Microbiological aspects of the traditional Travnik/Vlasic cheese manufactured in Bosnia and Herzegovina.

The microbiological aspects of traditional Travnik/Vlasic cheese was investigated. The cheese was made traditionally, from raw sheep milk at three small farms (A, B, C) on Mountain Vlasic. The microbiological quality of the cheese was examined during three stages of ripening (5, 30, 60 days) and followed during three seasons (3 years). Twenty-seven samples of cheese were collected and analyzed for the aerobic mesophilic count, yeasts and molds, coliforms and microorganisms from the group Staphylococcus spp. Average values determined for the number of investigated groups of microorganisms of all cheese samples throughout three different stages, seasons, and small farms were: aerobic mesophilic bacteria 8.03 log10 cfu·g-1, yeasts and molds 3.63 log10 cfu·g-1, coliforms 5.16 log10 cfu g-1, and microorganisms from the group Staphylococcus spp. 4.49 log10 cfu g-1. ANOVA showed that experimental factor ripening stage (days) had a significant effect on all testing parameters. Results obtained with this study indicate that hygiene during the production of traditional products must be increased in order to assure high quality of the final products.

Longitudinal dynamics of the bovine udder microbiota.

BACKGROUND: In recent years, the number of studies concerning microbiota of the intramammary environment has increased rapidly due to the development of high-throughput sequencing technologies that allow mapping of microbiota without culturing. This has revealed that an environment previously thought to be sterile in fact harbours a microbial community. Since this discovery, many studies have investigated the microbiota of different parts of the udder in various conditions. However, few studies have followed the changes that occur in the udder microbiota over time. In this study, the temporal dynamics of the udder microbiota of 10 cows, five with a low somatic cell count (SCC, SCC < 100,000 cells/mL) and five with a high SCC (SCC > 100,000 cells/mL), were followed over 5 months to gather insights into this knowledge gap. RESULTS: Analysis of the temporal changes in the microbial composition of milk from udders with a low SCC revealed a dynamic and diverse microbiota. When an imbalance due to one dominating genus was recorded, the dominant genus quickly vanished, and the high diversity was restored. The genera dominating in the samples with a high SCC remained the dominant genera throughout the whole sampling period. These cows generally displayed a heightened SCC or an intramammary infection in at least one quarter though-out the sampling period. CONCLUSION: Our results show that the bovine udder has a diverse microbiota, and that the composition and diversity of this community affects udder health with regards to SCC. Understanding what influences the composition and stability of this community has important implications for the understanding, control, and treatment of mastitis.

A core microbiota dominates a rich microbial diversity in the bovine udder and may indicate presence of dysbiosis.

The importance of the microbiome for bovine udder health is not well explored and most of the knowledge originates from research on mastitis. Better understanding of the microbial diversity inside the healthy udder of lactating cows might help to reduce mastitis, use of antibiotics and improve animal welfare. In this study, we investigated the microbial diversity of over 400 quarter milk samples from 60 cows sampled from two farms and on two different occasions during the same lactation period. Microbiota analysis was performed using amplicon sequencing of the 16S rRNA gene and over 1000 isolates were identified using MALDI-TOF MS. We detected a high abundance of two bacterial families, Corynebacteriaceae and Staphylococcaceae, which accounted for almost 50% of the udder microbiota of healthy cows and were detected in all the cow udders and in more than 98% of quarter milk samples. A strong negative correlation between these bacterial families was detected indicating a possible competition. The overall composition of the udder microbiota was highly diverse and significantly different between cows and between quarter milk samples from the same cow. Furthermore, we introduced a novel definition of a dysbiotic quarter at individual cow level, by analyzing the milk microbiota, and a high frequency of dysbiotic quarter samples were detected distributed among the farms and the samples. These results emphasize the importance of deepening the studies of the bovine udder microbiome to elucidate its role in udder health.

Production of organic flavor compounds by dominant lactic acid bacteria and yeasts from Obushera, a traditional sorghum malt fermented beverage.

Single and mixed starter cultures of lactic acid bacteria (LAB): Weissella confusa MNC20, Lactobacillus plantarum MNC21, Lactococcus lactis MNC24 and Lactobacillus fermentum MNC34 and yeasts: Issatchenkia orientalis MNC20Y and Saccharomyces cerevisiae MNC21Y were used to produce Obushera, a fermented sorghum beverage. Microbial counts, pH, sugars, organic acids, and volatile compounds in starter culture and spontaneous fermentations were monitored during 48 hrs. Maximum counts of LAB (8.4-9.4 log cfu g-1) and yeasts (7.5 ± 0.1 cfu g-1) starter cultures were attained in 6-48 hrs. Weissella confusa, Lc. lactis, and Lb. fermentum showed possible acid sensitivity while I. orientalis produced surface films. LAB starter cultures and their combinations with S. cerevisiae lowered pH from 5.83 to <4.5 (3.50-4.13) in a shorter time (12 hrs) than spontaneous fermentations (24 hrs). Lactococcus lactis and W. confusa metabolized glucose the fastest (p < .05) during the first 6 hrs. Lactobacillus fermentum, Lb. plantarum, and S. cerevisiae utilized glucose and maltose concurrently. Lactobacillus plantarum and S. cerevisiae additionally utilized fructose. S. cerevisiae metabolized sugars the fastest (p < .05) during the first 12-24 hrs. Lactobacillus plantarum and W. confusa produced the highest (p < .05) amounts of lactate (5.43 g kg-1) and diacetyl (9.5 mg kg-1), respectively. LAB also produced acetate, ethanol, acetaldehyde, acetone, and acetoin. Coculturing LAB with S. cerevisiae reduced (p < .05) lactate and diacetyl yield. Yeasts produced high amounts of acetaldehyde and methyl alcohols. Issatchenkia orientalis produced higher (p < .05) amounts of 2-methy-1-propanol and 3-methyl-1-butanol than S. cerevisiae. Combinations of LAB with S. cerevisiae produced a profile flavor compounds close to that of spontaneously fermented Obushera. These combinations can be adopted for controlled fermentation of Obushera and related fermented cereal products.

Enterotoxin Gene Profile and Molecular Characterization of Staphylococcus aureus Isolates from Bovine Bulk Milk and Milk Products of Tigray Region, Northern Ethiopia.

Staphylococcal food poisoning (SFP) is an important foodborne disease worldwide, and milk and milk products are commonly associated with SFP outbreaks. The objectives of this study were to investigate the distribution of staphylococcal enterotoxin (se) genes in Staphylococcus aureus from raw cow's milk and milk products and to assess their genetic background with the spa typing method. Of the 549 samples (297 bulk milk and 162 milk product samples) collected from Tigray region, Northern Ethiopia, 160 (29.1%) were positive for S. aureus, of which 82 (51%) were found to harbor se genes by a modified multiplex PCR. Nine se genes were identified: sea (n = 12), seb (n = 3), sec (n = 3), sed(n = 4), seg (n = 49), seh (n = 2), sei (n = 40), sej (n = 1), and tsst-1 (n = 24). The classical type of genes accounted for 27%. Of the 82 enterotoxigenic isolates, 41.5 and 12.4% harbored two or more se genes, respectively. The highest gene association was observed between sei and seg, whereas sea and seb were always found together with the new types of se genes. Altogether, 18 genotypes of toxin genes were identified, and 33% of the samples contained > 5 log CFU ml(-1) S. aureus. spa typing identified 22 spa types and three novel spa sequences, which showed the high genetic diversity of the isolates. No apparent relationship was observed between spa type and se genes. Of the 25 spa types, 13 (52%) were from raw milk, 3 (12%) from milk products, and 9 (36%) from both types of sample. Types t314 (20.7%,n = 17), t458 (18.3%, n = 15), and t6218 (9.8%, n= 8) were the most common spa types identified and were widely distributed in three of the eight studied localities. This is the first study from the Tigray region to report the high distribution of enterotoxigenic S. aureus with a diversified genetic background from dairy food. The study may provide valuable data for microbial food safety risk assessment, molecular epidemiology, and phylogenetic studies of S. aureus in Ethiopia.

The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods.

Obushera includes four fermented cereal beverages from Uganda namely: Obutoko, Enturire, Ekitiribita and Obuteire, whose microbial diversity has not hitherto been fully investigated. Knowledge of the microbial diversity and dynamics in these products is crucial for understanding their safety and development of appropriate starter cultures for controlled industrial processing. Culture-dependent and culture-independent techniques including denaturating gradient gel electrophoresis (DGGE) and mixed DNA sequencing of polymerase chain reaction (PCR) amplified ribosomal RNA genes were used to study the bacteria and yeast diversity of Obushera. The pH dropped from 6.0-4.6 to 3.5-4.0 within 1-2 days for Obutoko, Enturire and Obuteire whereas that of Ekitiribita decreased to 4.4 after 4 days. Counts of lactic acid bacteria (LAB) increased from 5.0 to 11.0 log cfug(-1) and yeasts increased from 3.4 to 7.1 log cfug(-1) while coliform counts decreased from 2.0 to <1 log cfug(-1) during four days of fermentation. LAB and yeast isolates were identified by rRNA gene sequence analysis. LAB isolates included: Enterococcus spp., Lactobacillus (Lb.) plantarum, Lb. fermentum, Lb. delbrueckii, Lactococcus lactis, Leuconostoc lactis, Streptococcus (S.) infantarius subsp. infantarius, Pediococcus pentosaceus and Weisella (W.) confusa. DGGE indicated predominance of S. gallolyticus, S. infantarius subsp. infantarius, Lb. fermentum, Lb. delbrueckii, W. confusa, Lb. reuteri, Fructobacillus spp., L. lactis and L. lactis. Yeast isolates included Clavispora lusitaniae, Cyberlindnera fabianii, Issatchenkia orientalis and Saccharomyces cerevisiae. DGGE indicated predominance of S. cerevisiae in Obutoko, Enturire and Obuteire and also detected Pichia spp. and I. orientalis in Obutoko. Obushera produced in the laboratory was initially dominated by Enterobacteriaceae and later by Lactococcus spp. Enterobacteriaceae and Bacillus spp. were also detected in Ekitiribita. Development of starters for Obushera may require combinations of LAB and S. cerevisiae for Obutoko, Enturire and Obuteire and LAB for Ekitiribita.

Influence of cofermentation by amylolytic Lactobacillus plantarum and Lactococcus lactis strains on the fermentation process and rheology of sorghum porridge.

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera, were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus (G'), loss modulus (G″), phase angle (δ), and complex viscosity (η*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly (P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials.

FT-IR spectroscopy for identification of closely related lactobacilli.

Fourier Transform Infrared (FT-IR) spectroscopy was used to analyse 56 strains from four closely related species of Lactobacillus, L. sakei, L. plantarum, L. curvatus and L. paracasei. Hierarchical Cluster Analysis (HCA) was used to study the clusters in the data, but in the dendrogram, the spectra were not differentiated into four separate clusters corresponding to species. When the data were analysed with Partial Least Squares Regression (PLSR), the strains were differentiated into four clusters according to species. It was also possible to recognise strains that were incorrectly identified by conventional methods prior to the FT-IR analysis. PLSR was used to identify strains from three of the species, and the results were compared to two other multivariate methods, Soft Independent Modelling of Class Analogy (SIMCA) and K-Nearest Neighbour (KNN). The three methods gave equally good identification results. The results show that FT-IR spectroscopy in combination with PLSR, or other multivariate methods, is well suited for identification of Lactobacillus at the species level, even in quite large data sets.

Evaluation of the robustness of FT-IR spectra of lactobacilli towards changes in the bacterial growth conditions.

In order to use Fourier Transform Infrared (FT-IR) spectroscopy for identification of microorganisms on a routine basis, it is important that the spectra are robust against small, uncontrollable variations in the bacterial growth conditions. In this study, the effect of small variations in growth temperature, growth time, growth medium and atmospheric conditions on the separation of Lactobacillus based on their FT-IR spectra was investigated. The resulting spectra were shown to be robust against the variations in the cultivation conditions, and the separation of both strains and species was unaffected. Larger variations in the growth medium influenced only the separation of strains. FT-IR spectroscopy for identification of lactobacilli therefore seems to be robust against small variations in the cultivation conditions.

Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley.

A fermented probiotic maize porridge with high energy density and low viscosity was prepared, using maize flour and barley malt. The porridge was fermented with four probiotic strains (grown separately): Lactobacillus reuteri, Lb. acidophilus (LA5 and 1748) and Lb. rhamnosus GG. These strains were inoculated at two levels; to obtain approx. 7 or 6 log cfu g(-1) in the porridge at 0 h. The porridge was fermented for 24 h at 37 degrees C, and analysed for viable cell count, pH, organic acids, volatile aromatic compounds and sugar content. The inoculated cell concentration was shown to be particularly important during the first hours of the fermentation period, showing a delayed production of most metabolites in porridge inoculated with approx. 6 log cfu g(-1). Most strains reached maximum cell count after 12-h fermentation (7.2-8.2 log cfu g(-1)), with a pH below 4.0. Depending on the strain, lactic acid was produced in amounts ranging from 1360 to 4000 mg kg(-1). Lb. reuteri metabolised succinate, while pyruvate and small amounts of diacetyl were detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748. High amounts of diacetyl (6 mg kg(-1)) and acetoin (27 mg kg(-1)) were detected in porridge inoculated with Lb. rhamnosus GG. Porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, contained acetaldehyde, while both Lb. reuteri and Lb. rhamnosus GG reduced the acetaldehyde to ethanol. Lb. reuteri utilised both maltose and glucose as carbohydrate sources, while Lb. acidophilus LA5, Lb. acidophilus 1748 and Lb. rhamnosus GG utilised only glucose.

Growth and metabolism of selected strains of probiotic bacteria in milk.

Growth and metabolism of five probiotic strains with well-documented health effects were studied in ultra-high temperature (UHT) treated milk, supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lactobacillus rhamnosus GG, Lactobacillus reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 72 h at 37 degrees C and the samples were analysed for pH, log cfu ml(-1), volatile compounds, organic acids and carbon dioxide. The strains reduced pH from 6.7 to between 3.9 and 4.4 after 24 h of incubation. All strains attained viable cell counts above 8.7-9.18 log cfu ml(-1) after 6-16 h of incubation. The two Lb. acidophilus strains showed a stable level of viable cells during 12-72 h of incubation but the three other strains showed a reduction of 0.4-1.1 log cfu ml(-1) from 24 to 72 h of incubation. However, all strains showed cell levels between 7.8 and 8.7 log cfu ml(-1) after 72 h of incubation. After 48 h of incubation, the amount of lactic acid produced varied according to strain from 6949 to 14,000 mg kg(-1) and acetic acid produced varied from 0 to 6901 mg kg(-1). Three of the strains metabolised citrate but only low amounts of diacetyl and acetoin were detected within strains, 0.2-0.8 and 6.5-10 mg kg(-1), respectively. Carbon dioxide produced varied from 221 to 3942 mg kg(-1) and was connected to the citrate-fermenting ability of the strain used and their carbohydrate fermentation pathway. Three of the strains produced detectable levels of acetaldehyde and the concentration varied from 9.4 to 12.6 mg kg(-1) after 24 h of incubation. All five probiotic strains showed very different profiles of metabolites during fermentation; however, the two Lb. acidophilus strains were the most alike. Our findings show the importance of controlling the fermentation time since the probiotic strains produced different amounts of metabolic products according to fermentation time.

The role of interaction between yeasts and lactic acid bacteria in African fermented milks: a review.

Yeasts are present in indigenous African fermented milks in numbers up to log 8 cfu g(-1), together with a varied lactic acid bacteria (LAB) flora, and therefore potentially contribute to product characteristics. However, interaction between yeasts and LAB in these products has received little notice. In studies of indigenous fermented milk in Zimbabwe and Uganda, many samples contained more than one species of yeast, but Saccharomyces cerevisiae was most commonly isolated. Other frequent isolates were other species of Saccharomyces and several species of Candida. Most yeast isolates were lactose-negative but usually galactose-positive. Some strains assimilated lactate and citrate. The growth in milk of strains of yeasts and LAB, isolated from naturally soured milk in Zimbabwe, and their interaction when selected pairs of strains were grown together has been studied. Interactions were shown by the significantly different amounts of certain metabolites produced, such as acetaldehyde and malty aldehydes, when co-cultures were compared to pure cultures. Preliminary sensory acceptance tests did not show, however, that milks made from a co-culture with Candida kefyr and LAB were preferable to the pure LAB culture. Further work is still needed to elucidate the reactions that may be taking place in fermented milk between varying LAB and yeast populations. The potential for use as starter cultures depends on various aspects, including the final product being prepared. The role of other microorganisms in naturally fermented milk also needs to be studied.