Imaging Flow Cytometry Demonstrates Physiological and Morphological Diversity within Treated Probiotic Bacteria Groups.

Probiotic bacteria can be introduced to stresses during the culturing phase as an alternative to the use of protectants and coating substances during drying. Accurate enumeration of the bacterial count in a probiotic formulation can be provided using imaging flow cytometry (IFC). IFC overcomes the weak points of conventional, commonly used flow cytometry by combining its statistical power with the imaging content of microscopy in one system. Traditional flow cytometers only collect the fluorescence signal intensities, while IFC provides many more steps as it correlates the data on the measured parameters of fluorescence light with digitally processed images of the analyzed cells. As an alternative to standard methods (plate cell counts and traditional flow cytometry) IFC provides additional insight into the physiology and morphology of the cell. The use of complementary dyes (RedoxSensorTM Green and propidium iodide) allows for the designation of groups based on their metabolic activity and membrane damage. Additionally, cell sorting is incorporated to assess each group in terms of growth on different media (MRS-Agar and MRS broth). Results show that the groups with intermediate metabolic activity and some degree of cellular damage correspond with the description of viable but nonculturable cells.

Co-encapsulation of Lactobacillus plantarum and bioactive compounds extracted from red beet stem (Beta vulgaris L.) by spray dryer.

Probiotic bacteria and bioactive compounds obtained from plant origin stand out as ingredients with the potential to increase the healthiness of functional foods, as there is currently a recurrent search for them. Probiotics and bioactive compounds are sensitive to intrinsic and extrinsic factors in the processing and packaging of the finished product. In this sense, the present study aims to evaluate the co-encapsulation by spray dryer (inlet air temperature 120 °C, air flow 40 L / min, pressure of 0.6 MPa and 1.5 mm nozzle diameter) of probiotic bacteria (L.plantarum) and compounds extracted from red beet stems (betalains) in order to verify the interaction between both and achieve better viability and resistance of the encapsulated material. When studying the co-encapsulation of L.plantarum and betalains extracted from beet stems, an unexpected influence was observed with a decrease in probiotic viability in the highest concentration of extract (100 %), on the other hand, the concentration of 50 % was the best enabled and maintained the survival of L.plantarum in conditions of 25 °C (63.06 %), 8 °C (88.80 %) and -18 °C (89.28 %). The viability of the betalains and the probiotic was better preserved in storage at 8 and -18 °C, where the encapsulated stability for 120 days was successfully achieved. Thus, the polyfunctional formulation developed in this study proved to be promising, as it expands the possibilities of application and development of new foods.

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality.

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 °C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics' viability decreases conversely with the drying temperature. At temperatures close to 120 °C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 °C are required to ensure the microbial cells' viability and shelf-life in the powdered preparation and survival during food processing and storage.

Programming bacteria for multiplexed DNA detection.

DNA is a universal and programmable signal of living organisms. Here we develop cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA-sensing mechanism and simple detection procedure could enable programmable DNA sensing for a wide range of applications.

The influence of almond's water activity and storage temperature on Salmonella survival and thermal resistance.

This study investigated the effects of inoculation method, water activity (aw), packaging method, and storage temperature and duration on the survival of Salmonella on almonds as well as their resistance to subsequent thermal treatments. Whole almond kernels were inoculated with a broth-based or agar-based growth Salmonella cocktail and conditioned to aw of 0.52, 0.43 or 0.27. Inoculated almonds with aw of 0.43 were treated with a previously validated treatment (4 h of dry heat at 73 °C) to determine the potential differences in heat resistance resulted from the two inoculation methods. The inoculation method did not significantly (P > 0.05) impact the thermal resistance of Salmonella. Inoculated almonds at aw of 0.52 and 0.27 were either vacuum packaged in moisture-impermeable mylar bags or non-vacuum packaged in moisture-permeable polyethylene bags before stored at 35, 22, 4, or -18 °C for up to 28 days. At selected storage intervals, almonds were measured for aw, analyzed for Salmonella population level, and subjected to dry heat treatment at 75 °C. Over the month-long storage of almonds, Salmonella populations remained almost unchanged (<0.2 log CFU/g) at 4 °C and -18 °C and declined slightly (<0.8 log CFU/g) at 22 °C and more substantially (1.6-2.0 log CFU/g) at 35 °C regardless of the inoculation method, packaging method, and almond aw. When stored at 35 °C, almonds with initial aw of 0.52 had significantly higher (P < 0.05) Salmonella reductions than those with initial aw of 0.27. Prior storage of almonds vacuum packaged in mylar bags at temperatures between -18 °C and 35 °C for 28 days affected their aw levels but did not significantly (P > 0.05) affect the subsequent thermal resistance of Salmonella at 75 °C regardless of almond aw and storage duration. Salmonella on almonds with higher aw was more sensitive to heat treatment than those with lower aw. To achieve >5 log CFU/g reductions of Salmonella, a dry heat treatment at 75 °C for 4 and 6 h was needed for almonds with initial aw of 0.52 and 0.27, respectively. When applying the dry heating technology for almond decontamination, the processing time needs to be determined based on initial aw of almonds regardless of storage condition or age of almonds within the current design frame.

Simple optical nanomotion method for single-bacterium viability and antibiotic response testing.

Antibiotic resistance is nowadays a major public health issue. Rapid antimicrobial susceptibility tests (AST) are one of the options to fight this deadly threat. Performing AST with single-cell sensitivity that is rapid, cheap, and widely accessible, is challenging. Recent studies demonstrated that monitoring bacterial nanomotion by using atomic force microscopy (AFM) upon exposure to antibiotics constitutes a rapid and highly efficient AST. Here, we present a nanomotion detection method based on optical microscopy for testing bacterial viability. This novel technique only requires a very basic microfluidic analysis chamber, and an optical microscope equipped with a camera or a mobile phone. No attachment of the microorganisms is needed, nor are specific bacterial stains or markers. This single-cell technique was successfully tested to obtain AST for motile, nonmotile, gram-positive, and gram-negative bacteria. The simplicity and efficiency of the method make it a game-changer in the field of rapid AST.

Synergistic effects of 915 MHz microwave heating and essential oils on inactivation of foodborne pathogen in hot-chili sauce.

Essential oil is a food additive with antimicrobial properties but with limitations due to strong organoleptic properties. However, thermal treatments can be applied to reduce essential oil content while ensuring antimicrobial activities in food matrices. In this study, the inactivation efficiency of essential oils on E. coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes in buffered peptone water (BPW) and hot-chili sauce was evaluated when coupled with 915 MHz microwave heating. Essential oils used in this study did not affect the dielectric properties and further heating rate of BPW and hot-chili sauce. The dielectric constant of BPW was 76.3 and dielectric loss factor was 30.9. In addition, it took 85 s to reach 100 °C for all samples. Among essential oils, synergistic microbial inactivation with microwave heating was observed from carvacrol (CL) and citral (CI), but not from eugenol (EU) and Carvone (CN). Specifically, CL and microwave heating (M) for 45 s showed the most effective inactivation (ca. 6 log reduction) for the pathogens in BPW. Similar trends were shown in hot-chili sauce. However, M + CI inactivation did not show synergistic effects in hot-chili sauce. Microwave heating time for hot-chilis sauce was 40 s. In propidium iodide uptake study, M + CL was found to cause most severe damage to cell membrane (758.5 of PI value for E. coli O157:H7) while M + CU and M + CN had little impact. In DiBAC4(3) test, CL resulted in the largest value (2.09 for E. coli O157:H7). These observations highlight that CL induces synergistic effects as it caused severe membrane damage along with destruction of membrane potential. The combined treatment did not show any significant difference in quality change compared to untreated hot-chili sauce (p > 0.05). The result indicates the potential application of CL and M combination for hot-chili sauce processes to ensure microbiological safety with acceptable quality.

Deuterium isotope probing (DIP) on Listeria innocua: Optimisation of labelling and impact on viability state.

An innovative approach, Raman microspectroscopy coupled with deuterium isotope probing (Raman-DIP), can be used to evaluate the metabolism of deuterated carbon source in bacteria and also to presume different anabolic pathways. This method requires the treatment of cells with heavy water that could affect the bacterial viability state at higher concentration. In this study, we evaluated the effect of heavy water incorporation on the viability state of Listeria innocua cells. We exposed the L. innocua suspensions to different heavy water concentrations (0%, 25%, 50% and 75%) from 30 minutes to 72 h of incubation times at 37°C. The total, viable and viable culturable populations were quantified by qPCR, PMA-qPCR and plate count agar respectively. We analyzed heavy water incorporation by Raman-DIP. The exposure of L. innocua cells to different concentrations of heavy water did not alter their cell viability to 24 h incubation time. In addition, the maximum intensity for C-D band, specific for the incorporation of heavy water, was reached after 2 h of exposure in a media containing 75% v/v D2O but an early detection of the labelling was possible at t = 1 h 30 min. In conclusion, the use of D2O as a metabolic marker was validated and can be developed for the detection of L. innocua cell viability state.

QMRA of beach water by Nanopore sequencing-based viability-metagenomics absolute quantification.

The majority of the current regulatory practices for routine monitoring of beach water quality rely on the culture-based enumeration of faecal indicator bacteria (FIB) to develop criteria for promoting the general public's health. To address the limitations of culture methods and the arguable reliability of FIB in indicating health risks, we developed a Nanopore metagenomic sequencing-based viable cell absolute quantification workflow to rapidly and accurately estimate a broad range of microbes in beach waters by a combination of propidium monoazide (PMA) and cellular spike-ins. Using the simple synthetic bacterial communities mixed with viable and heat-killed cells, we observed near-complete relic DNA removal by PMA with minimal disturbance to the composition of viable cells, demonstrating the feasibility of PMA treatment in profiling viable cells by Nanopore sequencing. On a simple mock community comprised of 15 prokaryotic species, our results showed high accordance between the expected and estimated concentrations, suggesting the accuracy of our method in absolute quantification. We then further assessed the accuracy of our method for counting viable Escherichia coli and Vibrio spp. in beach waters by comparing to culture-based method, which were also in high agreement. Furthermore, we demonstrated that 1 Gb sequences obtained within 2 h would be sufficient to quantify a species having a concentration of ≥ 10 cells/mL in beach waters. Using our viability-resolved quantification workflow to assess the microbial risk of the beach water, we conducted (1) screening-level quantitative microbial risk assessment (QMRA) to investigate human illness risk and site-specific risk patterns that might guide risk management efforts and (2) metagenomics-based resistome risk assessment to evaluate another layer of risk caused by difficult illness treatment due to antimicrobial resistance (AMR). In summary, our metagenomic workflow for the rapid absolute quantification of viable bacteria demonstrated its great potential in paving new avenues toward holistic microbial risk assessment.

Helicobacter pylori Dormant States Are Affected by Vitamin C.

Helicobacter pylori colonizes human gastric mucosa, overcoming stressful conditions and entering in a dormant state. This study evaluated: (i) H. pylori's physiological changes from active to viable-but-non-culturable (VBNC) and persister (AP) states, establishing times/conditions; (ii) the ability of vitamin C to interfere with dormancy generation/resuscitation. A dormant state was induced in clinical MDR H. pylori 10A/13 by: nutrient starvation (for VBNC generation), incubating in an unenriched medium (Brucella broth) or saline solution (SS), and (for AP generation) treatment with 10xMIC amoxicillin (AMX). The samples were monitored after 24, 48, and 72 h, 8-14 days by OD600, CFUs/mL, Live/Dead staining, and an MTT viability test. Afterwards, vitamin C was added to the H. pylori suspension before/after the generation of dormant states, and monitoring took place at 24, 48, and 72 h. The VBNC state was generated after 8 days in SS, and the AP state in AMX for 48 h. Vitamin C reduced its entry into a VBNC state. In AP cells, Vitamin C delayed entry, decreasing viable coccal cells and increasing bacillary/U-shaped bacteria. Vitamin C increased resuscitation (60%) in the VBNC state and reduced the aggregates of the AP state. Vitamin C reduced the incidence of dormant states, promoting the resuscitation rate. Pretreatment with Vitamin C could favor the selection of microbial vegetative forms that are more susceptible to H. pylori therapeutical schemes.

The Attenuation of Microbial Reduction in Blueberry Fruit Following UV-LED Treatment.

Ultraviolet-C (UV-C) irradiation is a well-recognized technology for improving blueberry postharvest quality, and previous literature indicates that it has the potential for dual-use as an antimicrobial intervention for this industry. However, the practicality and feasibility of deploying this technology in fresh blueberry fruit are significantly hindered by the shadowing effect occurring at the blossom-end scar of the fruit. The purpose of this study was to determine if treating the blueberry fruit within a chamber fitted with UV-Light Emitting Diodes (LEDs) emitting a peak UV-C at 275 nm could minimize this shadowing and result in improved treatment efficacy. Ten blueberry fruits were dip-inoculated with E. coli at a concentration of 105 CFU/mL and irradiated within the system at doses of 0, 1.617, 3.234, 9.702, and 16.17 mJ/cm2 (0, 30, 60, 180, and 300 s). Statistical analysis was performed to characterize the extent of microbial survival as well as the UV-C inactivation kinetics. A maximum of 0.91-0.95 log reduction was observed, which attenuated after 60 s of treatment. The microbial inactivation and survival were thus modeled using the Geeraerd-tail model in Microsoft Excel with the GInaFIt add-in (RMSE = 0.2862). Temperatures fluctuated between 23 ± 0.5°C and 39.5°C ± 0.5°C during treatment but did not statistically impact the treatment efficacy (P = 0.0823). The data indicate that the design of a UV-LED system may improve the antimicrobial efficacy of UV-C technology for the surface decontamination of irregularly shaped fruits, and that further optimization could facilitate its use in the industry.

Optimization of Plasma-activated Water and Validation of a Potential Surrogate for Salmonella for Future Egg Washing Processes.

Plasma-activated water (PAW) is considered a novel sanitizer for the food industry due to the antimicrobial mechanisms exhibited by reactive oxygen and nitrogen species. The plasma operation parameters can affect the chemistry of PAW and can therefore influence its microbial inactivation efficacy. This study statistically optimized the operating conditions of PAW (activation time, distance from nozzle, and volume of water) using response surface methodology. Two optimized conditions of PAW were identified for the inactivation of planktonic cells of the avirulent strain of Salmonella Typhimurium MHM112 providing a minimum reduction of 6.3 log. All three operating parameters significantly affected the physicochemical characteristics (pH, ORP, EC, nitrite, and nitrate) and microbial inactivation efficacy of PAW. Mixing of small batches using the two optimized conditions to obtain larger volumes did not significantly change the microbial inactivation. However, there were significant reductions in nitrite and nitrate concentrations in PAW due to the mixing of batches while the pH and ORP values remained unaffected. The storage of large volumes of PAW for 25 min at 40-46°C, which is the commercial egg washing temperature in the United States, did not significantly impact S. Typhimurium MHM112 inactivation or the physicochemical characteristics of PAW. A validation study using a cocktail of six pathogenic strains of Salmonella revealed no significant differences in inactivation between the avirulent S. Typhimurium MHM112 and the pathogenic strains, suggesting that the avirulent S. Typhimurium MHM112 may serve as a surrogate for sanitation of S. enterica at the optimized conditions of PAW. The results obtained from this study are useful for our long-term goal of evaluating PAW efficacy in surface egg washing to inactivate Salmonella.

Biointerface mineralization generates ultraresistant gut microbes as oral biotherapeutics.

Despite the fact that oral microecologics are effective in modulating the gut microbiome, they always suffer from multiple insults during the journey from manufacture to arrival at the intestine. Inspired by the protective mechanism of mineralization, we describe a cytocompatible approach of biointerface mineralization that can generate an ultraresistant and self-removable coating on bacterial surface to solve these challenges. Mineral coating endows bacteria with robust resistances against manufacture-associated oxygen exposure, ultraviolet irradiation, and 75% ethanol. Following oral ingestion, the coating is able to actively neutralize gastric acid and release encapsulated bacteria through spontaneous yet rapid double-decomposition reaction. In addition to acid neutralization, the generated calcium ions can trigger micellar aggregation of bile acid, enabling dual exemptions from the insults of gastric acid and bile acid to achieve uncompromised bacterial viability. Further supported by the therapeutic efficacy of coated bacteria toward colitis mice, biointerface mineralization provides a versatile platform for developing next-generation living oral biotherapeutics.

Encapsulation of Lactobacillus plantarum ELB90 by electrospraying in a double emulsion (W1/O/W2) loaded alginate beads to improve the gastrointestinal survival and thermal stability.

BACKGROUND: In the present study, the Lactobacillus plantarum ELB90 was encapsulated in double emulsion (W1/O/W2) loaded alginate beads (emulbeads) by electrospraying and compared with emulsion-free control beads. The viability of encapsulated and free cells was assessed by exposing them to thermal processing (65 °C for 30 min and 72 °C for 3 min) and simulated gastrointestinal conditions. The beads were characterized by optical, scanning electron, fluorescence, and confocal laser scanning microscopy, as well as Fourier transform infrared and gel strength analysis. RESULTS: After the intestinal stage of digestion, the survival rate of free bacteria was 38% [3.70 log colony-forming units (CFU) g-1 ], only increased to 43% and 53% for bare and chitosan-coated control beads, and it elevated the survival rate to 75% and 84% (8.70 log CFU g-1 ) for bare and chitosan-coated emulbeads, respectively. The presence of inulin increased gastrointestinal viability only in uncoated emulbeads. The bacteria loaded in emulbeads retained greater viability (5.90-6.90 log CFU g-1 ) against thermal treatment, compared to control beads (2.07-4.10 log CFU g-1 ) and free bacteria (2.57-3.11 log CFU mL-1 ). Encapsulation of L. plantarum ELB90 only in emulsion-free beads may not be appropriate for providing thermal stability. Inulin addition and chitosan-coating of the beads increased the size, and emulbeads presented smoother surfaces compared to emulsion-free beads. CONCLUSION: The contribution of a double emulsion into the gel matrix of electrosprayed alginate beads exhibited enhanced protection for probiotic bacteria that could be useful for the development of functional foods. © 2023 Society of Chemical Industry.

In-package cold plasma treatment for microbial inactivation in plastic-pouch packaged steamed rice cakes.

In-package atmospheric cold plasma (ICP) treatment was investigated as a method to inactivate microorganisms in Korean steamed rice cakes (SRCs) packaged in plastic pouches. The effect against Escherichia coli O157:H7 increased with increasing ICP treatment power and time and using nylon-containing pouches. Moreover, E. coli O157:H7 growth was effectively inhibited at 4 and 25 °C when SRCs were in a pouch filled with an O2-CO2 (70 % and 30 %) gas. Under optimal treatment power (30 W), treatment time (4 min), and headspace-to-SRC volume ratio (7:1) conditions, ICP effectively inactivated E. coli O157:H7, Bacillus cereus spores, Penicillium chrysogenum, and indigenous aerobic bacteria, as well as yeast and molds in SRCs packaged with air in the nylon/low density polyethylene pouch by 2.2 ± 0.2 log CFU/g, 1.4 ± 0.2 log spores/g, 2.2 ± 0.3 log spores/g, 1.1 ± 0.2 log CFU/g, and 1.0 ± 0.1 log CFU/g, respectively. Furthermore, post-treatment storage was effective in preventing the growth of E. coli O157:H7 in SRCs at 4 °C and 25 °C when the pouch was filled with N2-CO2 (50 % and 50 %) or O2-CO2 (70 % and 30 %). Collectively, these findings indicate that ICP treatment effectively decontaminates SRCs and represents a potential non-thermal microbial decontamination technology for SRCs in pouch packaging.

Exploring the roles of starch for microbial encapsulation through a systematic mapping review.

Microorganism encapsulation protects them from stressful conditions and assists in maintaining their viability, being especially beneficial when the carrier material is a renewable and biodegradable biopolymer, such as starch. Here, a systematic mapping was performed to provide a current overview on the use of starch-based systems for microbial encapsulation. Following well-established guidelines, a systematic mapping was conducted and the following could be drawn: 1) there was a significant increase in publications on microbial encapsulation using starch over the past decade, showing interest from the scientific community, 2) ionotropic gelation, emulsification and spray drying are the most commonly used techniques for starch-based microbial encapsulation, and 3) starch play important functions in the encapsulation matrix such as assisting in the survival of the microorganisms. The information gathered in this systematic mapping can be useful to guide researchers and industrial sectors on the development of innovative starch-based systems for microbial encapsulation.

Response of genes related to iron and porphyrin transport in Porphyromonas gingivalis to blue light.

BACKGROUND: Antimicrobial blue light (aBL) kills a variety of bacteria, including Porphyromonas gingivalis. However, little is known about the transcriptomic response of P. gingivalis to aBL therapy. This study was designed to evaluate the selective cytotoxicity of aBL against P. gingivalis over human cells and to further investigate the genetic response of P. gingivalis to aBL at the transcriptome level. METHODS: Colony forming unit (CFU) testing, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM) were used to investigate the antimicrobial effectiveness of blue light against P. gingivalis. The temperatures of the irradiated targets were measured to prevent overheating. Multiple fluorescent probes were used to quantify reactive oxygen species (ROS) generation after blue-light irradiation. RNA sequencing (RNA-seq) was used to investigate the changes in global gene expression. Following the screening of target genes, real-time quantitative polymerase chain reaction (RT-qPCR) was performed to confirm the regulation of gene expression. RESULTS: A 405 nm aBL at 100 mW/cm2 significantly killed P. gingivalis within 5 min while sparing human gingival fibroblasts (HGFs). No obvious temperature changes were detected in the irradiated surface under our experimental conditions. RNA-seq showed that the transcription of multiple genes was regulated, and RT-qPCR revealed that the expression levels of the genes RgpA and RgpB, which may promote heme uptake, as well as the genes Ftn and FetB, which are related to iron homeostasis, were significantly upregulated. The expression levels of the FeoB-2 and HmuR genes, which are related to hydroxyl radical scavenging, were significantly downregulated. CONCLUSIONS: aBL strengthens the heme uptake and iron export gene pathways while reducing the ROS scavenging pathways in P. gingivalis, thus improving the accumulation of endogenous photosensitizers and enhancing oxidative damage to P. gingivalis.

Identification of determinants for entering into a viable but nonculturable state in Vibrio alginolyticus by Tn-seq.

The viable but nonculturable (VBNC) state is a dormant state of nonsporulating bacteria that enhances survival in adverse environments. Systematic genome-wide research on the genetic basis of VBNC formation is warranted. In this study, we demonstrated that the marine bacterium Vibrio alginolyticus lost culturability but remained viable and entered into the VBNC state when exposed to low nutrient concentrations for prolonged periods of time. Using transposon-insertion sequencing (Tn-seq), we identified 635 determinants governing the formation of the VBNC state, including 322 genes with defective effects on VBNC formation and 313 genes contributing to entry into the VBNC state. Tn-seq analysis revealed that genes involved in various metabolic pathways were shown to have an inhibitory effect on VBNC formation, while genes related to chemotaxis or folate biosynthesis promoted entry into the VBNC state. Moreover, the effects of these genes on the formation of VBNC were validated with the growth of deletion mutants of eight selected genes under nutrient-limited conditions. Interestingly, fleQ and pyrI were identified as essential for entry into the VBNC state, and they affected the formation of the VBNC state independent of RpoE or ToxR regulation. Collectively, these results provide new insights into the mechanism of VBNC formation. KEY POINTS: • Vibrio alginolyticus has the ability to enter into the VBNC state under low nutrient conditions at low temperature. • The 635 determinants for entry into the VBNC state were systematically identified by transposon-insertion sequencing. • PyrI and FleQ were validated to play significant roles in the formation of the VBNC state.

The incorporation of peach gum polysaccharide into soy protein based microparticles improves probiotic bacterial survival during simulated gastrointestinal digestion and storage.

The objective of this research was to investigate the in vitro gastrointestinal digestion and storage properties of Lactobacillus plantarum 550 encapsulated in soy protein isolate (SPI) and peach gum polysaccharide (PG) through spray drying. High survival rates (>8.1 Log CFU/g) were obtained for all encapsulation formulas containing PG. Combination of SPI and PG showed positive effects on both gastric resistance and storage stability of cells. Among the formulas tested, sample of SPI:PG = 3:1 showed the highest survival (7.88 ± 0.12 Log CFU/g), corresponding to the strongest electrostatic interaction between SPI and PG. With PG content increasing, the storage stability of probiotic was also enhanced, as PG could reduce the moisture content within microcapsules as well as scavenge free radicals generated during storage. In conclusion, the current study demonstrates that SPI combined with PG may provide effective protection to cells not only during spray drying, but also during storage and gastrointestinal digestion.

Extended lag phase indicates the dormancy of biphenyl degrading Rhodococcus biphenylivorans TG9 under heat stress.

Microbial remediation is a green and sustainable technology, but harsh environmental conditions could lead to microbial dormancy, such as entering a viable but non-culturable (VBNC) state. However, the evidence of VBNC is controversial and limited. In this study, heat stress (60 °C), one of the leading challenges for mesophilic degrading bacteria, was mimicked to investigate the physiological response of Rhodococcus biphenylivorans TG9. After 2 h of heat stress, the culturable TG9 cell count decreased from 108 cells/mL to undetectable while the viable cell count was still 105 cells/mL. The biphenyl degradation efficiency of stressed TG9 dropped by 50% compared to that of cells at logarithmic phase. During heat stress, the respiratory activity of TG9 declined dramatically while the intracellular ATP level initially increased and then decreased. Notably, the corresponding indicators recovered when restored to 30 °C. These characteristics were in consistent with bacteria entering into VBNC state. Furthermore, fluorescence activated cell sorting together with single cell as seed culture detection verified the unculturability and viability of VBNC state of TG9 cells. Also, we found that single cells in VBNC state could resuscitate and regrowth with significantly extended lag phase (LP). Our results highlight the potential of TG9 for microbial remediation and hint LP duration as an indicator for survival state of bacteria.