Evolution of fermenting microbiota in tarhana produced under controlled technological conditions

The purpose of this study was to evaluate the evolution of lactic acid bacteria (LAB) and yeasts during the fermentation of tarhana produced with some pasteurised ingredients and carried out at 30 and 40 °C. The chemical parameters were those typical for tarhana production. Coliform bacteria were not detected during fermentation, while LAB and yeasts were in the range 10⁷-10⁸ colony forming units (CFU) g⁻¹. Plate counts showed an optimal development of both fermenting microbial groups and the differences in cell concentrations were not significant (P > 0.05). LAB were isolated during fermentation and grouped on the basis of phenotypic and polymorphic characteristics. LAB isolates were identified by a combined genetic approach consisting of 16S/23S rRNA intergenic spacer region (ITS) and partial 16S rRNA gene sequencing as Pediococcus acidilactici, Lactobacillus plantarum and Lactobacillus brevis. Hence, the pasteurisation of the vegetable ingredients, excluded wheat flour, enhanced the hygienic conditions of tarhana without influencing the normal evolution of LAB. However, the fermentation at 40 °C favoured pediococci, while the production at 30 °C was mainly characterised by lactobacilli. Yeasts, identified by the restriction fragment length polymorphism (RFLP) of the 5.8S ITS rRNA gene, were mainly represented by the species Saccharomyces cerevisiae in both productions.     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.     

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H′_L = 0.4 and H′_Y = 0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.     

Study of the behaviour of Lactobacillus plantarum and Leuconostoc starters during a complete wheat sourdough breadmaking process

The acidification properties, metabolic activity and technological performance of four individual Lactobacillus plantarum or Leuconostoc freeze-dried starters were investigated during a complete wheat sourdough breadmaking process including 0.2 g/100 g baker's yeast. Microbiological contents (lactic acid bacteria and yeasts), acidification characteristics (pH and total titratable acidity), soluble carbohydrates (maltose, glucose and fructose) and fermentative end-products (lactic and acetic acids, ethanol) contents were evaluated during both sourdough and corresponding bread dough fermentation. Biochemical and technological analysis of the resulting bread products are also presented. Some differences among strains in acidification properties and soluble carbohydrates availability were outlined both in sourdough and bread dough. Each individual Leuconostoc or Lb. plantarum starter was able to produce a characteristic fermentation and was found to ensure the production of breads with overall satisfactory acceptance.     

Evaluation of the functional potential of Weissella and Lactobacillus isolates obtained from Nigerian traditional fermented foods and cow's intestine

The characterisation of 24 lactic acid bacteria (LAB) isolates from Nigerian traditional fermented dairy foods, including some cow's intestine isolates, was conducted in order to select isolates for potential use as probiotics. LAB isolates were identified by partial sequencing the 16S rRNA gene as belonging to the species Lactobacillus paracasei, Lactobacillus brevis and mainly Weissella confusa. At the end of a characterisation process, 2 L. paracasei and 2 W. confusa isolates were selected, and their resistance to a simulated gastrointestinal digestion and their ability to adhere to eukaryotic cell lines were assessed. The survival to the simulated gastrointestinal passage was higher when bacterial suspensions were made in skimmed milk (2.0 ± 0.8 log units reduction) or at the simulated gastric juice pH 3 (2.7 ± 0.9 log units reduction) than at pH 2.0 (5.5 ± 0.7 log units reduction). Adhesion of LAB to both intestinal and vaginal epithelial models was comparable or higher than that of the reference Lactobacillus rhamnosus GG. However, some of the isolates increased the adhesion of the pathogen Escherichia coli LMG2092 to HT-29 and HeLa monolayers. Overall, isolates L. paracasei UI14 and W. confusa UI7 are good candidates for further studying potential benefits that support their use as probiotics. This is one of the few articles reporting the characterisation and the probiotic potential of Weissella, although more studies are needed in order to establish their safety for potential probiotic applications.     

Properties and acceptability of Som-fug, a Thai fermented fish mince, inoculated with lactic acid bacteria starters

Effect of inoculation of different lactic acid bacteria (LAB) on the fermentation and quality of Som-fug from bigeye snapper was investigated. Som-fug inoculated with Pediococcus acidilactici at 10⁴ CFU/g (PA104) had a greater acceptability than those inoculated with Lactobacillus plantarum and Pediococcus pentosaceus at either 10⁴ or 10⁶ CFU/g and the control (without inoculum). During fermentation, PA104 exhibited a higher rate of fermentation than the control as indicated by the greater rate of pH drop and lactic acid production (p<0.05). Based on pH desired (pH 4.5), the fermentation was completed within 48 and 36 h for the control and PA104, respectively. Due to higher acid production rate, the textural development of PA104 was more pronounced. Som-fug inoculated with LAB generally exhibited higher hardness and adhesiveness than the control (p<0.05), when assessed by texture profile analysis (TPA). From the result, inoculation with P. acidilactici resulted in a reduction of fermentation time and improved the quality of Som-fug. Therefore, P. acidilactici can be used as a potential starter for Som-fug fermentation.     

Enhancement of survival of probiotic and non-probiotic lactic acid bacteria by yeasts in fermented milk under non-refrigerated conditions

The effects of yeasts on the survival of probiotic and non-probiotic lactic acid bacteria (LAB) were studied in fermented milk under non-refrigerated conditions (30 °C) with a view to develop ambient-stable fermented milk with live LAB. Five yeasts tested (Saccharomyces bayanus, Williopsis saturnus var. saturnus, Yarrowia lipolytica, Candida kefyr and Kluyveromyces marxianus) enhanced the survival of Lactobacillus bulgaricus (but not Streptococcus thermophilus) in a mixed yoghurt culture in yoghurt by ~ 10² to 10⁵-fold. Seven yeasts examined (Candida krusei, Geotrichum candidum, Pichia subpelliculosa, Kloeckera apiculata, Pichia membranifaciens, Schizosaccharomyces pombe and Y. lipolytica) improved the survival of Lactobacillus rhamnosus in fermented milk by ~ 10³ to 10⁶-fold. W. saturnus var. saturnus enhanced the survival of Lactobacillus acidophilus, L. rhamnosus (probiotic) and Lactobacillus reuteri by up to 10⁶-fold, but the same yeast failed to improve the survival of Lactobacillus johnsonii (probiotic), S. thermophilus and L. bulgaricus in fermented milk. These results provide definitive evidence that yeasts possess stability-enhancing effects on LAB and that the specific effects of yeasts on LAB stability vary with yeasts as well as with LAB. However, the molecular mechanism of such interaction of yeasts with LAB remains to be found.     

Microorganisms associated with Maari, a Baobab seed fermented product

A microbiological study was carried out on Baobab fermented seeds (Maari) obtained from 4 different production sites in Burkina Faso (Mansila, Toulfé, Ouagadougou and Gorgadji).A total of 390 representative isolates comprising 251 aerobic mesophilic bacteria (AMB) and 139 lactic acid bacteria (LAB) were isolated and identified to species level using a combination of pheno- and genotypic methods including conventional morphological analysis, carbohydrate fermentation profiling, rep-PCR ((GTG)₅-fingerprinting) and 16S rRNA gene sequencing.The fermentation of Baobab seeds was initiated by the AMB identified as Bacillus subtilis (82% of AMB isolates) and Staphylococcus sciuri (18% of AMB isolates). No lactic acid bacteria were isolated at the beginning of the process. After 24 h fermentation time, Enterococcus faecium appeared in the fermenting seeds and remained until the end of the fermentation, as the predominant LAB.In Maari collected from retail outlets the AMB count ranged from 6.7 log10 CFU/g to 10 log10 CFU/g while the LAB load ranged from 4.4 log10 CFU/g to 9.9 log10 CFU/g. The AMB were identified as belonging to genus Bacillus (12 species), Staphylococcus (3 species) and one species of Aerococcus, Macrococcus, Leifsonia, Kurthia, Proteus, Acinetobacter and Globicatella, respectively. A putatively novel, previously undescribed Corynebacterium sp. was also found. E. faecium was the dominant LAB in all investigated retail samples except one sample dominated by Pediococcus acidilactici.     

Selection and Use of Phytate-Degrading LAB to Improve Cereal-Based Products by Mineral Solubilization During Dough Fermentation

ABSTRACT:  New producers of phytate-degrading enzymes, especially lactic acid bacteria (LAB), were used to improve mineral solubilization during dough fermentation. In all, among strains from different sources by microorganisms (150 lactic acid bacteria, 36 yeasts), 38 (24%) exhibited a clear zone around the colonies by hydrolyzing hexacalcium phytate contained in solid medium. When phytase-positive strains from plate assay were tested for phytase activity in liquid medium, 6 of the strains (37%) exhibited phytate-degrading activity in at least one of the 3 different media used. Of the LAB, the highest phytase values were found for  Enterococcus faecium  A86 (0.74 U/mL) and  Lactobacillus plantarum  H5 (0.71 U/mL). Two different starter cultures obtained by combinations of phytase-positive (phy+:  L. plantarum  H5 and L3,  Leuconostoc gelidum  A16, and  E. faecium  A86) or phytase-negative (phy−:  L. gelidum  LM249,  L. plantarum  H19, and  L. plantarum  L8) selected LAB strains, were used to measure mineral concentrations of iron, zinc, and manganese during dough fermentation. Although the 2 kinds of starter showed similar acidic values, the presence of phytate-degrading LAB strains increased mineral solubilization in comparison to the starter phy−.     

Isolation of lactic acid bacteria starters from <Emphasis Type="Italic">Jeung</Emphasis>-<Emphasis Type="Italic">pyun</Emphasis> for sourdough fermentation

Lactic acid bacteria (LAB) are key for the fermentation of sourdoughs to improve the quality and nutritive value of bread. The aim of this study was to isolate the LAB starter for sourdough fermentation from Jeung-pyun, a Korean traditional rice cake. Among the twenty two LAB screened, five isolates were selected based on exo-polysaccharide production. Among them, three isolates showed cell growth greater than 8.5 Log CFU/g, maximum increase in the volume of dough, and dextran concentration up to 0.16%. During the sourdough fermentation, pH and total titratable acidity (TTA) were changed, as the three isolates synthesized lactic acid and acetic acid with fermentation quotients less than 2.0. They were identified as Leuconostoc lactis EFEL005, Lactobacillus brevis EFEL004, and Le. citreum EFEL006. They displayed good fermentation properties (growth, dextran production, pH, and TTA) in dough and they are regarded as potential starters to be used in sourdough fermentation.     

Adaptability of lactic acid bacteria and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava and use of competitive strains as starters

The adaptability of lactic acid bacteria (LAB) and yeasts to sourdoughs prepared from cereals, pseudocereals and cassava was investigated using PCR-DGGE and bacteriological culture combined with rRNA gene sequence analysis. Sourdoughs were prepared either from flours of the cereals wheat, rye, oat, barley, rice, maize, and millet, or from the pseudocereals amaranth, quinoa, and buckwheat, or from cassava, using a starter consisting of various species of LAB and yeasts. Doughs were propagated until a stable microbiota was established. The dominant LAB and yeast species were Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus spicheri, Issatchenkia orientalis and Saccharomyces cerevisiae. The proportion of the species within the microbiota varied. L. paralimentarius dominated in the pseudocereal sourdoughs, L. fermentum, L. plantarum and L. spicheri in the cassava sourdough, and L. fermentum, L. helveticus and L. pontis in the cereal sourdoughs. S. cerevisiae constituted the dominating yeast, except for quinoa sourdough, where I. orientalis also reached similar counts, and buckwheat and oat sourdoughs, where no yeasts could be detected. To assess the usefulness of competitive LAB and yeasts as starters, the fermentations were repeated using flours from rice, maize, millet and the pseudocereals, and by starting the dough fermentation with selected dominant strains. At the end of fermentation, most of starter strains belonged to the dominating microbiota. For the rice, millet and quinoa sourdoughs the species composition was similar to that of the prior fermentation, whereas in the other sourdoughs, the composition differed.     

Taxonomic and molecular characterization of lactic acid bacteria and yeasts in nunu,a Ghanaian fermented milk product

Produced from raw unpasteurized milk, nunu is a spontaneously fermented yoghurt-like product made in Ghana and other parts of West Africa. Despite the importance of nunu in the diet of many Africans, there is currently only limited information available on the microorganisms associated with nunu processing. With the aim of obtaining a deeper understanding of the process and as a first step towards developing starter cultures with desired technological properties for nunu production, a microbiological characterization of nunu processing in three different towns in the Upper East region of Ghana, namely Bolgatanga, Paga and Navrongo, was carried out. Lactic acid bacteria (LAB) and yeasts associated with nunu processing were isolated and identified using a combination of pheno- and genotypic methods including morphological and carbohydrate fermentation tests, (GTG)₅-based rep-PCR, multiplex PCR, and 16S and 26S rRNA gene sequencing. The LAB counts during nunu processing increased from 4.5 ± 0.4 log cfu/ml at 0 h to 8.7 ± 1.8 log cfu/ml at 24 h of fermentation while yeasts counts increased from 2.8 ± 1.2 log cfu/ml at 0 h to 5.8 ± 0.5 log cfu/ml by the end of fermentation. Lactobacillus fermentum was the dominant LAB throughout the fermentations with Lactobacillus plantarum and Leuconostoc mesenteroides playing prominent roles during the first 6–8 h of fermentation as well. Less frequently isolated LAB included Lactobacillus helveticus, Enterococcus faecium, Enterococcus italicus, Weissella confusa and a putatively novel Lactococcus spp. The yeasts involved were identified as Candida parapsilosis, Candida rugosa, Candida tropicalis, Galactomyces geotrichum, Pichia kudriavzevii and Saccharomyces cerevisiae with P. kudriavzevii and S. cerevisiae being the dominant yeast species.     

Use of a starter culture of lactic acid bacteria in plaa-som, a Thai fermented fish

Plaa-som is a Thai fermented fish prepared from freshwater fish and various ingredients. In this study, two strains of lactic acid bacteria (LAB) isolated from natural plaa-som fermentation were used as starter cultures: Lactobacillus plantarum IFRPD P15 and Lactobacillus reuteri IFRPD P17. These strains were used as a mixed starter culture for plaa-som using an air-drying method (laminar airflow) with sterilized rice grains as the filler. This method produced a suitable starter culture, which was maintained at 4 °C for more than 20 weeks. LAB were the dominant bacteria in the starter culture and produced high acidity from 24 h until the end of fermentation. This resulted in decreased pH in plaa-som. L. plantarum IFRPD P15 was dominant as an acidity producer, whereas L. reuteri IFRPD P17 showed an ability to suppress and eliminate pathogenic bacteria such as Escherichia coli within 24 h. The use of a single starter culture for plaa-som resulted in incomplete suppression of pathogenic bacteria and elimination of E. coli. Thus, L. plantarum IFRPD P15 and L. reuteri IFRPD P17 have great potential for use as a mixed starter culture in plaa-som fermentation and may possibly help to reduce fermentation time.     

Prevalence and impact of single-strain starter cultures of lactic acid bacteria on metabolite formation in sourdough

Flavour of type II sourdoughs is influenced by the ingredients, processing conditions, and starter culture composition. It is, however, not fully clear to what extent different sourdough lactic acid bacteria (LAB) contribute to flavour. Therefore, two types of flour (rye and wheat) and different LAB starter culture strains were used to prepare sourdoughs, thereby leaving the yeast microbiota uncontrolled. All LAB starter culture strains tested were shown to be prevalent and to acidify the flour/water mixture to pH values between 3.1 and 3.9 after 24 h of fermentation. Multiple aldehydes, alcohols, ketones, and carboxylic acids were produced by the sourdough-associated microbiota throughout the fermentation period. Based on the organoleptic evaluation of breads produced with these sourdoughs, five LAB strains were selected to perform prolonged wheat and rye fermentations as to their capacity to result in an acidic (Lactobacillus fermentum IMDO 130101, Lactobacillus plantarum IMDO 130201, and Lactobacillus crustorum LMG 23699), buttermilk-like (Lactobacillus amylovorus DCE 471), or fruity flavour (Lactobacillus sakei CG1). Upon prolonged fermentation, higher metabolite concentrations were produced. For instance, L. sakei CG1 produced the highest amounts of 3-methyl-1-butanol, which was further converted into 3-methylbutyl acetate. The latter compound resulted in a fruity banana flavour after 48 h of fermentation, probably due to yeast interference. Rye fermentations resulted in sourdoughs richer in volatiles than wheat, including 3-methyl-1-butanol, 2-phenylethanol, and ethyl acetate.     

A review on traditional Turkish fermented non-alcoholic beverages: Microbiota,fermentation process and quality characteristics

Shalgam juice, hardaliye, boza, ayran (yoghurt drink) and kefir are the most known traditional Turkish fermented non-alcoholic beverages. The first three are obtained from vegetables, fruits and cereals, and the last two ones are made of milk. Shalgam juice, hardaliye and ayran are produced by lactic acid fermentation. Their microbiota is mainly composed of lactic acid bacteria (LAB). Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus paracasei subsp. paracasei in shalgam fermentation and L. paracasei subsp. paracasei and Lactobacillus casei subsp. pseudoplantarum in hardaliye fermentation are predominant. Ayran is traditionally prepared by mixing yoghurt with water and salt. Yoghurt starter cultures are used in industrial ayran production. On the other hand, both alcohol and lactic acid fermentation occur in boza and kefir. Boza is prepared by using a mixture of maize, wheat and rice or their flours and water. Generally previously produced boza or sourdough/yoghurt are used as starter culture which is rich in Lactobacillus spp. and yeasts. Kefir is prepared by inoculation of raw milk with kefir grains which consists of different species of yeasts, LAB, acetic acid bacteria in a protein and polysaccharide matrix. The microbiota of boza and kefir is affected from raw materials, the origin and the production methods.     

Investigation of Microbial Communities of Chinese Sourdoughs Using Culture‐Dependent and DGGE Approaches

Sourdough is typically characterized by the complex microbial communities mainly comprising of yeasts and lactic acid bacteria (LAB). The objective of this study was to explore the microbiota of Chinese traditional sourdoughs collected from different areas of China using culture‐dependent and denaturing gradient gel electrophoresis (DGGE) methods. A total of 131 yeasts, 2 molds, and 106 LAB strains were isolated and identified. Based on the culture‐dependent analysis, the populations of yeasts and LAB were at the level of 10⁵ to 10⁷ and 10⁶ to 10⁷ cfu/g, respectively. Similarly, the results of RT‐qPCR showed that the values of total yeasts and LAB populations were in the range of 10⁶ to 10⁷ and 10⁷ to 10⁸ copies/g, respectively. Using culture‐dependent method, a total of 7 yeasts, 2 molds and 7 LAB species were identified. Results showed that Saccharomyces cerevisiae and Lactobacillus plantarum were the predominant species among the yeasts and LAB microflora. Similarly, using PCR‐DGGE approach, 7 yeasts, 1 mold and 9 LAB species were detected. The yeast, S. cerevisiae, represented the predominant, while the yeast Candida tropicalis represented the subdominant species of the yeast community. Among the LAB community, Lactobacillus sanfranciscensis was the predominant species, while Lactococcus qarvieae, Enterococcus faecium, Lactobacillus delbrueckii and Enterococcus cecorum were among the less dominant species.     

Characterisation of the dry salted process for the production of the msayer, a traditional lemon aromatising condiment

Msayer is a dry salted lemon used as an aromatising condiment in many North-African food recipes. This preparation is obtained by adding coarse salt (10 g/100 g fresh lemons) to fresh cut lemons in containers kept under dark conditions away from air for around a month and a half. A survey of 52 days showed low initial uptake of citric acid and sugars from the formed liquid phase. Nevertheless, there were still sugars detected even at the end of the survey attesting for the low microbial activity as also shown by carbon dioxide production and microbial viable counts. Indeed, the microbial counts showed a steady decrease reaching 2 log units at the 52nd day of curing. Moulds were detected only at the beginning but at low numbers. No viable counts of lactic acid bacteria were observed on MRS medium despite detection of lactic acid (8 g/l). In contrast, yeasts seem to dominate in the msayer preparation. They count for almost all the microbial population especially after the first week. Six of these acid and halotolerant yeast isolates were identified, by sequence analysis of the amplified 5.8S rRNA and the two internal transcribed spacer regions, as Candida parapsislosis (2 isolates) and unclassified Saccharomycetales (4 isolates).     

Spontaneous fermentation of traditional sago starch in Papua New Guinea

Sago starch is an important dietary carbohydrate in lowland Papua New Guinea (PNG). An investigation was conducted to determine whether microbes play a role in its preservation using traditional methods. In 12 stored sago samples collected from PNG villages, lactic acid bacteria (LAB) were present (≥3.6 × 10⁴ cfu/g) and pH ranged from 6.8 to 4.2. Acetic and propionic acids were detected in all samples, while butyric, lactic and valeric acids were present in six or more. In freshly prepared sago, held in sealed containers in the laboratory at 30 °C, spontaneous fermentation by endogenous microflora of sago starch was observed. This was evident by increasing concentrations of acetic, butyric and lactic acids over 4 weeks, and pH reducing from 4.9 to 3.1: both LAB and yeasts were involved. Survival of potential bacterial pathogens was monitored by seeding sago starch with ∼10⁴/g of selected organisms. Numbers of Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus fell to <30/g within 7 days. Salmonella sp. was present only in low numbers after 7 days (<36/g), but Escherichia coli was still detectable after three weeks (>10²/g). Fermentation appeared to increase the storability and safety of the product.     

Isolation and characterisation of a novel Podoviridae-phage infecting Weissella cibaria N 22 from Nham, a Thai fermented pork sausage

A novel Podoviridae lactic acid bacteria (LAB) phage from Nham, a Thai fermented pork sausage, is reported. From a total of 36 samples, 41 isolates of LAB were obtained and employed as hosts for the isolation of phages. From these LAB, only one phage, designated Φ 22, was isolated. The lactic acid bacterial isolate named N 22, sensitive to phage Φ 22 infection was identified by an API 50 CHL kit and N 22’s complete sequence of the 16S rDNA sequence. BLASTN analysis of the 16S rDNA sequence revealed a 99% similarity to the 16S rDNA sequence of Weissella cibaria in the GenBank database. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of 92 × 50 nm and 27 nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage Φ 22 was not capable of infecting the other 40 isolates of LAB and referenced Weissella strains used. A one-step growth experiment showed that the latent period and burst size were estimated at 110 min and 55 phage particles/infected cell, respectively. Furthermore, the phage was infective over a wide range of pH (pH 5.0-8.0) and the D time of Φ 22 was calculated as 88 s at 70 °C and 15 s at 80 °C. Phage titers decreased below the detection limit (20 PFU/ml) after heating for more than 60 s at 80 °C, or 20 s at 90 °C or less than 10 s at 100 °C. The results from the study of Nham revealed that Φ 22 was active against the potential starter culture (W. cibaria N 22) for Nham fermentation. Phage infection could adversely affect the fermentation process of Nham by delaying acidification when using W. cibaria N 22 as a starter. However, the results from a sensory test revealed that the panelists did not detect any defects in the final products. This is the first report on the isolation of W. cibaria phage.     

Development and analysis of microbial characteristics of an acidulocomposting system for the treatment of garbage and cattle manure

An acidulocomposting system for the treatment of cattle manure with little emission of ammonia gas was developed, and the structure of its microbial community was investigated by denaturing gradient gel electrophoresis (DGGE) and clone library construction. An acidulocomposting apparatus (BC20, 20 L) was operated for 79 days to treat 2 kg (wet wt) of garbage per 1 or 2 days. On day 80 of operation, the substrate was changed from garbage to cattle manure (1 kg of beef cattle manure was added to the apparatus every 2 or 3 days), and the system continued operating from days 80 to 158. The compost in the vessel was under acidic conditions at pH 5.2–5.8, and ammonia emission was below the detectable level (< 5 ppm) throughout the period of cattle manure feeding. Total nitrogen and total carbon in the compost were 26–29 and 439 – 466 mg/g of dry weight, respectively, which are higher than those in general cattle manure compost. The main acids accumulated during operation were lactic and acetic. Sequencing analysis targeting the 16S rRNA gene revealed the stable dominance of the bacterial phylum Firmicutes, with a high proportion of the isolates belonging to the genus Bacillus. Using a culturing method with MRS agar, we isolated lactic acid bacteria (LAB) related to Pediococcus acidilactici, Weissella paramesenteroides, and Lactobacillus salivarius, indicating the existence of LAB in the system. These results indicate that acidulocomposting treatment of cattle manure is not accompanied by ammonia emission and that Bacillus and LAB may be the key components in the system.