GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production

The GABA shunt pathway involves three enzymes, glutamate decarboxylase (GAD), GABA aminotransferase (GAT) and succinate semialdehyde dehydrogenase (SSADH). These enzymes act in concert to convert glutamate (α‐ketoglutarate) to succinate. Deletion mutations in each of these genes in Saccharomyces cerevisiae resulted in growth defects at 45°C. Double and triple mutation constructs were compared for thermotolerance with the wild‐type and single mutant strains. Although wild‐type and all mutant strains were highly susceptible to brief heat stress at 50°C, a non‐lethal 30 min at 40°C temperature pretreatment induced tolerance of the wild‐type and all of the mutants to 50°C. The mutant strains collectively exhibited similar susceptibility at 45°C to the induced 50°C treatments. Intracellular reactive oxygen intermediate (ROI) accumulation was measured in wild‐type and each of the mutant strains. ROI accumulation in each of the mutants and in various stress conditions was correlated to heat susceptibility of the mutant strains. The addition of ROI scavenger N‐tert‐butyl‐α‐phenylnitrone (PBN) enhanced survival of the mutants and strongly inhibited the accumulation of ROI, but did not have significant effect on the wild‐type. Measurement of intracellular GABA, glutamate and α‐ketoglutarate during lethal heat exposure at 45°C showed higher levels of accumulation of GABA and α‐ketoglutarate in the uga1 and uga2 mutants, while glutamate accumulated at higher level in the gad1 mutant. These results suggest that the GABA shunt pathway plays a crucial role in protecting yeast cells from heat damage by restricting ROI production involving the flux of carbon from α‐ketoglutarate to succinate during heat stress. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparison of Selected Yersinia enterocolitica Indicator Tests for Potential Virulence

Three recently developed tests used to indicate virulence were performed on 34 strains of Yersinia enterocolitica. Agreement in virulence or nonvirulence prediction was found in (a) 29 of 31 strains when tested for calcium growth dependency at 35°C; (b) all 34 strains when tested for the ability to autoagglutinate in tissue culture medium at 35°C; and (c) among all 34 strains when tested for the presence of a 40–48 Mdal plasmid using a modification of the Hansen and Olsen method for plasmid analysis. The autoagglutination assay is recommended for testing potential pathogenicity of food-borne isolates. There are probably several virulence-determining factors for Y. enterocolitica, and no one in-vitro test will suffice. The analytical procedure described herein can be used to detect plasmids ranging in size from approximately 2 × 10⁶ to 3 × 10⁸ daltons.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16–1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M₁ replication defects of mak16–1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).     

Characterization of south african isolates of Salmonella enteritidis by phage typing, numerical analysis of RAPD-PCR banding patterns and plasmid profiles

Eleven of the 33 strains of Salmonella enteritidis (S.E.) included in this study belonged to phage type 34. Six strains belonged to phage type 14, six strains to phage type 4 and four strains to phage type 7. The remaining six strains belonged to phage types 35, 1, 24var (a variation of phage type 24), 9a, 1b and an unknown phage type. The majority of S.E. phage type 34 strains (eight of the 11) grouped at R2 > or =0.45 into one RAPD-PCR cluster with two strains of phage types 4, a strain of phage type 24var and a strain of phage type 9a, indicating that they consist of a genetically heterogeneous collection of strains. Two of the remaining three phage type 34 strains grouped into two different clusters, well separated from the other phage type 34 strains. One strain of phage type 34 was genetically diverse and did not cluster with any of the strains included in this study. Three of the phage type 14 strains grouped into cluster 11 at R2 > or =0.72, suggesting that they are genetically closely related. However, the remaining three strains of phage type 14 grouped into two separate clusters. Strains of phage types 7, 35, and 1 grouped in one cluster at R2 > or = 0.55. Our results clearly indicated that S.E. strains of the same phage type are not always genetically related. On the other hand, strains of a high genetic relatedness classified as different phage types. No specific plasmid profile could be linked to any of the phage types. Based on results obtained by LD50 virulence tests, strains containing the 38 MDa plasmid are more virulent compared to strains which do not contain the plasmid.     

Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast

Abstract: Campylobacter jejuni is the leading cause of bacterial diarrheal illness worldwide. Many strains are now becoming multidrug resistant. Apple‐based edible films containing carvacrol and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken. Retail chicken breast samples inoculated with D28a and H2a (resistant strains) and A24a (a sensitive strain) were wrapped in apple films containing cinnamaldehyde or carvacrol at 0.5%, 1.5%, and 3% concentrations, and then incubated at 4 or 23 °C for 72 h. Immediately after wrapping and at 72 h, samples were plated for enumeration of viable C. jejuni. The antimicrobial films exhibited dose‐ and temperature‐dependent bactericidal activity against all strains. Films with ≥1.5% cinnamaldehyde reduced populations of all strains to below detection at 23 °C at 72 h. At 4 °C with cinnamaldehyde, reductions were variable for all strains, ranging from 0.2 to 2.5 logs and 1.8 to 6.0 logs at 1.5% and 3.0%, respectively. Films with 3% carvacrol reduced populations of A24a and H2a to below detection, and D28a by 2.4 logs at 23 °C and 72 h. A 0.5‐log reduction was observed for both A24a and D28a, and 0.9 logs for H2a at 4 °C at 3% carvacrol. Reductions ranged from 1.1 to 1.9 logs and 0.4 to 1.2 logs with 1.5% and 0.5% carvacrol at 23 °C, respectively. The films with cinnamaldehyde were more effective than carvacrol films. Reductions at 23 °C were greater than those at 4 °C. Our results showed that antimicrobial apple films have the potential to reduce C. jejuni on chicken and therefore, the risk of campylobacteriosis. Possible mechanisms of antimicrobial effects are discussed. Practical Application: Apple antimicrobial films could potentially be used in retail food packaging to reduce C. jejuni commonly present on food.     

Thermoinducible Lysis of Temperature-Sensitive Streptococcus cremoris Strains

Temperature-sensitive Streptococcus cremoris SK11 appeared to harbor a temperate phage that was induced at the maximum cooking temperatures used in Cheddar cheese manufacture, i.e., 38 to 40°C. When incubated at 30°C in M17-lactose broth, followed by a shift to 40°C, lysis of the culture occurred within 2 h. Results were similar when S. cremoris SK11 was propagated in M17-lactose broth through a simultated Pearce activity test. In 11% solids reconstituted NDM, this strain also exhibited decreased cell numbers approximately 2 h after the temperature had reached 40°C in the Pearce activity test. To support the hypothesis that lysis of the cells was due to induction of phage, electron microscopy of the temperature-induced lysate revealed the presence of particles resembling phage heads and tails but only a few intact phage-like particles. Examination of other temperature-sensitive or temperature-insensitive strains of S. cremoris also suggested that lysis of temperature-sensitive strains occurred after cells were incubated at 40°C, but no lysis was observed for temperature-insensitive strains.     

Influence of Heating and Cooling Rates on Bacillus cereus Spore Survival and Growth in a Broth Medium and in Rice

Survival, spore germination, and growth of emetic and diarrheal type strains of Bacillus cereus were evaluated in broth and rice media during heating and cooling. Samples were heated to 80°C (20C°/hr or 40C°/hr) or 90°C (ca. 900C°/hr), prior to cooling to 10°C (5C°/hr or 10C°/hr). Following heating to 80°C, growth occurred during 5C°/hr cooling. After heating to 90°C, inactivation of three strains occurred during cooling from 90 to 80°C and again from 50 to 40°C. Great variability was observed among the responses of the four strains. Emetic strains exhibited greater survival than diarrheal strains. Rice reduced low temperature inactivation, and did not favor emetic strains. Significant two and three way interactions existed among media, strains, heating and cooling rates.     

Reduction of Listeria monocytogenes in cold‐smoked salmon by bacteriophage P100, nisin and lauric arginate,singly or in combinations

Three GRAS antimicrobials including, lauric arginate (LAE), bacteriophage P100 (phage P100) and bacteriocin nisin, were evaluated either singly or in combinations for the reduction of initial load of Listeria monocytogenes in cold‐smoked salmon (CSS). The stability of phage P100 in the presence of LAE (200 ppm) and nisin (500 ppm) or at 10× and 100× of these concentrations was determined at 4 °C or 30 °C for 24 h in a broth model. Phage P100 was found to be highly stable in the presence of these antimicrobial agents as plaque‐forming units (PFU) did not vary between control and antimicrobial‐treated phage. The survival of L. monocytogenes in the presence of phage P100, nisin and LAE showed remarkable reduction within 24 h both at 4 °C or 30 °C in broth. Treatment of CSS containing 3.5 log CFU cm^?2 L. monocytogenes with phage P100 (10⁸ PFU mL^?1), nisin (500 ppm) and LAE (200 ppm) showed strong listericidal action and reduced the L. monocytogenes by 2–3 log CFU cm^?2 after 24 h. Among the combined treatments, phage P100 + LAE or nisin + LAE exhibited the most listericidal action in which L. monocytogenes cells were reduced to undetectable level within 24 h in CSS.     

A Study on the Growth Potential of Staphylococcus aureus In Boletus edulis, A Wild Edible Mushroom, Prompted by a Food Poisoning Outbreak

A dish of Boletus edulis, a wild edible mushroom, in vinegar caused staphylococcal food poisoning in 13 of 35 diners in a restaurant. Enterotoxicosis was confirmed by detection of toxins A and D in the dish. Staphylococcus aureus growth potential in B. edulis was studied by inoculating fresh and frozen and thawed bolete with S. aureus strains VTTE 530, 757, 793 and 805 and storing for 3 days at 15 and/or 21°C. Essentially no staphylococcal growth was observed in frozen and thawed mushrooms contaminated with strains 530, 793 or 805 and stored at 15 or 21°C. In fresh B. edulis the same strains showed slight growth at 21°C. Frozen and thawed bolete inoculated with strains 530 and 757 (isolated from mushroom soup, nonenterotoxigenic) supported staphylococcal growth in 2 days at 21°C from a level of 4.8±10⁴ and 5.4±10³ to 2.0±10⁶ and 7.0±10₇ cfu/g, respectively. Enterotoxin was not detected in these samples.     

Thermal inactivation of the heat-resistant Lactococcus lactis bacteriophage P680 in modern cheese processing

The thermal inactivation of the highly thermo-resistant test phage (P680), was investigated in whey, whey protein concentrate (WPC) and whey cream. After a heat treatment at 90 °C for 15 min, only a 6-log reduction was obtained and the phage was still detectable in each medium. Kinetic parameters for the inactivation of the phage were calculated for temperatures ranging from 70 to 90 °C using a non-linear model. With the help of the parameters obtained, the lines of equal effects showing a 9-log inactivation of the phage were calculated. High temperature short time pasteurization was not sufficient for 9-log inactivation of phage P680 in skim milk, whey, WPC or whey cream. Temperature and time combinations ranging from 100 °C for 20 min to 140 °C for 2 s are necessary for a 9-log inactivation of P680.     

Extraordinary Heat Resistance of Talaromyces flavus and Neosartorya fischeri Ascospores in Fruit Products

Ascospores of three strains each of Talaromyces flavus, Neosartorya fischeri and Byssochlamys fulva/nivea were analyzed for resistance to thermal inactivation in five fruit-based (blueberry, cherry, peach, raspberry and strawberry) products. D_91°C values for two strains of T. flavus ranged from 2.9–5.4 min; D_88°C values ranged from 7.1–22.3 min. Ascospores of N. fischeri were somewhat less heat resistant; D_91°C values were < 2.0 min and D_88°C were 4.2–16.2 min. Ascospores of Byssochlamys spp. were considerably less heat resistant. The type of fruit product did not appear to substantially influence rates of thermal inactivation. No heat-resistant ascospores of T. flavus or N. fischeri, i.e., ascospores capable of surviving 15 min at 75°C, were formed on fruit products stored at 10°C for 137 days. However, T. flavus and N. fischeri formed ascospores on cherry substrate stored at 25°C within 65 and 137 days, respectively, that survived 15 min at 88°C.     

Growth and Toxin Production by Clostridium botulinum in Model Acldified Systems

TPGY medium was acidified to pH 4.20, 4.60, 5.00 and 5.40 with acetic or citric acid. The media were inoculated with spores from three strains of C. botulinum type A, B or E. Growth, pH, and toxin production under anaerobic conditions were monitored for 8 wk. Spores of C. botulinum types A and B were incapable of outgrowth and toxin production at pH 4.60 or below when incubated at 35°C. Spores of C. botulinum type E were capable of growth and toxin production at 26°C in citric acid acidified systems at pH 4.20. Growth and toxin production were not detected below pH 5.00 when acetic acid was used.     

Influence of Temperature on Germination and Growth of Spores of Emetic and Diarrheal Strains of Bacillus cereus in a Broth Medium and in Rice

Germination and growth of Bacillus cereus spores from emetic and diarrheal strains were measured in Trypticase soy broth (TSB) and in autoclaved rice/beef extract from 5°– 55°C. Growth for some strains occurred from 15°– 50°C, and little difference was noted between responses of diarrheal and emetic types or between media, except a higher maximum population was achieved in rice. Germination was more extensive in rice than in TSB at <15°C and was generally more extensive for diarrheal strains in either medium.     

Differences in thermotolerance of variousEscherichia coliO157:H7 strains in a salami matrix

Three strains ofEscherichia coliO157:H7 (ATCC 43895, Ent C9490 and 380–94) were inoculated into salami and heated in water baths at 50, 55 or 60°C. At intervals between 1 and 360 min, salami samples were removed from the water bath and examined for the presence of survivingE. coliO157:H7. Samples were directly plated onto sorbitol MacConkey (SMAC) agar, and onto tryptone soya agar (TSA) with SMAC overlay. The number of sub-lethally damaged cells in each sample was estimated from the differences between the resultant direct (uninjured cells only) and overlay (total recovery) counts. In samples heated at 50°C, the percentage of cell injury ranged from 71·8–88% for all strains. In samples heated at 55°C the percentage of sub-lethally damaged cells in strains ATCC 43895 and Ent C9490 was significantly higher (P< 0·001) at 97% than that observed in strain 380–94 (64%). Cell injury was not measured at 60°C. There were significant differences between the derived decimal reduction times (D-values) related to the different strains ofE. coliO157:H7, the heat treatment applied and the recovery/enumeration agars used. Significant interstrain differences (P< 0·05) in thermotolerance were noted. Strain Ent C9490 was significantly more heat resistant at 50°C and 60°C (D-values of 116·9 and 2·2 min, respectively), while at 55°C strain 380–94 was more thermotolerant (D-value of 21·9 min). The implications of these findings for the design of studies investigating the heat resistance ofE. coliO157:H7 in fermented meat environments are discussed.     

Practical application of bacteriophage in food manufacturing facilities for the control of Listeria sp.

Listeria monocytogenes is a foodborne pathogen with the ability to persist and form biofilm matrices in processing environments of food manufacturing facilities. Bacteriophages are bacterial viruses with host specific lethality. Published research on the application of phage to control Listeria sp. in manufacturing environments is limited. In this study, we have assessed the capacity of bacteriophage P100 (Listex™) to reduce incidence of Listeria sp. in the ready-to-eat (RTE) environment of refrigerated (4°C) and ambient (20°C) temperature facilities using two different application strategies. A moderate application applied as a single treatment every 24 hr over three days (2 × 10⁷ PFU/ml) and an intensified application applied once every 6 hr over a 24 hr period (1 × 10⁸ PFU/ml). Environmental nonfood contact surface (NFCS) samples were collected and analyzed for the presence of Listeria sp. before and after treatment. When the moderate treatment protocol was applied the incidence of positives decreased from 51.3 to 17.5% in the 4°C environment and from 67.5 to 23.1% in the 20°C production area. For the intensified phage treatment method, the initial positive rate in the 4°C environment ranged from 5 to 47.5%, with an overall 43% reduction in Listeria sp. In the 20°C facility, initial environmental Listeria sp. ranged from 15 to 50%, with an overall reduction of 32% after treatment with phage P100. Data indicate the application of Listeria specific phage P100 in RTE food production environments by either the moderate or intensified application method can reduce incidence and be considered an additional intervention strategy for controlling this pathogen on NFCS.     

Alteration of Talaromyces flavus Heat Resistance by Growth Conditions and Heating Medium Composition

A strain of Talaromyces flavus var. macrosporus (NRRL 13641) was isolated from spoiled fruit drink and identified. It had a heat resistance (l/k) (death rate constant or decimal reduction time) of 191 min at 80°C and 6 min at 90°C with a calculated z of 6.7°C. Several strains of T. flavus were tested to determine the effects on heat resistance of environmental conditions such as pH, organic acids, brix, and type of medium. Heat resistance increased with increased brix in the heating medium. Growth on solid medium compared with liquid medium resulted in greater heat resistance. Growth at 20°C and 35°C produced ascospores with higher heat resistance than growth at 25°C while those produced at 30°C had intermediate heat resistance. Five cultures differed significantly in heat resistance.     

Isolation and characterisation of lytic bacteriophages against Pseudomonas spp., a novel biological intervention for preventing spoilage of raw milk

Thirteen novel lytic bacteriophages against 13 Pseudomonas strains were isolated from local sewage and initially identified by morphology using a transmission electron microscope. PP1 and PP5 were identified as Pedoviridae and Cystoviridae, respectively; while the other 11 phages were identified as Leviviridae. Most phages showed high infectivity at either 4 °C or 25 °C, and the optimum pH range for phage infectivity was pH 5–7. A strong antimicrobial effect of the phage cocktail was evidenced by a 2-log reduction in Pseudomonas cell number of UHT milk inoculated with Pseudomonas, and a 1-log reduction in the psychrotrophic bacteria and total bacteria counts of raw milk at 4 °C over 5 d. A similar result was obtained at 25 °C over 8 h. Results indicated that the 13 phages with different morphological and physiological characteristics may have a potential application as biological preservative agent in raw milk.     

Bacteriocidal and Mutagenic Effects of Ozone on Shrimp(Penaeus monodon) Meat

We examined solubility and stability of ozone in shrimp-meat extract (SME), bacteriocidal effect of ozone on shrimp-meat microorganisms, mutagenicity of ozonated shrimp meat, and ozonolysis of DNA. The saturated concentration (1.4 mg-O₃/L) of ozone in SME was lower than in 2% saline or distilled water at 5 and 25°C. Upon standing for 25 min after ozonation, ozone exhibited the same decomposition rate (2.7%/min) in 5 and 25°C SME. Among 9 bacterial strains tested, Salmonella typhimurium was more resistant to ozone in shrimp meat. Mutagen was not detected in shrimp meat when it was ozonated in saline. Ozone in saline (less than 5 mg-O₃/L) could lyse M13 RF DNA in Escherichia coli and single-stranded DNA in phage M13 outside bacterial cell, within 30 min.     

Influence of Homofermentative Lactobacilli on Physicochemical and Sensory Properties of Cheddar Cheese

Cheddar cheeses were produced under pilot plant conditions using a commercial Streptococcus culture amended with one of 10 homofermentative Lactobacillus strains. During the ripening period, pH, acidity, salt, moisture, fat, texture, fissure formation, gas development and sensory status were evaluated. Lactobacillus treated cheese did not differ much from the control in pH and acidity but acidity increased substantially after draining and cheddaring. Lactobacillus numbers increased at all stages as compared with the uninoculated control. High quality Cheddar cheese was produced by L. casei-subsp-casei (119-10/62) and L. casei-subsp-pseudoplantarum (137-10/62) from 7 to 12 vats aged for 2 months at 15°C and for a further 10 months at 7°C or 15°C. Fissure formation was observed in cheese made with L. casei-subsp-rhamnosus, one of the four cultures of L. casei-subsp-casei (LH13) and two of the three strains of L. casei-subsp-pseudoplantarum (83-4-12/62 and L3E). Certain Lactobacillus strains produced cheese with slight flavor defects. Other strains, in particular L. casei-subsp-rhamnosus, contributed to high acidity (72 - 0.89° domic) and low pH (5.2) at salting.     

Lipolytic activities of two strains of Aspergillus aculeatus associated with the spoilage of two food sources

The production of extracellular lipolytic enzymes by 2 strains of Aspergillus aculearus associated with the deterioration of peanuts and a marketed margarine in Nigeria and the effects of temperature and pH on the activities of the enzymes were investigated. Lipolytic enzymes were detected within 2 days of incubation at 35 °C in 4 out of 6 natural oils and in 3 out of 4 synthetic glycerides used irrespective of strain type. The lipolytic enzymes of both strains hydrolysed both natural oils and synthetic glycerides to free fatty acids. There was correlation between the mycelium produced to the quantity of free fatty acids produced in medium within the first 8 days of incubation. There was a general increase in mycelium production with increase in incubation period. The enzyme activities were at peak at 30-35 °C and at pH 6-7.