Leuconostoc Species Research Articles

Characterization of the microbial diversity in yacon spontaneous fermentation at 20 °C

The prebiotic fructooligosaccharide content of yacon makes this root an attractive alternative for the supplementation of a variety of food products. The preservation of yacon by fermentation has been proposed as an alternative to increase the probiotic content of the root concomitantly with its shelf life. Thus the fermented yacon could have significant functional content. The objective of this research was to characterize the biochemistry and microbiology of spontaneous yacon fermentation with 2% NaCl and define the viability of the proposed process. The biochemical analysis of spontaneous heterolactic fermentation of yacon showed a progressive drop in pH with increased lactic and acetic acids, and the production of mannitol during fermentation. The microbial ecology of yacon fermentation was investigated using culture-dependent and culture-independent methods. Bacterial cell counts revealed a dominance of lactic acid bacteria (LAB) over yeasts, which were also present during the first 2days of the fermentation. Results showed that the heterofermentative LAB were primarily Leuconostoc species, thus it presents a viable method to achieve long term preservation of this root.

Effect of dissolved oxygen on redox potential and milk acidification by lactic acid bacteria isolated from a DL-starter culture

Milk acidification by DL-starter cultures [cultures containing Lactococcus lactis diacetylactis (D) and Leuconostoc (L) species] depends on the oxidation-reduction (redox) potential in milk; however, the mechanisms behind this effect are not completely clear. The objective of this study was to investigate the effect of dissolved oxygen on acidification kinetics and redox potential during milk fermentation by lactic acid bacteria (LAB). Fermentations were conducted by single strains isolated from mixed DL-starter culture, including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Leuconostoc mesenteroides ssp. cremoris, by the DL-starter culture, and by the type strains. High and low levels of oxygen were produced by flushing milk with oxygen or nitrogen, respectively. The kinetics of milk acidification was characterized by the maximum rate and time of acidification (Vamax and Tamax), the maximum rate and time of reduction (Vrmax and Trmax), the minimum redox potential (Eh7 final), and time of reaching Eh7 final (Trfinal). Variations in kinetic parameters were observed at both the species and strain levels. Two of the Lc. lactis ssp. lactis strains were not able to lower redox potential to negative values. Kinetic parameters of the DL-starter culture were comparable with the best acidifying and reducing strains, indicating their additive effects. Acidification curves were mostly diauxic at all oxygen levels, displaying 2 maxima of acidification rate: before (aerobic maximum) and after (anaerobic maximum) oxygen depletion. The redox potential decreased concurrently with oxygen consumption and continued to decrease at slower rate until reaching the final values, indicating involvement of both oxygen and microbiological activity in the redox state of milk. Oxygen flushing had a negative effect on reduction and acidification capacity of tested LAB. Reduction was significantly delayed at high initial oxygen, exhibiting longer Trmax, Trfinal, or both. Concurrently, anaerobic acidification rate maximum Vamax was decreased and Tamax was extended. Fermentation kinetics in nitrogen-flushed milk was not statistically different from that in untreated milk except for Lc. lactis ssp. lactis CHCC D2, which showed faster reduction time after nitrogen flushing. This study clarifies the relationship between the redox state in milk and acidification kinetics of the predominant subspecies in DL-starter cultures. This knowledge is important for dairies to ensure optimized, fast, and controlled milk fermentations, leading to greater standardization of dairy products.

Leuconostoc lactis: An unusual cause for bacteremia

Leuconostoc species, the common saprophytic bacteria are now emerging as one of the important opportunistic pathogens. There are different risk factors such as compromised immunity, use of vancomycin, parenteral nutrition, and contaminated surgical implanted devices to contribute the human infection. We here present one of the unusual fatal cases of bacteremia in a cancer patient who was infected with a vancomycin-resistant strain of L. lactis.

날치알을 첨가한 무말랭이 김치의 이화학적 성분 및 발효양상

날치알을 사용하여 무말랭이 김치를 제조하고 일반 무말랭이 김치와 일반성분, 무기질, 지방산, 핵산관련물질, 유리아미노산 함량 및 관능평가의 차이를 비교해 보고자 하였다. 또한, 35일간 저장하는 동안 일주일 간격으로 시료를 취하여 pH, 산도 및 젖산균의 변화를 측정하여 발효양상 비교 실험도 하였다. 일반 무말랭이 김치의 조단백질 및 조지방 함량은 각각 2.93% 및 0.16%이었고 날치알 무말랭이 김치의 경우는 각각 4.75% 및 0.36%로, 날치알을 첨가한 무말랭이 김치에서 조단백질 및 조지방 함량이 유의적으로 높게 나타났다. 철 및 칼슘 함량의 경우도 날치알을 첨가한 무말랭이 김치가 유의적으로 높은 값을 나타내었다. 핵산 관련물질 중에서도 정미성분과 관련이 있는 AMP 및 IMP의 함량이 날치알을 첨가한 무말랭이 김치에서 유의적으로 높은 함량을 나타내었다. 날치알 무말랭이 김치의 지방산 분석 결과, 포화지방산과 단일불포화지방산의 함량은 일반 무말랭이 김치보다 낮았으나 고도불포화지방산 함량은 일반 무말랭이 김치보다 5배 이상 높은 함량을 보였다. 특히 DHA의 함량은 일반 무말랭이 김치보다 약 23배 이상 높은 함량을 나타내었다. 일반 무말랭이 김치와 날치알 무말랭이 김치의 주요 유리아미노산은 arginine, proline, alanine, leucine 및 valine 이었고, glutamic acid와 glycine 함량은 유의적인 차이를 나타내었다. 날치알 무말랭이 김치의 관능평가 결과에서 일반 무말랭이 김치보다 외관, 향미, 맛 및 질감 면에서 유의적으로 높은 값을 나타내었다. 한편, 날치알 무말랭이 김치의 pH 및 산도의 변화를 보면, 레몬즙에 침지 처리한 날치알의 영향으로 인해 일반 무말랭이 김치보다 초기 pH는 낮고 초기 산도값은 높았으나, 저장 14일 이후부터는 동일한 발효양상을 보였다. Leconostoc sp. 및 Lactobacillus sp.의 경우는 저장 14일째에 최대값을 보인 후에 점차 감소하는 양상을 보였다. 본 연구결과에서 나타낸 것처럼, 날치알을 첨가한 무말랭이 김치는 조단백질 및 무기질 함량이 일반 무말랭이 김치에 비해 높고, 날치알의 첨가가 지방산과 유리아미노산 조성 및 함량에도 긍정적인 영향을 미쳐 식품영양학적으로 우수한 무말랭이 김치를 제조할 수 있음을 알 수 있었다. 또한, 발효기간 동안 일반 무말랭이 김치와 동일한 발효양상을 나타내는 것으로 볼 때, 날치알을 첨가한 무말랭이 김치는 정상적인 발효가 진행됨을 예측할 수 있었다. This study was conducted to compare the physicochemical composition and fermentation conditions of sliced, dried radish kimchi with flying fish roe (DFFR). The levels of crude protein, crude lipid, and crude ash in DFFR were higher than those in sliced, dried radish kimchi without flying fish roe (control). DFFR also contained higher levels of Fe and Ca, compared to the control. The inosine monophosphate (IMP) content of DFFR and control was 5.63 and 2.64 mg/100 g, respectively. The polyunsaturated fatty acid and DHA contents in DFFR were approximately 5 and 23 times higher than those in the control, respectively. The major free amino acids contained in these samples were arginine, proline, alanine, leucine, and valine. The number of cells belonging to the Leuconostoc species in DFFR was higher than that in the control. In sensory evaluation studies, DFFR scored the highest in terms of appearance, flavor, taste, and texture.

The Effect of Dextransucrase Gene Inactivation on Mannitol Production by Leuconostoc mesenteroides

Leuconostoc species, a group of hetero-fermentative lactic acid bacteria, can produce mannitol which has various applications in the food and medical industries. Sucrose is utilized by the microorganism to produce mannitol, dextran and other products. To improve mannitol yield, we hindered the dextran biosynthetic pathway by inactivating dextransucrase gene (dts) and then studied effects on mannitol production. The gene encoding dextransucrase from Leuconostoc mesenteroides CGMCC1.10327 isolated from kimchi was cloned and sequenced. We constructed a homologous recombination vector by using the method of gene splicing by overlap extension. The vector was transferred to L. mesenteroides and dts was inactivated by double-crossover gene replacement. A dts inactivated mutant strain DTS1 without antibiotic resistant marker was obtained. Fermentation results showed that yield of dextran in the mutant was decreased by 28.8 % and mannitol yield was increased by 15.3 %.

Diversity and probiotic potentials of lactic acid bacteria isolated from gilaburu, a traditional Turkish fermented European cranberrybush (Viburnum opulus L.) fruit drink

The aim of the present study was to characterize lactic acid bacteria (LAB) strains isolated from traditional fermented gilaburu fruit juice and their probiotic potential. The LAB counts of the fermented gilaburu fruit juice were in the range of 3.92–8.30logcfu/g. Total of 332 isolates belonging to Lactobacillus and Leuconostoc species were characterized from traditional fermented gilaburu juice by genotypic methods. It was also determined that the major LAB strains belong to Lactobacillus plantarum (173 isolates), Lactobacillus casei (52 isolates) and Lactobacillus brevis (24 isolates), while Lactobacillus buchneri, Lactobacillus parabuchneri, Lactobacillus pantheris, Leuconostoc pseudomesenteroides and Lactobacillus harbinensis were the least in isolated LAB strains. In terms of the probiotic potentials, Lb. plantarum strains were able to grow at pH2.5, but 3 of Lb. casei strains, one of each Lb. brevis and Lb. buchneri strains could not grow at the same pH. All selected LAB stains were resistant to bile salt at ≤0.3% concentration. While all the LAB species grew at 15°C, two Lactobacillus hordei strains could also grow at 45°C. The highest cell hydrophobicity degrees were for Lb. casei (G20a) and Lb. plantarum (G19e) as 87.5 and 86.0%, respectively. Listeria monocytogenes and Bacillus cereus were the most sensitive bacteria against the selected LAB strains, while Escherichia coli and Staphylococcus aureus were the most resistant. Again all the isolated LAB species were resistant to three antibiotics; kanamycin, streptomycin and vancomycin. Characterization and probiotic potentials of the LAB isolated from fermented gilaburu (Viburnum opulus) juice were studied first time, and further research needs to be done on their behaviors in similar food formulations as a probiotic.

Microbial successions are associated with changes in chemical profiles of a model refrigerated fresh pork sausage during an 80-day shelf life study.

Fresh pork sausage is produced without a microbial kill step and therefore chilled or frozen to control microbial growth. In this report, the microbiota in a chilled fresh pork sausage model produced with or without an antimicrobial combination of sodium lactate and sodium diacetate was studied using a combination of traditional microbiological methods and deep pyrosequencing of 16S rRNA gene amplicons. In the untreated system, microbial populations rose from 10(2) to 10(6) CFU/g within 15 days of storage at 4°C, peaking at nearly 10(8) CFU/g by day 30. Pyrosequencing revealed a complex community at day 0, with taxa belonging to the Bacilli, Gammaproteobacteria, Betaproteobacteria, Actinobacteria, Bacteroidetes, and Clostridia. During storage at 4°C, the untreated system displayed a complex succession, with species of Weissella and Leuconostoc that dominate the product at day 0 being displaced by species of Pseudomonas (P. lini and P. psychrophila) within 15 days. By day 30, a second wave of taxa (Lactobacillus graminis, Carnobacterium divergens, Buttiauxella brennerae, Yersinia mollaretti, and a taxon of Serratia) dominated the population, and this succession coincided with significant chemical changes in the matrix. Treatment with lactate-diacetate altered the dynamics dramatically, yielding a monophasic growth curve of a single species of Lactobacillus (L. graminis), followed by a uniform selective die-off of the majority of species in the population. Of the six species of Lactobacillus that were routinely detected, L. graminis became the dominant member in all samples, and its origins were traced to the spice blend used in the formulation.

Monitoring psychrotrophic lactic acid bacteria contamination in a ready-to-eat vegetable salad production environment

A study monitoring lactic acid bacteria contamination was conducted in a company producing fresh, minimally processed, packaged and ready-to-eat (RTE) vegetable salads (stored at 4°C) in order to investigate the reason for high psychrotrophic LAB levels in the products at the end of shelf-life. Initially, high microbial counts exceeding the established psychrotrophic thresholds (>107–108CFU/g) and spoilage manifestations before the end of the shelf-life (7days) occurred in products containing an assortment of sliced and diced vegetables, but within a one year period these spoilage defects became prevalent in the entire processing plant. Environmental sampling and microbiological analyses of the raw materials and final products throughout the manufacturing process highlighted the presence of high numbers of Leuconostoc spp. in halved and unseeded, fresh sweet bell peppers provided by the supplier. A combination of two DNA fingerprinting techniques facilitated the assessment of the species diversity of LAB present in the processing environment along with the critical point of their introduction in the production facility. Probably through air mediation and surface adhesion, mainly members of the strictly psychrotrophic species Leuconostoc gelidum subsp. gasicomitatum and L. gelidum subsp. gelidum were responsible for the cross-contamination of every vegetable handled within the plant.

Development of a genus-specific PCR combined with ARDRA for the identification of Leuconostoc species in kimchi

With Leuconostoc species being increasingly isolated from many fermented kimchi, a systematic identification of this genus is required, for which an amplified ribosomal DNA restriction analysis (ARDRA) has been developed. This method applies both polymerase chain reaction and restriction pattern analysis using a restriction enzyme. First, using specific primers that were designed from lactic acid bacteria (LAB) sequences, 217 bp specific to LAB was produced. Then, restriction analysis using the MspI was applied to identify these bacteria at the species level. This study demonstrates novel ARDRA technique for the identification of Leuconostoc species was successful in identifying according to the genomic species.

Sequence and comparative analysis of Leuconostoc dairy bacteriophages

Bacteriophages attacking Leuconostoc species may significantly influence the quality of the final product. There is however limited knowledge of this group of phages in the literature. We have determined the complete genome sequences of nine Leuconostoc bacteriophages virulent to either Leuconostoc mesenteroides or Leuconostoc pseudomesenteroides strains. The phages have dsDNA genomes with sizes ranging from 25.7 to 28.4kb. Comparative genomics analysis helped classify the 9 phages into two classes, which correlates with the host species. High percentage of similarity within the classes on both nucleotide and protein levels was observed. Genome comparison also revealed very high conservation of the overall genomic organization between the classes. The genes were organized in functional modules responsible for replication, packaging, head and tail morphogenesis, cell lysis and regulation and modification, respectively. No lysogeny modules were detected. To our knowledge this report provides the first comparative genomic work done on Leuconostoc dairy phages.

Designing of a novel dextransucrase efficient in acceptor reactions

Dextransucrase is produced by Leuconostoc, Streptococcus and Lactobacillus Species. The enzyme synthesizes dextran and acceptor products some of which act as prebiotics that are increasingly used in such industries as food, medicine, and cosmetics. B-512F Leuconostoc mesenteroides dextransucrase (DSR-S) is the preferred enzyme in commercial production of dextran and prebiotics. In the present work, a novel dextransucrase which is efficient in prebiotics production was designed. The enzyme was produced at optimal conditions in Escherichia coli by truncation and fusion to glutathione S-transferase (GST) in the gene from Leuconostoc mesenteroides B-512 FMC. The novel enzyme (MW: 119kDa) was active and carried out dextran biosynthesis and acceptor reactions effectively. The novel dextransucrase (fTDSR-S) was produced by truncating signal, variable, and the glucan-binding regions in the gene and fusion of gst gene at the 5′ end. fTDSR-S was characterized in detail and compared to the DSR-S. Truncation and fusion resulted in an increase in fTDSR-S biosynthesis in E. coli BL21 (DE3) by 35 fold. fTDSR-S leads to production of dextran as well as increased acceptor reactions. Due to GST fusion, it was possible to immobilize fTDSR-S covalently onto Eupergit C successfully. It was also found that the size of the active site of dextransucrase is 49 amino acids shorter than that reported previously in the literature.

Bacterial diversity of aMasi, a South African fermented milk product, determined by clone library and denaturing gradient gel electrophoresis analysis

In the present study, we investigated the bacterial diversity of aMasi, a traditional South African fermented milk product, by 16S rRNA clone library and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. Two hundred and eighty two clones from clone library were isolated and identified from aMasi, prepared from the milk of four cows from one herd in the EkuPindiseni Community, North West of Hluhluwe-iMfolozi Park in KwaZulu-Natal Province. The majority of the identified sequences corresponded to lactic acid bacteria (LAB), with the genus Lactococcus as major representative. The speciesLactococcus lactis accounted for 179 of the identified clones. In addition, several species of Lactobacillus, Leuconostoc and Enterococcus were detected. Furthermore, several clones belonging to Acinetobacter, Aeromonas and genera within the Enterobacteriaceaewere detected. It is important to note that human pathogens such as Klebsiella pneumoniae were identified in aMasi in the present study. Conversely, zoonotic bacteria such as Brucella abortus and Mycobacterium bovis were not detected in aMasi, although, they are present in the cattle population in the study area. Thirty (30) clones were identified as uncultured bacterial clones. Nine DGGE bands were successfully sequenced, of which four were classified as L. lactis with other bands belonging to lactobacilli, Clostridium acidurici, Enterobacter sp., Acinetobacter baumannii and an un-culturable bacterium. Even though there was some discrepancy between the two culture independent methods used to study the bacteriological community in aMasi, a general conclusion can be drawn,L. lactis may be considered as the dominant bacterium within a diverse bacterial community in this locally-produced dairy product. Key words: South Africa, aMasi, fermented milk, microbial diversity, clone library, denaturing gradient gel electrophoresis (DGGE).

Isolation and Characterization of a Cryptic Plasmid, pMBLR00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733

A cryptic plasmid, pMBLR00, from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733 was isolated, characterized, and used for the construction of a cloning vector to engineer Leuconostoc species. pMBLR00 is a rolling circle replication plasmid, containing 3,370 base pairs. Sequence analysis revealed that pMBLR00 has 3 open reading frames: Cop (copy number control protein), Rep (replication protein), and Mob (mobilization protein). pMBLR00 replicates by rolling circle replication, which was confirmed by the presence of a conserved double-stranded origin and single-stranded DNA intermediates. An Escherichia coli-Leuconostoc shuttle vector, pMBLR02, was constructed and was able to replicate in Leuconostoc citreum 95. pMBLR02 could be a useful genetic tool for metabolic engineering and the genetic study of Leuconostoc species.

Diversity of thermophilic bacteria in raw, pasteurized and selectively-cultured milk, as assessed by culturing, PCR-DGGE and pyrosequencing

Thermophilic lactic acid bacteria (LAB) species, such as Streptococcus thermophilus, Lactobacillus delbrueckii and Lactobacillus helveticus, enjoy worldwide economic importance as dairy starters. To assess the diversity of thermophilic bacteria in milk, milk samples were enriched in thermophilic organisms through a stepwise procedure which included pasteurization of milk at 63 °C for 30 min (PM samples) and pasteurization followed by incubation at 42 °C for 24 h (IPM samples). The microbial composition of these samples was analyzed by culture-dependent (at 42 °C) and culture-independent (PCR-DGGE and pyrosequencing of 16S rRNA gene amplicons) microbial techniques. The results were then compared to those obtained for their corresponding starting raw milk counterparts (RM samples). Twenty different species were scored by culturing among 352 isolates purified from the counting plates and identified by molecular methods. Mesophilic LAB species (Lactococcus lactis, Lactococcus garvieae) were dominant (87% of the isolates) among the RM samples. However, S. thermophilus and Lb. delbrueckii were found to be the dominant recoverable organisms in both PM and IPM samples. The DGGE profiles of RM and PM samples were found to be very similar; the most prominent bands belonging to Lactococcus, Leuconostoc and Streptococcus species. In contrast, just three DGGE bands were obtained for IPM samples, two of which were assigned to S. thermophilus. The pyrosequencing results scored 95 operational taxonomic units (OTUs) at 3% sequence divergence in an RM sample, while only 13 were encountered in two IPM samples. This technique identified Leuconostoc citreum as the dominant microorganism in the RM sample, while S. thermophilus constituted more than 98% of the reads in the IPM samples. The procedure followed in this study allowed to estimate the bacterial diversity in milk and afford a suitable strategy for the isolation of new thermophilic LAB strains, among which adequate starters might be selected.

Identification, typing, and functional characterization of Leuconostoc spp. strains from traditional, starter-free cheeses

Selected Leuconostoc strains are required as aroma producers in dairy starters. In this work, 42 Leuconostoc isolates from different raw milk traditional cheeses made without the addition of commercial starters were identified by molecular methods. These isolates were assigned to the species Leuconostoc citreum (24), Leuconostoc mesenteroides (13), and Leuconostoc lactis (5). Typing the isolates by repetitive extragenic profiling (Rep-PCR) identified 22 strains, out of which 14 were selected for further characterization based on their behavior in milk. The biochemical, technological, and food safety variables recorded were examined to select appropriate candidates for use as adjunct cultures. In agreement with the wide genetic diversity recorded by Rep-PCR, wide phenotypic biodiversity was seen among the species and strains in terms of enzyme and carbohydrate fermentation profile, acidification rate, the production of volatile compounds in milk, and growth under different pH, salt, and temperature conditions. No production of biogenic amines was recorded even when suitable amino acid precursors were present in the growth medium. All 14 strains proved to be susceptible or intrinsically resistant to a set of 16 antibiotics except for one Leuc. citreum strain that showed an atypical resistance to ciprofloxacin. The remaining 13 strains could be confidently used as components of adjunct cultures in the dairy industry.

Potential impact on cheese flavour of heterofermentative bacteria from starter cultures

Leuconostoc mesenteroides subsp. cremoris, Leuconostoc pseudomesenteroides and Lactobacillus danicus were grown to early stationary phase in MRS and a cheese based medium (CBM). Lb. danicus had lower aminopeptidase and aminotransferase activity after growth in CBM compared with growth in MRS. Lb. danicus showed aminotransferase activity on all 20 amino acids investigated after growth in CBM; growth in MRS gave higher activities but on fewer amino acids. Highest activity was on aromatic amino acids. Lb. danicus had generally higher and broader aminopeptidase and aminotransferase activity than the Leuconostoc species. When grown in CBM the aminotransferase activity was more similar between Lb. danicus and the leuconostocs. Lb. danicus and Leu. pseudomesenteorides strains had activity on substrates containing short chain fatty acids; activity on C8 and C12 was only seen for the leuconostocs. The results show that the potential role of heterofermentative bacteria in cheese flavour formation involves specific esterase and aminotransferase activities.

Selection of Acid-tolerant and Hetero-fermentative Lactic Acid Bacteria Producing Non-proteinaceous Anti-bacterial Substances for Kimchi Fermentation

Twenty-three strains of Leuconostoc species and 45 strains of Weissella species inhibiting the growth of Lactobacillus sakei, one of the most populous lactic acid bacteria in over-ripened kimchi, were isolated from kimchi in our previous study. Among these hetero-fermentative 68 strains, Leuconostoc mesenteroides CK0128, Weissella cibaria CK0633, and W. cibaria KK0797 exhibited a relatively high survival rate in MRS medium, which was adjusted to pH 4.3 using an acid mixture consisting of acetic and lactic acids, and produced a large amount of exopolysaccharides. The culture supernatants of 3 strains were fractionated by a molecular weight cutter and lyophilized. The fractions with a molecular weight smaller than 3,000 Da showed antagonistic activity against Staphylococcus aureus and Lb. sakei. The anti-bacterial substances were very stable to heat treatments (, 15 min) and active at acidic conditions below pH 5. -Amylase, lipase, and proteolytic enzymes (proteinase K and pepsin) did not affect their activities. These non-proteinaceous anti-bacterial substances inhibited the growth of several food pathogens.

Metatranscriptomic analysis of lactic acid bacterial gene expression during kimchi fermentation

Barcode-based 16S rRNA gene pyrosequencing showed that the kimchi microbiome was dominated by six lactic acid bacteria (LAB), Leuconostoc (Lc.) mesenteroides, Lactobacillus (Lb.) sakei, Weissella (W.) koreensis, Lc. gelidum, Lc. carnosum, and Lc. gasicomitatum. Therefore, we used completed genome sequences of representatives of these bacteria to investigate metatranscriptomic gene-expression profiles during kimchi fermentation. Total mRNA was extracted from kimchi samples taken at five time points during a 29day-fermentation. Nearly all (97.7%) of the metagenome sequences that were recruited on all LAB genomes of GenBank mapped onto the six LAB strains; this high coverage rate indicated that this approach for assessing processes carried out by the kimchi microbiome was valid. Expressed mRNA sequences (as cDNA) were determined using Illumina GA IIx. Assignment of mRNA sequences to metabolic genes using MG-RAST revealed the prevalence of carbohydrate metabolism and lactic acid fermentation. The mRNA sequencing reads were mapped onto genomes of the six LAB strains, which showed that Lc. mesenteroides was most active during the early-stage fermentation, whereas gene expression by Lb. sakei and W. koreensis was high during later stages. However, gene expression by Lb. sakei decreased rapidly at 25days of fermentation, which was possibly caused by bacteriophage infection of the Lactobacillus species. Many genes related to carbohydrate transport and hydrolysis and lactate fermentation were actively expressed, which indicated typical heterolactic acid fermentation. Mannitol dehydrogenase-encoding genes (mdh) were identified from all Leuconostoc species and especially Lc. mesenteroides, which harbored three copies (two copies on chromosome and one copy on plasmid) of mdh with different expression patterns. These results contribute to knowledge of the active populations and gene expression in the LAB community responsible for an important fermentation process.

Dynamics of starter, adjunct non-starter lactic acid bacteria and propionic acid bacteria in low-fat and full-fat Dutch-type cheese

The microbial dynamics of Dutch-type cheeses differing in starter (commercial DL starter or single strain of Lactococcus lactis ssp. cremoris), adjunct (Lactobacillus or Propionibacterium) and fat contents (10% or 28% fat) were investigated by culture-dependent and culture-independent analysis. The cheese microbiota was dominated by the adjunct Lactobacillus after 4 weeks of ripening and the fat content did not influence the microbial diversity. The Leuconostoc sp., presumably from the DL starter, was detected in cheeses made with added Lactobacillus plantarum and Lactobacillus rhamnosus and was not detected in cheese made with added Lactobacillus paracasei after 4 and 7 weeks. No Lactobacillus spp. were detected in cheese with added Propionibacterium, while Leuconostoc was the only species detected. In cheeses made with Lc. lactis ssp. cremoris as starter, the Lactobacillus microbiota was similar to the cheese milk microbiota after 24 h while after 4 weeks different species of Lactobacillus and Leuconostoc were detected.

Nature of Tomatoes Microflora under Storage

Aims: The aim of this study is to determine microbial load of tomatoes including microbial species it constitute under storage with particular reference to raw and canned tomatoes. Study Design: Random sampling of Tomatoes, from selected sources in Ondo State, Nigeria. Place and Duration of Study: Sample: Akungba-Akoko and some communities in Ondo State. Analysis at Microbiology Laboratory, Adekunle Ajasin University, Akungba-Akoko. August, 2011 to July, 2012. Methodology: Pour plate techniques were used to enumerate microbial load of the samples. Discrete isolated colonies from these sources were sub-cultured using streak plate method to get purified cultures for the study. The bacterial isolates were Gram stained while fungal species were examined using lactophenol cotton blue stain. Standard microbiological methods were subsequently used to identify the tomatoes microflora. Result: The bacterial load observed during the study ranged from 1 x 10 in CT3 to 38 x 10 in CT2 which are the canned sample sources. Similar range of 10 to 21 cfu/mL was recorded for the raw tomatoes. Microorganisms generally encountered include eight bacteria species such as, Leuconostoc Species, Pediococcus species, Staphylococcus species, Planococcus species, Micrococcus Species, Bacillus Species, Streptococcus species and Clostridium species. Similarly, three fungal species were encountered during the study. These include Saccharomyces species, Aspergillus species and Neurospora species. Conclusion: The microbial load and isolates from raw (local) tomatoes sources are at close range to those isolated from the canned tomatoes sources which still shows the Research Article American Journal of Experimental Agriculture, 3(1): 89-101, 2013 90 natural protective lactic acid components of this food source. Exposure of canned tomato to air gives room for deterioration by microorganisms as shown in this study. So, both raw and canned tomato should be well protected from contaminants to limit proliferation of spoilage organisms coupled with improvement of canning Technology. Similarly, the nature of microorganism encountered can be used to determine some food preservation systems in the study area.