Evidence of Viable But Non-Culturable state in Listeria monocytogenes by direct viable count and CTC-DAPI double staining

Viable but Non-culturable (VBNC) state in bacteria was detected originally in environmental microbiology studies. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and moreover the cells remain potentially pathogenic under favourable conditions. But the VBNC state has not been yet described in Listeria monocytogenes. Production of VBNC L. monocytogenes cells was studied using four strains resuspended in microcosm water. Various strains of L. monocytogenes were resuspended in filtered sterilized water adjusted to pH 6·0, incubated at 20°C and 4°C with or without NaCl supplementation (0%, 7%), with gentle shaking at 100 rpm. The culturability of starved cells suspensions was determined by spread plate count (PCA). The cells activity was measured by a Direct Viable Count technique and by CTC-DAPI double staining. Two strains of four (CNL 895807, Scott A) exhibited a VBNC state. They lost their culturability and maintained a cellular activity, even after 10 weeks of starvation in microcosm water.     

Dietary fiber,fructooligosaccharides, and physicochemical properties of homogenized aqueous suspensions of yacon (Smallanthus sonchifolius)

Yacon roots are a promising source of inulin­type fructans (35 g/100 g dry matter) with a total amount of dietary fiber of about 45 g/100 g dry matter. The polydispersity of the particle size distribution of yacon aqueous suspension was reduced with increasing the degree of homogenization due to two phenomena: aggregation of particles, and disruption of large particles. Non­homogenized yacon suspensions exhibited large cell clusters that were disrupted into small cell clusters, single cells, and aggregates. The most concentrated suspension exhibited mainly aggregates.The volume fraction of the suspensions decreased significantly with increasing the degree of homogenization. This was attributed to a denser packing of small particles and aggregates in between large particles thus allowing the water within the structure to be released.Yacon suspensions exhibited high elastic modulus (750 Pa) at low water insoluble solids content (0.9% WIS). Additional results suggested that inulin­type fructans contributed to the elastic properties of yacon suspensions.The yacon suspensions studied in this work can be considered as semi­concentrated suspensions, i.e. the plot elastic modulus of the suspensions versus concentration exhibited a region immediately after the transition region. Particle interactions seem to be of great importance in this region in the particular case of yacon suspension since the elastic modulus (G′) reached 750 Pa at low water insoluble solids content (< 1%).     

Suppressive effect of Tetragenococcus halophilus,isolated from fish-nukazuke,on histamine accumulation in salted and fermented fish

Occasionally, quantities higher than 1000 mg/kg of histamine (Hm) accumulate in salted and fermented fish products by the histidine decarboxylase of halophilic lactococci Tetragenococcus sp. In a total of 200 isolates from fish nukazuke (salted and fermented fish with rice bran), 13 strains produced Hm more than 200 μg/ml in 0.5% histidine containing broth, whereas 130 isolates produced absolutely no Hm. Among the strains, 22 strains suppressed the Hm production of the Hm-forming (HmF) strains. Both the HmF and Hm-suppressing (HmS) strains were identified as Tetragenococcus halophilus. To observe the Hm-suppressing effect, a specified quantity of live cells was needed. In the case of 10% NaCl salted sardine, inoculation with 3 log cells/g of a strain HmF-131 resulted in a significant Hm accumulation, 2800 μg/g in 30 days at 20 °C. The increase in Hm was clearly suppressed by 9 log live cells/g of strain HmS-129. These results suggest that HmS-129 can be used as a starter for salted and fermented fish products, enhancing food safety.     

Resistance of Campylobacter jejuni to heat and to pulsed electric fields

The resistance of Campylobacter jejuni NCTC 11351 to heat and to pulsed electric fields (PEF) was studied at different treatment intensities (temperatures between 52 and 60 °C, and electric field strengths between 15 and 35 kV/cm, respectively). The influence of the growth phase, the pH of the treatment medium and the presence of sodium pyruvate in the recovery medium was also examined. A model based on the Weibull distribution was used to describe the inactivation curves, and times for the first decimal reduction were calculated (δ values). C. jejuni cells did not increase their resistance to heat nor to PEF upon entrance into stationary phase. The acidification of the treatment medium from 7.0 to 4.5 caused a sensitization of C. jejuni to heat (δ value at 55 °C × 1/4); on the contrary, resistance to PEF was increased (δ value at 25 kV/cm × 2.5). The absence of pyruvate in the recovery medium prevented recovery of a high percentage of heat-treated cells, but did not affect PEF survival. Whereas C. jejuni can be considered a heat sensitive organism (δ value at 55 °C and buffer of pH 7.0 of 2 min, z value 4.40 °C), it showed a relatively high resistance to PEF as compared to other vegetative cells (δ value at 25 kV/cm and buffer of pH 7.0 of 7 pulses, z_PEF value 8.20 kV/cm). Results obtained in this investigation indicate that Campylobacter spp. should be taken into account for the design of PEF treatments for food hygienization.Industrial relevanceBefore PEF can be commercially implemented it is necessary to determine its efficacy on pathogenic microorganisms of interest in order to ensure safety of food. There is no data available about the resistance of C. jejuni to pulsed electric fields, although it is now recognised as the leading cause of bacterial food-borne gastroenteritis throughout the world. In this research we characterize the resistance to heat and to PEF of C. jejuni NCTC 11351. Physiological factors affecting its survival to both agents are also explored.     

Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk

Nine strains of non-starter Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk were selected for their anticlostridial activity. Resistance to thermisation (60 °C, 5 min) and pasteurisation (73 °C, 15 s) was investigated using a submerged-coil apparatus. MRS broth-grown cultures of all nine strains survived thermisation in buffer. The level of resistance to thermisation was strain dependent and lower for freshly grown cells (stationary phase cells) than for resting cells (freshly grown cells kept diluted 10-fold in MRS broth at 17 °C for 6 days). None of the nine Lb. paracasei strains survived or recovered after pasteurisation in buffer when grown in MRS broth, while seven of the nine strains survived pasteurisation in UHT whole milk when grown in milk. Identity of the strains was successfully confirmed during the experiments using repetitive-PCR analysis. The potential of Lb. paracasei strains to survive pasteurisation of cheese milk was demonstrated.     

Inactivation kinetics of Listeria monocytogenes,Salmonella enterica serovar Typhimurium,and Campylobacter jejuni in ready-to-eat sliced ham using UV-C irradiation

To investigate the applicability of UV-C irradiation on the inactivation of foodborne pathogenic bacteria in ready-to-eat sliced ham, UV-C treatment was evaluated. Irradiation dose required for 90% reduction of the populations of Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Campylobacter jejuni were determined to be 2.48, 2.39, and 2.18 J/m². Ready-to-eat sliced hams were inoculated with the pathogens and irradiated with UV-C light of 1000, 2000, 4000, 6000, and 8000 J/m². Microbiological data indicated that foodborne pathogen populations significantly (p < 0.05) decreased with increasing UV-C irradiation. In particular, UV-C irradiation at 8000 J/m² reduced the populations of L. monocytogenes, S. Typhimurium, and C. jejuni in the ham by 2.74, 2.02, and 1.72 log CFU/g. The results indicate that UV-C irradiation can be used as a microbial inactivation method for ready-to-eat sliced ham, and inactivation kinetics of the foodborne pathogens fit the Weibull model better than the first-order kinetics model.     

Comparison of multiple chemical sanitizers for reducing Salmonella and Escherichia coli O157:H7 on spinach (Spinacia oleracea) leaves

Effectiveness of multiple chemical sanitizers on the reduction of Salmonella spp. and Escherichia coli O157:H7 on spinach was compared. Fresh spinach (Spinacia oleracea) was inoculated with a bacterial suspension containing multiple strains of rifampin-resistant Salmonella and E. coli O157:H7. Inoculated spinach leaves were treated with a water wash or water wash followed by 2% L-lactic acid at 55 °C, peroxyacetic acid (80 mg/L), calcium hypochlorite (200 mg/L), ozonated water (mg/L) or ClO₂ gas (1.2 or 2.1 mg/L). The l-lactic acid produced a 2.7 log CFU/g reduction for E. coli O157:H7 and a 2.3 log CFU/g reduction for Salmonella, statistically significant compared to water wash alone (P < 0.05), which resulted in a reduction of 0.7 log CFU/g for both pathogens. These findings indicate that 2% l-lactic acid at 55 °C may be an effective treatment for reducing pathogens on spinach leaves.     

Stability of probiotic Lactobacillus plantarum in dry microcapsules under accelerated storage conditions

This study investigated the stability of freeze dried and fluid bed dried alginate microcapsules coated with chitosan containing model probiotic bacteria, Lactobacillus plantarum, during storage for up to 45 days at different water activities (0.11, 0.23, 0.40 and 0.70) and temperatures (4, 30 and 37 °C). The loss in cell viability was around 0.8 log in the case of fluid bed drying and around 1.3 in the case of freeze drying, with the former method resulting in dried capsules of smaller size (~ 1 mm vs 1.3 mm), more irregular shape, and with a rougher surface. In both cases, the water activity and water content were less than 0.25 and 10% w/w, respectively, which favours high storage stability. The storage stability studies demonstrated that as the water activity and temperature decreased the survival of the dried encapsulated cells increased. Considerably better survival was observed for fluid bed dried encapsulated cells compared to freeze dried encapsulated cells and freeze dried free cells with 10% sucrose (control), and in some cases, e.g. at 4 and 30 °C at water activities of 0.11, 0.23 and 0.40, there was more than 1 log difference after 45 days, with concentrations higher than 10⁸ CFU/g after 45 days of storage. The results indicate that fluid bed drying is an effective and efficient manufacturing method to produce probiotic containing capsules with enhanced storage stability.     

Destruction of Salmonella Enteriditis inoculated onto raw almonds by high hydrostatic pressure

The effects of continuous (50,000, 60,000 and 70,000 psi with holding times of 5 and 10 min) and discontinuous (oscillatory) (six cycles at 60,000 psi with a holding time of 20 s) high hydrostatic pressure (HHP) treatments on the viability of two Salmonella Enteriditis strains (FDA and PT30) inoculated onto raw almonds were evaluated at 25, 50, and 55 °C. Complete inactivation of the S. Enteriditis was achieved in 0.1% peptone water after continuous pressurization at 60,000 psi and 25 °C for 5 min. Continuous pressurization of raw almonds inoculated with S. Enteriditis at 60,000 psi and 50 °C for 5 min resulted in less than a log reduction (log₁₀ 0.83) of vegetative cells. The decimal reduction time using the continuous pressurization parameters was determined to be 9.78 min. A discontinuous process consisting of six cycles of pressurization at 60,000 psi and 50 °C for 20 s provided greater than a one log reduction (log₁₀ 1.27 for FDA and log₁₀ 1.16 for PT30) of the S. Enteriditis concentration. The low water activity (a_w) of the almonds was found to impart baroprotective attributes on the S. Enteriditis cells. When the almonds were directly suspended in water and then pressurized, a log₁₀ reduction of 3.37 was achieved. HHP of certain dry foods appears to be feasible if the food is directly suspended in the pressurizing medium (water).     

Development of probiotic beads similar to fish eggs

Probiotic foods are mainly restricted to dairy and soy products. This study aimed to develop a new probiotic beads similar to fish eggs, commonly used in oriental cuisine. Beads were produced by the extrusion encapsulation technique with calcium alginate, added to one of the following cultures: Lactobacillus rhamnosus GG ATCC 53103 and Bifidobacterium animalis DN-173 010 and stored for 30 days at 4 °C. The beads were characterized by the size, weight, morphology and viability of the probiotic strains in different storage temperatures and in simulated gastric juice adjusted to different pH values. The beads were also evaluated by a sensorial affective hedonic scale. The beads present a 2.8 mm diameter and a weight of 0.01 g (p > 0.05). Free and encapsulated cells were tolerant to pH 3.0. At pH 2.5 only of the encapsulated cells presented counts above 6 Log colony-forming units per gram (CFU/g). Beads containing L. rhamnosus showed higher viability 10⁷ CFU/g in storage for 30 days under refrigeration. The beads may be stored at abusive temperature for 5 h without loss of viability cells. The probiotic product developed showed an 82.2% acceptability index of overall characteristics and good market potential as a new probiotic product.     

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis,Streptococcus thermophilus and Lactobacillus acidophilus

High pressure processing (HPP) reduces the glycolytic activity of lactic acid bacteria (LAB) and provides a means to control further production of acidic metabolites in fermented dairy products during storage. However, there is limited information on the effects of HPP on specific enzymes of dairy starter bacteria responsible for the metabolism of lactose. The aim of this study was to determine pressure-induced inactivation of glycolytic enzymes in Lactococcus lactis subsp. lactis C10, Streptococcus thermophilus TS1 and Lactobacillus acidophilus 2400. Cultures were grown for 16 h in M17 or MRS broth containing 5% (w/v) lactose at pH 6.5 (maintained by addition of 10 M NaOH). The cells were harvested by centrifugation, washed and resuspended in 100 mM phosphate buffer (pH 6.5) and pressure-treated at 300 and 600 MPa (≤ 22 °C, 5 min). The ability of pressure-treated resting cells of Lactococcus, incubated with 5% (w/v) lactose at 30 °C, to ferment lactose was evaluated by determining titratable acidity (TA) during incubation. The activities of phospho-β-galactosidase (P-β-gal), β-galactosidase (β-gal) and lactate dehydrogenase (LDH) were determined in cell-free extracts of untreated and pressure-treated cells. Resting cells of Lactococcus treated at 600 MPa had a substantially lower rate of acidification than the controls and those treated at 300 MPa. Both P-β-gal and β-gal were significantly inactivated (p < 0.01) in the starter cultures treated at 300 or 600 MPa. The LDH in Lactococcus and Lactobacillus was highly resistant to pressure treatment at 300 MPa. In contrast, the LDH in Streptococcus was almost completely inactivated at ≥ 300 MPa.Industrial relevanceContinuing production of acidic metabolites in fermented dairy products during storage can be a technological challenge that adversely affects product quality. The current study demonstrates that high pressure processing (HPP) offers the potential of controlling this problem by inactivation of glycolytic enzymes in various mesophilic and thermophilic starter cultures. The findings of this research will assist in establishing optimised operating parameters for HPP treatment of cultured products to extend shelf-life, by reducing acid production during storage.     

Understanding high pressure-induced changes in wheat flour–water suspensions using starch–gluten mixtures as model systems

The effect of high pressure (HP) on wheat flour–water suspensions was investigated. Suspensions were treated for 10 min at 200–600 MPa. HP-treatment significantly increased the consistency of the flour suspensions, as studied by frequency sweep tests. Temperature sweeps revealed that HP-induced starch gelatinisation, with a sigmoidal-shaped correlation between degree of gelatinisation and treatment pressure. Analysis of protein solubility in different buffers indicated the HP-induced formation of urea-insoluble complexes and/or disulphide bonds. Furthermore, the effects of HP on the isolated components wheat starch and gluten were studied, and starch–gluten mixtures were used as a model system for flour. A negative effect of gluten on the consistency increase of starch suspension was observed. Comparing the rheological parameters of HP-treated wheat flour suspensions to those of starch suspensions, confirmed the weakening effect of gluten. However, the presence of gluten in flour could not fully explain the differences between starch and flour suspension.     

The influence of lactoperoxidase,heat and low pH on survival of acid-adapted and non-adapted Escherichia coli O157:H7 in goat milk

In hot climates where quality of milk is difficult to control, a lactoperoxidase (LP) system can be applied in combination with conventional preservation treatments at sub-lethal levels to inhibit pathogenic microbes. This study investigated the effect of combined heat treatments (55 °C, 60 °C and 72 °C) and milk acidification (pH 5.0) on survival of acid-adapted and non-adapted Escherichia coli O157:H7 strains UP10 and 1062 in activated LP goat milk. Heat treatment at 72 °C eliminated E. coli O157:H7. Acid-adapted strains UP10 and 1062 cells showed resistance to combined LP and heat at 60 °C in fresh milk. The inhibition of acid-adapted and non-adapted E. coli O157:H7 in milk following combined LP-activation, heat (60 °C) and milk acidification (pH 5.0) suggests that these treatments can be applied to reduce E. coli O157:H7 cells in milk when they occur at low numbers (<5 log₁₀ cfu mL^?1) but does not eliminate E. coli O157:H7 to produce a safe product.     

Oats protein isolate: Thermal,rheological, surface and functional properties

Oat protein isolate (OPI) was extracted in 0.015 N NaOH and acetylated or succinylated. The thermal analysis of the isolate showed a glass transition (T_g) at 43.4 °C and ΔC_p of 0.102 J/g/°C. The positive net charge of OPI and the positive or neutral charge of the modified OPI were apparent from the free capillary zone electrophoresis (FZCE) profiles. Acetylation significantly lowered foaming and emulsifying properties of OPI, while succinylation showed the highest foaming capacity, foam stability, and emulsion stability. Acetylated OPI showed the highest surface hydrophobicity compared to the other samples, while OPI was the most soluble of all. The water holding capacity of all samples analyzed was the same except for acetylated-crosslinked (ACXL). The surface tension test confirmed that unmodified and modified OPI possessed surface activity and the equilibrium surface tensions decreased sharply with increasing protein concentration and leveled off to a constant value. The elastic modulus, G′, for the acetylated OPI suspension exhibited the highest value, while the G′ of the crosslinked (XLOPI) had the lowest. The plateau of G′, was 2961 Pa, 920 Pa, 223 Pa, 41 Pa, and 1.8 Pa for the ACOPI, ACXL, SOPI, and XL, respectively.     

Simultaneous detection and enumeration of viable lactic acid bacteria and bifidobacteria in fermented milk by using propidium monoazide and real-time PCR

This study describes a procedure that allows specific detection and enumeration of viable bacteria in four species of lactic acid bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus casei subsp. casei and Lactobacillus acidophilus) and of Bifidobacterium lactis, mixed in fermented milk products. The procedure is based on the combined use of propidium monoazide (PMA), able to distinguish between viable and irreversibly damaged cells, with species-specific quantitative real-time PCR (RTi-PCR). Loss of viability of the species in a fermented milk through storage at 4 °C was similarly (P < 0.05) detected by PMA–RTi-PCR and selective plate counts. Furthermore, comparison of results obtained by both methods showed a Pearson linear correlation of 0.995. The enumeration of viable bacteria by PMA–RTi-PCR could be performed in 3 h, whereas enumeration by selective plate counts required three days. The procedure developed is a fast method for the identification, enumeration and discrimination of viability of lactic acid bacteria and bifidobacteria mixed in fermented milk products.     

Transglutaminase-induced caseinate gelation for the microencapsulation of probiotic cells

A novel method for the encapsulation of probiotic cells in foodgrade casein microcapsules was developed. The process is based on a transglutaminase-catalysed gelation of casein suspensions containing probiotic cells. Water insoluble, spherical capsules with a volume-based median diameter of 165 ± 23 μm resulted from the process. Encapsulation yields of 70 ± 15% and 93 ± 22% were achieved for Lactobacillus paracasei ssp. paracasei F19 and Bifidobacterium lactis Bb12, respectively. Analysis of living cell numbers after incubation of free and encapsulated probiotics in simulated gastric juice without pepsin at pH 2.5 and pH 3.6 (37 °C, 90 min) showed a protective effect due to microencapsulation under all conditions tested. The study indicates that transglutaminase-induced caseinate gelation can be applied to the microencapsulation of probiotics. Furthermore, it could be shown that an entrapment in a dense casein matrix can protect these microorganisms from damage due to pH-levels similar to those in the human stomach.     

Inactivation of bacterial pathogens on lettuce,sprouts, and spinach using hurdle technology

Effects of chemical treatment using slightly acidic electrolyzed water (SAEW), fumaric acid (FA), or calcium oxide (CaO) and physical treatment using ultrasonication (US), micro-bubbles (MB), or ultraviolet (UV) to inactivate bacterial pathogens Listeria monocytogenes, Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella spp. on lettuce, spinach, and sprouts were determined. Fresh produce inoculated with bacterial pathogens (~ 9 log CFU/mL) was immersed in distilled water (DW), SAEW, FA (0.5%), or CaO (0.2%) alone or in combination at 23 ± 2 °C for 3 min followed by treatment with US, MB for 3 min, or UV for 10 min. Effects of combined treatment on shelf-life of lettuce at 4 °C and 23 ± 2 °C were also determined in this study. Results revealed that the use of a combination of CaO + SAEW + FA + US exhibited significant reduction (p < 0.05) for bacterial pathogen on fresh produce compared to individual treatment or other combinations. CaO + SAEW + FA + US treatment exhibited highest reduction of E. coli O157:H7, S. aureus, L. monocytogenes and Salmonella spp. by 4.7, 4.9, 4.84 and 5.08 log CFU/g, respectively on lettuce as compared to spinach and sprouts. Microbial count reducing capability for combined treatment methods were ranked in the following order: SAEW + FA < CaO + SAEW + FA < CaO + SAEW + FA + US. However, introduction of US to CaO + SAEW + FA treatment resulted in little detrimental effect on the overall quality of lettuce. Moreover, CaO + SAEW + FA treatment effectively enhanced the shelf-life of lettuce stored at 4 °C and 23 ± 2 °C by about 6 days and 3 days, respectively as compared to control (DW treatment), with longer lag time (23.11 h on lettuce) for naturally occurring bacteria on fresh produce. These findings suggest that significant synergistic benefit could be obtained from combined sanitizer treatment to eliminate bacterial pathogens from fresh produce.     

Effect of modified atmosphere packaging,storage period,and storage temperature on the residual nitrate of sliced-pastırma,dry meat product,produced from fresh meat and frozen/thawed meat

The amount of nitrite in sliced-pastirma made, from fresh or frozen (which was stored at −18 °C for 240 days and then thawed at 10 °C for 24 h) M. Longissimus dorsi muscle was determined. Sliced-pastirma samples were stored in modified atmosphere packages (50% N₂ + 50% CO₂) at 4 and 10 °C for 150 days, and the amount of residual nitrite was measured after 0, 30, 60, 90, and 150 days of storage. The residual nitrite of pastirma samples made with frozen/thawed meat was higher than that of the pastirma made from fresh meat at both 0 day and at the end of the storage (150 days). The storage temperature (p < 0.01), storage period (p < 0.01) and the storage period × the storage temperature interaction (p < 0.01) had significant effects on the amount of the residual nitrite.     

Histamine accumulation and histamine-forming bacteria in Indian anchovy (Stolephorus indicus)

Accumulation of histamine, trimethylamine (TMA), and total volatile base nitrogen (TVB-N), as well as microbial population incidence in Indian anchovy (Stolephorus indicus) during storage in ice and at 15 and 35 °C were investigated. Histamine was as low as 1.9 mg/100 g in 15 days at ice storage, but it increased to 19.0 mg/100 g after 32 h at 15 °C. Histamine rapidly increased to 25.4 mg/100 g when stored at 35 °C for 8 h. TVB-N and TMA began to sharply increase after 11 days in ice storage, but abruptly increased after 16 and 8 h of storage at 15 and 35 °C, respectively. A high number of Enterobacteriaceae (10¹⁰–10¹¹ cfu/g) was detected and shown to be the dominant group of microbial flora during spoilage of Indian anchovy at both 15 and 35 °C. A total of 153 bacterial strains were selected from the prescreening step using various selective media. Only 75.8% of these selected isolates showed a positive reaction in Niven's differential medium, and 27.6% of the positive isolates were true histamine formers when confirmed by the enzymatic method. Prolific histamine formers were identified as Morganella morganii, Proteus vulgaris, and Enterobacter aerogenes, and produced high histamine content of 104.1–203.0 mg/100 ml. Optimum growth and histamine production of selected strains of these three species was at 35 °C in histamine evaluation broth (HEB) containing 0.5% NaCl, pH 5. E. aerogenes produced the highest histamine of 500 mg/100 ml at the optimum condition. All studied strains did not produce histamine at ⩾10% NaCl.     

Modeling the effect of natamycin,pine-resin and environmental factors on the growth and OTA production by Aspergillus carbonarius using response surface methodology

The effect of two antifungal compounds (natamycin, pine-resin), temperature and water activity, on the growth rate, lag phase duration and Ochratoxin A (OTA) production by three Aspergillus carbonarius isolates (Ac-28, Ac-29, and Ac-33), was studied by means of Response Surface Methodology (RSM) based on a Central Composite Design (CCD). Two different experimental designs were performed as a function of temperature (16.6–33.4 °C), water activity (0.90–0.97 a_w), natamycin (0–1000 ng ml^− 1) or pine-resin (0–2.61%, w/v) on a Synthetic Grape-juice Medium (SGM). OTA production was analyzed after 5, 10 and 15 days of incubation. A second-order polynomial model was fitted to each response parameter to assess the growth and OTA potential of all fungal isolates. Results showed that natamycin, a_w and temperature had significant effects on the lag phase duration of all isolates, as well as on OTA accumulation after 10 days of incubation for Ac-29 and 15 days for Ac-28 and Ac-33 isolates. The same results were obtained for OTA production after treatment with pine-resin. However, fungal growth rates were not statistically significant in both experiments, with the exception of Ac-29 and Ac-33 after treatment with pine-resin. Overall, high natamycin concentrations (800 and 1000 ng ml^− 1) delayed fungal growth depending on the environmental factors assayed. Moreover, treatment with pine-resin at 16.6 °C/0.94 a_w/1.1% w/v, as well as at 25 °C/0.90 a_w/1.1% w/v, completely inhibited fungal growth up to 15 days of incubation.