Anise essential oil immobilized in chitosan microparticles: a novel bactericidal material for food protection.

Foodborne infections in humans are one of the major concerns of the food industries, especially for minimally processed foods (MPF). Thereby, the packaging industry applies free chlorine in the sanitization process, ensuring the elimination of any fecal coliforms or pathogenic microorganisms. However, free chlorine's propensity to react with organic matter, forming toxic compounds such as trihalomethanes and haloacetic acid. Therefore, the present work aimed to synthesize a novel organic biomaterial as an alternative to free chlorine. Chitosan microparticles were produced, with Pimpinella anisum (anise) essential oil immobilized in the biopolymer matrix (MPsQTO). The characterization of this biomaterial was done through GC-MS/MS, FT-IR, and SEM. Antimicrobial assays proved that the MPsQTO presented antibacterial activity for Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, and Bacillus subtilis at 300 µL mL-1 of concentration. The fluorescence microscope also showed the MPsQTO targets the cytoplasmatic membrane, which is responsible for cell death in the first minutes of contact. Studies with the mutant B. subtilis (amy::pspac-ftsZ-gfpmut1) and the Saccharomyces cerevisiae D7 also proved that the biomaterial did not affect the genetic material and did not have any mutagenic/carcinogenic effect on the cells. The sanitization assays with pumpkin MPF proved that the MPsQTO is more effective than free chlorine, increasing the shelf-life of the MPF. Consequently, the novel biomaterial proposed in this work is a promising alternative to traditional chemical sanitizers.

Whole-Genome Sequencing-Based Characterization of Listeria Isolates from Produce Packinghouses and Fresh-Cut Facilities Suggests Both Persistence and Reintroduction of Fully Virulent L. monocytogenes.

The contamination of ready-to-eat produce with Listeria monocytogenes (LM) can often be traced back to environmental sources in processing facilities and packinghouses. To provide an improved understanding of Listeria sources and transmission in produce operations, we performed whole-genome sequencing (WGS) of LM (n = 169) and other Listeria spp. (n = 107) obtained from 13 produce packinghouses and three fresh-cut produce facilities. Overall, a low proportion of LM isolates (9/169) had inlA premature stop codons, and a large proportion (83/169) had either or both of the LIPI-3 or LIPI-4 operons, which have been associated with hypervirulence. The further analysis of the WGS data by operation showed a reisolation (at least 2 months apart) of highly related isolates (<10 hqSNP differences) in 7/16 operations. Two operations had highly related strains reisolated from samples that were collected at least 1 year apart. The identification of isolates collected during preproduction (i.e., following sanitation but before the start of production) that were highly related to isolates collected during production (i.e., after people or products have entered and begun moving through the operation) provided evidence that some strains were able to survive standard sanitation practices. The identification of closely related isolates (<20 hqSNPs differences) in different operations suggests that cross-contamination between facilities or introductions from common suppliers may also contribute to Listeria transmission. Overall, our data suggest that the majority of LM isolates collected from produce operations are fully virulent and that both persistence and reintroduction may lead to the repeat isolation of closely related Listeria in produce operations. IMPORTANCE Listeria monocytogenes is of particular concern to the produce industry due to its frequent presence in natural environments as well as its ability to survive in packinghouses and fresh-cut processing facilities over time. The use of whole-genome sequencing, which provides high discriminatory power for the characterization of Listeria isolates, along with detailed source data (isolation date and sample location) shows that the presence of Listeria in produce operations appears to be due to random and continued reintroduction as well as to the persistence of highly related strains in both packinghouses and fresh-cut facilities. These findings indicate the importance of using high-resolution characterization approaches for root cause analyses of Listeria contamination issues. In cases of repeat isolation of closely related Listeria in a given facility, both persistence and reintroduction need to be considered as possible root causes.

Evaluation of calcium hydroxide, calcium hypochlorite, peracetic acid, and potassium bicarbonate as citrus fruit sanitizers.

Xanthomonas citri (X. citri) is a quarentenary plant pathogen and the causal agent of the citrus canker. X. citri forms biofilms and remains fixed on the surface of plant tissues, especially on leaves and fruits. Considering this, all the citrus fruits have to be sanitized before they can be commercialized. NaOCl is the main sanitizer used to decontaminate fruits in the world. Due to its toxicity, treatment with NaOCl is no longer accepted by some Europe Union countries. Therefore, the aim of this work was to evaluate potassium bicarbonate (KHCO3), calcium hydroxide (Ca(OH)2), calcium hypochlorite (Ca(OCl)2) and peracetic acid (CH3CO3H) as alternatives to NaOCl for the sanitization of citrus fruit. By monitoring cell respiration and bacterial growth, we determined that peracetic acid and calcium hypochlorite exhibit bactericidal action against X. citri. Time-response growth curves and membrane integrity analyses showed that peracetic acid and calcium hypochlorite target the bacterial cytoplasmatic membrane, which is probably responsible for cell death in the first minutes of contact. The simulation of the sanitization process of citrus fruit in packinghouses showed that only peracetic acid exhibited a performance comparable to NaOCl. Among the tested compounds, peracetic acid constitutes an efficient and safer alternative to NaOCl.

Survival of Salmonella enterica and shifts in the culturable mesophilic aerobic bacterial community as impacted by tomato wash water particulate size and chlorine treatment.

Particulates of harvest debris are common in tomato packinghouse dump tanks, but their role in food safety is unclear. In this study we investigated the survival of Salmonella enterica and the shifts in relative abundance of culturable mesophilic aerobic bacteria (cMAB) as impacted by particulate size and interaction with chlorine treatment. Particulates suspended in grape tomato wash water spanned a wide size range, but the largest contribution came from particles of 3-20 µm. Filtration of wash water through 330 µm, applied after 100 mg/L free chlorine (FC) wash, reduced surviving cMAB by 98%. The combination of filtration (at 330 µm or smaller pore sizes) and chlorinated wash also altered the cMAB community, with the survivors shifting toward Gram-positive and spore producers (in both lab-simulated and industrial conditions). When tomatoes and harvest debris inoculated with differentially tagged Salmonella were washed in 100 mg/L FC for 1 min followed by filtration, only cells originating from harvest debris survived, with 85 and 93% of the surviving cells associated with particulates larger than 330 and 63 µm, respectively. This suggests that particulates suspended in wash water can protect Salmonella cells from chlorine action, and serve as a vector for cross-contamination.

Prevalence of Listeria spp. in produce handling and processing facilities in the Pacific Northwest.

Listeria monocytogenes is a significant concern for the produce industry; however, there is limited information to support the practical decision-making to mitigate this risk. This study investigated the prevalence of Listeria spp. and L. monocytogenes in seven produce handling and processing (PHP) facilities in the Pacific Northwest. PHP facilities were defined as facilities that receive raw agricultural commodities and further handle, pack, wash, or process prior to distribution into the retail sector. Environmental swabs (n = 50/facility) were collected in high-risk areas (e.g., near raw product entry points) from seven PHP facilities over two visits. Listeria spp. were isolated using modified ISO 11290-1 method and speciated with Microgen® Listeria-ID. Listeria spp., including L. monocytogenes, were found in 5/7 PHP. Prevalence of Listeria spp. ranged from 2% to 26% in these five facilities. Drains, entry areas, and portable equipment consistently tested positive for Listeria spp. during active production. Two additional sampling rounds (n = 50/round) were conducted in the highest prevalence facility (Facility #1). Overall, Listeria spp. were detected in 44/150 (29.3%) swabs collected from Facility #1. This study demonstrated the high prevalence of Listeria spp. near raw product entry points across PHP facilities.

Effects of packinghouse operations on the flavor of 'Orri' mandarins.

Mandarins have a delicate flavor and are easy to peel and easy to consume. However, they are relatively perishable and suffer from flavor deterioration after harvest. The goal of the current study was to examine the effects of commercial packinghouse operations on the flavor of 'Orri' mandarins. For that purpose, we collected fruit from four different points along a commercial citrus packing line: (1) directly from the harvest bin, (2) after application of a hot (53°C) fungicide treatment for 30 s, (3) after waxing, and (4) after waxing and after the fruit had passed through a hot-air drying tunnel (37°C) for 2 min. The collected fruit were stored for 3 or 6 weeks at 5°C and then kept for five more days under shelf-life conditions at 22°C. The observed results indicate that the hot fungicide treatment had no effect on flavor quality. However, the waxing and waxing +drying treatments resulted in significant increases in ethanol accumulation, lower flavor-acceptability scores, and increased off-flavors. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that the waxing and waxing +drying treatments resulted in particular increases in the levels of alcohol and ethyl ester volatiles; whereas levels of other aroma volatiles (i.e., esters, aldehydes, monoterpenes, and sesquiterpenes) decreased after storage in all fruit samples. Overall, the waxing process in commercial citrus packinghouses increased ethanol and ethyl ester volatile levels and harmed flavor acceptability. These findings demonstrate the need to identify new wax formulations that do not hamper fruit-flavor quality.

Genetic Diversity of Listeria monocytogenes Isolated From Three Commercial Tree Fruit Packinghouses and Evidence of Persistent and Transient Contamination.

Whole genome analysis was performed on 501 isolates obtained from a previous survey which recovered 139 positive environmental sponge samples (i.e., up to 4 isolates per sample) from a total of 719 samples collected at 40 standardized sites in 3 commercial apple packinghouse facilities (i.e., P1, P2, and P3) over 3 successive seasons in a single production year. After excluding duplicated isolates, the data from 156 isolates revealed the clonal diversity of L. monocytogenes and allowed the detection of transient contamination, persistent contamination, and cross-area transmission events. Facility P2 with the poorest sanitary conditions had the least diversity (Shannon's index of 0.38). P2 contained a Clonal Complex (CC) 554, serogroup IVb-v1 strain that persisted throughout the year and spread across the entire facility, a singleton Sequence Type (ST) 1003, lineage III strain that persisted through two seasons and spread across two areas of the facility, and 3 other clones from transient contaminations. P1 and P3, facilities with better sanitary conditions, had much higher diversity (i.e., 15 clones with a Shannon's index of 2.49 and 10 clones with a Shannon's index of 2.10, respectively) that were the result of transient contamination. Facilities P1 and P3 had the highest incidence (43.1%) of lineage III isolates, followed by lineage I (31.3%) and lineage II (25.5%) isolates. Only 1 isolate in the three facilities contained a premature stop codon in virulence gene inlA. Fourteen samples yielded 2-3 clones per sample, demonstrating the importance of choosing appropriate methodologies and selecting a sufficient number of isolates per sample for studying L. monocytogenes diversity. Only 1 isolate, belonging to CC5 and from facility P3, contained a known plasmid, and this was also the only isolate containing benzalkonium chloride tolerance genes. The persistent CC554 strain did not exhibit stronger sanitizer resistance than other isolates and did not contain any confirmed molecular determinants of L. monocytogenes stress resistance that were differentially present in other isolates, such as genes involved in sanitizer tolerance, heavy metal resistance, biofilm-forming, stress survival islet 1 (SSI-1), stress survival islet 2 (SSI-2) or Listeria genomic island (LGI2).

Prevalence and Distribution of Listeria monocytogenes in Three Commercial Tree Fruit Packinghouses.

A 2-year longitudinal study of three tree fruit packinghouses was conducted to determine the prevalence and distribution of Listeria monocytogenes. Samples were collected from 40 standardized non-food-contact surface locations six different times over two 11-month production seasons. Of the 1,437 samples collected, the overall prevalence of L. monocytogenes over the course of the study was 17.5%. Overall prevalence did not differ significantly (p > 0.05) between each year. However, values varied significantly (p ≤ 0.05) within each production season following packing activity levels; increasing in the fall, peaking in early winter, and then decreasing through spring. L. monocytogenes was most often found in the packing line areas, where moisture and fruit debris were commonly observed and less often in dry cold storage and packaging areas. Persistent contamination was attributed to the inability of water drainage systems to prevent moisture accumulation on floors and equipment during peak production times and uncontrolled employee and equipment traffic throughout the facility. This is the first multiyear longitudinal surveillance study to compare L. monocytogenes prevalence at standardized sample sites common to multiple tree fruit packinghouses. Recommendations based on our results will help packinghouse operators to identify critical areas for inclusion in their L. monocytogenes environmental monitoring programs.

Listeria monocytogenes Survival on Peaches and Nectarines under Conditions Simulating Commercial Stone-Fruit Packinghouse Operations.

Recent recalls of stone fruit due to potential Listeria contamination and associated foodborne outbreaks highlight the risk for pathogen transmission through stone-fruit consumption. Particularly, surface contamination of fruits increases the risk for cross-contamination of produce during processing and storage. This highlights the need for quality control in stone fruits intended for consumption. To develop effective food safety practices, it is essential to determine the critical factors during stone-fruit processing that influence Listeria survival. Therefore, this study evaluated the ability of Listeria to survive on peaches and nectarines under simulated stone-fruit loading and staging, waxing and fungicide application and storage conditions. The results of our study indicate that current stone-fruit handling conditions do not favor Listeria growth. However, once fruit is contaminated, Listeria can survive on the fruit surface in significant numbers under current processing conditions. Therefore, there is a need to develop and implement preventive controls at the stone-fruit packinghouse to prevent Listeria contamination and deter pathogen persistence.

Chlorine Concentration and the Inoculation of Tomato Fruit in Packinghouse Dump Tanks.

Chlorine concentrations (pH 6 to 7 and 22 to 27°C) that killed arthrospores (spores) of Geotrichum candidum or sporangioles (spores) of Rhizopus stolonifer, causal agents of sour rot and Rhizopus rot, respectively, in moving water within 30 to 45 s did not prevent these pathogens from inoculating wounded tomatoes (Lycopersicon esculentum) in a water flume containing chlorine and spores. Free chlorine concentrations of 20 or 25 mg/liter were lethal to spores of G. candidum within 30 s in most in vitro tests, whereas spores of R. stolonifer were slightly less sensitive. Wounded tomatoes placed in a flume with free chlorine at 30 mg/liter and then exposed to spores for 1 min developed about 50% less decay incidence during storage at 24°C for 6 days than did fruit exposed to spores and water alone. In the absence of chlorine, incidence averaged 57% (range, 15 to 95%) for R. stolonifer and 38% (range, 17 to 58%) for G. candidum. Sporadic sour rot lesions were observed among fruit that had been treated with free chlorine at 75 mg/liter, whereas chlorine at up to 180 mg/liter failed to completely protect fruit from Rhizopus rot. A water-soluble dye rapidly penetrated wounds on tomato fruit. The dye framed the outlines of cells at the wound surface and appeared to penetrate into a few intercel-lular spaces. Application of 1% sodium hypochlorite decolorized the dye on the wound surface, whereas deposits located below the wound surface remained blue. Thus, spores suspended in moving water can escape the action of chlorine if carried into intercellular spaces by diffusion or by capillary movement of cell sap and water.

Beneficiamento pós-colheita de lima ácida 'Tahiti' afeta sua qualidade e conservação / Postharvest packing process of 'Tahiti' acid lime affects their quality and conservation

RESUMO: O objetivo deste trabalho foi determinar os efeitos das etapas de beneficiamento pós-colheita de lima ácida 'Tahiti' na sua qualidade e conservação. Foram utilizados frutos colhidos por torção, em estádio de maturação fisiológica e beneficiados de acordo com o procedimento para frutos de exportação. Foram estabelecidos cinco pontos de coleta de frutos ao longo do processo de beneficiamento, os quais correspondem aos tratamentos estudados: "Campo"- coleta diretamente na planta; "Recepção"- na área de recepção da unidade de beneficiamento; "Repouso"- após a primeira etapa do beneficiamento; "Beneficiamento completo"- após o beneficiamento completo; e "Centro de comercialização"- na recepção do centro de comercialização. Foram conduzidas análises físicas e químicas durante 20 dias de armazenamento a 22±2ºC e 70±5% de UR. Os frutos que passaram pelo processo de beneficiamento completo e os que foram coletados no centro de comercialização apresentaram maior perda de massa fresca, maior incidência de oleocelose, menor porcentagem de frutos em condição de comercialização ao final do período de armazenamento, escurecimento da polpa e maior degradação de ácido ascórbico. A qualidade e conservação dos frutos de lima ácida 'Tahiti' foram prejudicadas pela movimentação, manipulação e injúrias mecânicas que ocorrem durante as etapas de beneficiamento pós-colheita.

ABSTRACT: This study aimed to determine the effects of postharvest packing line stages of 'Tahiti' acid lime on their quality and conservation. Fruits were harvested by twisting in physiological maturation stage and packed according to the procedure for export fruits. Five sampling points were set throughout the packing line, which correspond to the treatments studied: "Field"- sampling directly in the plant; "Reception"- collection in the reception area of the packinghouse; "Rest"- collection after the first stage of the packing process; "Complete packing"- collection at the end of the packing process; "Marketing center" and collects at the reception area of the marketing center. Physical and chemical analyzes were conducted for 20 days of storage at 22±2ºC and 70±5% RH. Fruits that have gone through the complete packing process and those collected in the marketing center had higher fresh mass loss, higher incidence of oleocellosis, lower percentage of fruit on condition of marketing at the end of the storage period, pulp browning and higher degradation of ascorbic acid. Quality and conservation of 'Tahiti' lime fruits were affected by movement, handling and mechanical injuries that occur during postharvest processing.