JP2001333766A - New lactic acid bacterium effective on infectious disease and lactobacillus preparation comprising lactic acid bacterium as main ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a Lactobacillus casei producing an antibacterial substance having growth inhibiting actions on microorganisms and moreover having hypotoxicity actions on pathogenic bacteria, a lactobacillus preparation compris ing the lactic acid bacterium as a main ingredient, and a health care agent, a prophylactic agent and a therapeutic agent to infectious diseases for human, animals and plants. SOLUTION: The Lactobacillus casei produces the antibacterial substance exhibiting growth inhibiting actions and safening actions on microorganisms. The Lactobacillus casei has the action safening hemolysis when brought into contact with a pathogenic bacterium exhibiting the hemolysis and the Lactobacillus casei has the action promoting the mutation when brought into contact with a pathogenic bacterium spontaneously exhibiting S-R mutation. The lactobacillus preparation comprises the Lactobacillus casei.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a Lactobacillus casei which produces an antibacterial substance which has not only a growth inhibitory effect on microorganisms but also an attenuating effect on pathogenic bacteria.
llus casei), a lactic acid bacterium preparation containing the lactic acid bacterium as a main component, and a health agent, a preventive agent and a therapeutic agent for infectious diseases of humans, animals and plants.

[0002]

BACKGROUND OF THE INVENTION The history of antibiotics was described in 1928 in the laboratory of St. Mary's Hospital in London. Fleming (AF
leming) around a blue mold that happened to grow on a petri dish,
It begins by observing that the growth of staphylococci is blocked. It was the beginning of the development of the miracle drug "penicillin". Until then, I had to be scared just before the enemies were invisible.

[0003] Since history, tremendous epidemics have killed many people, attacked the army, destroyed social organizations and affected the rise and fall of civilization. Especially feared were plague. Called the Black Death, it was a true death, with the skin of the whole body becoming dark purple and dying. In medieval Europe, pandemics often occur, and it is said that one-quarter of the population of Europe at that time died from plague. It is a historical fact that the social system and spiritual structure that governed at that time collapsed due to the repeated epidemics, and this spread to immigrants to the US continent.

[0004] With the discovery of antibiotics, humans have taken the first step towards a glorious victory in the fight against pathogens. Since then, the concept of an all-purpose antibiotic has emerged in both the field of drug development and the field of therapy, and to this day it has been inherited not only in bacteriology but also as a source of medicine. In fact, it seemed that a variety of antibiotics, discovered and developed one after another, have freed us from disease caused by bacteria. However, with its dramatic effects, magic bullets and tobacco antibiotics,
Now the shadows have begun to appear.

[0005] That is, since antibiotics have been widely used as chemotherapeutic agents, bacteria that resist the drug have been found one after another mainly in hospitals. Therefore, it was necessary to develop new antibiotics that were effective against these resistant bacteria, and much effort was put into them. However, even with the emergence of new antibiotics, resistant bacteria will soon emerge when their administration begins. The use of the drug itself kills susceptible organisms and encourages the growth of surviving resistant organisms. Therefore, no matter how much new antibiotics are developed, the emergence of resistant bacteria is inevitable, and there is a continuing play of drugs and bacteria. Bacteria have been able to withstand the drastic changes in the earth's environment since their birth in the primordial sea some 300 million years ago, and have acquired drug resistance by making use of the "art of transformation" that has been cultivated for their survival. This mechanism is roughly divided into the following patterns. a) Inactivate antibiotics by hydrolyzing or producing enzymes that modify side chains. b) It changes qualitatively and reduces the binding affinity of the antibacterial agent. c) Eliminate the effects of antimicrobial agents by increasing the amount of target that is inhibited. d) suppress the permeability of the drug into cells. And multidrug-resistant bacteria that are now a major problem in the medical field, such as Mycobacterium tuberculosis, MRSA (Methicillin Resistance Staphyl)
ococcus Aureus), VRE (Vancomysin Resistance Enterococcus), Pseudomonas aeruginosa, etc., and the human side is in fact on the verge of defeat.

Again, in Japan, with the aging society, the necessity of disease prevention has been re-recognized, and now the gaze is focused on "preventive medicine", and instead of treating after becoming ill, The idea of preventing illness and protecting one's own health is becoming mainstream. Nevertheless, our consciousness, including in the medical setting, still cannot escape from the equation of infectious disease = antibiotics. This is actually a problem. As many as 100 trillion intestinal bacteria inhabit the intestine, weighing as much as 1 kg, far more than 60 trillion human cells. And over the years, epidemiological studies and studies have revealed a deep involvement in human and animal health and disease, and experts have identified the gut flora as one of the key organs. Among them, the genus Lactobacillus and Bifidobacterium, which are called good bacteria, have various beneficial effects, including the ability to normalize and enhance immune function, but antibiotics These are destroyed by the administration of. Elderly people in particular are not immune to decline in immunity as they age, otherwise they become infected →
Administration of antibiotics → destruction of intestinal microflora (rapid reduction of good bacteria, emergence of bacterial alternation phenomenon, side effects) → further reduction of immunity → vicious cycle to infectious diseases. The 120-year-old theory of human life is another dream.

[0007] For this reason, the first conceivable means is to stop using messy antibiotics and use them in a planned manner. For example, a drug called ethambutol is frequently used recently for multidrug-resistant Mycobacterium tuberculosis. The drug was not recently discovered, but was already developed and used in the 1950's, but had been avoided because of significant side effects (such as kidney damage). Although ethambutol may not be resistant to bacteria, it is rarely used because it has not been used so often. Even now, the current situation in which drugs that have been abandoned in terms of side effects must be used without interruption is a struggle between humans and bacteria, and there is a glimpse of the struggle on the human side.

When the use of a particular antibiotic is discontinued for a long period of time, a large number of bacteria are disarmed and do not show resistance to the antibiotic.
In some cases, R plasmids involved in acquiring resistance are deleted. Multidrug-resistant bacteria make enzymes that destroy or inactivate many antibiotics. This property is of great advantage where antibiotics are present. However, in the absence of antibiotics, the production of many of these destructive enzymes with useless longs lags the competition for survival with light susceptible bacteria. Therefore, gradually, these enzymes are no longer produced, and they are transformed into susceptible bacteria.

Similar to ethambutol, the use of chloramphenicol has been greatly restricted in terms of side effects. Before restriction, the majority of Shigella bacteria found in Japan were resistant to kuromai, but after more than 10 years since use was restricted, it became less than 10%. From these facts,
Avoiding antibiotic abuse will reduce resistant bacteria. Antibiotics also regain their effect. Careful use of medicine is of utmost importance in solving the problem of resistant bacteria.

[0010] The next step is to change the "force-based policy" of eradicating large amounts of antibiotics against bacteria. Bacteria called pathogens are among the vast majority of bacteria a very minority, a small number actually infect, and an exceptionally few develop from infection to infectious disease. The overwhelming majority of bacteria are harmless or beneficial. Killing the whole bacterium for some outlaw pathogens is like using a nuclear weapon to kill an individual.

[0011] The creatures living on this earth live in mutual influence. Remember, whether competitive or symbiotic, the result is a biologically harmonious condition on Earth. Very few pathogens have historically seen their virulence changing over time. At first, it is customary that an intensely pathogenic bacterium (or virus) appears, becomes a pandemic, and is gradually replaced by a weaker one. Both Y. pestis, V. cholerae (Asian type → Eltor type), Shigella (Shiga type → Flexinel type) and syphilis spirochetes are also remarkable. The reason for this is that the infected person also mobilizes various biological defense functions and responds,
Bacteria change over the life of the species.

[0012] The virulent bacterium breeds offspring by intercepting nutrients with humans and animals as dwellings. When the host dies, that is the end. The former and the child are gone. On the other hand, a weakly virulent bacterium does not kill the host, so progeny prosper with prolonged nutrition. Many of the new virulent pathogens that suddenly attack humans originally coexisted in animals, which accidentally attach to humans and cause them to be poisoned and killed by a drastic environmental change. This is the starting point for the outbreak of the new virulent epidemic. Among them, pathogens that have been mutated into attenuated forms adapted to the environment by gene transfer etc. occupy a large number due to the instinct of the survival of the species. Bacteria are unicellular organisms, but much smarter and warmer than humans. Bacteria are trying to coexist and co-prosper with humans (hosts).
You should step up and reach out without refusing it. In other words, in the natural world, it is only necessary to lend a hand to the progress of gradually changing from virulent to low or non-toxic strains. What is the facilitator, and if so, where?

As can be seen from the past outbreaks of plague and cholera, and recent outbreaks of food poisoning caused by O-157 school lunches in Sakai City, even if bacteria enter the body in the same way, all members will be affected. I did not get sick. In addition, some people become ill if they get sick, while others remain mild. What do these facts tell? Is this simply a problem that can be addressed by the strength of the individual's immunity, the number of invading bacteria, and the timing of infection?

[0014]

As described above, the harmful effects of conventional antibiotics, that is, drug-resistant bacteria, drug allergy,
Solving and eliminating various problems related to side effects and abnormalities of the indigenous flora has been demanded as a current countermeasure against infectious diseases.

[0015]

As described above, the largest site of the relationship between humans and bacteria is in the intestine. 100 kinds for people
Trillions of enormous quantities of intestinal bacteria are settled. Perhaps since the birth of human beings, intestinal bacteria and humans have been coexisting. It has been handed down to this day. There is always a reason for what exists. The fact that there are far more than 60 trillion human gut bacteria in the intestinal tract is a natural thing to consider as an integral part of human life. Without killing the invading pathogens separately, they may be acting to make them mild and to reduce or eliminate their toxicity. For example, it is like putting a villain in a correctional facility, rehabilitating the heart with the right mind, or pulling out the poisonous snake's fangs to make it just a non-toxic snake. Whether these special bacteria are colonized in the intestine, and whether or not the intestinal environment is livable for such bacteria, are affected by the invasion of pathogens into the body. The present inventors have focused on the bacterial group that constitutes intestinal bacteria based on the inference that even if the infection is established, it may be a major reason that there is a great difference in the course of the infection.

Among them, lactic acid bacteria, which have been established as non-pathogenic and have been handed down from the era of Metnikov, or since ancient times, have been passed down for generations as being good for the body, ie, non-pathogenic, have a high affinity for mucosa in the intestine, The research started by focusing on the genus Lactobacillus, which has a very wide range of nature, including the oral cavity and the vagina, and energetically isolates and investigates it. It is known that some strains of the genus Lactobacillus produce antibiotics,
Some crystals have already been extracted and named. However, they seemed to have been buried in third-generation antibiotics without a valid reputation and never returned. The value of conventional antibiotics that focus on killing bacteria was probably class C. From another point of view, it may have been a property that could be a super-A class. Therefore, among the genus Lactobacillus, Acidophilus
(L. acidophilus), Casei (L. casei), Brevis (Lb
revis), L. salivalius, plantarum
We started by rewashing the antibiotics produced by bacteria that have colonization in the intestine, such as (L. plantarum) and fermentum (L. fermentum). And as a result, the casei species require "attenuated toxicity antibiotics"
The present inventors have found out that there is a strain producing, and confirmed the efficacy and effect thereof, and finally accomplished the present invention.

That is, Lactobacillus casei (Lactobacillus casei) is characterized by producing an antibacterial substance having a growth inhibiting action and a toxicity reducing action on microorganisms.
i) wherein Lactobacillus casei preferably has resistance to a known antibiotic, and the known antibiotic is a β-lactam antibiotic, an aminoglucoside antibiotic, a macrolide antibiotic. It is particularly preferable that at least one or two or more of a substance, a tetracycline antibiotic, a quinolone antibiotic and a glycopeptide antibiotic be used, and that Lactobacillus casei is FERM
Particularly preferred is at least one or more of BP-6972, FERM BP-6971 and FERM BP-6973.

A second aspect of the present invention is a Lactobacillus casei, which has an action of reducing the hemolytic property of a pathogenic bacterium when brought into contact with the pathogenic bacterium, the lactobacillus casei being a known antibiotic. It is preferable that the known antibiotics are β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolone antibiotics, and glycobiotics. It is particularly preferred that at least one or two or more peptide-based antibiotics are used, and Lactobacillus casei is FERM BP-697.
2. It is particularly preferred that at least one or two or more of FERM BP-6971 and FERM BP-6973 are used.

A third aspect of the present invention is a Lactobacillus casei, which has an action of promoting the mutation when brought into contact with a pathogenic bacterium which spontaneously exhibits the SR mutation. Preferably, the known antibiotics are β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolone antibiotics. It is particularly preferred that at least one or two or more of the substance and the glycopeptide antibiotic are used, and Lactobacillus casei is used as the FERM BP-
At least one of 6972, FERM BP-6971 and FERM BP-6973
It is particularly preferred that the number of species is two or more.

A fourth aspect of the present invention is a lactic acid bacterium preparation characterized by comprising Lactobacillus casei as a main component, which produces an antibiotic having a growth inhibitory effect and a toxicity attenuating effect on microorganisms, wherein Lactobacillus casei is known. Preferably, the known antibiotics are β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolone antibiotics. It is particularly preferred that at least one or two or more substances and glycopeptide antibiotics are used.
Particularly preferred is at least one or more of FERM BP-6972, FERM BP-6971 and FERM BP-6973.

A fifth aspect of the present invention is a lactic acid bacterium preparation comprising lactobacillus casei as a main component, which has an action of reducing the hemolytic property of a pathogenic bacterium when brought into contact with the pathogenic bacterium, It is preferred that Lactobacillus casei has resistance to known antibiotics, wherein the known antibiotics are β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics,
At least one or two of tetracycline antibiotics, quinolone antibiotics and glycopeptide antibiotics;
It is particularly preferred that the Lactobacillus casei is FERM BP-6972, FERM BP-6971 and FERM BP-69.
It is particularly preferred that at least one or two or more of 73 are used.

A sixth aspect of the present invention is a lactic acid bacterium preparation containing lactobacillus casei as a main component, which has an action of promoting the mutation when brought into contact with a pathogenic bacterium which spontaneously exhibits the SR mutation. Oh, Lactobacillus
Preferably, casei has resistance to known antibiotics, and the known antibiotics are β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics, Particularly preferred is at least one or more of quinolone antibiotics and glycopeptide antibiotics, and Lactobacillus casei is FERM BP-6972, FERM BP-6971 and FE
Particularly preferred is at least one or two or more of RM BP-6973.

A seventh aspect of the present invention is that Lactobacillus casei, which produces an antibiotic having a growth inhibitory action and attenuating action on microorganisms, reduces the hemolytic property of the pathogenic bacterium when brought into contact with the pathogenic bacterium. Lactobacillus casei having an action and Lactobacillus casei having an action of accelerating the mutation when brought into contact with a pathogenic bacterium exhibiting S-R mutation spontaneously. Characterized by
A human, animal, or plant health or infectious disease preventive or therapeutic agent, wherein Lactobacillus casei is FERM BP-
At least one of 6972, FERM BP-6971 and FERM BP-6973
It is particularly preferred that the number of species is two or more.

An eighth aspect of the present invention is that Lactobacillus casei, which produces an antibiotic having a growth inhibitory action and attenuating action on microorganisms, reduces the hemolytic property of the pathogenic bacterium when brought into contact with the pathogenic bacterium. Lactobacillus casei having an action, and a lactobacillus preparation containing any one of Lactobacillus casei having a function of promoting the mutation when brought into contact with a pathogenic bacterium which spontaneously exhibits the SR mutation, A human, animal or botanical health agent, a prophylactic agent for an infectious disease or a therapeutic agent for an infectious disease, comprising Lactobacillus casei, comprising FERM P-17148, FERM P-17149 and FERM P-17149.
Particularly preferred is at least one or more of FERM P-17150, and the known antibiotics are β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolones It is preferable that at least one kind or two or more kinds of antibiotics and glycopeptide antibiotics be used. Incidentally, FERM BP-6972, FERM BP-6971 and FERM BP-69 used in the present invention
6973 is a strain of Lactobacillus casei deposited on January 18, 1999 as a mutant strain of Lactobacillus casei and deposited at the Patent Microorganisms Depositary Center of the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry of Japan. is there.

[0025]

BEST MODE FOR CARRYING OUT THE INVENTION Lactobacillus casei according to the present invention is a strain isolated by the following procedure. That is, using a combination of a selective medium and a non-selective medium of the genus Lactobacillus at 28 ° C. and 37 ° C. under anaerobic and aerobic conditions, samples from human and animal intestines, oral cavity, grass leaves, Fruit, agricultural products,
Lactobacillus sp. Was isolated from samples such as soil. A modified LBS medium is used as a selective medium for Lactobacillus, a BCP medium and 1 L (pH 7.
In 2), 5 g of casamino acid, 3 g of yeast extract, 10 g of lactose, 4 g of CaCO ₃ , ₂ g of K ₂ HPO ₄ , MgSO
₄ · 7H ₂ O1g, Chlorella CGF1g, sodium deoxycholate 3g, 0.2% BTB5ml, agar 15
g can be exemplified. The isolated genus Lactobacillus is described in the taxonomy authority book "Bergey's Manual
of Determinative Bacteriology 9 Edition ".

Antibiotic-producing ability of the isolated Lactobacillus
About 1g of peptone 8g, meat extract 2
g, yeast extract 2 g, lactose 8 g, trehalose 2
g, Chlorella CGF 3 g, L-cystine 1 g, K_TwoHP
O_Four2g, MgSO_Four・ 7H _TwoO1g, NaCl1g,
CaCO_Three5 g, diammonium citrate 2 g, MnS
O_Four・ XH_TwoO0.3g, FeSO_Four・ 7H_TwoO0.0
3g and 15g of agar in the medium.
We made various kinds of plate media, and isolated bacteria in the center with a diameter of 1
Aerobic and anaerobic when applied to a 0-15mm circle
Pre-culture at 37 ° C and 28 ° C for 72 hours
Staphylococcus (Staphylococcus) is a representative of Gram-positive bacteria.
aureus) and Streptococcus pyogenes,
As a representative of the rum-negative bacteria, Salmonella
ritidis) and Klebsiella pneumoniae
Spread as test bacteria by cross-segmentation method and culture under aerobic condition
Continued. After 48 hours, how much growth of test bacteria is stopped
By observing whether or not antibiotics have been
Determine the presence and degree of liveness and determine only the producing strain.
Selected. There are various strains producing antibiotics.
Existed over "species". Also described in this specification
All pathogens were sold by Medic Co., Ltd.
Things.

[0027] If it affects not only growth inhibition but also a gene that controls toxin production, it is considered that it acts across the boundaries of "genus". Therefore, using the antibacterial spectrum as an index, only strains having a wide antibacterial spectrum were picked up. Therefore, in addition to the strains used for the above-described antibiotic production studies, enterococci (Enterococcus sp.) And Bacillus s.
p.), Clostridium (Clostridium sp.), Corynebacterium (Corynebacterium sp.), Mycobacterium (Micobacte
rium sp.), Listeria sp. .), Enterobacter sp., Pseudomonas (Pseud
omonas sp.) and Bacteroides (Bacteroides sp.), as fungi, Candida (Candida sp.), Cryptococcus (Cryptococcus sp.), Trichophyton (Trichopon)
hyton sp.) and Aspergillus (Aspergillus sp.).

First, the hemolytic properties of the lactobacilli produced broad-spectrum antibiotics were examined for their reduced toxicity. Many bacterial exotoxins are hemolytic, albeit to varying degrees. Therefore, when examining the ability to produce antibiotics, the bacteria on the side close to the lactic acid bacteria of the pathogenic bacteria that had grown by the cross-segmentation method on the plate medium were picked, and smeared again by the cross-segmentation method. The bacteria close to the lactic acid bacteria are picked again several times, and the pathogens that have been exposed many times to the antibiotics produced by the lactic acid bacteria are applied to the blood agar medium (using sheep and horse blood) and independent. The colonies were allowed to form colonies, the degree of hemolysis was observed, and the degree of toxicity reduction or toxin production was measured as compared to the original strain. At this time, MRSA (Staphylococcucu
s aureus), Streptococcus pyogene
s), Streptococcus pneumoniae, Pseudomonas aeruginosa (Pse
udomonas aeruginosa) and Shigella flexneri were employed.

Next, the SR mutation was examined. That is, Escherichia coli O-157 and Salmonella enteritidis were individually cultured and mixed with lactic acid bacteria, and the results were compared.
The medium composition used for the culture was 10 g of meat extract, 10 g of peptone, 2 g of glucose, 2 g of NaCl, 2 g of CaCO 3 in 1 L.
_The pH was adjusted to 7.2 containing 1 g. The culture was performed anaerobically at 37 ° C, and subculture was repeated every 72 hours. Each time, the cells were diluted and applied to a plate medium, and the bacterial growth and appearance were changed to S-type (original) or toxic, resulting in toxicity. It was observed whether the R type was attenuated or the ratio thereof.

The results of culture of the lactic acid bacterium FERM BP-6973 of the present invention which produces attenuated toxicity antibiotics with the above-mentioned pathogenic bacteria associated with FERM BP-6973 are shown in Tables 1, 2, 3 and 4. Table 1 shows E. coli O
In the table showing the results of single culture of -157, the number of bacteria was almost constant at 4 to 5 × 10 ⁹ cells / ml throughout the 20 passages, and no R type appeared during this time. On the other hand, in the mixed culture of E. coli O-157 and FERM BP-6973,
As is clear from the above, although the change in the number of bacteria of FERM BP-6973 is small, the change in the number of bacteria of Escherichia coli O-157 is large.
The R-type emerged from around the generation, and as the successive passages were repeated,
The percentage of R-type increased, and all bacteria formed R-type at the 18th generation. The S-type was never resurrected even if the passage was continued thereafter.

[0031]

[Table 1]

[0032]

[Table 2]

Table 3 shows the results of single culture of Salmonella enteritidis, wherein the number of bacteria was 3-5 × 10 ⁹ cells / cell.
ml, R-type appears spontaneously at the 14th generation,
The ratio became 5% in the 20th generation, and the maximum value was recorded at 23% in the 50th generation while increasing and decreasing with each passage, but the ratio of the R type after that was around 20%. On the other hand, Table 4 is a table showing the results of mixed culture of Salmonella enteritidis and FERM BP-6973. As the number of passages increases, the percentage of R-type increases, and 50% in the 10th generation and 90% in the 20th generation. ,
It reached 100% in the 47th generation. When the passage was continued thereafter, the S-type was sometimes resurrected, but the ratio was less than 1%. After the 70s, the S-type completely disappeared and did not recover again.

[0034]

[Table 3]

[0035]

[Table 4]

Next, in order to determine whether the toxicity was actually attenuated, the test animal was administered. First, the original stock 1
00% lethal dose was determined and pathogenic bacteria and R
One of the pathogenic bacteria mutated into the type was administered at a bacterial count corresponding to the lethal dose of the original strain, and the course of the symptoms was observed in detail. After the administration, a mouse showing a process almost the same as that of the non-administered mouse was finally selected, and only the casei species remained. The representative stock is FERM BP-6972, FERM
BP-6971 and FERM BP-6973. Table 5 shows an example of the experimental results. In addition, it became clear that the results of hemolysis and SR mutation had a high correlation with the results of animal experiments.

[0037]

[Table 5]

The culture medium for cultivating Lactobacillus casei of the present invention contains tryptophan, lysine and amino acid as amino acids.
Leucine, cystine, as vitamins, vitamin B _1, vitamin C, folic acid, calcium pantothenate,
As a mineral, zinc, molybdenum, and manganese are found to be deeply involved in the ability to produce attenuated antibiotics, and then the appropriate amount of the accelerating substance is added to the medium components to form. By repeating the selection of colonies, stronger strains could be collected.

Furthermore, if the Lactobacillus casei of the present invention can impart resistance to a commonly used antibiotic, it can be used in combination with the antibiotic, and a new therapeutic field which has not existed in the past has been developed. It will expand.
Among the currently widely used antibiotics, AB penicillin, cefpirome and the like as β-lactam antibiotics, streptomycin as aminoglycoside antibiotics,
Fradiomycin, etc., as macrolide antibiotics, erythromycin, oleandomycin, etc., as tetracycline antibiotics, tetracycline,
Select oxytetracycline, minomycin, etc., quinolone antibiotics, norfloxacin, toscloxin, etc., and glycopeptide antibiotics, vancomycin, etc., and use the sensitive disk (manufactured by Showa Disk Co., Ltd.) according to the usual method. Created a resistance strain. It is generally said that Lactobacillus is highly sensitive to antibiotics and resistant strains are unlikely to appear.However, by appropriately using an appropriate amount of the above-described amino acids, vitamins, and minerals that effectively act on Lactobacillus casei of the present invention, FERM Resistant strains such as BP-6972, FERM BP-6971 and FERM BP-6973 were also successfully created.

[0040]

Next, the present invention will be described in detail with reference to examples, but the gist of the present invention is not limited to these examples. Needless to say, an arbitrary number of bacteria can be obtained by changing the production process or using a known extender or excipient. In addition, as for the dosage form of each preparation, a usual dosage form such as a powder, a granule, and a capsule can be appropriately adopted together with a suitable excipient.

(Example 1) Skim milk 100 per liter
g of trehalose and 1 g of trehalose were inoculated with the lactic acid bacterium of the present invention (FERM BP-6972, to which no resistance was imparted), and cultured under facultative anaerobic conditions at 37 ° C. for 72 hours.
Next, the culture solution (yogurt) was freeze-dried in a vacuum in a conventional manner to obtain 510 g of a bacterial preparation. When the number of bacteria was measured,
It was 3 × 10 ⁹ pieces / g. The preparation obtained here contains freeze-dried cells and a substance produced by the cells.

Example 2 8 g of peptone, 3 g of meat extract, 2 g of yeast extract, 7 g of lactose, 3 g of trehalose, 2 g of sodium acetate, 2 g of KH ₂ PO _4, 2 g of CH ₂ PO in 1 L
aCO ₃ 5 g, MgSO ₄ .7H ₂ O 0.5 g, MnS
_{O 4 · xH 2 O0.1g, FeSO} 4 · 7H 2 O0.0
The lactic acid bacterium of the present invention (multidrug-resistant FERM BP-6972) was added to 10 L of a pH 7.2 medium containing 2 g and 1 g of Tween 80.
Was inoculated and cultured at 37 ° C. for 72 hours under facultative anaerobic conditions. After the culture, the culture was filtered with paper to remove CaCO _3, and 2 L of the filtrate was refrigerated and stored, and the residual liquid was centrifuged to obtain 9.2 g of bacterial mass. The bacterial mass was thoroughly washed with 500 ml of physiological saline, centrifuged again, and collected. The purified cells obtained were added to a protective solution consisting of 50 g of skim milk, 500 ml of soy milk, 40 g of trehalose, and 2 g of taurine. After stirring, the mixture was freeze-dried in a vacuum in a conventional manner to obtain 154 g of a bacterial preparation. When the number of bacteria was measured, it was 2 × 10 ¹⁰ cells / g (cells + protective agent). Among the preparations obtained here, the freeze-dried cells contain the cells and a protective agent,
The number of bacteria is 2 × 10 ¹⁰ cells / g, and the culture solution contains bacterial cells and bacteria-producing substances, and the number of bacteria is 3 × 10 ⁹ cells / m 2.
l.

(Test Example 1) Fifteen patients having the same causative bacterium of the infectious disease and exhibiting similar symptoms were divided into three groups of five each of acute colitis, acute cystitis and acute bronchitis. The treatment method of each group is as follows. In the A-group, only 1000 mg / day of the antibiotic was administered for 5 days, and in the B-group, 500 mg / day of the antibiotic was administered for 5 days, and the antibiotic produced in the same manner as in Example 2 was used. Material resistant
FERM BP-6972 was administered 2 g / day of a freeze-dried bacterial preparation having a bacterial count of 5 × 10 ⁹ cells / g for 10 days, and in group C, 1000 mg / day of only antibiotics was first administered to alleviate acute symptoms. At that time (usually for 2 to 3 days), and 2 g / day of a freeze-dried bacterial preparation having 5 × 10 ⁹ cells / g of the antibiotic-resistant FERM BP-6972 produced by the method of Example 2 It was administered for 10 days. The treatment results of each group over 10 days were averaged and shown in Table 6. As is clear from Table 6, as a method of coping with acute infectious disease, in addition to conventional antibiotic administration, by using the lactic acid bacteria preparation of the present invention in combination, or by using the lactic acid bacteria preparation of the present invention after antibiotic administration. Can reduce the dosage of antibiotics,
Symptom reduction, alleviation, and quicker recovery, no side effects to be felt, less disturbance of intestinal microflora, etc.Compared to conventional antibiotics, bacterial replacement phenomenon, resistant bacteria Considering the above problems, the merits are enormous even for medical sites and patients. In addition to the above-mentioned acute infections, intestinal infections known as food poisoning, ENT infections such as otitis media,
It has been demonstrated that various acute infections such as postoperative infections show high therapeutic effects.

[0044]

[Table 6]

(Test Example 2) A test was conducted in the field of chronic infectious diseases, for which the difficulty of treatment is constantly described despite the presence of powerful antibiotics. In the A-group, only the antibiotic was administered, and in the B-group, the antibiotic was used together with the freeze-dried bacterial preparation prepared in the same manner as in Example 2;
-Groups receive antibiotics first, then
The freeze-dried bacterial preparation produced in the same manner as in Example 2 was administered, and the D-group was administered only the freeze-dried bacterial preparation produced in the same manner as in Example 2. In the test,
The optimal antibiotic was administered depending on the causative bacteria and symptoms. In the case of the A-group, the daily dose is 12 mg or more and 1000 mg in the case of the A-group.
The amount was set to 4 times / day in each case with a half amount of the A-group. Administration of the lactic acid bacteria preparation of the present invention is basically freeze-dried cells,
1 × 10 ⁹ / g, 2 g / day, but increased slightly depending on the symptoms. In addition, depending on the location of the disease, Example 2
, 3 × 10 ⁹ cells / ml were used. Some of the treatment results were extracted and shown in Tables 7 to 10. Among the lactic acid bacteria preparations of the present invention, FD-6972R, FD-6971R and F
D-6973R is FERM BP-6972, FERM BP-6971 and F respectively
The antibiotic-resistant lyophilized preparation, Br-6972R, Br-69, produced in the same manner as in Example 2 using ERM BP-6973
71R and Br-6973R are FERM BP-6972 and FERM B, respectively.
A culture solution produced by the same method as in Example 2 using P-6971 and FERM BP-6973, FD-6972S, FD-6971S and
FD-6973S was a freeze-dried preparation produced in the same manner as in Example 1 using FERM BP-6972, FERM BP-6971 and FERM BP-6973, respectively, and Br-6972S, Br-6971S and
Br-6973S is a culture solution produced by the same method as in Example 2 using FERM BP-6972, FERM BP-6971 and FERM BP-6973, respectively. As is evident from these tables, treatment with antibiotics alone did not improve treatment results, and even for chronic infections that had relied on surgery, the lactic acid bacteria preparation of the present invention was used alone or in combination with other drugs. Good results were obtained. If the causative organism was still excreted even after the administration was completed, the causative organism was isolated and tested. In most cases, it was confirmed that the toxicity was reduced. Chronic infections other than the above, for example, cecumitis, urethritis, vaginitis,
It was also tested for acne, etc.
The lactic acid bacteria preparation of the present invention was proved to be effective.

[0046]

[Table 7]

[0047]

[Table 8]

[0048]

[Table 9]

[0049]

[Table 10]

(Test Example 3) In order to examine the relationship between the change of the intestinal bacterial flora and health, a representative 3 of the lactic acid bacteria of the present invention was used.
Strains, namely FERM BP-6972, FERM BP-6971 and FERM B
A formulation was prepared by mixing equal amounts of each of the freeze-dried cells of P-6973, and 2 g (5 × 10 ⁸ cells / g) thereof was taken daily, and the fate of the intestinal flora was measured over time. . Bifidobacterium as a representative of good bacteria
Of Lactobacillus and Lactobacillus / Feces 1
g as a representative of bad bacteria, Clostridium (Clos
tridiumu perfrigens) and Bayonela
The number of bacteria of (Vellonella) / g of feces was measured. In healthy subjects, as shown in Table 11, after 6 months from the administration, the amount of Bacterium bacterium increased by 100% and that of Lactobacillus increased by 400%.
In contrast, Clostridium decreased by 80% and Bayonela decreased by 60%. Twelve months after taking the difference widened. In the sick, as shown in Table 12, six months after taking Bifidobacterium was 50%.
%, Lactobacillus increased by 200%, Clostridium decreased by 60%, and Bayonela decreased by 75%. Twelve months after taking the difference widened.

[0051]

[Table 11]

[0052]

[Table 12]

Examination of the intestinal flora of normal and diseased subjects before taking them showed that healthy subjects had an average of 220% more good bacteria and only 22.5% had bad bacteria as compared to the weak. Did not. This data shows that the current state of the intestinal flora is an indicator of current health status, that is, how the intestinal flora is deeply involved in health. In fact, all the healthy volunteers improved their health, and those with disabilities recovered their health over time.

Further, as a new finding, by taking this drug, healthy persons take a pattern in which the number of good bacteria increases first, and as the number of good bacteria increases, the number of bad bacteria decreases gradually and forever. It was also found that the sick people take a pattern in which bad bacteria started to decrease first, and then good bacteria gradually increased. A questionnaire survey was conducted on all 40 people three months after the administration, and the following responses were obtained. a) I have no more colds than before. In addition, even if a cold is caught, the process is light. b) Injuries are healed quickly. c) The bowel movement abnormalities (diarrhea, constipation) were greatly improved. d) The atopy is improved. In addition, it did not come out. e) My family did not suffer from food poisoning. f) The skin became shiny and the makeup paste became better. The spots and wrinkles became thin. g) My blood color has improved and I have more confidence in my health.

Test Example 1, Test Example 2 and Test Example 3
The fact indicates that the lactic acid bacteria preparation of the present invention can greatly contribute to the prevention and treatment of infectious diseases. In addition, the intestinal environment becomes beautiful by the effect of improving the intestinal flora in the true sense,
As a result, it spreads to the whole body, and the function of the living body is activated, and it is thought that it leads to health promotion, health recovery, and constitution improvement. In addition, an experiment was conducted in which a commercially available lactic acid bacterium was administered, but the number of good bacteria increased and the number of bad bacteria decreased as compared to before administration, but the rate of change was slightly smaller in each case. Was. According to the results of the survey,
Most people did not feel that they were too healthy. The first reason may be due to the production of attenuated antibiotics.

(Test Example 4) A test group was prepared by lyophilizing the antibiotic-sensitive FERM BP-6972 manufactured by the method of Example 2 to 2 × 10 ⁷ cells for 2 months every day using a sonde. The mice were orally administered to mice (ICR system, manufactured by CLEA Japan), feces were collected once a week, and various intestinal bacteria per 1 g of feces were quantified. As a control group of mice, a standard strain of Lactobacillus casei (ATCC
393) and a group without bacteria were set. In the test group, as compared to the non-administered group and the ATCC strain group, the numbers of bacteria of Bacterium bacterium and Lactobacillus increased over time, and a decrease in natural course was suppressed. Conversely, with respect to Clostridium and Bayonella, the number of bacteria decreased in the test group. Around 9 days after the start of the administration, a slight difference in the number of bacteria was already observed, and a remarkable difference was observed two months after the start of the administration.

From the above results, the friend leukemia virus was infected two months after the administration of live bacteria when the difference in the balance of intestinal bacteria between the mice in the test group and the mice in the control group became remarkable. As a result, a survival curve showing a statistically significant survival prolongation effect was obtained in the test group of females in the group with less viral infection.

In an experiment in which Salmonella was orally infected, a large dose (1 × 10 ⁷ cells / mouse) was used.
In each case, all died, but the test group showed prolonged survival as in the case of inoculation with the friend leukemia virus. In the case of the low dose group (2 × 10 ⁶ mice / mouse), all mice eventually died in the control group, but the mortality was only 20% in the test.

(Test Example 5) 300 female white leghorn females, 10 months old, were divided into three groups, A, B, and C. A group was fed with a standard feed to which antibiotics were added, and B group was fed with the standard feed described above. The lyophilized bacterial preparation produced in the same manner as in Example 1 was
A mixture of 10 ⁷ / g of feed was given. In group C, 1 × 10 ⁷ freeze-dried bacterial preparations / g of feed prepared in the same manner as in Example 1 were added to a feed without antibiotics, fed and fed for 3 months. Table 13 shows the results. During the breeding period, no chickens died from the infection. In group A, some chickens showed diarrhea and were unhealthy, but none in groups B and C.
The number of eggs laid was in the order of group C> group B> group A, and the weight per egg was also in this order, and this result is considered to reflect the health condition of the chicken.

[0060]

[Table 13]

(Test Example 6) A test was conducted using four small fish net cages, each of which cultivates about 1,000 juvenile hamachi larvae having an average fish weight of about 90 g. A mixture of a commercially available mixed feed for yellowtail and minced sardine at a ratio of 2: 1 was used as a feed. The control group was given only the mixture, the test group 1 was supplemented with a tetracycline antibiotic (10 mg / kg of the diet), and the test group 2 was reduced to one-half the amount of the tetracycline antibiotic in Example 2. 1 × 10 ⁸ cells / g of feed were added to the feed depending on the culture solution. Next, in the three test sections, 1 × 1 of the culture solution of Example 2 was used.
0 ⁸ / bait g only were added, respectively 2 months were fed. The average water temperature during the test period was 22-24 ° C. Table 13 shows the fish weight and growth rate, as well as disease incidence and mortality. When the culture solution of Example 2 was added to the feed, the growth was fast and the number of mortality was small as compared with the normal culture method (feed + antibiotics). In other words, it became clear that the amount of the antibiotic to be added can be reduced by half, and it is sufficient to add the lactic acid bacteria preparation of the present invention to the feed without adding the antibiotic.

[0062]

[Table 14]

In the piglet breeding experiment, results similar to those in the above test example, that is, the group administered with the lactic acid bacterium preparation of the present invention showed the same growth and morbidity as the group administered with the antibiotics. Was.

(Test Example 7) 40 g of chemical fertilizer (N: 8%,
P: 5%, K: 5%) and 10 g of soluble phosphorus fertilizer. On top of that, humus was mixed with the soil of the field at a ratio of 20%, and 10 L of the soil was mixed with 8 g of formic lime to a height of 8 minutes. At the beginning of November, 10 plant seedlings of strawberry of the reddish varieties were planted in each planter, and liquid fertilizer was applied weekly for one month from two weeks later. At the same time, a freeze-dried bacterial preparation produced in the same manner as in Example 2 was dissolved in water (1 × 10 ⁸ cells / ml) in three planters in the first test section.
The whole seedling was sprayed by spraying. Planter 3 in Test 2
The culture solution of Example 2 was diluted 300 times with water and sprayed. During this time, only water was sprayed on three planters in the control plot. When the buds came out in March of the following year,
Divided into three groups, Group A again sprayed the liquid fertilizer and the lactic acid bacteria preparation every week. Group B sprayed spores of mildew in addition to this. After spraying the spores of the mildew disease, the C group diluted the prescribed amount of the antifungal agent (Sumirex, manufactured by Sumitomo Chemical Co., Ltd.) in the first week and sprayed it evenly by spraying. Sprayed. For 1-2 months after flowering, the fruits were harvested sequentially from red ripe fruits. Table 15 shows the results of Group A. As is clear from Table 15, Test 1 in which the lactic acid bacteria preparation of the present invention was sprayed
Strawberries in the plot and the test 2 plot did not cause any disease and grew quickly, and 220-240 g of high quality first grade strawberries could be harvested per strain. On the other hand, the strawberry in the control plot was 2
Strawberries, which had 0% of downy mildew and escaped, also had so-called normal grades. In the case of the group B to which the spores of the causative fungus of the gray mold disease were sprayed, the strawberry in the control group was almost completely eliminated, and the strawberry that could be eaten could not be harvested. On the other hand, in the test 1 section, 10% of strawberries were ill and could not be harvested, and in the test 2 section, only 6%. In addition, the harvested strawberries were the first-class products which were equal to those of the first and second test groups of Group A. In Group C, the incidence of illness was less than 2% in all plots, with the help of pesticides. In addition, both plots seemed to be slightly inferior in both yield and quality as compared to the group A. From the results of this test, by using the lactic acid bacterium preparation of the present invention, it was possible to prevent the occurrence of disease with a considerable probability without using pesticides, and also to obtain a high yield of high-quality strawberries. I knew I could get it. Furthermore, a test of spraying the lactic acid bacterium preparation of the present invention on a strawberry suffering from a disease was also conducted, and the therapeutic effect was confirmed.However, from the onset of the disease, the earlier the disease was cured, the better it was. No effect was observed.

[0065]

[Table 15]

In addition to the above strawberries, vegetables such as komatsuna, spinach and tomato, fruit trees such as grape and mandarin, mushrooms such as mushrooms, shiitake mushrooms, houseplants such as pothos, flowers such as chrysanthemums and chamomile For herbs such as, a comparative test of their quality and disease incidence was conducted, but all showed superior results compared to the control group.

Next, for humans, animals and plants,
By subjecting the lactic acid bacteria preparation of the present invention to a test, comparing the results obtained and the conventionally known antibiotics from various angles,
Table 16 shows the concept table. The table clearly shows how excellent the lactic acid bacteria preparation of the present invention is.

[0068]

[Table 16]

[0069]

The invention of penicillin and subsequent antibiotics has eliminated many of the infectious diseases that have been afflicted since human birth. Because penicillin's effects on infections are so dramatic that it has magically cured many illnesses that had previously died, medicine may now cure all illnesses. I came to think about it. This overconfidence and illusion sparked the "myth of medicine," and the era of drug flooding and abuse came. As a result, the medicinal effect is a form of contradictory drug evil,
It produced various side effects and caused many victims. Side effects are usually an individual problem, but resistant bacteria born as side effects are not limited to individual problems, but are released to the environment, increase offspring, and become part of the shared system of the entire environment. Become. The danger is not limited to local and national dwellers, but has spread globally, affecting people all over the world. This is especially fatal for an elderly society whose immunity declines with age. It is clear in the course of the fight against pathogens to date that the only way to minimize damage in the future is to remove the horns and tusks of the demon and pick up the sticks to help them. From the viewpoint of "eye for eyes" and "teeth for teeth", as a natural enemy, there is no bacterium as good as the lactic acid bacterium of the present invention. "Attenuated toxicity antibiotics" produced by the lactic acid bacteria
This is because, unlike conventional antibiotics, it is different from mere growth inhibition by directly acting on the toxin-producing ability of the pathogenic bacterium, and has the effect of reducing or eliminating its toxicity.

Since ancient times, human beings have been tremendously benefited by microorganisms, especially lactic acid bacteria, both inside and outside the living body.
The history of the research is French Pasteur (1845-1895)
Inspired by the Russian physiologist Mechinnikov's theory of longevity, many studies and clinical applications have been made since then. However, it did not gain true practicality. This was because the epidemiological findings did not match what the experiment revealed. Recently, it has been elucidated that lactic acid bacteria play a number of important roles, such as activation of immune functions and intestinal purification, as biological regulation functions due to progress in intestinal bacteriology.
Here, the existence of lactic acid bacteria having a new function became clear, ushering in a new era of utilizing lactic acid bacteria.

The lactic acid bacterium of the present invention becomes a leader of the intestinal microflora due to its characteristics, builds up the intestine in which good intestinal bacteria are overwhelmingly majority, and strongly supports the vital activity of living organisms together with these good bacteria. It also has an effect. Therefore, administration of the lactic acid bacteria preparation of the present invention reaffirms the importance of preventive medicine. It also changes the treatment method for infectious diseases. Thus, the abuse of antibiotics will be eliminated and the dosage will be minimal. There is no doubt that the lactic acid bacteria preparation of the present invention will be the gospel in the coming aging society. In addition, the emergence of this drug has taken the first step toward realizing an ideal society in which all bacteria that live can coexist and co-prosper, as opposed to pathogenic bacteria, rather than hostile bacteria. It will be recognized for a long time.

[Brief description of the drawings]

Fig. 1 Growth test of strawberry using planter (a) Group A: spraying liquid fertilizer and lactic acid bacteria preparation every week (b) Group B: spraying liquid fertilizer and lactic acid bacteria preparation together with gray mold spores every week (c) Group C: Spraying antifungal agent on the first and second week after spraying gray spores

[Explanation of symbols]

 1. Test 1 zone 2. Test 2 section 3. Control plot 4. 4. Sprayed gray mold spores Spray inhibitor

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A23K 1/16 304 A23K 1/16 304B A61K 35/74 A61K 35/74 A A61P 31/04 A61P 31/04 171 171 // (C12N 1/20 (C12N 1/20 A C12R 1: 245) C12R 1: 245) F term (reference) 2B022 AA01 AB11 AB17 AB20 2B150 AA03 AA05 AA08 AB03 AC06 DD12 DD26 4B065 AA30X AC10 AC14 CA43 CA44 CA47 4C087 AA01 AA02 BC57 CA09 MA01 NA14 ZB35 ZC61 4H011 AA01 AA02 AA03 AA04 BA01 BB21 BC07 BC08 BC22 BC23 DA02 DA13 DC08 DD03 DE15

Claims (15)

[Claims] 1. A Lactobacillus casei, which produces an antibacterial substance having a growth inhibitory action and a toxicity attenuating action on microorganisms. 2. A Lactobacillus casei, which has an action of reducing the hemolytic property of a pathogenic bacterium when brought into contact with the pathogenic bacterium. 3. A Lactobacillus casei bacterium having an action of promoting the mutation when brought into contact with a pathogenic bacterium which spontaneously exhibits an SR mutation. 4. The Lactobacillus casei according to claim 1, wherein the Lactobacillus casei has resistance to known antibiotics. 5. The known antibiotic according to claim 4, wherein the known antibiotic is β-
Lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics,
Lactobacillus casei, which is at least one or more of a quinolone antibiotic and a glycopeptide antibiotic. 6. Lactobacillus casei according to any one of claims 1 to 5, wherein the Lactobacillus casei is FERM BP-6972,
FERM BP-6971 and FERMBP-697
3. Lactobacillus casei, which is at least one kind or two or more kinds of 3. 7. A lactic acid bacterium preparation comprising, as a main component, Lactobacillus casei which produces an antibiotic having a growth inhibitory action and a toxicity attenuating action on microorganisms. 8. A lactic acid bacterium preparation comprising, as a main component, Lactobacillus casei having an action of reducing the hemolytic property of a pathogenic bacterium when brought into contact with the pathogenic bacterium showing hemolytic properties. 9. A lactic acid bacterium preparation comprising, as a main component, Lactobacillus casei having an action of accelerating the mutation when brought into contact with a pathogenic bacterium which spontaneously exhibits an SR mutation. 10. A lactic acid bacterium preparation, wherein the Lactobacillus casei according to any one of claims 7 to 9 is mainly composed of Lactobacillus casei having resistance to known antibiotics. . 11. The known antibiotic according to claim 10, wherein
Lactobacillus casei, which is at least one or more of β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolone antibiotics and glycopeptide antibiotics, as a main component A lactic acid bacterium preparation characterized in that: 12. Lactobacillus casei according to any one of claims 7 to 11 is FERM BP-697.
2, FERM BP-6971 and FERM BP-
69. A lactic acid bacterium preparation comprising as a main component at least one or two or more types of Lactobacillus casei of 6973. 13. A lactobacillus preparation comprising Lactobacillus casei as a main component according to any one of claims 7 to 12, characterized in that it is a health agent for humans, animals or plants and a preventive agent for infectious diseases. Or a therapeutic agent for infectious diseases. 14. A health agent for humans, animals or plants, comprising a lactic acid bacterium preparation containing Lactobacillus casei as a main component according to any one of claims 7 to 12 and a known antibiotic. Agent for preventing or treating infectious diseases. 15. The known antibiotic according to claim 14, wherein
β-lactam antibiotics, aminoglucoside antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolone antibiotics and glycopeptide antibiotics are characterized by at least one or more, Health agents, preventive agents for infectious diseases or therapeutic agents for infectious diseases of humans, animals or plants.