Antimicrobial Activity Against MRSA
By Iichiroh Ohhira

Lactic acid bacteria, a physiologically related group of gram-positive bacteria, produce a variety of compounds with antimicrobial activity, and they are termed bactcriocin. Bacteriocin is generally defined as extracellularly released peptide or protein that shows a bacteriocidal activity against species closely related to the bacteriocin producing strain.' It has now become evident, however, that many bacteriocins from lactic acid bacteria show somewhat broader spectrum of activity, affecting also more distantly related species. In fact, bacteriocin producing lactic acid bacteria appears to interfere with the growth of the food born pathogen Listerta monocytogenes during fermentation process. 35 Researchers have long discussed the potential application of bactcriocins in prevention and even in treatment of infectious diseases.

Bioassay-directed screening for anticrobial activity against methicillin resistant Staphylococcus aureus (MRSA) led us to the culture broth of Enterococcusfaecalis TH 10, which was isolated from a Malaysian fermentation food, Tempeh. The anti MRSA component was readily extracted with ethyl acetate from the culture broth at pH 3, and the extract retained the activity when incubated overnight with various proteolytic enzymes. In addition, the extract showed poor antimicrobial activity against lactic acid bacteria. including Enterococcus faecalis. These properties strongly suggest that the active compound is distinct from bacteriocin, an extracellularly released species closely related to the bacteriocin producing strain. Efiaecalis has been reported to produce bacteriocin that has strong hemolytic activity on mammalian erythrocytes, but the active component of the TH 10 strain did not mediate the lysis of rabbit and human erfthrocytes.

Lactic acid bacteria, a physiologically related group of gram-positive bacteria, produce a variety of compounds with antimicrobial activity, and they are termed bactcriocin. Bacteriocin is generally defined as extracellularly released peptide or protein that shows a bacteriocidal activity against species closely related to the bacteriocin producing strain.' It has now become evident, however, that many bacteriocins from lactic acid bacteria show somewhat broader spectrum of activity, affecting also more distantly related species. In fact, bacteriocin producing lactic acid bacteria appears to interfere with the growth of the food born pathogen Listerta monocytogenes during fermentation process. 35 Researchers have long discussed the potential application of bactcriocins in prevention and even in treatment of infectious diseases.

In recent years, there has been a dramatic increase in the incidence of hospital associated (nosocomial) infections caused by Staphylococcus aureus strains that are resistant to multiple antibiotics, and these strains are collectively termed methicillin resistant S_ aureus (MRSA). The incidence of MRSA outbreak is particularly high in Japan where the cases are almost four times that reported in Europe. 6 Although the threat to patient care posed by such organisms has stimulated continuing efforts to search for potent anti MRSA agents, there has been a growing concern that the pharmaceutical industry may no longer be able to develop novel antibiotics sufficiently quickly.

To exploit the potential of antimicrobial metabolite of lactic acid bacteria for the control and chemotherapy of MRSA, we carried out bioassay directly screening of microorganisms insolated from fermented foods and beverages. Several strains were found to produce anti crobial activity in their culture broth, which inhibited the in vitro growth of MRSA on agar medium. Enterococcusfaecalis TH 10, an isolate from a Malaysian fermentation food, Tempeh, was especially effective among the lactic acid bacteria isolated More interestingly, the active component was readily extracted with ethyl acetate, and it retained the activity when treated with proteohlic enzymes.

To see whether the anti MRSA compound falls on the category of bacteriocin, we investigated various properties of the product of Efiaecalis TH 10 strain: proteolytic treatment using various proteases, and antimicrobial activity against closely related lactic acid bacteria. Hemolytic activity was also assayed for the comparison with previously reported bacteriocin/hemolysin produced by Efiaecalis.

Materials and Methods

1. Materials. Reagents were purchased from the following suppliers: proteinase K, V8 protease, achromopeptidase were from Wako Pure Chemical Industries, Ltd. and trypsin was from Signia Chemical Company. Other chemicals used were analytical grade reagents. Methicillin resistant Staphylococcus aureus typeII strain was used in this study.

2. Culture of Enterococcus faecalis TH 10. A stock culture of Efiaecalis TH 10 was inoculated on 10 nd of the seed medium containing 1.5% polypepton, 0.5% yeast extract, 0.25% NaCl, 1.0% glucose, 0.05% sodium thioglycolate, 0.025 % Lcystine, 0.01% sodium suffitc, 0.01% sodium carbonate (pH 6.6 before sterilization). After incubation at 37°C for 24 hr., the broth was transferred to one liter of the same medium, and cultivation was carried out at 37°C for 8 days.

3. Extraction of Active Component. The fermentation broth was centrifuged at 4000 xg for 20 min at 4°C to remove cells, and the supernatant solution was extracted with ethyl acetate at pH 3.0. The solvent layer was dried over anhydrous NA2SO4, and concentrated under reduced pressure. The residue was dissolved in 2 ml of 2 N NaOH solution, neutralized to pH 7, and the 60 p I portion was loaded on a paper disk. After the paper disk was dried, bioassay was performed by placing the disk on an agar plate and seeded with suspension of test organism. After incubation at 300C for 24 h, a zone of growth inhibition around the disk was presumed to indicate the presence of active compound.

4. Proteolysis Treatment. The ethyl acetate extract (10 mg in dry weight) was treated overnight at 37°C with 0. 1 mg of proteinase K, V8 proteasc, trypsin, and achromopeptidase, respectively in 100 y L of 20 mM potassiurn phosphate buffer at pH 7.5, loaded on paper disks and dried for the bioassay. The amount of the proteolytic enzymes used corresponds to 2.2 units for proteinase K, 1, 100 units for trypsin, 2.2 units for V8 protease, and 100 units for achromopeptidase.

5. Hemolpis Assay. Hemolysis assay was performed with rabbit and human erythrocytes as described for hemolysin/bacteriocin of Efiaecalis.

Results and Discusssion

1. Properties of anti-MRSA component in the culture broth of E.faecalis TH 10. Enterococcusfaecalis TH 10, an isolate from a Malaysian fermentation food Tempeh, was especially effective among the lactic acid bacteria tested. The active component was readily extracted with ethyl acetate from the culture broth of the TH 10 strain at pH 3. Ile extract showed potent growth inhibition against MRSA, and a moderate activity against methicillin sensitive S.aureus (Table). Since the extract contains various compounds in it, the result does not prove that an identical substance inhibited MRSA and methicillin sensitive S.aureus. It is rather more important to show inhibitory activity against the lactic acid bacteria tested except for two species, Streptococcus salivarius and Pediococcus addilactid. Enterococcus species were not inhibited by the extract, either. This antimicrobial spectrum indicates that the anti MRSA component appears to be distinct from bacteriocin molecules that are usually inhibitory against closely related lactic acid bacteria.

Proteolytic enzymes are frequently used for conventional diagnosis for bacteriocin production; proteolytic enzymes catalyze hydrolysis of protein or peptide like components, and cause dramatic decrease in the bacteriocidal activity. We found that the ethyl acetate extract retained the anti MRSA activity after the treatment with V8 protease, proteinase Y, trypsin, and achromopeptidase. These proteolytic enzymes alone do not affect the growth of MRSA. The results suggest that the active component is not a peptide or a protein, but it rather appears to be an acidic low molecular weight substance, which is extracted with ethyl acetate at pH 3.

Thus, the active component produced in the culture broth of Efiaecalis TH 10 strain appears to differ from bacteriocins, a protein or peptide line antimicrobial metabolite with potent activity against species closely related to the bacteriocin producing strain.

2. Hemolysis Assay.In previous papers, E.faecafis has been isolated from natural food samples such as traditional French cheese~, mozzarella cheese, and foods in a retail supermarket. The species has also been recognized as human pathogens with multiple antibiotic resistance and high level aminoglycoside resistant". These clinical isolates of Efiaecalis are reported to produce bactcriocin that mediates the lysis of a broad range of gram positive bacteria , and the molecule also acts as a hemolysin that effectively lyses human, rabbit and horse erythrocytes'. The anti MRSA component of the T14 10 strain, however, failed to mediate the lysis of human and rabbit erythrocytes, indicating that the anti MRSA compound is distinct from ever reported bacteriocins produced by E.faecafis.

Our present study shows that E.faecalis produces a potent anti-microbial product against MRSA, whose identity appears to be a low molecular weight substance. Racemic lactate did not show antimicrobial activity against MRSA even when 30mg of the sodium salt was loaded on the paper disk, so the commonly distributed metabolite of lactic acid bacteria can be ruled out as the candidate for the active substance. Further study is in progress to purify and characterize the active substance using silica gel column, activated charcoal, and anion-exchange resin, and the results will be reported soon. In the curren situation where the discovery of novel antimicrobial agents is becoming increasingly difficult, the present study suggests that food-born lactic acid bacteria may offer some potential applicability in chemotherapy and control of MRSA

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Bacteriocidal and Antimicrobial Activity Against MRSA
By Iichiroh Ohhira 1996

In recent years, there has been a dramatic increase in the incidence of hospital-associated (nosocomial) infections caused by Staphylococcus aureus strains that are resistant to multiple antibiotics; some strains now demonstrate resistance to as many as 20 antimicrobial compounds, including antiseptics and disinfectants.

These strains are collectively termed methicillin resistant S. aureus (MRSA). The incidence of MRSA out-break varies throughout the world , but it is particularly high in Japan where the cases are almost four times that reported in Europe . Although the threat to patient care posed by such organisms has stimulated continuing efforts to search for potent anti-MRSA agents , there has been a growing concern that the pharmaceutical industry may no longer be able to develop novel antibiotics sufficiently quickly. It is also pointed out, however, that most of the antimicrobial compounds, to which MRSA shows intransigent resistance, has been produced by soil bacteria such as Streptomyres and Bacillus genera and fungi, Penicillium and Cephalosporium, thus novel anti-MRSA agents may be discovered from other microorganisms that have not been exammed so intensively.

Lactic acid bacteria, a physiologically related group of Gram positive bacteria, produce a variety of compounds with antimicrobial activity. Some of these are proteins or pcptides, and they are termed bactcriocin. Bacteriocin is generally defined as cxtracellularly released peptide or protein that shows a bacteriocidal activity against those closely related to the producer species. However, it has now become evident that many bacteriocins from lactic acid bacteria have somewhat broader spectrum of activity, affecting also more distantly related species. in fact, bacteriocin-producing lactic acid bacteria appear to interfere with the growth of the food-borne pathogen Listeria monocytogenes during fermentation process. Researchers have long discussed the potential application of bacteriocins in prevention and even in treatment of various infectious diseases.

To exploit the potential of bacteriocins for the control and chemotherapy of MRSA, bioassay directed screening of lactic acid bacteria was earned out. Several strains were found to produce antimicrobial activity in their culture broths, which inhibited the in vitro growth of MRSA on agar medium.

Enterococcusfaecalis THIO, an isolate from a Malaysian fermentation food, tempeh, was especially among the lactic acid bacteria tested. More interestingly, the active component was readily extracted by ethyl acetate at pH 3. The present communication describes the cultivation of E. faecalis THIO, extraction of anti-MRSA component, and its inhibitory spectruni to various lactic acid bacteria.

A stock culture of E. faecalis THIO was inoculated in 10 nil of the seed medium containing 1.5% polypeptone, 0.5% yeast extract 0.25% NaCl, 1.01/6 glucose, 0.05% sodium thioglycolate, 0.025% L-cystine, 0.01% sodiurn carbonate (pH 6.6 before sterilization.) After incubation at 37 degrees centigrade for 24 hours, the broth was tansferred to one liter of the same medium and cultivation was carried out at 37 degrees centigrade for eight days. The fermentation broth was centrifuged to remove cells, and the supernatant solution was extracted with ethyl acetate of pH3. The solvent layer was dried over anhydrous Na2SO4. and concentrated under reduced pressure. The residue was dissolved with 2 in of 2 N NaOH solution, and neutralized to pH 7. Bioassay was performed by placing paper disks saturated with the solution on a nutrient agar plate and seeded with suspension of test organism. After incubation at 310 degrees centigrade for 24 hours, a zone of growth inhibition around the disk was presumed to indicate the presence of active compound. The minimum inhibitory dosage was determined by the dilution method with 2-fold dilution.

Bacteriocidal activity of the ethyl acetate extract was tested using MRSA and a variety of lactic acid bacteria as test organisms. Contradictory to the common properties of bacteriocins, the extract did not show potent activity against closely related lactic acid bacteria. The growth inhibition was potent against MRSA, while most of the lactic acid bacteria tested were not affected. Streptococcus salivarius and Pediococcus acidilactici were affected, but only when the dosage was twice the amount required for the inhibition of MRSA. In addition, we found that the active component retained the bacteriocidal activity against MRSA when it was treated with various proteases such as protcinase K, V8 protcase, trypsin and achromopeptidase.

The extract of minimum inhibitory dosage was treated at 37 degrees centigrade overnight with 0.1 in of each proteolytic enzyme, and the activity was assayed as described above. The results suggest that the active component is not peptide or a protein. It rather appears to be an acidic low molecular weight substance, since it was extracted with ethyl acetate of pH 3.

E. faecalis has been isolated from natural food samples such as traditional French cheese , mozzarella cheese and foods in a retail supermarket. However, in most of the cases the species has been recognized as human pathogens with multiple antibiotic-resistance and high-level aminoglycoside resistance. Many clinical isolates of Enterococcus faecafts have been reported to produce bacteriocin which mediates the lysis of a broad range of Gram-positive bacteria , and the molecule also acts as a hemolysin which effectively lyses human, rabbit and horse erythrocytes. The active component of the THIO strain, however, failed to show hemolytic activity toward human and rabbit erythrocytes.

Further study is in progress to purify and characterize the active substance using silica gel column, activated charcoal and anionexchange resin and the results will be reported soon. In the current situation where the discovery of new antin crobial agents are becoming increasingly difficult, the present study suggests that investigation of lactic acid bacteria may offer some potential applicability to chemotherapy of MRSA.

Acknowledgement from the research team:
This work was supported by a grant from Bioactive Research Institute. The authors are grateful to Dr. Taku Miyarnoto, Faculty of Agriculture, Okayama University, for providing us various lactic acid bacteria.

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Upper Gastrointestinal Tract Conditions as Affected by Probiotics

Antioxidant Property of OM-X Probiotic Capsules. Bioactive components of the human diet, such as antioxidants, contribute a significant "flinction" to several plant-based food products. Antioxidants are very important to human health because they play a vital role in reducing the risk of "radical-related" oxidative damage associated with many clinical conditions and degenerative diseases.

By Iichiroh Ohhira

Antioxidant Property of OM-X Probiotic Capsules. Bioactive components of the human diet, such as antioxidants, contribute a significant "flinction" to several plant-based food products. Antioxidants are very important to human health because they play a vital role in reducing the risk of "radical-related" oxidative damage associated with many clinical conditions and degenerative diseases.

Current 20th Century research focuses on the potential to influence, by dietary means, the functional level of antioxidants in the body. Western research has emphasized the importance of the established antioxidant micronutrients including vitamins A, C, E and beta-carotene. Evidence tends to argue in favor of the possible protective mechanisms underlying the value of these nutrients in reducing the risk of several important degenerative diseases especially cancer, cardiovascular disease and ocular cataracts.

As has been establish by scientific research conducted by a team of Japanese research scientists, headed by Dr. lichiroh Ohhira (the award-winning microbiologist most celebrated as the inventor of OM-X Probiotic Capsules), consumption of OM-X Probiotic Capsules on a regular basis will enhance the capability of the human body to produce badly needed antioxidants. Antioxidants are particularly important because of the role they play in combating certain human degenerative conditions. Worldwide, only OM-X Probiotic Capsules provide the ingredients isolated by Dr. Ohhira and his team of scientists.

Upper Gastrointestinal Tract Conditions and OM-X. The role of probiotics like OM-X in the prevention and treatment of certain conditions have now become evident from research on the microflora of the upper gastrointestinal tract with special reference to the lactic acid bacteria group (LAB). In fact, many research scientists are now convinced that probiotic products, such as OM-X, can assist in the treatment of certain diseases.

The normal microflora of the mouth, stomach and duodenum are a rich ecosystem of enormous complexity containing a large number of species of bacteria. The esophagus and mouth have similar bacterial populations. In a "fasting" condition, the stomach and duodenum contain very few microorganisms and those that are present are mainly derived from the oral cavity and throat The total population and species in the entire ecosystem show dramatic variations along the gastrointestinal tract with the highest concentration of microorganisms residing in the colon.

During fasting, the gastric juice contains only small numbers of bacteria and yeast ranging from 100 to 100,000 per ml. The predominant bacterial group found in the stomach and duodenum include Streptococci, Lactobacillus sp., Veillonelta sp. and Clostridium perfringens.

After a meal, the bacterial counts in the gastric juice increase 100 to 1000 fold. This dramatic increase in transient bacterial counts will return to baseline levels within one to two hours as a result of a decrease in gastric juice volume and pH, as well as the effects of gastric motility.

A wide variation of bacterial types occurs among individuals, however, the number of species and population of bacteria are relatively stable in healthy adults. Within the upper gastrointestinal tract, the normal established "resident" bacterial microflora may be altered by bacteria introduced into the body as a normal part of food ("transient" microflora) or as contaminants ("accidental" microflora). In the upper gastrointestinal tract, these transient bacteria have a much greater effect on the resident microflora because of the lower numbers of the latter being present.

In spite of its stability, the intestinal microflora can vary enormously in the stomach and duodenum dependent on host factors such as level of gastric acid secretion, bile salts, and mucous in the intestinal wall.

In addition, medications, diet, infections, age, stress and climate can also alter the microflora. The contents of microflora may also be influenced by bacterial interaction such as antagonism or symbiosis. Adaptation of intestinal microflora can occur to most substances that enter the intestines from the oral tract or the biliary system. This adaptation occurs within several days with the ability of intestinal microflora to metabolize these substances. Gastric acid inhibits the growth of microorganisms with the stomach of patients having no gastric acid showing an increased number of bacteria.

Lactic acid bacteria (LAB) including lactobacillus, leuconostoc, lactococcus, pediococcus, and bifidobacterium are found throughout the gastrointestinal tract. The predominant population of lactic acid bacteria in the upper gastrointestinal tract is the lactobacillus species. Lactobacilli may colonize the mucosal surface of the duodenum as well as the stomach. For this to occur, lactobacilli must possess certain properties including adhesion, competitive exclusion ability and bacterial inhibitor production.

Only OM-X Probiotic Capsules provide live lactic acid bacteria that is "cultured" by Dr. Ohhira to be 6.25 times stronger than any naturally occurring lactic acid bacteria. Consequently, the "bad" bacteria in the upper gastrointestinal tract is destroyed or "inhibited" by the presence of the "good" bacteria contained in OM-X. Also, the special strains of OM-X's lactic acid bacteria, specifically TH 10, which is isolated from the Malaysian delicacy tempeh, are capable of inhibiting all harmful pathogenic bacteria living in the human "gut" including the MRSA (methicillin-resistant Staphylococcus aureus).

Clearly, OM-X Probiotic Capsules, as developed by Dr. Ohhira, are the world's finest probiotic products. Only OM-X recognizes the challenges associated with maintaining and strengthening the microflora contained in the human "gut" so that the resident "good" bacteria make up at least 85 percent of the bacteria living in the "gut" and not more than 15 percent of "bad" bacteria reside in the human body.

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The Safety of Capsules Containing Lactic Acid Bacilli

Products containing lactic acid bacteria have long been available in the market. They may be in the form of food such as yogurt (thairoo, dadih) or they may be marketed in pharmaceutical formulations such as capsules and liquids. In the last decade or so, with increasing interest in natural foods, the number of such products has increased.

By Iichiroh Ohhira

The Safety of Capsules Containing OM-X Lactic Acid Bacilli

Abstract

The population in Malaysia uses various types of health and food supplements. These products are considered safe and are used without and any concern for their toxicity. Among the products used as health supplements are ptducts that contain latic acid bacteria. This project studed the acute and subacute toxicity of a product containing minerals, herbs, vitamins and live lactic acid bacteria, on Sprague-Dawley rats. Acute toxicity was tested 24 hours after a single dose and subacute toxicity was studied 24 hours after 7 days of daily dosing. The parameters that were studied were alanine aminotrasferase (Alt, SGPT), aspartate aminotrasferase (ASTm SGOT), serum uream ratios of weight of kidney and liver weight to body weight and percentages in body weights. The contents of capsules of the product (6,8 or 10 capsules for acute studies, the leavel of ALT in the rats treated with the contents of capule was lower than controls. There were no significant changes in the other parameters of the rats in the treatment groups as compared to controls. There were no significant differences in all the parametrs between rats in the treatment groups as compared to controls in the subacute studies. Sprague-Dawley rats fed with high doses of the product did not show signs of toxicity in the parameters that were studied.

Products containing lactic acid bacteria have long been available in the market. They may be in the form of food such as yogurt (thairoo, dadih) or they may be marketed in pharmaceutical formulations such as capsules and liquids. In the last decade or so, with increasing interest in natural foods, the number of such products has increased.

Although the use of such products either as food or as health supplements is quite widespread, there is not much information available regarding the presence or absence of toxicity of such preparations. Some may be tempted to say "but, these are natural products!" however; it must be borne in mind that some of the most powerful toxins known to man are natural substances. Therefore, natural does not equal safe. In this study we looked at the acute and sub-acute toxicity of OM-X capsules, a product containing live lactic acid bacilli, minerals, heibs and vitamins.

Materials and Methods

OMX Malaysia Sdn Bhd supplied OM-X capsules.

Male Sprague Dawley rats weighing about 220-250g and aged between 53 to 58 weeks were purchased from the Institute of Medical Research and housed in the pharmacology laboratory of the Pharmacy Department, Faculty of Allied Health Sciences, UKM. The ALT, AST and serum urea assays were carried out using the Reflotron dry chemistry analyzer and the corresponding test strips purchased from Roche Diagnostics Sdn Bhd. Blood was collected using heparinised capillary tubes.

The contents of the OM-X capsules were suspended in corn oil and the animals were fed using a syringe and a feeding needle. In the acute toxicity studies, the animals were fed one dose of the product and then they were sacrificed after 24 hours. There were 4 groups of animals; they were the controls that were fed just corn oil, the treatment group that were fed the contents of 6 capsules, 8 capsules or 10 capsules. Each group had 5 rats.

In the sub-acute toxicity studies, the animals were fed the product once a day for 7 days and then sacrificed on the 8th day. In these studies also, there were 4 groups of rats, the control group that was fed corn oil, the treatment groups that were fed the contents of 6 capsules, 10 capsules or 12 capsules.

The animals were sacrificed by cervical dislocation 24 hours after the last dose was fed. The viscera were opened and the liver and kidneys removed rinsed, blotted dry and weighed. Blood was collected in heparinised tubes and a drop placed on the test strips and the readings obtained from the Reflotron dry chemistry analyzer.

"The parameters that were looked at were body weight, kidney weight, liver weight, ALT, AST and serum urea. The animals were also observed for any visible physical changes.

Results

In the acute toxicity studies, there was a significant decrease as compared to controls in the ALT levels of the rats fed with OM-X capsules. However, no dose-related effects were observed. There was no significant differences in ALT levels between rats in the control group and treatment groups in the sub-acute studies. No differences were observed in ALT levels between rats in the control group and treatment groups in the sub-acute studies.

No differences were observed in AST levels of rats between any of the groups in both the acute as well as sub-acute toxicity studies. No differences were observed in serum urea levels of rats between any of the groups in both the acute as well as the sub-acute toxicity studies. The ratio of weight of kidney and liver to body weight was not different between all groups of rats in both the acute as well as the sub-acute toxicity studies.

In the acute toxicity studies, there was a trend towards a decrease in body weight in the treatment group rats as compared to the rats in the control group. In the sub-acute studies, however, the trend was towards an increase in body weight of the rats in the treatment groups as compared to the control rats, after 7 days of treatment. However, these differences were not statistically significant.

There were some changes in the fur of the animals' fed the higher doses of the product. Diarrhea was seen in the rats fed with corn oil (control) after the first day of feeding.

Discussion

The sale of health supplements in Malaysia was until recently on the increase. Consumers with extra cash to spend did not mind spending a little extra on health supplements "hoping" that they would help to keep them healthy. Another reason for the popularity of health supplements was that they were considered as "natural" and therefore safer than the synthetic drugs and vitamins. This opinion stems from a belief that all things chemical or synthetic are toxic and harmful. Such generalization is not safe because some of the most toxic substances are "natural", such as botulinum toxin and aflatoxin.

Products containing lactic acid bacteria have been available for a long time. Initially they were home produced products, but more recently they have been made into pharmaceutical dosage forms such as capsules. One such product is the OM-X capsule that contains a mixture of minerals, vitamins) and herbs and live lactic acid bacteria.

The Ministry of Health approves the product as a health supplement. Among the effects that have been reported for the product include action against MRSA, restoration of gut flora, treatment of hypercholesterolemia, hepatic encephalopathy and gut infections. There have been anecdotal reports of patients, in certain instances, taking more than a few capsules a day. Being a "natural" product, the possible toxicity of the product is often not considered especially since the ingredients have been part of our native foods. However, large numbers of live bacteria in a capsule can have different effects from the amounts found in foods and so it is important to study the toxicity of these products.

Our results show that there is no significant toxicity of OM-X capsules in rats, even at the extremely high doses that were used. While an increase in the levels of the hepatic enzymes ALT and AST are indicators of liver damage, a reduction in the levels of activity of these enzymes do not necessarily mean a beneficial effect on the liver. The absence of any effect on serum urea levels indicates that there may not be any toxic effect on the kidney. These results taken together with an absence of changes seen in the ratio of kidney and liver weight to body weight as compared to controls indicates an absence of toxicity in the kidney and liver.

The rats in the treatment groups as compared to the control rats showed a reduction in body weight in the acute toxicity studies, while they increased in body weight in the sub-acute toxicity studies. This trend may indicate that in the first day of feeding with the contents of the OM-X capsules the rats may not have eaten as well as the control rats, but, with repeated feeding an adaptation was made, such that after 7 days they were eating as well as the controls.

The results of this study show that, for the parameters that were studied, products containing live lactic acid bacteria, such as OM-X capsules, do not produce toxicity in Sprague-Dawley rats.

References

Aim, L 1982. Effect of Fermentation on Lactose, Glucose and Galactose Content in Milk and Suitability of Fermented Milk Products for Lactose

Intolerant Individuals. J. Dairy Sci., 65: 346-352

Gillilan). S. E., Nelson, C. R. & Maxwell. C. 1985. Assimilation ofCholesterol by Lactobacillus Acidophilus. Appl. Environ. Microbiol., 49: 377-381.

Gotz, V., Romankiemcz. J. A.. Moss, J. &Mwwy, H. W. 1979. Prophylaxis against Ampicillin-Associated Diairhea with a Lactobacillus Preparation. AmerJ. Hasp. Pharm., 36: 754.

Macbeth, W. A. A. G? Kass, E. H. &McDennolt. W. V. Jr. 1965. Treatment of Hepatic Encephalopathy by Alteration of Intestinal Flora with Lactobacillus acidophilus. Lancit., i: 399-403

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Properties of Lactic Acid Bacteria From Fermented Foods

One hundred eighty-nine strains of lactic acid bacteria were isolated from the following 16 traditional fermented foods in Southeast Asia: alcohol drinks, side-dish foods and seasoning foods.

By Iichiroh Ohhira P.h.D. 1999

Summary

One hundred eighty-nine strains of lactic acid bacteria were isolated from the following 16 traditional fermented foods in Southeast Asia: (1) alcohol drinks (2 samples), (2) side-dish foods (5 samples) and (3) seasoning foods (9 samples).

They were physiologically characterized and classified into 4 genera and 15 species. The distribution of dominant lactic acid bacteria identified were investigated in the present study. The results were as follows.

1. Out of the 189 strains from 16 samples, the incidences oiLactobacillus, Enterococcus, Leuconostoc and Pedwcoccus were 36%, 25%, 35% and 4%, respectively.

2. Dominant species were Leuconostoc "mesenteroides subs. mesenteroides (39 strains), which was followed by Lactobacillus plantarum (36 strains), Enterococcus faecium (31 strains) and Enterococcus faecalis (26 strains). These species were detected at high frequencies in seasoning foods and were also found in many samples.

3. It was presumed that the distribution of lactic acid bacteria was due to the constituent ingredients and used for the traditional processing of fermented foods such as plant- and animal-origin foods. Therefore, samples processed by animal-origin ingredients showed higher amounts of lactic acid bacterial counts and species in contrast with plant-origin samples.

From traditional fermented foods in Southeast Asia, 4 genera, 15 species, and 189 strains of lactic acid bacteria were isolated and identified. Forty-six strains of lactic acid bacteria were examined for salt-acid- and heat-tolerances, acid production activity, protein hydrolysis, and aroma-producing activity.

Pedicoccus halophilus and Leuconostoc paramesenteroides could be grown in 10% NcCI added broth medium. Lactobacillus casei subsp. pseudoplantarum, Ped. Halophilus, and Fed. Halophilus, Leuc. mesenteroides and Enterococcus faecalis had been active at 45?C.

Acid production activities ofEnt. bovis, Ent. faecalis, Ent. faecium Leuc. mesenteroides were high and Ent. bovis which showed that highest activity produced 0.42% lactic acid after 24 hours in broth medium.

The strains, which showed high protein hydrolysis, were Enterococcus faecalis No. 10 and Ent. faecalis No. 15 isolated from Tempeh. Ent. faecalis No. 15 also showed diacetyl-acetoin production by creatine test and only this strain had positive activity among all strains on the aroma production.

Introduction

Presently, as many as several thousand fermented foods are said to be in existence and are classified as beverages, fruits and processed foods, such as cereal, dairy food, fish, vegetables, beans, meat, starch-added crops and others'. It may be safely said that the majority of each fermented food, the number of which is estimated to reach several thousand kinds, is the fruit of several thousand-year histories.

Since securing fresh foodstuffs at the dawn of human history was influenced by seasons, the period for hunting and collecting food was limited. Early man desperately collected resources for survival, and storage techniques were devised for the purpose of securing seasonal fresh food for a long period". One of those early-man storage techniques was fermented food, which cleverly utilized a natural phenomenon termed "fermentation". A large number of natural fermented foods were found to have beeri eaten in Mesopotamia, Egypt and China where the World's Big Three civilizations were born.

Among food microorganisms, lactic acid bacteria, which is the particular focus of this research paper, are related to the group of useftil microorganisms, and through fermentation, they individually or in cooperation with other lactic acid bacteria, add various value to food". As well as fungi and yeast, lactic acid bacteria also are deeply related to human history and have made a great contribution to food presentation, and the improvement of the flavor of food.

Therefore, we selected 16 fermented foods, including 2 alcohol drinks, 5 side-dish foods, and 9 sauce foods, from among the traditional fermented foods widely distributed in Southeast Asia. After selection, these fermented foods were isolated, and we then identified the kind of lactic acid bacteria contained in the fermented foods. We then examined the distribution of the lactic acid bacteria and their biochemical properties. The names of the lactic acid bacteria described in this research are described in accordance with Bergey 's Manual of Systematic Bacteriology (Vol. 2).

Producing Process of Samples

The researcher collected samples during the period between July 21 and August 4, 1987 from private houses and domestic factories. The samples were collected throughout all of the states in western Malaysia. The samples were flown to Japan and preserved in the laboratory at 5?C until they were used in experiments, in order to avoid any changes in their quality. Further, it is sufficient to say that each sample mainly followed that of sauce foods for want of space.

1. Alchol Drinks
Coconut wine and rice wine were used as samples. They were obtained in an Indian area in Jahor in western Malaysia on July 24, 1987, and in lpoh, also in western Malaysia on August 1, 1987, respectively.

2. Side-dish foods.
The samples of Dadih, Dosai and Idii, Tape and Tempeh were obtained in George Town (Pinang), Kuala Lumpur (Selangor), a market in Malacca (Melaka) in-western Malaysia, respectively.

Sauce foods.
The following 9 samples, classified as sauce foods, were used: marine fries, Belachan (made from shrimp and salt), Badu, Chinchaluk, limnetic fish, Pekasam (made from salt), Trassi (made from marine fish, soybeans and cereals), Kicap (the principle ingredient of which is salt), Tauco.Tempoyak (made from durian and minute salt), Sambal belachan (made from Balachan and onion pepar). All of the samples were obtained in domestic factories, markets and private houses in Kelantan, Perak and Selangor in western Malaysia.

1. Belachan is a sauce food made by adding suitable amount of salt (5 ~ 20%) to its principal ingredient?shrimp (2 ~ 3 cm) that were caught in a suburban area of Nelaka, Malaka in western Malaysia, where the mouth of the river and coastline intersect each other. The usual ratio between shrimp and salt widely range from 10:1 to 5:1, depending on individual taste. After being caught, the; shrimp are spread on wooden boards to be exposed to the sun and dried for several hours. They remain in the sun until their moisture rate falls to around 50%. Then, salt is added to the shrimp in accordance with the individual's favorite ratio and mixed thoroughly. This mixture is put into wooden barrels or other appropriate containers, and stationary fermentation is employed for approximately I week, at natural room temperature. In some areas, some rice powder is added to the mixture. After being suitably fermented, the paste-like mixture is poured into molds, and thereafter solidified and dried in the shade until finished. For this product, containers such as plastic packets are used for packing. Incidentally, a sample of Belachan produced on July 22, 1987, without rice powder, was used in this research.

2. Badu was obtained in a domestic factory in Kota Bahru, Kelatan in western Malaysia. Its constituent ingredients are marine fries of Stolophorus genera and salt water whose concentration is between 10% and 20%. Although the concentration of salt differs depending on the preferences in each area, the most common ratio between fries and salt water is 1:2. Some factories add other ingredients, including the tamarind and coconut sugar, depending on the wishes of the factory's owner. Next, this mixture is transferred into earthen bottles or round concrete tanks and permitted to ferment in the shade. After fermenting for approximately 6 months, with stirring every few days during the fermentation period, the upper part of the solution becomes dark brown, transparent liquid in its final stage. This liquid is then withdrawn, boiled, filtrated and finally bottled into product. Also, tomato juice may be added to give it more flavor and taste. Incidentally, the sample used in this research was the undiluted solution obtained on July 26, 1987 in a factory just before the finishing process.

3. Chinchaluk was produced in a domestic factory in the suburbs of Malacca, Malaka in western Malaysia and was obtained just before it completed the finishing process on July 22, 1987. The ingredients of Chinchaluk are shrimp (mainly marine shrimp, but limnetic shrimp are used in some areas), rice powder and salt. Initially, the shrimp (the principal ingredient) are washed in tap or natural water and dried in the sun for 2 ~ 3 hours. Thereafter, salt water, whose salt concentration is between 10% and 20%, and suitable amounts of rice powder are added and mixed. The basic ratio of its combination is 5:5:1. Finally, this mixture is poured into bottles or large earthen jars and permitted to ferment for approximately I month at natural room temperature.

4. The Kicap sample was a mixture of unrefined soy prepared in a domestic factory in the suburbs of Malacca, Malaka in western Malaysia, which was obtained during the middle of May 1987. Kicap is produced by washing soybeans (grown in the vicinity of the factory) in pure water and boiled in an iron pot, until soft. After being strained, they are refrigerated at normal temperature for 12- 18 hours. Then, the boiled beans and crushed wheat are mixed in a ratio of 2:1. The mixture is then spread on bamboo blind (sized 100 x 200 x 5 cm) in a dark place. Wild and useful bacteria, yeast and fungi, stuck to the bamboo blind (because of the bamboo's limited use for this special purpose) become moldy after I week ~ 10 days. Thus, the fermentation process proceeds and the mixture softens. It is poured into prepared bottles or round concrete tanks, and salt water is added with a salt content of 10% ~ 20%. After several months ofstational fermentation in the outdoors, the upper part and the liquefied part of the mixture is strained, boiled and bottled. Saccharine and tomato juice may be added to give it more flavor and taste.

5. The Tauco sample was produced and obtained in a domestic factory in the suburbs of Kuala Lumpur, Selangor in western Malaysia during the middle of April 1987. Tauco consists of soybeans (usually produced in the vicinity of the factory), crushed barley and/or wheat, steamed rice and salt water with a 20% salt content. Initially, the soybeans and rice are steamed, and then the crushed barley and/or wheat are added and mixed thoroughly. The mixture is then spread on a special bamboo blind (sized 100 x 200 x 5 cm) in a dark room at normal room temperature. Useful bacteria, which are stuck to the bamboo blind (due to its limited use for this special purpose) becomes moldy after I week ~ 10 days and the steamed soybeans begin to soften. Salt water whose concentration is 20% salt is added to the soften mixture in a ratio of 1:1 or 1:2. The mixture is put into bottles or round concrete tanks and mixed thoroughly. After being thoroughly mixed, the mixture is sealed but regularly stirred. After 3 ~ 6 months of stational fermentation, the product is finished.

Distribution of Lactic Acid Bacteria in Traditional Fermented Foods

Isolation and identification tests for lactic acid bacteria using 16 traditional fermented foods widely ranging in Southeast.

Belachan, with a pH of 8.75 was the highest pH value among the samples collected, and conversely, Dosai, with a pH of 3.62, was the lowest. The pH value of the rest of the samples mostly ranged between pH4 and pH6. Side-dish foods and alcohol drinks, whose salt content are low, showed generally low pH values, whereas the pH values of sauce foods tended to be a little higher when compared to the former. Budu showed the highest salt concentration of 8.66%, whereas Tape showed the lowest value of 0.16%. Some sauce foods are high in salt concentration due to the necessity of preservation. The long-term fermentation of the samples with higher salt concentrations, such as Budu (8.66%), Kicap (5.82%) and Cincaluk (4.65%), is considered to inhibit their production of acid in a short period. However, it is presumed that lactic acid bacteria serve to lower the pH value of the food gradually, which improves the preservation of food along with the synergism of salt.

Incidentally, there is a general trend that the number of lactic acid bacteria distributed in plant-origin samples exceeded animal-origin samples. Furthermore, lactic' acid bacteria in a wide range of samples are detected especially when the sample contains various sorts of both animal and plant ingredients. It is presumed that the reason for this propensity is that lactic acid bacteria, which are peculiaf to the ingredients, seem to have a deep connection with the fermentation process.

It is meaningful for food processing to select strains with outstanding properties by screening for the biochemical properties of lactic acid bacteria from functional qualities of metabolism and cultivation. From this viewpoint, the screening for chloride-resisting lactobacillus and acid-resisting lactobacillus , and moreover, the strains with outstanding functional qualities such as acid productivity"', proteolytic effect, fat resolving effect, fragrance productivity, and antimicrobial component productivity' " have been in progress. Furthermore, in a research into bacteria aimed to increase lactic acid bacteria's range of use, it is essential to screen for lactic acid bacteria with special saccharide fermentativeness or heat-resisting effect in order to search for the ones that are applicable to a wide range of fermented materials. These researches are all vital in the sense that functional properties in lactic acid bacteria improve preservative effect and add flavor and taste.

Geographically, Southeast Asia belongs to the tropical region and various traditional fermented foods?salted in most cases?are utilized there due to the climatic; peculiarity of the region. It is presumed that those lactic acid bacteria with unique, strong fermentation activity exist in natural fermented foods originating in this unique, tropical environment. Therefore, the cultural and metabolic properties of 46 strains of typical lactic acid bacteria isolated from Southeast Asian traditional fermented foods were assayed. With regard to the cultural properties, researches were made into salt-tolerance, acid-tolerance and heat-tolerance. With regard to the metabolic properties, research was conducted into acid-producing effect, proteolytic effect and flavor-producing effect. The various strains of lactic acid bacteria, with outstanding properties, were searched to examine their usefulness.

1. Salt-tolerance, acid tolerance and heat-tolerance isolates. In a salt tolerance test performed in a medium containing 5% sodium chloride, all the strains showed vigorous growth. However, the only strains whose growth was confirmed in mediums containing 10% sodium chloride were Fed. halophilus, the strains identified with Leuc. paramesenteroides and the ones related to Ent. faecalis and Ent. faecium. No strains showed their viability in mediums containing 15% and 20% sodium chloride. Although it is stated in the new edition otBergey", that Fed. halophilus are viable in the medium with 15% sodium chloride, that viability was not confirmed in this test. The reason for this result is presumed to be linked to the decline in the salt-tolerance of Fed. halophilus that was influenced by having been cultured in the medium, Idii, which contains a low percentage of salt.

Based on the findings from the acid-tolerance tests, all sample strains were not viable in a medium at pH3.0. Satisfactory growth of L. casei subsp. pseudoplantarum, Fed. halophilus and Leuc. lactis was observed at pH3.5, and some of the strains related to L plant arum and Ent. faecalis showed their viability at this same pH level. However, no other strains were found to be viable. Leuc. mesenteroides subsp. mesenteroides and Leuc. paramesenteroides were not viable at pH4.0, while some strains related to L. plantarum, Ent. faecalis and Ent. faecium were viable, but some were not viable. The growth was confirmed in all the other strains in the said condition. At pH4.5, all of the strains demonstrated their viability. Judging from the above results, around pH3.0 is estimated to be the limit of acid-tolerance of lactic acid bacteria.

In heat-tolerance tests, all strains excluding Ent. lactis confirmed their viability at 40?C. Fed. halophilus, Leuc. mesenteroides subsp. mesenteroides, Leuc. paramesenteroides, and Ent. faecium were viable in the tests at 45?C. However, a mixture of viable strains and non-viable strains were observed in L. plantarum and the strains identified with Ent. faecalis. Furthermore, no sample strains proved their viability at 50?C or 55?C. Although it is sated in the new edition of Bergy" that some lactic acid bacteria such as L. delbrueckii, L. helveticus, Ent. thermophilus and Fed. acidilactici are viable at 50?C, these strains did not show their viability in this test, and the limit of heat-tolerance was determined to be around 45?C.

2. Acid productivity of isolates. After a 24-hour cultivation of the samples, each strain was determined in mediums with and without some sodium chloride. The acidity having ranges of similar values in non-bacteria mediums, the acid productivity of each sample lactic acid bacteria was estimated from the results of acidity produced. First, the strain that demonstrated the highest acid productivity in skimmed milk medium without sodium chloride was Ent. bovis isolated from Dosai, and its value marked 0.42% after 24 hours. Observing by species, Ent. faecalis showed a tendency of producing the most numerous acid and chaversely, L. plantarum showed a tendency of producing the least. The outstanding acid productivity ofEnt. faecalis had also been mentioned in a report by the group of Sasaki.

The species that demonstrated high acid productivity in skimmed milk mediums with 5% sodium chloride were Leuc. mesenteroides subsp. mesenteroides, Leuc. paramesenteroides and Ent. faecium, whereas the ones that demonstrated low acid productivity were L. plantarum, Ent. lactis and Fed. halophilus.

Among the isolated and identified 189 strains of lactic acid bacteria, the species most often detected was 39 strains of Leuc. mesenteroides subsp. mesenteroides, which was followed by 36 strains of L. plantarum, 31 strains of Ent. faecium and 26 strains of Ent. faecalis. Great numbers of these species were distributed especially in sauce foods. Leuc. mesenteroides subsp. mesenteroides, L plantarum and Ent. faecium were all isolated from sauce foods, and Ent. faecalis was isolated from 4 sauce foods and a side-dish food.

The species of lactic acid bacteria that are distributed in rather various samples were Ent. faecalis, Leuc. mesenteroides subsp. mesenteroides and L. plantarum. These species had been reported to be widely distributed both in the animal and plant kingdoms'", and this research resulted in the same consequence. The following is the comparisons of species between the strains isolated and identified in this research and those already previously researched and reported.

Firstly, with regard to the group of alcohol drinks, the report of L. plantarum and Leuc. mesenteroides from coconut wine had been published previously, but L. casei subsp. casei was an originally isolated species identified in this research. Also, L. coryniformis subsp. coryniformis and Fed. pentosaceus were first isolated from rice wine in this research, whereas the report on the distribution of L. casei and Leuc. mesenteroides from rice wine was published previously.

The species that have been previously isolated from Dadih, one of the side-dish foods, are Ent. lactis subsp. lactis, Ent. faecalis, Ent. lactis subsp. diacetilactis, Ent. thermophilus, and L. acidophilus'. However, the isolation of Leuc. lactis had not been previously reported. With regard to Dosai, L. delbrueckii, Ent. lactis subsp. lactic and Leuc. lactis had been previously identified. However, Ent. bovis and L. coryniformis subsp. coryniformis, which were identified in this research, had not been reported on previously. Although L. mesenteroides, Ent. faecalis and Ped. cerevisiae had been isolated from Idii by another researcher, the isolation ofL. casei subsp. pseudoplantarum and Ped. halophilus had not been previously reported. Few researchers into the lactic acid bacteria in Tape have been made, yet the isolation of Leuconostoc genera and Lactobacillus genera have been reported. However, the identification of the level to species was first performed in this research and the identification of L. casei subsp. pseudoplantarum was reported. Very few researches into the lactic acid bacteria in Tempeh have been made, and with regard to other bacteria, only Rhizopus genera have been researched , but no reports on the isolation ofE. faecalis have been made previously.

With regard to the group of sauce foods, reports had not been made on isolation or identification of lactic acid bacteria from Belachan. Although Pediococcus gensra and Streptococcus genera had been isolated from Bagun and fish sauce, which are similar samples to Belachan, at the level of genus", the isolation of their species had not been performed. As a result of this test, Leuc. mesenteroides subsp. mesenteroides and L. plantarum were isolated. Similarly, only a few reports on the isolation or identification from Budu have been made. Only the report on the isolation otPediocuccus genera and Streptococcus genera from fish sauce, a similar sample to Budu, had been made, as mentioned earlier, Ent. faecalis and L. plantarum were first isolated and identified in this test. Also, reports on the screening ofCinchuluk, a similar sample to Belachan and Budu, had not been found. As a result of this research, Ent. faecalis and Leuc. mesenteroides subsp. mesenteroides have been isolated. Although a few screenings for the microorganisms in Kicap have been performed, researches into Japanese unrefined soy ' , have been in existence. In these reports, L. delbrueckii, a species related to lactic acid bacteria, had been isolated. In consequence of this test, Ent. faecalis and Leuc. paramesenteroides have been isolated and identified.

Researches into Pekasam, or its similar sample, lzushi, have not been made before, but as a result of this research, Ent. faecium and Leuc. mesenteroides subsp. mesenteroides were isolated. There had not been any researches into the microorganisms in Sambal Belachan, but thi test cast the first light on this sample, and as a result, Ent. faecalis, Ent. faecium, Ent. gallinarum, L casei subsp. casei and L. Casei subsp. rhamnosus were isolated and identified. There have not been any reports on the screening for the lactic acid bacteria in Tauco, a sample that bears resemblance to unrefined soy and also to Kicap. Leuc. mesenteroidesi subsp. mesenteroides, and L. coryniformis subsp. coryniformis were isolated as a consequence of this research. Research reports on the microorganisms in Tempoyak have not been made but Ent. faecium, Leuc. mesenteroides subsp. mesenteroides and L. plantarum were isolated and identified because of this test. Although the study in the microorganisms in Torassi had not been done, the distribution of Ent. faecium and Leuc mesenteroides subsp. mesenteroides in the sample became clear after this test.

Biochemical Propertiies of Isolates

From the above results, the amount of acid formed by Ent. bovis, Ent.faecalis, Ent. faecium, Leuc. mesenteroides subsp. mesenteroides and Leuc. paramesenteroides tended to be high when they were cultivated in the mediums without sodium chloride but the amount tended to decrease dramatically when the said species were cultivated in mediums with 5% sodium chloride. All the said species being viable in the presence of 5% sodium chloride, the decline in the amount of formed acid is presumed to have originated in the decline in the acidogenesis activity of bacteria affected by sodium chloride. The species that possess a tendency of steady acidogenesis without being affected by 5% sodium chloride were Ent. faecium, and Leuc. mesenteroides subsp. mesenteroides. On the other hand, the amount of acid formed by Ped. halophilus, which demonstrated its possessing acid tolerance, tended to be relatively low compared to other species. The vegetation of the said species in the skimmed milk mediums is presumed to have had a relation to this result.

3. Proteolytic activity of isolates. The strain that demonstrated the strongest proteolytic activity in a skimmed milk medium with no salt was Ent.faecalis, isolated from Tempeh, and the amount of free tyrosine increase was 1.32mg/5ml. Ent. faecium isolated from Tempeh and Tempoyak followed it. The rest of the strains demonstrated proteolytic activity in a range of 0.01 to 0.2mg/5ml. In skimmed milk mediums with 5% salt, the values of all strains ranged between 0.01 and 0.15/5ml.

The group of Sasaki performed similar tests using lactic acid bacteria isolated from milk such as Ent. faecatis, Ent. lactis, Leuc mesenteroides subsp. mesenteroides, Leuc. mesenteroides subsp. dextranicum, L. acidophilus, L. casei subsp. casei, L. plantarum and Lfermentum. In consequence, the strains that increased free tyrosine by O.I lmg/5ml or above influenced by proteolytic activity were Ent. faecalis, Ent. lactis, Leuc. mesenteroides subsp. mesenteroides, L acidophilus, L. casei subsp. casei and the strains related to L. plantarum. Some strains among Ent. faecalis and Ent. lactis were reported to have increased free tyrosine by 0.2mg/5ml or above. In any case, like the result of the experiments performed by the group of Sasaki, the proteolytic activity of 2 strains of Ent. faecalis isolated from Tempeh was outstanding in this research.

4. Aroma productivity of isolates. Among 189 lactic acid bacteria strains isolated from 16 samples of traditional Southeast Asian fermented foods, creatine tests were performed using 46 selected typical strains. Consequently, the only strain that aroma productivity turned positive was a strain related to Ent. faecalis isolated from Tempeh. There had been reports on the aroma productivity of the said strain by Hammer, the group of Shermann and the group of Saski, and this experiment got similar results. The rest of sample strains were all negative in aroma productivity tests.

The group of Saskai performed similar experiments using 256 strains isolated from non-processed Japanese milk, and they reported that a number of species such as Ent. faecalis, Ent. lactis, Leuc mesenteroides subsp. dextranicum, L. acidophilus, L. casei subsp. casei and L. plantarum, isolated from lactic acid bacteria, were positive in creatine tests. The results in this test using sample species such as Ent. lactic. L. casei subsp. casei and L. plantarum were as mentioned earlier. Also, the group of Sasaki has commented that Lactobacillus genera are the highest in the aroma productivity. 19 strains related to Lactobacillus genera were also examined in this test and all of the results were negative. The difference of mediums, the cultural composition used in the tests and the time taken for cultivation may have affected these results.

Conclusions First, isolation and identification tests for lactic acid bacteria using 2 alcohol drinks, 5 side-dish foods and 9 sauce foods, totaling 16 samples among typical Southeast Asian fermented foods, were performed. Consequently, 4 genera, 15 species, and 189 strains were isolated and identified from the said samples. Also, lactic acid bacteria isolated from all samples were showed to be their principle bacteria, and the structural species distributed in each sample possessed peculiarity.

Next, biochemical property tests using typical 46 isolates from lactic acid bacteria were performed, and in each test in salt-acid and heat-tolerance, Ped halophilus was found to be viable at pH3.5, 45?C and 19% if salt concentration. Ent. bovis, isolated from Dosai, demonstrated the highest acid productivity of all strains. On the other hand, Ent. faecalis, isolated from Tempeh, was found to possess strong proteolytic activity, and one of those strains was also positive in aroma productivity. These results suggested their potential applicability in increasing flavor and taste.

According to the review by Kozaici, Southeast Asian fermented foods are treasuries of unknown microorganisms with the high possibility of fresh discovery that they are ideal targets for searching for useful microorganisms. In conclusion, greater promotion of explications of microorganisms and searches of usefill strains in fermented foods in this area is expected to be carried out.

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Lactic Acid Bacteria Keeping You Healthy Within

Although we cannot see it with the human eye, another world flourishes all around us, populated by billions of bacteria. From our earliest days, we are taught to fear these microorganisms, viewing them as the cause of disease, infection and suffering. A large part of our modem medicine is based on the "germ theory''-the idea that germs cause diseases. And the only way to be cured of diseases is to kill the germs usually with a potent pharmacological "magic bullet".

By Iichiroh Ohhira

"Maintaining a Balance Between the Good and Bad Flora in Our Intestines is Vital For Good Health. Here's How Live Lactic Acid Bacteria Can Help."

Although we cannot see it with the human eye, another world flourishes all around us, populated by billions of bacteria. From our earliest days, we are taught to fear these microorganisms, viewing them as the cause of disease, infection and suffering. A large part of our modem medicine is based on the "germ theory''-the idea that germs cause diseases. And the only way to be cured of diseases is to kill the germs usually with a potent pharmacological "magic bullet"

Manufacturing companies make billions, selling a variety of disinfectants, antiseptic and personal hygiene products all designed to keep "germs" at bay. But is this the best solution? Do we really want to "kill" and rid ourselves of all bacteria?

While it is true that bacteria can cause health problems, there is another side to the story. Now is the time to hear that other side and give the "good" bacteria some long-overdue favorable publicity! The Battle of the Bugs

Inside your gut (especially in the large intestine or colon) rages a never-ending bacterial battle. Hundreds of different types of bacteria fight to establish dominance in the warm, moist environment so conducive to their growth. In fact, the average human large intestine plays host to over 400 different species of bacteria. With numbers that outnumber the cells in your body, gut bacteria serve several important digestive functions. These functions include breaking down fiber or other food residues, producing some of the E vitamins and breaking-down and destroying some toxic chemicals that may have been ingested with your food.

In the "ideal" body consuming the "ideal" diet, the different strains of bacteria compete with each other and hold each other in check, preventing the excessive proliferation of any one strain. In the real world, though, problems do occur. Many different internal and external factors have an impact on your gut bacteria. These include your diet, your mental and emotional state, environmental and food toxins, and frequent use of drugs such as contraceptive pills and antibiotics. Did you know that if you live in a fairly constant state of anger and frustration, the walls of your intestinal tract secrete a different type of mucus, which encourages the proliferation of a less desirable breed of bacteria?

The Good Guys and the Bad Guys

What happens when the less desirable types of bacteria multiply out of control? Well, huge colonies of "bad guys" usually mean ever decreasing colonies of "good guys" and that can spell problems for you, including intestinal irritation, cramps and spasm, excessive flatulence, chronic diarrhea, diarrhea alternating with constipation and a seriously compromised immune system. As the friendly strains of bacteria diminish, it also leaves the way open for fungus and yeast living in the gut to propagate. Severe yeast overgrowth can cause a variety of unpleasant symptoms that we call the Candida Syndrome. Yeast overgrowth in the gut can be responsible for chronic exhaustion, skin problems, multiple allergies, chronic vaginal thrush, digestive and bowel problems, PMS and menstrual irregularities, to name only a few.

So, what can you do to encourage strong, healthy, friendly bacteria colonies? The answer is plenty! One of the traditional ways to achieve this has been the regular consumption of fermented foods. In China, Japan, India and Eastern Europe, fermented products have been dietary staples for centuries.

The fermentation process allows certain bacteria from the air and local surroundings to grow in the food and subsequently, in the gut of those people who consume it. This is good!

Yeast Syndrome

Vaginal thrush, a condition of excessive yeast in the vagina, is an ongoing and irritating problem for many women, especially resulting from pregnancy and the use of contraceptive pills. High estrogen levels in pregnant women or women using contraceptive pills, alter the acidity of the vaginal mucus, making it more hospitable for the proliferation of yeast organisms. Often these yeast organisms are originally transferred from bowel movements. Many women suffering from vaginal thrush find that taking friendly live lactic acid bacteria to change the type of gut bacteria greatly decreases the frequency of vaginal thrust attacks. Research has verified that OM-X Probiotic Capsules contain the type of live lactic add bacteria beneficial to women suffering from vaginal thrush.

Whenever you take antibiotics, you will benefit greatly from also using friendly live lactic acid bacteria during the course of the antibiotic medication and for a couple of weeks afterwards. Antibiotics tend to kill off the "good" as well as the "bad" gut bacteria, which often results in diarrhea and, in women, vaginal thrush infections. Using friendly live lactic acid bacteria, such as those contained in OM-X Probiotic Capsules, re-establishes the friendly gut bacteria and prevents these common antibiotic side effects.

Acidophilus for Kids

Infants and children can also benefit from the regular intake of live lactic acid bacteria, especially those suffering from digestive disorders, infant colic, loose stools, excessive flatulence or constipation.

Babies and children have strains of friendly bacteria in their guts that differ from those in an adult They also need friendly bacteria strains that can only be found in balanced live lactic acid bacteria supplements such as OM-X Probiotic Capsules.

So now, you see that bacteria can work for you as well as against you! In many, many instances, bacteria are your friends!

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Inhibitory Effects of Lactic Acid Bacteria On Some Pathogens

Lactic acid bacteria are referred to as probiotics in scientific literature by Lilley and Stillwell (1965). Paricer (1974) redefined it as organisms and substances that contribute to the intestinal microbial balance. The latest and most accurate description of probiotics, like lactic acid bacteria based food products, was presented by Fuller (1989) as a live microbial feed supplement beneficial to the host by improving the microbial balance within its body. This balance is achieved by its inhibitory effect upon the harmful pathogenic microorganisms.

By Iichiroh Ohhira

Abstract

Lactic acid bacteria are referred to as probiotics in scientific literature by Lilley and Stillwell (1965). Paricer (1974) redefined it as organisms and substances that contribute to the intestinal microbial balance. The latest and most accurate description of probiotics, like lactic acid bacteria based food products, was presented by Fuller (1989) as a live microbial feed supplement beneficial to the host by improving the microbial balance within its body. This balance is achieved by its inhibitory effect upon the harmful pathogenic microorganisms.

This paper presents some background on the scientific research and development of one such encapsulated product, which has become a commercial reality. Branded under the label Ohhira Mountain fruits eXtract (OM-X) it is fermented by 12 strains of specially isolated and cultured lactic acid bacteria from the genera Lactobacillius, Bifidobacteria, Streptoccus and Enteroccus. The substrate consists of mountain fruits, plants, herbs and mountain spring water and the fermentation process is done under natural conditions. The final capsule has a total count of 59 million colony forming units (cfu) of lactic acid bacteria. The high level of organic acids in the final fermented product further contributes to its antimicrobial property.

One of the strains used in the OM-X capsule is Enteroccus faecalis TH 10 isolated from the local food delicacy tempeh. This strain has shown inhibition on methicillin resistant Staphylococcus aureus (MRSA). The active inhibitory component was extracted with ethyl actetate at pH3 and did not show growth inhibition against the various lactic acid bacteria. Similar results were also obtained on E. coil 0157.

All strains of the lactic acid bacteria placed in the food supplement were found to be active against Helicobacter pylori. While more work is in progress, these findings establish the effectiveness of the product as a functional food with significant antimicrobial properties.

Introduction

Lactic acid bacteria have shown to play an important physiological role in the human gastrointestinal (Gl) tract Microbial interactions represent the main force in the homeostasis of the bacteria flora in the Gl tract. Combined with the host this microflora form a unique ecosystem in which the complex interactions can either be synergistic or to antagonistic depending on their strain identify, population levels and metabolic activity. The healthy survival of the host is determined by these interactions. The ecosystem is destabilized as a result of gastrointestinal disorders and other endogenous components such as enzymes, hormones and immunoglobulins can influence it.

The importance of maintaining a beneficial microbial balance in the Gl tract is vital to the stability of its ecosystem and the optimal health of the host.

The colon is also known to contain up to 3.5 pounds of bacteria of which some 700 types can give rise to ahmentary toxemia, which only 20 types are harmless. Toxemia results from harmfill bacteria.

OM-X probiotics contain specially isolated lactic acid bacteria that can inhibit the activity of several pathogenic bacteria and thereby overcome this toxemia. Ultimately this can bring about a favorable balance of beneficial or synergistic bacteria over the harmfill ones. This paper examines one such product that has been developed through a period of intensive research and development. OM-X can serve as an effective functional food or health supplement to bring about this favorable coionic microbial balance.

Branded under the label OM-X (Ohhira Mountain fruits eXtract) this paper presents its scientific rationale for it's antimicrobial efficacy and how it consequently benefits the host.

Methods of Production

The lactic acid bacteria are mass-produced for encapsulation by a natural fermentation process. The substrate used consists of mountain fruits, plants, herbs and pure mountain spring water. Specially isolated and cultured strains of lactic acid bacteria are used. No other additives are used.

The inoculum consists of four major groups comprising six strains of Lactobacillus, three strains of Bifidobacteria, one strain of Streptococcus and two strains of Enterococcus.

The substrate is allowed to be fermented naturally with the inoculum for five years after which the end product in paste form is extracted for live encapsulation. At the end of the fermentation process the final product has a high level of organic acids produced by the 12 stains in the inoculum.

The shelf life of the bacteria in the capsule has been monitored and found to be viable for a period of five years.

An analysis of the metabolites in the final product has shown its efficacy by way of its functional ingredients as a functional food. The 18 types ofamino acids, five minerals and nine vitamins were determined by qualitative analysis.

Biosafety

For quality control the final product is subjected to qualitative analysis and quantitative estimation for the presence of any heavy metals. Arsenic, lead, copper, tin and mercury were tested.

The levels of these parameters were far below the accepted or permitted limit of international standards (Food Regulation Act, 1985). For instance the lead level is 0.07 ppm compared to the allowed standard of 2ppm. Copper present is 0.53 ppm compared to the standard of 300 ppm. It is also reassuring to note that mercury, cadmium and antimony are not detected.

This ensured the biosafety of the fermented end product.

Antimicrobial Properties of OM-X Lactic Acid Bacteria

The most significant finding is the inhibitory activity of these specially isolated strains of lactic acid bacteria against three pathogenic bacteria - methicillin resistant Staphylococcus aureus (MRSA), Helicobacter pylori and Escherichia coli 0157.

MRSA is a challenge to medical practitioners as it is involved in nosocomial infections worldwide. During an outbreak it has been shown to play havoc in hospitals. Antibiotics lead to only partial control during a widespread infection. The emergence of vancomycin resistant Staphylococcus aureus is also of great concern.

One of the 12 strains used in the capsule - Enterococcus faecalis THIO - demonstrated inhibition on the MRSA.

This strain is used in the fermentation process and thrives well within the capsule. Originally isolated from the local Malaysian delicacy - tempeh - the strain was cultured and used as the inoculum for the production of the capsules. The strain was tested for its antagonism against MRSA. For qualitative test, a paper disc bioassay technique was used. Zones of inhibition were noted in comparison to the control.

Further investigations revealed that the active component of TH 10 extracted in ethyl acetate at pH3 retained its activity after treatment with various proteases. The bacteriocidal activity of the ethyl acetate extract was tested using MRSA and a variety of related lactic acid bacteria strains as the test organisms (Table 4). Contradictory to the common properties ofbacteriocins, the extract