Lai-Yu Kwok

Angiotensin-converting enzyme inhibitory activity of Lactobacillus helveticus strains from traditional fermented dairy foods and antihypertensive effect of fermented milk of strain H9

Hypertension is a major global health issue which elevates the risk of a large world population to chronic life-threatening diseases. The inhibition of angiotensin-converting enzyme (ACE) is an effective target to manage essential hypertension. In this study, the fermentation properties (titratable acidity, free amino nitrogen, and fermentation time) and ACE-inhibitory (ACEI) activity of fermented milks produced by 259 Lactobacillus helveticus strains previously isolated from traditional Chinese and Mongolian fermented foods were determined. Among them, 37 strains had an ACEI activity of over 50%. The concentrations of the antihypertensive peptides, Ile-Pro-Pro and Val-Pro-Pro, were further determined by ultra performance liquid chromatography with quadrupole-time-of-flight mass spectrometry. The change of ACEI activity of the fermented milks of 3 strains exhibiting the highest ACEI activity upon gastrointestinal protease treatment was assayed. Fermented milks produced by strain H9 (IMAU60208) had the highest in vitro ACEI activity (86.4 ± 1.5%), relatively short fermentation time (7.5 h), and detectable Val-Pro-Pro (2.409 ± 0.229 µM) and Ile-Pro-Pro (1.612 ± 0.114 µM) concentrations. Compared with the control, a single oral dose of H9-fermented milk significantly attenuated the systolic, diastolic, and mean blood pressure of spontaneously hypertensive rats (SHR) by 15 to 18 mmHg during the 6 to 12 h after treatment. The long-term daily H9-fermented milk intake over 7 wk exerted significant antihypertensive effect to SHR, but not normotensive rats, and the systolic and diastolic blood pressure were significantly lower, by 12 and 10 mmHg, respectively, compared with the control receiving saline. The feeding of H9-fermented milk to SHR resulted in a significantly higher weight gain at wk 7 compared with groups receiving saline, commercial yogurt, and captopril. Our study identified a novel probiotic L. helveticus strain originated from kurut sampled from Tibet (China), which is a valuable resource for future development of functional foods for hypertension management.

High-throughput sequencing for the detection of the bacterial and fungal diversity in Mongolian naturally fermented cow’s milk in Russia

BackgroundTraditional fermented dairy products are major components of the typical Mongolian diet since ancient times. However, almost all the previous studies on the microbial composition of traditional Mongolian fermented dairy products analyzed food samples from the Chinese Mongolian region and Mongolia but not the Russian Mongolian region. In this study, the bacterial and fungal community diversity of nineteen naturally fermented cow’s milk (NFCM) samples from local Mongolian families residing in Kalmykia and Chita of Russia was investigated with pyrosequencing.ResultsFirmicutes and Ascomycota were the predominant phyla respectively for bacteria and fungi. The abundance of the bacterial phylum Acidobacteria was considerably different between the samples from the two regions. At genus level, Lactobacillus and Pichia were the predominating bacterial and fungal genera, respectively, while six bacterial genera significantly differed between the Kalmykia (enrichment of Aeromonas, Bacillus, Clostridium, Streptococcus, Vogesella) and Chita (enrichment of Lactococcus) samples. The results of principal coordinate analysis (PCoA) based on the bacterial or fungal composition of the Kalmykia and Chita samples revealed a different microbiota structure between the samples collected in these two locations. The redundancy analysis (RDA) identified 60 bacterial and 21 fungal OTUs as the key variables responsible for such microbiota structural difference.ConclusionsOur results suggest that structural differences existed in the microbiota of NFCM between Kalmykia and Chita. The difference in geographic environment may be an important factor influencing the microbial diversity of NFCM made by the Mongolians in Russia.

454 pyrosequencing reveals changes in the faecal microbiota of adults consuming Lactobacillus casei Zhang

Probiotics are believed to help to maintain a healthy balance of the human gut microbiota. Lactobacillus casei Zhang (LcZ) is a novel potential probiotic isolated from the naturally fermented food koumiss. To better understand the impact of this potential probiotic on human intestinal microbiota, 24 subjects were randomly recruited for a longitudinal study: the subjects were required to consume LcZ for 28 days, and faecal samples were collected prior to, during and after the LcZ consumption phase. Alterations in the gut microbiota were monitored using 454 pyrosequencing and quantitative polymerase chain reaction(q-PCR) technologies. We found that the consumption of LcZ significantly altered the composition of intestinal microbiota (P < 0.001) and the gut microbiota diversity. Further analysis at the genus level revealed a positive correlation between LcZ and Prevotella, Lactobacillus, Faecalibacterium, Propionibacterium, Bifidobacterium and an unidentified genus from Bacteroidaceae and Lachnospiraceae and a negative correlation between LcZ administration and the presence of Clostridium, Phascolarctobacterium, Serratia, Enterococcus, Shigella and Shewanella. Furthermore, these changes were confirmed by q-PCR data.

Evaluation of bacterial contamination in raw milk, ultra-high temperature milk and infant formula using single molecule, real-time sequencing technology.

The Pacific Biosciences (Menlo Park, CA) single molecule, real-time sequencing technology (SMRT) was reported to have some advantages in analyzing the bacterial profile of environmental samples. In this study, the presence of bacterial contaminants in raw milk, UHT milk, and infant formula was determined by SMRT sequencing of the full length 16S rRNA gene. The bacterial profiles obtained at different taxonomic levels revealed clear differences in bacterial community structure across the 16 analyzed dairy samples. No indicative pathogenic bacteria were found in any of these tested samples. However, some of the detected bacterial species (e.g., Bacillus cereus, Enterococcus casseliflavus, and Enterococcus gallinarum) might potentially relate with product quality defects and bacterial antibiotic gene transfer. Although only a limited number of dairy samples were analyzed here, our data have demonstrated for the first time the feasibility of using the SMRT sequencing platform in detecting bacterial contamination. Our paper also provides interesting reference information for future development of new precautionary strategies for controlling the dairy safety in large-scale industrialized production lines.

Investigation of bacterial and fungal diversity in tarag using high-throughput sequencing

This is the first study on the bacterial and fungal community diversity in 17 tarag samples (naturally fermented dairy products) through a metagenomic approach involving high-throughput pyrosequencing. Our results revealed the presence of a total of 47 bacterial and 43 fungal genera in all tarag samples, in which Lactobacillus and Galactomyces were the predominant genera of bacteria and fungi, respectively. The number of some microbial genera, such as Lactococcus, Acetobacter, Saccharomyces, Trichosporon, and Kluyveromyces, among others, was found to vary between different samples. Altogether, our results showed that the microbial flora in different samples may be stratified by geographic region.

Assessment of the bacterial and fungal diversity in home-made yoghurts of Xinjiang, China by pyrosequencing.

BACKGROUND The aim of this study was to gain a deeper knowledge of the bacterial and fungal community diversity in local home-made yoghurts from Zhaosu and Tekesi counties, Xinjiang, China, where a relatively high proportion of the ethnic minority population resides. RESULTS The bacterial and fungal community diversity in 22 home-made yoghurt samples was analyzed by pyrosequencing. This approach revealed the presence of six bacterial and two fungal phyla, comprising 69 bacterial and 20 fungal genera respectively, among all samples. Firmicutes and Ascomycota were the dominant phyla and Lactobacillus and Saccharomyces the predominant genera of bacteria and fungi respectively. Based on redundancy analysis, the bacterial diversity in samples from the two counties differed mainly because of eight responsive operational taxonomic units of Lactobacillus. However, no obvious difference existed in the fungal population of samples from the two counties. CONCLUSION The results indicated that the microbial community of home-made yoghurts varied with their geographical origin and manufacturing process. The study showed that pyrosequencing could provide a better understanding of microbiological diversity as well as useful information for future studies on the selection of starter cultures for yoghurts.

High-throughput sequencing reveals microbial community diversity of Tibetan naturally fermented yak milk

Naturally fermented yak milk (NFYM) is a traditional Tibetan fermented product that contains a rich microbial community. This study used high-throughput pyrosequencing to investigate the bacterial and fungal community diversity of 16 NFYM samples from the villages Geda and Ningzhong in Tibet. Pyrosequencing produced a total of 112,173 high quality bacterial 16S rRNA gene sequences and 90,980 fungal high quality reads from the NFYM. Sequence analysis grouped the high quality gene sequences into eleven bacterial and five fungal phyla, of which Firmicutes and Ascomycota dominated. At the genus level, Lactobacillus and Saccharomyces were the dominating bacteria and fungi, respectively. Principal coordinate analysis (PCoA) based on weighted and unweighted UniFrac values revealed no significant differences in the structure of bacterial and fungal microbiota. However, multivariate analysis of variance (MANOVA) based on unweighted UniFrac distances showed significant differences (P < 0.05) between the bacterial and fungal populations of the two NFYM samples from different villages. After the redundancy analysis (RDA), 49 key responding operational taxonomic units (OTUs) could be identified. Among them, 7 OTUs (Acinetobacter, unidentified Bacteroidetes, Lactobacillus, unidentified Proteobacteria, Streptococcus, Pantoea, and unidentified Firmicutes) were enriched in the Geda village samples, while 42 OTUs (mainly Massilia, Propionibacterium, Lactococcus, Leuconostoc, and Enterococcus) were enriched in the Ningzhong village samples. The present work provided insights into the microbiome of Tibetan NFYM and showed that the microbiota composition may be stratified by their geographic regions.

Characterization of angiotensin-converting enzyme inhibitory activity of fermented milk produced by Lactobacillus helveticus

Hypertension affects up to 30% of the adult population in most countries. It is a known risk factor for cardiovascular diseases, including coronary heart disease, peripheral artery disease, and stroke. Owing to the increased health awareness of consumers, the application of angiotensin-converting enzyme (ACE)-inhibitory peptides produced by Lactobacillushelveticus to prevent or control high blood pressure has drawn wide attention. A total of 59 L. helveticus strains were isolated from traditional fermented dairy products and the ACE-inhibitory activity of the fermented milks produced with the isolated microorganisms was assayed. The ACE-inhibitory activity of 38 L. helveticus strains was more than 50%, and 3 strains (IMAU80872, IMAU80852, and IMAU80851) expressing the highest ACE-inhibitory activity were selected for further studies. Particularly, the gastrointestinal protease tolerance and thermostability of the ACE-inhibitory activity in the fermented milks were assessed. Based on these 2 criteria, IMAU80872 was found to be superior over the other 2 strains. Furthermore, IMAU80872 exhibited a high in vitro ACE-inhibitory activity at the following fermentation conditions: fermentation temperature at 40°C, inoculation concentration of 1×10(6) cfu/mL, and fermentation for 18h. Finally, by using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis, we observed changes of the metabolome along the milk fermentation process of IMAU80872. Furthermore, 6 peptides were identified, which might have ACE-inhibitory activity. In conclusion, we identified a novel ACE-inhibitory L. helveticus strain suitable for the production of fermented milk or other functional dairy products.

A Perspective Study of Koumiss Microbiome by Metagenomics Analysis Based on Single-Cell Amplification Technique

Koumiss is a traditional fermented dairy product and a good source for isolating novel bacteria with biotechnology potential. In the present study, we applied the single-cell amplification technique in the metagenomics analysis of koumiss. This approach aimed at detecting the low-abundant bacteria in the koumiss. Briefly, each sample was first serially diluted until reaching the level of approximately 100 cells. Then, three diluted bacterial suspensions were randomly picked for further study. By analyzing 30 diluted koumiss suspensions, a total of 24 bacterial species were identified. In addition to the previously reported koumiss-associated species, such as Lactobacillus (L.) helveticus. Lactococcus lactis. L. buchneri, L. kefiranofaciens, and Acetobacter pasteurianus, we successfully detected three low-abundant taxa in the samples, namely L. otakiensis. Streptococcus macedonicus, and Ruminococcus torques. The functional koumiss metagenomes carried putative genes that relate to lactose metabolism and synthesis of typical flavor compounds. Our study would encourage the use of modern metagenomics to discover novel species of bacteria that could be useful in food industries.

Effects of microencapsulated Lactobacillus plantarum LIP-1 on the gut microbiota of hyperlipidaemic rats

The in vivo effects of administering free and microencapsulated Lactobacillus plantarum LIP-1 cells (2·0×109 colony-forming units/d) were evaluated in high-fat-diet-induced hyperlipidaemic rats. Results from real-time quantitative PCR targeting to LIP-1 cells showed a higher colon colonisation count of LIP-1 in the rats receiving microencapsulated cells compared with free cells (P<0·05). Moreover, the microencapsulated LIP-1 treatment resulted in a more obvious lipid-lowering effect (P<0·05). Meanwhile, their faecal samples had significantly less lipopolysaccharide-producing bacteria (especially Bilophila, Sutterella and Oscillibacter) and mucosa-damaging bacteria (Bilophila and Akkermansia muciniphila), whereas significantly more SCFA-producing bacteria (P<0·05) (namely Lactobacillus, Alloprevotella, Coprococcus, Eubacterium and Ruminococcus) and bacteria that potentially possessed bile salt hydrolase activity (Bacteroides, Clostridium, Eubacterium and Lactobacillus), and other beneficial bacteria (Alistipes and Turicibacter). Further, Spearmans correlation analysis showed significant correlations between some of the modulated gut bacteria and the serum lipid levels. These results together confirm that microcapsulation enhanced the colon colonisation of LIP-1 cells, which subsequently exhibited more pronounced effects in improving the gut microbiota composition of hyperlipidaemic rats and lipid reduction.