Lactobacillus delbrueckii subsp. bulgaricus F17 and Leuconostoc lactis H52 supernatants delay the decay of strawberry fruits: a microbiome perspective.

Strawberries are vulnerable to physical injuries and microbial invasion. To explore if beneficial lactic acid bacteria can improve the shelf life and edible quality of postharvest strawberry fruits, the effects of Lactobacillus delbrueckii subsp. bulgaricus (ital.) F17 (F17) and Leuconostoc lactis (ital.) H52 (H52) inoculation on the strawberry microbial community structure and saleable characteristics were examined by bacterial 16S rRNA and fungal ITS sequencing techniques. Lactobacillus (ital.) F17 and Leuconostoc lactis (ital.) H52 isolated from the traditional fermented yak milk in the Qinghai-Tibetan Plateau were used as the potential probiotic inocula. Samples from treated strawberries stored at 25 °C for 0, 12, 24, 48, and 72 hours were analyzed for their pH, weight loss percentage, decay percentage, total soluble solid content (SSC) and microbial counts, and for microbiome community diversity and canonical correspondence analysis. The results showed that F17 and H52 did not only significantly reduce the weight loss and decay percentage of strawberry fruits, but also delayed the decrease of the total SSC and pH (P < 0.05). In addition, F17 and H52 significantly inhibited the growth and colonization of aerobic mesophilic bacteria, yeast, mold and coliform bacteria. In particular, by comparing the microbiota composition of the samples, F17 significantly inhibited Pantoea, Mycospherella, unclassified_Pleosporales, Aureobasidium and Phoma at the genus level, whereas H52 inhibited Bacillus, Streptophyta, Mycospherella, Aureobasidium and Phoma. Moreover, analysis of alpha and beta diversity revealed that F17 and H52 had a significantly greater inhibitory effect on bacterial species compared to fungi. The results of canonical correspondence analysis revealed that the total SSC and pH were positively correlated with bacteria, whereas the decay percentage, weight loss percentage and total SSC were positively associated with fungi. Additionally, Podosphaera, Hanseniaspora, Botrytis and unclassified_Pleosporales were positively correlated with strawberry fruit decay and weight loss percentage. As a general result, Lactobacillus F17 and Leuconostoc lactis H52 have the potential to promote biological preservation, which is economically important to reduce the loss due to strawberry spoilage.

Using Goat's Milk, Barley Flour, Honey, and Probiotic to Manufacture of Functional Dairy Product.

Stirred yogurt manufactured using probiotic culture which usually called Rayeb milk in the Middle East region is one of the most important functional fermented milk products. To increase the health and functionality properties to this product, some ingredients like fruits, cereal, and whey protein are used in production. This study was carried out to prepare functional Rayeb milk from goat's milk, barley flour (15%) and honey (4%) mixtures using ABT culture. Also, vanilla and cocoa powder were used as flavorings. Adding barley flour and honey to goat's milk increased curd tension and water-holding capacity and decreased coagulation time and susceptibility to syneresis. The values of carbohydrate, total solids, dietary fiber, ash, total protein, water soluble nitrogen, total volatile fatty acids, unsaturated fatty acids, oleic, linoleic, α-linolenic acids, and antioxidant activity were higher in Rayeb milk supplemented with barley flour and honey than control. The viabilities of Lactobacillus acidophilus and Bifidobacterium lactis Bb12 (Chr. Hansen's Lab A/S) increased in fortified Rayeb milk. The recommended level of 107 cfu g-1 of bifidobacteria as a probiotic was exceeded for these samples. Addition of vanilla (0.1%) or cocoa powder (0.5%) improved the sensory properties of fortified Rayeb milk.

The changes of proteins fractions shares in milk and fermented milk drinks.

BACKGROUND: The aim of this research was to observe the changes which take place in the electrophoretic picture of milk proteins after pasteurisation and inoculation with different starter cultures (both traditional and probiotic). After incubation, the yoghurt, kefir, acidified milk, fermented Bifidobacterium bifidum drink and Lactobacillus acidophillus drink were chilled for 14 days to observe the changes which occurred. METHODS: The research materials were raw and pasteurised milk, as well as fermented milk- based drinks. The raw milk used for research came from Polish Holstein-Fresian black and white cows. The milk was sampled 3 times and divided into 5 parts, each of which was pasteurised at 95°C for 10 min and then cooled for inoculation: yoghurt to 45°C, kefir and acidified milk to 22°C and drinks with Bifidobacterium bifidum and Lactobacillus acidophillus to 38°C. Milk was inoculated with lyophilised, direct vat starter cultures, in an amount equal to 2% of the working starter. For the production of fermented drinks, the subsequent starters were applied: "YC-180" Christian Hansen for yoghurt, "D" Biolacta-Texel-Rhodia for kefir, CH-N--11 Christian Hansen for acidified milk, starter by Christian Hansen for the probiotic Bifidobacterium bifidum milk, starter by Biolacta-Texel-Rhodia for the probiotic Lactobacillus acidophillus milk. The analyses were conducted in raw, pasteurised and freshly fermented milk as well as in milk drinks stored for 14 days. The total solid content was estimated by the drying method; the fat content by the Gerber method; the lactose content by the Bertrand method; the protein content by the Kjeldahl method with Buchi apparatus; the density of milk was measured with lactodensimeter; acidity with a pH-meter; and potential acidity by Soxhlet-Henkl method (AOAC, 1990). The electrophoretic separation of proteins in raw and pasteurised milk, as well as in freshly produced milk drinks and those stored for 14 days, was performed with SDS-PAGE (on polyacrylamid gel) basing on procedure described by Laemmli (1970). RESULTS: It was shown that, in comparison with raw milk, the pasteurised milk had smaller amounts of αs-, ß- and κ-casein, whereas the shares of γ-casein and peptides were greater, and there were no changes in immunoglobulin, α-lactalbumin or ß-lactoglobulin levels, which indicated that hydrolysis of caseins had occurred. In all freshly fermented milk drinks, a drop in αs- and ß-casein was observed relative to raw milk. An increase in peptides and γ-casein was also noticed (with the exception of acidified milk). There were differences in α-lactalbumin and ß-lactoglobulin levels between the different drinks: raw, pasteurised or freshly fermented milk. It was shown that kefir, compared to the other drinks, had the lowest levels of αs- and ß-casein, α-lactalbumin and of peptides, as well as the highest level of γ-casein, which is evidence of an increased rate of hydrolysis in that drink. It was stated that, during the storage of fermented milk drinks, the levels of lactoferrin, serum albumin and peptides significantly increased whereas the content of κ-casein diminished. The proportions of serum albumin and lactoferrin in fermented milk drinks increased relative to raw milk and/or after storage, which is evidence of aggregation of proteins of low molecular mass into bigger conglomerates. CONCLUSIONS: The observed differences between fermented milks, including during chilled storage, in the amounts of individual proteins proves the different proteolytic abilities of starter cultures used in fermented milk production. α-lactoalbumin and ß-lactoglobulin are, besides caseins, the most allergenic milk proteins. So, kefir, because of its low α-lactoalbumin content, and Bifidobacterium bifidum milk, with the lowest content of ß-lactoglobulin, were the most advantageous and least allergenic drinks examined.

Modelling and predicting the simultaneous growth of Escherichia coli and lactic acid bacteria in milk.

Modelling and predicting the simultaneous competitive growth of Escherichia coli and starter culture of lactic acid bacteria (Fresco 1010, Chr. Hansen, Hørsholm, Denmark) was studied in milk at different temperatures and Fresco inoculum concentrations. The lactic acid bacteria (LAB) were able to induce an early stationary state in E. coli The developed model described and tested the growth inhibition of E. coli (with initial inoculum concentration 10(3) CFU/mL) when LAB have reached maximum density in different conditions of temperature (ranging from 12 ℃ to 30 ℃) and for various inoculum sizes of LAB (ranging from approximately 10(3) to 10(7) CFU/mL). The prediction ability of the microbial competition model (the Baranyi and Roberts model coupled with the Gimenez and Dalgaard model) was first performed only with parameters estimated from individual growth of E. coli and the LAB and then with the introduced competition coefficients evaluated from co-culture growth of E. coli and LAB in milk. Both the results and their statistical indices showed that the model with incorporated average values of competition coefficients improved the prediction of E. coli behaviour in co-culture with LAB.

Microbial cell-free extracts as sources of enzyme activities to be used for enhancement flavor development of ewe milk cheese.

Freeze-dried cell-free extracts (CFE) from Lactobacillus casei LC01, Weissella cibaria 1XF5, Hafnia alvei Moller ATCC 51815, and Debaryomyces hansenii LCF-558 were used as sources of enzyme activities for conditioning the ripening of ewe milk cheese. Compared with control cheese (CC), CFE did not affect the gross composition and the growth of the main microbial groups of the cheeses. As shown through urea-PAGE electrophoresis of the pH 4.6-soluble nitrogen fraction and the analysis of free AA, the secondary proteolysis of the cheeses with CFE added was markedly differed from that of the CC. Compared with CC, several enzyme activities were higher in the water-soluble extracts from cheeses made with CFE. In agreement, the levels of 49 volatile compounds significantly differentiated CC from the cheeses made with CFE. The level of some alcohols, ketones, sulfur compounds, and furans were the lowest in the CC, whereas most aldehydes were the highest. Each CFE seemed to affect a specific class of chemical compounds (e.g., the CFE from H. alvei ATCC 51815 mainly influenced the synthesis of sulfur compounds). Apart from the microbial source used, the cheeses with the addition of CFE showed higher score for acceptability than the control cheese. Cheese ripening was accelerated or conditioned using CFE as sources of tailored enzyme activities.

Yeast species and genetic heterogeneity within Debaryomyces hansenii along the ripening process of traditional ewes' and goats' cheeses.

The yeasts present during the ripening process of ewes' and goats' cheeses produced in a small traditional dairy in Mediterranean Spain were isolated and identified. Five hundred and thirty strains pertaining to eleven yeast species representing eight genera were identified using molecular methods. Debaryomyces hansenii was the yeast species most frequently isolated in all cheeses. Other yeast species commonly found in dairy products were present at the first maturing weeks. Two yeast species Trichosporon coremiiforme and Trichosporon domesticum have been reported in cheeses for the first time, and Meyerozyma guilliermondii has been newly isolated from goats' cheeses. Yeast species composition changed greatly along the process; although, D. hansenii dominated at the end of ripening in all cheeses. Most yeast isolates were able to hydrolyze casein and fatty acid esters. One hundred and eighty seven D. hansenii isolates were genotyped by PCR amplification of M13 satellites and an UPGMA dendrogram was constructed. The majority of isolates were grouped in 5 clusters while 7 profiles were represented by 1-3 isolates. These results demonstrate the genetic heterogeneity present in D. hansenii strains isolated from raw milk cheeses.

Qualidade da água utilizada na higienização dos tanques de granelização de leite cru do município de Rio Pomba, MG / Quality of water used in cleaning tanks of raw milk from Rio Pomba, MG

O objetivo deste trabalho foi avaliar a qualidade da água utilizada na higienização dos tanques de resfriamento de leite cru do Município de Rio Pomba, MG. Amostras de água provenientes de 15 tanques de expansão individuais e comunitários foram coletadas as septicamente no período de maio a agosto de 2010 e transportadas para os laboratórios de análise físico-química e microbiologia do IF Sudeste MG, campus Rio Pomba. Foram realizadas análises microbiológicas de micro-organismos mesófilos aeróbios, coliformes termotolerantes e Escherichia coli e análises físico-químicas de pH, dureza, cloro residual, alcalinidade, cloretos, cor e turbidez. Verificou-se que a contagem de micro-organismos mesófilos aeróbios variou entre < 1,0 x 101 UFC/mL estimado e 1,2 x 103 UFC/mL, que 53,3% e 33,3%das amostras apresentaram, respectivamente, coliformes termo tolerantes e E. coli. Constatou-se também que 26,6% das amostras encontravam-se com valores de pH abaixo do recomendado. Além disso, verificou-se que todas as amostras apresentaram valores abaixo de 50 mg/L de CaC03 em relação à dureza e ausência de cloro residual. Em relação à alcalinidade, constatou-se variação entre 8 mg/L e 44 mg/L de CaC03. Na análise de cor, observou-se que todas as amostras de água apresentaram 5 unidades Hansen e em relação à turbidez, constatou-se que todas as amostras apresentaram-se dentro do limite estabelecido pela legislação. Portanto, torna-se imprescindível o tratamento da água a fim de obter leite de qualidade, bem como a implantação de programas que visem o treinamento dos produtores para aplicação de boas práticas higiênicas na propriedade rural.

The aim of this study was to evaluate the quality of water used in cleaning of cooling tanks of rawmilk from Rio Pomba, Minas Gerais, Brazil. Water samples from 15individual and community expansion tanks were aseptically collected during the period from May to August 2010 and transported to the physical chemistry and microbiology laboratories of IF Sudeste MG. It was developed microbiologicalanalysis of mesophilic aerobic microorganisms, coliforms and Escherichia coli and physical chemical analyses of pH, hardness, residual chlorine, alkalinity, chloride, color and turbidity. It was verifiedthat count of mesophilic aerobic micro-organisms ranged from <1.0 x 10' CFU/mL estimated to 1.2 x 103CFU/mL, and that 53.3% and 33.3% of samples presented fecal coliform and E. coli, respectively. It wasalso found that 26.6% of samples presented pH values below recommendation. Besides, it was verifiedthat all samples had values below 50 mg/L CaCO3 in relation to hardness and absence of residual chlorine. In relation to alkalinity, it was detected variation between 8 mg/L to 44 mg/L CaCO3. In color analysis, it was found that all samples of water showed five Hansen units for turbidity, been within the limits of Brazil legislation. Therefore, it is essential to treat water in order to obtain milk of better quality, as well as implementation of programs that provide training to producers to implement good hygienic practices at the farm.

Isolation and identification of the microbiota of Danish farmhouse and industrially produced surface-ripened cheeses.

For studying the microbiota of four Danish surface-ripened cheeses produced at three farmhouses and one industrial dairy, both a culture-dependent and culture-independent approach were used. After dereplication of the initial set of 433 isolates by (GTG)5-PCR fingerprinting, 217 bacterial and 25 yeast isolates were identified by sequencing of the 16S rRNA gene or the D1/D2 domain of the 26S rRNA gene, respectively. At the end of ripening, the cheese core microbiota of the farmhouse cheeses consisted of the mesophilic lactic acid bacteria (LAB) starter cultures Lactococcus lactis subsp. lactis and Leuconostoc mesenteorides as well as non-starter LAB including different Lactobacillus spp. The cheese from the industrial dairy was almost exclusively dominated by Lb. paracasei. The surface bacterial microbiota of all four cheeses were dominated by Corynebacterium spp. and/or Brachybacterium spp. Brevibacterium spp. was found to be subdominant compared to other bacteria on the farmhouse cheeses, and no Brevibacterium spp. was found on the cheese from the industrial dairy, even though B. linens was used as surface-ripening culture. Moreover, Gram-negative bacteria identified as Alcalignes faecalis and Proteus vulgaris were found on one of the farmhouse cheeses. The surface yeast microbiota consisted primarily of one dominating species for each cheese. For the farmhouse cheeses, the dominant yeast species were Yarrowia lipolytica, Geotrichum spp. and Debaryomyces hansenii, respectively, and for the cheese from the industrial dairy, D. hansenii was the dominant yeast species. Additionally, denaturing gradient gel electrophoresis (DGGE) analysis revealed that Streptococcus thermophilus was present in the farmhouse raw milk cheese analysed in this study. Furthermore, DGGE bands corresponding to Vagococcus carniphilus, Psychrobacter spp. and Lb. curvatus on the cheese surfaces indicated that these bacterial species may play a role in cheese ripening.

Culture-independent detection of microorganisms in traditional Slovakian bryndza cheese.

In order to investigate the microflora of Slovakian bryndza cheese (a cheese containing unpasteurized or pasteurized ewes' milk component) by a culture-independent method, DNA was extracted directly from 7 bryndza samples and analysed by an innovative method. Using the universal prokaryotic and fungal primers, ribosomal DNA internal transcribed spacer (ITS) regions with variable length were amplified. The standard universal reverse primer L1 aligning to bacterial 23s rDNA was found unsuitable for some lactic acid bacteria and other species based on in silico analysis. Therefore, L1 primer was replaced by a combination of novel primers GplusR and GminusR aligning to the adjacent, more conserved DNA region. The amplification profiles were visualised by both standard electrophoresis and by fluorescent capillary gel electrophoresis. From representative samples, major amplicons were excised from the gel, cloned and sequenced. Sequencing revealed that the samples contained Lactobacillus delbrueckii, Lactobacillus brevis, Streptococcus thermophilus, Lactococcus lactis, Lactococcus raffinolactis, Streptococcus macedonicus, Leuconostoc pseudomesenteroides, Debaromyces hansenii, Mucor fragilis, Yarrowia lipolytica and Galactomyces geotrichum. These results represent an extension of the knowledge on the microflora of Slovakian bryndza cheese. The introduced automated ribosomal DNA intergenic spacer analysis of the bacterial and fungal genomes proved to be very effective in the application of studying microflora of cheese.

Modelling the effect of the starter culture on the growth of Staphylococcus aureus in milk.

The competitive growth of a starter culture of lactic acid bacteria (Fresco 1010, Chr. Hansen, Hørsholm, Denmark) and Staphylococcus aureus was studied in milk. The lactic bacteria (LAB) were able to induce an early stationary state in S. aureus. The developed model highlights that the growth of S. aureus is inhibited when the LAB have reached a critical density. The model was tested in different conditions of temperature (from 12 degrees to 25 degrees C), for various inoculum sizes of LAB and S. aureus. The results show that the model accurately quantifies the kinetics of S. aureus as a function of the starter culture.

Stability of microbial communities in goat milk during a lactation year: molecular approaches.

The microbial communities in milks from one herd were evaluated during 1-year of lactation, using molecular methods to evaluate their stability and the effect of breeding conditions on their composition. The diversity of microbial communities was measured using two approaches: molecular identification by 16S and 18S rDNA sequencing of isolates from counting media (two milks), and direct identification using 16S rDNA from clone libraries (six milks). The stability of these communities was evaluated by counting on selective media and by Single Strand Conformation Polymorphism (SSCP) analysis of variable region V3 of the 16S rRNA gene and variable region V4 of the 18S rRNA gene. One hundred and eighteen milk samples taken throughout the year were analyzed. Wide diversity among bacteria and yeasts in the milk was revealed. In addition to species commonly encountered in milk, such as Lactococcus lactis, Lactococcus garvieae, Enterococcus faecalis, Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus equorum, Micrococcus sp., Kocuria sp., Pantoea agglomerans and Pseudomonas putida, sequences were affiliated to other species only described in cheeses, such as Corynebacterium variabile, Arthrobacter sp., Brachybacterium paraconglomeratum, Clostridium sp. and Rothia sp. Several halophilic species atypical in milk were found, belonging to Jeotgalicoccus psychrophilus, Salinicoccus sp., Dietza maris, Exiguobacterium, Ornithinicoccus sp. and Hahella chejuensis. The yeast community was composed of Debaryomyces hansenii, Kluyveromyces lactis, Trichosporon beigelii, Rhodotorula glutinis, Rhodotorula minuta, Candida pararugosa, Candida intermedia, Candida inconspicua, Cryptococcus curvatus and Cryptococcus magnus. The analyses of microbial counts and microbial SSCP profiles both distinguished four groups of milks corresponding to four periods defined by season and feeding regime. The microbial community was stable within each period. Milks from winter were characterized by Lactococcus and Pseudomonas, those from summer by P. agglomerans and Klebsiella and those from autumn by Chryseobacterium indologenes, Acinetobacter baumanii, Staphylococcus, Corynebacteria and yeasts. However, the composition of the community can vary according to factors other than feeding. This study opens new investigation fields in the field of raw milk microbial ecology.

Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese.

The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64+/-1 log(10) cfu ml(-1) in milk to 0.65+/-1 log(10) cfu g(-1) in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein. G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.

Microbial succession of Debaryomyces hansenii strains during the production of Danish surfaced-ripened cheeses.

Surface-ripened cheeses of the Danbo type were analyzed for the presence of yeasts with special emphasis on Debaryomyces hansenii. Samples were taken from pasteurized milk, brine, and inoculation slurries and from cheese surfaces during ripening at a Danish dairy. D. hansenii was found to be the dominant yeast species throughout the ripening period, whereas other yeast species such as Trichosporon spp., Rhodotorula spp., and Candida spp. were found in minor concentrations during early stages of cheese ripening. Mitochondrial DNA RFLP was used to show that several strains of D. hansenii were present from the onset of ripening. Thereafter, a microbial succession among the strains took place during the ripening. After 3 d of ripening, only one strain was found. This particular strain was found to be dominant in 16 additional batches of surface-ripened cheeses. We investigated the cause of the observed microbial succession by determining the variation in strains with regard to their ability to grow on lactate and at different pH and NaCl concentrations. The strains were shown to vary in their ability to grow on lactate. In a full factorial design at three levels with factor levels close to the actual levels on the cheese surface, differences in pH and NaCl tolerances were observed. The dominant strain was found to be better adapted than other strains to the environmental conditions existing in surface-ripened cheeses during production [e.g., lactate as the main carbon source, pH 5.5 to 6.0 and NaCl concentrations of 7 to 10% (wt/vol)].

Pigment producing yeasts involved in the brown surface discoloration of ewes' cheese.

The appearance of a brown surface discoloration on Portuguese ewes' cheese has never previously been reported on. The regular occurrence of this defect over the past few years has caused serious financial losses to producers, which has led to growing interest in its study. This paper describes a preliminary approach to the problem, based on the hypothesis that pigment producing yeasts are involved. From a group of 51 yeast strains isolated from a number of brown cheese rinds, it was possible to distinguish four pigment producing groups: group I (12 strains), produced an extracellular brown pigment from tyrosine and alkalised the tested media; group II (21 strains), produced a diffusible, reddish-brown pigment from resorcinol and alkalised the tested media; group III (three strains), alkalised the tested media without producing any pigments; group IV (15 strains), neither produced pigments nor alkalised the media. Yarrowia lipolytica and Candida catenulata type strains were also tested and their behaviour was similar to the strains in groups I and IV, respectively. The Filobasidiella neoformans type strain was distinct from all the other groups. The identification methods used for some strains in groups I, II and III suggest that Yarrowia lipolytica species is common to all strains in group I, and that Debaryomyces hansenii is present in both groups II and III. A study of the effect of several metal ions on the production of the brown pigment from tyrosine indicated Mn2+ to be a strong activator. Evidence is provided suggesting that the browning process may be related to tyrosine Yarrowia lipolytica metabolism.