Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.     

Fermentation characteristics and transit tolerance of probiotic Lactobacillus casei Zhang in soymilk and bovine milk during storage

Lactobacillus casei Zhang is a novel strain that was screened out of koumiss collected in Inner Mongolia, and our previous research showed that L. casei Zhang has health benefits such as cholesterol-reducing and immunomodulating effects. The fermentation characteristics of L. casei Zhang in soymilk and bovine milk and the transit tolerance of L. casei Zhang in fermented milk products during refrigerated storage for 28 d were assessed. A faster decrease in pH and faster growth of L. casei Zhang during fermentation were observed in soymilk compared with bovine milk at various inoculation rates, probably because of the low pH buffering capacity of soymilk. The fermented bovine milk samples had much higher final titratable acidity (TA) values (between 0.80 and 0.93%) than the soymilk samples (between 0.40 and 0.46%). Dramatic increases in TA values in the fermented soymilk samples during storage were observed, and the TA values of the fermented soymilk samples changed from <0.56% to values between 0.86 and 0.98%. On the other hand, only slight increases in TA were observed in the bovine milk samples during the 28 d of storage. The survival rates of freshly prepared cultures of L. casei Zhang in simulated gastric juice at pH 2.0 and 2.5 were 31 and 69%, respectively, and the delivery of L. casei Zhang through fermented soymilk and bovine milk significantly improved the viability of L. casei Zhang in simulated gastric transit. Lactobacillus casei Zhang showed good tolerance to simulated gastric juice and intestinal juice in the fermented soymilk and bovine milk samples, and maintained high viability (>10⁸ cfu/g) during storage at 4°C for 28 d. Our results indicated that both soymilk and bovine milk could serve as vehicles for delivery of probiotic L. casei Zhang, and further research is needed to elucidate the mechanism of the change in pH and TA of L. casei Zhang in fermented milk samples during fermentation and storage and to understand the difference between soy- and milk-based systems.     

Temporal gene expression and probiotic attributes of Lactobacillus acidophilus during growth in milk

Lactic acid bacteria have been used as starter strains in the production of fermented dairy products for centuries. Lactobacillus acidophilus is a widely recognized probiotic bacteria commonly added to yogurt and used in dietary supplements. In this study, a whole genome microarray was employed to monitor gene expression of L. acidophilus NCFM cells propagated in 11% skim milk during early, mid and late logarithmic phase, and stationary phase. Approximately 21% of 1,864 open reading frames were differentially expressed at least in one time point. Genes differentially expressed in skim milk included several members of the proteolytic enzyme system. Expression of prtP (proteinase precursor) and prtM (maturase) increased over time as well as several peptidases and transport systems. Expression of Opp1 (oligopeptide transport system 1) was highest at 4 h, whereas gene expression of Opp2 increased over time reaching its highest level at 12 h, suggesting that the 2 systems have different specificities. Expression of a 2-component regulatory system, previously shown to regulate acid tolerance and proteolytic activity, also increased during the early log and early stationary phases of growth. Expression of the genes involved in lactose utilization increased immediately (5 min) upon exposure to milk. The acidification activity, survival under storage conditions, and adhesion to mucin and Caco-2 tissue culture cells of selected mutants containing insertionally inactivated genes differentially expressed in the wild-type strain during growth in milk were examined for any potential links between probiotic properties and bacterial growth and survival in milk. Some of the most interesting genes found to be expressed in milk were correlated with signaling (autoinducer-2) and adherence to mucin and intestinal epithelial cells, in vitro.     

Short communication: Biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.     

Elaboration of a probiotic oblea from whey fermented using Lactobacillus acidophilus or Bifidobacterium infantis

A novel probiotic product was developed, which was formulated as an oblea (wafer-type dehydrated traditional Mexican dessert) using goat sweet whey fermented with Bifidobacterium infantis or Lactobacillus acidophilus. To obtain the probiotic oblea, the fermented whey was formulated with prebiotic carbohydrates (inulin and resistant starch) and gelatin, and the preparation was poured onto a polytetrafluoroethylene-coated nonstick baking pan, dried in a convection oven, and finally dehydrated at a low relative humidity and room temperature (23 ± 2°C). The amounts of prebiotic carbohydrates and gelatin to be used in the formulation were determined by a factorial experimental design. An untrained sensory panel evaluated 3 quality characteristics (film formation, homogeneity, and smoothness) in the final product. Three different drying temperatures were tested, namely, 40, 55, and 70°C. Bacterial survival at each temperature was determined by viable plate-counting. The best formulation, based on the quality characteristics tested, consisted of 58.33% (vol/vol) of fermented whey, 8.33% (vol/vol) of 6% (wt/vol) resistant starch dispersion, 16.66% (vol/vol) of 15% (wt/vol) inulin solution, and 16.66% (vol/vol) of a 10% (wt/vol) gelatin solution. Drying at 55 ± 2°C for 2.66 ± 0.22 h allowed for concentrations of probiotic bacteria above 9 log₁₀ cfu/g, which is above the minimum concentration required in a probiotic product.     

In vitro assessment of the gastrointestinal transit tolerance of taxonomic reference strains from human origin and probiotic product isolates of Bifidobacterium

Next to health promoting effects, the functional aspect of probiotic strains also involves their capacity to reach the colon as viable metabolically active cells. The present study aimed to assess the potential of 24 probiotic product isolates and 42 human reference strains of Bifidobacterium to survive gastrointestinal transit under in vitro conditions. The survival capacity of exponential and stationary phase cultures upon exposure to gastric and small intestinal juices was determined using a recently developed microplate-based assay in combination with the LIVE/DEAD BacLight Bacterial Viability kit. All 66 strains tested displayed a considerable loss in viability during exposure to an acidic pepsin containing solution (pH 2.0). Among the 10 taxa tested, cultures of B. animalis ssp. lactis appeared to be most capable to survive gastric transit. Although to a lesser extent, the presence of bile salts also affected the viability of most of the strains tested. Except for 3 strains, all 66 strains showed bile salt hydrolase activity using an agar-based assay. In contrast, the bifidobacterial strains used in this study appeared to possess a natural ability to survive the presence of pancreatin (pH 8.0). Although the effect was not significant, a slightly enhanced tolerance to gastrointestinal transit was observed when cells were in the stationary phase, especially when exposed to acid, compared with cells being in the exponential phase. Survival in the gastrointestinal tract appeared to be largely strain-dependent and hence implies that different strains will likely display a different behavior in functionality. The assay used in this study allows an initial assessment of strains for use as probiotic cultures prior to selecting potential candidate strains for further investigation in vivo.     

Short communication: Survival of the characteristic microbiota in probiotic fermented camel,cow, goat,and sheep milks during refrigerated storage

The objective of this study was to monitor the viability during storage of Lactobacillus acidophilus LA-5 (A), Bifidobacterium animalis ssp. lactis BB-12 (B), and Streptococcus thermophilus CHCC 742/2130 (T) in probiotic cultured dairy foods made from pasteurized camel, cow, goat, and sheep milks fermented by an ABT-type culture. The products manufactured were stored at 4°C for 42 d. Microbiological analyses were performed at weekly intervals. Streptococcus thermophilus CHCC 742/2130 was the most numerous culture component in all 4 products both at the beginning and at the end of storage. The viable counts of streptococci showed no significant decline in fermented camel milk throughout the entire storage period. The initial numbers of Lb. acidophilus LA-5 were over 2 orders of magnitude lower than those of Strep. thermophilus CHCC 742/2130. With the progress of time, a slow and constant decrease was observed in lactobacilli counts; however, the final viability percentages of this organism did not differ significantly in the probiotic fermented milks tested. The cultured dairy foods made from cow, sheep, and goat milks had comparable B. animalis ssp. lactis BB-12 counts on d 0, exceeding by approximately 0.5 log₁₀ cycle those in the camel milk-based product. No significant losses occurred in viability of bifidobacteria in fermented camel, cow, and sheep milks during 6 wk of refrigerated storage. In conclusion, all 4 varieties of milk proved to be suitable raw materials for the manufacture of ABT-type fermented dairy products that were microbiologically safe and beneficial for human consumption. It was suggested that milk from small ruminants be increasingly used to produce probiotic fermented dairy foods. The development of camel milk-based probiotic cultured milks appears to be even more promising because new markets could thus be conquered. It must be emphasized, however, that further microbiological and sensory studies, technology development activities, and market research are needed before such food products can be successfully commercialized.     

Genetic relatedness and virulence factors of bovine Staphylococcus aureus isolated from teat skin and milk

The objective of this study was to assess the role of teat skin colonization in Staphylococcus aureus intramammary infections (IMI) by evaluating genetic relatedness of Staph. aureus isolates from milk and teat skin of dairy cows using pulsed-field gel electrophoresis and characterizing the isolates based on the carriage of virulence genes. Cows in 4 known Staph. aureus-positive herds were sampled and Staph. aureus was detected in 43 quarters of 20 cows, with 10 quarters positive in both milk and skin (20 isolates), 18 positive only in milk, and 15 only on teat skin. Quarters with teat skin colonized with Staph. aureus were 4.5 times more likely to be diagnosed with Staph. aureus IMI than quarters not colonized on teat skin. Three main clusters were identified by pulsed-field gel electrophoresis using a cutoff of 80% similarity. All 3 clusters included both milk and skin isolates. The majority of isolates (72%) belonged to one predominant cluster (B), with 60% of isolates in the cluster originating from milk and 40% from teat skin. Genotypic variability was observed within 10 pairs (formed by isolates originating from milk and teat skin of the same quarter), where isolates in 5 out of the 10 pairs belonged to the same cluster. Forty-two virulence factors were screened using PCR. Some virulence factors were carried more frequently by teat skin isolates than by milk isolates or isolates from quarters with high somatic cell counts. Isolates in the predominant cluster B carried virulence factors clfA and clfB significantly more often than isolates in the minor clusters, which may have assisted them in becoming predominant in the herds. The present findings suggest that teat skin colonization with Staph. aureus can be an important factor involved in Staph. aureus IMI.     

Short communication: Development of a direct in vivo screening model to identify potential probiotic bacteria using Caenorhabditis elegans

Caenorhabditis elegans is an accepted model host to study host-bacteria interactions in the gut, in addition to being a simple model with which to study conserved aspects of biological signaling pathways in intestinal environments, because these nematode worms have similar intestinal cells to those of humans. Here, we used C. elegans to develop a new in vivo screening system for potential probiotic lactic acid bacteria (LAB). Initially, critical colonization ability of LAB strains isolated from Korean infant feces was screened in the worm intestinal tract over a period of 5 d. Furthermore, we investigated host health-promoting activities, including longevity-extending effects and immune-enhancing activities against foodborne pathogen infection. We identified 4 LAB strains that were highly persistent in the nematode gut and that significantly prolonged the longevity of C. elegans and improved the survival of C. elegans in response to infection by Staphylococcus aureus. The 4 LAB strains we identified showed resistance to acid and bile conditions, assimilated cholesterol, and were able to attach to a mucus layer. The 4 LAB isolates were identified as Lactobacillus plantarum using 16S rRNA sequencing analysis. Taken together, we developed a direct in vivo screening system using C. elegans to study host health-promoting LAB. Our system is simple, rapid, cost-effective, and reliable, and we anticipate that this system will result in the discovery of many more potential probiotic bacteria for dairy foods.     

Short communication: Viability of culture organisms in honey-enriched acidophilus-bifidus-thermophilus (ABT)-type fermented camel milk

The aim of this research was to monitor the survival during refrigerated storage of Lactobacillus acidophilus LA-5 (A), Bifidobacterium animalis ssp. lactis BB-12 (B), and Streptococcus thermophilus CHCC 742/2130 (T) in cultured dairy foods made from camel and, for comparison, cow milks supplemented with black locust (Robinia pseudoacacia L.) honey and fermented by an acidophilus-bifidus-thermophilus (ABT)-type culture. Two liters of dromedary camel milk and 2 L of cow milk were heated to 90°C and held for 10 min, then cooled to 40°C. One half of both types of milk was fortified with black locust honey at the rate of 5.0% (wt/vol), whereas the other half was devoid of honey and served as a control. The camel and cow milks with and without honey were subsequently inoculated with ABT-5 culture and were fermented at 37°C until a pH value of 4.6 was reached. Thereafter, the probiotic fermented milks were cooled to 15°C in ice water and were each separated into 18 fractions that were transferred in sterile, tightly capped centrifuge tubes. After 24 h of cooling at 8°C (d 0), the samples were stored at refrigeration temperature (4°C). Three tubes of all 4 products (i.e., fermented camel and cow milks with and without honey) were taken at each sampling time (i.e., following 0, 7, 14, 21, 28, and 35 d of storage), and the counts of characteristic microorganisms and those of certain spoilage microbes (yeasts, molds, coliforms, Escherichia coli) were enumerated. The entire experimental program was repeated twice. The results showed that addition of black locust honey at 5% to heat-treated camel and cow milks did not influence the growth and survival of starter streptococci during production and subsequent refrigerated storage of fermented ABT milks. In contrast, honey improved retention of viability of B. animalis ssp. lactis BB-12 in the camel milk-based product during storage at 4°C up to 5 wk. No spoilage organisms were detected in any of the samples tested in this study. In conclusion, supplementation of cultured dairy foods, especially those made from camel milk, with honey is recommended because honey is a healthy natural sweetener with a variety of beneficial microbiological, nutritional, and sensory properties.     

Molecular characteristics and virulence potential of Listeria monocytogenes isolates from Chinese food systems

In this study, we examined Listeria monocytogenes isolates from Chinese food sources in an attempt to gain further insights on the molecular characteristics and virulence potential of this important foodborne pathogen. Of the 88 L. monocytogenes food isolates recovered, 42 (47.7%) were of serovars 1/2a or 3a; 23 (26.1%) of serovars 1/2b or 3b; 15 (17.0%) of 1/2c or 3c; 6 (6.8%) of serovars 4b, 4d or 4e; and 2 (2.2%) of serovars 4a or 4c. In contrast to inlAB locus conserved in all serovars, internalin cluster between ascB and dapE varies with different serovars, with inlC2DE, inlGC2DE and inlGHE predominantly in serovars 1/2b or 4b, serovar 1/2a and serovar 1/2c. While inlF existed in all the inlGHE- and inlGC2DE-containing isolates but 17.4% of those having inlC2DE, lmo2026 existed in all the inlGHE-containing isolates but 20.0% of those bearing inlGC2DE, suggesting that inlF might have co-evolved with inlGC2DE and inlGHE while lmo2026 with inlGHE only. With the exception of serovar 4a isolate, most serovar isolates demonstrated remarkable ability to form plaques on L929 cells and produced significant mouse mortality irrespective of the internalin gene organization and whether an intact actA gene is present or not. These results indicate that majority of these food isolates may have the potential to cause human diseases if ingested via contaminated foods. Given that serovar 4b accounts for nearly half of human clinical listeriosis cases documented, the relative low proportion of serovar 4b food isolates suggests that this serovar is probably more tolerant of the adverse conditions in the host's stomach and/or more efficient in entering host cells than serovars 1/2a, 1/2b and 1/2c.     

Development of a real-time RT-PCR method for enumeration of viable Bifidobacterium longum cells in different morphologies

Viability of probiotic bacteria is traditionally assessed by plate counting which has several limitations, including underestimation of cells in aggregates or chains morphology. We describe a quantitative PCR (qPCR)-based method for an accurate enumeration of viable cells of Bifidobacterium longum NCC2705 exhibiting different morphologies by measuring the mRNA levels of cysB and purB, two constitutively expressed housekeeping genes. Three primer-sets targeting short fragments of 57-bp of cysS and purB and one 400-bp fragment of purB were used. Cell quantification of serially diluted samples showed a good correlation coefficient of R² 0.984 ± 0.003 between plate counts and qRT-PCR for all tested primer sets. Loss of viable cells exposed to a lethal heat stress (56 °C, 10, 20 and 30 min) was estimated by qRT-PCR and plate counts. No significant difference was observed using qRT-PCR targeting the 400-bp fragment of purB compared to plate counts indicating that this fragment is a suitable marker of cell viability. In contrast, the use of the 57-bp fragments led to a significant overestimation of viable cell counts (18 ± 3 and 7 ± 2 fold for cysB and purB, respectively). Decay of the mRNA fragments was studied by treatment of growing cells with rifampicin prior qRT-PCR. The 400-bp fragment of purB was faster degraded than the 57-bp fragments of cysB and purB. The 400-bp fragment of purB was further used to enumerate viable cells in aggregate state. Cell counts were more than 2 log₁₀ higher using the qRT-PCR method compared to plate counts.     

Comparative antioxidative characteristics of polysaccharide-enriched extracts from natural sclerotia and cultured mycelia in submerged fermentation of Inonotus obliquus

The potential antioxidant property of polysaccharide-enriched extracts from the natural fungal sclerotia and the cultured mycelia in submerged fermentation of Inonotus obliquus was evaluated using three antioxidant assays. The extracts from both the natural sclerotia and cultured mycelia including extra- and intra-cellular extracts were effective in scavenging hydroxyl radicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and in inhibiting lipid peroxidation. The content and type of polysaccharide present in the extracts from the natural sclerotia and the cultured mycelia were different. Both the extra- and intra-cellular extracts from the mycelia of I. obliquus with a higher polysaccharide content demonstrated a stronger free radical scavenger activity against hydroxyl radical and a greater inhibition of lipid peroxidation, shown as much lower IC50 values, than those from the natural sclerotia.     

Rumen pH and fermentation characteristics in dairy cows supplemented with Megasphaera elsdenii NCIMB 41125 in early lactation

This study investigated the effect of using the lactate-utilizing bacterium Megasphaera elsdenii NCIMB 41125 as a probiotic supplement on rumen fermentation and pH in dairy cows in the immediate postcalving period. Fourteen multiparous rumen-fistulated Holstein cows, blocked according to 305-d milk yield in the previous lactation, were used in a randomized complete block design. From d 1 to 28 postcalving, cows were fed ad libitum a total mixed ration with a forage to concentrate ratio of 392:608 and a starch concentration of 299 g/kg of dry matter. Treatments consisting of a minimum of 10¹⁰ cfu of Megasphaera elsdenii NCIMB 41125 or autoclaved M. elsdenii (placebo) were administered via the rumen cannula on d 3 and 12 of lactation (n = 7 per treatment). Mid-rumen pH was measured every 15 min, and eating and ruminating behaviors were recorded for 24 h on d 2, 4, 6, 8, 11, 13, 15, 17, 22, and 28. Rumen fluid for volatile fatty acid and lactic acid analysis was collected at 11 time points on each of d 2, 4, 6, 13, and 15. Yields of milk and milk protein and lactose were similar, but milk fat concentration tended to be higher in cows that received the placebo. Time spent eating and ruminating and dry matter intake were similar across treatments. Ruminal lactic acid concentrations were highly variable between animals, and no cases of clinical acidosis were observed. Both treatment groups had rumen pH <5.6 for more than 3 h/d (a commonly used threshold to define subacute ruminal acidosis), but the length of time with rumen pH <5.6 was markedly reduced in the days immediately after dosing and fluctuated much less from day to day in cows that received M. elsdenii compared with those that received the placebo. Ruminal total volatile fatty acid concentrations were similar across treatments, but the acetate:propionate ratio tended to be smaller in cows that received M. elsdenii. Despite the lack of a measurable treatment effect on ruminal lactic acid concentration, supplementation of early lactation dairy cows with lactate-utilizing M. elsdenii altered the rumen fermentation patterns in favor of propionate, with potential benefits for energy balance and animal productivity.     

Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses

Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.     

Prevotella bryantii 25A used as a probiotic in early-lactation dairy cows: effect on ruminal fermentation characteristics, milk production, and milk composition

Ingestion of high levels of rapidly fermented carbohydrates after parturition often leads to the production of excessive quantities of organic acids that may exceed the buffering capacity of the rumen and cause pH to drop. Ruminal acidosis results in animal discomfort, anorexia, depression, decreased digestibility, and decreased milk production. In the present study, we examined the effects of daily addition of cells of a newly isolated strain of Prevotella bryantii (25A) to the rumen of 12 ruminally cannulated cows in early lactation. This strain was selected based on earlier in vitro studies that indicated its ability to grow rapidly, compete for starch, and produce organic acids other than lactate. After calving, all cows received increasing amounts of an energy-dense diet containing barley grain, corn silage, and grass silage in a 40:60 forage-to-concentrate ratio. Animals were blocked according to milk production from their previous lactation. Treatments (control and P. bryantii) were distributed among cows within the same block. Cows were fed once a day. Six cows were given a daily dose of P. bryantii (2 × 10¹¹ cells/dose), administered directly with a syringe through the rumen cannula, from 3 wk prepartum up to 7 wk postpartum. Rumen fluid was sampled before feeding and at 2 and 3 h postfeeding on wk 1, 2, 3, 4, 6, and 7 postpartum. Feed intake and milk yield were recorded daily and milk composition was recorded 2 d/wk, up to wk 7 of lactation. Feed intake was similar between control and treated cows. Prevotella bryantii did not change milk production, but milk fat tended to be greater in treated cows compared with control cows (3.9 vs. 3.5%). Rumen pH was similar between the 2 groups and differed across sampling times, being higher before feeding (6.3) as opposed to 2 h (5.9) and 3 h (5.7) postfeeding. Rumen lactate concentration was similar before feeding between control and treated cows; however, 2 to 3 h after feeding, lactate concentration was lower in cows receiving P. bryantii compared with control cows (0.7 vs. 1.4 mM). This difference was maintained throughout the experimental period. Concentration of NH₃-N was greater in treated cows than in control cows (174 vs. 142 mg/L). Acetate (65.5 vs. 57.8 mM), butyrate (12.7 vs. 10.5 mM), and branched-chain C4 fatty acid (0.90 vs. 0.75 mM) concentrations were greater in postfeeding samples of treated cows compared with control cows. Supplementing early-lactating cows with P. bryantii 25A increased ruminal fermentation products and milk fat concentration. Because signs of subacute ruminal acidosis were not observed in either treated or control cows, no conclusions can be made about possible protection against acidosis by P. bryantii.     

Short communication: Detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johne's disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohn's disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohn's disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil.     

Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP₆ degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP₆ degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P₅ or D/L-Ins(1,2,3,4,6)P₅; D/L-Ins(1,2,3,4)P₄; to finally Ins(1,2,3)P₃ and D/L-Ins(1,2,4)P₃/D/L-Ins(1,3,4)P₃. This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.     

The production of surfactin during the fermentation of cheonggukjang by potential probiotic Bacillus subtilis CSY191 and the resultant growth suppression of MCF-7 human breast cancer cells

Bacillus subtilis CSY191, the potential probiotics and surfactin-like compound producer, was isolated from doenjang (Korean traditional fermented soybean paste).The survival rate of this strain appeared to be the 58.3% under artificial gastric conditions after 3 h at pH 3.0. Surfactin was purified from the strain CSY191. Three potential surfactin isoforms were detected, with protonated masses of m/z 1030.7, 1044.7, and 1058.71. These different structures were detected in combination with Na⁺, K⁺ and Ca²⁺ ions by MALDI-TOF mass spectrometry. Upon 500 MHz ¹H NMR analysis, the surfactin isoforms had identical amino acids (GLLVDLL) and hydroxy fatty acids (of 13-15 carbons in length). The MTT assay showed that surfactin inhibited growth of MCF-7 human breast cancer cells in a dose-dependent manner, with an IC₅₀ of approximately 10 μg/ml at 24 h. Additionally, the surfactin contents, during cheonggukjang fermentation with strain CSY191, increased from 0.3 to 48.2 mg/kg over 48 h of fermentation, while the level of anticancer activity increased from 2.6- to 5.1-fold.     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.