Quantitative proteomic analysis of Neosartorya pseudofischeri ascospores subjected to heat treatment.

Neosartorya pseudofischeri, a heat- resistant fungus, was isolated from contaminated blueberry juice in our laboratory. To obtain a better understanding of the cellular process of heat- resistant fungus, a quantitative proteomic investigation employing stable isotope labeling by peptide demethylation was conducted on changes in intracellular proteins of N. pseudofischeri ascospores subjected to heat treatment at 93 °C for 0, 1 or 8 min. In total, 811 proteins were identified and quantified. Using the normalized ratio of protein abundance, proteins that changed more than two- fold after heat treatment were identified as significantly increased or decreased proteins and grouped into four clusters based on their quantitative changes. Decreased proteins were found mainly involved in the central carbon metabolism, heat stress responses, reactive oxygen intermediates elimination and translation events. A group of proteins in relation to toxicant degradation and antibiotic neutralization linking to environmental adaptability and tolerance of heat- resistant, was also identified. These findings provide insights into protein changes of N. pseudofischeri ascospores and lay foundations for further investigations on heat- resistant molds using targeted quantitative approaches. SIGNIFICANCE OF THE RESEARCH: Heat- resistant fungi can survive pasteurization processes, and subsequently germinate and grow to cause spoilage of food products, leading to significant economic losses for the food industry and potential health risk for the consumers. To obtain a better understanding of the cellular process of heat- resistant fungi, a quantitative proteomic approach employing stable isotope labeling by peptide demethylation has been used to investigate the intracellular protein changes of N. pseudofischeri ascospores isolated from blueberry juice and subjected to heat treatment. 150 significantly changed proteins were grouped into four clusters based on their quantitative changes. The significant decrease in protein abundance in response to heat treatment revealed possible mechanism that N. pseudofischeri ascospores could survive the heat treatment. This is the first proteomic profile report for N. pseudofischeri. These findings provide insights into protein changes of N. pseudofischeri and lay foundations for further investigations on heat- resistant fungi using targeted quantitative approaches to evaluate the efficiency of thermal treatment for processed food products.

Effect of natural antimicrobial agent (MicroGARD) combined with edible coating (NatureSeal) treatment on fresh-cut butternut squash.

To improve fresh-cut produce quality and shelf life, 0.5% or 1.0% MicroGARD® 730 (MG) as a natural alternative to synthetic chemical preservatives, 2.5% NatureSeal® (NS) product (vitamin/mineral-based blends), 0.5% MG combined with 2.5% NS, and 1% MG combined with 2.5% NS were used to treat fresh-cut butternut squash (Cucurbita moschata). The 240 g samples were put into food grade bags and stored at 4 or 7 °C. Microbial population, including aerobic plate counts (APCs), yeast and molds, total coliforms, and quality parameters, including head space O2 /CO2 concentration in package, pH, soluble solids, color, and conductivity, were evaluated after 0, 3, 6, 9, 12, and 20 days of storage. Results showed that after 6 days of storage at 7 °C, APC of check and control samples reached to 2.6 × 108 and 1.5 × 107 CFU/g, respectively; while they were kept at 104 CFU/g (3 to 4 log reduction) in the squash samples treated with 0.5% or 1% MG combined with NS at 7 °C. Similar results were found on squash samples stored at 4 °C for 9 days. The cut squash treated with MG combined with NS had APC ≤ 107 CFU/g at 4 °C for about 20 days compared to 9 days in controls or 0.5% MG-treated samples, and 12 days in 1% MG-treated or NS-treated samples, respectively. Considering overall quality and extended shelf life, MG combined with NS was recommended to apply to cut squashes stored at 4 °C. PRACTICAL APPLICATION: This research provided useful information and practical treatment application for developing fresh-cut produce with good quality and extended shelf life up to 20 days at 4 °C.

Microbial Quality and Shelf Life of Blueberry Purée Developed Using Cavitation Technology.

Blueberry purée was developed using hydrodynamic cavitation technology. The product was made from entire blueberries without adding any food additives. In this study, microbial reduction following each processing stage (at the industry setting) and after product pasteurization at 86, 88, 90, 92, 94, and 96 °C was investigated. Microbial quality including total plate counts, yeast and molds, and heat-resistant molds counts was determined. Shelf life of pasteurized products stored for up to 24 weeks at room temperature were assessed for microbial quality, soluble solids (°Brix), titratable acidity (citric acid %), pH, viscosity (cP) and flow rate (cm/30 s). Our results indicated that heat-resistant molds, initially present in frozen blueberries with counts at 2.03 log CFU/200g, were totally inactivated at 94 to 96 °C with 1 to 2 min holding time. Shelf life study showed that no product spoilage was caused by bacteria, yeasts and heat-resistant molds along with non-significant changes of textural characteristics. This study provided useful information for the food industry to develop variety of fruit purée products with no wastes of fruit materials. PRACTICAL APPLICATION: This study provides useful information for the food industry to develop safe liquid food products using cavitation technology without wasting any raw materials.

Influence of culture media, pH and temperature on growth and bacteriocin production of bacteriocinogenic lactic acid bacteria.

There has been continued interest in bacteriocins research from an applied perspective as bacteriocins have potential to be used as natural preservative. Four bacteriocinogenic lactic acid bacteria (LAB) strains of Lactobacillus curvatus (Arla-10), Enterococcus faecium (JFR-1), Lactobacillus paracasei subsp. paracasei (JFR-5) and Streptococcus thermophilus (TSB-8) were previously isolated and identified in our lab. The objective of this study was to determine the optimal growth conditions for both LAB growth and bacteriocins production. In this study, various growth conditions including culture media (MRS and BHI), initial pH of culture media (4.5, 5.5, 6.2, 7.4 and 8.5), and incubation temperatures (20, 37 and 44 °C) were investigated for LAB growth measured as optical density (OD), bacteriocin activity determined as arbitrary unit and viability of LAB expressed as log CFU ml-1. Growth curves of the bacteriocinogenic LAB were generated using a Bioscreen C. Our results indicated that Arla-10, JFR-1, and JFR-5 strains grew well on both MRS and BHI media at growth temperature tested whereas TSB-8 strain, unable to grow at 20 °C. LAB growth was significantly affected by the initial pH of culture media (p < 0.001) and the optimal pH was found ranging from 6.2 to 8.5. Bacteriocin activity was significantly different in MRS versus BHI (p < 0.001), and the optimal condition for LAB to produce bacteriocins was determined in MRS broth, pH 6.2 at 37 °C. This study provides useful information on potential application of bacteriocinogenic LAB in food fermentation processes.

Antimicrobial activity of bacteriocin-producing lactic acid bacteria isolated from cheeses and yogurts.

The biopreservation of foods using bacteriocinogenic lactic acid bacteria (LAB) isolated directly from foods is an innovative approach. The objectives of this study were to isolate and identify bacteriocinogenic LAB from various cheeses and yogurts and evaluate their antimicrobial effects on selected spoilage and pathogenic microorganisms in vitro as well as on a food commodity.LAB were isolated using MRS and M17 media. The agar diffusion bioassay was used to screen for bacteriocin or bacteriocin-like substances (BLS) producing LAB using Lactobacillus sakei and Listeria innocua as indicator organisms. Out of 138 LAB isolates, 28 were found to inhibit these bacteria and were identified as strains of Enterococcus faecium, Streptococcus thermophilus, Lactobacillus casei and Lactobacillus sakei subsp. sakei using 16S rRNA gene sequencing. Eight isolates were tested for antimicrobial activity at 5°C and 20°C against L. innocua, Escherichia coli, Bacillus cereus, Pseudomonas fluorescens, Erwinia carotovora, and Leuconostoc mesenteroides subsp. mesenteroides using the agar diffusion bioassay, and also against Penicillium expansum, Botrytis cinerea and Monilinia frucitcola using the microdilution plate method. The effect of selected LAB strains on L. innocua inoculated onto fresh-cut onions was also investigated.Twenty percent of our isolates produced BLS inhibiting the growth of L. innocua and/or Lact. sakei. Organic acids and/or H2O2 produced by LAB and not the BLS had strong antimicrobial effects on all microorganisms tested with the exception of E. coli. Ent. faecium, Strep. thermophilus and Lact. casei effectively inhibited the growth of natural microflora and L. innocua inoculated onto fresh-cut onions. Bacteriocinogenic LAB present in cheeses and yogurts may have potential to be used as biopreservatives in foods.

Antimicrobial effects of a commercial fermentation product on fresh-cut onions.

The antimicrobial effects of a commercial fermentation product (CFP) added to fresh-cut onions was assessed. Diced onions without treatment were packed in sealed and vented single-pinhole polyethylene bags, which served as controls. Treated and control samples were then stored at 4 and 7°C. Changes in the microbial loads including total aerobic bacteria, yeasts and molds, and coliforms were determined. Quality parameters including O(2)-CO(2) concentration, pH, soluble solids, and conductivity were also evaluated following 0, 3, 7, 10, 14, and 18 days of storage. After 10 days of storage at 4°C, CFP-treated samples had significantly lower microbial loads (P < 0.001), and aerobic plate counts, yeast and mold counts, and coliform counts were lower by 2.5, 3.2, and 1.4 log, respectively, compared with the controls. The addition of 0.5 or 1% CFP maintained the shelf life of fresh-cut onions for up to 14 and 18 days, respectively, compared with 10 days for the control. In addition, it was determined that storage temperature played an important role in maintaining the quality of fresh-cut onions. Significant (P ≤ 0.05) differences in microbial loads and product quality were found between 4 and 7°C in all treatments.

Effects of ozone treatment on Botrytis cinerea and Sclerotinia sclerotiorum in relation to horticultural product quality.

Botrytis cinerea and Sclerotinia sclerotiorum are fungal pathogens that cause the decay of many fruits and vegetables. Ozone may be used as an antimicrobial agent to control the decay. The effect of gaseous ozone on spore viability of B. cinerea and mycelial growth of B. cinerea and S. sclerotiorum were investigated. Spore viability of B. cinerea was reduced by over 99.5% (P < 0.01) and height of the aerial mycelium was reduced from 4.7 mm in the control to less than 1 mm after exposure to 450 or 600 ppb ozone for 48 h at 20 degrees C. Sporulation of B. cinerea was also substantially inhibited by ozone treatments. However, ozone had no significant effect on mycelial growth of S. sclerotiorum in vitro. Decay and quality parameters including color, chlorophyll fluorescence (CF), and ozone injury were further assessed for various horticultural commodities (apple, grape, highbush blueberry, and carrot) treated with 450 ppb of ozone for 48 h at 20 degrees C over a period of 12 d. Lesion size and height of the aerial mycelium were significantly reduced by the ozone treatment on carrots inoculated with mycelial agar plugs of B. cinerea or S. sclerotiorum. Lesion size was also reduced on treated apples inoculated with 5 x 10(6) spores/mL of B. cinerea, and decay incidence of treated grapes was reduced. The 450 ppb ozone for 48 h treatment had no significant effect on color of carrots and apples or on CF of apples and grapes. Ozone, an environmentally sound antimicrobial agent, inactivates microorganisms through oxidization and residual ozone spontaneously decomposes to nontoxic products. It may be applied to fruits and vegetables to reduce decay and extend shelf life.

Effect of hexanal vapor on the growth of postharvest pathogens and fruit decay.

The effect of the natural volatile hexanal was studied as an antifungal agent on the major postharvest fungal pathogens Botrytis cinerea, Monilinia fructicola, Sclerotinia sclerotiorum, Alternaria alternata, and Colletotrichum gloeosporioides. The antifungal effect of hexanal vapor was dependent on concentration and treatment duration, but sensitivity of the pathogens varied. All spores of B. cinerea and M. fructicola were killed after exposure to 900 microL/L for 12 h at 20 degrees C, and almost all were killed after a 24-h exposure to 450 microL/L. Only moderate numbers of spores were killed at a concentration of 200 microL/L. Mycelial growth of S. sclerotiorum on agar was completely inhibited after a 12-h exposure to 900 microL/L, but only slight inhibition occurred at 450 microL/L and none at 200 microL/L. Mycelium of A. alternata and C. gloeosporioides appeared more sensitive, with strong inhibition occurring after a 12-h exposure at 450 microL/L. Similar trends in spore viability and mycelial growth were observed at 7 degrees C. The antifungal effect of hexanal vapor was further tested on raspberry fruit naturally infected with B. cinerea and on peach fruit inoculated with spores of M. fructicola. Decay was markedly reduced in raspberry and almost completely controlled in peach after exposure to 900 microL/L hexanal vapor for 24 h. The potential of hexanal for postharvest decay control is discussed.