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.

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.

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.

Draft Genome Sequence of Enterococcus hirae Strain INF E1 Isolated from Cultured Milk.

Here, we present the draft genome of Enterococcus hirae INF E1, found as a contaminant in cultured milk and studied for its ability to metabolize milk fat globule membrane glycoconjugates.