Raw milk microbiota is complex and influenced by many factors that facilitate the introduction of undesirable microorganisms. Milk microbiota is closely related to the safety and quality of dairy products, and it is therefore critical to characterize the variation in the microbial composition of raw milk. In this cross-sectional study, the variation in raw milk microbiota throughout the year (n = 142) from three farms in China was analyzed using 16S rRNA amplicon sequencing, including α and β diversity, microbial composition, and the relationship between microbiota and milk quality parameters. This aimed to characterize the contamination risk of raw milk throughout the year and the changes in quality parameters caused by contamination. Collection month had a significant effect on microbial composition; microbial diversity was higher in raw milk collected in May and June, while milk collected in October and December had the lowest microbial diversity. Microbiota composition differed significantly between milk collected in January–June, July–August, and September–December (p < 0.05). Bacterial communities represented in raw milk at the phylum level mainly included Proteobacteria, Firmicutes and Bacteroidota; Pseudomonas, Acinetobacter, Streptococcus and Lactobacillus were the most common genera. Redundancy analysis (RDA) found strong correlations between microbial distribution and titratable acidity (TA), fat, and protein. Many genera were significantly correlated with TA, for example Acinetobacter (R = 0.426), Enhydrobacter (R = 0.309), Chryseobacterium (R = 0.352), Lactobacillus (R = −0.326), norank_o__DTU014 (R = −0.697), norank_f__SC-I-84 (R = −0.678), and Subgroup_10 (R = −0.721). Additionally, norank_f__ Muribaculaceae was moderately negatively correlated with fat (R = −0.476) and protein (R = −0.513). These findings provide new information on the ecology of raw milk microbiota at the farm level and contribute to the understanding of the variation in raw milk microbiota in China.

The impact of five human milk oligosaccharides (HMOs)—2′-fucosyllactose (2FL), 3′-sialyllactose (3SL), 6′-sialyllactose (6SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT)—on the gut microbiota and short-chain fatty acid (SCFA) metabolites in infants aged 0–6 months was assessed through in vitro fermentation. Analyses of the influence of different HMOs on the composition and distribution of infant gut microbiota and on SCFA levels were conducted using 16S rRNA sequencing, quantitative real-time PCR (qPCR), and gas chromatography (GC), respectively. The findings indicated the crucial role of the initial microbiota composition in shaping fermentation outcomes. Fermentation maintained the dominant genera species in the intestine but influenced their abundance and distribution. Most of the 10 Bifidobacteria strains effectively utilized HMOs or their degradation products, particularly demonstrating proficiency in utilizing 2FL and sialylated HMOs compared to non-fucosylated neutral HMOs. Moreover, our study using B. infantis-dominant strains and B. breve-dominant strains as inocula revealed varying acetic acid levels produced by Bifidobacteria upon HMO degradation. Specifically, the B. infantis-dominant strain yielded notably higher acetic acid levels than the B. breve-dominant strain (p = 0.000), with minimal propionic and butyric acid production observed at fermentation’s conclusion. These findings suggest the potential utilization of HMOs in developing microbiota-targeted foods for infants.

An accurate method that rapidly detects the number of viable but nonculturable (VBNC) Cronobacter sakazakii was developed by combining propidium bromide with quantitative LAMP (PMA-QLAMP). The gyrB gene was the target for primers design. The optimal PMA treatment conditions were determined to eliminate the DNA amplification of 10⁸ CFU/mL of dead C. sakazakii without affecting any viable C. sakazakii DNA amplification. Compared with the DNA of 24 strains of common non-C. sakazakii strains found in raw milk and dairy products, the DNA of only six C. sakazakii strains from different sources was amplified using PMA-QLAMP. The ability of PMA-QLAMP to quantitatively detect non-dead C. sakazakii in a 10% powdered infant formula (PIF) solution was limited to 4.3 × 10² CFU/mL and above concentrations. Pasteurizing 10⁶ CFU/mL viable C. sakazakii yielded the maximum ratio of the VBNC C. sakazakii. PMA-QLAMP-based detection indicated that, although approximately 13% of 60 samples were positive for viable C. sakazakii, the C. sakazakii titers in these positive samples were low, and none entered the VBNC state under pasteurization. PMA-QLAMP showed potential as a specific and reliable method for detecting VBNC-C. sakazakii in pasteurized raw milk, thereby providing an early warning system that indicates potential contamination of PIF.

To investigate the changes in the intestinal flora in the Chinese elderly with cardiovascular disease (CVD) and its correlation with the metabolism of trimethylamine (TMA), the intestinal flora composition of elderly individuals with CVD and healthy elderly individuals was analyzed using 16S rRNA sequencing, the TMA levels in the feces of elderly were detected using headspace–gas chromatography (HS-GC), and four kinds of characterized TMA-producing intestinal bacteria in the elderly were quantified using real-time fluorescence quantitative polymerase chain reaction (qPCR). The results showed that Firmicutes, Actinobacteria, Proteobacteria, Bacteroidetes, and Verrucomicrobia are the dominant microorganisms of the intestinal flora in the Chinese elderly. And there were significant differences in the intestinal bacteria composition between healthy elderly individuals and those with CVD, accompanied by a notable difference in the TMA content. The richness and diversity of the intestinal flora in the elderly with CVD were higher than those in the healthy elderly. Correlation analysis indicated that certain significantly different intestinal flora were associated with the TMA levels. Our findings showed a significant difference in TMA-producing intestinal flora between healthy elderly individuals and those with CVD. The TMA levels were found to be positively and significantly correlated with Klebsiella pneumoniae, suggesting that this bacterium is closely linked to the production of TMA in the elderly gut. This may have implications for the development and progression of CVD in the elderly population.

Raw milk is the primary material for all dairy products, making it imperative to characterize and monitor its microbial composition to ensure product quality. To investigate microbial contamination from pasture to processing facilities and establish microbial traceability systems, eighty-seven raw milk samples were collected from a dairy farm in Shijiazhuang of China in August. The raw milk samples were categorized into three experimental groups based on: sampling sites along the production chain (manual milking, buffer tank, filter, refrigeration tank, milk truck, and plant factory), sampling time (before dawn, morning, noon, and afternoon), and refrigeration storage (0-72 h at 12 h intervals). The microbiota were evaluated by 16S rRNA sequencing. The results identified Pseudomonas, Lactobacillus, and Prevotella as the predominant bacterial genera across all sampling conditions. The α-diversity (Shannon and Chao1) and β-diversity analysis jointly revealed significant differences in microbial communities of raw milk samples. Specifically, raw milk collected from milk truck showed distinct bacterial communities compared with upstream collecting points, while morning-collected samples showed marked compositional differences from other time points. These findings were consistently supported by cluster heatmap analysis. In addition, the relative abundance of Pseudomonas in raw milk decreased but Lactococcus and Serratia increased with refrigerated time (P < 0.05). This inverse relationship was further evidenced in cooccurrence network showing a strong negative correlation between Lactococcus, Serratia, and Pseudomonas. These results indicated where and when (after being transported to milk truck and in the morning) we need to alert owing to potential contamination in raw milk. Our results also suggested that psychrotrophic bacteria in raw milk should be paid attention, especially Pseudomonas during early refrigerated storage and Serratia during late refrigerated storage.

Currently, no effective method exists to detect and monitor fermentation probiotics and evaluate the quality of inactivated fermented milk. Therefore, in this study, a fluorescence quantitative loop-mediated isothermal amplification (FQ-LAMP) method was developed to detect Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The specificity of LAMP primers for L. bulgaricus and S. thermophilus was verified using S-type amplification curves and a single peak at approximately 88.568°C and 83.704°C of the melting curves, respectively. The lowest quantification limits of FQ-LAMP for the two strains in inactivated fermented milk were 8.1×10³CFU/g (170fg/μL) and 6.8×10³CFU/g (170fg/μL), respectively. FQ-LAMP was used to analyse 40 inactivated fermented milk samples from six randomly selected brands. The logarithmic concentration of S. thermophilus in all products was between 7.482 and 8.936. The logarithmic concentration of L. bulgaricus ranged from 4.590 to 8.277, with no detectable L. bulgaricus in three samples. FQ-LAMP has the potential as a rapid, specific, and accurate method for detecting and monitoring L. bulgaricus and S. thermophilus in inactivated fermented milk during their shelf life.