Paralactobacillus selangorensis gen. nov., sp. nov. is described. This organism, isolated from a Malaysian food ingredient called chili bo, is an obligatory homofermentative, rod-shaped lactic acid bacterium. The G+C content is 46.1-46.2+/-0.3 mol%. Earlier 16S rRNA studies showed that this organism constitutes a new taxon distantly related to the Lactobacillus casei-Pediococcus group. A phenotypic description that distinguishes Paralactobacillus selangorensis from other genera of lactic acid bacteria is presented. The type strain of Paralactobacillus selangorensis is LMG 17710T.
Tempoyak is a traditional Malaysian fermented condiment made from the pulp of the durian fruit (Durio zibethinus). Salt is sometime added to proceed fermentation at ambient temperature. In various samples obtained from night markets, lactic acid bacteria (LAB) were the predominant microorganisms, ranging from log 8.4 to log 9.2 cfu g(-1). No other microorganisms were present to such a level. These samples contained reduced amount of saccharose, glucose and fructose but increased amount of D- and L-lactic acid and acetic acid compared with samples of non-fermented durian fruit. Sixty-four isolates of LAB were divided into five groups by use of a few phenotypic tests. A total of 38 strains of LAB were selected for comparison by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their whole cell protein patterns with a SDS-PAGE database of LAB. These strains were also examined for their carbohydrate fermentation patterns by use of API 50 CH. Isolates belonging to the Lactobacillus plantarum group were shown to be the predominant members of the LAB flora. In addition, isolates belonging to the Lactobacillus brevis group, Leuconostoc mesenteroides, Lactobacillus mali, Lactobacilus fermentum and an unidentified Lactobacillus sp. were also observed. A high degree of diversity among isolates belonging to the Lb. plantarum group was demonstrated by analysis of their plasmid profiles.
The effect of heat-treatment on the internal temperature of raw cockles (Anadara granosa) and survival of their intrinsic flora of Vibrio spp. as well as of inoculated V. cholerae 0139 was examined. The cockles were purchased from markets in Malaysia and had an average weight including shells of 8.90+/-2.45 g. In one experiment heatpenetration of individual cockles was examined. Cockles weighing < 8 g (including shell) exhibited maximum internal temperatures of between 50 and 75 degrees C when heated in water at 99 degrees C for 10 s and 71-93 degrees C when heated for 30 s. Cockles weighing > 12 g exhibited maximum internal temperatures between 42 and 58 degrees C when heated in water at 99 degrees C for 10 s and 56-69 degrees C when heated for 30 s. In another experiment, heat-treatment of 10 cockles treated as a group at 99 degrees C for 10 or 30 s resulted in reduction of levels of intrinsic Vibrio spp. (enumerated directly on thiosulphate-citrate-bile salt sucrose agar; TCBS) from 5.73 to 3.15 log cfu g(-1) or below 1 log cfu g(-1), respectively. The levels of Vibrio spp. after heat-treatment decreased with an increase in numbers of cockles grouped together during treatment. In a third experiment V. cholerae 0139 was inoculated into cockles and subjected to heat-treatment at 99 degrees C for 0, 10, 15, 20, 25 or 30 s. The levels of Vibrio spp. in uninoculated, non-heat-treated cockles was 4.89 log cfu g(-1) on TCBS, and the predominant species were V. parahaemolyticus and V. alginolyticus. V. cholerae 0139 inoculated into cockles with an average weight of 13.5+/-1.90 g (including shell) decreased for samples examined immediately after heat-treatment from 6 log cfu g(-1) initially to 3.5 log cfu g(-1) after 25 s and < 1 log cfu g(-1) (TCBS) after 30 s of heat-treatment. The most probable number method by enrichment in alkaline peptone water gave in general within 1 log unit higher counts than TCBS direct enumeration. TCBS direct enumeration and MPN counts were up to 2.38 or 1.30 log units higher, respectively, for samples heat-treated for 20 s or longer and stored for 6 h at 30 degrees C before examination, than for samples heat-treated for same periods of time and examined immediately. This study shows that a mild heat-treatment of cockles for up to 25 s is inadequate to ensure a large reduction in numbers of Vibrio spp., including V. cholerae 0139.
Lactic acid bacteria (LAB) are the predominant micro-organisms in tempoyak, a Malaysian acid-fermented condiment. In a study on the diversity of LAB in this product, three isolates could not be identified using SDS-PAGE of whole-cell proteins or API 50 CH. The taxonomic position of the three isolates was clarified in the present study. 16S rDNA sequencing classified a representative strain in the genus Lactobacillus, clearly separated from all known species, and most closely related to the Lactobacillus reuteri phylogenetic group. DNA-DNA hybridization experiments and an extensive phenotypic description confirm that the strains represent a single and separate novel species among the obligately heterofermentative lactobacilli. The three isolates are distinguished at the intra-species level by plasmid profiling, pulsed-field gel electrophoresis of macro-restriction fragments and biochemical features. The name Lactobacillus durianis sp. nov. is proposed for the novel taxon and the type strain is LMG 19193T (= CCUG 45405T).
Isolation of bacteriocinogenic lactic acid bacteria (LAB) from the Malaysian mould-fermented product tempeh and characterization of the produced bacteriocin(s).
Three lactic acid bacterial (LAB) strains obtained from a Malaysian acid-fermented condiment, tempoyak (made from pulp of the durian fruit), showed analogous but distinct patterns after screening by SDS-PAGE of whole-cell proteins and comparison with profiles of all recognized LAB species. 16S rRNA gene sequencing of one representative strain showed that the taxon belongs phylogenetically to the genus Leuconostoc, with its nearest neighbour being Leuconostoc fructosum (98 % sequence similarity). Biochemical characteristics and DNA-DNA hybridization experiments demonstrated that the strains differ from Leuconostoc fructosum and represent a single, novel Leuconostoc species for which the name Leuconostoc durionis sp. nov. is proposed. The type strain is LMG 22556(T) (= LAB 1679(T) = D-24(T) = CCUG 49949(T)).
The predominant microbial flora of a specific Malaysian food ingredient, chili bo (containing 9% ground dried chilies, 0.6% acetic acid, and 5 to 10% cornstarch, wt/vol) stored for up to 25 days at 28°C without added benzoic acid (product A) and with 7,000 ppm of added benzoic acid (product B) was examined. Aerobic plate counts for both products were initially 6.2 to 6.5 log CFU/g increasing to 8.5 log CFU/g for product A after 4 days. Aerobic plate counts for product B did not increase during storage. Lactic acid bacteria (LAB) counts increased in product A from 4.8 log CFU/g to 8.3 log CFU/g and in product B from 2.1 log CFU/g to 7 .6 log CFU/g after 17 days. Growth of yeast occurred in product A. Both products exhibited spoilage after 1 to 2 days of storage at 28°C indicated as accumulation of gas bubbles. In addition surface growth of molds (product A) or whitish discoloration (product B) was observed later in storage. For product A the predominant isolates were LAB, Bacillus pumilus , Bacillus subtilis , Staphylococcus spp., and yeasts. B. pumilus and B. subtilis predominated initially whereas the other types of microorganisms predominated after 25 days of storage. B. pumilus and B. subtilis were also predominant in product B, but after 25 days of storage a homofermentative LAB was found in higher numbers (7.6 log CFU/g). Isolates of heterofermentative LAB but not homofermentative LAB or B. pumilus or B. subtilis were able to produce gas during growth in chili bo sterilized by autoclaving at l2l°C for 15 min. Growth of heterofermentative LAB, B. pumilus , and B. subtilis was inhibited by acidifying agents, a nisin-containing supernatant, or incubation at low temperatures.
Ninety-two strains of lactic acid bacteria (LAB) were isolated from a Malaysian food ingredient, chili bo, stored for up to 25 days at 28 degreesC with no benzoic acid (product A) or with 7,000 mg of benzoic acid kg-1 (product B). The strains were divided into eight groups by traditional phenotypic tests. A total of 43 strains were selected for comparison of their sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) whole-cell protein patterns with a SDS-PAGE database of LAB. Isolates from product A were identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus farciminis, Pediococcus acidilactici, Enterococcus faecalis, and Weissella confusa. Five strains belonging to clusters which could not be allocated to existing species by SDS-PAGE were further identified by 16S rRNA sequence comparison. One strain was distantly related to the Lactobacillus casei/Pediococcus group. Two strains were related to Weissella at the genus or species level. Two other strains did not belong to any previously described 16S rRNA group of LAB and occupied an intermediate position between the L. casei/Pediococcus group and the Weissella group and species of Carnobacterium. The latter two strains belong to the cluster of LAB that predominated in product B. The incidence of new species and subspecies of LAB in chili bo indicate the high probability of isolation of new LAB from certain Southeast Asian foods. None of the isolates exhibited bacteriocin activity against L. plantarum ATCC 14917 and LMG 17682.