Antioxidant' Effects on Gastrointestianl Tract

Antioxidant' Effects on Gastrointestianl Tract

Antioxidant Property of Probiotic Capsules contain bioactive components of the human diet, such as antioxidants, which contribute a significant "function" to several plant-based food products. 

By Koshy Phillip, Ph.D. 1998

Bioactive components of the human diet, such as antioxidants, contribute a significant "funiction" 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, an award-winning microbiologist, consumption of Dr. Ohhira's OM-X 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 Dr. Ohhira's OM-X provide the ingredients isolated by Dr. Ohhira and his team of research scientists.

The role of probiotics like Dr. Ohhira's 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. In fact, many research scientists are now convinced that probiotic products, such as Dr. Ohhira's 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., Veillonella sp. and Clostridiumperfringens.

After a meal, the bacterial counts in the gastric juice increase 100 to 1000 folds. 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 alter the microflora. The content 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 thestomach of patients having no gastric acid exhibiting an increased number of bacteria.

Lactic acid bacteria 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 Dr. Ohhira's OM-X 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 Dr. Ohhira's OM-X. Also, the special strains of Dr. Ohhira's 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 (methicillinresistant Staphylococcus aureus).

Clearly, Dr. Ohhira's OM-X, as developed by Dr. Ohhira, are the world's finest probiotic product. Only Dr. Ohhira's 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.

Development of Lactic Acid as a Health Food Supplement or Probiotic

Development of Lactic Acid as a Health Food Supplement or Probiotic

Medical practitioners have come to recognize that a disturbance of the microbiological flora in patients can lead to sickness and a daily supplement of the friendly lactobacilli bacteria can overcome such unhealthy conditions. 

By Iichiroh Ohhira

The term "probiotic" was first used in scientific literature by Lilley and Stillwell (1965) to refer to substances secreted by one microorganism which stimulated the growth of another in contrast to how antibiotics perform.

However, it took on a different terminology when Sperti (1971) used the term "probiotic" to describe tissue extracts that stimulated microbial growth. 

Paricer (1974) defined it as organisms and substances that contribute to the intestinal microbial balance. The most recent and accurate description of probiotics was undertaken by Fuller (1989) who redefined it as "a live microbial feed supplement beneficial to the host (man or animal by improving the microbial balance within its body. "

Medical practitioners have come to recognize that a disturbance of the microbiological flora in patients can lead to sickness and a daily supplement of the friendly lactobacilli bacteria can overcome such unhealthy conditions.

In addition, the gastrointestinal tract contains food in different stages of digestion, digestive ferments, liquids and solid waste. Within the gut are also wide ranges of microbes that may be either harmfill or beneficial. The beneficial ones assist in the breakdown of food while they also manufacture vitamins essential to the body.

Under both healthy and sick conditions, several different types of bacteria compete or fight with each other to establish dominance in the warm and moist environment of the alimentary canal that serves as an ecosystem for their survival and propagation. The average human large intestine harbors over 400 different special of bacteria with a total population far outnumbering even the number of human cells in the body.

The "friendly" gut bacteria serve several functions?breakdown of fiber and other food residues, production of B vitamins and at times decompose toxic chemicals ingested with food to harmless end products.

Under ideal conditions of health and diet, the different strains of bacteria or microflora compete and check the excessive number of any one strain. And a healthy condition can be achieved if a balance is maintained between the "good" and "bad" bacteria in the ratio of 85 percent to 15 percent.

But this condition or ratio can alter and even reverse due to several external factors such as diet, adverse environmental conditions, physiological stress and over-use of certain drugs such as antibiotics and contraceptive pills leading to a sick condition. A healthy condition can be ensured if the level of good or friendly bacteria is maintained at a level of 85 percent.

This may be achieved in several ways including a healthy lifestyle. Since time immemorial, ancient civilizations have documented that man has been known to consume fermented foods such as yogurt, curds, fermented soybeans, buttermilk and fermented cabbage or sauerkraut

The fermentation process has permitted certain bacteria from the air to grow in these foods and subsequently in the gut of those consuming it. The most prevalent microorganisms in most yogurts?Lactobacillus bulgaricus or Lactobacillus acidophilus?is also found in the human gut apart from other friendly ones such as Lactobacillus casei and Bifidobacteria. But such foods with its lower level of useful lactic acid bacteria have not been able to entirely counteract the unhealthy body conditions contributed by a modem lifestyle and extreme environmental conditions. Hence, modem biotechnology has made it possible to innovate novel concentrated forms of microbial food supplements such as OM-X capsules each of which contains as many as 59 million live lactic acid bacteria.

As a superior biotechnology product, its inventor Dr. lichiroh Ohhira succeeded to concentrate 12 different strains of live lactic acid bacteria from four groups namely Lactobacillus, Streptococcus, Enterococcus and Bifidobacterium within a single OM-X capsule.

The scientifically tested and proven innovation has produced wide-ranging beneficial health effects on those consuming the capsules.

Research and epidemiological studies to show that lactic acid bacteria inhibited many different ailments further support the rationale for these supplements. Fermented dairy products or starter cultures used in the dairy industry have shown to inhibit chemically induced colon tumors in laboratory rodents.

Research on both humans and animals also suggest that the effect of diet was mediated by altering metabolic activity of the intestinal bacteria (Goldin and Gorbach, 1976; 1984). Oral L. acidophilus supplements to the diet of rats also lowered the level ofcarcinogenic amines excreted in feces after feeding procarcinogen precursors to these animals.

Further oral supplement of diet with viable L. acidophilus of human origin, which is bile resistant, led to a significant decline of three different fecal bacterial enzymes (Goldin and Goibach, 1977; 1984). This decrease in the fecal bacterial enzyme activity observed in both humans and rats included beta glucuronidase, azoreductase and nitroreductase. All these enzymes catalyze the conversion of procarcinogens to proximal carcinogens in the large bowel leading to colon cancer.

Similarly, the effects of lactic acid bacteria on elevated plasma cholesterol have also been documented. Although there are currently a number of drugs available to lower plasma cholesterol, it would be preferable to achieve the reduction through non-pharmacological agents such as probiotics.

Several studies also show that lactic acid bacteria inhibit the in vitro multiplication of Candida albicans that can cause many health problems. Under certain circumstances, some lactic acid producing bacteria form detectable amounts of harmful bacteria especially pathogenic Gram-negative types. Hydrogen peroxide may also be involved in the activation of the lactoperoxidase-thiocyanate system in the gut. In this system, lactoperoxidase combines with hydrogen peroxide and then oxidizes thiocyanate to an intermediary oxidation product, which can inhibit bacterial growth and may be bacterioddal at a low pH. 

Most recently, it has also been suggested that beneficial bacteria can stimulate the immune system thereby increasing resistance to diseases.

In Vitro Efficacy of OM-X Capsules Against Helicobacter Pylori

In Vitro Efficacy of OM-X Capsules Against Helicobacter Pylori 

Report on Preliminary Work on OM-X Capsules 

By John Lambert 1998

Introduction: Helicobacter pylori play an important role in peptic ulcer disease and gastric carcinoma. Infection occurs v~vidwkk (50 percent to 80 percent in some areas). About 35 percent of the adult Australian population are infected and 15 peTccrt havc clinically significant symptoms. Current therapy for the treatment of H. pylori infection is about 80 percent effective, e.xpcnsi?.v and associated with side effects. New treatments that are safe, cost effective, reduce and/or replace the use of allopathic drug(s). a%vid the development of bacterial resistance and are simple to administer are urgently required for the treatment of H. pylori infection.

The Gastrointestinal Research Group at Monash University (Monash Medical Centre and Frankston Mornington Perunsula Hospital) had shown that probiotic micro-organisms (Lactic Acid bacteria or LAB) possess anti-microbial activity against H. pvlori in invitro (Ref 1,2&3). Our initial in vitro study prompted us to determine the fate of H. pylori in the presence of ingredients of OM-X capsules.

From the literature provided on the OM-X capsule, it should be noted that this capsule is a concentrate of wild fruits, herbs. scauted and 12 strains of Lactic Acid Bacteria. Each capsule contains 18 arnino acids, 9 vitamins, 5 minerals and 59 million live lactic acid bacteria from four groups namely Lactobacillus, Streptococcus, Enterococcus and Bifidobacterium from various fermented foods. Its inventor, Dr. Iichiroh Ohhira from Japan, has shown that Enterococcus faecalis TH10 has bactercidal activity against Methicillin-Resistant Staphylococcus aureus. The active component is not a peptide or a protein.

The preliminary work was to investigate the first objective of our project proposal submitted by OMX Marketing Australia Pty. Ltd. (Dated July 19, 1996).

Objectives:The broad objective is to establish the feasibility of OM-X probiotic capsules use as adjunct therapy for the treatment of H. pylori infection.

The first phase of the Specific objective was to - determine the spectrum of antimicrobial activity of OM-X capsules against H. Plori in vitro.

Materials and Supplies Used in the Research

• OM-X capsules (supplied by OMX Marketing Australia Pty. Ltd.). 
• CM-2B media (as recommened by OMX company). 
• All-purpose medium with Tween (Difco) (APT Agar and APT broth). 
• APT Agar and APT broth with 0.3% Biotin (Sigma) 
• De Man, Rogosa and Sharpe Agar and broth (Oxoid) (MRS agar and MRS broth). 
• Rogosa Agar (Oxoid) 
• Selective media for Helicobacter - Colombia agar plate supplemented with 5% defribinated horse blood containg the following antibiotics" Cufsulodin 5mgl^-1, Trimethoprim 5mb^-1, Vancomycin 10mg^-1, and Amphotericin B %mgl^-1 (H. pylori selective supplement, Oxoid)(HPA plate). 
• Wilkins-Chalren agar (Oxoid) supplemented with 5% horse blood (WCA). 
• Wilkins-Chalren agar (Oxoid) broth supplement 5% Horse blood and 0.5% cyclodextrin (WCACB).
• Brain heart infusion broth (Oxoid (BHI broth). 
• Camp Gas Pak (Oxoid). 
• Two type strains (NCTC 11637 and 11638) and two clinical isolates of H. pylori.

A. Estimation of Viable Count of OM-X Capsule: 
One OM-X capsule was cut with a streile blade. The content of one capsule was dissolved in 9 ml of the following media: APT broth, APT broth with biotin, MRS broth, MRS broth with biotin and in a Sterile distilled water for 6 to 18 hours and incubated microaerophilically (Camp Fas Pak) on a shaker at 35 degrees centigrade. THe viability count was made on the respective plater (for example, culture from MRS broth was spotted on MRS Agar plate) by serial dilution in duplicate. THe plates were incubated for two days at 35 dgrees centigrade under microaerophilic condiion. Total number of CFU.ML of sample was calculated by standard technique.

B. Inhibitory Activity of OM-X Against H. Pylori Bactericidal activity of OM-X was determined by the loss of viability of H. pylori, the well diffusion assay and Spot on lawn assays.

The Well Diffusion Assay:One OM-X capsule was dissolved and incubated as described above. The assay was performed on a WCA media swabbed over with a four different H. pylori strains (@10^t cfu/ml). A well was cut using a sterile metal borer and was filled with 100ul-dissolved OM-X capsule product from different broth. APT broth, APT broth with biotin, MRS broth, MRS broth with biotin and 3% lactic acid was used as a control. Plates were incubated at 35 degrees centrigrade for four days under microaerophilic conditions. The size of the inhibitory zone was measured with Vernier calipers.

The Spot on Lawn Assay:This assay was performed in a similar way as described above except that the 20ul of dissolved product from the OM-X capsule was spotted on a lawn of H. pylori culture onto SCA media.

Viability Assay was performed in two ways:

(A) A one Mcfarland (@10^7 cfu/ml) suspension of the spiral shape and motile H. oykir straub was made in 9 ml of SCACB and BHI broth. The product of one OM-X capsule was added into the respective broth media and incubated microaerophillically on a shaker at 35 degrees centigrade for 24 hours. The viability count was made by serial dulution in sterile distilled waer in duplicate and plating on HPA plae to determine the number of viable cells. Plates were incubated as described above. Other biochemical test including urease, oxidase, and gram staining were also performed.

(B) The OM-X capsule was dissolved first in a 9 ml of SCACB or BHI broth for 18 hours and then spiral shape and motile H. pylori strain (@10^7 cfu/ml) was added. After incubation estimation of viability, count was done as described above.

WCACB and BHI broth with H. pulori strain alone (@10^7 cfu/ml) was used as a control. All test were repeated three times in duplicate. 

ph and L-Lactic Acid Testing: THe ph was measured using and insertion pH meter and L-Lactic acid was measured on a DuPont Dimension discrete auto-analyzed (Dupont, Wilmington, U.S.A.). 

Results:

A. Total Viable Count from OM-X Capsule 
Several media were used to determine the total viable number of organisms present in and OM-X capsule. It was found that an OM-X capsule dissolved for 18 hours in APT with/or without biotin gives good recovery of the organisms from the OM-X capsule. The results are the mean of duplicate tests. THe colonies recovered from the OM-X capsule dissolved in water and plated on CM-2B media were very tiny compared to APT or MRS media.

B. Inhibitory Activity of OM-X capsule Against H. pylori: 
Well Diffusion Assay: The average inhibitory zone of four different strains of H. pylori strains tested. The capsule was dissolved in various media either for 6 hours or for 18 hours. However, there was no difference in the size of the Inhibitory zone from test samples dissolved for two different time intervals. APT with/or without biotin, MRS with/or without biotin (without OM-X capsule) didn't show any inhibition against H. pylori strains.

Spot on lawn assay: Did not show any inhibition of H. pylori.

A.When one McFarland of H. pylori strain and product of OM?X capsule was incubated together for one day there was only one log cycle decrease in number of H. pylori cells counted from the BPA plate. From the HPA plate, biochemical tests - rapid urease oxidase and Gram staining - were performed to confirm the presence of H. pylori. The rapid Urease test directly from the broth media (SCACB and BHI) was also positive and Gram staining showed gram negative spiral bacilli along with gram positive bacilli and cocci from the capsule. The results were similar for all the stains tested both in SCACB and BHI.

B. When an OM?X capsule dissolved 18 hours prior to addition of H. pylori cells and count was made after further incubation, there was 3?4 log cycle decreased in viable cells on HPA plate. The rapid Urease test directly from the broth media was negative and gram staining showed very few spiral gram-negative bacilli along with gram-positive bacilli and cocci. The WCACB and BHI broth contained H. pylori strain (without OM-X capsule) alone grew well after incubation.

Influences of Lactic Acid Bacteria on Bone Structure

Influences of Lactic Acid Bacteria on Bone Structure

In order to excercise heavy training, one has to develope one's body strong enough to endure heavy training. At the same time, heavy excercises lead to lack of protein in the body and affect protein syntheses and production of blood. 

By Iichiroh Ohhira 1998

The Influences of human taking Lactic Acid Bacteria (OM-X)

1. INTRODUCTION

Athletic kinetic anemia seen in a great number of athletes suggests many health problems on athletes (3, 22, 29). Athletic kinetic anemia is thought to be resulted from a variety of chemical and physical causes, and among those, many cases are iron deficiency anemia seemingly resulted from lack of iron(13, 22, 29). Most of athletes tend to force themselves in heavy training to enforce their sports ability. As a result, they suffer many biologically physical changes (10, 13, 15, 22, 29).

In order to excercise heavy training, one has to develope one's body strong enough to endure heavy training. At the same time, heavy excercises lead to lack of protein in the body and affect protein syntheses and production of blood. Therefore athelets have to consider the nutrition, source of energy for sports, in their dietis(8, 10, 13, 21, 22, 27, 30, 31). Training curriculums should be suited to individual physical features and designed to improve physical function effectively. In training for track and field in long distance runners, there are basically endurance training, interval & repetition training, build?up training and others.

In any way of training, the aims of training by repeting are to enlarge V02 (the volume of taking oxygen) and improve the ability of enduring carbon dioxide at contraction of the muscles (4?16, 23, 25, 26, 33, 34). However, there is a possibility training depending on its combination will be a great burden to the body when overloaded to the athletes. Then, in order to execute trainings effectively, athletes make body conditioning which is not to overload ones' weight to their lower body, reducing %Fat (percent body fat) by changing ones' weight^, 18?20). The "body conditioning" aims to make athlete's body suited to sports by improving and controling one's dietary habit. This leads thier diet unbalanced to low calory diet mainly with low protein (8, 10, 13, 15, 22, 24, 27, 29). Atheletes' low calory diet would, not only decrease thier energy when playing, but also effect tissue cell metabolism by delaying protein syntheses in the body which is a basic life metabolism and affect in many ways to body construction which is the base of life preservation (8, 10, 13, 22, 30).

This affect makes great changes in function of blood corpuscles production which has an important role in life preservation (1,22). These vicious circles can result in an increase of kinetic anemia (8, 22, 30). Moreover low calory diet irregulates nutritious absorbption to the body because of unbalanced diet and, at the same time, affects on metabolic hindrance of, for exapmie, storange of calcium in the bones (22, 30). This tendency is seen a lot in long distance or marathon runners in track and field, especially in increasing numbers for female long distance runners (8, 22, 30). Then, how do they manage their energy supply and nutrition to maintain their physical strength durable to trainings?

The situation nowadays is to take supplementary medicine for nutrition that is lacking for energy or biometabolism (3, 8, 13, 22, 30). However, nutrition supply with supplementary medicine can be done temporarily but not develope fundamentaly that leads direct to development of bio -function. Because mechanism of basic life metabolism that our human beings obtain is something held originally in structure of biometabolism. Therefore, even chemical medicines for lacked nutrition are supplied, it would not result in improving metabolic function fundamentally (22, 29?32).

Then, we focused on lactic acid bacteria which are believed to activate (28) tissue cells with great amount of amino acid and others. Lactic acid bacteria are one of the bacteria fundamentally existed in our body (30,31), and they have a role to promote biometabolism more effectively. However, people of modem days are under the condition that the activity of many enterobacterial living in our body is inhibited or depressed in relation to food taken leading to the situation that enterobacterial cannot exhibit their role (30, 32). In such situation of society changes, many kinds of lactic acid bacterial products are sold under the catchphrase that the bacteria are taken alive helping activate tissue cells. However, many of them taken from the mouth would die (22, 32) about 80% to 90% before being absorbed to the body in the stomoch by strong acid there.

We focused on the vegetable fermented food with its proteolytic power is 6.25 times stronger than those bacteria of same genus and species which our cooperate researcher, Ohhira discovered in Malaysia in 1976(6). In 1979, Ohhira succeeded in encapsuling the vegetable fermented food in result of searching an appropriate way to have the above food absorbed alive to the body with many experiments. That is the lactic acid bacteria product "OM-X" shown at Fig.1. Encapsuled OM-X are absorbed to the small intestines within 12 to 15 minutes when one has an empty stomach.

That is the reason why OM-X reach to the small instestines and get absorbed to the body without being killed by strong stomach acid apart from other lactic acid bacteria products. Also, OM-X is a totalized lactic acid bacteria product not only with a great amount of arnino acid and lactic acid bacteria, but also with iron, phosphorus, mineral as potassium, and a variety of vitamins (7). These vegetable fermented bacteria are considered to be taken in the body, absobed in the small intestines, activate with enterobacterial, and exhibit its benefit in tissue cell activity. Especially, in production of red corpuscles, those contained in OM-X, iron, potassium, mineral as phosphorus, a variety of vitamins and amino acid are believed to activate totally in the body and affect changes in blood production promoting its action.

Also, sodium and potassium are substances that affect muscle contraction metabolism and affect muscle contraction at playing sports (1, 2, 8, 17). Ingestion of OM-X is thought to activate physical kinetic functions athletes fundamentally possess toward physical changes caused from today's diet and therefore improve their sports ability.

This time we experimented in aim of investigating on V02max (the maximum amount of oxygen ingestion) after the ingestion of OM-X for male university track and field athletes specializing in long distance running.

II. Methods

1. Subjects 
Subjects are 6 male long distance runners belonging to a track and field team at K University. Their ages are 18.9+0.8. Their performance history of track and field is three to six years since they started competition. The reason is that this test is to set on a wide range of athletes differ in their competitive ability and experience.

2. Procedures 
In our experiment we compared two occasions, before and after of OM'X ingestion as experimental data taken before and after its ingestion.

We had subjects take lactic acid bacteria product OM-X three capsules (1.5g) a day at one has an empty stomach before sleep at night. The reason why we had them take at their stomach empty is, in case we had them take at when something is being digested in the stomach, we worried a range of live bacteria in OM-X which are live to be killed (28) by strong acid of the stomach. Moreover the reason why we appointed their OM-X ingestion before sleep at night is that we thoght that absorption rate(3, 17, 22, 32) before one's sleep at night is the highest in relation to metabolism cycle of hormone metabolism absorption.

Hb (hemoglobin in blood) was measured in blood collected through earlobes before the test with FUJI DRI-CHEM EDC5500 (product of FUJI MEDICAL SYSTEMS CO,.LTD.)

V02max and ECG (electrocardiogram) were measured with a metabolic nutrition monitor of Morgan Medical, Ltd. (formally P.K.Morgan, Ltd.), the United Kingdoms (Benchmark exercise test system) and a kinetic load system of GE Marquett Medical System, U.S.A. (CASE 15). The way to collect breath gas was with breath by breath. Kinetic load was executed by protocol developed from Buruce protocol by the writer, Kawakami. Kinetic strength was tested with gradual loading way that increases the strength in every three minutes. We stated the VO2max of the subjects at the amount when we had them run all-out.

III. Results 

1. Before Taking OM-X 
The body's reaction on the occasion of Max (maximum kinetic load) before the ingestion of OM-X. HR (heart rate) at Max is about 2.91 times greater than that of 62.3 ?9.1beats/min. at rest. V02 was about 5 times greater than that of 542?179.4ml/mii. VCO2 (carbon dioxide production) was about 8.09 times greater than that of 434?110.4ml/iiin. at rest. Ventilation amount was about 6.62 times greater than that of 15.03 ?3.67L/min. at rest. Q (cardiac output) was about 3.03 times greater than that of 6.7?1.48L/mia. LA (lactic acid in blood) showed about 8.12 times greater than that of 1.13?0.26mmol/L. VO2max before ingestion was 47.85?7.36ml/kg.min. VO2max per weight varied greatly because of the difference of subject's sports ability. Table 3 shows the body reaction of the point crossed by V02 and VC02. Biometabolism at AT was as follows : HR, about 97.6% against Max ; VE, about 95.7% ; V02, about 74.7% ; VC02, about 61.4% ; Q, about 82.0% ; LA, about 43.1% and LA on AT was around 3mmol/L.

The influence during maximal exercise 2 to 3 weeks after taking OM-X. HR at Max showed about 3.11 times greater than that of 59.0?9.0beats/min. at rest. V02 was about 3.95 times greater than that of 889.83 ?272.03ml/min. at rest. VCO2 was about 5.35 stronger than that of 235.52ml/mia. at rest. VE was about 4.61 times greater than that of 25.64 ?7.33L/mm. at rest. Q was about 2.6 times great than that of 9.44 ? 1.93L/min. at rest. LA showed about 5.56 times greater than that of 1.77?0.38mmol/L. Fig. 4 shows the relation to lactate during maximal exercise before and after taking OM-X. At this point, V02inax was 62.07 ?8.77ml/kg. mil. We found here also the difference in average deviation toward the average amount due to diversity of subjects' athletic ability. Table 5 shows the influence on the AT after taking OM-X. Fig. 3 indicates the relation between V02 and VC02 during maximal exercise after taking OM-X.

For AT, it is revealed that the data in after taking OM-X cross at higher point compared to the data in before taking OM-X. Biometabolism at this point was as follows : HR, about 98.8% against Max ; VE, about 69.1% ; VOi about 78.6% ; VCO:,, about 72.1% ; Q, about 84.9% ; LA, about 52.6%. LA at AT showed around 4mmol/L. This concludes that after taking OM-X make muscle contraction maximal point higher than before taking OM-X.

IV. Discussion

Lactic acid bacteria products, since they activate enterobacterial, recently attract public attention as health food that has good inflences on various harmful influences caused from modern diet. It is said that lactic acid bacteria, especially, give influences on enterobacterial that promote resolution and absorption of nutrition in the intestinal tracts.

This test is to investigate on the influences of lactic acid bacteria OM-X on the V02inax which is believed to be an index of excercise ability for athletes. In other words, we tried to reveal the effect ingredients of OM-X have toward respiratory circulation metabolism and muscles contraction metablism. 

To explicate the influences of taking OM-X on the V02inax means to confirm on the Hb change of red corpuscles that deliver oxygen and carbon deoxide, and also to study on lactic acid bacteria resolution in blood and carbon deoxide resistant being influenced during muscle contraction while exercise. This type of study was done mainly on an experiment expecting to improve body functions by administrating substances or medicine that affect the subjected organs artificially as in the following methods: enforcing muscles by administering sex hormone products as steroid hormone in sports science, increasing V02 by changing blood ingredients artificially by doping in blood, or exhibiting higher exercise ability by administering medicine that affect on erethism (iS, 22). This is called doping in aim of improving athletes' exercise condition. These dopings somehow give harmful inflence on the body (22). Today's athletes are over loaded in the strength of training to the utmost of the body to improve more than ones' ability even they practice effective and individually suited training as scientific trainings are progressed (2, 8, ]5, 26, 34). That leads athletes to think to train ones' body tolerable to hard training, maintain and improve body function.

This also leads to improve body function in respiratory circulation metabolism and muscle contraction metabolism (2,8,15,34). Moreover, to execute trainings smoothly, they need to make ones' bodies to be ready to do so and lighten a burden to the body by body conditioning. Body conditioning is subjected to create the body suited to each sport and control one's weight by limiting diet. Weight control here means to reduce body weight and make the body move smoothly by decreasing unwanted %Fat for athletes. This lightens a weight burden to the lower body which works for exercise and is a theory of training body of athletes to reduce accidents by consumption energy loss or weight (2, 8, 15). However, limiting athletes' diet also limits wanted nutrition for body maintance and leads to serious inconveniences at bio maintainance because it decreases energy metabolism as active body and basic strength (72, 22, 29, 32). Limiting diet for athletes invites kinetic anemia and affects muscle contraction metabolism at exercise and production of bones because it lacks nutrition needed for energy in muscle contraction metabolism at exercise, nutrition needed to maintain bio tissue cells, nutrition to synthesize protein in the body and producing blood, and nutrition in relation to calcium metabolism in production of bones. Under such circumstances, athletes who repeat hard trainings need to think about how to absorb taken food effectively to the body.

Also they need to resolve nutrition taken to the body effectively and synthesize as nutrition source. Food taken from the mouth are delivered through the gullet, digested in the stomach and more than 90% of them are absorbed to the body through the small intestines (22, 29, 32 ). Nutrition absorbed in the body is delivered from the small intestines to the liver through a portal vein, synthesized as nutrition source, preserved, and supplied to each targeted organ of the body (22 ).

Syntheses of nutrition source are chemically exercised with various amino acid, lactic acid bacteria, and enterobacterial livining in the body (30, 32). Then we have to focus on the effective way of absorption of nutrition one intakes. It is to search for the way to activate enterobacterial in relation to absorption activity of nutrition in the small intestines and the way to help act enterobacterial.

The latter means to supply the necessary nutrition to have bacterial activation, which is livining in the intestinal tract as enterobacterial. Or athletes should not intake substances that inhibit and block the growth and activity of enterobacterial. We see the nutrition source (30, 32) that activate enterobacterial is to replenish various lactic acid bacteria including amino acid. Because dietary changes of modern people arc transferred from vegetable food to animal food in material and taste. It leads to the situation that limits and supresses the opportunity of vigorous action of enterobacterial with contamination of food addicts (30, 32). These dietary changes alter a basic cycle in biometabolism we the human beings basically obtain and change basic function of life metabolism creatures fundamentally obtain (30, 32 ).

These changes in life metabolism are causes of manifection of sickness of living habit issued these days or malignant tumor or malignant new creature, and other manifection of modem sickness thoght to be caused from many metabolism hindrances (20, 27, 29). Toward this, we think it is necessary to activate each bacteria living fundamentally in the intestinal tract or in the body and promote the activity of enterobacterial which absorbe energy source food for body mentainance as nutrition effectively. At the same time, we must look for the way to increase beneficial bacteria livining in the intestinal tract. In other words, we should supply nutrition source that bacteria as enterobacteria can become active and give opportunities for that action (30, 32 ).

Lactic acid bacteria product OM-X which we tested in this research is vegetable fermented food that contains 20 kinds of amino acids (of these, 8 kinds are essential amino acids), 9 kinds of vitamins, 5 kinds of minerals and 12 kinds of lactic acid bacteria in one capsule of 500 mg. Notably, the ratio of iron, potassium and sodium are very much ideal in relation to minerals for biometabolism, also, it is believed to act beneficially for tissue cell activity and muscle contraction move (29?32). Because basical function of life metabolism in human beings is to activate oxygen taken from respiration organs and nutrition taken from digestive organs, having cells activate by maintaining tissue cells of the whole body (22, 29, 32). The basic body structure for tissue cell activity depends on the constituent parts of blood supplied from circulation organs to the body (22, 29). Blood supplies oxygen, nutrition and other substances necessary for every targeted organ or tissue cells of the body and maintain body structural cells and organs (22).

Especially, ingredients necessary for blood syntheses are amino acid and vitamin which promote appropriate mineral as iron and protein syntheses (29, 32). On this experiment we determined that the amount of VE, SV which is the cardiac output at a beat from the heart per minute before taking OM-X showed higher amount (P<0.05) than that of after taking OM-X, and Hb of red corpuscles, carrier of oxygen showed about 8.4% increase in before and after taking OM-X (P< 0.05). These changes in the body are derived from changes in red corpuscles production and early syntheses of red corpuscles are being done. 

This is in the result that Hb is being increased in a very short period of time and early in consideration of existing logic (3, 8, 13, 32, 34) of red corpuscles production, and it is such an incredible phenomenon. Primarily life expectancy of red corpuscles is said to be between about 100 to 120 days, they are renewed simulteneously when about 50ml of them in blood are destroyed a day, keeping homeostasis of blood constituent parts (24, 29, 31, 32, 34). Production of red corpuscles differentiates to proerythroblast with influence of erythropoetin, a kind of hormone a stem cell of bone marrow exists in the renal, then matures to blood corpuscles as they grow(8, 22, 32). 

Main ingredients that relate to Hb syntheses of red blood cells are iron and protein (30). Iron absorbed in the body is sent to the liver connecting with protein namely transferrin and reserved as a substance called ferrin, or sent to bone marrow or other tissue and become an ingredient to synthesize heme protein like Hb or myoglobin (8, 30). Kinetic anemia affects on activity of blood cell production cell with lack of iron and delay of protein syntheses. 

We consider that we can solve it by promoting the activity of liver cells and liver function, which will raise body absorption rate of nutrition necessary for blood cell production and by promoting tissue cell metabolism by facilitating protein syntheses (2.S, 30). Especially, majority of iron absorbed in the body will not be excreted outside the body. They will be recycled to be charged in the reticuloendothelial system of the liver and spleen. We may say that the blood cell production will be synthesized in a rapid cycle when heme protein syntheses are promoted and reserved iron will be activated at the same time. 

Judging from the above, though this result differs from existing red corpuscles production and destruction metabolism theory (27, 22, 30), there is no wonder of early red corpuscle syntheses with Hb if hem protein syntheses are promoted variationally. That is, blood cell production structure or the place of blood production is different from the former cycle or place in blood production organ. Red corpuscles production of adults is majorly synthesized by red bone marrow. However, in case Hb volume is increased in a short period as in this experimental result, and in case the circumstance allows to promote heme protein syntheses, it is likely that there is an early blood synthesis in organs like the liver and spleen, other than in the red bone marrow (28, 30). Because we have a result of blood cells production in the liver and spleen in thew fetal period or in the infant and early childhood period (22, 29). Therefore, we note that production of blood is promoted in organs other than the bone marrow under the condition that there exists a circumstance in the body that acts beneficially to the red corpuscle syntheses for adults, too(22,29?32).

We may say that it is because a line of ingredients OM -X holds and enterobacterial living in the body act each other in promotion of syntheses of red blood corpuscles (30).

The reason to it is that ingredients of OM-X, iron, mineral like phosphorus and potassium and many kinds of vitamins affect active promotion on bacteria, enterobacterial, and boost syntheses of heme protein which is the base of Hb production of red corpuscles. Then, each kind of amino acid and lactic acid bacteria activate bacteria in the body and blood cell syntheses are promoted (8, 22). We can judge its synthetic mechanism as follows : ingredients of OM-X and each kinds of bacteria living in the body act each other to promote absorption of nutrition by the intestinal tract, and they are sent to the liver by a portal vein, early production of red corpuscles are made because syntheses of blood cells are promoted. Increasing volume of Hb is the most infulencial matter to V02i-nax and improve athletic ability as aerobic exercised?, 15). Also, the increase ofV02max improve oxygen supply ability and tolerance ability of carbon dioxide at muscle contraction becuase resolving function of lactic acid in the blood is being promoted (6, 13).

Strengthening of tolerance ability of carbon dioxide at muscle means the continuing posibility of muscle contraction at higher athletic strength (8, 13?15). Substances that help muscle contraction are seen potassium and sodium which control oxygen supply and muscle contraction, and appropriate ratio of mineral as phosphorus (30). For potassium, 899mg/100g of them is included in the ingredients ofOM-X which we used here. We believe that affects beneficially to helping muscle contraction (30). 178mg/100g of phosphorus and 58.6mg/100g of sodium are extremely contained in appropriate ratio, we may suggest that they affect beneficially to the syntheses of adenosine triphosphate, ATP, which allows muscle acts automatically (8, 22, 30). 

We find it as the result of improvement of athletic ability which aerobic exercise ability is widened in relation to an increase of AT, as high as up to about 600ml/mii. after taking OM-X at exercise (8). This is also of the result that it affects to the tolerance of carbon dioxide which dicide the maximum point of muscle contraction exercise. Moreover, we may say that as production volume of LA at AT shows higher amount in after taking OM-X than before and as VOamax also shows great number, the tolerance of carbon dioxide is clearly accelerated at muscle contraction exercise (8).

These changes of biofunction at exercise are derived from the changes of Hb volume in red corpuscles in the component of blood. It is considered that ingredients of OM-X relate in great deal the increase of V02max and the promoting act of resolving LA at exercise. The ingredients of OM-X are amino acid, lactic acid bacteria, mineral and vitamin. These work beneficially to blood production act in the body because they act totally with enterobacterial, conform with each kinds of bacteria in the intestinal tract, and promote protein syntheses cells construcy. As the result, Hb syntheses of red corpuscles are promoted, and blood cells are produced early. And ingredients of OM-X also contain vitamin B12 (0.07?/100g), folic acid (4?/100g), and vitamin E. We think these also affect (28) early blood production at the same time.

All these things make it clear that intake of lactic acid bacteria OM-X increases V02max, the index of athletic ability to athletes (8). We may say that ingredients of OM-X, lactic acid bacteria, amino acid and mineral affect red corpuscle production and lead to early blood production(28). Also, we can recognize that this mechanism has a big key to the bacteria activity in enterobacterial (30).

This experiment was resulted in adminestering 1.5g of OM-X to athletes of different weight, Our next themes we plan to solve step by step are the relation between the volme of OM-X intake and body type, difference in sexes, or the relation between intake period and diet, and the role and mechanism of lactic acid bacteria product OM-X intake to the body. We believe that this is to investigate changes of biometabolism occured from modern diet and habit of living and to study the basic working of life metabolism.

V. Summary

This experiment was tested in aim to study the influences on the V02 of athlete taking lactic acid bacteria product OM-X. It was made clear that V02 max has a dramatic increase after taking OM-X. We hereby report we see other changes in bio-function during exercises loaded.

The major results obtained are as follows:

1. The numerical volume of Hb increases about 8.4% (g/dl) after taking OM-X (p<0.05)

2. After taking OM-X, the AT point under V02 exercise test indicates about 600ml/min. higher point than before. (p<0.05)

3. The numerical volume of LA at Max does not indicate significant differences between before taking OM-X and after.

4. The numerical volume of V02max per weight increases about 29.7% after taking OM-X. (p<0.05)

5. These results lead to the identification that the intake of lactic acid bacteria product OM-X has significant influences on the V02max of long distance runners in track and field. We may say that a variety of ingredients in OM-X act totally to enterobacterial in the body and promote Hb synthese of red corpuscles. We may also say that early increase of Hb promotes resolving ability of lactic acid in blood while exercising, improves tolerance of carbon dioxide, and enables to maintain muscle contraction exercise in stronger strength

Inhibitory Effects of Lactic Acid Bacteria On Some Pathogens

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 did not show potent growth inhibition or activity against the various closely related lactic acid bacteria. However the growth inhibition against MRSA was evident while most of the lactic acid bacteria 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 it was found that the active component retained the bactericidal activity against MRSA when it was treated with various proteases such as proteinase K, V8 protease, trypsin and achromopeptidase. These results suggest that the active component is not a peptide or protein. Further work is in progress to determine more details on its physical and biochemical characteristics which is beyond the present scope of this paper.

But it is important to note that the active component of TH10 did not show any hemolytic activity towards human and rabbit erythrocytes.

Inhibition of Helicobacter Pylori

The 12 strains of lactic acid bacteria in the OM-X capsule showed antimicrobial activity against H. pylori in vitro as shown by loss of viability of the latter.

A well diffusion assay technique was used. One OM-X capsule was dissolved and incubated in All-purpose Medium with Tween (APT) broth with or without biotin for good recovery of the organisms from the capsule. The colonies recovered from the capsule dissolved in water and then plated on APT agar and de Man, Rogosa and Sharpe (MRS) agar showed decrease in the number of colony forming units of the H. pylori strains (NCTCI 1637 & NCTCI 1638).

This preliminary results explains how viable lactic acid bacteria contained in live capsule based probiotic can bring about much relief to those suffering from peptic ulcers and gastritis by inhibiting the H. pylori infection.

Growth Inhibition on E.Coli 0157

The component from THIO strain was extracted with ethyl acetate and then neutralized by regulating the pH. Then variable rates (0.75, 1.5,3.0,6.0, 12 and 24 mg) of the lactic acid bacteria extract were introduced on dipped paper discs into petri dishes containing cultured viable colonies of E. coli 0157m several replicates. These petri dishes were incubated at 37 degrees centigrade. Growth inhibitions of the 0157 strain were monitored by observing the diameter of zones of inhibitions obtained around the paper discs.

Results indicated that inhibition of 0157 were obtained at all amounts of 3.0 mg and above of the extract. Consistent results were obtained on repetition. Inhibitory effects were increased with more amount of the extract.

The substance acting on the 0157 strain is initially made out to be of low molecular weight and further research is being carried out to specify its structure and mechanism of activity.

Mode of Action

At this stage the explanation that can be offered is the action of the different bacteriocins and the lowering of pH by the OM-X inhibiting the proliferation of the antagonistic microorganisms. With the five years fermentation and low pH achieved it is found that the acidolin produced by the LactobacUlus acidophilus is particularly active against bacteria and viruses. This evidence is farther supported by earlier research ofHamdan and Mikolajcik (1974). These makes OM-X lactic acid bacteria highly effective in terms of its antimicrobial property as well a functional food supplement.

References

Lilley, D. M. and StiUwell, R. H. (1965). Probiotics: growth promoting factors produced by Microorganisms. Science 147 747-748

Parker, R. B. (1974). Probiotics, the other half of the antibiotic story. Anim. Nutr. Health 29:4-8

Fuller, R. (1989). Probiotics in man and animals. J. Appl. Bacteriol. 66: 365-378

Hamdan, 1. Y. and Mikolajcik, E. M. (1974). Acidolin: an antibiotic produced by Lactobacillus acidophilus. Journal of Antibiotics 8: 631-636

Lactic Acid Bacteria Keeping You Healthy Within

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!

Properties of Lactic Acid Bacteria From Fermented Foods

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.