Viable Bacteria Research Articles

Effect of Encapsulation of Lactobacillus casei in Alginate-Tapioca Flour Microspheres Coated with Different Biopolymers on the Viability of Probiotic Bacteria.

To realize the health benefits of probiotic bacteria, they must withstand processing and storage conditions and remain viable after use. The encapsulation of these probiotics in the form of microspheres containing tapioca flour as a prebiotic and vehicle component in their structure or shell affords symbiotic effects that improve the survival of probiotics under unfavorable conditions. Microencapsulation is one such method that has proven to be effective in protecting probiotics from adverse conditions while maintaining their viability and functionality. The aim of the work was to obtain high-quality microspheres that can act as carriers of Lactobacillus casei bacteria and to assess the impact of encapsulation on the viability of probiotic microorganisms in alginate microspheres enriched with a prebiotic (tapioca flour) and additionally coated with hyaluronic acid, chitosan, or gelatin. The influence of the composition of microparticles on the physicochemical properties and the viability of probiotic bacteria during storage was examined. The optimal composition of microspheres was selected using the design of experiments using statistical methods. Subsequently, the size, morphology, and cross-section of the obtained microspheres, as well as the effectiveness of the microsphere coating with biopolymers, were analyzed. The chemical structure of the microspheres was identified by using Fourier-transform infrared spectrophotometry. Raman spectroscopy was used to confirm the success of coating the microspheres with the selected biopolymers. The obtained results showed that the addition of tapioca flour had a positive effect on the surface modification of the microspheres, causing the porous structure of the alginate microparticles to become smaller and more sealed. Moreover, the addition of prebiotic and biopolymer coatings of the microspheres, particularly using hyaluronic acid and chitosan, significantly improved the survival and viability of the probiotic strain during long-term storage. The highest survival rate of the probiotic strain was recorded for alginate-tapioca flour microspheres coated with hyaluronic acid, at 5.48 log CFU g-1. The survival rate of L. casei in that vehicle system was 89% after storage for 30 days of storage.

The exploration of pasteurization processes and mechanisms of inactivation of Bacillus cereus ATCC 14579 using radio frequency energy

Radio frequency (RF) heating has been utilized to investigate sterilization techniques, but the mechanism of sterilization via RF heating, particularly on Bacillus cereus (B. cereus), has not been thoroughly examined. In this paper, sterilization processes and potential bactericidal mechanisms of B. cereus using RF were investigated. The best heating and sterilization efficiency was achieved at (Electrode gap 130 mm, conductivity of bacterial suspension 0.1 S/m, volume of bacterial suspension 40 mL). Heating a suspension of B. cereus to 90 °C in 80 s using RF reduced the number of viable bacteria by 4.87 logarithms. At the cellular level, there was a significant leakage of nucleic acids and proteins from the bacterial cells. Additionally, the integrity of the cell membrane was severely damaged, with a decrease in ATP concentration of 2.08 mM, Na, K-ATPase activity to 10.7 (U/109 cells), and Ca, Mg-ATPase activity to 11.6 (U/109 cells). At the molecular level, transcriptomics analysis showed that RF heating of B. cereus to 65 °C produced 650 more differentially expressed genes (DEGs) compared with RF heating to 45 °C. The GO annotation analysis indicated that the majority of differentially expressed genes (DEGs) were predominantly associated with cellular components. KEGG metabolic analysis showed enrichment in microbial metabolism in diverse environments, etc. This study investigated the potential bactericidal mechanism of B. cereus using RF, and provided some theoretical basis for the research of the sterilization of B. cereus.

Shield-armed probiotic delivery system based on co-deposition of poly-dopamine and poly-l-lysine helps Lactiplantibacillus plantarum relieve hyperuricemia

Hyperuricemia (HUA) is a disease characterized by an abnormal metabolism of purine. Lactic acid bacteria (LAB) have attracted much attention for their safe and effective treatment of HUA by inhibiting xanthine oxidase (XOD) and regulating gut microbiota. However, the effectiveness of probiotics can be compromised by the harsh environment of the gastrointestinal tract. In preliminary experiments, Lactiplantibacillus plantarum DY1, which is generally regarded as safe (GRAS), can lower uric acid. We have devised a straightforward and efficient technique for encapsulating DY1 using a coating comprising polydopamine (PDA) co-deposited with poly-l-lysine (PLL) to obtain DY1@PDLL. TEM, SEM, FT-IR and DLS tests showed that DY1 was successfully coated. Incubate at SGF or SIF for 3 h, the number of viable bacteria of free probiotics and DY1@PDLL decreased by 0.92 and 0.46 log cfu/mL, 1.66 and 0.66 log cfu/mL, respectively. The fluorescence intensity of the intestines of the DY1@PDLL treated mice was 3.96 times that of free probiotic. Notably, DY1@PDLL can reduce the uric acid levels of HUA mice by 31.63 % and free probiotics by 18.72 % (≈1.69 times). DY1@PDLL could also regulate gut microbiota and serum metabolic profile. These findings unequivocally highlight the remarkable potential of DY1@PDLL as an exceptional oral probiotic delivery system.

PSV-12 Evaluating the effect of lyoprotectants in preserving community structure and bacterial viability in the lyophilized fecal community of pigs

Abstract Fecal microbiota transplantation (FMT) stands as a potential method for enhancing pig health. FMT has progressed from the basic delivery of fresh stool, to lyophilized preparations which is beneficial for sample storage, but can damage bacterial cells. This has necessitated the need for lyoprotectants to preserve bacterial viability. However, the ideal lyoprotectant for retaining community structure and viability remains unclear. Furthermore, DNA-based microbial profiling methods such as 16S rRNA and metagenomics sequencing do not distinguish live and dead bacteria, prompting the need for other methods of distinguishing live and dead members of the bacterial community after lyophilization. PMA-seq (propidium monoazide treatment followed by 16S rRNA gene amplicon sequencing) has been shown to differentiate between live and dead bacteria in microbial communities. To refine FMT in pigs, we sought to ascertain the impact of four lyoprotectants on bacterial viability and fecal microbiome structure. Fecal samples from six pigs were treated with mannitol, maltodextrin, trehalose, maltodextrin-trehalose mix (2.5% each) as lyoprotectants or left untreated. Lyophilized fecal samples underwent PMA treatment prior to 16S rRNA sequencing to capture the active community, and total and viable bacterial cells number were quantified. One-way ANOVA was used to analyze total and viable bacterial cells, and richness, while PERMANOVA was used to compare beta diversity. Results revealed no initial differences in total, viable bacteria, or community structure (P > 0.05). However, untreated, and mannitol-treated groups showed significantly reduced total and viable bacterial cells post-lyophilization (P < 0.01), unlike the other lyoprotectants (P > 0.05). Mannitol and the untreated group also displayed decreased richness and beta diversity shifts (P < 0.05). Overall, lyophilization with mannitol or no lyoprotectant notably impacted pig fecal microbiota community structure, while maltodextrin, trehalose, and maltodextrin-trehalose effectively preserved bacterial viability and structure.

179 Charting the evolution of rumen microbial models from past to present

Abstract This review delves into the intricate realm of ruminal microbes, shedding light on their complexity beyond previously recognized dimensions. Existing publications have probed the essential facets of these microorganisms on a finer scale than previously acknowledged. Mathematical models have emerged to simulate the behavior of approximately 200 distinct bacterial species, 25 genera of ciliate protozoa, and five genera of anaerobic fungi, constituting about 10% of the total viable bacteria in the rumen. However, the expansive diversity and density of rumen microbes challenge the complete characterization of species or genera in relation to substrates or end products. Consequently, continuous revision and re-engineering of mathematical models become imperative. In the context of ruminants, prevalent models often categorize bacteria into fiber-fermenting and nonfiber-fermenting groups. An additional subgroup featuring hyper-ammonia-producing bacteria has been proposed, but its explicit integration into nutrition models remains unexplored. Furthermore, incorporating a more mechanistic evaluation of protozoa and fungal growth may be warranted to improve nutrition models. The time horizon and intervals at which models are constructed present another critical consideration, with feed efficiency variations among ruminants believed to be closely tied to differences in the ruminal microbiota. Addressing immediate concerns, this review underscores the need for a comprehensive exploration of factors influencing microbial growth in the rumen. Several indices have been suggested and adopted to predict microbial efficiencies, but they may provide simplification for practical application. Thus, a mechanistic approach should be considered to remove dependencies between estimates and improve predictive accuracy and precision. While artificial intelligence holds promise in elucidating gaps within empirical and mechanistic models, the generation of accurate, vetted datasets is paramount. Furthermore, this review posits that factors such as recycled nitrogen, endotoxins, and microbial population dynamics (i.e., cross-feeding and competition) may be limiting our understanding of microbial growth, suggesting the necessity of modeling these intricacies concurrently to achieve a more nuanced comprehension. This multifaceted approach aims to propel the understanding of ruminal microbial dynamics into new frontiers, paving the way for more informed and effective modeling in the future.

Physicochemical, enzymatic and fermentation modifications improve resistant starch levels and prebiotic properties of porang (Amorphophallus oncophyllus) flour

SummaryPorang tubers (Amorphophallus oncophyllus) are one of the Araceae family plants, which naturally contain resistant starch (RS). The RS is able to provide health impacts such as reducing the glycaemic index (GI), preventing the formation of gallstones and cardiovascular disease, and increasing mineral absorption. This research aims to improve the RS and prebiotic properties of porang flour through physical, chemical, enzymatic and microbiological modifications. Research methods include modification with physical treatment of autoclaving‐cooling one and two cycles (AC‐1S and AC‐2S), microwave‐cooling (MWC), heat moisture treatment (HMT), annealing (ANN), chemical treatment with acid hydrolysis (HA), enzymatic treatment with pullulanase debranching (DP) and microbiological treatment with combined heating and cooling fermentation (FAC). The results showed that physical, chemical, enzymatic and fermentation modification techniques increased the characteristics of RS and the prebiotic properties of porang flour. The best modification method for porang flour was obtained in the DP treatment with the morphological characteristics of sharp‐surfaced granules, total starch 39.81%, amylose content 3.73%, amylopectin content 36.08%, reducing sugar content 16.31%, power digestibility 43.81%, very rapidly digestible starch (VRDS) 8.59%, rapidly digestible starch (RDS) 11.08%, slowly digestible starch (SDS) 23.60%, RS 56.73%, resistance to gastric acid 98.60%, lactic acid bacteria (LAB) viability 11.87 log cfu/ml, prebiotic effect 3.07, prebiotic index 2.46 and prebiotic activity 1.77.

BD Vacutainer™ Urine Culture & Sensitivity Preservative PLUS Plastic Tubes Minimize the Harmful Impact of Stressors Dependent on Temperature and Time Storage in Uropathogenic Bacteria.

Background: Urinary tract infection is a worldwide health problem. According to the Clinical Laboratory Improvement Amendments and the European Urinalysis Guideline, urine samples should be tested within 2 h of collection. Thus, using chemical preservatives that guarantee the pre-analytical conditions is a practical tool. However, the effects of temperature and storage time as uropathogenic bacteria stressors are unclear. Methods: Gram-negative and -positive ATTC strains, E. coli, P. mirabilis, E. faecalis, and S. aureus, were used in this study. Strains in liquid media were stored at 4, 25, and 37 °C for 0, 2, 12, 24, and 48 h in tubes with and without preservatives. Then, reactive oxygen species (ROS) levels, viable but non-culturable bacteria (VBNC), and bacteria growth were analyzed. Results: A high ROS level was associated with the presence of VBNC and dead bacteria with low CFU counts, but a low ROS level increased the CFU number, depending on temperature and storage time in tubes without preservatives (boric acid, sodium borate, and formate). The BD Vacutainer™ Urine Culture & Sensitivity Preservative PLUS Plastic Tubes (C&S-PP) prevent this ROS increase, maintaining the CFU number for longer. Conclusions: C&S-PP tubes minimize the stressor effects (temperature and time storage) on uropathogenic bacteria when stored, improving the pre-analytical conditions of cultures realized by the clinical laboratory.

Comparative Analysis of Antibacterial Efficacy and Flow Characteristics of Three Root Canal Sealers.

Oral microflora containing microorganisms is responsible for the majority of orodental diseases as well as post-endodontic treatment failure. Even in the ideal root canal treatment cases, a small number of viable bacteria will remain in the dentinal tubule hence come the role of root canal sealers, which must offer and possess some degree of antimicrobial activity alongside adequate flowability to ensure excellent sealing of all lateral dentinal tubule canals to prevent the possibility of future reinfection. The present study aimed to examine the antibacterial efficacy against Enterococcus faecalis (E. faecalis) using an agar diffusion test (ADT) at different time intervals, as well as the flow characteristics of three different root canal sealers: Endofill (Produits Dentaires SA,Vevey, Switzerland), AH Plus (Dentsply De Trey GmbH, Konstanz, Germany), and the newly introduced GuttaFlow2 (Coltène/Whaledent, Altstätten, Switzerland). The antibacterial activity and flow characteristics of three root canal sealers, Endofill, AH Plus, and GuttaFlow2, were tested using ADT at three different time intervals (24 hours, 48 hours, and seven days). For this purpose,E. faecalis strains were used as the pathogen in this study. Flow characteristics were done using the standard flow test protocol recommended for endodontic sealers. Data were analyzed using one-way ANOVA and the Tukey post hoc test, with a p-value < 0.05 considered statistically significant. The results for antibacterial activity showed a statistically significant difference between Endofill and the other groups (p < 0.05). The antibacterial activity of Endofill increased over time from 24 hours to 48 hours and seven days. AH Plus demonstrated antibacterial activity only within the first 24 hours of mixing, while GuttaFlow2 showed no inhibition zones against E. faecalis. Regarding the flow test results, the Endofill group recorded the lowest flow values compared to GuttaFlow2 and AH Plus, which was statistically significant (p < 0.05). There was no statistically significant difference between GuttaFlow2 and AH Plus for flow values. Endofill demonstrated the highest antibacterial activity at all time intervals, while GuttaFlow2 showed no antimicrobial activity. AH Plus exhibited antimicrobial effects only within the first 24 hours of mixing. In terms of flow values, Endofill had the lowest flow, whereas GuttaFlow2 and AH Plus had the highest flow values.

Effects of Chemically and Green Synthesized Zinc Oxide Nanoparticles on Shelf Life and Sensory Quality of Minced Fish (Pangasius hypophthalmus).

The purpose of this study was to investigate the effect of chemically and green synthesized zinc oxide nanoparticles (ZnO-NPs) on the shelf life and sensory quality of fish meat. In this study, ZnO-NPs were synthesized by employing the colloidal chemistry (CZnO-NPs) and green synthesis (GZnO-NPs) methods, and they were also characterized to assess their morphology. The synthesized ZnO-NPs, ZnO, and zinc acetate (ZnA) were used for the preservation and fortification of fish (Pangasius hypophthalmus) meat at 20 mg/kg of Zn. In a six-day storage study at 4 °C, the fish samples were evaluated for their sensory attributes (color and odor), physicochemical quality (pH and total volatile base nitrogen), oxidative changes (thiobarbituric acid-reactive substances and peroxide value), and microbial loads at 0, 3, and 6 days of storage. The fortification of raw fish with the synthesized CZnO-NPs produced better sensory attributes (color and odor) and maintained a pH non-conducive to microbial growth throughout the entire storage period compared with the control, ZnO, and ZnA-fortified samples. The GZnO-NPs largely did not provide any added advantage over CZnO-NPs but sometimes responded better than the control, ZnO, and ZnA samples. Oxidative status and total volatile base nitrogen were lower for CZnO-NPs in refrigerated fish compared with the other treatments. The ZnO-NP-fortified fish had the lowest counts of total viable bacteria, coliforms, Staphylococcus spp., and Vibrio spp. Hence, the fortification of fish with synthesized CZnO-NPs is promising as a food additive to reduce microbial spoilage and lipid peroxidation of fish in storage.

Efficacy and safety of Bacteroides fragilis BF839 for pediatric autism spectrum disorder: a randomized clinical trial.

The clinical utility of Bacteroides fragilis in treating autism spectrum disorder (ASD) remains unclear. Therefore, this randomized, double-blind, placebo-controlled study aimed to explore the therapeutic effects and safety of B. fragilis BF839 in the treatment of pediatric ASD. We examined 60 children aged 2-10 years diagnosed with ASD, and participants received either BF839 powder (10 g/bar with ≥106 CFU/bar of viable bacteria, two bars/day) or placebo for 16 weeks. The primary outcomes was Autism Behavior Checklist (ABC) score. The secondary outcomes were Childhood Autism Rating Scale (CARS), Social Responsiveness Scale (SRS), Normal Development of Social Skills from Infants to Junior High School Children (S-M), Gastrointestinal Symptom Rating Scale (GSRS) scores, and fecal microbiome composition. Assessments were performed on day 0 and at weeks 8 and 16. Compared with the placebo group, the BF839 group showed significant improvement in the ABC body and object use scores at week 16, which was more pronounced in children with ASD aged <4 years. Among children with a baseline CARS score ≥30, the BF839 group showed significant improvements at week 16 in the ABC total score, ABC body and object use score, CARS score, and GSRS score compared to the placebo group. Only two patients (6.67%) in the BF839 group experienced mild diarrhea. Compared with baseline and placebo group levels, the BF839 group showed a significant post-intervention increase in abundance of bifidobacteria and change in the metabolic function of neuroactive compounds encoded by intestinal microorganisms. BF839 significantly and safely improved abnormal behavior and gastrointestinal symptoms in children with ASD.

Bacterial accumulation dynamics in runoff from extreme precipitation

Extreme precipitation can significantly influence the water quality of surface waters. However, the total amount of bacteria carried by rainfall runoff is poorly understood. Here, thirty rainfall scenarios were simulated by artificial rainfall simulators, with designed rainfall intensity ranging from 19.3 to 250 mm/h. The instantaneous concentration ranges of R2A, nutrient agar (NA) culturable bacteria, and viable bacteria in runoff depended on the types of underlying surfaces. The instantaneous bacterial concentrations in runoff generated by forest lands, grasslands and bare soil were: R2A culturable bacteria = 104.5–6.3, 104.5–6.1, 104.0–5.3 colony-forming units (CFU)/mL, NA culturable bacteria = 104.0–6.0, 103.9–5.8, 103.2–4.9 CFU/mL, and viable bacteria = 106.4–8.0, 107.0–8.9, 106.4–7.6 cells/mL. Based on the measured bacterial instantaneous concentration in runoff, cumulative dynamic models were established, and the maximum amount of culturable bacteria and viable bacteria entering water sources were estimated to be 109.38–11.31 CFU/m2 and 1011.84–13.25 cells/m2, respectively. The model fitting and the bacterial accumulation dynamics were influenced by the rainfall types (p < 0.01). Surface runoff from the underlying surface of forest lands and grasslands had a high microbial risk that persisted even during the “Drought-to-Deluge Transition”. Bacterial accumulation models provide valuable insight for predicting microbial risks in catchments during precipitation and can serve as theoretical support for further ensuring the safety of drinking water under the challenge of climate change.

204. ALTERATIONS IN LOCAL OESOPHAGEAL MICROBIOTA AND OUTCOMES AFTER OESOPHAGECTOMY FOR OESOPHAGO-GASTRIC CANCER: A PILOT STUDY

Abstract Oesophageal cancer is a global health burden, with an estimated 600,000 new cases and 550,000 deaths per year. It is the 9th most common cancer yet is ranked fifth for cancer mortality. The current gold standard curative strategy includes oesophagectomy, which is a morbid procedure. In particular, anastomotic leaks are a feared complication postoperatively that result in significant morbidity. Studies of patients undergoing colorectal cancer surgery show that alterations in the local microbiota can contribute to complications including anastomotic leak. Furthermore, some pathogenic bacterial species have been implicated in shorter recurrence-free survival periods after curative oncologic surgery. Unlike bacterial culture, which requires viable bacteria to be preserved from the time of sampling through to processing, 16S RNA sequencing can be performed on both fresh and fixed tissues. However, there are few studies using either tissue source and to date, none that compare results from the same patient. The aims of this pilot study were to: 1. Assess the feasibility of testing archived histological specimens (i.e. formalin fixed) that in turn facilitates retrospective analysis of patients with known clinical outcomes (i.e. anastomotic leaks) 2. Identify any difference (variance, reduce or over-abundance) between the microbiota of patients who suffered anastomotic leak and those who did not 3. Generate preliminary data to inform sample sizes, statistical methods, workflow and cost of a future prospective trial Using an established Upper GI Cancer Surgery Database, we performed propensity score matching to identify 15 patients who had a clinically evident anastomotic leak to be matched with 15 patients who did not have an anastomotic leak. Known predictors of anastomotic leak (e.g. neoadjuvant radiotherapy, smoking status, COPD, chronic kidney disease, diabetes mellitus, chronic liver disease) have been controlled for. Fresh frozen and formalin-fixed samples for the same patients were available. The formalin-fixed tissue was comprised of the "anastomotic doughnuts" created at the time of the circular stapled anastomosis. This tissue was chosen because of its immediate proximity to the anastomosis. All samples were analysed using 16S RNA sequencing to obtain a full microbial profile. Sequencing data was analysed by scientific investigators blinded to the clinical outcome (i.e. anastomotic leak vs. no leak) using a pre-existing workflow and bioinformatics pipeline.

Human milk microbiota, oligosaccharide profiles, and infant gut microbiome in preterm infants diagnosed with necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a severe intestinal disease of very preterm infants with mother's own milk (MOM) providing protection, but the contribution of the MOM microbiota to NEC risk has not been explored. Here, we analyze MOM of 110 preterm infants (48 NEC, 62 control) in a cross-sectional study. Breast milk contains viable bacteria, but there is no significant difference in MOM microbiota between NEC and controls. Integrative analysis between MOM microbiota, human milk oligosaccharides (HMOs), and the infant gut microbiota shows positive correlations only between Acinetobacter in the infant gut and Acinetobacter and Staphylococcus in MOM. This study suggests that NEC protection from MOM is not modulated through the MOM microbiota. Thus, "'restoring" the MOM microbiota in donor human milk is unlikely to reduce NEC, and emphasis should instead focus on increasing fresh maternal human milk intake and researching different therapies for NEC prevention.

The revealing of the Cyto-genotoxic properties (Allium and MTT) and the effect of chicken meat quality of characterized zein-eugenol nanofibers

Electrospun zein-based eugenol nanofibers (ZEnF) with diameters (148.19–631.52 nm) were fabricated. Thermal degradation was found as <15 % until 300 °C while the nanofiber diffraction pattern presented three main peaks among the 5o and 45o positions. ZEnF was not only evaluated as non-toxic to cells but also possessed anticancer characteristics revealing with the MCF-7 cell line at 800 μg/mL (reduction: 18.08 %) and 1600 μg/mL (reduction: 41.64 %). Allium tests revealed that ZEnF did not have any adverse impact on the health status (chromosomes-DNA) of exposed organisms. Following the nanofiber coating for chicken meat parts (thigh and breast), it was observed up to 1.25 log CFU/g limitation in total viable bacteria counts (p < 0.05). The sensory score (difference: 3.64 in 10 points scoring on the 6th day of the cold storage) and odor score of chicken meat samples were found to be as higher than control samples (p < 0.05).

Endoprotein-activating DNAzyme assay for nucleic acid extraction- and amplification-free detection of viable pathogenic bacteria

Pathogenic bacteria in food or environment, can pose threats to public health, highlighting the requirement of tools for rapid and accurate detection of viable pathogenic bacteria. Herein, we report a sequential endoprotein RNase H2-activating DNAzyme assay (termed epDNAzyme) that enables nucleic acid extraction- and amplification-free detection of viable Salmonella enterica (S. enterica). The direct detection allows for a rapid detection of viable S. enterica within 25 min. Besides, the assay, based on sequential reporting strategy, circumvents internal modifications in the DNAzyme's active domain and improve its catalytic activity. The multiple-turnover DNAzyme cutting and the enhanced catalytic activity of DNAzyme render the epDNAzyme assay to be highly sensitive, and enables the detection of 190 CFU/mL and 0.1% viable S. enterica. The assay has been utilized to detect S. enterica contamination in food and clinical samples, indicating its potential as a promising tool for monitoring pathogen-associated biosafety.

Kinetic study of the thermal inactivation of Weizmannia coagulans during food thermal processing

Weizmannia coagulans has attracted attention due to its remarkable health benefits for human, but the dynamic changes of its viable bacteria during thermal processing have been less reported. In this study, a predictive model for the survival of Weissmanella coagulans during thermal processing of food was developed and validated during the processing of coffee, tea, instant noodles, calcium milk biscuits, muffin cake and steamed buns. The kinetics of heat inactivation activities of Weizmannia coagulans VHProbi C08 and Weizmannia coagulans GBI-30, 6086 at 85, 95, 105, 110 and 115 °C were investigated, and their coefficients of determination were greater than 0.91 and 0.87, and the root-mean-square errors were less than 0.64 and 0.43, respectively. The z-values of VHProbi C08 and GBI-30, 6086 were obtained by Bigelow model fitting as 36.1 °C and 36.9 °C, respectively. The developed prediction model was applied to the thermal processing of six food products and the measured values were all within ±0.5 Log10 (CFU/mL) of the predicted values, indicating high prediction accuracy. The model predicts the survival of Weissmanella coagulans simply by obtaining the initial number of viable bacteria and the change in temperature. These suggested that the model can be used as an effective tool to evaluate the stability of Weizmannia coagulans in food thermal processing.

Precise, Sensitive Detection of Viable Foodborne Pathogenic Bacteria with a 6-Order Dynamic Range via Digital Rolling Circle Amplification.

The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one. The one-target-one-amplicon characteristic of dRCA enables high sensitivity and a broad quantitative detection range, conferring a detection limit of 10 CFU/mL and a dynamic range of 6 orders. dRCA can detect rare viable bacteria, even at a proportion as low as 0.1%, which is 50 times more sensitive than the live/dead staining method. The high sensitivity for detecting viable bacteria accommodates dRCA for assessing sterilization efficiency. Based on the assay, we found that, for pasteurization, slightly elevating the temperature to 68 °C can reduce the heating time to 10 min, which may minimize nutrient degradation caused by high-temperature exposure. The assay can serve as a precise tool for estimating the contamination by viable pathogenic bacteria and assessing sterilization, which facilitates food safety control.

Pacific white shrimp (Litopenaus vannamei) treated with chitooligosaccharide-catechin conjugate with the aid of prior pulse electric field and vacuum impregnation: Quality and microbial diversity during refrigerated storage

Quality of Pacific white shrimp (PWS) soaked in chitooligosaccharide-catechin (CHOS-CAT) conjugate at varying concentrations (1.0 and 2.0%, w/v) and those treated with CHOS-CAT conjugate at aforementioned concentrations with the aid of pulsed electric field (PEF) and vacuum impregnation (VI) was studied when stored at 4 °C for 15 days. Among all treatments, a lower melanosis score of 7 was found in the samples when pre-treated with PEF, followed by soaking in CHOS-CAT conjugate at 2% (w/v) with VI (CC-PV-2) than other samples after 15 days of storage (p < 0.05). Total viable bacteria, psychrotrophic bacteria, presumptive Pseudomonas, Vibrio parahaemolyticus, other Vibrio sp., H2S-producing bacteria, and Enterobacteriaceae counts of CC-PV-2 were lower than other samples at the end of storage (day 15). CC-PV-2 exhibited the lowest TVB-N and TMA-N contents, PV, and TBARS value when compared to the control and other treated samples throughout the storage (p < 0.05). Polyunsaturated fatty acids were retained at a substantial amount in the CC-PV-2 at the end of storage. In addition, next generation sequencing revealed a low percentage of spoilage bacteria such as Shewanella and Pseudomonas and pathogenic bacteria namely Vibrio sp. in the CC-PV-2. Also, CHOS-CAT conjugate showed hydrogen bonding with various targeted proteins of bacteria, as elucidated using molecular docking, verifying its bacterial inhibitory potential. CC-PV-2 had a shelf-life of at least 12 days, while the control could be kept only for 3 days. Overall, the application of CHOS-CAT conjugate with the aid of PEF and VI led to the enhanced quality and extended shelf-life of refrigerated PWS.

Synergistic effects of arginine and fluoride on human dental biofilm control

ObjectivesThe aim of this study was to quantitatively and comprehensively investigate the combined effects of arginine and fluoride on the suppression of pathogenicity using an in situ biofilm model and next-generation sequencing (NGS). MethodsUsing the in situ model, dental biofilms were formed and the viable bacterial counts and arginine activity in the arginine- and fluoride-containing dentifrice and control groups were measured. We also compared their effects on the bacterial microbiota and predictive functional factors in the control, arginine (arg), and arginine + fluoride (argF) groups using NGS analysis. ResultsCompared to the control treatment, the use of 8 % arginine and 1450 ppm fluoride toothpaste resulted in significantly high oral NH4+ concentrations without affecting the number of viable bacteria (P < 0.05). NGS analysis revealed that the oral microbiota of the control, arg, and argF groups were significantly different. Heat map analysis of the predicted functional factors revealed that the arg group had different properties from the other groups and activated specific substrate metabolic pathways; contrastingly, argF treatment inhibited the activity of these pathways and prevented an increase in the abundance of bacterial genera that utilize substrates such as sucrose, suggesting the synergistic effect of arginine and fluoride. ConclusionsThis study indicates that the combination of arginine and fluoride has a synergistic effect on the bacterial microbiota and pathogenicity of dental biofilms compared with arginine alone. Clinical significanceOur findings suggest that the combination of arginine and fluoride could be used as an effective prebiotic and may inhibit the growth of bacteria associated with dental diseases.

Quantifying the viability of lactic acid bacteria using ratiometric fluorescence assays

Quantifying the viability of lactic acid bacteria (LAB) has become an important task in evaluating the quality, function and storage condition of LAB products. Propidium iodide (PI), as a DNA-binding dye that penetrates only dead bacteria, has been widely used for LAB viability assessments. However, high fluorescence background from live LAB was frequently occurred during PI-based staining, which led to the low sensitivity in probing minor amounts of dead LAB in a dead/live mixture. Here, we found that PI could be adsorbed nonspecifically in LAB periplasm, which contributed to the high fluorescence background of live LAB. CaCl2 was used to reduce this nonspecific adsorption and increase the fluorescence ratio of dead to live LAB. Combined with SYTO 9 (a membrane permeable dye) for double-staining, a ratiometric fluorescent method that could probe dead LAB at a ratio as low as 0.2 % was established. Using Lacticaseibacillus casei as a LAB model, an allometric correlation between the ratiometric fluorescence and theoretical ratio of live to dead LAB was acquired, with a correlation coefficient R2 of 0.993. The as-developed method was successfully applied to the viability assessment of LAB stored in different environments. We believe it would have practical applications in LAB food industry for quality management and control.