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.

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.

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.

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.

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.

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.

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.