Fermentation Characteristics of Fermented Milk with Streptococcus thermophilus CICC 6063 and Lactobacillus helveticus CICC 6064 and Volatile Compound Dynamic Profiles during Fermentation and Storage.

The lactic acid bacteria Streptococcus thermophilus and Lactobacillus helveticus are commonly used as starter cultures in dairy product production. This study aimed to investigate the characteristics of fermented milk using different ratios of these strains and analyze the changes in volatile compounds during fermentation and storage. A 10:1 ratio of Streptococcus thermophilus CICC 6063 to Lactobacillus helveticus CICC 6064 showed optimal fermentation time (4.2 h), viable cell count (9.64 log10 colony-forming units/mL), and sensory evaluation score (79.1 points). In total, 56 volatile compounds were identified and quantified by solid-phase microextraction and gas chromatography-mass spectrometry (SPME-GC-MS), including aldehydes, ketones, acids, alcohols, esters, and others. Among these, according to VIP analysis, 2,3-butanedione, acetoin, 2,3-pentanedione, hexanoic acid, acetic acid, acetaldehyde, and butanoic acid were identified as discriminatory volatile metabolites for distinguishing between different time points. Throughout the fermentation and storage process, the levels of 2,3-pentanedione and acetoin exhibited synergistic dynamics. These findings enhance our understanding of the chemical and molecular characteristics of milk fermented with Streptococcus thermophilus and Lactobacillus helveticus, providing a basis for improving the flavor and odor of dairy products during fermentation and storage.

Development of a single nucleotide polymorphism-based strain-identified method for Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 using pan-genomics analysis.

The health benefits conferred by probiotics is specific to individual probiotic strains, highlighting the importance of identifying specific strains for research and production purposes. Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 are exceedingly valuable for commercial use with an excellent mixed-culture fermentation. To differentiate these 2 strains from other S. thermophilus and L. delbrueckii ssp. bulgaricus, a specific, sensitive, accurate, rapid, convenient, and cost-effective method is required. In this study, we conducted a pan-genome analysis of S. thermophilus and L. delbrueckii ssp. bulgaricus to identify species-specific core genes, along with strain-specific SNPs. These genes were used to develop suitable PCR primers, and the conformity of sequence length and unique SNPs was confirmed by sequencing for qualitative identification at the strain level. The results demonstrated that SNPs analysis of PCR products derived from these primers could distinguish CICC 6038 and CICC 6047 accurately and reproducibly from the other strains of S. thermophilus and L. delbrueckii ssp. bulgaricus, respectively. The strain-specific PCR method based on SNPs herein is universally applicable for probiotics identification. It offers valuable insights into identifying probiotics at the strain level that is fit-for-purpose in quality control and compliance assessment of commercial dairy products.

Fermentation characteristics and postacidification of yogurt by Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047 at optimal inoculum ratio.

This study aimed to investigate the symbiosis between Streptococcus thermophilus CICC 6038 and Lactobacillus delbrueckii ssp. bulgaricus CICC 6047. In addition, the effect of their different inoculum ratios was determined, and comparison experiments of fermentation characteristics and storage stability of milk fermented by their monocultures and cocultures at optimal inoculum ratio were performed. We found the time to obtain pH 4.6 and ΔpH during storage varied among 6 inoculum ratios (1:1, 2:1, 10:1, 19:1, 50:1, 100:1). By the statistical model to evaluate the optimal ratio, the ratio of 19:1 was selected, which exhibited high acidification rate and low postacidification with pH values remaining between 4.2 and 4.4 after a 50-d storage. Among the 3 groups included in our analyses (i.e., the monocultures of S. thermophilus CICC 6038 [St] and Lb. bulgaricus CICC 6047 [Lb] and their cocultures [St+Lb] at 19:1), the coculture group showed higher acidification activity, improved rheological properties, richer typical volatile compounds, more desirable sensor quality after the fermentation process than the other 2 groups. However, the continuous accumulation of acetic acid during storage showed that acetic acid was more highly correlated with postacidification than d-lactic acid for the Lb group and St+Lb group. Our study emphasized the importance of selecting an appropriate bacterial consortium at the optimal inoculum ratio to achieve favorable fermentation performance and enhanced postacidification stability during storage.

Single-cell rapid identification, in situ viability and vitality profiling, and genome-based source-tracking for probiotics products.

Rapid expansion of the probiotics industry demands fast, sensitive, comprehensive, and low-cost strategies for quality assessment. Here, we introduce a culture-free, one-cell-resolution, phenome-genome-combined strategy called Single-Cell Identification, Viability and Vitality tests, and Source-tracking (SCIVVS). For each cell directly extracted from the product, the fingerprint region of D2O-probed single-cell Raman spectrum (SCRS) enables species-level identification with 93% accuracy, based on a reference SCRS database from 21 statutory probiotic species, whereas the C-D band accurately quantifies viability, metabolic vitality plus their intercellular heterogeneity. For source-tracking, single-cell Raman-activated Cell Sorting and Sequencing can proceed, producing indexed, precisely one-cell-based genome assemblies that can reach ~99.40% genome-wide coverage. Finally, we validated an integrated SCIVVS workflow with automated SCRS acquisition where the whole process except sequencing takes just 5 h. As it is >20-fold faster, >10-time cheaper, vitality-revealing, heterogeneity-resolving, and automation-prone, SCIVVS is a new technological and data framework for quality assessment of live-cell products.

Inoculum size of co-fermentative culture affects the sensory quality and volatile metabolome of fermented milk over storage.

Lacticaseibacillus paracasei PC-01 is a probiotic candidate isolated from naturally fermented yak milk in Lhasa, Tibet, and it has been shown to possess excellent milk fermentation properties. This study used Lacticaseibacillus paracasei PC-01 as a co-fermentation strain to investigate the effect of inoculum size with a commercial starter in milk fermentation on the product flavor and profile of volatile metabolites over 28 d of cold storage. Lacticaseibacillus paracasei PC-01 was allowed to ferment in pasteurized milk with or without the commercial starter (YF-L904) at 42°C until the pH decreased to 4.5. The finished fermented milks were stored at 10°C for 28 d. Milk samples were taken at hour 0 (before fermentation) and then at d 1, 14, and 28 of cold storage. Different inoculum sizes of Lacticaseibacillus paracasei PC-01 had no significant effect on pH or titratable acidity during storage of fermented milk. Viable counts of strain PC-01 continued to increase during cold storage of the fermented milk. Generally, as storage of fermented milk proceeded, the overall sensory quality score decreased in all groups. However, the overall sensory scores of PC-01-M were generally higher than those of other groups, suggesting that a medium dose of Lacticaseibacillus paracasei PC-01 had the most obvious effect of slowing the decline in sensory quality of fermented milk during storage. Changes in sensory scores and consumer preferences were accompanied by increases in both the quantity and variety of key volatile metabolites in fermented milk during fermentation, post-ripening (d 1), and storage. Major differentially abundant metabolites, including acetaldehyde, methyl ketones, medium-chain and short-chain fatty acids, 2,3-butanedione, and acetoin, were enriched in fermented milks rated highly in the sensory evaluation. Our data confirmed that the inoculum size of co-fermentative culture affected the sensory quality and volatile metabolome of fermented milk over storage, and an optimal range of co-fermentative culture was titrated in this work.

Comparison of Meconium Microbiome in Dizygotic and Monozygotic Twins Born by Caesarean Section (CS).

The early-life microbiota triggers life-long effects on physiological functions and health disorders. Previous studies in adult twins or animal models have revealed associations between host genetics and the harmonious microbiota. However, such associations may be obscured by the fact that each intra-pair of twins will continually encounter various environmental factors as they grow up. Here, we collected the meconium samples from nineteen dizygotic pairs (DZ, n = 38) and nine monozygotic pairs (MZ, n = 18) with cesarean delivery, and 16S rRNA gene sequencing was performed to profile the microbiome at birth. Diversity analysis showed that alpha diversity was not significantly different between two groups, whereas beta diversity of MZ twins was significantly lower than that of either DZ twins or unrelated individuals (i.e., randomly selected individual pairs of non-twinship) (p < 0.05). Two groups had very similar microbial classifications but different relative abundances of certain taxa including more Firmicutes (p = 0.05, Wilcoxon test) at the phylum level and lower abundances of five genera (p < 0.05) in DZ group compared to MZ group, including Rheinheimera, Proteus, SMB53, Sphingobium, and Megamonas. Co-occurrence analysis in each group showed slightly more complicated microbial interactions in DZ than MZ twins, although 22 shared bacterial genera co-existed in two groups, with both Rheinheimera and Megamonas having different centralities in their respective co-occurrence networks. Mean intra-class correlation coefficient (ICC) were also significantly higher for MZ (0.312) compared to DZ twins (0.138) (p < 0.05). The predicted microbial gene functions related to carbohydrate were higher in DZ group, whereas folding, sorting, degradation, cell motility pathways and energy metabolism were markedly over-represented in the microbiota of MZ group. In summary, our study uncovered that microbial diversity and components of the meconium microbiome between DZ and MZ twins were partially consistent with that in singleton neonates by cesarean delivery, but several distinctions related to the heritability supported genetic contributions to intestinal microbiome in early life.

Bacillus aquiflavi sp. nov., isolated from yellow water of strongly flavored Chinese baijiu.

A Gram-stain-positive, strictly aerobic and rod-shaped bacterium, designated as 3 H-10T, was isolated from a yellow water sample collected from the manufacturing process of strong flavor Chinese baijiu in Yibin region of Sichuan province (PR China). Oval endospores were formed at the subtermini of cells with swollen sporangia. The isolate was able to grow at temperatures of 20-45 °C (optimum growth at 37 °C), at pH 6.0-10.0 (optimum growth at pH 8.0) and in the presence of 0-2 % (w/v) NaCl (optimum growth with 0 % NaCl). Ribose was the major cell-wall sugar, and meso-diaminopimelic acid (meso-DAP) was the diagnostic amino acid. The main polar lipids of 3 H-10T included diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). MK-7 was predominant menaquinone and iso-C15 : 0 (60.7 %) was the major fatty acid. Comparisons of 16S rRNA gene sequence indicated that 3 H-10T was most closely related to Bacillus mesophilus SA4T (96.30 %), Bacillus ginsengihumi Gsoil 114T (96.27 %) and Bacillus shackletonii LMG 18435T (96.27 %). The average nucleotide identity (ANI) values between strain 3 H-10T and the three type strains mentioned above were 69.56, 70.19 and 70.67 %, respectively. The genomic DNA G+C content was 35.4 mol%. On the basis of its phenotypic, chemotaxonomic and phylogenetic properties, strain 3 H-10T represents a novel species of the genus Bacillus, for which the name Bacillus aquiflavi sp. nov. is proposed. The type strain is Bacillus aquiflavi 3 H-10T (=CICC 24755T=JCM 33703T).

Cell concentration, viability and culture composition of airborne bacteria during a dust event in Beijing.

Airborne bacteria were measured when a dust storm passed Beijing in spring 2012 with a focus on cell concentration, viability and TSA- and R2A-cultured strain composition. The concentration varied at an order of 107cells/m3 with dust loading (demonstrated with PM10) and they had a very close correlation (RT2=0.91, p<0.01). At the time of highest PM10 of 652µg/m3, the bacterial concentration reached 1.4×108cells/m3, which was larger than that before and after the dust event by one order. Bacterial viability, the ratio of number concentration of viable cells to total cells, was 32%-64% and smaller in the dust plume than that before the dust arrival. Bacterial strains from the culture ranged between 2.5×104 and 4.6×105CFU/m3 and no correlation with PM10 was determined. Their composition was different before and after the dust arrival according to 16S rRNA gene sequences and strains belong to Actinomycetes and Firmicutes were the majority in the dust samples.

Universal Indicators for Oil and Gas Prospecting Based on Bacterial Communities Shaped by Light-Hydrocarbon Microseepage in China.

Light hydrocarbons accumulated in subsurface soil by long-term microseepage could favor the anomalous growth of indigenous hydrocarbon-oxidizing microorganisms, which could be crucial indicators of underlying petroleum reservoirs. Here, Illumina MiSeq sequencing of the 16S rRNA gene was conducted to determine the bacterial community structures in soil samples collected from three typical oil and gas fields at different locations in China. Incubation with n-butane at the laboratory scale was performed to confirm the presence of "universal microbes" in light-hydrocarbon microseepage ecosystems. The results indicated significantly higher bacterial diversity in next-to-well samples compared with background samples at two of the three sites, which were notably different to oil-contaminated environments. Variation partitioning analysis showed that the bacterial community structures above the oil and gas fields at the scale of the present study were shaped mainly by environmental parameters, and geographic location was able to explain only 7.05% of the variation independently. The linear discriminant analysis effect size method revealed that the oil and gas fields significantly favored the growth of Mycobacterium, Flavobacterium, and Pseudomonas, as well as other related bacteria. The relative abundance of Mycobacterium and Pseudomonas increased notably after n-butane cultivation, which highlighted their potential as biomarkers of underlying oil deposits. This work contributes to a broader perspective on the bacterial community structures shaped by long-term light-hydrocarbon microseepage and proposes relatively universal indicators, providing an additional resource for the improvement of microbial prospecting of oil and gas.

High efficiency transformation of stevioside into a single mono-glycosylated product using a cyclodextrin glucanotransferase from Paenibacillus sp. CGMCC 5316.

Stevioside is a non-caloric, natural, high-intensity sweetener. However, the bitter aftertaste of stevioside restricts its utilization for human consumption and limits its application in the food industry. In this study, a high efficiency enzymatic modification system was investigated to improve stevioside taste quality. A cyclodextrin glucanotransferase (CGTase) producing strain Paenibacillus sp. CGMCC 5316 was isolated from Stevia planting soil. With starch as glycosyl donor, this CGTase can transform stevioside into a single specific product which is an isomer of rebaudioside A and identified as mono-glycosylated stevioside. The taste of stevioside is improved noticeably by generating mono-glycosylated stevioside, which possesses a sucrose-like taste and has sweetness increased significantly by 35.4%. Next, the parameters influencing CGTase production were optimized. Compared to initial conditions, CGTase activity increased by 214.7% under optimum conditions of 3.9 g/L starch, 17.9 g/L tryptone, and 67.6 h of culture time, and the transglycosylation rate of stevioside was remarkably increased by 284.8%, reaching 85.6%. This CGTase modification system provides a promising solution for improving the sweetness and taste quality of stevioside. The efficiency of CGTase transformation can be greatly increased by optimizing the culture conditions of Paenibacillus sp. CGMCC 5316.

Effects of growth stage and fulvic acid on the diversity and dynamics of endophytic bacterial community in Stevia rebaudiana Bertoni leaves.

The aim of this study was to learn the interactions among the endophytic bacteria, the plant growth, the foliar spray of fulvic acid, and the accumulation of steviol glycosides in the leaves of Stevia rebaudiana. Metagenomic DNA was extracted from the Stevia leaves at different growth stages with or without the fulvic acid treatment; and the diversity of endophytic bacteria in Stevia leaves was estimated by pyrosequencing of 16S rRNA genes. As results, Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were found to be the dominant phyla despite the growth stages and fulvic acid application. Stevia growth stages strongly regulated composition of endophytic community. The genera Agrobacterium (12.3%) and Erwinia (7.2%) dominated in seedling stage were apparently declined in the vegetable and initial flowering stages, while Sphingomonas and Methylobacterium increased in mature leaves at harvest time, which showed that the mature leaves of Stevia preferred to accumulate some certain endophytic bacteria. Sphingomonas and Methylobacterium constituted an important part of the core endophytic community and were positively correlated with the stevioside content and UGT74G1 gene expression, respectively; while Erwinia, Agrobacterium, and Bacillus were negatively correlated with the stevioside accumulation. Fulvic acid treatment accelerated the variation of endophytes along the growth stages and increased the steviol glycosides content. This is the first study to reveal the community composition of endophytic bacteria in the Stevia leaves, to evidence the strong effects of growth stage and fulvic acid application on the endophytes of Stevia, and to demonstrate the correlation between the endophytic bacteria and the steviol glycosides accumulation.

Novosphingobium tardum sp. nov., isolated from sediment of a freshwater lake.

A Gram-negative, non-motive, aerobic and non-spore-forming strain 16-28-2(T) isolated from freshwater sediment of Taihu Lake was characterized by using a polyphasic approach. The optimum growth conditions were found to be as follows: 28 °C, pH 6.5 and 0-0.5 % NaCl in YG liquid medium. The major fatty acids were identified to be summed feature 3 (consisting of C16:1 ω7c and/or C16:1 ω6c), summed feature 8 (consisting of C18:1 ω7c and/or C18:1 ω6c), C14:0 2-OH, C17:1 ω6c, C16:0 and C18:1 ω7c 11-methyl (>5 %). Strain 16-28-2(T) was found to contain diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and sphingoglycolipid as the major polar lipids; and ubiquinone 10 (Q-10) as the major respiratory quinone. DNA G+C content of strain 16-28-2(T) was 63.5 mol % (Tm). A phylogenetic study of 16S rRNA gene indicated that strain 16-28-2(T) is a member of the genus Novosphingobium, with the highest 16S rRNA gene sequence similarity of 96.3 % with Novosphingobium lentum MT1(T) and below 96 % with the other Novosphingobium species. On the basis of the phylogenetic, phenotypic analyses and biochemical characterization, we suggest that strain 16-28-2(T) is a novel species in the genus Novosphingobium, for which the name Novosphingobium tardum sp. nov. is proposed. The type strain of N. tardum is 16-28-2(T) (=CGMCC 1.12989(T) =NBRC 110956(T)).

Synergy of crude enzyme cocktail from cold-adapted Cladosporium cladosporioides Ch2-2 with commercial xylanase achieving high sugars yield at low cost.

BACKGROUND: The efficiency and cost of current lignocellulosic enzymes still limit the large-scale production of cellulosic ethanol in industry. Residual lignin after pretreatment severely depresses the activity of polysaccharide hydrolases and the h ydrolysis of holocellulose. If we include in hydrolase mixture construction the ligninase involved in lignin degradation, which mainly includes laccase, manganese peroxidases (MnP) and lignin peroxidase (LiP), it is feasible that this could greatly improve the fermentable sugars yield. RESULTS: The psychrophilic lignocellulosic enzymes system of Cladosporium cladosporioides Ch2-2 including ligninase and polysaccharide hydrolases was suitable for selective delignification and efficient saccharification of biomass with wide thermal adaptability. The purified laccase was optimally active at 15°C and pH 3.5, exhibiting high thermostability over a broad range of temperatures (between 4 and 40°C). In addition, manganese-independent peroxidase (MIP), a special type of ligninase with the capacity to oxidize dimethyl phthalate (DMP) in the absence of H2O2 and Mn(2+), was optimally active at 20°C and pH 2.5, exhibiting high thermostability over a broad range of temperatures (4 and 28°C), while depressed completely by Fe(2+) and essentially unaffected by EDTA. Synergy between Ch2-2 crude enzymes and commercial xylanase obviously enhanced biomass hydrolysis, which could take the place of expensive commercial cellulase mixture. The maximum value of synergistic degree reached 4.7 at 28°C, resulting in 10.1 mg/mL reducing sugars. CONCLUSIONS: The psychrophilic enzymes system of C. cladosporioides Ch2-2 with a different synergistic mechanism has huge potential for the enhancement of biomass hydrolysis at mesophilic and low temperatures. The application scope of the lignocellulosic enzyme cocktail could be greatly enlarged by optimizing the operation conditions specific to the characteristics of ligninase.