at-3~C for 36 hours to prepare a_product. ~
(3) The product thus prepared was administered to patients twice a day, at morning and evening, for a continuous 4 days.
The results of the deodoriæation test are shown in Table 4, Table 5 and Table 6. Table 4 shows the results of testing wherein Lactobacillus clearans was used as the Lactobacillus strain. Table 5 shows the results of testing using Lactobacillus deodorans, and Table ~ using an equally mixed strain of Lactobacillus sulfurica and Lactbacillus nitrosus, as the Lactobacillus strain.
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Deodorant Effects:
The deodorant effects shown in Tables 4, 5 and
The effect of ~ strain was larger and more significant in the case of Lactobacillus deodorans.
That is, the use of ~ strain, a Streptococcus faecalis lO strain producing antibiotics, showed a very significant effect against offensive odor not only by oral administration but also by application to the.female pubic region. ~
~he results obtained are shown in Example 6.
r EXAMPLE 3 (Preparation of biodeodorizer?
15 Preparation of Yoqurt: -(l) Preparation of yogurt by ferment,ation of Lactobacillus clearans and Streptococcus faecalis:
A medium for seed cells (containing 30 g of skimmed milk and 3.g o-f calcium carbonate and having a pH
20 of 7.0) was divided into two and strilized. Then, the above strains were inoculated to each medium, respectively, and cultivated at 37C for 24 hours to obtain seed cells of Lactobacillus clearans and seed cells of Streptococcus faecalis. (The number of cells was 1.5 x 109 cells/ml for 25 Lactobacillus clearans and 2 x lO9 cells/ml for StrePtococcus - 3~ -faecalis.) On the other hand, 200 ml of a medium for yogurt consisting of skimmed milk 100 g, sucrose 100 g and agar 2 g, was sterilized.
To the medium for yogurt so obtained, 10 ml of the seed cells of Lactobacillus clearans and 0.8 ml of .
the seed cells of Streptococcus faecalis were added.
Then, cultivation was effected at 37C for 24 hours to prepare yogurt~
- The yogurt prepared contained 2~5 x 109 cells~ml of Lactobacillus clearans and 2.5 x 108 cells/ml of Streptococcus faecalis. The ratio of Lactobacillus clearans to Streptococcus faecalis was 10:1 (by weight~
:
- This yogurt was administered to man and animals.
(2) Preparation of yogurt by fermentation of Lactobacillus clearans, Streptococcus faecalis and Bifidobacterium:
.. .. _ Seed cells of Lactobacillus clearans and Streptococcus f~ecalis were obtained by preparing a medium for seed cells, inoculating each strain~to the medium and cultivating at 37C for 24 hours. Separately, Bifiobacterium was inoculated to a medium for seed cells consisting of skimmed milk 30 g cysteine 1 g, Vitamin B2 1 mg,calcium pantothenate 8 mg and biotin 0.1 mg, and cultivated at 37C for 72 hours.
On the other hand, 200 ml of a medium for yogurt consisting of skimmed milk 100 g, sucrose 100 g, agar 2 g lZ 7a~4~2 and cystein 1 g, was sterilized and subjeeted to sterile filtration. Then, 1 mg of vitamin ~2~ 8 mg of calcium pantothenate and 0.1 mg of biotin were added to the medium.
To the medium thus obtained, 10 ml of the seed cells of Bifidobacterium was added, and cultivation was effected at 37C for 36 hours.
Then, 10 ml of the seed cells of Lactobacillus clearans and 0.8 ml of the seed cells of Strep ococcus faecalis were further added simultaneously to the culture medium, and cultivation was further continued at 37C for 36 hours to prepare yogurt.
The yogurt thus prepared contained 2 x 109 cells/ml of Lactbacillus clearans, 2 x 108 cells/ml of Stre~tococcus faecalis and 3 x 109 cells/ml of Bifidobacterium. The ratio of Lactobacilius clearans: Streptococcus faecalis:
Bifidobacterium - 10 : 1 : 15 (by weight).
EXAMPLE 4 (PreDaration of biodeodorizer) Preparation of Wet Cell Preparations :
To MRS-lactose medium consisting of meat extract 10 g, peptone 10 g, yeast extract 5 g, lactose 20 g, Iween 80 1 ml, KH2Po4 2 g, sodium acetate 5 g, ammonium citrate 2 g, MgSO27H2O 0.2 g and MnSO4 4H20 0.05 g, Lactobacillus clearans, Streptococcusu faecalis and ~acillus were inoculated separately, and pure cultivation of each strain was effected at 37C for 72 hours. After completion ~Z~744~2 of the cultivation, cells were collected by centrifugation.
IThe yield was l g for Lactobacilus clearans, 1.2 g for StreDtococcus faecalis and 1 g for Bacillus, per lO00 ml.) In the czse of Bifidobacterium, cysteine l g, ~itamin B~ l mg, calciu~. pantothenate 8 mg and biotin 0.1 mg were added to MRS-lactose medium and the cultivation was effected at 37C
for 96 hours. After completion of cultivation, cells were collected by centrifugation. ~The yield was 0.5 g per lO00 ml.
(a) Preparations containing Lactobacillus clearans and Streptococcus faecalis:
By mixing lO g of the above collected cells of Lactobacillus clearans with l g of those of Streptococcus faecalisr a preparation was formed. The preparation was admixed with a feed stuff. (For chickens, 0.5 g of-the preparation was added to l kg of the feed stuff, and for pigs, 0.8 g of the preparation was added to l kg of the feed stuff.) (b) Preparations containing Lactobacillus clearans, Streptococcus faecalis and Bacillus:
By mixing 10 g of the above collected cells of Lactobacillus clearans, l g of those of Streptococcus faecalis and lO0 g of those of Bifidobacterium together, a preparation was formed. The preparation was admixed wi,h a feed stuff (for example, in an amount of 0.6 g per l kg of a feed stuff for chickens, and in an amount of 1 g per 1 kg of a feed stuff for pigs).
(c) Preparations containing Lactobacillus clearans, Stre~tococcus faecalis~ Bifidobacterium and Bacillus:
- By mixing 10 g of the above collected cells of Lacto~acillus clearans, 1 g of those of StxePtococcus faecalis, 100 g of those of Bifidobacterium and 0.5 g of those of Bacillus together~ a preparation was formed.
The preparation was added to a feed stuff (for example, in an amount of 0O6 g per 1 Kg of a feed stufr for chickens and in an amount of 1 g per 1 kg of a feed stuff for pits).
Pre~aration of Dry Cell PreParations:
(1) Semi-dry cell preparations:
To the above-described MRS~lactose medium and the above-described medium for Bifidobacterium, Lactobacillus clearans, Stre~tococcus faecalis and Bifidobacterium were inoculated individually, and pure cultivation of each strain was effected. To 10 g of each of the wet cells collected by centrifugation 300 g of starch dried by heating to have a moisture content of 0.2% and 1 g of cystine were ad~ed and mixed to form a wet preparation having finally a moisture content of 2~ or less.
(a) Preparations containing Lactobacillus clearans and StreDtococcus faecalis:
.
By mixing 10 g of the Lactobacillus clearans thus dried and coated, with 1 g of the StreDtococcus lZ'~448Z
faecalis thus dried and coated, a semi-dry preparation was formed~ The preparation was put into a vial, sealed under nitrogen gas and stored in a cold and dark place.
(b) Preparations containing Lactobacillus clearans, Streptococcus faecalis and Bifidobacterium:
By mixing 10 g of the Lactobacillus clearans thus dried and coated, 1 g of the Stre~tococcus faecalis dried and coated and 100 g of the Bifidobacterium dried and coated, together, a semi-dry preparation was formed.
The preparation was put into a vial, sealed under nitrogen gas and stored in a cold and dark place.
(2) Dry cell preparations:
.
To the above-described MRS-lactose medium and the above-described medium for Bifidobacterium, Lactobacillus clearans, Streptococcus faecalis and Bifidobacterium were individually inoculated, and pure cultivation of each strain was effected~ Ten g of each of the wet cells collected by centrifugation was dispersed in 100 ml of a solution containing 1% of lactose and 0.1% of cystine, and the solution was then freeze-dried.
(a) Preparations containing Lactobacillus clearans and strePtococcus faecalis:
By mixing 10 g of the live cell preparation thus obtained of Lactobacillus clearans with 1 g of the live cell preparation thus obtained of Stre~tococcus faecalis, a dry - 4~ -cell preparation was formedO The preparation was filled in a capsule and storedO
(b) Preparatlons_cQntaini~g Lactabacillus.clearans~
Streptococcus faecalis and Biid~bacteLium:
S By m.ixing lO g.Qf the Lactohacillus_clear~s obtained above, 1 g of the StrePtococcus faecalis obtained above and 100 g of the Bifidobacterium obtained abover together, a dry cell preparation was formed. The preparation was processed into tablets and stored in a vial.
Procedures:
(1) Mixed culture of Lactobacillus clearans, Lactobacillus deodorans or an equally mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus and/or Bacillus genus was effected using a medium consisting of skimmed milk 100 g, sucrose 100 g and agar 2 g. First, the medium was sterilized at 100C for 30 minutes, ~nter-mittently for 3 days. Then, the above strains were inoculated to the medium simultaneously in a desired cell number ratio, and culture was effected at 37C
for 24 hours to prepare a product.
(2) Mixed culture of the strains used in ~1) above with Bifidobacterium was effected in the above-mentioned medium to which cystine 1 g, vitamin B2 1 mg, calcium pantothenate 8 mg and biotin 0.1 mg were added under - 4~ -1~744~Z
sterile conditions, Bifidobacterium was cultivated previouqly at 37C for 36 hours~ and then the other strains were inoculated simultaneously and the cultiuatio~ ~as co~tinued further-at-3~C for 36 hours to prepare a_product. ~
(3) The product thus prepared was administered to patients twice a day, at morning and evening, for a continuous 4 days.
The results of the deodoriæation test are shown in Table 4, Table 5 and Table 6. Table 4 shows the results of testing wherein Lactobacillus clearans was used as the Lactobacillus strain. Table 5 shows the results of testing using Lactobacillus deodorans, and Table ~ using an equally mixed strain of Lactobacillus sulfurica and Lactbacillus nitrosus, as the Lactobacillus strain.
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Deodorant Effects:
The deodorant effects shown in Tables 4, 5 and
6 are those evaluated as to the following four items:
1) Whether the deodorant effect appeared rapidly or not, i.e~ "rate".
2) Whether the deodorant power was strong or not, i.e.
"strength".
3) For how many days did deodorant effect continue, i.e. "period'l.
4) Whether the feeling on evacuation was good or not, i.e. "feeling".
With regard to each of the above items, when more than half of the patients judged that the effect was a remarkable one, the symbol ~ was used. When more than half of the patients judged that tne effect was present, the symbol O was used. When more than half of the patients judged that there was no effect, the symbol ~ was used.
Explanation of Ta~le 4:
The effect of the combined use of Lactobacill~s clearans with the other strains is shown in Table 4.
I Combined use of two kinds of strains:
(1) Either of ~ strain and ~ strain produced plural antibiotics very well, though there was difference between these two strains in growth rate and ~ strain was superior to the ~ strain in rate. Both the ~Z7g4~2 strain and ~ strain did not produce antibiotics, though their rate of growth and multiplying was superior than that of the ~ strain.
Ex~eriment No. 1: The deodorant effect of Lactobacillus .
clearans plus the ~ strain was faster and stronger than tha~ of the single use of Lactobacillus clearans.
Also the duration time of the effect was somewhat longer than the time for the single use, and two-thirds of the patients expressed a good feeling on evacuation.
Expe ment No. 2: The deodorant effect of Lactobacillus clearans plus the ~ strain was stronger than that of Lactobacillus clearans only. However, no lengthening of the duration was recognized with the combined use.
Experiments Nos. 3 and 4: The deodorant effect of _ Lactobacillus clearans plus the ~ strain or Lactobacillus clearans plus the ~ strain was noted to be almost the same as that of the Lactobacillus clearans onlyr As is apparent from the above Experiments Nos. 1 - 4, the deodorant effect of Lactobacillus clearans is improved by the ccmbined use with Streptococcus faecalis capable of producing antibiotics, although no improvement was brought about by the combined use with Streptococcus faecalis not capable of producing antibiotics.
~Z7~4~2 l2) Ex~eriments Nos. 5 and 6: In the case of Lactobacillus clearans plus the ~ strain or Lactobacillus clearans plus the ~ strain, lengthening of the deodorant period as compared with the single use of Lactobacillus clearans was no~ed by one-third of the patients subjected to the test. However, thus lengthening was ~ot recognized by two-thirds of them.
In view of the past experience in biological testing, it can be said that such results of effectiveness amounting to 30~ or so show that the combined i5 quite useful.
(33 The A strain is a Bacillus coa~ulans and has an excellent property as a seed cell for yogurt ~see U.S. Patent No. 4~210,672). The B strain is a Bacillus subtilis and has an excellent property as a seed cell for fermented soybeans. It is commercially available, Experiments Nos~ 7 and 8: In the case of Lactobacillus clearans plus the A strain or Lactobacillus clearans plus, the B strain, no report that the deodorant effect was stronger or the deodorant period was longer, than the single use of Lactobacillus clearans, was received~
The results of examination by gas chromatography also did not show any significant difference from the single use of Lactobacillus clearans.
-1~:749~82 II Combined use of three kinds of strains:
., . .. . .... . _ (1) Combined use with StrePtococcus faecalis producing antibiotlcs and Bifidobacter1um:
From the experimental results, the effect attained by using the ~ strain was considered almost the same as that attained by using the ~ strain (though the ~ strain was somewhat superior to the ~ strain~.
Therefore, consideration about the experimental results obtained by using the ~ strain is omitted from the following explanation.
Experiments Nos. 9 and 10: In the case of the cmbined .
use of Lactbacillus clearans with the ~ strain and the ~ strain or the ~ strain, almost 70% of the test subjects reported that the effect was stronger and continued longer than the case of Lactobacillus clearans only and the feeling on evacuation was not unpleasant.
Among those people who recognized thus effects, 20%
reported that the combined use of these three strains was more effective than the combined use of Experiment No. 1 (Lactobacillus clearans plus the ~ strain), 30% reported that there was no difference from the combined use of Experiment No. 1 and the remaining 50 reported that the combined use of the three strains was rather inferior to the combined use of Experiment No. 1. The effect was mild and continued for a long time.
Ex~eriments Nos. 1? ,18,21 and 22: In the case of combined use of Lactobacillus clearans with the strain or the ~ strain and the ~ strain or the ~ strain,.the effect was the same as the cases of Experiments Nos. 3 and 4. No report that the deodorant effect was strengthened or the period was lengthened was received.
From the results of the experiments as described above, 50% of the test subjects reported that when Bifidobacterium was added to Lactobacillus clearans and Streptococcus faecalis producing antibiotics, the deodorant effect attained was superior to the single use of Lactbacillus clearans.
Further~ 50~ of the test subjects reported that the effect of the combined use of these three strains was the same as, or better than, that of the combined use of Lactobacillus clearans with Stre~tococcus faecalis producing antibiotics. Only 15% of the test subjects reported that the combined use of the two strains was better than that of the three strains.
The combined use with Streptococcus faecalis not producing antibiotics was found to be not so effective.
(2) Combined use with StrePtococcus fa calis producing antibiotics and Bacillus:
Experiments Nos. 11 and 12: In the case of the combined ~Z'74482 use of Lactobacillus clearans with the ~ strain and .. .. _ . _ _ . . _ _ the A strain or the B strain, the deodorant effect was stronger than the single use of Lactobacillus clearans for more than 50~ of the test subjects. In some test subjects, however, the effect was less than that attained by the combined use of Lactobacillus clearans and the ~ strain or of Lactobacillus clearans, the ~ strain and the ~ or the ~ strain.
Experiments Nos. 19 and 20: Neither combination was effective r From the above experimental results, it was found that - the deodorant effect attained by adding a digestive Bacillus genus to Lactobacillus clearans and StrePtococcus faecalis producing antibiotics was similar to that attained without such addition.
Further, the combined use with Streptococcus faecalis not producing antibiotics was found to be not so effective.
(3) Combined use with Bifidobacterium and Bacillus:
Ex~eriments Nos. 25,26,27 and 28: In either combination, almost ~0% of the test subjects recognized a lengthening of the deodorant period. About 20% of those test subjects who recognized the lengthening reported that the strength was however weak, compared with the single use of Lactobacillus clearans.
lZ744132 [III] Combined use of four kinds of strains:
(1) Combined use with Stre~tococcus faecalis, Bifidobacterium and Bacillus Ex~eriments Nos. 29,30,31 and 32- In the càse of the combined use of Lactobacillus clearans with the ~
strain, the ~ strain or the ~ strain and the A
strain or the B strain, the deodorant effect was sometimes rather weak compared with the case of the combined use of Lactobacillus clearans and the ~
strain. ~owever, the period of duration was longest in the case of the combined use of these four kinds of strainsc Ex eriments Nos. 37,38,39,40,41,42,43 and 44: In the P, . _ _ case of the combined use of Lactobacillus clearans with StrePtococcus faecalis not producing antibiotics, Bifidobacterium and Bacillus, the deodorant effect was not superior to the single use of Lactobacillus clearans.
From the results of these experiments, it was found that, by addition of Bifidobacterium and Bacillus to Lactobacillus clearans and Streptococcus faecalis producing antibiotics, a mild deodorant effect continued for the longest period although a remarkable change was not recognized in other respects, as compared with the cases in which Bifidobacterium and ~acillus were not added.
iL~7448Z
Conclusions:
Summarizing the effects attained by the combined use of Lactobacillus clearans with various strains, it is apparent from the above results that combinations of 1) Lactobacillus clearans and StrePtococeus faecalis producing antibiotics, 2) Lactobacillus clearans, Streetococcus faeealis producing antibiotics, and Bifidobacterium, .
3) Lactobacillus clearans, Streptococcus faecalis producing antibiotics, and Bacillus, and 4) Lactobacillus clearans, Streptococcus faecalis producing antibioties, Bifidobacterium and Bacillus r show better and/or longer deodorant effects, compared with the single use of Lactobacillus clearans.
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O ~ ~ ~ ~ o Z ~ rl 1`7 ~ ~ r~ ~ ~r ~r lZ~744~2 Explanation of Table 5 and Table 6:
. _ Table 5 shows the results of deodorant tests using Lactobacillus deodorans instead of Lactobacillus clearans, and Table 6 shows the results of deodorant tests using a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus, instead of Lactobacillus clearans.
From the results of experiments using Lactobacillus deodorans, instead of Lactobacillus clearans, almost the same tendency as in Table 4 was noted.
- Here, it is to be noted that the deodorant effect shown by the symbol ~ in the cases where Lactobacillus plus the ~ strain were used is different in the degree of deodorizing feces, from that shown by the same symbol ~ in the cases where Lactobacillus clearans plus the ~ strain were used. For example, when administration of Lactobacillus clearans only was stopped, the offensive odor which had been reduced to half of original revived soon, while when Lactobac llus clearans was administered in combination with the ~ strain, such revival ceased at a quarter of the criginal and the influence of the administration remained for three days after the stoppage thereof. In contrast, when Lactobacillus deodorans was administered alone, the offensive odor was removed completely for a while.
However, when Lactbacillus deodorans was administered in combination with the ~ strain, the time necessary for 127g4BZ
reaching complete deodorization was shortened and the effect continued, in many cases, for 6 - 10 days after stopping the administration D
In connection with the revival or the odor of feces, the state of intestinal bacteria of the test subjects was examined. When Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used, no change in intestinal bacteria was observed~
Even when Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used in combination with the ~ strain, the intestinal bacteria was restored to the original state within 5 days or so.
However, when Lactobacillus deodorans was used, 15 days or so were generally required for restoration and the intestinal bacteria sometimes showed a change (reduction) in many cases.
In the case,the restoration was not complete in some cases.
The difference of effects exerted on the intestinal bacteria by Lactobacillus clearans and Lactobacillus deodorans is a characteristic thereof~
Table 6 shows that, when a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used, almost the same results as the case of using Lactbacillus clearans were obtained with respect to rate strength and period of the deodorant effect.
~2~7a~3z As shown in Tables 4 - 6, when two, three or four kinds of the strains according to the present invention were administered to man in combination, there were many experimental sections where a definite difference could hardly be reco~nized under the above-mentioned standard of judgement. Therefore, the degrees of the growth of chickens and pigs were also investigated.
The strains were added to feed stuff in the following amount.
For chickens: Each strain was added in an amount of 0.2 g as dry cells containing extending agents, per 1 kg of usual assorted feed for chickens.
For pigs: Each strain was added in an amount of 0.4 g as dry cells containing extending agents, per 1 kg of usual assorted feed for pigs.
The investigation was effected mainly by comparing the weight and the state of flesh of chickens or pigs after the administration, with those before the administration.
Thus, it was found that, in the experimental sections where Lactobacillus deodorans, Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used in combination with -Bifidobacterium ( ~ strain or ~ strain) or in combination with Bacillus (A strain or B strain) and also in the 12~44~2 experimental sections where Lactbacillus deoaorans, Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used in combi-nation with Streptococcus fa~calis and Bifidobacterium .
or Bacillus or in combination with Streptococcus faecalis, Bifidobacterium and Bacillus, the feeding effici~oncy was improved by 10% or so, compared with th~ cases wherein Lactobacillus deodorans, Lactobacillus cl arans or a mixed strain of Lac_obacillus sulfurica and Lactbacillus 0 nitrosus was added solely to the feed stuff.
~n absorbent cotton well dipped in a solution of 5 mq - of chlorohexidine hydrochloride in 250 ml of water was inserted into the vagina of a patient, in whose vagina Lactbacillus was reduced to one-hundredth or less of the normal value, three-times for 15 minutes each. Then, another cotton well dipped in a culture solution of each of Lactobacillus clearans, Lactobacillus clearans plus the ~ strain, Lactobacillus deodorans, and Lactobacillus deodorans plus the ~ strain, which were provided with resistance against 5 y chlorohexidine hydrochloride solution, was inserted into the vagina of the above patient, and the absorbent cotton was exchanged for new one, five times at intervals of one hour. The test was carried out with 15 persons for each test section.
12744E~Z
At the same time, the prudendaor vulvo of the patient was treated three-times with a disinfectant to control contaminated bacteria, and then absorbent cotton well dipped in the culture solution or each of the above-mentioned strains was applied to the prudenda.
The treatment as above was continued for one week. After 2 days, the presence of many Lactobaci;lus was observed in the vagina, almost without exception or vulvo.
However, after a further 5 days, the survival ratio of Lactobacillus in those test sections where Lactobacillus clearans was used in combination with the ~ strain was higher than that of the test sections where the ~ strain was not used in combination, although reduction of Lactbacillus as noted before the start of the test was observed in some patients treated with Lactobacillus clearans.
As regards the ratio of removiny offensive odor, a distinct difference was recognized between the test sections where the ~ strain was used in combination and the test sections where the ~ strain was not used in combination. (The deodorant effect was superior in the former test sections to the latter test sections.) In case of a sole use of Lactobacillus deodorans, deodorizing efficiency and survival count is extraordinary superior to those of Lactobacillus clearans. Further addition of the ~ strain shows more solid results.
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The same results were obtained when using Lactobac_llus clearans, Lactobacillus deodor ns, a mixture of Lactobacillus nitrosus and Lactobacillus sulfurica, and _ _ Stre~tocuccus faecalis other than the IFO Nos. 14253, 14450, 14257, 14255, 14452 and 14453.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modification can be made therein without de-parting from the splrit and scope of thereof.
1) Whether the deodorant effect appeared rapidly or not, i.e~ "rate".
2) Whether the deodorant power was strong or not, i.e.
"strength".
3) For how many days did deodorant effect continue, i.e. "period'l.
4) Whether the feeling on evacuation was good or not, i.e. "feeling".
With regard to each of the above items, when more than half of the patients judged that the effect was a remarkable one, the symbol ~ was used. When more than half of the patients judged that tne effect was present, the symbol O was used. When more than half of the patients judged that there was no effect, the symbol ~ was used.
Explanation of Ta~le 4:
The effect of the combined use of Lactobacill~s clearans with the other strains is shown in Table 4.
I Combined use of two kinds of strains:
(1) Either of ~ strain and ~ strain produced plural antibiotics very well, though there was difference between these two strains in growth rate and ~ strain was superior to the ~ strain in rate. Both the ~Z7g4~2 strain and ~ strain did not produce antibiotics, though their rate of growth and multiplying was superior than that of the ~ strain.
Ex~eriment No. 1: The deodorant effect of Lactobacillus .
clearans plus the ~ strain was faster and stronger than tha~ of the single use of Lactobacillus clearans.
Also the duration time of the effect was somewhat longer than the time for the single use, and two-thirds of the patients expressed a good feeling on evacuation.
Expe ment No. 2: The deodorant effect of Lactobacillus clearans plus the ~ strain was stronger than that of Lactobacillus clearans only. However, no lengthening of the duration was recognized with the combined use.
Experiments Nos. 3 and 4: The deodorant effect of _ Lactobacillus clearans plus the ~ strain or Lactobacillus clearans plus the ~ strain was noted to be almost the same as that of the Lactobacillus clearans onlyr As is apparent from the above Experiments Nos. 1 - 4, the deodorant effect of Lactobacillus clearans is improved by the ccmbined use with Streptococcus faecalis capable of producing antibiotics, although no improvement was brought about by the combined use with Streptococcus faecalis not capable of producing antibiotics.
~Z7~4~2 l2) Ex~eriments Nos. 5 and 6: In the case of Lactobacillus clearans plus the ~ strain or Lactobacillus clearans plus the ~ strain, lengthening of the deodorant period as compared with the single use of Lactobacillus clearans was no~ed by one-third of the patients subjected to the test. However, thus lengthening was ~ot recognized by two-thirds of them.
In view of the past experience in biological testing, it can be said that such results of effectiveness amounting to 30~ or so show that the combined i5 quite useful.
(33 The A strain is a Bacillus coa~ulans and has an excellent property as a seed cell for yogurt ~see U.S. Patent No. 4~210,672). The B strain is a Bacillus subtilis and has an excellent property as a seed cell for fermented soybeans. It is commercially available, Experiments Nos~ 7 and 8: In the case of Lactobacillus clearans plus the A strain or Lactobacillus clearans plus, the B strain, no report that the deodorant effect was stronger or the deodorant period was longer, than the single use of Lactobacillus clearans, was received~
The results of examination by gas chromatography also did not show any significant difference from the single use of Lactobacillus clearans.
-1~:749~82 II Combined use of three kinds of strains:
., . .. . .... . _ (1) Combined use with StrePtococcus faecalis producing antibiotlcs and Bifidobacter1um:
From the experimental results, the effect attained by using the ~ strain was considered almost the same as that attained by using the ~ strain (though the ~ strain was somewhat superior to the ~ strain~.
Therefore, consideration about the experimental results obtained by using the ~ strain is omitted from the following explanation.
Experiments Nos. 9 and 10: In the case of the cmbined .
use of Lactbacillus clearans with the ~ strain and the ~ strain or the ~ strain, almost 70% of the test subjects reported that the effect was stronger and continued longer than the case of Lactobacillus clearans only and the feeling on evacuation was not unpleasant.
Among those people who recognized thus effects, 20%
reported that the combined use of these three strains was more effective than the combined use of Experiment No. 1 (Lactobacillus clearans plus the ~ strain), 30% reported that there was no difference from the combined use of Experiment No. 1 and the remaining 50 reported that the combined use of the three strains was rather inferior to the combined use of Experiment No. 1. The effect was mild and continued for a long time.
Ex~eriments Nos. 1? ,18,21 and 22: In the case of combined use of Lactobacillus clearans with the strain or the ~ strain and the ~ strain or the ~ strain,.the effect was the same as the cases of Experiments Nos. 3 and 4. No report that the deodorant effect was strengthened or the period was lengthened was received.
From the results of the experiments as described above, 50% of the test subjects reported that when Bifidobacterium was added to Lactobacillus clearans and Streptococcus faecalis producing antibiotics, the deodorant effect attained was superior to the single use of Lactbacillus clearans.
Further~ 50~ of the test subjects reported that the effect of the combined use of these three strains was the same as, or better than, that of the combined use of Lactobacillus clearans with Stre~tococcus faecalis producing antibiotics. Only 15% of the test subjects reported that the combined use of the two strains was better than that of the three strains.
The combined use with Streptococcus faecalis not producing antibiotics was found to be not so effective.
(2) Combined use with StrePtococcus fa calis producing antibiotics and Bacillus:
Experiments Nos. 11 and 12: In the case of the combined ~Z'74482 use of Lactobacillus clearans with the ~ strain and .. .. _ . _ _ . . _ _ the A strain or the B strain, the deodorant effect was stronger than the single use of Lactobacillus clearans for more than 50~ of the test subjects. In some test subjects, however, the effect was less than that attained by the combined use of Lactobacillus clearans and the ~ strain or of Lactobacillus clearans, the ~ strain and the ~ or the ~ strain.
Experiments Nos. 19 and 20: Neither combination was effective r From the above experimental results, it was found that - the deodorant effect attained by adding a digestive Bacillus genus to Lactobacillus clearans and StrePtococcus faecalis producing antibiotics was similar to that attained without such addition.
Further, the combined use with Streptococcus faecalis not producing antibiotics was found to be not so effective.
(3) Combined use with Bifidobacterium and Bacillus:
Ex~eriments Nos. 25,26,27 and 28: In either combination, almost ~0% of the test subjects recognized a lengthening of the deodorant period. About 20% of those test subjects who recognized the lengthening reported that the strength was however weak, compared with the single use of Lactobacillus clearans.
lZ744132 [III] Combined use of four kinds of strains:
(1) Combined use with Stre~tococcus faecalis, Bifidobacterium and Bacillus Ex~eriments Nos. 29,30,31 and 32- In the càse of the combined use of Lactobacillus clearans with the ~
strain, the ~ strain or the ~ strain and the A
strain or the B strain, the deodorant effect was sometimes rather weak compared with the case of the combined use of Lactobacillus clearans and the ~
strain. ~owever, the period of duration was longest in the case of the combined use of these four kinds of strainsc Ex eriments Nos. 37,38,39,40,41,42,43 and 44: In the P, . _ _ case of the combined use of Lactobacillus clearans with StrePtococcus faecalis not producing antibiotics, Bifidobacterium and Bacillus, the deodorant effect was not superior to the single use of Lactobacillus clearans.
From the results of these experiments, it was found that, by addition of Bifidobacterium and Bacillus to Lactobacillus clearans and Streptococcus faecalis producing antibiotics, a mild deodorant effect continued for the longest period although a remarkable change was not recognized in other respects, as compared with the cases in which Bifidobacterium and ~acillus were not added.
iL~7448Z
Conclusions:
Summarizing the effects attained by the combined use of Lactobacillus clearans with various strains, it is apparent from the above results that combinations of 1) Lactobacillus clearans and StrePtococeus faecalis producing antibiotics, 2) Lactobacillus clearans, Streetococcus faeealis producing antibiotics, and Bifidobacterium, .
3) Lactobacillus clearans, Streptococcus faecalis producing antibiotics, and Bacillus, and 4) Lactobacillus clearans, Streptococcus faecalis producing antibioties, Bifidobacterium and Bacillus r show better and/or longer deodorant effects, compared with the single use of Lactobacillus clearans.
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O ~ ~ ~ ~ o Z ~ rl 1`7 ~ ~ r~ ~ ~r ~r lZ~744~2 Explanation of Table 5 and Table 6:
. _ Table 5 shows the results of deodorant tests using Lactobacillus deodorans instead of Lactobacillus clearans, and Table 6 shows the results of deodorant tests using a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus, instead of Lactobacillus clearans.
From the results of experiments using Lactobacillus deodorans, instead of Lactobacillus clearans, almost the same tendency as in Table 4 was noted.
- Here, it is to be noted that the deodorant effect shown by the symbol ~ in the cases where Lactobacillus plus the ~ strain were used is different in the degree of deodorizing feces, from that shown by the same symbol ~ in the cases where Lactobacillus clearans plus the ~ strain were used. For example, when administration of Lactobacillus clearans only was stopped, the offensive odor which had been reduced to half of original revived soon, while when Lactobac llus clearans was administered in combination with the ~ strain, such revival ceased at a quarter of the criginal and the influence of the administration remained for three days after the stoppage thereof. In contrast, when Lactobacillus deodorans was administered alone, the offensive odor was removed completely for a while.
However, when Lactbacillus deodorans was administered in combination with the ~ strain, the time necessary for 127g4BZ
reaching complete deodorization was shortened and the effect continued, in many cases, for 6 - 10 days after stopping the administration D
In connection with the revival or the odor of feces, the state of intestinal bacteria of the test subjects was examined. When Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used, no change in intestinal bacteria was observed~
Even when Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used in combination with the ~ strain, the intestinal bacteria was restored to the original state within 5 days or so.
However, when Lactobacillus deodorans was used, 15 days or so were generally required for restoration and the intestinal bacteria sometimes showed a change (reduction) in many cases.
In the case,the restoration was not complete in some cases.
The difference of effects exerted on the intestinal bacteria by Lactobacillus clearans and Lactobacillus deodorans is a characteristic thereof~
Table 6 shows that, when a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used, almost the same results as the case of using Lactbacillus clearans were obtained with respect to rate strength and period of the deodorant effect.
~2~7a~3z As shown in Tables 4 - 6, when two, three or four kinds of the strains according to the present invention were administered to man in combination, there were many experimental sections where a definite difference could hardly be reco~nized under the above-mentioned standard of judgement. Therefore, the degrees of the growth of chickens and pigs were also investigated.
The strains were added to feed stuff in the following amount.
For chickens: Each strain was added in an amount of 0.2 g as dry cells containing extending agents, per 1 kg of usual assorted feed for chickens.
For pigs: Each strain was added in an amount of 0.4 g as dry cells containing extending agents, per 1 kg of usual assorted feed for pigs.
The investigation was effected mainly by comparing the weight and the state of flesh of chickens or pigs after the administration, with those before the administration.
Thus, it was found that, in the experimental sections where Lactobacillus deodorans, Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used in combination with -Bifidobacterium ( ~ strain or ~ strain) or in combination with Bacillus (A strain or B strain) and also in the 12~44~2 experimental sections where Lactbacillus deoaorans, Lactobacillus clearans or a mixed strain of Lactobacillus sulfurica and Lactobacillus nitrosus was used in combi-nation with Streptococcus fa~calis and Bifidobacterium .
or Bacillus or in combination with Streptococcus faecalis, Bifidobacterium and Bacillus, the feeding effici~oncy was improved by 10% or so, compared with th~ cases wherein Lactobacillus deodorans, Lactobacillus cl arans or a mixed strain of Lac_obacillus sulfurica and Lactbacillus 0 nitrosus was added solely to the feed stuff.
~n absorbent cotton well dipped in a solution of 5 mq - of chlorohexidine hydrochloride in 250 ml of water was inserted into the vagina of a patient, in whose vagina Lactbacillus was reduced to one-hundredth or less of the normal value, three-times for 15 minutes each. Then, another cotton well dipped in a culture solution of each of Lactobacillus clearans, Lactobacillus clearans plus the ~ strain, Lactobacillus deodorans, and Lactobacillus deodorans plus the ~ strain, which were provided with resistance against 5 y chlorohexidine hydrochloride solution, was inserted into the vagina of the above patient, and the absorbent cotton was exchanged for new one, five times at intervals of one hour. The test was carried out with 15 persons for each test section.
12744E~Z
At the same time, the prudendaor vulvo of the patient was treated three-times with a disinfectant to control contaminated bacteria, and then absorbent cotton well dipped in the culture solution or each of the above-mentioned strains was applied to the prudenda.
The treatment as above was continued for one week. After 2 days, the presence of many Lactobaci;lus was observed in the vagina, almost without exception or vulvo.
However, after a further 5 days, the survival ratio of Lactobacillus in those test sections where Lactobacillus clearans was used in combination with the ~ strain was higher than that of the test sections where the ~ strain was not used in combination, although reduction of Lactbacillus as noted before the start of the test was observed in some patients treated with Lactobacillus clearans.
As regards the ratio of removiny offensive odor, a distinct difference was recognized between the test sections where the ~ strain was used in combination and the test sections where the ~ strain was not used in combination. (The deodorant effect was superior in the former test sections to the latter test sections.) In case of a sole use of Lactobacillus deodorans, deodorizing efficiency and survival count is extraordinary superior to those of Lactobacillus clearans. Further addition of the ~ strain shows more solid results.
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The same results were obtained when using Lactobac_llus clearans, Lactobacillus deodor ns, a mixture of Lactobacillus nitrosus and Lactobacillus sulfurica, and _ _ Stre~tocuccus faecalis other than the IFO Nos. 14253, 14450, 14257, 14255, 14452 and 14453.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modification can be made therein without de-parting from the splrit and scope of thereof.
Claims (22)
1. A biodeodorizer to assimilate odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixtures thereof, said biodeodorizer comprising in combination (a) at least one Lactobacillus selected from the group consisting of:
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
2. The biodeodorizer of Claim 1, wherein the pro-portion of Lactobacillus to Streptococcus faecalis is about 150:1 to about 0.01:1.
3. The biodeodorizer of Claim 1, additionally including (c) a Bifidobacterium and/or a Bacillus.
4. The biodeodorizer of Claim 3, wherein the biodeodorizer comprises about 5 to about 15 parts by weight of Lactobacillus, about 10 to 100 parts by weight of a Bifidobacterium and about 0.05 to 0.15 parts by weight of a Bacillus per part by weight of Streptococcus faecalis.
5. The biodeodorizer of Claim 4, wherein said Bacillus is Bacillus coagulans or Bacillus subtilis.
6. The biodeodorizer of Claim 1, wherein said bio-deodorizer is a live bacterial preparation.
7. The biodeodorizer of Claim 1, wherein said bio-deodorizer additionally includes a pharmaceutically acceptable excipient.
8. An active feed stuff comprising a feed stuff and the biodeodorizer of Claim 1.
9. The biodeodorizer of Claim 1 in suppository form.
10. The biodeodorizer of Claim 1 in an absorbent cotton form.
11. A process to prepare a biodeodorizer comprising mixing (a) at least one Lactobacillus selected from the group consisting of:
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
12. The process of Claim 11, wherein the proportion of Lactobacillus to Streptocuccus faecalis is about 150:1 to about 0.01:1.
13. The process of Claim 11, additionally including (c) a Bifidobacterium and/or a Bacillus.
14. The process of Claim 13, wherein the biodeodorizer comprises about 5 to about 15 parts by weight of Lactobacillus, about 10 to 100 parts by weight of a Bifidobacterium and about 0.05 to 0.15 part by weight of a Bacillus per part by weight of Streptococcus faecalis.
15. The process of Claim 14, wherein said Bacillus is Bacillus coagulans or Bacillus subtilis.
16. The process of Claim 11, wherein said biodeodorizer is a live bacterial preparation.
17. The process of Claim 11, wherein said bio-deodorizer additionally includes a pharmaceutically acceptable excipient.
18. The process of Claim 11, wherein said Lactobacillus deodorans, Lactobacillus clearans, Lactobacillus sulfurica and Lactobacillus nitrosus are cultivated under a presence of at least one compound selected from the group consisting of odoriferous compound containing sulfur atoms, odoriferous compound containing nitrogen atoms, odoriferous compound containing carbon atoms and a sulfur containing-amino acid.
19. A process to prepare a biodeodorizer comprising reducing the moisture content of the wet cells of (a) at least one Lactobacillus selected from the group consisting of:
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics, said microorganisms collected by centrifugation, mixing the cells with excipients and drying in vacuum.
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics, said microorganisms collected by centrifugation, mixing the cells with excipients and drying in vacuum.
20. A process to prepare a biodeodorizer comprising dispersing wet cells of (a) at least one Lactobacillus selected from the group consisting of:
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics, said microorganisms collected by centrifugation in a solution of excipients and freeze-drying the resulting dispersion.
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics, said microorganisms collected by centrifugation in a solution of excipients and freeze-drying the resulting dispersion.
21. A process to prepare a biodeodorizer comprising mixing strains obtained by the processes of Claims 19 and 20.
22. A process for deodorizing in vitro comprising administrating a biodeodorizer comprising (a) at least one Lactobacillus selected from the group consisting of:
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
(i) Lactobacillus deodorans, which is promoted in growth thereof by odoriferous compounds containing sulfur atoms and/or nitrogen atoms, and has an ability to assimilate completely the odorifrous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, (ii) Lactobacillus clearans, which is not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms, or mixture thereof and has an ability to decrease the odoriferous compounds containing sulfur atoms and/or odoriferous compounds containing nitrogen atoms, and (iii) a mixed strain of Lactobacillus sulfurica which has an ability to decrease the odoriferous compounds containing sulfur atoms and Lactobacillus nitrosus which has an ability to decrease the odoriferous compounds containing nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
nitrogen atoms, which are each not promoted in growth thereof by odoriferous compounds containing sulfur atoms, nitrogen atoms, carbon atoms or mixture thereof; and (b) Streptococcus faecalis having the ability to produce antibiotics.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000486901A CA1274482A (en) | 1985-07-16 | 1985-07-16 | Biodeodorizer and the process thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000486901A CA1274482A (en) | 1985-07-16 | 1985-07-16 | Biodeodorizer and the process thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1274482A true CA1274482A (en) | 1990-09-25 |
Family
ID=4130989
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000486901A Expired - Fee Related CA1274482A (en) | 1985-07-16 | 1985-07-16 | Biodeodorizer and the process thereof |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1274482A (en) |
-
1985
- 1985-07-16 CA CA000486901A patent/CA1274482A/en not_active Expired - Fee Related
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