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  • Bacterial and fungal diversity in Laphet, traditional fermented tea leaves in Myanmar, analyzed by culturing, DNA amplicon-based sequencing, and PCR-DGGE methods
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-16
    Bo Bo; Seul-Ah Kim; Nam Soo Han

    Laphet is a traditional fermented food in Myanmar, made from tea leaves (Camellia sinensis) under anaerobic condition. We performed microbial diversity analyses on 14 Laphet products collected from different locations in Myanmar. Amplicon-based sequencing results revealed Lactobacillus and Acetobacter were abundant bacteria and Candida, Pichia, Cyberlindnera, and Debaryomyces were abundant yeast. Using selective media, eight species of lactic acid bacteria and nine species of yeast were isolated; Lactobacillus plantarum and L. collinoides were dominant bacteria and Pichia manshurica, Candida boidinii, and Cyberlindnera jadinii were major yeasts. PCR-DGGE analysis confirmed that most of the dominant bacterial and yeast species found in culture dependent analysis were present in Laphet samples. Microbial diversity and pH of Laphet were different between samples from tea plantation area and local markets due to possible differences in incubation time periods. When tannase activity was tested, 25 among 29 bacterial isolates and two among 36 yeast isolates showed positive activities. These findings provide new insights into microbial diversity of Laphet and increased our understanding of the core bacterial and yeast species involved in the manufacture of Laphet.

  • Inactivation by osmotic dehydration and air drying of Salmonella, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, hepatitis A virus and selected surrogates on blueberries
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-15
    Xi Bai; Matteo Campagnoli; Sophie Butot; Thierry Putallaz; Lise Michot; Sophie Zuber

    Osmotically dehydrated and air dried berry fruits are used as ingredients for the production of yoghurts, chocolates, cereal bars and mixes, ice creams and cakes and are often subjected to mild thermal treatments only, posing questions around their microbiological safety. As osmotic dehydration methods and parameters vary considerably within the industry and minimally processed high quality fruits are increasingly sought, the scope of this study was to determine which temperatures are required for the inactivation of relevant bacteria and viruses during osmotic dehydration of berries, using blueberries as a model berry in a thawed state to mimic common industrial practices. Additionally, we studied the inactivation of osmotic dehydration at 23 °C, sometimes referred to “cold infusion” followed by air drying at 100 °C to determine the microbiological safety achieved by this combined treatment. Four pathogens (Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes and hepatitis A virus (HAV)) and five surrogates (Enterococcus faecium, Escherichia coli P1, Listeria innocua, murine Norovirus (MNV) and bacteriophage MS2) were inoculated on blueberries and reductions were measured after different treatment combinations. After osmotic dehydration of bacterial strains at 40 °C no survivors were detected on blueberries, with the exception of E. faecium. Inactivation of the viruses at 45 °C showed no survivors for MS2 and mean reductions of 1.5 and 3.4 log10 median tissue culture infectious dose (TCID50)/g for HAV and MNV, respectively. Similarly, in the sugar solution at 40 °C, no survivors were observed, with the exception of E. faecium and the three viruses. The combined process (osmotic dehydration at 23 °C followed by air-drying at 100 °C) achieved an >6 log reduction of all tested bacterial strains and MS2. For HAV and MNV, 2.6 and >3.4 log10 TCID50/g were measured. In summary, the present study shows that osmotic dehydration appears an efficient control measure for the control of L. monocytogenes, S. enterica and E. coli O157:H7 if carried out at 40 °C or at 23 °C and followed by air-drying at 100 °C. Based on the results generated with MNV, the combined treatment is also expected to reduce human Norovirus (NoV) but does not appear to be sufficient to fully control HAV. The results contribute to a better management of the microbial safety of osmotically dehydrated and dried berries and especially the results generated for the viruses emphasize that within a robust food safety management system, safety must be assured through the entire food supply chain and therefore must start at primary production with the implementation of Good Agricultural Practices (GAP).

  • Development and evaluation of pullulan-based composite antimicrobial films (CAF) incorporated with nisin, thymol and lauric arginate to reduce foodborne pathogens associated with muscle foods
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-15
    Abdelrahim H.A. Hassan; Catherine N. Cutter

    A novel composite antimicrobial film (CAF), made from a pullulan-based biopolymer and polyethylene (PE) was developed and evaluated for controlling pathogens associated with muscle foods. Initially, CAFs were developed by incorporating thymol (T), nisin (N) and/or lauric arginate (LAE) into the pullulan layer and layering it on top of PE. The antimicrobial activity of the resulting CAFs was evaluated against cocktails of Shiga toxin-producing E. coli (STEC), Salmonella spp., Listeria monocytogenes (L. monocytogenes) and Staphylococcus aureus (S. aureus) in disk diffusion assays (DDAs). CAFs containing N were ineffective, while those containing T were effective for inhibiting the pathogens in DDAs. However, CAFs made with them did not exhibit desirable physical and mechanical properties since solvents (HCl and ethanol, respectively) interfered with the binding of pullulan to PE. Conversely, CAFs made with 0.5, 1 and 2.5% LAE maintained proper physical and mechanical characteristics and inhibited the four bacterial pathogens in DDAs. Based on these preliminary results, cocktails consisting of approximately 8 log10 CFU/ml of STEC, Salmonella, L. monocytogenes, or S. aureus were experimentally-inoculated onto raw beef, raw chicken breast, or ready-to-eat (RTE) turkey breast to obtain approximately 6.6 log10 CFU/cm2, aseptically transferred to CAFs containing 0.5, 1, or 2.5% LAE that were made into sachets/bags, vacuum packaged, sealed, and remaining microbial populations determined up to 28 days of refrigerated storage (4 °C). By day 28, CAFs containing 0.5, 1, and 2.5% LAE reduced: STEC by 1.13, 1.33 and 2.88 log10 CFU/cm2 respectively, on raw beef; Salmonella by 2.03, 2.12 and 3.01 log10 CFU/cm2 respectively, on raw chicken breast; L. monocytogenes by 1.12, 1.81 and 3.56 log10 CFU/cm2 respectively, on RTE turkey breast; and S. aureus by 0.68, 2.02 and 3.43 log10 CFU/cm2, respectively, on RTE turkey breast. CAFs may be of interest to the meat and poultry industry to control foodborne pathogens associated with these food products.

  • Genome based safety assessment for Bacillus coagulans strain LBSC (DSM 17654) for probiotic application
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-13
    Dina B. Saroj; Anil K. Gupta

    The present study on Bacillus coagulans strain LBSC (DSM 17654) describes the use of whole genome sequencing, in correlation with the phenotypic properties to assess the safety of the strain. Analysis of the 16S rRNA sequence of the B. coagulans strain LBSC (DSM 17654), showed 100% homology with 99% coverage with B. coagulans strain HM-08. BLAT (BLAST Like Analysis Tool) analysis for whole genome comparison with B. coagulans ATCC 7050, B. coagulans HM-08 and B. coagulans Slac showed 96%, 99% and 99% sequence identity respectively. Whole genome sequencing results demonstrated a single scaffold of 36,35,902 bp and 3331 coding sequences. Gene ontology segregated the proteins as those with molecular function, cellular component and biological process of the predicted genes from assembled genome. Risk associated sequences like antibiotic resistance genes, biogenic amine producing genes, virulence factor genes and other safety related genes were identified with focus on horizontal gene transfer and its non-functionality. The absence of mobile elements in the vicinity of the genes, render it non-transferable and non-toxic phenotypic properties confirm the non-functionality of the genes. Absence of functional genes of concern and confirmation of absence of mobile elements in the vicinity of other non-clinically significant genes indicated no safety concern. The absence of complete and functional prophage sequences which are deleterious for the genome stability and presence of CRISPR system which are advantageous for genome stability by acting as a barrier to entry of foreign DNA elements indicated the stability of the genome. The molecular approach used in this study satisfies the requirements for the safety assessment of the probiotic strain which could indicate it to be potentially safe.

  • Use of yeasts from different environments for the control of Penicillium expansum on table grapes at storage temperature
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-11
    L.A. Rodríguez Assaf; L.P. Pedrozo; M.C. Nally; V.M. Pesce; M.E. Toro; L.I. Castellanos de Figueroa; F. Vazquez

    A wide range of fresh fruits and vegetables is attacked by Penicillium species causing diseases during their postharvest handling. Many of these species are psychrotrophic and they are able to cause food spoilage at refrigeration temperature as happens with table grapes. After the harvest, grape bunches are stored inside boxes with SO2 generator pads to reduce the contamination with fungal conidia. However, SO2 residues are dangerous to people allergic to sulfites and they negatively affect the quality of fresh fruit. Biological control of phytopathogens with microbial antagonists naturally present on fruit surfaces could be helpful against postharvest diseases. The present study aimed to select native yeasts isolated from fermentation microenvironments and the surface of refrigerated grapes for their use in the biological control of P. expansum on table grapes stored in cold rooms. Non-pathogenic and pathogenic Penicillium species were isolated, and the four most aggressive pathogen isolates were identified as Penicillium expansum. Twenty yeast isolates identified as Aureobasidium pullulans, Cryptococcus magnus, Metschnikowia pulcherrima and Rhodotorula glutinis presented positive antagonistic activity against Penicillium expansum; they controlled the development of at least one of the fungi, significantly reducing the disease incidence. The results showed that three antagonistic yeasts (M. pulcherrima 22, 36 and 43) reduced the disease incidence and severity of all 4 P. expansum isolates. It was also found that the fruit surface is not the only source for isolation of biological control agents. Microenvironments with different stress conditions could be a promising source to isolate antagonistic microorganisms.

  • Inhibition of Cronobacter sakazakii in reconstituted infant formula using triglycerol monolaurate and its effect on the sensory properties of infant formula
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-10
    Song Zhang; Jian Xiong; Wenyong Lou; Zhengxiang Ning; Denghui Zhang; Jiguo Yang

    Cronobacter sakazakii (C. sakazakii) is an opportunistic foodborne pathogen in infant formula. This study was designed to explore the inhibitory effect of TGML on C. sakazakii in reconstituted infant formula (RIF). Firstly, the growth curve of C. sakazakii in RIF treated by TGML and the effect of different temperatures (4, 10, 21, 30 and 37 °C), pH values (5, 6, 7, 8 and 9) and ionic strengths (25, 50, 100, 200, 400 and 800 mM) on its activity were assessed. The results showed that the inhibitory effect of TGML on C. sakazakii was dose-dependent, and 1, 2 and 5 μg/mL TGML delayed the visible growth of pathogen by 4, 12 and 24 h, respectively. Storage temperature above or below room temperature enhanced the bioactivity of TGML. And a decrease in pH also increased the antibacterial effect of TGML. However, the effect of ionic strength on its activity was not obvious. Subsequently, the antibacterial effect of TGML in physiological gastric acid and simulated gastric juice in vitro was further explored. We found that only 5 μg/mL TGML could inhibit the growth of pathogen below the infectious dose (10,000 CFU in total) in simulated gastric juice during the whole gastric emptying period (3.5–21 h), weaker than its antibacterial effect in physiological gastric acid and room temperature culture. Finally, the effect of TGML and the above environmental factors on the color and aroma of infant milk was evaluated by a 12-person panel. The results revealed that TGML did not affect the sensory flavor of milk, and the color and odor scores of infant milk under different environmental conditions did not show any significant differences. Therefore, it is concluded that TGML has a good inhibitory effect on C. sakazakii in RIF and a high sensory acceptability for consumers. Adjusting the temperature or lowering the pH enhances its bacteriostatic activity. However, the presence of infant gastric juice can impair the bioactivity of TGML. Overall, this study will provide some new ideas for controlling and eliminating the potential risk of C. sakazakii infection during infant feeding.

  • Assessment of the microbiological quality and safety of marinated chicken products from Greek retail outlets
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-09
    Anastasia E. Lytou; Chrissanthi T. Renieri; Agapi I. Doulgeraki; George-John E. Nychas; Efstathios Z. Panagou

    The prevalence of three pathogens in marinated chicken products and the evaluation of their quality by microbiological and sensory analysis were assessed. Eighty (80) samples obtained from several meat retail markets in Greece were analyzed for the presence of Campylobacter spp., Salmonella and Listeria monocytogenes. Concerning Campylobacter, rep-PCR and species specific PCR were applied for the differentiation and identification of isolates, respectively. The samples were subsequently stored aerobically at 4 °C for 5 days. Microbiological analysis, sensory assessment and HPLC analysis were carried out for the evaluation of spoilage microorganisms, sensory quality and the presence of preservatives (potassium sorbate and sodium benzoate). Τhe prevalence of Campylobacter spp., Salmonella, and Listeria monocytogenes was 50%, 11% and 44%, respectively. In the case of Campylobacter, from a total of 40 isolates, 27 were identified as Campylobacter coli, 4 as Campylobacter jejuni, whereas the remaining 9 belonged to unidentified Campylobacter species. Pseudomonas spp. was the dominant spoilage microbial genus in 43% of the samples, while in 31% of them a co-dominance of Pseudomonas spp. and Brochothrix thermosphacta was observed. Total aerobic counts increased to 7.0 log CFU/g at the 1st, 2nd or 3rd day of storage in 71% of the samples, while sensory analysis showed that 80% of the samples were characterized as spoiled after 3, 4 or 5 days. The presence of preservatives was confirmed in 31% of the samples and slightly affected the microbiological profile. In conclusion, the obtained data demonstrated the occurrence of foodborne pathogens and allowed the acquisition of an overall view about the microbiological quality of marinated chicken products.

  • High prevalence of multidrug resistant S. aureus-CC398 and frequent detection of enterotoxin genes among non-CC398 S. aureus from pig-derived food in Spain
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-08
    Olouwafemi Mistourath Mama; Liliana Morales; Laura Ruiz-Ripa; Myriam Zarazaga; Carmen Torres

    Methicillin-resistant Staphylococcus aureus (MRSA) CC398 is a livestock-associated (LA) lineage, mainly detected in swine. Its dissemination via the food-chain could be a food-safety issue. This work aimed to study the diversity of S. aureus lineages in pork-products, to determine the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) of lineage CC398, and to study the antimicrobial resistance phenotype/genotype and the virulence traits of recovered isolates. One hundred and one samples of pig-derived food were collected in Northern Spain for S. aureus isolation. Antibiotic resistance profile was analysed, and associated resistance genes were screened by PCR. Detection of CC398 lineage, spa-type, multilocus sequence-type (ST), virulence factors, immune evasion cluster (IEC) genes, and phage ΦSa3 integrase was performed by PCR/sequencing. The prevalence of S. aureus and MRSA among pig-derived food was 33.6% and 21.8%, respectively. Thirty-nine S. aureus isolates were recovered and attributed to 19 spa-types and 12 STs, ST398 being the predominant lineage (n = 25; 64%). MRSA-CC398 isolates (n = 23) were mainly spa-t011 (n = 16) and 82.6% were multidrug-resistant (MDR). All MRSA-CC398 were tetracycline-resistant and IEC-negative and four hosted either eta, tst or sea gene. The two MSSA-CC398 isolates detected were spa-t5452, IEC-positive, and were resistant to penicillin (blaZ) and erythromycin/clindamycin (inducible) (ermT with/without ermC + msrA). Among the 14 non-CC398 isolates, only two were MRSA (ST8, PVL-positive, enterotoxin-positive, IEC-negative). The 12 isolates MSSA included two of CC45 IEC-positive. CC398 lineage is prevalent among S. aureus of pig-derived food (both MRSA and MSSA), LA-MRSA-CC398/t011 being the clone most represented. The presence of the IEC-positive MSSA-CC398 and MSSA-CC45 in food products highlights the potential implication of handlers in transmission of foodborne pathogens. Moreover, given the high frequency of MDR isolates and virulence genes detected, hygienic practices should be improved to limit the dissemination risk of S. aureus via the food chain.

  • Identification of antimicrobial resistant bacteria from plant-based food products imported into Canada
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-08
    Dongyun Jung; Joseph E. Rubin

    The role of plant-based foods in the epidemiology of antimicrobial resistance has been inadequately studied. In this investigation, resistant organisms from vegetables, fruits and spices imported into Canada were identified and characterized. A total of 143 products imported from primarily Asian and African countries were purchased from international markets in Saskatoon, Saskatchewan. Samples were selectively cultured for bacterial species where resistance is known to be emerging. The proportions of samples positive for each organism were as follows: E. coli (n = 13, 9.1%), Salmonella spp. (n = 2, 1.4%), ESBL producing Enterobacter spp. (n = 2, 1.4%) and K. pneumoniae (n = 2, 1.4%), S. aureus (n = 7, 4.9%) and Enterococcus spp. (n = 66, 46.2%). Antimicrobial minimum inhibitory concentrations were determined by broth micro-dilution and agar-dilution. Based on the susceptibility of each organism, isolates were screened for resistance genes (β-lactamases and plasmid mediated quinolones resistance determinants) by PCR. Extended-spectrum β-lactamase producing Enterobacteriaceae and methicillin resistant S. aureus (MRSA) were identified from 6/143 (4.2%) and 2/143 (1.4%) of samples respectively. The qnrB, qnrS and aac(6′)-Ib-cr plasmid mediated quinolone resistance determinants were identified in 2/143 (1.4%) of samples tested. None of the Enterobacteriaceae isolates were resistant to meropenem or colistin. Similarly, all Enterococcus isolates remained susceptible to ampicillin, penicillin and vancomycin. Finding multi-drug resistant bacteria which are frequently isolated from human infections is concerning, although the contribution of the global food trade to the dissemination of resistance remains cryptic. These results suggest that imported plant-based foods may be an underappreciated source of clinically relevant resistant organisms. Further study is required to address these gaps in our understanding of the epidemiology of resistance, and the magnitude of the risk posed to human health by these organisms.

  • Detection of hepatitis E virus (rabbit genotype) in farmed rabbits entering the food chain
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-03
    Ewelina Bigoraj; Iwona Kozyra; Ewa Kwit; Artur Rzeżutka

    Hepatitis E virus (HEV) infects humans and many animal species. The rabbit HEV has been found in farmed, wild and pet rabbits as well as in human patients suggesting zoonotic transmission. Although the routes of human infection with rabbit strains are unclear a foodborne transmission is suggested especially when asymptomatically infected animals could enter the food chain. The aims of the study were an evaluation of the prevalence of HEV infections in slaughtered rabbits, identification of the virus genotype(s) and assessment of their genetic relatedness to other zoonotic HEV strains. A pair of blood and liver samples (n = 482) were collected from meat rabbits of different breeds slaughtered at the age of 2.8 to 6 months. The animals originated from 20 small-scale and 4 large-scale commercial farms operating in Poland. The presence of anti-HEV antibodies in animals was detected by the use of a recomWell HEV IgG (human) ELISA kit (Mikrogen Diagnostik) adapted to rabbit sera. The isolation of HEV and sample process control virus (feline calicivirus) RNA from homogenates of liver destined for food and virus-positive sera was performed using a QIAamp® Viral RNA Mini Kit (Qiagen). A one-step real-time reverse transcription PCR method containing a target-specific internal amplification control was used for detection of HEV. The (sub)genotype of detected rabbit HEV strains was identified based on sequence analysis of the ORF2 and ORF2/3 virus genome fragments. Anti-HEV antibodies were detected in 29 (6%) out of 482 rabbit sera samples collected from animals raised only on the small-scale rabbit farms. Four sera were also positive for HEV RNA. Viral RNA was detected in 72 (14.9%) animal livers. Analysing ELISA and PCR results using Student's t-test, there were significant differences observed in the frequency of HEV infections between rabbits from small-scale and commercial farms (t = 2.675, p = 0.015 < 0.05 for ELISA and t = 2.705, p = 0.014 < 0.05 for PCR). All detected virus strains were identified as HEV gt3 ra subtype. The results of this study provide data on the occurrence of HEV infections in rabbits entering the food chain, suggesting that a risk of foodborne HEV infection due to consumption of contaminated meat and liver exists. In this light, the presence of rabbit HEV in food animals is pertinent as an issue of food safety and the surveillance of these animals for emerging or re-emerging viruses.

  • Inheritance of winemaking stress factors tolerance in Saccharomyces uvarum/S. eubayanus × S. cerevisiae artificial hybrids
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-03
    Origone Andrea Cecilia; González Flores Melisa; Rodríguez María Eugenia; Querol Amparo; Lopes Christian Ariel

    Stress has been defined as any environmental factor that impairs the growth of a living organism. High concentrations of ethanol, sugars and SO2 as well as temperature variations occurring during winemaking processes are some recognized stress factors that yeasts must overcome in order to avoid stuck or sluggish fermentations. At least two of these factors -sugar and ethanol concentrations- are strongly influenced by the global warming, which become them a worry for the future years in the winemaking industry. One of the most interesting strategies to face this complex situation is the generation of hybrids possessing, in a single yeast strain, a broader range of stress factors tolerance than their parents. In the present study, we evaluated four artificial hybrids generated with S. cerevisiae, S. uvarum and S. eubayanus using a non-GMO-generating method, in their tolerance to a set of winemaking stress factors. Their capacity to overcome specific artificial winemaking situations associated with global warming was also analyzed. All four hybrids were able to grow in a wider temperature range (8–37 °C) than their parents. Hybrids showed intermediate tolerance to higher ethanol, sugar and sulphite concentrations than their parents. Additionally, the hybrids showed an excellent fermentative behaviour in musts containing high fructose concentrations at low temperature as well as under a condition mimicking a stuck fermentation.

  • Galactomyces geotrichum mold isolated from a traditional fried cottage cheese produced omega-3 fatty acids
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2020-01-02
    Anna Grygier; Kamila Myszka; Wojciech Juzwa; Wojciech Białas; Magdalena Rudzińska

    Thirty nine strains of Galactomyces geotrichum molds were isolated from a traditional fried cottage cheese and production of polyunsaturated fatty acids (PUFA) was assessed. Among them eleven strains produced an extracellular lipids enriched in n-6 and n-3 PUFA. The extracellular lipids produced by G. geotrichum strain 38 contained the highest amounts of total PUFA (24.3%), with the highest contribution of n-3 fatty acids (17.9%), where α-linolenic, eicosapentaenoic, docosapentaenoic and docosahexaenoic acids were the main contributors. To obtain maximal production of PUFA, composition of the medium consisted of 10 g/L rapeseed oil, 5 g/L yeast extract, 0.05 g/L K2HPO4, 0.17 g/L MgSO4, 0.015 g/L MnSO4, 0.015 g/L ZnSO4, 0.05 g/L FeSO4, and 10 mg/L vitamin B12. The optimal growth conditions at 30 °C involve: aeration at 1.5 vvm (volume of air per volume of broth per minute) at pH 6.5. The cheese produced under described conditions contained higher amount of n-3 PUFA (0.25 mg/g cheese) in comparison to control (0.01 mg/g). α-Linolenic acid predominated among n-3 fatty acids. Galactomyces geotrichum is a natural microflora of dairy products, and could be used to enrich food/cheese in deficient omega-3 lipids.

  • Different carbon sources result in differential activation of sigma B and stress resistance in Listeria monocytogenes
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-30
    Natalia Crespo Tapia; Amber L. Dorey; Cormac G.M. Gahan; Heidy M.W. den Besten; Conor P. O'Byrne; Tjakko Abee

    Listeria monocytogenes is an important food-borne pathogen that is ubiquitous in the environment. It is able to utilize a variety of carbon sources, to produce biofilms on food-processing surfaces and to survive food preservation–associated stresses. In this study, we investigated the effect of three common carbon sources, namely glucose, glycerol and lactose, on growth and activation of the general stress response Sigma factor, SigB, and corresponding phenotypes including stress resistance. A fluorescent reporter coupled to the promoter of lmo2230, a highly SigB-dependent gene, was used to determine SigB activation via quantitative fluorescence spectroscopy. This approach, combined with Western blotting and fluorescence microscopy, showed the highest SigB activation in lactose grown cells and lowest in glucose grown cells. In line with this observation, lactose grown cells showed the highest resistance to lethal heat and acid stress, the highest biofilm formation, and had the highest adhesion/invasion capacity in Caco-2-derived C2Bbe1 cell lines. Our data suggest that lactose utilisation triggers a strong SigB dependent stress response and this may have implications for the resistance of L. monocytogenes along the food chain.

  • Transmission of antimicrobial resistant non-O157 Escherichia coli at the interface of animal-fresh produce in sustainable farming environments
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-30
    Ayanna Glaize; Eduardo Gutierrez-Rodriguez; Irene Hanning; Sandra Díaz-Sánchez; Chris Gunter; Arnoud H.M. van Vliet; Wes Watson; Siddhartha Thakur

    The interaction of typical host adapted enteric bacterial pathogens with fresh produce grown in fields is complex. These interactions can be more pronounced in co-managed or sustainable farms where animal operations are, by design, close to fresh produce, and growers frequently move between the two production environments. The primary objectives of this study were to 1) determine the transmission of STEC or enteric pathogens from small and large animal herds or operations to fresh produce on sustainable farms in TN and NC, 2) identify the possible sources that impact transmission of AMR E. coli, specifically STEC on these systems, and 3) WGS to characterize recovered E. coli from these sources. Samples were collected from raw and composted manure, environment, and produce sources. The serotype, virulence, and genotypic resistance profile were determined using the assembled genome sequences sequenced by Illumina technology. Broth microdilution was used to determine the antimicrobial susceptibility of each isolate against a panel of fourteen antimicrobials. The prevalence of E. coli increased during the summer season for all sources tested. ParSNP trees generated demonstrated that the transmission of AMR E. coli is occurring between animal feeding operations and fresh produce. Ten isolates were identified as serotype O45, a serotype that is associated with the “Big Six” group that is frequently linked with foodborne outbreaks caused by non-O157 E. coli. However, these isolates did not possess the stx gene. The highest frequency of resistance was detected against streptomycin (n = 225), ampicillin (n = 190) and sulfisoxazole FIS (n = 140). A total of 35 (13.7%) isolates from two TN farms were positive for the blaCMY (n = 5) and blaTEM (n = 32) genes. The results of this study show the potential of AMR E. coli transmission between animal feeding operations and fresh produce, and more studies are recommended to study this interaction and prevent dissemination in sustainable farming systems.

  • Epiphytic bacteria from withered grapes and their antagonistic effects on grape-rotting fungi
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-29
    Marilinda Lorenzini; Giacomo Zapparoli

    The bacterial community in the surface of withered grapes, which are partially dehydrated in the post-harvest period to produce Italian passito wine, has been seldom investigated. Fifty epiphytic bacterial strains isolated from withered berries were identified and characterized. Genera such as Bacillus, Brevibacillus, Curtobacterium, Micrococcus, Pseudomonas and Staphylococcus have been identified by comparative sequence and phylogenetic analyses of 16S rRNA gene sequences. Bacillus was predominant and several taxa within this genus have been recognized. All isolates were characterized by PCR fingerprinting and assayed for osmotic tolerance, motility and antifungal activity. Several Bacillus strains displayed antagonistic effects on grape-rotting fungi such as Botrytis cinerea, Penicillium expansum and Aspergillus uvarum. The other strains were weakly or non-antagonistic on these fungi. Assay on antagonistic interactions among bacteria was also carried out. Bacillus strains, which exhibit swimming and swarming motility, have the potential to colonize the grape surface and to compete with their neighbours for space and resources. The occurrence of these isolates could reduce the contamination of fungal pathogens during grape withering. Epiphytic antagonistic bacteria could potentially be of interest for fungal biocontrol in the post-harvest processing of fruit and vegetables.

  • Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-28
    Saidou Harouna; Indira Franco; Juan J. Carramiñana; Arturo Blázquez; Inés Abad; María D. Pérez; Miguel Calvo; Lourdes Sánchez

    Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10–102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10–102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.

  • Detection of Shiga toxin producing Escherichia coli (STEC) in beef products using droplet digital PCR
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-28
    Joseph A. Capobianco; Mike Clark; Astrid Cariou; Adélaïde Leveau; Sophie Pierre; Pina Fratamico; Terence P. Strobaugh; Cheryl M. Armstrong

    Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of an E. coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx+, eae+ genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx+, eae+ genotype.

  • Transcriptome analysis of non-ochratoxigenic Aspergillus carbonarius strains and interactions between some black aspergilli species
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-27
    Gemma Castellá; M. Rosa Bragulat; Riccardo Aiese Cigliano; F. Javier Cabañes

    Aspergillus carbonarius consistently produces large amounts of ochratoxin A (OTA), a mycotoxin with nephrotoxic effects on animals and humans. In the present study, we analyzed the transcriptional changes associated to OTA production in three atypical non-ochratoxigenic strains of A. carbonarius. In addition, in vitro interactions between ochratoxigenic strains of A. carbonarius and A. niger and non-ochratoxigenic strains of A. carbonarius and A. tubingensis were studied in order to evaluate their potential for controlling OTA production. RNA-seq analysis revealed that there are 696 differentially expressed genes identified in the three non-OTA-producing strains, including 280 up-regulated and 333 down-regulated genes. A functional and gene ontology enrichment analysis revealed that the processes related to metabolic and oxidation processes, associated with functions such as oxidoreductase and hydrolase activity were down regulated. All the genes related with OTA biosynthesis in A. carbonarius were the most down-regulated genes in non-ochratoxigenic strains. We also showed that these strains possess a deleterious mutation in the AcOTApks gene required for OTA biosynthesis. Moreover, one of these strains gave the best control of OTA production resulting in an OTA reduction of 98–100% in co-inoculation with an ochratoxigenic strain of A. niger and an OTA reduction of 79–89% with an ochratoxigenic strain of A. carbonarius. Results of this study provided novel insights into the knowledge of the OTA biosynthetic pathway in these non-ochratoxigenic wild strains, and showed the biocontrol potential of these strains.

  • Identification of toxigenic fungal species associated with maize ear rot: Calmodulin as single informative gene
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-26
    Antonia Susca; Alessandra Villani; Antonio Moretti; Gaetano Stea; Antonio Logrieco

    Accurate identification of fungi occurring on agrofood products is the key aspect of any prevention and pest management program, offering valuable information in leading crop health and food safety. Fungal species misidentification can dramatically impact biodiversity assessment, ecological studies, management decisions, and, concerning toxigenic fungi, health risk assessment, since they can produce a wide range of toxic secondary metabolites, referred to as mycotoxins. Since each toxigenic fungal species can have its own mycotoxin profile, a correct species identification, hereby attempted with universal DNA barcoding approach, could have a key role in mycotoxins prevention strategies. Currently, identification of single marker for species resolution in fungi has not been achieved and the analysis of multiple genes is used, with the advantage of an accurate species identification and disadvantage of difficult setting up of PCR-based diagnostic assays. In the present paper, we describe our strategy to set up a DNA-based species identification of fungal species associated with maize ear rot, combining DNA barcoding approach and species-specific primers design for PCR based assays. We have (i) investigated the appropriate molecular marker for species identification, limited to mycobiota possibly occurring on maize, identifying calmodulin gene as single taxonomically informative entity; (ii) designed 17 sets of primers for rapid identification of 14 Fusarium, 10 Aspergillus, 2 Penicillium, and 2 Talaromyces species or species groups, and finally (iii) tested specificity of the 17 set of primers, in combination with 3 additional sets previously developed.

  • Elucidating antimicrobial mechanism of nisin and grape seed extract against Listeria monocytogenes in broth and on shrimp through NMR-based metabolomics approach
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-26
    Xue Zhao; Lin Chen; Ji'’en Wu; Yun He; Hongshun Yang

    Nisin and grape seed extract (GSE) have been widely used as food preservatives; however, the mechanism against pathogens at molecular level has not been well elucidated. This work aimed to investigate their antimicrobial effect against Listeria monocytogenes and to elucidate the mechanism by NMR-based metabolomics. Nisin exhibited enhanced in vitro antilisterial effect when combined with GSE (4.49 log CFU/mL reduction). Marked changes in cell membrane permeability was observed in the combination group using confocal laser scanning microscopy; this was verified by increased leakage of protein and nucleic acid. The underlying antimicrobial mechanism was revealed by NMR coupled with multivariate analysis. Significant decreases in threonine, cysteine, ATP, NADP, adenine were observed, whereas a few of metabolites such as lactic and γ-aminobutyric acids (GABA) increased after nisin-GSE treatment (P < 0.05). Pathway analysis further manifested that the nisin-GSE inhibited the survival of L. monocytogenes by blocking the TCA cycle, amino acid biosynthesis and energy-producing pathway. Lastly, nisin and GSE were applied to shrimp and binary combination showed remarkably antilisterial activity (1.79 log CFU/g reduction). GABA shunt and protein degradation from shrimp compensated the unbalanced glycolysis and amino acid metabolism by providing energy and carbon source for L. monocytogenes inoculated on shrimp. Thus, they were more tolerant to nisin and GSE stresses as compared to the broth-grown culture.

  • Culturable bacteria resident on lettuce might contribute to accumulation of human noroviruses
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-26
    Danlei Liu; Zilei Zhang; Ningbo Liao; Songyan Zou; Haoxuan Tang; Peng Tian; Glenn M. Young; Qingping Wu; Dapeng Wang

    Human noroviruses (HuNoVs) are the primary non-bacterial pathogens causing acute gastroenteritis worldwide. Attachment and invasion of HuNoVs are thought to involve histo-blood group antigens (HBGAs). Romaine lettuce, which is usually consumed raw, is a common food-related vehicle for HuNoVs transmission. This study investigated the possibility that bacteria resident on the surface of lettuce leaves contribute to norovirus adherence to this food. To test this hypothesis, bacteria were isolated from romaine lettuce and screened to evaluate whether they produced any polysaccharides with structures resembling HBGAs. Twenty-seven bacterial isolates were screened and 18, belonging to 13 different genera, were found to produce HBGAs-like polysaccharides that were recognized by monoclonal antibodies specific to type A, B, H and Lewis a, b, x and y. One bacterial isolate, belonging to the genus Pseudomonas was further investigated because it produced polysaccharides with the widest range of HBGA types, including type B, H and Lewis a, b and x. The Pseudomonas HBGAs-like polysaccharides were found to be extracellular and their production was enhanced when the bacteria were cultured in oligotrophic medium. HuNoVs capture assays revealed that GI.1, GI.8, and GII.2, GII.3, GII.4, GII.6, GII.12, GII.17 genotypes can be bind to Pseudomonas HBGAs-like polysaccharides. The direct evidence of bacterial production HBGAs-like polysaccharides demonstrates one possible mechanism driving accumulation of HuNoVs on lettuce.

  • Mega-plasmid found worldwide confers multiple antimicrobial resistance in Salmonella Infantis of broiler origin in Russia
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-26
    Alexandra N. Bogomazova; Veronika D. Gordeeva; Ekaterina V. Krylova; Irina V. Soltynskaya; Ekaterina E. Davydova; Olga E. Ivanova; Alexander A. Komarov

    Plasmids which are the mobile part of the bacterial genome can acquire and carry over genes conferring antimicrobial resistance, thus contributing to rapid adaptation of bacterial community to human-defined environment. In 2014, Israeli scientists have reported a large conjugative mega-plasmid pESI (plasmid for emerging S. Infantis) that provides multiple drug resistance (MDR) of Salmonella Infantis isolated from broilers. Later, very similar pESI-like plasmids have been found in Salmonella isolated from poultry in the United States, Italy, Switzerland, Hungary, and Japan. Here we report detection of pESI-like plasmids in Salmonella Infantis isolated from chicken food products in Russia. Whole genome sequencing of three MDR isolates revealed pESI-like plasmids in all three cases. These plasmids have such typical pESI features as a locus for siderophore yersiniabactin, a cluster of IncI1 conjugative genes, a cluster of type IV pilus genes, and three toxin-antitoxin modules. The pESI-like plasmids carry from two to five resistance genes in each isolate. In total, we observed six antimicrobial resistance genes associated with pESI-like plasmids (aadA1, blaCTX-M-14, dfrA14, sul1, tetA/tetR, tetM). Besides plasmid genes of antimicrobial resistance, all three MDR isolates of S. Infantis harbor a mutation in chromosomal gene gyrA (p.S83Y or p.D87Y) that is associated with resistance to fluoroquinolones. In addition, we performed a comparative bioinformatics meta-analysis of 25 pESI-like plasmids hosted by S. Infantis from the USA, Europe, Latin America, Israel, and Japan. This analysis identified a 173 kB sequence that is common for all pESI-like plasmids and carries virulence operons and toxin-antitoxin modules.

  • The effects of gelatin-CMC films incorporated with chitin nanofiber and Trachyspermum ammi essential oil on the shelf life characteristics of refrigerated raw beef
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-24
    Maryam Azarifar; Babak Ghanbarzadeh; Mahmoud Sowti Khiabani; Afshin Akhondzadeh Basti; Ali Abdulkhani

    The effects of gelatin-carboxymethyl cellulose (Gel-CMC) based films containing chitin nano fiber (CHNF) and Trachyspermum ammi essential oil (Ajowan), on the shelf life extension of the raw beef at refrigerated temperature (4 °C) over a 12-day period were evaluated. Ajowan essential oil (AJEO) and CHNF were added to the films at 0.24, 0.64 and 1% v/v; and 2 and 4 wt%, respectively. The microbiological (total viable count, psychrotrophic count, Pseudomonas spp., Staphylococcus aureus, lactic acid bacteria, molds and yeasts), the chemical (pH, thiobarbituric acid and total volatile basic nitrogen), color and sensory properties of the packaged samples were evaluated periodically. Bacteria grew the most quickly in the control samples, followed by those wrapped with the Gel-CMC films; The lowest microbial counts, the least change in the chemical properties and the highest sensory scores after 12 days of storage were obtained for the samples wrapped in the films incorporated with 1% AJEO and 4 wt% CHNF.

  • Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-23
    Mengyue Guo; Wenjun Jiang; Meihua Yang; Xiaowen Dou; Xiaohui Pang

    Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%–99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%–85.51%, 0.20%–29.11%, and 0.11%–32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.

  • High prevalence of mcr-1 encoding colistin resistance and first identification of blaCTX-M-55 in ESBL/CMY-2-producing Escherichia coli isolated from chicken faeces and retail meat in Tunisia
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-16
    Bilel Hassen; Mohamed Salah Abbassi; Laura Ruiz-Ripa; Olouwafemi M. Mama; Abdennaceur Hassen; Carmen Torres; Salah Hammami

    Avian industries have been reported as an important contributor in the worldwide spread of antibiotic resistance owing to some particular practices especially the overuse of antibiotics. Thus in this study, we aimed to characterize extended-spectrum-beta-lactamase (ESBL) and acquired-AmpC-beta-lactamase (aAmpC)-producing Escherichia coli isolates from chicken faeces and raw meat in Tunisia. During the year 2018, 286 faecal chicken swabs and 47 raw chicken meat samples were collected and processed to recover cefotaxime-resistant E. coli. Antimicrobial susceptibility was performed by disk-diffusion and/or broth-microdilution. blaTEM, blaSHV, blaCTX-M, and blaCMY genes were investigated by PCR/sequencing. Genes encoding resistance to colistin (mcr-1 to mcr-8), tetracycline (tetA/tetB), sulfonamide (sul1/sul3), and chloramphenicol (cmlA), were analysed by PCR. Class 1 integrons were investigated by PCR/sequencing. Phylogenetic groups of all isolates were determined. PFGE and MLST were performed for representative isolates. PCR-based replicon typing was performed in mcr1-harbouring isolates. Cefotaxime-resistant E. coli was detected in 22.4% (64/286) and 63.8% (30/47) of faeces and meat samples, respectively. Ninety isolates were ESBL-producers and harboured the genes: blaCTX-M-1 +/− blaTEM-1 (n = 65), blaCTX-M-55 +/− blaTEM-1 (n = 21), blaCTX-M-14 (n = 1), and blaSHV-12 (n = 3). The blaCMY-2 gene was detected in four ESBL-negative isolates. Isolates belonged to phylogroups D (50%), A (36.2%), B1 (9.6%), and B2 (4.3%). Fifty-four were colistin-resistant and 52 carried the mcr-1 gene. The tetA, sul1/sul3 and cmlA genes were detected among resistant isolates and 76 harboured class 1 integrons. MLST analysis revealed 13 sequence types (STs). The isolates were classified into 28 PFGE types. The IncP, IncFIB, and IncI1 replicons were detected among mcr-1-positive strains. We report a high frequency of ESBL-producers and colistin-resistant E. coli in chicken and derived food and the detection for the first time of blaCTX-M-55 in poultry in Tunisia.

  • Monitoring of Lactobacillus sanfranciscensis strains during wheat and rye sourdough fermentations by CRISPR locus length polymorphism PCR
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-16
    Esther Rogalski; Rudi F. Vogel; Matthias A. Ehrmann

    Lactobacillus (L.) sanfranciscensis is a competitive key species in sourdough fermentations. However, the principles involved in establishing the commonly observed phenomenon of strain dominance are unresolved. This has been studied little because the methods for fast and reliable differentiation of strains and their monitoring during fermentation are tedious and cannot be done with large numbers of isolates. In this contribution, we present a strain-specific, PCR-based typing method that uses length heterogeneities of the clustered regularly interspaced short palindromic repeats (CRISPR) loci as they occur in the genomes of different strains. In silico analysis of 21 genomes revealed 14 different CRISPR genotypes. We then designed a primer set to simultaneously detect different strains in a multiplex PCR assay designated CRISPR locus length polymorphism PCR (CLLP-PCR). The usefulness of this method was evaluated in lab-scale sourdough fermentations conducted with rye and wheat flours. First, the flour was mixed with water to a dough yield of 200. Then each dough was inoculated with four different L. sanfranciscensis strains (TMW 1.1150, TMW 1.392, TMW 1.2142, and TMW 1.2138) at levels of 109 cfu/g each. Sourdoughs were propagated at 28 °C for 5 days by back slopping 5% to the flour mass every 24 h. Samples were collected each day; DNA was isolated, and the presence of strains was detected qualitatively in the sourdoughs with PCR. L. sanfranciscensis TMW 1.392 became dominant as early as 2 days into the fermentation and remained the only detectable strain for the rest of the sampling period. CLLP-PCR proved to be useful in investigating the assertiveness of different strains of L. sanfranciscensis in sourdoughs. Therefore, CLLP-PCR may be used as a tool to investigate assertiveness of microorganisms in food fermentations at the strain level.

  • Microbial interaction between Salmonella enterica and main postharvest fungal pathogens on strawberry fruit
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-14
    J. Ortiz-Solà; A. Valero; I. Viñas; P. Colás-Medà; M. Abadias

    The microbial interaction between Salmonella enterica and the main postharvest fungal pathogens of strawberries was evaluated. Inoculation of fungal suspension was done 2 (D2) and 1 (D1) day(s) before and at the same time (D0) as S. enterica. Fruits were stored at 20 °C and 4 °C. At both temperatures, Botrytis cinerea and Rhizopus stolonifer caused a decrease in S. enterica population. Treatments where the mould was inoculated (D2, D1 and D0) achieved a significant logarithmic reduction (P < 0.05) of S. enterica populations after 48 h (20 °C) and 14 days (4 °C) compared to fungal-uninoculated fruits (CK). Regarding temperature, average reductions were significantly higher at 4 °C (3.38 log10 CFU/wound) than at 20 °C (1.16 log10 CFU/wound) (P < 0.05). Average reductions comprising all treatments were 1.91 and 0.41 log10 CFU/wound for B. cinerea and R. stolonifer at 20 °C, and 3.39 and 3.37 log10 CFU/wound for B. cinerea and R. stolonifer at 4 °C. A linear log10 model was fitted in order to predict the inactivation rate (kmax, log10 CFU/h) of S. enterica. Inactivation rates were higher at 20 °C for D2 treatments than at 4 °C throughout the running time. The main inactivation rate was obtained for B. cinerea at 20 °C (0.160 ± 0.027/h), which was found to have stronger inhibitory activity against S. enterica than R. stolonifer. Univariate analysis ANOVA was carried out to evaluate the effect of different external variables on the inhibition of S. enterica. Results found that single effects were significant (P < 0.05) except for the pH. The inhibitory effect caused by the action of moulds in conjunction with some environmental factors could indicate the potential interactions between strawberry fungal pathogens and S. enterica.

  • Moulds and their secondary metabolites associated with the fermentation and storage of two cocoa bean hybrids in Nigeria
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-14
    Taye O. Akinfala; Jos Houbraken; Michael Sulyok; Abiodun R. Adedeji; Adegboyega C. Odebode; Rudolf Krska; Chibundu N. Ezekiel

    Fungi and mycotoxin contamination of cocoa beans during fermentation and storage may constitute a hazard in the cocoa value chain and risk to consumers of its products. In this study, fungal profile and secondary metabolite patterns in two cocoa bean hybrids, F and T series, during fermentation and storage were determined. Additionally, secondary metabolite production by the recovered fungi in the beans was examined in culture media. Fungal isolates spanned six genera and eight species: Aspergillus niger, A. tamarii, Paecilomyces variotii, Penicillium citrinum, Pseudopithomyces palmicola, Simplicillium sp., Talaromyces atroroseus and Talaromyces sp. In both hybrids, Aspergilli (38%) dominated the other fungi while more than one half of all the fungal isolates were from the beans in storage. Among the diverse secondary metabolites produced in media by the isolates were uncommon compounds, e.g. aspulvinone E produced by A. niger, aspterric acid by P. variotii, scalusamid A and sydowinin A by P. citrinum, norlichexanthone and siccanol by Simplicillium, and fallacinol and orsellinic acid by Talaromyces. The strains of P. citrinum produced up to 372 mg/kg citrinin. Forty-four fungal metabolites were quantified in both bean hybrids across the various processing stages, with about 86% occurring in the fermented beans stored for 30 days. The nephrotoxic citrinin, which was not previously reported in cocoa beans worldwide, was the only mycotoxin found in the fermented beans at overall mean concentration of 368 μg/kg. Additionally, its metabolite, dihydrocitrinone, was detected in fermented and stored beans. Consumption of freshly fermented cocoa beans may result in citrinin exposure. Appropriate fungal and mycotoxin control measures are proposed.

  • Quantification of Salmonella enterica transfer between tomatoes, soil, and plastic mulch
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-13
    Jennifer Todd-Searle; Loretta M. Friedrich; Ruth A. Oni; Kenneth Shenge; Jeffrey T. LeJeune; Shirley A. Micallef; Michelle D. Danyluk; Donald W. Schaffner

    Tomatoes have been linked to Salmonella outbreaks in the United States (US). Plasticulture systems, that combine raised beds, plastic mulch, drip irrigation and fumigation, are common in commercial staked fresh tomato production in the US. The US FDA Produce Safety Rule prohibits the distribution of any produce covered by the rule (including fresh market tomatoes) that drops to the ground before harvest. This research was undertaken to better characterize the risks posed by tomatoes that touch plastic mulch or soil immediately before or during harvest. Research was conducted in three states (Florida, Maryland, and Ohio). Each state utilized tomatoes from their state at the point of harvest maturity most common in that state. Each state used indigenous soil and plastic mulch for transfer scenarios. New plastic mulch obtained directly from the application roll and used plastic mulch that had been present on beds for a growing season were evaluated. A five-strain cocktail of Salmonella enterica isolates obtained from tomato outbreaks was used. Mulch (new or used), soil, or tomatoes were spot inoculated with 100 μl of inoculum to obtain a final population of ~6 log CFU/surface. Items were either touched to each other immediately (1–2 s) after inoculation (wet contact) or allowed to dry at ambient temperature for 1 h or 24 h (dry contact). All surfaces remained in brief (1–5 s) or extended (24 h) contact at ambient temperature. Transfer of Salmonella between a tomato and plastic mulch or soil is dependent on contact time, dryness of the inoculum, type of soil, and contact surface. Transfer of Salmonella to and from the mulch and tomatoes for wet and 1 h dry inocula were similar with mean log % transfers varying from 0.7 ± 0.2 to 1.9 ± 0.1. The transfer of Salmonella between soil or plastic mulch to and from tomatoes was dependent on moisture with wet and 1 h dry inocula generally yielding significantly (p < 0.05) higher transfer than the 24 h dry inoculum. Results indicate that harvesting dry tomatoes significantly (p < 0.05) reduces the risk of contamination from soil or mulch contact. Transfer to tomatoes was generally significantly greater (p < 0.05) from new and used plastic mulch than from soil. If contamination and moisture levels are equivalent and contact times are equal to or <24 h before harvest, significantly (p < 0.05) more Salmonella transfers to tomatoes from mulch than from soil. Our findings support that harvesting tomatoes from soil has similar or lower risk than harvesting from plastic mulch.

  • Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoebae in lettuce samples by next generation sequencing and other molecular techniques
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-12
    Laura Moreno-Mesonero; Irene Hortelano; Yolanda Moreno; Mª. Antonia Ferrús

    Vegetables are one of the sources from which Helicobacter pylori can be acquired. This bacterium infects >50% of the global population and is a recognized type I human carcinogen. H. pylori enters into the viable but non-culturable state when it is in the environment, and therefore the use of molecular techniques is much convenient for its detection. Free-living amoebae (FLA) are protozoans found in vegetables. They are transmission vehicles for amoeba-resistant bacteria, among which H. pylori is included. The aim of this study is to study the occurrence and viability of H. pylori from lettuce samples, H. pylori internalized into FLA and the microbiome of FLA isolated from these samples. Special focus was pointed to human pathogenic bacteria. H. pylori was not directly detected in any lettuce sample by means of molecular techniques and neither by culture. However, intra-amoebic H. pylori DNA was detected by means of PMA-qPCR in 55% of the samples and viable intra-amoebic H. pylori cells in 25% of the samples by means of DVC-FISH technique. When FLA microbiome was studied, 21 bacterial genera were part of FLA microbiome in all samples. Helicobacter genus was detected as part of the FLA microbiome in two samples. Other bacteria of public health interest such as Aeromonas sp., Arcobacter sp., Legionella sp., Mycobacterium sp., Pseudomonas sp. and Salmonella sp. were detected as part of FLA microbiome along the analysed samples. This study demonstrates for the first time that H. pylori is internalized as well as alive inside FLA isolated from vegetables. Moreover, this study shows that FLA promote H. pylori detection in environmental samples. In addition, as far as we are aware, this is the first study which studies the microbiome of FLA isolated from vegetables. Among the FLA microbiome, bacteria of public health interest were detected, pointing out that FLA are carriers of these pathogens which can reach humans and cause a public health concern.

  • Circulation of hepatitis E virus (HEV) and/or HEV-like agent in non-mixed dairy farms could represent a potential source of infection for Egyptian people
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-12
    Ibrahim M. Sayed; Amal A. Elkhawaga; Mohamed A. El-Mokhtar

    Hepatitis E virus (HEV) infection is endemic in many developing countries and becomes of interest in the developed countries. Several animals are sources of HEV infection to humans. Recently, HEV was detected in the milk of cows in China, this data comes up with the probability of HEV transmission to humans via ingestion of contaminated milk. In Egypt, contaminated water and residing in rural communities are risk factors for HEV infection, while limited data is available on the zoonotic HEV transmission. Since pigs, wild boars, camels are not common in Egypt, we investigated if cows and/or cow milk represent a risk factor for HEV transmission in the Assiut governorate. Milk samples (n = 480), collected from Assiut city and 12 non-mixed dairy farms distributed in the rural communities, were tested for HEV markers such as anti-HEV IgG, HEV RNA, and HEV Ag. All milk samples collected from Assiut city (n = 220) were negative for HEV markers. Also, milk samples collected from 11 farms (n = 220) were negative for HEV markers. While, in one farm, we could detect anti-HEV IgG in 8 out of 40 samples (20%), HEV RNA and HEV Ag were detectable in 1 out of 40 samples (2.5%). However, we could not detect the HEV markers in the stool from anti-HEV IgG positive cows. Surprisingly, phylogenetic analysis of the isolated virus revealed it belonged to HEV-3 subtype 3a. Importantly, when cows from the positive farm were retested 1 month later, we observed an increase in the number of animals that were positive for anti-HEV IgG (10/40, 25%). In addition, the level of anti-HEV IgG was significantly higher in the milk of these cows in the second collection than the samples of the first collection suggesting ongoing infection on this farm. In conclusion: we reported that HEV-3 and/or HEV like agent was detected in the milk of the cow distributed in rural communities of Assiut governates. Investigation of the cow milk should be done to assess if the cow milk is a risk factor for HEV transmission for Egyptian people, especially in rural communities.

  • Assessment of chemical composition and sensorial properties of ciders fermented with different non-Saccharomyces yeasts in pure and mixed fermentations
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-10
    Jianping Wei, Yuxiang Zhang, Yuwei Wang, Hongmei Ju, Chen Niu, Zihan Song, Yahong Yuan, Tianli Yue

    This work presents the attempt to enhance the flavor complexity of cider fermented by different non-Saccharomyces species. Pichia kluyveri and Hanseniaspora vineae pure cultures were used as reference ciders. Mixed cultures between all 4 species gave 5 fermentations, where Hanseniaspora uvarum or Torulaspora quercuum were included for apple juice fermentation. Chemical composition and sensorial properties of all ciders were studied. The results indicated that the growth of P. kluyveri and H. vineae were interreacted and also affected by H. uvarum and T. quercuum. H. vineae was more capable of consuming sugar than P. kluyveri. Ciders from the single culture fermentation with P. kluyveri (Pk), as well as from mixed fermentation with P. kluyveri and H. uvarum (Pk-Hu), had high residual sugar, sugar/acid ratio, and glucose-fructose consumption ratio. Large shifts in the consumption and production of organic acids and polyphenols among all ciders were observed. The calculation of the relative odor activity value (rOAV) showed that 17 volatile compounds had an rOAV >1 in at least one sample, and acetate esters and ethyl esters were the groups with the highest number of volatile compounds of importance to the cider aroma. Among these 17 compounds, 3-methylbutyl acetate, 2-methylbutyl acetate, ethyl hexanoate, ethyl octanoate, and β-damascenone exhibited high rOAVs in some ciders and might contribute fruity, floral, and sweet features to the cider aroma. Besides, the tropical fruity aroma from 3-methylbutyl acetate was only perceived in Pk and Pk-Hu. The partial least squares regression (PLSR) analysis revealed that acetate esters contributed positively to the roasted and cooked odor of all ciders. This is the first study evaluating simultaneous fermentation of two non-Saccharomyces yeasts to produce cider, which provides new insights into cider production.

  • Analysis of rpoB polymorphism and PCR-based approaches for the identification of Leuconostoc mesenteroides at the species and subspecies level
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-09
    Annamaria Ricciardi, Livia V. Storti, Teresa Zotta, Giovanna E. Felis, Eugenio Parente

    Leuconostoc mesenteroides includes the subsp. cremoris, subsp. dextranicum, subsp. mesenteroides and subsp. jonggajibkimchii, but the identification at the subspecies level using current phenotypic and/or genotypic methods is still difficult. In this study, a polyphasic approach based on the analysis of rpoB gene polymorphism, Multiplex-PCR and phenotypic tests was optimised and used to identify a collection of Leuc. mesenteroides strains at the species and subspecies levels. The annotation of published Leuc. mesenteroides genomes was also revised. A polymorphic region of rpoB gene was effective in separating Leuc. mesenteroides strains at the species (rpoB-species-specific-PCR) and subspecies (phylogenetic comparison) levels. Multiplex-PCR discriminated the subsp. mesenteroides from subsp. cremoris, but strains of uncertain attribution were found among subsp. dextranicum and subsp. jonggajibkimchii. Most of phenotypic features were not suitable for subspecies discrimination. Our assays may provide a rapid and reliable identification of subsp. mesenteroides and subsp. cremoris strains in fermented foods. The discrimination of subsp. dextranicum and subsp. jonggajibkimchii suffered from several limitations (e.g. low number of available strains and genomes, phenotypic profile close to subsp. mesenteroides, discrepancy between genotypic and phenotypic traits) and further investigations are needed to clarify their delineation and taxonomical position.

  • Characterization of class 1 integrons harboring blaVEB-1 in Vibrio parahaemolyticus isolated from ready-to-eat foods in China
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-09
    Tao Lei, Jumei Zhang, Fufeng Jiang, Min He, Haiyan Zeng, Moutong Chen, Rui Pang, Haoming Wu, Shi Wu, Juan Wang, Yu Ding, Qingping Wu

    The aim of this study is to investigate the prevalence of integrons and integron-associated antibiotic resistance in V. parahaemolyticus strains collected from RTE foods in China, and to carry out a comprehensive analysis on the molecular characterization of V. parahaemolyticus strains carrying blaVEB-1-positive class 1 integron. Of the 51 V. parahaemolyticus strains isolated from RTE food samples, none of the isolates was found to carry integrase genes intI2 and IntI3. However, all 51 strains were positive to integrase gene intI1, and only 2 of 51 (3.92%) intI1-positive isolates yielded polymerase chain reaction (PCR) products of gene cassette amplification. Sequence data and BLAST analysis indicated the gene cassette arrays of class 1 integron in VP007 is dfrA14-blaVEB-1-aadB, while the gene cassette arrays of class 1 integron in V187 is blaVEB-1-aadB-arr2-cmlA-blaOXA-10-aadA1. Antimicrobial susceptibility testing showed that the two V. parahaemolyticus isolates harboring class 1 integrons exhibited multi-drug resistance to various antibiotics. S1-PFGE and Southern blot analysis confirmed the class 1 integron harboring blaVEB-1 gene in V187 was located on the plasmid of ~175 kb and transferrable to the recipient strain by conjugation. This is the first detection of class 1 integrons harboring the ESBL gene blaVEB-1 in V. parahaemolyticus. To the best of our knowledge, this is also the first report of VEB-producing V. parahaemolyticus from RTE foods. Our findings revealed that class 1 integron on conjugative plasmid contributes significantly to the dissemination of VEB-producing V. parahaemolyticus, which warrants further investigation because of the public health threat it poses.

  • Metabolism of biosynthetic oligosaccharides by human-derived Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-09
    Laura Ruiz-Aceituno, Maria Esteban-Torres, Kieran James, F. Javier Moreno, Douwe van Sinderen

    This work aimed to investigate the ability of two human-derived bifidobacterial strains, i.e. Bifidobacterium breve UCC2003 and Bifidobacterium longum NCIMB 8809, to utilize various oligosaccharides (i.e., 4-galactosyl-kojibiose, lactulosucrose, lactosyl-oligofructosides, raffinosyl-oligofructosides and lactulose-derived galacto-oligosaccharides) synthesized by means of microbial glycoside hydrolases. With the exception of raffinosyl-oligofructosides, these biosynthetic oligosaccharides were shown to support growth acting as a sole carbon and energy source of at least one of the two studied strains. Production of short-chain fatty acids (SCFAs) as detected by HPLC analysis corroborated the suitability of most of the studied novel oligosaccharides as fermentable growth substrates for the two bifidobacterial strains, showing that acetic acid is the main metabolic end product followed by lactic and formic acids. Transcriptomic and functional genomic approaches carried out for B. breve UCC2003 allowed the identification of key genes encoding glycoside hydrolases and carbohydrate transport systems involved in the metabolism of 4-galactosyl-kojibiose and lactulosucrose. In particular, the role of β-galactosidases in the hydrolysis of these particular trisaccharides was demonstrated, highlighting their importance in oligosaccharide metabolism by human bifidobacterial strains.

  • Contribution of non-Saccharomyces yeasts to wine volatile and sensory diversity: A study on Lachancea thermotolerans, Metschnikowia spp. and Starmerella bacillaris strains isolated in Italy
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-05
    Renato L. Binati, Wilson J.F. Lemos Junior, Giovanni Luzzini, Davide Slaghenaufi, Maurizio Ugliano, Sandra Torriani

    Saccharomyces cerevisiae starter cultures are largely used in winemaking to repress the wild microorganisms and achieve more predictable and desired outcomes. Notwithstanding, alternative microbial resources received increasing attention for their potential to produce wines with more distinctive and typical features. Our previous survey revealed a great inter- and intra-species diversity in an extensive collection of non-Saccharomyces wine yeasts from multiple regions of Italy. This study aimed to explore the detected biodiversity evaluating the quality of wines obtained by sequential inoculation of specific selected strains of the collection (Lachancea thermotolerans or Metschnikowia spp. or Starmerella bacillaris), and S. cerevisiae EC 1118. Fermentations of natural grape must at laboratory scale were followed by microbiological, chemical and sensorial analysis of the wines. The results indicated that each yeast species and strain exerted a distinctive impact on the wine, giving final products clearly separated with Principal Component Analysis. In particular, L. thermotolerans contributed producing relevant amounts of lactic acid and had the highest potential to reduce ethanol content; the presence of S. bacillaris increased the level of glycerol, and, remarkably, reduced acetaldehyde and total SO2; Metschnikowia spp. promoted the formation of higher alcohols and esters., and reduced volatile phenols. The sensory analysis based on the orthonasal aroma confirmed the separation between the wines obtained with the sequential fermentations and the control with single inoculation of EC 1118, although the three non-Saccharomyces species used could not be clearly distinguishable by the panelists. This study indicates that the use of selected native non-Saccharomyces strains in conjunction with S. cerevisiae positively modulates some relevant chemical parameters, and improves the aromatic intensity of wine, therefore justifying investments in alternative yeasts as co-starter cultures.

  • A mathematical modeling approach to the quantification of lactic acid bacteria in vacuum-packaged samples of cooked meat: Combining the TaqMan-based quantitative PCR method with the plate-count method
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-02
    Wiaslan Figueiredo Martins, Daniel Angelo Longhi, Gláucia Maria Falcão de Aragão, Beatriz Melero, Jordi Rovira, Ana M. Diez
  • Effect of interacting conditions of water activity, temperature and incubation time on Fusarium thapsinum and Fusarium andiyazi growth and toxin production on sorghum grains
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-02
    G.A. Pena, M. Sulyok, S.N. Chulze

    This study examined the effect of interacting conditions of water activity (aW, 0.995, 0.98 and 0.95) and temperature (15, 25 and 30 °C) on growth rate of two Fusarium thapsinum and one F. andiyazi strains isolated from sorghum in Argentina. In addition, the effect of interacting conditions (aW × temperature × incubation time (7, 14, 21 and 28 days)) on mycotoxin production (moniliformin (MON), fusaric acid (FA) and fusarin C (FUS C)) on a sorghum grain substrate was evaluated. Statistical analysis showed that aW and temperature significantly affected growth of both species, mainly the aW. Incubation time significantly influenced mycotoxin production by both species as well, mostly for FA. Maximum growth rates of the F. thapsinum strains were obtained at the highest aW (0.995) and 25 °C and growth rate decreased as aW and temperature were reduced. The same growth profile was observed for F. andiyazi RCFA09 (maximum growth rates at 0.995–25 °C). Mycotoxin production by both species was detected at the highest aW levels whereas at 0.95 aW only low amounts of MON were produced by F. thapsinum. Maximum MON and FUS C production by both F. thapsinum strains was observed at 0.995 aW and 25–30 °C after 28 days of incubation. Also, F. thapsinum strains showed maximum FA production at the highest aW and temperature but after 14 days; after this incubation time toxin levels significantly decreased. The responses to aW and temperature of F. andiyazi were similar to that of F. thapsinum strains in relation to FA and FUS C production. Maximum levels of FA were detected at the highest aW after 14 days of incubation at 25–30 °C. Fusarin C was produced at all assayed temperatures but maximum levels were detected at 30 °C and 0.995 aW after 28 days of incubation. Two-dimensional profiles on the interactions of aW by temperature were developed from these data to identify conditions that indicate a significant risk from MON, FA and FUS C accumulation on sorghum grains. The results of this study suggest that sorghum grains could be colonized by these species and toxin production can occur, especially during development stages under field conditions at high water activity of grains or during grain storage if the drying process is slow or deficient. To our knowledge, this study described for the first time FUS C production by F. thapsinum and F. andiyazi under interacting conditions of aW, temperature and incubation time on sorghum grains.

  • Inactivation of Listeria monocytogenes during dry-cured ham processing
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-02
    Raquel Montiel, Ángela Peirotén, Sagrario Ortiz, Daniel Bravo, Pilar Gaya, Joaquín V. Martínez-Suárez, Julio Tapiador, Manuel Nuñez, Margarita Medina

    The effect of Serrano and Iberian dry-cured ham processing and ripening on Listeria monocytogenes inactivation at the surface of whole hams was investigated. Salted hams were surface inoculated (6 log CFU) with a cocktail of 4 L. monocytogenes strains isolated from environment and products of a meat industry. Serrano and Iberian hams were ripened for 16 and 24 months, respectively. A decrease of at least 4.6 log units on the surface of Serrano ham was recorded after 4 months for L. monocytogenes counts, which remained under the detection limit thereafter. L. monocytogenes declined by >5 log units on the surface of Iberian ham during the first 9 months and was not detected afterwards. The higher nitrite content of Serrano ham might have accelerated the decrease of the pathogen. This study validates the inactivation of L. monocytogenes on the surface of whole dry-hams during extended ripening.

  • Influence of pulp on the microbial diversity during cupuassu fermentation
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-02
    Simone Ramos, Marcela Salazar, Leandro Nascimento, Marcelo Carazzolle, Gonçalo Pereira, Tiago Delforno, Maristela Nascimento, Tiago de Aleluia, Renata Celeghini, Priscilla Efraim

    Cupuassu (Theobroma grandiflorum Schum) is a fruit belonging to the same genus as cocoa and, through seed fermentation, a chocolate-like product called “the cupulate” is obtained. The pulp is removed from the seeds before fermentation because its abundance hinders the process. Unlike cocoa, little is known about the microbial diversity involved in cupuassu fermentation. The goal of this study was to explore the use of next-generation sequencing to identify the yeasts and bacteria communities involved in cupuassu seed fermentation on three different pulp concentrations (0, 7.5, and 15%) as well as two turning schemes on the microbial growth. In order to do that, a massive sequencing of the 16S and ITS4 rRNA region (S) using the Illumina MiSeq Platform identified some genera of bacteria and yeasts, respectively, in the fermentation environment. Taxonomic analyses of both communities, especially at the genus level, revealed a predominance of yeasts such as Pichia and Hanseniaspora, and bacteria such as Acetobacter and Lactobacillus. A predominance of bacteria over yeasts diversity was observed in the experiments with higher pulp concentrations (15%). The physicochemical analysis showed that fermentation of samples with 15% pulp exhibited longer fermentation times, the highest temperatures, and elevated production of organic acids such as acetic acid, a precursor of flavor. In addition, the turning applied every 24 h to the mass slightly favored the formation of flavor precursors. It seems that the abundance and composition of cupuassu pulp, rich in organic compounds, can influence the diversity of some populations of yeasts. Some of those compounds identified in previous studies are terpenes with antimicrobial activity. More studies will be necessary to confirm if the presence of terpenes compounds in the cupuassu pulp exert some inhibitory action on microorganism diversity.

  • Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-12-02
    Marilia Miotto, Clarissa Barretta, Sylvia O. Ossai, Helen Silvestre da Silva, Airton Kist, Cleide Rosana Werneck Vieira, Salina Parveen

    The present study compared different concentrations of propidium monoazide (PMA), time of exposure to light and different light intensities to determine the optimal conditions for the quantification of viable Escherichia coli in cell suspension and in food matrix. The influence of cell density and the effectiveness of PMA in viable but non-culturable (VBNC) E. coli cells were evaluated and also applied in food matrix. For that purpose, different concentrations of PMA (20 μM, 40 μM, 50 μM, 60 μM and 80 μM) under different times of exposure (5 min, 10 min, 15 min, 20 min and 30 min) to lights of different intensities (500 W and 650 W) were evaluated. After determining the optimal conditions, the PMA-qPCR methods were applied to different compositions of live and heat-killed E. coli suspensions (v:v; 0:1; 1:0; 1:1) in concentrations ranging from 3 Log to 7 Log CFU/mL. The same dilutions were prepared with E. coli in VBNC state and applied in food matrix. The results obtained from qPCR, PMA-qPCR and plate counts were compared. The results suggested that a PMA treatment of 50 μM PMA for 15 min under 650 W light intensity was optimal under our conditions. For E. coli cell suspensions, the amplification of heat-killed cells was inhibited greatly by PMA when concentrations were ≤ 5 Log CFU/mL. For the samples of oyster inoculated with heat-killed cells, E. coli was not detected by PMA-qPCR in concentrations ≤4 Log CFU/g. Regarding the results with VBNC state, we considered the PMA-qPCR method to be applicable for enumerating E. coli VBNC cells in oyster samples. Based on our findings, we further recommend the use of PMA-qPCR with the aim of reducing the amplification of dead cells for improving its performance, since false-positives could still occur depending on the level of E. coli in the sample. The application of the PMA-qPCR for quantification of bacteria, compared to the use of culture-dependent methods, is quite promising. However, further studies are recommended, especially using different food matrices.

  • Effects of temperature on paocai bacterial succession revealed by culture-dependent and culture-independent methods
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-29
    Dongdong Wang, Gong Chen, Yao Tang, Heng Li, Wenxi Shen, Meng Wang, Shuliang Liu, Wen Qin, Qisheng Zhang

    Paocai is a widely consumed Chinese traditional fermented vegetable product. To understand the effect of temperature on paocai fermentation flora, the bacterial community structure of paocai fermented at 10 °C, 15 °C, 25 °C and 35 °C was analyzed by culture-dependent and culture-independent methods. The results showed that increasing the fermentation temperature in a certain range is beneficial for rapid paocai acid production and shortening of the maturity period. Illumina Miseq sequencing was performed on 56 samples at different fermentation process temperatures using a culture-independent method. A total of 1,964,231 high-quality reads of 16S rRNA V3-V4 regions were obtained, and they were divided into 405 operational taxonomic units (OTUs) and identified as 213 bacterial genera. The bacterial diversity decreased with the progression of fermentation, and some spoiled samples had an increased diversity. The culture-independent method found that at 10 °C, Lactococcus appeared at the start of fermentation, Leuconostoc and Weissella appeared in the middle of fermentation, and Lactobacillus and Leuconostoc dominated fermentation in the late stage. At 15 °C, Lactococcus started fermentation, Leuconostoc appeared in the middle stage, and Lactobacillus was dominant in the late stage. At 25 °C, Lactococcus started fermentation, Weissella and Lactobacillus appeared in the middle stage, and Lactobacillus dominated fermentation in the late stage. Finally, at 35 °C, Lactococcus, Weissella, and Lactobacillus started fermentation, Weissella and Lactobacillus appeared in the middle stage, and Lactobacillus dominated fermentation in the late stage. A total of 647 strains of bacteria were isolated by culture-dependent methods and were divided into 12 genera and 19 species by randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and 16S ribosomal RNA gene (rDNA) sequencing technology. More types of bacteria were isolated in the early stage of fermentation. At 10 °C, Lactococcus lactis began fermentation, and Lactobacillus brevis and Leuconostoc mesenteroides dominated acid production in the middle and late stages of paocai fermentation. At 15 °C, L. lactis initiates fermentation, while Lactobacillus plantarum dominates the acid fermentation of paocai. At 25 °C and 35 °C, there were a large number of Enterobacteriaceae bacteria in the start-up fermentation stage, and L. plantarum was dominant after 1–2 days of fermentation. Redundancy analysis (RDA) found that the lower the temperature, the more bacterial species that are produced, and the higher the temperature and the longer the time, the more obvious are the effects of L. plantarum on paocai. The results of dominant bacteria studied by culture-dependent and culture-independent methods are similar. The results indicate that most of the dominant microorganisms in the paocai fermentation system are culturable. This discovery can provide data and physical support for modernization and regulation of different types of paocai production.

  • New advances on the Brettanomyces bruxellensis biofilm mode of life
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-28
    Manon Lebleux, Hany Abdo, Christian Coelho, Louise Basmaciyan, Warren Albertin, Julie Maupeu, Julie Laurent, Chloé Roullier-Gall, Hervé Alexandre, Michèle Guilloux-Benatier, Stéphanie Weidmann, Sandrine Rousseaux

    The wine spoilage yeast Brettanomyces bruxellensis can be found at several steps in the winemaking process due to its resistance to multiple stress conditions. The ability to form biofilm is a potential resistance strategy, although it has been given little attention so far for this yeast. In this work, the capacity to form biofilm and its structure were explored in YPD medium and in wine. Using microsatellite analysis, 65 isolates were discriminated into 5 different genetic groups from which 12 strains were selected. All 12 strains were able to form biofilm in YPD medium on a polystyrene surface. The presence of microcolonies, filamentous cells and extracellular polymeric substances, constituting the structure of the biofilm despite a small thickness, were highlighted using confocal and electronic microscopy. Moreover, different cell morphologies according to genetic groups were highlighted. The capacity to form biofilm in wine was also revealed for two selected strains. The impact of wine on biofilms was demonstrated with firstly considerable biofilm cell release and secondly growth of these released biofilm cells, both in a strain dependent manner. Finally, B. bruxellensis has been newly described as a producer of chlamydospore-like structures in wine, for both planktonic and biofilm lifestyles.

  • Prevalence, virulence, antimicrobial resistance, and molecular characterization of fluoroquinolone resistance of Vibrio parahaemolyticus from different types of food samples in China
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-28
    Tao Lei, Fufeng Jiang, Min He, Jumei Zhang, Haiyan Zeng, Moutong Chen, Rui Pang, Shi Wu, Lei Wei, Juan Wang, Yu Ding, Qingping Wu

    Vibrio parahaemolyticus is the leading cause of foodborne bacterial poisoning in China. The aim of this research is to conduct a study on the prevalence, virulence, and antimicrobial resistance of V. parahaemolyticus from different types of food samples in 12 different cities of China. Since fluoroquinolones are the major choice of treatment for V. parahaemolyticus infections, the genetic basis for fluoroquinolone resistance in V. parahaemolyticus were also investigated. V. parahaemolyticus was detected in 163 of the 784 food samples collected from 12 different cities in China, resulting in a prevalence of 20.79%. The prevalence of V. parahaemolyticus in ready-to-eat (RTE) food (4.96%) was much lower than those of shrimp (32.62%) and fish (22.00%). Virulence gene screening showed that 44 (27.00%) V. parahaemolyticus strains carried at least one virulence gene. Four isolates from shrimp and three isolates from fish contained both the virulence genes tdh and trh. In addition, the trh was firstly detected in one isolate collected from RTE food. All isolates exhibited relatively high resistance rates to ampicillin (82.21%), gentamicin (19.63%), and tetracycline (14.11%), while <10% of strains were resistant to ciprofloxacin (4.91%), levofloxacin (4.91%), and tetracycline (4.29%). Eight fluoroquinolone-resistant V. parahaemolyticus were selected to determine the molecular basis for fluoroquinolone resistance. These eight isolates belonged to three different types according to enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). A Ser83Ile substitution in GyrA was deteted in seven fluoroquinolone-resistant strains, except V209 which harbored a Ser83Phe substitution in GyrA. Moreover, A Ser85Leu substitution in ParC was found in five isolates (V52, V53, V61, V163, and V209). Plasmid-mediated quinolone resistance (PMQR) genes were detected in all eight fluoroquinolone-resistant V. parahaemolyticus strains. This is the first report of Ser83Phe substitution in GyrA, qnrD and qnrS1 in V. parahaemolyticus. The information generated in this study will provide valuable information for risk assessment of V. parahaemolyticus infections and future control of antibiotic-resistant V. parahaemolyticus species in China.

  • Inhibitory effect of a combination with novel jumbo bacteriophages ΦMV-1 and ΦMV-4 on Morganella morganii subsp. morganii growth and histamine accumulation
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-27
    Shogo Yamaki, Soya Kuronuma, Yuji Kawai, Koji Yamazaki

    Histamine (scombroid) poisoning is a foodborne illness caused by ingestion of histamine-contaminated seafood; therefore, inhibition of the growth of histamine-producing bacteria is key for it prevention. Infection of pathogenic bacteria by bacteriophages (phages) is being developed to prevent multiple foodborne illnesses. Here, we describe the inhibitory effect of a phage mixture on growth and histamine accumulation of Morganella morganii subsp. morganii, the primary causative agent of histamine poisoning in fish meat. We isolated novel two phages, ΦMV-1 and ΦMV-4, which infected M. morganii subsp. morganii strains tested in this study. ΦMV-1 and ΦMV-4 belong to family Myoviridae. Pulsed-field gel electrophoresis revealed that these phages are jumbo bacteriophages with large genomes. The latent period, rise period and burst size of ΦMV-1 were 30 min, 60 min, and 224 PFU per infected cell, respectively, and those of ΦMV-4 were 60 min, 50 min, and 62 PFU per infected cell, respectively. A mixture of ΦMV-1 and ΦMV-4 effectively prevented regrowth of M. morganii subsp. morganii after phage treatment, suggesting that the phage mixture treatment is more effective for inhibition of growth and histamine accumulation by M. morganii subsp. morganii than single phage treatment. Treatment with phage mixture inhibited growth and histamine accumulation by M. morganii subsp. morganii in canned and fresh tuna. The phage mixture might be an effective way to prevent growth of the histamine producer and accumulation of histamine in seafood.

  • Evaluation of yeasts from Ecuadorian chicha by their performance as starters for alcoholic fermentations in the food industry
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-26
    Nubia Grijalva-Vallejos, Agustín Aranda, Emilia Matallana

    Yeasts involved in the spontaneous fermentation of traditional beverages like chicha (indigenous Andean beer) may have the potential to be used as starter cultures to improve the quality and microbiological safety of these products, but also as non-conventional alternatives to other food alcoholic fermentations. In this research, we isolated, identified and characterised yeast strains from four Ecuadorian chichas made by using four different raw materials: rice (RC), oat (OC), grape (GC) and a mixture of seven corn varieties (yamor, YC). Finally, 254 yeast isolates were obtained and identified by molecular methods. Eleven yeast genera and 16 yeast species were identified with relatively few isolates belonging to Saccharomyces cerevisiae (9.1% belonging to 6 strains) and Torulaspora delbrueckii (18.6% belonging to 2 strains). In order to select good candidates for fermentative starter production, different analyses were performed. The results of the stress response tests showed a wide variability between species and strains, and identified some yeasts displaying high stress tolerance, similarly to commercial wine strains. Amylase production was screened as being indicative of the capacity to degrade and ferment starch-rich substrates. A Cryptococcus sp. isolate showed the highest amylase activity. The growth rate and fermentative capacity in molasses medium was measured for three S. cerevisiae, T. delbrueckii and Candida sp. strains as tests for yield and performance in biomass production. Based on their excellent behaviour, three S. cerevisiae strains and one T. delbrueckii strain were selected for further analyses, including dehydration tolerance and invertase activity as additional desired traits for chicha starters. All the S. cerevisiae strains exhibited high invertase activity and one also displayed high resistance to dehydration. The yeasts selected in this study can thus be suitably used as dry starters for the microbiologically controlled production of traditional beverages, and also for other alcoholic fermentations.

  • A study of surface moulds and mycotoxins in Croatian traditional dry-cured meat products
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-26
    Manuela Zadravec, Nada Vahčić, Dragan Brnić, Ksenija Markov, Jadranka Frece, Relja Beck, Tina Lešić, Jelka Pleadin

    Xerophilic species of Aspergillus, Penicillium and Eurotium genera from surfaces of dry-cured traditional meat products (TMPs) can cause mycotoxin contamination during uncontrolled household processing. The aim of this study was to investigate into surface moulds growing on Croatian prosciuttos and fermented sausages produced in different climate regions using different technologies (n = 160), and to relate the occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) to their presence. The results revealed the Penicillium (79%) to be the dominating contaminating mould, while Aspergillus (11%), Eurotium (7%) and Mucor (4%) species were present in a significantly lower number of isolates, with higher prevalence and greater diversity in prosciuttos than in sausages, relative of the production technology and regional climate. OTA contamination (14% of samples) was significantly more frequent than that with AFB1 (8% of samples), with OTA concentration rising to the maximal 6.86 μg/kg, whereas AFB1 concentrations were slightly higher than, or around, the limit of quantification of the method in use, with the maximal value of 1.92 μg/kg. The presence of AFB1 in absence of toxicogenic moulds, observed in some samples, can be attributed to contaminated spices used in TMP production or an indirect contamination via a carry-over effect.

  • Microbiome convergence following sanitizer treatment and identification of sanitizer resistant species from spinach and lettuce rinse water
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-23
    Ganyu Gu, Andrea Ottesen, Samantha Bolten, Yaguang Luo, Steven Rideout, Xiangwu Nou

    Fresh produce, as a known or suspected source of multiple foodborne outbreaks, harbors large populations of diverse microorganisms, which are partially released into wash water during processing. However, the dynamics of bacterial communities in wash water during produce processing is poorly understood. In this study, we investigated the effect of chlorine (FC) and peracetic acid (PAA) on the microbiome dynamics in spinach and romaine lettuce rinse water. Treatments with increasing concentrations of sanitizers resulted in convergence of distinct microbiomes. The resultant sanitizer resistant microbiome showed dominant presence by Bacillus sp., Arthrobacter psychrolactophilus, Cupriavidus sp., and Ralstonia sp. Most of the FC and PAA resistant bacteria isolated from spinach and lettuce rinse water after sanitation were gram positive spore forming species including Bacillus, Paenibacillus, and Brevibacillus spp., while several PAA resistant Pseudomonas spp. were also isolated from lettuce rinse water. Inoculation of foodborne pathogens altered the microbiome shift in spinach rinse water under PAA treatment, but not in lettuce rinse water or FC treated samples. These inoculated foodborne pathogens were not isolated among the sanitizer resistant strains.

  • Competitive yeast action against Aspergillus carbonarius growth and ochratoxin A production
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-23
    Paschalitsa Tryfinopoulou, Antonia Chourdaki, George-John E. Nychas, Efstathios Z. Panagou

    The aim of the present study was to investigate the interaction between 67 different yeast isolates and 3 wild isolates of Aspergillus carbonarius originated from Greek vineyards and characterized by high ochratoxigenic potential. The selected fungi were used either as single cultures or combined in a mixed culture. Yeasts and fungi were grown as mono-cultures and co-cultures in solid (MEA, CYA) and liquid (CY broth) media, grape berries and sterilized grape juice. Fungal growth was monitored by means of colony area measurements. The model of Baranyi and Roberts was further fitted to growth data to provide estimates of the colony area growth rate (cm2/day). Moreover, OTA analysis was undertaken for CY broth and agar as well as for grape berries and juice, on the 8th and 15th days of incubation at 25 °C. A significant reduction in fungal growth rate, final colony size and toxin production was observed in both liquid and solid media by the different yeast species assayed. The most competitive strains belonged to Saccharomyces, Pichia, Metschnikowia, Dekkera and Rhodotorula genera. Similar results were obtained from inoculated grape berries and grape juice. Specifically, Saccharomyces cerevisiae Y33 resulted in a decrease in fungal colony area of >90% and 93% after 3 and 4 days of co-culture, respectively. Similar results were obtained for OTA, where toxin concentration of the highest producer (A. carbonarius F3) was reduced from 14,983 and 31,565 ng/mL at 8 and 15 days, respectively, to 5 ng/mL and below detection limit (1 ng/g) when co-cultured with S. cerevisiae Y33. The results of this study could provide a pool of yeast species that must be further investigated for potential application as biological control agents at pre- and post-harvest level in wine and grape juice processing.

  • Presence of Anisakidae in commercial fish species imported into the Belgian food markets: A systematic review and meta-analyses
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-21
    E. Mercken, I. Van Damme, A. Serradell, S. Gabriël

    Anisakidae are marine zoonotic nematodes with most commercial fish species as intermediate hosts. Both public health risks and socio-economic problems are attributed to these larvae. Despite these concerns, the occurrence of Anisakidae in commercial fish species in Belgium remains unknown. Therefore, the main objective of this systematic review was to look into studies assessing the prevalence and intensity (level of infection) of Anisakidae in countries importing fish to the Belgian market. The databases of PubMed, Web of Science, Cordis, Google Scholar, Google, African Journals online and Asia Journals online were searched. Main eligibility criteria were: fish species consumed in Belgium; studies conducted in one of the main importing countries; and the availability of prevalence data. From the original 519 identified studies, 83 were included with data from Spain, Germany, Chile, Denmark, Turkey, France, China, England, Belgium, Norway, Iceland, Senegal and Sweden. Overall results show a widespread occurrence of Anisakidae with a high variability in prevalence between fish species and fishing sea. Cod (Gadus morhua) and Atlantic salmon (Salmo salar), the most consumed fish species in Belgium, have a mean prevalence of 33% and 5% respectively. Of all investigated fishing zones, fish caught in the Northeast Atlantic has the highest rate of infection (98%). Furthermore, higher prevalences were found when looking at the viscera (mean prevalence 59%) compared to the muscle (29%) and with superior techniques such as enzymatic digestion or UV press (46%) compared to candling, the routine method (23%). Farmed fish were found to be the least infected (2%) but were still not Anisakidae free. The widespread presence of Anisakidae and the associated food safety implications indicate the need to further investigate the presence of Anisakidae in fish in the Belgian market.

  • An evaluation of Lux technology as an alternative methodology to determine growth rates of Listeria in laboratory media and complex food matrices
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-13
    L. Nyhan, M. Begley, N. Johnson, M. Callanan

    Listeria monocytogenes is a foodborne pathogen which is a significant challenge in food production, particularly for ready-to-eat (RTE) products. Incidence of Listeria in food can be reduced by the application of multiple preservative factors or “hurdles”, which include acids, water activity and salts. Studying the growth of Listeria in complex foods is often reliant on laborious plate-counting techniques, therefore alternative methodologies are required. In this study we investigated the use of bioluminescence produced by chromosomally integrated genes encoding luciferase and its substrate to determine microbial growth rates in media and complex food matrices. Five Listeria innocua strains, used as a non-pathogenic surrogate for L. monocytogenes, were transformed with plasmid pPL2luxPhelp, resulting in a collection of Lux-tagged strains. Three test matrices (BHI broth, zucchini purée and béarnaise sauce) were adjusted, testing various combinations of pH (4.7–5.3), water activity (0.96 & 0.98) and undissociated acetic and propionic acid concentrations (0-2 mM). Adjusted matrices were inoculated with a cocktail of Lux-tagged strains and growth monitored over time by both bioluminescence and viable plate counts. Specific growth rates were calculated using both the bioluminescence and plate count data and compared. Statistical analysis showed that specific growth rates determined by bioluminescence did not significantly differ from those determined by plate counts (t-test, P > 0.05), while measurements of bias and accuracy showed good agreement between growth rates determined by both methods. Although plate counts remain the method of choice for detection of very low specific growth rates, this study demonstrates the potential of bioluminescence as a rapid alternative to plate counts for determining microbial growth rates in complex foods.

  • Analysing the impact of the nature of the nitrogen source on the formation of volatile compounds to unravel the aroma metabolism of two non-Saccharomyces strains
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-11
    Pauline Seguinot, Audrey Bloem, Pascale Brial, Emmanuelle Meudec, Anne Ortiz-Julien, Carole Camarasa

    Even though non-Saccharomyces yeasts were regarded as spoilage microorganisms for a long time, their abilities to improve and diversify the aromatic profile of wines is now well recognized. Consequently, their use in combination with S. cerevisiae strains during winemaking has attracted substantial attention over the last decade. However, our limited understanding of the metabolism and physiology of these species remains a barrier to promoting efficient exploitation of their full potential. In this study, we further explored the metabolism involved in the production of fermentative volatile compounds of two commercial non-Saccharomyces strains, T. delbrueckii Biodiva™ and M. pulcherrima Flavia®, in comparison with the reference wine yeast S. cerevisiae Lalvin EC1118®. After growing these strains in the presence of 24 different N-compounds, particular attention was paid to the influence of the nitrogen source on the profile of aroma compounds synthesized by these yeasts (higher alcohols and acids, medium-chain fatty acids and their acetate or ethyl esters derivatives). A comprehensive analysis of the dataset showed that these three species were able to produce all the fermentative aromas, regardless of the nitrogen source, demonstrating the key contribution of the central carbon metabolism to the formation of volatile molecules. Nevertheless, we also observed some specific phenotypic traits for each of the strains in their assimilation capacities for the various nitrogen nutrients as well as in their response to the nature of the nitrogen source in terms of the production of volatile molecules. These observations revealed the intricacy and interconnection between the networks involved in nitrogen consumption and aroma production. These differences are likely related to the genetic backgrounds of the strains. Overall, this study expands our understanding of the metabolic processes responsible for the formation of volatile compounds during wine fermentation and their variations according to species and the nature of the nitrogen source. This knowledge provides a new platform for the more efficient exploitation of non-Saccharomyces strains during winemaking, improving the management of the fermentation.

  • Optimization of Salmonella detection in garlic, onion, cinnamon, red chili pepper powders and green tea
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-11
    Virginie Barrere, Elizabeth Tompkins, Marcia Armstrong, Patrick Bird, Benjamin Bastin, Lawrence Goodridge

    Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30 mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23 mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in <24 h in the modified (BPW + L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.

  • Optimisation and evaluation of an automated system for extraction of viral RNA from oysters
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-07
    Sofia Persson, Louise Nybogård, Magnus Simonsson, Ronnie Eriksson

    The NucliSENS MiniMAG (Minimag) system from bioMérieux is widely used for extraction of viral RNA from oysters and is included as informative material in the ISO method for quantification of hepatitis A virus (HAV) and norovirus genogroups I and II (GI and GII) in food (ISO 15216-1:2017). However, the system is no longer on sale within the EU and alternative methods are therefore needed. We optimised and evaluated an automated benchtop system for extraction of viral RNA from oysters artificially contaminated with HAV, norovirus GI, norovirus GII and mengovirus, using the same reagents and a similar protocol as with the Minimag method. Using the automated system instead of Minimag increased measured viral concentration by on average 1.3 times, suggesting that the automated system extracts viral RNA more efficiently than Minimag. A drawback with the automated system was that it displayed higher variability in measured concentration for mengovirus. The median viral recovery was 17%, 37%, 44% and 41% for samples extracted with the automated system and 15%, 27%, 34% and 23% for samples extracted with Minimag for HAV, norovirus GI, norovirus GII and mengovirus, respectively. All samples displayed <75% inhibition in RT-qPCR when extracted with the automated system or Minimag. Together, these results suggest that the automated system can be a suitable alternative to Minimag in analysis of HAV, norovirus GI and norovirus GII in oysters. However, verification using naturally contaminated oysters is needed before it can be used for food safety control purposes.

  • Comparison of survival and heat resistance of Escherichia coli O121 and Salmonella in muffins
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-07
    Minto Michael, Jennifer Acuff, Keyla Lopez, Daniel Vega, Randall Phebus, Harshavardhan Thippareddi, Lakshmikantha H. Channaiah

    This study was conducted to validate a simulated commercial baking process for plain muffins against E. coli O121 (isolated from the recent illness outbreak associated with flour), and compare the thermal inactivation parameters (D- and z-values) of cocktails of four isolates of E. coli O121 and three serovars of Salmonella (Newport, Typhimurium, and Senftenberg) in muffin batter. Flour samples were spray inoculated with the E. coli O121 or Salmonella cocktails, dried back to the pre-inoculation weight to achieve ~7 log10 CFU/g, and used to prepare muffin batter. For the muffin baking validation study using E. coli O121, muffin batter was baked at 375 °F (190.6 °C) oven temperature for 21 min followed by 30 min of ambient cooling. The E. coli O121 population decreased by >7 log10 CFU/g in muffins by 17 min of baking, and was completely eradicated after 21 min of baking and ambient cooling. The D-values of E. coli O121 and Salmonella cocktails in muffin batter at 60, 65 and 70 °C were 42.0 and 38.4, 7.5 and 7.2, and 0.4 and 0.5 min, respectively; whereas the z-values of E. coli O121 and Salmonella were 5.0 and 5.2 °C, respectively.

  • Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented chia sourdough
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-06
    Stefania Dentice Maidana, Cecilia Aristimuño Ficoseco, Daniela Bassi, Pier Sandro Cocconcelli, Edoardo Puglisi, Graciela Savoy, Graciela Vignolo, Cecilia Fontana

    Chia, is a gluten-free, rich in proteins, oilseed that is “on trend” as an alternative ingredient in food production, adding nutritional value. As a reservoir of natural biodiversity, lactic acid bacteria development, during spontaneous chia flour fermentation (sourdough) for 10 days, were investigated by culturing and high throughput sequencing (HTS). Culture-dependent analysis showed a rapid increase in total LAB numbers from the second day of sourdough refreshment. Taxonomical identification of LAB isolates by rep-PCR and further 16S rRNA sequencing was performed. Besides Among identified LAB by culture-dependent approach, species from genus Enterococcus were the most abundant; Lactococcus (Lc. lactis), Lactobacillus (L. rhamnosus) and Weissella (W. cibaria) species were also isolated. By HTS, twelve OTUs belonging to LAB genera were identified during chia sourdough fermentation with an increased Lactobacillus diversity. Enterococcus (E.) faecium, E. mundtii, W. cibaria and L. rhamnosus were detected as dominant species in the final propagation stages while Bacillus and Clostridium were mostly present during first fermentation stages. The investigation of biotechnological and safety traits (acidification ability, protein hydrolysis, exopolysaccharides production, antimicrobial activity and antibiotic resistance) of 15 representative LAB strains was performed. Strains characterization led to the selection of Lc. lactis CH179, L. rhamnosus CH34 and W. cibaria CH28 as candidates to be used as novel functional starter culture for gluten-free chia fermented products. As far as we know, this is the first study providing information on the molecular inventory of LAB population during spontaneous fermentation of chia sourdough.

  • Tracking bacteriome variation over time in Listeria monocytogenes-positive foci in food industry
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-06
    Pedro Rodríguez-López, Juan José Rodríguez-Herrera, Marta López Cabo

    The variation in microbial composition over time was assessed in biofilms formed in situ on selected non-food and food contact surfaces of meat and fish industries, previously identified as Listeria monocytogenes-positive foci. First, all samples were analysed for the detection and quantification of L. monocytogenes using ISO 11290-1 and ISO 11290-2 norms, respectively. Although the pathogen was initially detected in all samples, direct quantification was not possible. Psychrotrophic bacteria counts were among resident microbiota in meat industry samples (Meanmax = 6.14 log CFU/cm2) compared to those form fish industry (Meanmax = 5.85 log CFU/cm2). Visual analysis of the biofilms using epifluorescence microscopy revealed a trend to form microcolonies in which damaged/dead cells would act as anchoring structures. 16S rRNA gene metagenetic analysis demonstrated that, although Proteobacteria (71.37%) initially dominated the bacterial communities at one meat industry location, there was a dramatic shift in composition as the biofilms matured, where Actinobacteria (79.72%) became the major phylum present in later samples. This change was largely due to an increase of Norciardiaceae, Micrococcaceae and Microbacteriaceae. Nevertheless, for the other sampling location, the relative abundance of the dominating phylum (Firmicutes) remained consistent over the entire sampling period (Mean = 63.02%). In fish industry samples, Proteobacteria also initially dominated early on (90.69%) but subsequent sampling showed a higher diversity in which Bacteroidetes and Proteobacteria were the most abundant phyla accounting for the 48.04 and 37.98%, respectively by the last sampling period. Regardless of the location, the community profiles of the endpoint samples were similar to those reported previously. This demonstrated that in a given industrial setting there is a trend to establish a determinate biofilm structure due to the environmental factors and the constant incoming microbiota. This information could be used to improve the existing sanitisation protocols or for the design of novel strategies.

  • Growth behaviour and thermal inactivation of E. coli O157:H7 and Salmonella spp. in ground lean camel meat
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-06
    Tareq M. Osaili, Anas A. Al-Nabulsi, Dinesh Kumar Dhanasekaran, Fayeza Hasan, Sowmya Rao, Hera Fatima, Mutamed Ayyash, Richard Holley, Reyad S. Obaid

    This study aimed to evaluate growth behaviour of Escherichia coli O157:H7, Salmonella spp., and background microbiota in ground lean camel meat during refrigerated storage for up to 7 d, and determine thermal inactivation parameters (D and z-values) of the pathogens in the meat. Fresh ground camel meat samples were individually inoculated with two isolates of each E. coli O157:H7 strains and Salmonella serovars. Inoculated and uninoculated samples were stored aerobically at 4 °C and abusive temperature (10 °C) for 1, 4 and 7 d. Inoculated samples were also heat-treated in a circulating water bath at 55–65 °C. Upon storage for 7 d at 4 °C, the population of E. coli O157:H7 and Salmonella cells decreased significantly in samples. In contrast, samples stored at 10 °C showed significant increases in microbial populations. With storage, the populations of mesophilic lactic acid bacteria, mesophilic total plate counts, yeasts and molds, Pseudomonas spp., as well as Enterobacteriaceae increased significantly. E. coli O157:H7 strains showed average D-values at 57.5 to 65 °C ranging from 1.8 to 0.067 min while Salmonella spp. at 55 to 62.5 °C showed D-values ranging from 2.2 to 0.057 min. The z-values of E. coli O157:H7 strains were 4.6 and 5.0 °C and were 5.1 and 5.5 °C for Salmonella spp. This study provides information to assist food industry and retail meat processors to enhance safety and storage quality of camel meat and facilitate validation of thermal processing of camel meat products.

  • Sanitization of packaging and machineries in the food industry: Effect of hydrogen peroxide on ascospores and conidia of filamentous fungi
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-05
    Nicoletta Scaramuzza, Cigarini Massimo, Paola Mutti, Elettra Berni

    In the food industry, sterilization of packaging and filling machines by hydrogen peroxide (HP) is a widespread practice. Its effectiveness is usually tested by means of inactivation tests on selected test microorganisms that were any case chosen without taking into account that food products could be also spoiled by microorganisms presumably resistant to HP. For this reason, the aim of this work was to assess the resistance of different ascospore-forming moulds (Talaromyces bacillisporus, Aspergillus hiratsukae, Chaetomium globosum) to HP, in order to find the most resistant to this kind of chemical stress, and to compare their resistance with that registered for other moulds, including test microorganism Aspergillus brasiliensis ATCC 16404. Tests were carried out from 50 to 60 °C on spores or conidia, depending on the strain, either by immersing inoculated strips (aluminium, tin-plate, HDPE, PET) in HP, or by directly inoculating cells in the sanitizing medium. In both tests, T. bacillisporus proved the most resistant strain, followed by A. hiratsukae, C. globosum and A. brasiliensis at all temperatures tested. In test without a supporting material, D values of T. bacillisporus varied from 6 to 23 s. In test with metallic or plastic strips, D values of T. bacillisporus were higher on plastic materials, compared to those obtained on metallic ones up to 53 °C, whereas at higher temperatures D values proved similar. For A. hiratsukae, D values were similar if different materials were compared, except for D50 on aluminium and HDPE, which proved slightly higher (3.1–3.4 s) than those obtained on tin-plate and PET (2.7–2.8 s). Analogously, ascospores of C. globosum behaved in a similar way if different materials were compared, except for D50 values that varied in a wide range (from 2.9 s on tin-plate to 4.0 s on HDPE). A. brasiliensis was rapidly inactivated by the synergistic effect of heat and hydrogen peroxide, so for this strain it was not possible to calculate any D value. Based on the results obtained in this paper, tested ascospore-forming moulds proved to be sensibly more resistant to HP than other heat-sensitive strains tested, their D values always being significantly higher, regardless of the strain considered and the supporting material assessed. Ascospore-forming moulds could be furtherly investigated, as for practical purposes they seemed most suitable as target microorganisms than heat-sensitive microorganisms such as Aspergillus niger ATCC 6275 or Aspergillus brasiliensis ATCC 16404, their use during bio-validations of sanitizing processes on machineries used for refrigerated products (pH > 4.5) or non-refrigerated acid products (pH ≤ 4.5) leading to more performing results.

  • Properties and potential food applications of lauric arginate as a cationic antimicrobial
    Int. J. Food Microbiol. (IF 4.006) Pub Date : 2019-11-05
    Qiumin Ma, P. Michael Davidson, Qixin Zhong

    Lauric arginate (LAE, ethyl-Nα-lauroyl-L-arginate hydrochloride) is synthesized from food components lauric acid and L-arginine and is quickly hydrolyzed to lauric acid and L-arginine in vivo. The antimicrobial properties and low toxicity are the basis for approval as a generally recognized as safe (GRAS) preservative at a level of up to 200 ppm in certain food products in the United States such as meat, poultry, and cheese and a safe food preservative up to 225 ppm in the European Union. These developments have generated great interest to apply LAE to improve the safety and quality of food products. In the present review, physicochemical and toxicological properties are first discussed. Antimicrobial properties and mechanisms of LAE in microbiological media, and antimicrobials used in combination with LAE aiming to achieve synergistic activities are then reviewed. The physical basis of reduced antimicrobial activities of LAE in food matrices is discussed, and studies applying LAE in meat, poultry, dairy, produce, and low-moisture foods and food-contact surfaces are summarized. Antimicrobial properties of LAE in emulsion systems and potential packaging films are also discussed for potential novel applications to improve the application in food systems. Finally, the possible impact of LAE on food sensory properties is reviewed along with some perspectives on research needs in the science and technology of LAE for use as a food antimicrobial preservative.

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