By Iichiroh Ohhira P.h.D. 1999
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.
Dr.
Iichiroh Ohhira, Ph.D, a distinguished professor and research scientist
who has lectured at universities in Malaysia, Korea, and Japan, has
authored or coauthored more than 20 scientific research studies on
lactic acid bacteria. 
