Classification of Common Food Lipid Sources Regarding Healthiness Using Advanced Lipidomics: A Four-Arm Crossover Study.

Prospective studies have failed to establish a causal relationship between animal fat intake and cardiovascular diseases in humans. Furthermore, the metabolic effects of different dietary sources remain unknown. In this four-arm crossover study, we investigated the impact of consuming cheese, beef, and pork meat on classic and new cardiovascular risk markers (obtained from lipidomics) in the context of a healthy diet. A total of 33 young healthy volunteers (23 women/10 men) were assigned to one out of four test diets in a Latin square design. Each test diet was consumed for 14 days, with a 2-week washout. Participants received a healthy diet plus Gouda- or Goutaler-type cheeses, pork, or beef meats. Before and after each diet, fasting blood samples were withdrawn. A reduction in total cholesterol and an increase in high density lipoprotein particle size were detected after all diets. Only the pork diet upregulated plasma unsaturated fatty acids and downregulated triglycerides species. Improvements in the lipoprotein profile and upregulation of circulating plasmalogen species were also observed after the pork diet. Our study suggests that, within the context of a healthy diet rich in micronutrients and fiber, the consumption of animal products, in particular pork meat, may not induce deleterious effects, and reducing the intake of animal products should not be regarded as a way of reducing cardiovascular risk in young individuals.

Cyberlindnera jadinii yeast as a protein source in early- to mid-lactation dairy cow diets: Effects on feed intake, ruminal fermentation, and milk production.

We examined the effects of substituting soybean meal with either yeast protein from Cyberlindnera jadinii or barley in concentrate feeds on feed intake, ruminal fermentation products, milk production, and milk composition in Norwegian Red (NRF) dairy cows. The concentrate feeds were prepared in pellet form as soy-based (SBM; where soybean meal is included as a protein ingredient), yeast-based (YEA; soybean meal replaced with yeast protein), or barley-based (BAR; soybean meal replaced with barley). The SBM contained 7.0% soybean meal on a dry matter (DM) basis. This was replaced with yeast protein and barley in the YEA and BAR concentrate feeds, respectively. A total of 48 early- to mid-lactation [days in milk ± standard deviation (SD): 103 ± 33.5 d] NRF cows in their first to fourth parity and with initial milk yield of 32.6 kg (SD = 7.7) were allocated into 3 groups, using a randomized block design, after feeding a common diet [SBM and good-quality grass silage: crude protein (CP) and neutral detergent fiber (NDF) content of 181 and 532 g/kg of DM, respectively] for 14 d (i.e., covariate period). The groups (n = 16) were then fed one of the dietary treatments (SBM, YEA, or BAR) for a period of 56 d (i.e., experimental period). The concentrate feeds were offered in split portions from 3 automatic feeders using electronic identification, with ad libitum access to the same grass silage. Dietary treatments had no effect on daily silage intake, total DM intake, or total NDF intake. Dietary CP intake was lower and starch intake was higher in the BAR group compared with the other groups. Ruminal fluid pH, short-chain volatile fatty acid (VFA) concentrations, acetate-to-propionate ratio, and non-glucogenic to glucogenic VFA ratio were not affected by dietary treatments. No effects of the dietary treatments were observed on body weight change, body condition score change, milk yield, energy-corrected milk yield, milk lactose and fat percentages, or their yields. In conclusion, yeast protein can substitute conventional soybean meal in dairy cow diets without adverse effect on milk production and milk composition, given free access to good-quality grass silage.

Characterization of Bacillus cereus sensu lato isolates from milk for consumption; phylogenetic identity, potential for spoilage and disease.

This study addresses the biodiversity of Bacillus cereus group population present along the value chain of milk for consumption. The B. cereus population did not grow and remained mainly unaltered during storage of milk at 4 °C while storage at a suboptimal temperature at 8 °C (representative of a broken cold chain) caused a major shift in its composition. Mesophilic strains dominated the B. cereus population in raw milk and after storage at 4 °C, while psycrotrophic strains dominated after storage at 8 °C. All psycrotrophic and mesophilic isolates (n = 368) demonstrated high spoilage potentials of the milk components. Fifteen out of 20 mesophilic isolates but only two out of 40 psychrotrophic isolates, exhibited vero cell toxicity. No genes encoding the emetic toxin cereulide were detected in the genomes of 100 milk isolates while 14 of them harbored the enterotoxin genes cytK1/cytK2. Both psycrotrophic and mesophilic isolates carried the enterotoxin genes nheA and hblA. Together, the results provide insight into the composition and properties, of the B. cereus population present in milk along the value chain and during storage at optimal refrigerated temperature and at suboptimal temperature. This knowledge is useful in the dairy industry's work to assure high quality products and for risk assessment.

Manufacture and characterization of acid-coagulated fresh cheese made from casein concentrates obtained by acid diafiltration.

This study aimed to investigate the production of acid-coagulated fresh cheese by using slightly acid diafiltered (DF) microfiltered (MF) casein concentrates (8% protein). Three different acidifying agents were tested during DF: carbon dioxide, lactic acid, and citric acid. Fresh cheese was manufactured using acid-DF casein concentrates, or casein concentrates DF with just water, and compared with cheese manufactured using MF casein concentrates without DF. The fresh cheeses were characterized for composition, rheological, and sensorial properties. Acid-DF casein concentrates improved acidification kinetics during cheesemaking and reduced casein leakage to cheese whey, compared with cheese from regular MF casein concentrate. Among the rheological properties investigated in this study, the storage modulus of the fresh cheese was higher when DF of the casein concentrate was performed with nonacidified DF water or when DF water was acidified with citric acid. However, fresh cheese made from casein concentrate diafiltered with DF water acidified by citric acid was most liked in a sensory ranking test.

Minor acidification of diafiltration water using various acidification agents affects the composition and rennet coagulation properties of the resulting microfiltration casein concentrate.

Cheese made from microfiltration (MF) retentate may suffer from textural defects due to a high Ca concentration. The reduction of colloidal minerals by the acidification of milk before MF at pH below 6.0 has been well documented in the literature. This process, however, creates less valuable side streams to the MF process and induces changes in the casein micelles that negatively affect their coagulation properties. The objective of this study was to determine whether a minor reduction in pH by using different acidifiers in the diafiltration (DF) water could induce changes in composition and renneting properties of the MF retentate. A 2-stage filtration process was used, with the first designed to increase the casein concentration to 8% and the second to slightly reduce the casein concentrate by 0.1 pH unit by DF, without influencing the total protein concentration. Four acidifying agents were tested during DF: lactic acid, hydrochloric acid, citric acid, and carbon dioxide. Diafiltration with water was used as a reference. At the start of DF, the retentates of acid DF had a slightly reduced pH, with an average of 0.09, whereas the pH of the reference retentate increased by an average of 0.07 unit. The reference retentate regained its starting pH by the end of DF. The carbonated retentate gradually increased in pH during processing, whereas the pH of the lactic, hydrochloric, and citric acid retentates remained constant. The permeate from the lactic acid and carbonated treatments had a reduced whey protein content compared with the reference. The total P and inorganic phosphate were lowered in the retentate by using carbonation. The total amount of Mg and Na were lowered in the retentate by using citric acid. The ionic Ca content in the retentate increased with use of lactic or hydrochloric acid. The type of acidifier used reduced the rennet clotting time. Combined acidified diafiltration with a slight reduction affects the permeate composition and improves the retentate clotting time despite the minimal mineral modification.

Application of a novel amplicon-based sequencing approach reveals the diversity of the Bacillus cereus group in stored raw and pasteurized milk.

Members of the Bacillus cereus sensu lato (B. cereus group) are spore-forming organisms commonly associated with spoilage of milk and dairy products. Previous studies have shown, by using 16S marker gene sequencing, that the genus Bacillus is part of the core microbiota of raw bovine milk and that some members of this genus are able to grow during sub-optimal storage (8 °C) of pasteurized consumption milk. Here, the composition of this genus in pasteurized consumption milk samples, collected from two dairies, over a one-year period and stored at 4 or 8 °C up to the end of shelf life is uncovered. Our results show that the B. cereus group is the dominant Bacillus group in stored consumption milk. By applying a new marker gene sequencing approach, several dominating phylogenetic clusters were identified within the B. cereus group populations from the milk samples. There was a higher phylogenetic diversity among bacteria from milk stored at 8 °C compared to milk stored at 4 °C. Sampling period and the dairy the samples were collected from, also significantly influenced the diversity, which shows that the B. cereus group population in consumption milk is heterogeneous and subjected to temporal and spatial changes. The new approach applied in this study will facilitate the identification of isolates within the B. cereus group, of which some are potential spoilage bacteria and pathogenic contaminants of milk and dairy products.

Bulk tank raw milk microbiota differs within and between farms: A moving goalpost challenging quality control.

Microbial contamination of bovine raw milk often occurs at the farm. To acquire a deeper knowledge of the microbiota of farm tank milk, we studied milk from 45 farms situated in 2 geographical areas in Norway. Each farm was visited on 3 different occasions, with at least 2 wk between visits. We combined both bacterial cell counts and a sequence variant inference method of amplicon-based high-throughput sequencing to achieve a high-resolution overview of the microbiota in each sample. Compositional variation of the farm milk microbiota was shown in relation to the 2 areas, between the farms and between the sampling times. Despite the near constant level of bacteria enumerated in milk from each individual farm, the dominant microbiota differed significantly between the samplings. The predominant microbiota was dominated by spoilage genera, such as Pseudomonas and Bacillus, as well as the dairy fermentation genus Lactococcus and mastitis-causing organisms (Streptococcus). Analysis of the identified sequence variants within these genera showed that the populations of Pseudomonas and Lactococcus in milk had similar composition between the farms, but that Bacillus and, in particular, Streptococcus populations changed between collection days from the same farm and between farms and geographical areas. Furthermore, the levels and composition of Bacillus and Paenibacillus were different between the 2 geographical areas. The results presented here provide new insight into the farm milk microbiota and show that this microbiota is a dynamic community highly subject to variation.

Optimization of protein fractionation by skim milk microfiltration: Choice of ceramic membrane pore size and filtration temperature.

The objective of this study was to investigate how ceramic membrane pore size and filtration temperature influence the protein fractionation of skim milk by cross flow microfiltration (MF). Microfiltration was performed at a uniform transmembrane pressure with constant permeate flux to a volume concentration factor of 2.5. Three different membrane pore sizes, 0.05, 0.10, and 0.20µm, were used at a filtration temperature of 50°C. Furthermore, at pore size 0.10µm, 2 different filtration temperatures were investigated: 50 and 60°C. The transmission of proteins increased with increasing pore size, giving the permeate from MF with the 0.20-µm membrane a significantly higher concentration of native whey proteins compared with the permeates from the 0.05- and 0.10-µm membranes (0.50, 0.24, and 0.39%, respectively). Significant amounts of caseins permeated the 0.20-µm membrane (1.4%), giving a permeate with a whitish appearance and a casein distribution (αS2-CN: αS1-CN: κ-CN: ß-CN) similar to that of skim milk. The 0.05- and 0.10-µm membranes were able to retain all caseins (only negligible amounts were detected). A permeate free from casein is beneficial in the production of native whey protein concentrates and in applications where transparency is an important functional characteristic. Microfiltration of skim milk at 50°C with the 0.10-µm membrane resulted in a permeate containing significantly more native whey proteins than the permeate from MF at 60°C. The more rapid increase in transmembrane pressure and the significantly lower concentration of caseins in the retentate at 60°C indicated that a higher concentration of caseins deposited on the membrane, and consequently reduced the native whey protein transmission. Optimal protein fractionation of skim milk into a casein-rich retentate and a permeate with native whey proteins were obtained by 0.10-µm MF at 50°C.

The composition and functional properties of whey protein concentrates produced from buttermilk are comparable with those of whey protein concentrates produced from skimmed milk.

The demand for whey protein is increasing in the food industry. Traditionally, whey protein concentrates (WPC) and isolates are produced from cheese whey. At present, microfiltration (MF) enables the utilization of whey from skim milk (SM) through milk protein fractionation. This study demonstrates that buttermilk (BM) can be a potential source for the production of a WPC with a comparable composition and functional properties to a WPC obtained by MF of SM. Through the production of WPC powder and a casein- and phospholipid (PL)-rich fraction by the MF of BM, sweet BM may be used in a more optimal and economical way. Sweet cream BM from industrial churning was skimmed before MF with 0.2-µm ceramic membranes at 55 to 58°C. The fractionations of BM and SM were performed under the same conditions using the same process, and the whey protein fractions from BM and SM were concentrated by ultrafiltration and diafiltration. The ultrafiltration and diafiltration was performed at 50°C using pasteurized tap water and a membrane with a 20-kDa cut-off to retain as little lactose as possible in the final WPC powders. The ultrafiltrates were subsequently spray dried, and their functional properties and chemical compositions were compared. The amounts of whey protein and PL in the WPC powder from BM (BMWPC) were comparable to the amounts found in the WPC from SM (SMWPC); however, the composition of the PL classes differed. The BMWPC contained less total protein, casein, and lactose compared with SMWPC, as well as higher contents of fat and citric acid. No difference in protein solubility was observed at pH values of 4.6 and 7.0, and the overrun was the same for BMWPC and SMWPC; however, the BMWPC made less stable foam than SMWPC.

Seasonal dynamics of bulk milk somatic cell count in grazing Norwegian dairy goats.

Somatic cell count (SCC) is a widely used indicator of milk quality in the dairy industry. It is a relatively good indicator of udder health in dairy cows, but strongly confounded by non-infection factors, including season, in dairy goats. This study's objectives were to estimate the variation in SCC explained by season and stage of lactation. We also investigated associations between SCC and other milk quality parameters routinely measured, including total bacterial count, fat, protein, and lactose content, in different seasons and stages of lactation. In this observational study, we included results from the routine analyses of bulk milk samples (n = 5,180) collected every third day in 88 Norwegian goat herds through one year. Herd information was collected via a questionnaire and from the dairy herd recording system. The herds had a notable increase in bulk SCC associated with the pasture season. The median bulk SCC was 470,000 cells/mL in deliveries from the indoor spring period and 1,100,000 cells/mL in the pasture season. During the indoor fall season, when most goat herds are in late lactation, the median SCC was 940,000 cells/mL. The combined effect of the season and herd stage of lactation explained 53.5% of the variation in bulk milk SCC. Fat and protein contents varied significantly with season and stage of lactation, and the association with SCC was non-significant for fat content and explained less than 3% of the variation in protein content. Lactose content was associated with SCC, stage of lactation, and season. Total bacterial count was associated with SCC and the indoor seasons. Determination of normal seasonal variation of goat bulk milk SCC is necessary to establish thresholds for defining milk as abnormal and unacceptable for further processing and improving the usefulness of SCC as a tool for milk quality improvement in dairy goats. The results suggest that SCC thresholds for taking action to improve the milk quality should be adjusted for season and stage of lactation.

Database selection for shotgun metaproteomic of low-diversity dairy microbiomes.

The metaproteomics field has recently gained more and more interest as a valuable tool for studying both the taxonomy and function of microbiomes, including those used in food fermentations. One crucial step in the metaproteomics pipeline is selecting a database to obtain high-quality taxonomical and functional information from microbial communities. One of the best strategies described for building protein databases is using sample-specific or study-specific protein databases obtained from metagenomic sequencing. While this is true for high-diversity microbiomes (such as gut and soil), there is still a lack of validation for different database construction strategies in low-diversity microbiomes, such as those found in fermented dairy products where starter cultures containing few species are used. In this study, we assessed the performance of various database construction strategies applied to metaproteomics on two low-diversity microbiomes obtained from cheese production using commercial starter cultures and analyzed by LC-MS/MS. Substantial differences were detected between the strategies, and the best performance in terms of the number of peptides and proteins identified from the spectra was achieved by metagenomic-derived databases. However, extensive databases constructed from a high number of available online genomes obtained a similar taxonomical and functional annotation of the metaproteome compared to the metagenomic-derived databases. Our results indicate that, in the case of low-diversity dairy microbiomes, the use of publically available genomes to construct protein databases can be considered as an alternative to metagenome-derived databases.

Two-year investigation of spore-formers through the production chain at two cheese plants in Norway.

Spore-forming bacteria are the most complex group of microbes to eliminate from the dairy production line due to their ability to withstand heat treatment usually used in dairy processing. These ubiquitous microorganisms have ample opportunity for multiple points of entry into the milk chain, creating issues for food quality and safety. Certain spore-formers, namely bacilli and clostridia, are more problematic to the dairy industry due to their possible pathogenicity, growth, and production of metabolites and spoilage enzymes. This research investigated the spore-forming population from raw milk reception at two Norwegian dairy plants through the cheesemaking stages until ripening. Samples were collected over two years and examined by amplicon sequencing in a culture independent manner and after an anaerobic spore-former enrichment step. In addition, a total of 608 isolates from the enriched samples were identified at the genus or species level using MALDI-TOF analysis. Most spore-forming isolates belong to the genera Bacillus or Clostridium, with the latter dominating the enriched MPN tubes of raw milk and bactofugate. Results showed a great variation among the clostridia and bacilli detected in the enriched MPN tubes. However, B. licheniformis and C. tyrobutyricum were identified in all sample types from both plants throughout the 2-year study. In conclusion, our results shed light on the fate of different spore-formers at different processing stages in the cheese production chain, which could facilitate targeted actions to reduce quality problems.

Composition and fate of heat-resistant anaerobic spore-formers in the milk powder production line.

Anaerobic spore-forming bacteria are a continuous threat to the dairy industry due to their ability to withstand processing conditions, such as those during heat treatment. These ubiquitous microorganisms have ample opportunity for multiple entry points into the milk chain, creating food quality and safety issues. Certain spore-formers, namely bacilli and clostridia, are more problematic due to their ability to spoil dairy products and pathogenicity. In this study, we investigated how milk treatment and milk powder production influenced the composition and survival of anaerobic spore-formers. Samples were obtained on three different days (replicate blocks) during the production of dairy powders and examined in a culture-dependent manner using the most probable number method coupled with 16S rRNA amplicon sequencing and metagenomic analysis of the enriched samples. Results revealed that the milk separation greatly affected the spore-former presence and composition which were detected along the entire production line from raw material to milk powders. Throughout the various points of the production line, the occurrence of species belonging to the Bacillus cereus sensu lato was higher than that of clostridia. Sequence variants (SVs) belonging to the anaerobic spore-forming genus Clostridium were taxonomically assigned to two SVs groups and were detected in all three replicate blocks. A total of 19 metagenome-assembled genomes were recovered from nine enrichments. Four near-complete and two medium-quality genomes were found in raw milk/milk powder samples and further assigned as Clostridium tyrobutyricum and Clostridium diolis, which may constitute a problem in the finished dairy product. In conclusion, our findings highlight spore-formers' importance on dairy quality and may aid in their intervention and control in the dairy production line.

Basic taste sensitivity, eating behaviour, food propensity and BMI of preadolescent children: How are they related?

Background: Taste sensitivity has been reported to influence children's eating behaviour and contribute to their food preferences and intake. This study aimed to investigate the associations between taste sensitivity, eating behaviour, food frequency and BMI (Body Mass Index) in preadolescents. Methods: Preadolescents' taste sensitivity was measured by detection threshold of sweetness (sucrose), sourness (citric acid), saltiness (sodium chloride), bitterness (caffeine, quinine), and umami (monosodium glutamate). In addition, the Child Eating Behaviour Questionnaire (CEBQ), the Food Propensity Questionnaire (FPQ) measuring food frequency, and the children's body weight and height were completed by the parents. A total of 69 child-parent dyads participated (preadolescents mean age =10.9 years). Results: Taste sensitivity to caffeine bitterness was significantly associated with eating behaviour in food responsiveness, emotional overeating, and desire to drink. The preadolescents who were less sensitive to caffeine bitterness had higher food responsiveness scores. Those who were less sensitive to caffeine bitterness and to sweetness had higher emotional overeating scores. In addition, preadolescents who were less sensitive to sourness and bitterness of both caffeine and quinine demonstrated to have higher scores in desire to drink. There was no association between taste sensitivity and FPQ, but significant differences were observed across preadolescents' BMI for FPQ of dairy food items, indicating higher consumption of low-fat milk in the overweight/obese compared to the underweight/normal-weight subjects. There was no significant difference in taste sensitivity according to BMI. Preadolescents' eating behaviour differed across BMI, demonstrating a positive association between BMI and food approach, and a negative association between BMI and food avoidance. Conclusions: This study contributes to the preliminary understanding of the relationships between taste sensitivity and eating behaviour in preadolescents. The results may be used to develop effective strategies to promote healthy eating practices by considering taste sensitivity in preadolescents.

Whole genome sequencing reveals possible host species adaptation of Streptococcus dysgalactiae.

Streptococcus dysgalactiae (SD) is an emerging pathogen in human and veterinary medicine, and is associated with several host species, disease phenotypes and virulence mechanisms. SD has traditionally been divided into the subspecies dysgalactiae (SDSD) and subsp. equisimilis (SDSE), but recent molecular studies have indicated that the phylogenetic relationships are more complex. Moreover, the genetic basis for the niche versatility of SD has not been extensively investigated. To expand the knowledge about virulence factors, phylogenetic relationships and host-adaptation strategies of SD, we analyzed 78 SDSD genomes from cows and sheep, and 78 SDSE genomes from other host species. Sixty SDSD and 40 SDSE genomes were newly sequenced in this study. Phylogenetic analysis supported SDSD as a distinct taxonomic entity, presenting a mean value of the average nucleotide identity of 99%. Bovine and ovine associated SDSD isolates clustered separately on pangenome analysis, but no single gene or genetic region was uniquely associated with host species. In contrast, SDSE isolates were more heterogenous and could be delineated in accordance with host. Although phylogenetic clustering suggestive of cross species transmission was observed, we predominantly detected a host restricted distribution of the SD-lineages. Furthermore, lineage specific virulence factors were detected, several of them located in proximity to hotspots for integration of mobile genetic elements. Our study indicates that SD has evolved to adapt to several different host species and infers a potential role of horizontal genetic transfer in niche specialization.

Longitudinal Study of the Bulk Tank Milk Microbiota Reveals Major Temporal Shifts in Composition.

Introduction of microbial contaminations in the dairy value chain starts at the farm level and the initial microbial composition may severely affect the production of high-quality dairy products. Therefore, understanding the farm-to-farm variation and longitudinal shifts in the composition of the bulk tank milk microbiota is fundamental to increase the quality and reduce the spoilage and waste of milk and dairy products. In this study, we performed a double experiment to study long- and short-term longitudinal shifts in microbial composition using 16S rRNA gene amplicon sequencing. We analyzed milk from 37 farms, that had also been investigated two years earlier, to understand the stability and overall microbial changes over a longer time span. In addition, we sampled bulk tank milk from five farms every 1-2 weeks for up to 7 months to observe short-term changes in microbial composition. We demonstrated that a persistent and farm-specific microbiota is found in bulk tank milk and that changes in composition within the same farm are mostly driven by bacterial genera associated with mastitis (e.g., Staphylococcus and Streptococcus). On a long-term, we detected that major shift in milk microbiota were not correlated with farm settings, such as milking system, number of cows and quality of the milk but other factors, such as weather and feeding, may have had a greater impact on the main shifts in composition of the bulk tank milk microbiota. Our results provide new information regarding the ecology of raw milk microbiota at the farm level.

Microbial diversity of consumption milk during processing and storage.

Bovine milk contains a complex microbial community that affects the quality and safety of the product. Detailed knowledge of this microbiota is, therefore, of importance for the dairy industry. In this study, the bacterial composition of consumption milk was assessed during different stages in the production line and throughout the storage in cartons by using culturing techniques and 16S rRNA marker gene sequencing. Monthly samples from two dairies were analyzed to capture the seasonal variations in the milk microbiota. Although there was a core microbiota present in milk samples from both dairies, the composition of the bacterial communities were significantly influenced by sampling month, processing stage and storage temperature. Overall, a higher abundance of operational taxonomic units (OTUs) within the order Bacillales was detected in samples of raw and pasteurized milk from the spring and summer months, while Pseudomonadales and Lactobacillales OTUs were predominant in the winter months. OTUs belonging to the order Lactobacillales, Pseudomonadales, Clostridiales and Bacillales were significantly more abundant in milk samples taken immediately after pasteurization compared to raw milk samples. During storage of milk in cartons at 4°C, the bacterial composition remained stable throughout the product shelf life, while storage at 8°C significantly increased the abundance of OTUs belonging to the genus Bacillus and the plate count levels of presumptive Bacillus cereus. The knowledge obtained in this work will be useful to the dairy industry during their quality assurance work and risk assessment practices.

Detection and quantification of Bacillus cereus group in milk by droplet digital PCR.

Droplet digital PCR (ddPCR) is one of the newest and most promising methods for the detection and quantification of molecular targets by PCR. Here, we optimized and used a new ddPCR assay for the detection and quantification of the Bacillus cereus group in milk. We also compared the ddPCR to a standard qPCR assay. The new ddPCR assay showed a similar coefficient of determination and a better limit of detection compared to the qPCR assay during quantification of the target molecules in the samples. However, the ddPCR assay has a limitation during quantification of a high number of target molecules. This new assay was then tested for the quantification of the B. cereus group in 90 milk samples obtained over three months from two different dairies and the milk was stored at different temperatures before sampling. The ddPCR assay showed good agreement with the qPCR assay for the quantification of the B. cereus group in milk, and due to its lower detection limit more samples were detected as positive. The new ddPCR assay is a promising method for the quantification of target bacteria in low concentration in milk.

Effect of a cheese rich in angiotensin-converting enzyme-inhibiting peptides (Gamalost(®)) and a Gouda-type cheese on blood pressure: results of a randomised trial.

BACKGROUND: High blood pressure (BP) is the leading risk factor for global disease burden, contributing to 7% of global disability adjusted life years. Angiotensin converting enzyme (ACE)-inhibiting bioactive peptides have the potential to reduce BP in humans. These peptides have been identified in many dairy products and have been associated with significant reductions in BP. OBJECTIVE: The objective of this trial was to examine whether a cheese rich in ACE-inhibiting peptides (Gamalost(®)), or a standard Gouda-type cheese could lower BP. DESIGN: A total of 153 healthy participants were randomised to one of three parallel arms: Gamalost(®) (n=53, 50 g/day for 8 weeks), Gouda-type cheese (n=50, 80 g/day for 8 weeks), and control (n=50). BP and anthropometric measurements were taken at the baseline and at the end, with an additional BP measurement midway. Based on BP at baseline, participants were categorised as having optimal BP (<120/<80 mmHg), normal-high BP (120-139/80-89 mmHg), or being hypertensive (>140/>90 mmHg). Questionnaires about lifestyle, health, and dietary habits were completed at baseline, midway and end. RESULTS: In total, 148 participants (mean age 43, 52% female) completed the intervention. There were no differences among the three groups in relevant baseline characteristics. BP was reduced in the entire study population, but the cheese groups did not differ from control. However, in a subgroup of participants with slightly elevated BP, BP at 4 weeks of intervention seemed to be borderline significantly more reduced in the Gamalost(®) group compared with the control group (Dunnett test: diastolic BP -3.5 mmHg, 95% confidence interval (CI) -7.3, 0.4, systolic BP: -4.3 mmHg, 95% CI -9.8, 1.1). CONCLUSION: An intention-to-treat analysis of the data showed no cheese effect upon BP compared to control, but Gamalost(®) seemed to have a small, non-significant lowering effect on diastolic BP after 4 weeks in people with a normal-high BP.

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.