Kefir Grains Change Fatty Acid Profile of Milk during Fermentation and Storage.

Several studies have reported that lactic acid bacteria may increase the production of free fatty acids by lipolysis of milk fat, though no studies have been found in the literature showing the effect of kefir grains on the composition of fatty acids in milk. In this study the influence of kefir grains from different origins [Rio de Janeiro (AR), Viçosa (AV) e Lavras (AD)], different time of storage, and different fat content on the fatty acid content of cow milk after fermentation was investigated. Fatty acid composition was determined by gas chromatography. Values were considered significantly different when p<0.05. The highest palmitic acid content, which is antimutagenic compost, was seen in AV grain (36.6g/100g fatty acids), which may have contributed to increasing the antimutagenic potential in fermented milk. Higher monounsaturated fatty acid (25.8 g/100g fatty acids) and lower saturated fatty acid (72.7 g/100g fatty acids) contents were observed in AV, when compared to other grains, due to higher Δ9-desaturase activity (0.31) that improves the nutritional quality of lipids. Higher oleic acid (25.0 g/100g fatty acids) and monounsaturated fatty acid (28.2g/100g fatty acids) and lower saturated fatty acid (67.2g/100g fatty acids) contents were found in stored kefir relatively to fermented kefir leading to possible increase of antimutagenic and anticarcinogenic potential and improvement of nutritional quality of lipids in storage milk. Only high-lipidic matrix displayed increase polyunsaturated fatty acids after fermentation. These findings open up new areas of study related to optimizing desaturase activity during fermentation in order to obtaining a fermented product with higher nutritional lipid quality.

Probiotic potential of selected lactic acid bacteria strains isolated from Brazilian kefir grains.

A total of 34 lactic acid bacteria isolates from 4 different Brazilian kefir grains were identified and characterized among a group of 150 isolates, using the ability to tolerate acidic pH and resistance to bile salts as restrictive criteria for probiotic potential. All isolates were identified by amplified ribosomal DNA restriction analysis and 16S rDNA sequencing of representative amplicons. Eighteen isolates belonged to the species Leuconostoc mesenteroides, 11 to Lactococcus lactis (of which 8 belonged to subspecies cremoris and 3 to subspecies lactis), and 5 to Lactobacillus paracasei. To exclude replicates, a molecular typing analysis was performed by combining repetitive extragenic palindromic-PCR and random amplification of polymorphic DNA techniques. Considering a threshold of 90% similarity, 32 different strains were considered. All strains showed some antagonistic activity against 4 model food pathogens. In addition, 3 Lc. lactis strains and 1 Lb. paracasei produced bacteriocin-like inhibitory substances against at least 2 indicator organisms. Moreover, 1 Lc. lactis and 2 Lb. paracasei presented good total antioxidative activity. None of these strains showed undesirable enzymatic or hemolytic activities, while proving susceptible or intrinsically resistant to a series of clinically relevant antibiotics. The Lb. paracasei strain MRS59 showed a level of adhesion to human Caco-2 epithelial cells comparable with that observed for Lactobacillus rhamnosus GG. Taken together, these properties allow the MRS59 strain to be considered a promising probiotic candidate.

Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese.

The Brazilian market for dairy products made from goat milk is increasing despite the seasonality of production and naturally small milk production per animal, factors that result in high-priced products and encourage fraud. In Brazil, no official analytical method exists for detecting adulteration of goat dairy products with cow milk. The aim of this study was to design a strategy to investigate the adulteration of frescal (fresh) goat cheeses available in the Rio de Janeiro retail market, combining analysis of cheese composition and the perception of adulteration by consumers. Commercial goat cheeses were tested by using a duplex PCR assay previously designed to authenticate cheeses, by targeting the mitochondrial 12S ribosomal RNA genes of both species simultaneously. The PCR test was able to detect 0.5% (vol/vol) cow milk added during goat cheese formulation. The analysis of 20 locally produced goat cheeses (20 lots of 4 brands) showed that all were adulterated with cow milk, even though the labels did not indicate the addition of cow milk. To estimate the ability of consumers to perceive the fraudulent addition of cow milk, a triangle test was performed, in which cheeses formulated with several different proportions of goat and cow milk were offered to 102 regular consumers of cheese. Detection threshold analysis indicated that almost half of the consumers were able to perceive adulteration at 10% (vol/vol) cow milk. Effective actions must be implemented to regulate the market for goat dairy products in Brazil, considering the rights and choices of consumers with respect to their particular requirements for diet and health, preference, and cost.

Microbiological and chemical characteristics of Brazilian kefir during fermentation and storage processes.

The microbial community composition and chemical characteristics of a Brazilian milk kefir sample produced during its manufacturing and refrigerated storage were investigated by culture-dependent and -independent methods and HPLC. Lactococcus lactis ssp. cremoris and ssp. lactis, Leuconostoc mesenteroides, Acetobacter lovaniensis, and Saccharomyces cerevisiae were isolated, whereas the detected bands on denaturing gel gradient electrophoresis corresponded to Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacillus parakefiri, and S. cerevisiae. After fermentation, lactic acid bacteria were present at levels of 10 log units, whereas acetic acid bacteria and yeast were present at levels of 7.8 and 6 log units, respectively. The lactic acid bacteria and yeast counts remained constant, whereas acetic acid bacteria counts decreased to 7.2 log units during storage. From fermentation to final storage, the pH, lactose content and citric acid of the kefir beverage decreased, followed by an increase in the concentrations of glucose, galactose, ethanol, and lactic, acetic, butyric, and propionic acids. These microbiological and chemical characteristics contribute to the unique taste and aroma of kefir. This research may serve as a basis for the future industrial production of this beverage in Brazil.

Assessment of the microbial diversity of Brazilian kefir grains by PCR-DGGE and pyrosequencing analysis.

The microbial diversity and community structure of three different kefir grains from different parts of Brazil were examined via the combination of two culture-independent methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and pyrosequencing. PCR-DGGE showed Lactobacillus kefiranofaciens and Lactobacillus kefiri to be the major bacterial populations in all three grains. The yeast community was dominated by Saccharomyces cerevisiae. Pyrosequencing produced a total of 14,314 partial 16S rDNA sequence reads from the three grains. Sequence analysis grouped the reads into three phyla, of which Firmicutes was dominant. Members of the genus Lactobacillus were the most abundant operational taxonomic units (OTUs) in all samples, accounting for up to 96% of the sequences. OTUs belonging to other lactic and acetic acid bacteria genera, such as Lactococcus, Leuconostoc, Streptococcus and Acetobacter, were also identified at low levels. Two of the grains showed identical DGGE profiles and a similar number of OTUs, while the third sample showed the highest diversity by both techniques. Pyrosequencing allowed the identification of bacteria that were present in small numbers and rarely associated with the microbial community of this complex ecosystem.

Prevalência de tuberculose bovina em animais e rebanhos abatidos em 2009 no estado de Mato Grosso, Brasil / Prevalence of bovine tuberculosis in herds and animals slaughtered in 2009 in the State of Mato Grosso, Brazil

Estimou-se a prevalência de tuberculose em bovinos, e em seus respectivos rebanhos, abatidos em 2009 no estado de Mato Grosso, utilizando como diagnóstico confirmatório o exame bacteriológico e o molecular a partir de fragmentos de tecidos lesionados. Nos sete abatedouros selecionados, detentores de serviço de inspeção federal (SIF), foram inspecionados 41.193 bovinos, sadios ao exame ante mortem, procedentes de 492 rebanhos originários de 85 (60%) municípios mato-grossenses. Um total de 198 carcaças apresentaram lesões suspeitas. Apenas três carcaças (3/198) apresentaram lesões confirmadas como tuberculosas pelos diagnósticos laboratoriais. A prevalência aparente de tuberculose bovina em animais e rebanhos abatidos no estado de Mato Grosso foi de 0,007% [IC 95% = -0,001%; 0,016%] e 0,61% [IC 95% = -0,08%; 1,30%], respectivamente. O estado do Mato Grosso possui, naturalmente, um status sanitário considerado de baixa prevalência.

The prevalence of bovine tuberculosis in cattle, and its herds, slaughtered in 2009 in the state of Mato Grosso, Brazil, was estimated using bacteriological analysis and molecular test, from fragments of injured tissues as well as direct DNA templates. 41,193 cattle, which appeared healthy in the ante mortem examination, from seven selected slaughterhouses, under Brazilian federal inspection services (SIF), were inspected. The animals were from 492 herds located in 85 (60%) different cities of Mato Grosso. A total of the 198 carcasses had suspicious lesions. Three carcasses (3/198) had lesions that were found to be tuberculous in laboratory diagnosis. The apparent prevalence of bovine tuberculosis in the total number of animals and in herds slaughtered in Mato Grosso was 0.007% [IC 95% = -0.001%; 0.016%] and 0.61% [IC 95% = -0.08%; 1.30%], respectively. The sanitation status demonstrated in Mato Grosso indicates the progress in this state toward the eradication of bovine tuberculosis.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members.

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members

Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37 percent of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89 percent similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.

Shared control of maltose and trehalose utilization in Candida utilis.

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 mol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast.

Shared control of maltose and trehalose utilization in Candida utilis

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 æmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70 percent of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed

Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae.

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar beta-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed.

Catabolite inactivation of trehalose synthesis during growth of yeast on maltose

1. The effects of catbolite inactivation upon the trehalose pathway linked to maltose utilization were investigated in Saccharomyces cerevisiae. Mutant strains devoid of UDPG-trehalose synthase activity were used in this study. 2. Trehalose accumulation was also susceptible to catabolite inactivation as has been reported for the carrier protein, one of the components of the maltose system. Reversibility was only achieved when incubation with glucose did not exceed 5 min and was dependent upon protein sunthesis