Rapid and non-destructive microbial quality prediction of fresh pork stored under modified atmospheres by using selected-ion flow-tube mass spectrometry and machine learning.

Volatile organic compounds (VOCs) indicative of pork microbial spoilage can be quantified rapidly at trace levels using selected-ion flow-tube mass spectrometry (SIFT-MS). Packaging atmosphere is one of the factors influencing VOC production patterns during storage. On this basis, machine learning would help to process complex volatolomic data and predict pork microbial quality efficiently. This study focused on (1) investigating model generalizability based on different nested cross-validation settings, and (2) comparing the predictive power and feature importance of nine algorithms, including Artificial Neural Network (ANN), k-Nearest Neighbors, Support Vector Regression, Decision Tree, Partial Least Squares Regression, and four ensemble learning models. The datasets used contain 37 VOCs' concentrations (input) and total plate counts (TPC, output) of 350 pork samples with different storage times, including 225 pork loin samples stored under three high-O2 and three low-O2 conditions, and 125 commercially packaged products. An appropriate choice of cross-validation strategies resulted in trustworthy and relevant predictions. When trained on all possible selections of two high-O2 and two low-O2 conditions, ANNs produced satisfactory TPC predictions of unseen test scenarios (one high-O2 condition, one low-O2 condition, and the commercial products). ANN-based bagging outperformed other employed models, when TPC exceeded ca. 6 log CFU/g. VOCs including benzaldehyde, 3-methyl-1-butanol, ethanol and methyl mercaptan were identified with high feature importance. This elaborated case study illustrates great prospects of real-time detection techniques and machine learning in meat quality prediction. Further investigations on handling low VOC levels would enhance the model performance and decision making in commercial meat quality control.

Mechanistic insights into the adaptive evolvability of spore heat resistance in Bacillus cereus sensu lato.

Wet heat treatment is a commonly applied method in the food and medical industries for the inactivation of microorganisms, and bacterial spores in particular. While many studies have delved into the mechanisms underlying wet heat killing and spore resistance, little attention has so far been dedicated to the capacity of spore-forming bacteria to tune their resistance through adaptive evolution. Nevertheless, a recent study from our group revealed that a psychrotrophic strain of the Bacillus cereus sensu lato group (i.e. Bacillus weihenstephanensis LMG 18989) could readily and reproducibly evolve to acquire enhanced spore wet heat resistance without compromising its vegetative cell growth ability at low temperatures. In the current study, we demonstrate that another B. cereus strain (i.e. the mesophilic B. cereus sensu stricto ATCC 14579) can acquire significantly increased spore wet heat resistance as well, and we subjected both the previously and currently obtained mutants to whole genome sequencing. This revealed that five out of six mutants were affected in genes encoding regulators of the spore coat and exosporium pathway (i.e. spoIVFB, sigK and gerE), with three of them being affected in gerE. A synthetically constructed ATCC 14579 ΔgerE mutant likewise yielded spores with increased wet heat resistance, and incurred a compromised spore coat and exosporium. Further investigation revealed significantly increased spore DPA levels and core dehydration as the likely causes for the observed enhanced spore wet heat resistance. Interestingly, deletion of gerE in Bacillus subtilis 168 did not impose increased spore wet heat resistance, underscoring potentially different adaptive evolutionary paths in B. cereus and B. subtilis.

Influence of traditional dehulling on mycotoxin reduction and GC-HRTOF-MS metabolites profile of fermented maize products.

Contamination with mycotoxins has been a worldwide food safety concern for several decades, and food processing has been suggested as a potential method to mitigate their presence. In this study, the influence of traditional dehulling (TD) on the mycotoxin reduction and metabolites profile of fermented white maize products obtained via natural and three controlled fermentation methods (involving Lactobacillus fermentum, Lactobacillus plantarum, and their mixed cultures) was examined. Gas chromatography coupled with high resolution time-of-flight mass spectrometry (GC-HRTOF-MS) and ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were employed. TD brought the levels of fumonisin B1 (FB1) and B2 (FB2) in the white maize below the regulatory limit set by the European Union (EU) for maize consumed by humans. While TD increased the concentration of several mycotoxins in the fermented maize products obtained from other studied fermentation methods, it primarily reduced aflatoxin B1 (AFB1), FB1, deoxynivalenol, and 15-acetyldeoxynivalenol in the L. plantarum-fermented products. By tempering the dehulled maize, a solid-state fermentation process began. This was used in TD to make it easier to remove the pericarp. GC-HR-TOF-MS metabolomics revealed that TD brought about the generation of 12 additional compounds in the dehulled maize though some metabolites in the whole maize were lost/biotransformed. The fermented dehulled maize products obtained from the four studied fermentation procedures contained fewer compounds than the fermented whole maize products. Overall, the analysis showed that all fermented maize (whole and dehulled) produced had varied nutritional metabolites and mycotoxin concentrations below the EU maximum level, except for fermented maize obtained from mixed strains (AFB1 + AFB2 > 4.0 g/kg).

Addition of (bio)surfactants in the biofiltration of hydrophobic volatile organic compounds in air.

The removal of volatile organic compounds (VOCs) in air is of utmost importance to safeguard both environmental quality and human well-being. However, the low aqueous solubility of hydrophobic VOCs results in poor removal in waste gas biofilters (BFs). In this study, we evaluated the addition of (bio)surfactants in three BFs (BF1 and BF2 mixture of compost and wood chips (C + WC), and BF3 filled with expanded perlite) to enhance the removal of cyclohexane and hexane from a polluted gas stream. Experiments were carried out to select two (bio)surfactants (i.e., Tween 80 and saponin) out of five (sodium dodecyl sulfate (SDS), Tween 80, surfactin, rhamnolipid and saponin) from a physical-chemical (i.e., decreasing VOC gas-liquid partitioning) and biological (i.e., the ability of the microbial consortium to grow on the (bio)surfactants) point of view. The results show that adding Tween 80 at 1 critical micelle concentration (CMC) had a slight positive effect on the removal of both VOCs, in BF1 (e.g., 7.0 ± 0.6 g cyclohexane m-3 h-1, 85 ± 2% at 163 s; compared to 6.7 ± 0.4 g cyclohexane m-3 h-1, 76 ± 2% at 163 s and 0 CMC) and BF2 (e.g., 4.3 ± 0.4 g hexane m-3 h-1, 27 ± 2% at 82 s; compared to 3.1 ± 0.7 g hexane m-3 h-1, 16 ± 4% at 82 s and 0 CMC), but a negative effect in BF3 at either 1, 3 and 9 CMC (e.g., 2.4 ± 0.4 g hexane m-3 h-1, 30 ± 4% at 163 s and 1 CMC; compared to 4.6 ± 1.0 g hexane m-3 h-1, 43 ± 8% at 163 s and 0 CMC). In contrast, the performance of all BFs improved with the addition of saponin, particularly at 3 CMC. Notably, in BF3, the elimination capacity (EC) and removal efficiency (RE) doubled for both VOCs (i.e., 9.1 ± 0.6 g cyclohexane m-3 h-1, 49 ± 3%; 4.3 ± 0.3 g hexane m-3 h-1, 25 ± 3%) compared to no biosurfactant addition (i.e., 4.5 ± 0.4 g cyclohexane m-3 h-1, 23 ± 3%; hexane 2.2 ± 0.5 g m-3 h-1, 10 ± 2%) at 82 s. Moreover, the addition of the (bio)surfactants led to a shift in the microbial consortia, with a different response in BF1-BF2 compared to BF3. This study evaluates for the first time the use of saponin in BFs, it demonstrates that cyclohexane and hexane RE can be improved by (bio)surfactant addition, and it provides recommendations for future studies in this field.

Inclusion of microbiological food safety as a novel aspect in life cycle assessments of food production.

The life cycle assessment (LCA) methodology currently covers a limited number of human health-related impact categories. Microbiological food safety is an essential aspect for the selection of an appropriate food production system and has been neglected in the LCA so far. A framework for the inclusion of a microbiological food safety indicator, expressed as disability-adjusted life year (DALY) value of the consumed food product to the human health damage category (end-point) was created, and applied in a case study model on the cooked-chilled meals as the ready-to-eat meals can be associated with the occurrence of foodborne illness cases and outbreaks. This study suggests a framework for the inclusion of microbiological risk caused by Bacillus cereus associated with the consumption of ready-to-eat meals (in Belgium) in the LCA. The results indicated that the microbiological risk of one package of the investigated ready-to-eat meal was 1.95 × 10-6 DALY, and the obtained DALY value was included as an impact category in the LCA methodology. Inclusion of other categories of food safety (including chemical safety hazards, pesticide residues, heavy metals, and mycotoxins) in LCA could be done in the same fashion.

Prevalence and growth potential of Listeria monocytogenes in innovative, pre-packed, plant-based ready-to-eat food products on the Belgian market.

In recent years, pre-packed ready-to-eat (RTE) food products on the Belgian market have shifted to a more plant-based composition due to a variety of reasons, including consumer concerns about health, animal welfare, and sustainability. However, similar to animal-based RTE foods, plant-based RTE foods can be susceptible to the presence and outgrowth of Listeria monocytogenes (L. monocytogenes). Three innovative, pre-packed, plant-based RTE food product categories on the Belgian market were identified based upon data gaps regarding the prevalence and growth potential of this pathogen. These were vegetarian and vegan deli sandwich slices (category 1), fresh-cut (mixes of) leafy vegetables (category 2), and multi-ingredient salad bowls (category 3). Reports on associated listeriosis outbreaks and recalls were collected and a comprehensive literature review on the prevalence of L. monocytogenes (i.e. detection in 25 g food) was performed. In addition, the prevalence of L. monocytogenes was also determined through an exploratory retail survey of ca. 50 different RTE products of each category. A batch was considered positive if L. monocytogenes was detected in a food item, either on the day of purchase, at the end of shelf life, or both. During the retail survey, L. monocytogenes was not detected in category 2 (0 out of 51 batches), while 1 out of 51 and 6 out of 48 batches were found positive for respectively category 1 and 3. The observed L. monocytogenes concentration did not exceed 10 CFU/g at any point in time in any batch. Furthermore, challenge tests were performed to determine the growth potential of L. monocytogenes in nine pre-packed, plant-based RTE food products (two to four different products of each category, and three different batches per product). After inoculation, products were stored for half of their shelf life at 7 °C and half of their shelf life at 9 °C (simulation of resp. retail and consumer storage). In six of the nine challenge tests executed, growth of L. monocytogenes was supported (i.e. growth potential ≥0.50 log10 CFU/g during shelf life). The highest growth potential was observed for fresh-cut iceberg lettuce (3.60 log10 CFU/g in 9 days), but a large variation regarding the growth potential of L. monocytogenes was noted both between and within the three studied pre-packed, plant-based RTE food product categories. This variation was mainly caused by differences in product composition, physicochemical product characteristics, present (competitive) microbiota such as lactic acid bacteria, applied preservation techniques, and shelf life.

Degradable Plasma-Polymerized Poly(Ethylene Glycol)-Like Coating as a Matrix for Food-Packaging Applications.

Currently, there is considerable interest in seeking an environmentally friendly technique that is neither thermally nor organic solvent-dependent for producing advanced polymer films for food-packaging applications. Among different approaches, plasma polymerization is a promising method that can deposit biodegradable coatings on top of polymer films. In this study, an atmospheric-pressure aerosol-assisted plasma deposition method was employed to develop a poly(ethylene glycol) (PEG)-like coating, which can act as a potential matrix for antimicrobial agents, by envisioning controlled-release food-packaging applications. Different plasma operating parameters, including the input power, monomer flow rate, and gap between the edge of the plasma head and substrate, were optimized to produce a PEG-like coating with a desirable water stability level and that can be biodegradable. The findings revealed that increased distance between the plasma head and substrate intensified gas-phase nucleation and diluted the active plasma species, which in turn led to the formation of a non-conformal rough coating. Conversely, at short plasma-substrate distances, smooth conformal coatings were obtained. Furthermore, at low input powers (<250 W), the chemical structure of the precursor was mostly preserved with a high retention of C-O functional groups due to limited monomer fragmentation. At the same time, these coatings exhibit low stability in water, which could be attributed to their low cross-linking degree. Increasing the power to 350 W resulted in the loss of the PEG-like chemical structure, which is due to the enhanced monomer fragmentation at high power. Nevertheless, owing to the enhanced cross-linking degree, these coatings were more stable in water. Finally, it could be concluded that a moderate input power (250-300 W) should be applied to obtain an acceptable tradeoff between the coating stability and PEG resemblance.

Effect of inoculum type, packing material and operational conditions on the biofiltration of a mixture of hydrophobic volatile organic compounds in air.

The emission of volatile organic compounds (VOCs) into the atmosphere causes negative environmental and health effects. Biofiltration is known to be an efficient and cost-effective treatment technology for the removal of VOCs in waste gas streams. However, little is known on the removal of VOC mixtures and the effect of operational conditions, particularly for hydrophobic VOCs, and on the microbial populations governing the biofiltration process. In this study, we evaluated the effect of inoculum type (acclimated activated sludge (A-AS) versus Rhodococcus erythropolis) and packing material (mixture of compost and wood chips (C + WC) versus expanded perlite) on the removal of a mixture of hydrophobic VOCs (toluene, cyclohexane and hexane) in three biofilters (BFs), i.e., BF1: C + WC and R. erythropolis; BF2: C + WC and A-AS; and BF3: expanded perlite and R. erythropolis. The BFs were operated for 374 days at varying inlet loads (ILs) and empty bed residence times (EBRTs). The results showed that the VOCs were removed in the following order: toluene > cyclohexane > hexane, which corresponds to their air-water partitioning coefficient and thus bioavailability of each VOC. Toluene is the most hydrophilic VOC, while hexane is the most hydrophobic. BF2 outperformed BF1 and BF3 in each operational phase, with average maximum elimination capacities (ECmax) of 21 ± 3 g toluene m-3 h-1 (removal efficiency (RE): 100 %; EBRT: 82 s), 11 ± 2 g cyclohexane m-3 h-1 (RE: 86 ± 6 %; EBRT: 163 s) and 6.2 ± 0.9 g hexane m-3 h-1 (RE: 96 ± 4 %; EBRT: 245 s). Microbial analysis showed that despite having different inocula, the genera Rhodococcus, Mycobacterium and/or Pseudonocardia dominated in all BFs but at different relative abundances. This study provides new insights into the removal of difficult-to-degrade VOC mixtures with limited research to date on biofiltration.

Selected-ion flow-tube mass spectrometry for the identification of volatile spoilage markers for fresh pork packaged under modified atmospheres.

Microbial behavior during meat storage leads to the generation of volatile organic compounds (VOCs) and unpleasant off-odors. This study focused on a novel real-time analytical method, selected-ion flow-tube mass spectrometry (SIFT-MS), to monitor VOC quality and identify spoilage indicators for fresh pork stored under different packaging atmospheres (air, 70/0/30, 70/30/0, 5/30/65, 0/30/70 - v/v% O2/CO2/N2) at 4 °C. A comprehensive selection methodology was used to identify compounds with good instrumental data quality as well as a strong relationship with microbial growth and olfactory rejection. Based on the volatolome quantified by SIFT-MS, storage periods and conditions can be discriminated using multivariate statistics. Acetoin (or ethyl acetate) represented a significant pork quality marker for high-O2 conditions, whereas ethanol, 3-methylbutanal and sulfur compounds can indicate the anaerobic storage progress. Considering the applicability in monitoring different VOC profiles, SIFT-MS is expected to be promising in many storage scenarios to improve analytical efficiency and ensure reliability.

The potential of UVC decontamination to prolong shelf-life of par-baked bread.

The effect of UVC (254 nm) treatment on the mould-free shelf-life of par-baked wholemeal, rye and six-grain bread was examined. Currently, these breads are par-baked, wrapped in high-density polyethylene (HDPE)-foil and transported or stored at room temperature for a couple of days before being full-baked and sold/consumed. Generally, after five days, these breads show signs of mould spoilage. A shelf-life increase in one or more days would already offer immense economical and logistic benefits for the baker or retailer. In this study, the parameters fluence rate (irradiation intensity), fluence (UV dose), distance to the UV-lamp (DTL) and number of layers of a common wrapping HDPE-foil (20 µm) were diversified. The breads were subjected to a UVC treatment (0-2502 mJ/cm²), packed and stored at room temperature for a period of 15 days (21.5 ± 0.8°C). Similar as for the breads, agar plates with mould spores of Aspergillus niger, Aspergillus montevidensis and Penicillium roqueforti were UVC treated (0-1664 mJ/cm²) and checked daily for visible mould growth during 15 days (25°C). Aspergillus niger showed the strongest resistance towards UVC, a fluence of 800 mJ/cm² was needed to inhibit growth during 15 days of storage, whereas for P. roqueforti and A. montevidensis, respectively, UV levels of 291 and 133 mJ/cm² were found sufficient. Furthermore, the shelf-life of wholemeal, rye and six-grain bread can be prolonged from 5 to 6, 8 and 9 days, respectively, using 2502 mJ/cm². The effect of higher UVC dosage on shelf-life reached a maximal level and was strongly impacted by the wide spread on data of mould-free shelf-life. The main factors influencing the potential of UV decontamination were the rough bread surface, differences in DTL, the possibility of post-contamination and UV permeability of packaging materials.

Non-Thermal Plasma Decontamination Using a Multi-Hollow Surface Dielectric Barrier Discharge: Impact of Food Matrix Composition on Bactericidal Efficacy.

The non-thermal plasma (NTP) treatment of food products as an alternative for thermal processing has been investigated over the last few years. This quasi-neutral gas contains a wide variety of reactive oxygen and nitrogen species (RONS), which could be lethal for bacterial cells present in the product. However, apart from only targeting bacteria, the RONS will also interact with components present in the food matrix. Therefore, these food components will protect the microorganisms, and the NTP treatment efficiency will decrease. This effect was investigated by supplementing a plain agar medium with various representative food matrix components. After inoculation with Escherichia coli O157:H7 (STEC) MB3885, the plates were treated for 30 s by a multi-hollow surface dielectric barrier discharge (MSDBD) generated in either dry air or air at 75% humidity, at constant power (25.7 ± 1.7 W). Subsequently, the survival of the cells was quantified. It has been found that the addition of casein hydrolysate (7.1 ± 0.2 m%), starch (2.0 m%), or soybean oil (4.6 m%) decreased the inactivation effect significantly. Food products containing these biomolecules might therefore need a more severe NTP treatment. Additionally, with increasing humidity of the plasma input gas, ozone levels decreased, and the bactericidal effect was generally less pronounced.

Extension of growth/no growth predictive models for the preservation of low-acid pasteurized sauces by incorporating water activity and model validation in sauces.

Combined preservation methods have awakened a growing interest in low-acid pasteurized sauces under ambient storage, aiming to produce more 'natural' foods with enhanced microbial stability. However, limited information and predictive models are available to assess the microbial stability of this kind of products, for which the spoilage is mainly caused by acid-tolerant spore-forming spoilage bacteria (ATSSB). In this study, a set of growth/no growth (G/NG) models developed previously (Sun et al., 2021a) for spores of two ATSSB strains (Bacillus velezensis and Bacillus subtilis) from pasteurized sauces were upgraded to incorporate the effect of water activity (aw). The growth from heat-treated spores (80 °C, 10 min) was assessed during three months in Nutrient Broth under 320 combinations of storage temperatures (22 and 30 °C), aw (0.95 and 0.965), pH (4.4-5.6), total acetic acid (0.0-0.3 % (w/w)), and total lactic acid (0.00-1.00 % (w/w)). Twelve replicates were tested for each combination. After merging the dataset collected previously at aw 0.98, a set of 4-variate (4V) models were developed for each strain under a single temperature after 30, 60 and 90 days separately. Additionally, the developed models were validated by challenge tests in different industrially produced low-acid pasteurized sauces for three months. The results showed that lowering aw significantly enhanced the antimicrobial effect of decreased pH and the addition of acetic and lactic acid against the ATSSB spores, while the enhancement was less evident at high acetic acid concentrations. In the challenge tests, the developed models exhibited accurate predictions for the sauces, with the % correct-predicted values ≥90 %. The developed models can be used in microbiological risk assessments or the innovation of preservation strategies for low-acid pasteurized sauces.

Predictive modelling of the radial growth of Aspergillus flavus and Fusarium proliferatum on paddy and white rice (Oryza sativa).

The growth of Aspergillus flavus 01 (AF01) and Fusarium proliferatum 01 (FP01) was studied on paddy and white rice, using a full factorial design with five temperature levels (20, 25, 30, 35 and 40 °C) and five points of water activity (0.80, 0.85, 0.90, 0.95 and 0.99 aw). The maximum radial growth rates (µmax, mm.day-1) and lag times (λ, days) of both fungal strains were estimated by linear regression. When the obtained values were compared with the prediction of reported predictive models for growth of A. flavus 01 and F. proliferatum 01, none of the reported models was able to describe the growth as determined by water activity and temperature adequately. Therefore, new secondary models were developed to describe the obtained fungal radial growth rate of both strains on paddy and white rice as a function of temperature and water activity. The results showed that optimum radial growth rate of AF01 and FP01 were estimated at 0.99 aw/35 °C and 0.99 aw/30 °C, respectively. Both strains failed to grow at 0.80, 0.85 and 0.90 aw at all observed temperatures (except for AF01 at 0.90aw/35 °C). Multi-factorial cardinal and General Polynomial models were the best models to describe combined influence of water activity and temperature on fungal growth rate on both matrices with the bias factors of 0.771-1.10 and accuracy factors of 1.102-1.297. These values should be concerned during storage to mitigate fungal growth on paddy and white rice.

Non-thermal plasma inactivation of Salmonella Typhimurium on different matrices and the effect of selected food components on its bactericidal efficacy.

Non-thermal plasma (NTP) is known as an effective source of a variety of reactive species generated in the gas phase. Nowadays, NTP is gaining increasing interest from the food industry as a microbial inactivation technique. In the present study the effect of inoculation method and matrix on inactivation of Salmonella Typhimurium was examined by treating spread plated agar (2.2 log CFU/sample inactivation by NTP), spot inoculated agar (1.9 log CFU inactivation), glass beads (1.3 log CFU inactivation) and peppercorn (0.2 log CFU inactivation). Furthermore, multiple agar matrices supplemented with low and high concentrations of a certain food component (casein, starch, sunflower oil, vitamin C, sodium pyruvate or grinded peppercorns) were inoculated and treated to determine the effect of those components on NTP efficiency. Although starch, vitamin C and sodium pyruvate had no significant influence on the inactivation degree, the presence of 10% casein (2.1 log CFU/sample less inactivation compared to tryptone soy agar (TSA)), 10% pepper (2.1 log CFU less inactivation) or 1% and 10% sunflower oil (1.6 and 2.1 log CFU less inactivation, respectively) in TSA demonstrated the protective effect of these substances for NTP treatment. These experiments led to the conclusion that low inactivation on produce seemed not to arise from the inoculation method nor from the shape of the produce, but is the result of the food matrix.

Streptomyces Strains Promote Plant Growth and Induce Resistance Against Fusarium verticillioides via Transient Regulation of Auxin Signaling and Archetypal Defense Pathways in Maize Plants.

Driven by climate change, Fusarium ear rot (FER) caused by Fusarium verticillioides occurs frequently in maize worldwide. In parallel, legislative regulations and increasing environmental awareness have spurred research on alternative FER biocontrol strategies. A promising group of bacterial control agents is Streptomyces species due to their metabolic versatility. However, insights into the molecular modes of action of these biocontrol agents are often lacking. This study aims at unraveling the biocontrol efficacy of Streptomyces rhizobacterial strains against F. verticillioides. We first assessed the direct antagonism of four Streptomyces strains ST02, ST03, ST07, and ST08. Then, a profile of 16 genes associated with intrinsic plant defense signaling was assessed in maize plants. Both in vitro and in vivo data showed that the biocontrol strain ST03 perfectly suppressed the growth of F. verticillioides. High inhibition efficacy was also observed for extracellular compounds in the supernatant secreted by this strain. Especially, for maize cobs, the biocontrol strain ST03 not only inhibited the proliferation of F. verticillioides but also significantly repressed fungal fumonisin production 7 days after inoculation. On maize plants, the direct antagonism was confirmed by a significant reduction of the fungal DNA level in soils when co-applied with F. verticillioides and strain ST03. In terms of its action on plants, strain ST03 induced downregulation of auxin responsive genes (AUX1, ARF1, and ARF2) and gibberellic acid (GA)-related gene AN1 even in the absence of F. verticillioides at early time points. In leaves, the biocontrol strain induced the expression of genes related to salicylic acid (SA), and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-mediated pathways, and pathogenesis-related proteins in the presence or absence of the pathogen. Interestingly, the biocontrol strain significantly promoted plant growth even in the presence of F. verticillioides. All of which demonstrated that the Streptomyces strain ST03 is a promising FER biocontrol and a growth-promoting candidate.

Modeling the combined effect of temperature, pH, acetic and lactic acid concentrations on the growth/no growth interface of acid-tolerant Bacillus spores.

The application of minimal processing technologies has led to increased spoilage incidents in low-acid pasteurized sauces due to the outgrowth of acid-tolerant spore-forming spoilage bacteria (ATSSB). Controlling the germination and subsequent growth of ATSSB spores is vital to enhance the ambient storage stability of pasteurized sauces. This study developed and validated a set of growth/no growth (G/NG) models for spores of two ATSSB strains (Bacillus velezensis and Bacillus subtilis subsp. subtilis) isolated from pasteurized sauces. The G/NG data at two levels of temperature (22 and 30 °C) were collected in Nutrient Broth (aw = 0.98 adjusted with NaCl) by a full factorial design with five equidistant levels of pH (4.4-5.6), four concentrations of total acetic acid (0.0-0.3% (w/w)), and four concentrations of total lactic acid (0.00-1.00% (w/w)). The growth, starting from heat-treated (10 min 80 °C) spores, of each strain was assessed under 160 combinations by regular optical density measurements during three months. Twelve replicates were made for each combination. The developed models demonstrate that without organic acids even the lowest pH (4.4) allows a high growth possibility of the ATSSB spores, while acetic and lactic acids exhibit a significant antibacterial activity, which can be enhanced at decreased pH. The growth starting from B. subtilis spores can be inhibited for at least three months with 1.0% (w/w) total lactic acid in the water phase at both temperatures, which was not the case for B. velezensis, while 0.3% acetic acid achieves a full inhibition on both strains at 22 °C. With a combination of 0.3% acetic acid and 0.7% lactic acid, no growth should occur in the investigated range. This research is one of the first studies exploring the feasibility of ambient storage for low-acid pasteurized sauces eliminating preservatives such as benzoic and sorbic acids, and proves the synergistic effect of decreased pH and the presence of acetic and lactic acids on inhibiting bacterial growth from ATSSB spores.

Molecular Insights into Defense Responses of Vietnamese Maize Varieties to Fusarium verticillioides Isolates.

Fusarium ear rot (FER) caused by Fusarium verticillioides is one of the main fungal diseases in maize worldwide. To develop a pathogen-tailored FER resistant maize line for local implementation, insights into the virulence variability of a residing F. verticillioides population are crucial for developing customized maize varieties, but remain unexplored. Moreover, little information is currently available on the involvement of the archetypal defense pathways in the F. verticillioides-maize interaction using local isolates and germplasm, respectively. Therefore, this study aims to fill these knowledge gaps. We used a collection of 12 F. verticillioides isolates randomly gathered from diseased maize fields in the Vietnamese central highlands. To assess the plant's defense responses against the pathogens, two of the most important maize hybrid genotypes grown in this agro-ecological zone, lines CP888 and Bt/GT NK7328, were used. Based on two assays, a germination and an in-planta assay, we found that line CP888 was more susceptible to the F. verticillioides isolates when compared to line Bt/GT NK7328. Using the most aggressive isolate, we monitored disease severity and gene expression profiles related to biosynthesis pathways of salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), benzoxazinoids (BXs), and pathogenesis-related proteins (PRs). As a result, a stronger induction of SA, JA, ABA, BXs, and PRs synthesizing genes might be linked to the higher resistance of line Bt/GT NK7328 compared to the susceptible line CP888. All these findings could supply valuable knowledge in the selection of suitable FER resistant lines against the local F. verticllioides population and in the development of new FER resistant germplasms.

Effect of temperature, CO2 and O2 on motility and mobility of Anisakidae larvae.

Anisakidae, marine nematodes, are underrecognized fish-borne zoonotic parasites. Studies on factors that could trigger parasites to actively migrate out of the fish are very limited. The objective of this study was to assess the impact of different environmental conditions (temperature, CO2 and O2) on larval motility (in situ movement) and mobility (migration) in vitro. Larvae were collected by candling or enzymatic digestion from infected fish, identified morphologically and confirmed molecularly. Individual larvae were transferred to a semi-solid Phosphate Buffered Saline agar, and subjected to different temperatures (6 ℃, 12 ℃, 22 ℃, 37 ℃) at air conditions. Moreover, different combinations of CO2 and O2 with N2 as filler were tested, at both 6 °C and 12 °C. Video recordings of larvae were translated into scores for larval motility and mobility. Results showed that temperature had significant influence on larval movements, with the highest motility and mobility observed at 22 ℃ for Anisakis spp. larvae and 37 ℃ for Pseudoterranova spp. larvae. During the first 10 min, the median migration of Anisakis spp. larvae was 10 cm at 22 ℃, and the median migration of Pseudoterranova spp. larvae was 3 cm at 37 ℃. Larval mobility was not significantly different under the different CO2 or O2 conditions at 6 °C and 12 ℃. It was concluded that temperature significantly facilitated larval movement with the optimum temperature being different for Anisakis spp. and Pseudoterranova spp., while CO2 and O2 did not on the short term. This should be further validated in parasite-infected/spiked fish fillets.

Bacillus weihenstephanensis can readily evolve for increased endospore heat resistance without compromising its thermotype.

Proper elimination of bacterial endospores in foods and food processing environment is challenging because of their extreme resistance to various stresses. Often, sporicidal treatments prove insufficient to eradicate the contaminating endospore population as a whole, and might therefore serve as a selection pressure for enhanced endospore resistance. In the sporeforming Bacillus cereus group, Bacillus weihenstephanensis is an important food spoilage organism and potential cereulide producing pathogen, due to its psychrotolerant growth ability at 7 °C. Although the endospores of B. weihenstephanensis are generally less heat resistant compared to their mesophilic or thermotolerant relatives, our data now show that non-emetic B. weihenstephanensis strain LMG 18989T can readily and reproducibly evolve to acquire much enhanced endospore heat resistance. In fact, one of the B. weihenstephanensis mutants from directed evolution by wet heat in this study yielded endospores displaying a > 4-fold increase in D-value at 91 °C compared to the parental strain. Moreover, these mutant endospores retained their superior heat resistance even when sporulation was performed at 10 °C. Interestingly, increased endospore heat resistance did not negatively affect the vegetative growth capacities of the evolved mutants at lower (7 °C) and upper (37 °C) growth temperature boundaries, indicating that the correlation between cardinal growth temperatures and endospore heat resistance which is observed among bacterial sporeformers is not necessarily causal.

Post-harvest contamination of maize by Fusarium verticillioides and fumonisins linked to traditional harvest and post-harvest practices: A case study of small-holder farms in Vietnam.

Together with conducive climatic factors, poor pre-harvest practices of ethnic small-holder farmers are a major cause of the contamination of maize by Fusarium verticillioides and fumonisins. The proliferation of this field pathogen and the accumulation of its mycotoxins in post-harvest maize caused by ethnic post-harvest practices of subsistence farms have received little attention. Therefore, this study aimed to evaluate the impact of traditional harvest and post-harvest practices on the proliferation of F. verticillioides and fumonisins contamination in post-harvest maize of two ethnic groups: Ede and Kinh from the central highlands of Vietnam. In parallel with analysis, a survey on harvest and post-harvest practices of these farmers was conducted from late December 2017 to early January 2019. As a result, four effective post-harvest practices at mitigating the contamination were defined: (1) removal of damaged cobs at harvest, (2) transport of maize home after harvest, (3) shelling maize away from fields, and (4) drying maize on cement yards. These practices were better implemented by Kinh households than Ede households reducing the post-harvest contamination of maize with F. verticillioides and fumonisin B1 (FB1), FB2, and FB3. Nevertheless, there is still room for improvement with respect to inadequate open-air drying method, poor storage infrastructure, and poor moisture content management as these correlated to the proliferation of F. verticillioides. Finally, the presence of fumonisins together with aflatoxins in some samples at the storage phase might cause a severe impact on human health.