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Preservatives from food—For food: Pea protein hydrolysate as a novel bio‐preservative against Escherichia coli O157:H7 on a lettuce leaf

ABSTRACT: Abstract Fresh‐cut fruits and vegetables are becoming particularly popular as healthy fast‐food options; however, they present challenges such as accelerated rates of decay and increased risk for contamination when compared to whole produce. Given that food safety must remain paramount for producers and manufacturers, research into novel, natural food preservation solutions which can help to ensure food safety and protect against spoilage is on the rise. In this work, we investigated the potential of using a novel protein hydrolysate, produced by the enzymatic hydrolysis of Pisum sativum (PSH), as a novel bio‐preservative and its abilities to reduce populations of Escherichia coli O157:H7 after inoculation on a lettuce leaf. While unhydrolyzed P. sativum proteins show no antimicrobial activity, once digested, and purified, the enzymatically released peptides induced in vitro bactericidal effects on the foodborne pathogen at 8 mg/ml. When applied on an infected lettuce leaf, the PSH significantly reduced the number of bacteria recovered after 2 hr of treatment. PSH may be preferred over other preservation strategies based on its natural, inexpensive, sustainable source, environmentally friendly process, nontoxic nature, good batch to batch consistency, and ability to significantly reduce counts of E. coli both in vitro and in a lettuce leaf. In this work, we investigated the potential to use Pisum sativum hydrolysate (PSH) as a novel bio‐preservative and its abilities to reduce populations of Escherichia coli O157:H7 in a lettuce leaf model. PSH may be preferred over other preservation strategies based on its natural, inexpensive, sustainable source, environmentally friendly process, nontoxic nature, good batch to batch consistency and ability to significantly reduce counts of E. coli both in vitro and in a lettuce leaf model.

SUBMITTER: Mohan N

PROVIDER: S-EPMC8565202 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study examined the biological and food safety relevance of leaf lesions for potential invasion of food pathogens into the plant tissue (internalization). This was done by determining the role of artificial leaf damage in terms of damaged leaf area on proliferation of E. coli O157:H7 gfp+. In a two-factorial experiment, unwashed fresh baby leaf spinach (Spinacia oleracea L.) was subjected to four damage levels (undamaged, low, moderate, high damage; factor 1) and three incubation intervals (0, 1, 2 days post-inoculation; factor 2). Individual leaves were immersed for 15 s in a suspension loaded with E. coli O157:H7 gfp+ (106 CFU × mL-1). The leaves were analyzed individually using image analysis tools to quantify leaf area and number and size of lesions, and using confocal laser scanning and scanning electron microscopy to visualize leaf lesions and presence of the introduced E. coli strain on and within the leaf tissue. Prevalence of E. coli O157:H7 gfp+ was assessed using a culture-dependent technique. The results showed that size of individual lesions and damaged leaf area affected depth of invasion into plant tissue, dispersal to adjacent areas, and number of culturable E. coli O157:H7 gfp+ directly after inoculation. Differences in numbers of the inoculant retrieved from leaf macerate evened out from 2 days post-inoculation, indicating rapid proliferation during the first day post-inoculation. Leaf weight was a crucial factor, as lighter spinach leaves (most likely younger leaves) were more prone to harbor E. coli O157:H7 gfp+, irrespective of damage level. At the high inoculum density used, the risk of consumers' infection was almost 100%, irrespective of incubation duration or damage level. Even macroscopically intact leaves showed a high risk for infection. These results suggest that the risk to consumers is correlated with how early in the food chain the leaves are contaminated, and the degree of leaf damage. These findings should be taken into account in different steps of leafy green processing. Further attention should be paid to the fate of viable, but non-culturable, shiga-toxigenic E. coli on and in ready-to-eat leafy vegetables.

Project description:Leafy green vegetables, such as lettuce, have been increasingly implicated in outbreaks of foodborne illnesses due to contamination by Escherichia coli O157:H7. While E. coli can survive in soils, colonize plants, and survive on produce, very little is known about the interaction of E. coli with the roots of growing lettuce plants. In these studies, a combination of microarray analyses and surface enhanced Raman spectroscopy (SERS) were used to gain a comprehensive understanding of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots and compared to E. coli K12 using a hydroponic system (HS) which we have reported in the previous studies. Using microarray, after three days of interaction with lettuce roots, 94 and 109 genes of E. coli O157:H7 were significantly up-regulated and down-regulated at least 1.5 fold, respectively. Only 8 genes were also found in the E. coli K12 up-regulated genes. No genes were found in the down-regulated genes clusters between those two strains. For E. coli O157:H7, forty out of the 94 up-regulated genes (43%) were involved in protein synthesis and were highly repressed compared to 40 out of 193 (23%) E. coli K12 up-regulated genes associated with protein synthesis. The wildtype of E.coli O157:H7 colonized two log CFU per root less compared to E. coli K12. Genes involved in biofilm modulation (bhsA and ybiM) were significantly up-regulated in E. coli O157:H7 and curli production (crl and csgA) were found important for E. coli K12 to attach to lettuce roots in the previous studies. BhsA mutant of E. coli O157:H7 was impaired in the colonization of lettuce roots. The SERS spectra of E. coli K12 and O157 controls (cells without interacting with roots) were very similar. The spectra of E. coli K12 and O157 exposed to the hydroponic system (HS) showed some differences in the nucleic acid, protein, and lipid regions compared with controls. The spectra of E. coli K12 HS cells exhibited significant differences compared to spectra from E. coli O157 HS cells in the RNA and protein regions. The overall band intensity of amide regions declined for E. coli O157 HS cells, while it increased for E. coli K12 HS cells. The intensity of the RNA bands of E. coli K12 HS cells were also found much higher than those of E. coli O157 HS cells. These findings were in agreement to our Microarray data. Our microarray and SERS data showed that E. coli K12 and O157:H7 behavior dramatically differently in colonizing on lettuce roots. Compared to K12, E. coli O157:H7 colonized less efficiently on lettuce roots.

Project description:Escherichia coli O157:H7 (EcO157) infections have been recurrently associated with produce. The physiological state of EcO157 cells surviving the many stresses encountered on plants is poorly understood. EcO157 populations on plants in the field generally follow a biphasic decay in which small subpopulations survive over longer periods of time. We hypothesized that these subpopulations include persister cells, known as cells in a transient dormant state that arise through phenotypic variation in a clonal population. Using three experimental regimes (with growing, stationary at carrying capacity, and decaying populations), we measured the persister cell fractions in culturable EcO157 populations after inoculation onto lettuce plants in the laboratory. The greatest average persister cell fractions on the leaves within each regime were 0.015, 0.095, and 0.221%, respectively. The declining EcO157 populations on plants incubated under dry conditions showed the largest increase in the persister fraction (46.9-fold). Differential equation models were built to describe the average temporal dynamics of EcO157 normal and persister cell populations after inoculation onto plants maintained under low relative humidity, resulting in switch rates from a normal cell to a persister cell of 7.7 × 10-6 to 2.8 × 10-5 h-1 Applying our model equations from the decay regime, we estimated model parameters for four published field trials of EcO157 survival on lettuce and obtained switch rates similar to those obtained in our study. Hence, our model has relevance to the survival of this human pathogen on lettuce plants in the field. Given the low metabolic state of persister cells, which may protect them from sanitization treatments, these cells are important to consider in the microbial decontamination of produce.IMPORTANCE Despite causing outbreaks of foodborne illness linked to lettuce consumption, E. coli O157:H7 (EcO157) declines rapidly when applied onto plants in the field, and few cells survive over prolonged periods of time. We hypothesized that these cells are persisters, which are in a dormant state and which arise naturally in bacterial populations. When lettuce plants were inoculated with EcO157 in the laboratory, the greatest persister fraction in the population was observed during population decline on dry leaf surfaces. Using mathematical modeling, we calculated the switch rate from an EcO157 normal to persister cell on dry lettuce plants based on our laboratory data. The model was applied to published studies in which lettuce was inoculated with EcO157 in the field, and switch rates similar to those obtained in our study were obtained. Our results contribute important new knowledge about the physiology of this virulent pathogen on plants to be considered to enhance produce safety.

Project description:BackgroundPresently available chemical based synthetic preservative have emerged with various side effects, so the aspiration of natural and side effect free novel preservative has been greatly increased. As the natural preservative exhibit poor side effect with improved preservative efficacy. The recent development in computational studies leads advancement in drug designing and discovery of novel glucosamine-6-phosphate synthase (G-6-P synthase) inhibition based natural antimicrobial preservatives. Here, selected aesculin derivatives were screened for G-6-P synthase inhibition via docking study and evaluated for antioxidant, antimicrobial, preservative efficacy as well stability study.ResultsModified aesculin derivatives were designed, synthesized and showed potent G-6-P synthase inhibition with remarkable antimicrobial, antioxidant, preservative efficacy and stability study. The molecular docking with target pdb id 1moq from G-6-P synthase resulted with better dock score and energy for compound 1 as compared to standard drugs streptomycin, ciprofloxacin, ampicillin and fluconazole, that supported the wet lab results. Among the synthesized compounds, the compound 1 possessed good antioxidant activity as compared to standard L-ascorbic acid. The resultant data for antimicrobial activity of aesculin derivatives revealed compound 1 as the most potent antimicrobial compound as compared to the standard drugs streptomycin, ciprofloxacin, ampicillin and fluconazole. While compound 2 showed better antimicrobial activity as compared to streptomycin, ciprofloxacin, ampicillin. The preservative efficacy test for compound 1 in aloe vera juice and white lotion USP has been showed the log CFU/mL values within the prescribed limit of USP standard and results were comparable to standard sodium benzoate, ethyl paraben and propyl paraben. Compound 1 has been found to be within prescribed limit of stability study over six month.ConclusionCompound 1 showed the potent G-6-P synthase inhibitory, antioxidant, antimicrobial, preservative efficacy and stability study results as compared to standard drugs taken. The results have found comparable to molecular docking results, and this final compound may be used as new preservatives for food and pharmaceutical products. Moreover, the mechanistic insight into the docking poses was also explored by binding interactions of aesculin derivatives inside the pdb id 1moq. These results also supported the results for novel synthesized G-6-P synthase inhibitors.

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Preservatives from food—For food: Pea protein hydrolysate as a novel bio‐preservative against Escherichia coli O157:H7 on a lettuce leaf

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