Antimicrobial activity of various essential oils and their application in active packaging of frozen vegetable products.

Essential oils (EOs) have potential utility as clean-label food preservatives due to their antimicrobial and antioxidant properties. In this study, various EOs were screened for their antimicrobial activities against Listeria grayi in vitro. The susceptibility of L. monocytogenes to select EOs was compared with that of L. grayi. The effectiveness of the selected EOs in inhibiting the growth of L. grayi on vegetable products was also investigated. The results showed that cinnamon and oregano EOs and carvacrol were effective in the vapor phase in inhibiting the growth of L. grayi as well as L. monocytogenes, with the susceptibility of L. monocytogenes to cinnamon EO being slightly higher than that of L. grayi. The packaging of green peppers with cellulose stickers impregnated with cinnamon EO at 556 µL/Lheadspace reduced the Listeria count to 1 log CFU/g after 2 days of storage as compared to 7.5 log CFU/g for controls.

Effects of salt concentration on the quality of paocai, a fermented vegetable product from China.

BACKGROUND: Paocai is a traditional Chinese fermented vegetable food. As the most important ingredient, salt has crucial effects on the bacterial community and volatile compounds of paocai. To demonstrate the effects of salt on the fermentation of paocai, the bacterial composition and volatile compounds were investigated using high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS). RESULTS: The salt had no significant effects on the bacterial community at the phylum level. Proteobacteria and Bacteroidetes gradually decreased during the fermentation, and Firmicutes gradually increased as the dominant bacteria in the late stage of fermentation. At the genus level, Lactobacillus and Lactococcus gradually increased in relative abundance during the fermentation and became the dominant bacteria in paocai. High salt levels can contribute to the growth of Lactobacillus, which became the dominant genus in paocai. The salt concentration affected the profiles of volatile compounds in paocai after fermentation. A total of 42 volatile components were detected by GC-MS, among which phenols, aldehydes, and nitriles were the main ones. A high salt concentration will increase the volatile compound content, mainly aldehydes and alcohols, and improve the flavor of paocai. At the same time, the electronic tongue analysis also showed that a high salt concentration made a major contribution to the flavor of paocai. CONCLUSIONS: These data are helpful to elucidate the effects of salt on the quality of paocai and contribute to improving the quality and reducing the use of salt. © 2021 Society of Chemical Industry.

Contribution of autochthonous yeasts with probiotic potential to the aroma profile of fermented Guajillo pepper sauce.

BACKGROUND: Three yeast strains with probiotic potential, Hanseniaspora opuntiae, Pichia kudriavzevii, and Wickerhamomyces anomalus were inoculated in the fermentation of Guajillo chilli pepper (Capsicum annuum L.) sauce, and the different aroma profiles were investigated. Using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis and gas chromatography-olfactometry (GCO), flavour compound production was evaluated during the fermentation of the Guajillo chilli pepper sauces. RESULTS: A total of 78 volatile compounds were identified during the yeast fermentation of the sauce. Most aldehydes and terpenes detected were present at the beginning of the fermentation, indicating a Guajillo chilli pepper origin. Among the 34 active aroma compounds detected by GCO, propanoic acid (cheesy), 3-methylbutanoic acid (sharp, cheese), ethyl 2-methylbutanoate (fruity), and 6-methyl-5-hepten-2-one (strong, citrus) were identified as key aroma contributors produced by the inoculation of the yeasts. A different aroma profile was produced by probiotic yeast. Hanseniaspora opuntiae produced an aroma profile with herbal and green notes based on high production of aldehydes, ketones, and acetic acid. Pichia kudriavzevii and W. anomalus produced fruity, green-herbal, and cheesy notes based on ester compounds, alcohol and branched-chain acids production although, the production of propanoic acid by W. anomalus increased the cheesy character in the sauces. CONCLUSION: The aroma profile of fermented chilli pepper sauces depends not only on the chili pepper varieties used but also on the fermentation process as a source of aroma compounds. The use of probiotic yeast can be used to improve and diversify the aroma profile of fermented chilli pepper sauces. © 2020 Society of Chemical Industry.

Fate of Spoilage and Pathogenic Microorganisms in Acidified Cold-Filled Hot Pepper Sauces.

Consumption of spicy foods and hot sauces is currently a popular trend worldwide. Shelf-stable acidified sauces are commonly hot-filled to ensure commercial sterility, but cold-fill-hold processes might also be suitable if microbial safety and stability are ensured. For this study, model acidified hot pepper sauces were developed and characterized. The effects of sauce pH and of two different organic acids on the survival of Pichia manshurica and Lactobacillus curvatus isolated from contaminated commercial hot sauces and on pathogenic Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes were assessed. Full factorial designs with three levels for pH (3.2, 3.5, and 3.9) and two for organic acid (citric and acetic) were used to determine the effects of these factors and their interactions on the survival of the microorganisms. Commercially sterile sauces were independently inoculated and kept at ambient temperature. Microbial counts were determined at different sampling times, depending on the treatment evaluated. Sauces acidified to pH 3.2 with citric or acetic acid were inoculated with cocktails of five strains or serotypes of the three pertinent pathogens, and inactivation curves were determined. Trials were performed in triplicate. A greater than 5-log reduction of P. manshurica and L. curvatus was achieved in less than 6 h in sauces adjusted to pH 3.2 with acetic acid. Greater than 5-log reductions of pathogenic bacteria were achieved 0.5 h after inoculation in sauces acidified to pH 3.2 with acetic acid. In contrast, at least 48 h was required to guarantee the same inactivation for the most tolerant pathogen when citric acid was used. Thus, a cold-fill-hold process may be a suitable alternative for acidified hot pepper sauces. Based on survival of the microorganisms evaluated in this study, microbial safety and stability can be achieved by adjusting the pH to 3.2 or less by the addition of acetic acid.

Comparative Effects of Thermal, High Hydrostatic Pressure, and UV-C Processing on the Quality, Nutritional Attributes, and Inactivation of Escherichia coli, Salmonella, and Listeria Introduced into Tiger Nut Milk.

HIGHLIGHTS: Thermal and nonthermal methods can support a 5-log CFU reduction of model bacteria introduced into tiger nut milk. Thermal treatment of tiger nut milk results in significant loss of protein, antioxidants, and quality properties. HHP or UV-C treatment of tiger nut milk retains quality and nutritional characteristics. HHP or UV-C are suitable for the pasteurization of tiger nut milk.

Occurrence of Cronobacter Spp. in Ready-to-Eat Vegetable Products, Frozen Vegetables, and Sprouts Examined Using Cultivation and Real-Time PCR Methods.

Environmental matrices and food products are hypothesized to be sources of Cronobacter spp. The severity of neonatal infections, increasing number of cases in elderly and immunocompromised individuals, as well as isolation of Cronobacter spp. from clinical materials demands that more attention should be paid to Cronobacter spp. detection and occurrence of the bacteria in food products. Here, a total of 175 samples of ready-to-eat vegetables, frozen vegetables, and sprouted seeds were collected during a period of 1 year and examined for the presence of Cronobacter spp. using a cultivation method with two different sample preparations and real-time polymerase chain reaction (qPCR). In total, Cronobacter spp. were detected in 22.3% of tested samples using cultivation. In comparison, direct qPCR detected Cronobacter spp. in 37.7% of these samples (p < 0.01; Fisher's exact test) and the numbers of genome equivalents per gram reached 108 in some samples of sprouts. Cronobacter spp. were isolated from 51.4%, 37.2%, and 5.2% samples of sprouts, frozen vegetables, and cut green leaves/salads, respectively. Using qPCR, the most frequently contaminated sample types were sprouts (91.4%) and frozen vegetables (60.5%), whereas the rate of positivity for cut green leaves/salads was, in comparison, only 8.2% (p < 0.01; χ2 -test for independence). PRACTICAL APPLICATION: This study provided valuable information on the occurrence of Cronobacter spp. in ready-to-eat vegetables using cultivation and qPCR. Cronobacter spp. are emerging opportunistic pathogens that can be present in food of plant origin. Cronobacter spp. were isolated from sprouts, frozen vegetables, and cut green leaves/salads, and the numbers of genome equivalents per gram reached 108 in some samples of sprouts.

16S rRNA Gene Primer Validation for Bacterial Diversity Analysis of Vegetable Products.

High-throughput sequencing of the 16S rRNA gene enhances understanding of microbial diversity from complex environmental samples. The 16S rRNA gene is currently the most important target in bacterial evolution and ecology studies, particularly for determination of phylogenetic relationships among taxa, exploration of bacterial diversity in a given environment, and quantification of the relative abundance of taxa at various levels. However, some parts of the conserved region of the bacterial 16S rRNA gene are similar to the conserved regions of plant chloroplasts and eukaryotic mitochondria. Therefore, if DNA contains a large amount of nontarget DNA, this nontarget DNA can be coamplified and consequently produce useless sequence reads. We experimentally assessed the primer pair 335f/769r and the widely used bacterial primer pair SD (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21). The primer pair 335f/769r was examined for its ability to amplify bacterial DNA in plant and animal feed samples by using the single-strand confirmation polymorphism method. In our present study, these primer pairs were validated for microbial community structure analysis with complex food matrices by using next-generation sequencing. The sequencing results revealed that the primer pair 335f/769r successfully resulted in fewer chloroplast and mitochondrial sequence reads than generated by the universal primer pair SD and therefore is comparatively suitable for metagenomic analyses of complex food matrices, particularly those that are rich in plant DNA. Additionally, some taxonomic groups were missed entirely when only the SD primer pair was used.

Complete genome analysis of Lactobacillus fermentum SK152 from kimchi reveals genes associated with its antimicrobial activity.

Research findings on probiotics highlight their importance in repressing harmful bacteria, leading to more extensive research on their potential applications. We analysed the genome of Lactobacillus fermentum SK152, which was isolated from the Korean traditional fermented vegetable dish kimchi, to determine the genetic makeup and genetic factors responsible for the antimicrobial activity of L. fermentum SK152 and performed a comparative genome analysis with other L. fermentum strains. The genome of L. fermentum SK152 was found to comprise a complete circular chromosome of 2092 273 bp, with an estimated GC content of 51.9% and 2184 open reading frames. It consisted of 2038 protein-coding genes and 73 RNA-coding genes. Moreover, a gene encoding a putative endolysin was found. A comparative genome analysis with other L. fermentum strains showed that SK152 is closely related to L. fermentum 3872 and F-6. An evolutionary analysis identified five positively selected genes that encode proteins associated with transport, survival and stress resistance. These positively selected genes may be essential for L. fermentum to colonise and survive in the stringent environment of the human gut and exert its beneficial effects. Our findings highlight the potential benefits of SK152.

A novel probiotic Bacillus siamensis B44v isolated from Thai pickled vegetables (Phak-dong) for potential use as a feed supplement in aquaculture.

The use of probiotic bacteria to control bacterial infection in farmed fish is of clear practical interest. The aims of this study were to isolate and select a probiotic Bacillus sp. and to evaluate the effects of its supplementation on the growth and disease resistance of hybrid catfish. Bacillus siamensis strain B44v, selectively isolated from Thai pickled vegetables (Phak-dong), displayed a high potential as a probiotic in catfish culture. This bacterium produced a bacteriocin-like substance and exhibited a broad-spectrum antibacterial activity inhibiting both Gram-positive and Gram-negative bacteria, especially the fish pathogens Aeromonas hydrophila and Streptococcus agalactiae. The susceptibility to all 14 antibiotics tested implies its less possibility to be the antibiotic-resistant bacterium. Bacillus siamensis strain B44v possessed interesting adhesion properties, as shown by its high percentages of hydrophobicity (64.8%), auto-agglutination (73.8%), co-aggregation (67.2% with A. hydrophila FW52 and 63.5% with S. agalactiae F3S), and mucin binding (88.7%). The strain B44v survived simulated gastrointestinal conditions and produced protease and cellulase enzymes. Hybrid catfish (C. macrocephalus × C. gariepinus) were employed in the feed-trial experiments. Fish fed diet containing strain B44v (107 CFU/g feed) displayed not only no mortality but also growth improvement. At the end of the feed trial, fish were challenged by an intraperitoneal injection of Aeromonas hydrophila FW52. The Bacillus siamensis strain B44v fed fish survived (75.0%; p < 0.05) better than the controls (36.7%; p < 0.05) after a two week challenge. These collective results present for the first time the potential of Bacillus siamensis strain B44v for use as a bacterial probiotic in aquaculture.

Development of a quantitative fluorescence single primer isothermal amplification-based method for the detection of Salmonella.

Food-borne disease caused by Salmonella has long been, and continues to be, an important global public health problem, necessitating rapid and accurate detection of Salmonella in food. Real time PCR is the most recently developed approach for Salmonella detection. Single primer isothermal amplification (SPIA), a novel gene amplification technique, has emerged as an attractive microbiological testing method. SPIA is performed under a constant temperature, eliminating the need for an expensive thermo-cycler. In addition, SPIA reactions can be accomplished in 30 min, faster than real time PCR that usually takes over 2h. We developed a quantitative fluorescence SPIA-based method for the detection of Salmonella. Using Salmonella Typhimurium genomic DNA as template and a primer targeting Salmonella invA gene, we showed the detection limit of SPIA was 2.0 × 10(1)fg DNA. Its successful amplification of different serotypic Salmonella genomic DNA but not non-Salmonella bacterial DNA demonstrated the specificity of SPIA. Furthermore, this method was validated with artificially contaminated beef. In conclusion, we showed high sensitivity and specificity of SPIA in the detection of Salmonella, comparable to real time PCR. In addition, SPIA is faster and more cost-effective (non-use of expensive cyclers), making it a potential alternative for field detection of Salmonella in resource-limited settings that are commonly encountered in developing countries.

FoodMicrobionet: A database for the visualisation and exploration of food bacterial communities based on network analysis.

Amplicon targeted high-throughput sequencing has become a popular tool for the culture-independent analysis of microbial communities. Although the data obtained with this approach are portable and the number of sequences available in public databases is increasing, no tool has been developed yet for the analysis and presentation of data obtained in different studies. This work describes an approach for the development of a database for the rapid exploration and analysis of data on food microbial communities. Data from seventeen studies investigating the structure of bacterial communities in dairy, meat, sourdough and fermented vegetable products, obtained by 16S rRNA gene targeted high-throughput sequencing, were collated and analysed using Gephi, a network analysis software. The resulting database, which we named FoodMicrobionet, was used to analyse nodes and network properties and to build an interactive web-based visualisation. The latter allows the visual exploration of the relationships between Operational Taxonomic Units (OTUs) and samples and the identification of core- and sample-specific bacterial communities. It also provides additional search tools and hyperlinks for the rapid selection of food groups and OTUs and for rapid access to external resources (NCBI taxonomy, digital versions of the original articles). Microbial interaction network analysis was carried out using CoNet on datasets extracted from FoodMicrobionet: the complexity of interaction networks was much lower than that found for other bacterial communities (human microbiome, soil and other environments). This may reflect both a bias in the dataset (which was dominated by fermented foods and starter cultures) and the lower complexity of food bacterial communities. Although some technical challenges exist, and are discussed here, the net result is a valuable tool for the exploration of food bacterial communities by the scientific community and food industry.

Proteolysis in tempeh-type products obtained with Rhizopus and Aspergillus strains from grass pea (Lathyrus sativus) seeds.

BACKGROUND: Tempeh is a food product obtained from legumes by means of solid-state fermentation with Rhizopus sp. Our previous research proved that mixed-culture inoculum may also be successfully applied. The objective of present research was to study the proteolytic activity of R. microsporus var. chinensis and A. oryzae during tempeh-type fermentation of grass pea seeds, and the effect of inoculum composition on the protein level and in vitro protein bioavailability in products. METHODS: Fermentation substrate were soaked and cooked grass pea seeds. Material was mixed with single- or mixed-culture inoculum, and incubated in perforated plastic bags at 30°C for 32 hrs. In the products, the proteolytic activity (pH 3, 5 and 7), glucosamine, total protein and free amino acids levels, as well as protein in vitro bioavailability and degree of protein hydrolysis were obtained. RESULTS: The significant correlation was found between glucosamine content and proteolytic activity in grass pea seeds fermented with Rhizopus or Aspergillus. The activities of Rhizopus proteases were higher than Aspergillus ones, which corresponded with the degree of seed protein hydrolysis. Both strains showed the highest activity of protease at pH 3. Tempeh made with pure culture of Rhizopus had 37% protein of 69% in-vitro bioavailability. Mixed-culture fermentation improved nutritional parameters of products only when the dose of Aspergillus spores in the inoculum was equal and lower than that of Rhizopus. This process resulted in higher in-vitro bioavailability of protein, slightly more efficient protein hydrolysis and higher level of free amino acids, as compared to standard tempeh. CONCLUSIONS: The activity of A. oryzae in tempeh-type fermentation is beneficial as long as it does not dominate the activity and/or growth of Rhizopus strain.

The impact of biopreservatives and storage temperature in the quality and safety of minimally processed mixed vegetables for soup.

BACKGROUND: The combined effects of bioactive agents (tea tree essential oil, propolis extract and gallic acid) and storage temperature on the microbiological and sensory quality of fresh-cut mixed vegetables for soup (celery, leek and butternut squash) were studied with the objective of preserving its quality and safety. RESULTS: Refrigeration temperature was confirmed as the main factor to limit the growth of spoilage and pathogenic microorganisms. Biopreservatives applied on mixed vegetables were effective only when combined with optimal refrigeration temperature (5 °C). Bioactive compounds showed slight effectiveness in controlling the microbiota present in mixed vegetables, although coliforms were greatly reduced by gallic acid and propolis treatments, achieving 0.5-2 log unit reductions during storage. Also, these agents showed antimicrobial activity against endogenous Escherichia coli and inoculated E. coli O157:H7, exerting a bacteriostatic effect and reducing population counts by 0.9-1.2 log CFU g(-1) at 10 days of refrigerated storage. The combination of propolis treatment with refrigerated storage conditions effectively preserved the sensory quality and prolonged the sensory shelf life of fresh-cut mixed vegetables by 3 days. CONCLUSION: The use of natural agents such as propolis extract to preserve the quality and safety of mixed vegetables for soup might be an interesting option to address the concerns of the consumer about the use of synthetic chemical antimicrobials potentially harmful to health.