Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for the Rapid Detection of Toxigenic Aspergillus flavus and A. carbonarius in Nuts.

Aspergillus species create major postharvest problems due to the food losses caused by their mere presence and the hazardous mycotoxins they produce, such as aflatoxin B1 (AFB1) and ochratoxin A (OTA). These mycotoxins are mainly produced by A. flavus and A. carbonarius, respectively. In this study, we developed a rapid detection method for the two aforementioned species based on loop-mediated isothermal amplification (LAMP). The primers were designed to target genes belonging to the mycotoxin clusters pks and aflT for A. carbonarius and A. flavus, respectively. Result visualization was carried out in real time via the detection of fluorescent signals. The method developed showed high sensitivity and specificity, with detection limits of 0.3 and 0.03 pg/reaction of purified DNA of A. carbonarius and A. flavus, respectively. The assays were further implemented on inoculated nuts, including pistachios and almonds, after one-step crude DNA extraction. These tests revealed a detection level of 0.5 spore/g that shows the effectiveness of LAMP as a rapid method for detecting potentially toxigenic Aspergillus spp. directly in food. The validation of the assays included tests on a larger scale that further confirmed their sensitivity and specificity, as well as enabling the production of ready-to-use LAMP prototype kits. These kits are easy to use and aim to simplify the screening of food samples in order to monitor the presence of specific Aspergillus contaminations.

Characterization of the effect of pomegranate crude extract, and its post-harvesting preservation procedures, on redox tone, cellular growth and metabolic profile of MDA-MB-231 cell line.

BACKGROUND: Pomegranate is known for its beneficial properties due to its high content in antioxidants and might constitute a natural option for preventing and treatment of different pathologies including cancer. Since mitochondria are involved in tumorigenesis through ROS production and modulation of oxidative metabolism, we investigated the biological effects of pomegranate on cellular redox state, proliferation and metabolism in the breast cancer cell line MDA-MB-231 (MDA). METHODS: MDA were treated for 24 h with graded concentration of filtered Pomegranate juice (PJ) and tested for metabolic Flux Analysis with XFe96 Extracellular Flux Analyzer, for proliferation using the xCELLigence System Real-Time Cell Analyzer and for intracellular ROS content by Confocal Microscopy Imaging. RESULTS: Cells-treatment with freshly prepared pomegranate juice (PJ) resulted in a significant reduction of the intracellular ROS content already at the lower concentration of PJ tested. Additionally, it enhanced mitochondria respiration, and decreased glycolysis at high concentrations, inhibiting at the same time cell proliferation. As pomegranate is a seasonal fruit, assessment of optimum storage conditions preserving its bio-active properties was investigated. Our results indicated that storage conditions under controlled atmosphere for 30 days was able to enhance mitochondrial respiration at the same extent than freshly extracted PJ. Conversely, freezing procedure, though retaining the antioxidant and cell-growth inhibitory property, elicited an opposite effect on the metabolic profile as compared with fresh extract. CONCLUSION: Overall, the results of our study, on the one hand, confirms the preventive/therapeutic potential of PJ, as well as of its post-harvested processing, for cancer management. On the other hand, it highlights the intrinsic difficulties in attaining mechanistic insights when a multiplicity of effects is elicited by a crude mixture of bio-active compounds.

Prevention of Chilling Injury in Pomegranates Revisited: Pre- and Post-Harvest Factors, Mode of Actions, and Technologies Involved.

The storage life of pomegranate fruit (Punica granatum L.) is limited by decay, chilling injury, weight loss, and husk scald. In particular, chilling injury (CI) limits pomegranate long-term storage at chilling temperatures. CI manifests as skin browning that expands randomly with surface spots, albedo brown discoloration, and changes in aril colors from red to brown discoloration during handling or storage (6-8 weeks) at <5-7 °C. Since CI symptoms affect external and internal appearance, it significantly reduces pomegranate fruit marketability. Several postharvest treatments have been proposed to prevent CI, including atmospheric modifications (MA), heat treatments (HT), coatings, use of polyamines (PAs), salicylic acid (SA), jasmonates (JA), melatonin and glycine betaine (GB), among others. There is no complete understanding of the etiology and biochemistry of CI, however, a hypothetical model proposed herein indicates that oxidative stress plays a key role, which alters cell membrane functionality and integrity and alters protein/enzyme biosynthesis associated with chilling injury symptoms. This review discusses the hypothesized mechanism of CI based on recent research, its association to postharvest treatments, and their possible targets. It also indicates that the proposed mode of action model can be used to combine treatments in a hurdle synergistic or additive approach or as the basis for novel technological developments.

Is pomegranate husk scald during storage induced by water loss and mediated by ABA signaling?

BACKGROUND: Husk scald (HS) is a physiological disorder limiting the marketability of pomegranate fruit during long-term storage. Herein we propose that HS is triggered by water loss and mediated by ABA signaling. Therefore, pomegranate fruit were stored at three different storage temperatures (3.5, 7 and 11°C) and 96.5% ± 2.3% relative humidity (RH) evaluating weight loss (WL), abscisic acid (ABA), respiration rate (RR), total phenolics (TP), total anthocyanin (TA), antioxidant activity (AA), exocarp electrolyte leakage (EL), malondialdehyde (MDA), color attributes, browning index and visual quality of fruit. RESULTS: HS appeared after 3 months of storage at 11 °C, less at 7°C and non-present at 3.5°C. Incidence of HS occurred along with higher WL, RR, EL, MDA, and ABA content. Conversely, TP, TA and AA decreased significantly. WL increased with water vapor pressure deficit (VPD) at higher temperatures. After 93 days, 11 °C fruit reached ~10% WL while ABA increased to ~150 µg kg-1 . However, 3.5 and 7 °C fruit, reached 5 and 7% WL while ABA was ~31 and 75 µg kg-1 , respectively. CONCLUSION: Herein, we propose a mechanistic model of HS development where water stress induces ABA as a primary signaling molecule that triggers the HS response mediated by reactive oxygen species (ROS). Accumulation of ROS induces phenolic biosynthesis and oxidative stress promotes loss of membrane compartmentalization that induces phenolic degradation. Ultimately, husk scalding becomes visible due to the oxidation of phenolics into brown pigments. © 2022 Society of Chemical Industry.

Quality of Goji Berry Fruit (Lycium barbarum L.) Stored at Different Temperatures.

Goji berries are widely known for their outstanding nutritional and medicinal properties; they are usually found in the market as dried fruit or as juice because the fruit has a short shelf-life, and little information is available about its postharvest behavior at low temperatures. This study aimed to determine the storage performance of goji berry fruit by evaluating physicochemical, and sensorial attributes during storage at three different temperatures (0, 5, and 7 °C) for 12 days in a range that has not been extensively studied before. In addition, fruit respiration and ethylene production rates were also measured at the three temperatures. Fruit stored at 0 °C showed the lowest respiration rate and ethylene production (5.8 mg CO2 kg-1h-1 and 0.7 µg C2H4 kg-1h-1, respectively); however, at this temperature, the incidence and severity of pitting and electrolytic leakage were the highest. In contrast, 5 °C was found to be the best storage temperature for goji berry fruit; the fruit appeared fresh and healthy, had the highest scores during sensory analysis with an acceptable general impression, and had the lowest amount of damage attributable to chilling injury, with 17.1% fruit presenting with shriveling, 12.5% pitting, 6.7% mold, and 35% electrolytic leakage on day 9 of storage. Storage of goji berries at 7 °C resulted in the lowest marketability and the highest incidence of decay. Significant differences were also found in the phytochemical attributes, vitamin C content, soluble solid content (SSC), titratable acidity (TA), SSC/TA ratio, total polyphenol content, 2,2-diphenylpicrylhydrazy (DPPH), and anthocyanin content. This study revealed that a storage temperature of 5 °C for 9 days is recommended to maintain the quality of fresh goji berry. Thus, broadening the existing knowledge of the postharvest behavior of fresh goji berries; our results can help improve the commercial life of goji berries and ensure high-quality attributes throughout distribution.

Pomegranate Husk Scald Browning during Storage: A Review on Factors Involved, Their Modes of Action, and Its Association to Postharvest Treatments.

The pomegranate (Punica granatum L.), which contains high levels of health-promoting compounds, has received much attention in recent decades. Fruit storage potential ranges from 3 to 4 months in air and from 4 to 6 months in Controlled Atmospheres (CA) with 3-5% oxygen and 10-15% carbon dioxide. Storage life is limited by decay, chilling injury, weight loss (WL), and husk scald. In particular, husk scald (HS) limits pomegranate long-term storage at favorable temperatures. HS appears as skin browning which expands from stem end towards the blossom end during handling or long-term storage (10-12 weeks) at 6-10 °C. Even though HS symptoms are limited to external appearance, it may still significantly reduce pomegranate fruit marketability. A number of postharvest treatments have been proposed to prevent husk scald, including atmospheric modifications, intermittent warming, coatings, and exposure to 1-MCP. Long-term storage may induce phenolic compounds accumulation, affect organelles membranes, and activate browning enzymes such as polyphenol oxidases (PPO) and peroxidases (POD). Due to oxidation of tannins and phenolics, scalding becomes visible. There is no complete understanding of the etiology and biochemistry of HS. This review discusses the hypothesized mechanism of HS based on recent research, its association to postharvest treatments, and their possible targets.

Comparison Performance of Visible-NIR and Near-Infrared Hyperspectral Imaging for Prediction of Nutritional Quality of Goji Berry (Lycium barbarum L.).

The potential of hyperspectral imaging for the prediction of the internal composition of goji berries was investigated. The prediction performances of models obtained in the Visible-Near Infrared (VIS-NIR) (400-1000 nm) and in the Near Infrared (NIR) (900-1700 nm) regions were compared. Analyzed constituents included Vitamin C, total antioxidant, phenols, anthocyanin, soluble solids content (SSC), and total acidity (TA). For vitamin C and AA, partial least square regression (PLSR) combined with different data pretreatments and wavelength selection resulted in a satisfactory prediction in the NIR region obtaining the R2pred value of 0.91. As for phenols, SSC, and TA, a better performance was obtained in the VIS-NIR region yielding the R2pred values of 0.62, 0.94, and 0.84, respectively. However, the prediction of total antioxidant and anthocyanin content did not give satisfactory results. Conclusively, hyperspectral imaging can be a useful tool for the prediction of the main constituents of the goji berry (Lycium barbarum L.).

Evaluation of Quality and Storability of "Italia" Table Grapes Kept on the Vine in Comparison to Cold Storage Techniques.

The aim of the study was to compare the quality of table grapes (cv. Italia) held on the vine compared to grapes stored in cold rooms with or without modified-atmosphere packaging (MAP). The grapes were harvested from 12 plants in 2 vineyards in the same area, differing for the age of the plant. Four- and a fourteen-year-old vines were cultivated with the "Apulia tendone" system. After the first harvest, grapes were divided into small clusters and used for storage treatments in air and in MAP. Samples of 400 g were packaged in polypropylene (PP) trays sealed with a polypropylene/polyamide (PP/PA) film with 20% CO2 in air. MAP and control samples were then stored in the same cold room at 0 °C. Initially and after 8, 21, and 28 days, grapes stored in air and MAP were compared to fresh harvested grapes, stored on the plants. Quality attributes included color, texture, maturity index, phenols, antioxidant activity, sugars, organic acids, sensory parameters, and volatile compounds. The results obtained demonstrated that grapes held on the plant and in MAP showed better quality in terms of appearance scores compared to grapes stored in air. In particular, the application of high CO2 contributed to reduce the deterioration rate of the clusters, minimizing weight loss, and delaying degradation processes, and this particularly for grapes from the 14-year-old vine, where grapes held on the plant degraded faster than grapes in the younger vines. Most volatile compounds did not change their concentration with the storage treatment, except for ethyl acetate and ethanol, which increased in MAP at the end of storage, and to some compound responsible for green odor. In conclusion, keeping the grapes on the plant can be considered a good agronomic practice to preserve the quality, whereas MAP can be applied to better maintain postharvest quality of the product throughout storage and distribution.

Screening of Lactic Acid Bacteria for the Bio-Control of Botrytis cinerea and the Potential of Lactiplantibacillus plantarum for Eco-Friendly Preservation of Fresh-Cut Kiwifruit.

Botrytis cinerea, responsible for grey mold, represents the first biological cause of fruit and vegetable spoilage phenomena in post-harvest. Kiwifruit is a climacteric fruit particularly prone to this mold infestation during storage. Lactic acid bacteria (LAB) are food-grade bacteria that can synthesize several metabolites with antimicrobial activity and are, therefore, suggested as promising and eco-friendly resources for the bio-control of molds on fruits and vegetables. In this work, we propose the screening of a collection of 300 LAB previously isolated from traditional sourdoughs for their ability to counteract in vitro the growth of Botrytis cinerea CECT 20973. Only 2% of tested LAB strains belonging to Lactiplantibacillus plantarum species, exerted a strong antagonism against B. cinerea. The cell-free supernatants were partially characterized and results clearly indicated that high levels of lactic acid contributed to the antagonistic activity. PAN01 and UFG 121 cell-free supernatants were investigated as potential bio-control agents in a preliminary in vivo assay using freshly cut kiwifruits as a food model. The application of cell-free supernatants allowed to delay the growth of B. cinerea on artificially contaminated kiwifruits until two weeks. The antagonistic activity was greatly affected by the storage temperature (25 °C and 4 °C) selected for the processed fruits, suggesting the importance to include microbial-based solution in a broader framework of hurdle technologies.

Microbial-based Biocontrol Solutions for Fruits and Vegetables: Recent Insight, Patents, and Innovative Trends.

BACKGROUND: Fruits and vegetables are susceptible to colonisation by undesired microflora, which, in pre- and post-harvest conditions, negatively impact the quality of these products, leading to a reduction of yield, shelf-life, and marketability. In the few last years, the use of microbial Biological Control Agents (BCAs) has assumed international relevance in order to control harmful microorganisms, as a promising alternative to chemical interventions. OBJECTIVE: The purpose of this review is to discuss the microbial-based solutions applicable for the biocontrol of the main microbial spoilers, phytopathogens, and human food-borne pathogens affecting fruits and vegetables during their production and storage. RESULTS: A comprehensive overview of the scientific literature investigating the effectiveness of BCA-based products available on the market is provided, as well as of the most recent patents protecting biotechnological applications in this field. Innovative trends are discussed, with a particular focus on the integration of BCAs to minimise spoilage phenomena and microbiological risks adopting combined approaches. CONCLUSION: This study underlines the growing interest about biocontrol strategies to counteract the growth of spoilage and/or pathogenic microorganisms indicating that in the next years a considerable increase of commercial products and patents will be developed worldwide to exploit innovative biotechnological solutions in the sector.

Botrytis cinerea and Table Grapes: A Review of the Main Physical, Chemical, and Bio-Based Control Treatments in Post-Harvest.

Consumers highly appreciate table grapes for their pleasant sensory attributes and as good sources of nutritional and functional compounds. This explains the rising market and global interest in this product. Along with other fruits and vegetables, table grapes are considerably perishable post-harvest due to the growth of undesired microorganisms. Among the microbial spoilers, Botrytis cinerea represents a model organism because of its degrading potential and the huge economic losses caused by its infection. The present review provides an overview of the recent primary physical, chemical, and biological control treatments adopted against the development of B. cinerea in table grapes to extend shelf life. These treatments preserve product quality and safety. This article also focuses on the compliance of different approaches with organic and sustainable production processes. Tailored approaches include those that rely on controlled atmosphere and the application of edible coating and packaging, as well as microbial-based activities. These strategies, applied alone or in combination, are among the most promising solutions in order to prolong table grape quality during cold storage. In general, the innovative design of applications dealing with hurdle technologies holds great promise for future improvements.

Effect of organic agronomic techniques and packaging on the quality of lamb's lettuce.

BACKGROUND: This research focused on the effect of organic production systems on initial quality and postharvest performance of lamb's lettuce leaves stored in air or under modified atmosphere at refrigerated temperature. Different strategies of organic soil fertility management were compared under the same environmental conditions: (i) a simplified organic production system based on organic commercial fertilizers to recover crop uptake (SB); (ii) an organic production system based on organic matter amendment mainly supplied by animal manure (AM); and (iii) an organic production system based on organic matter amendment supplied by green waste compost (AC). Fully developed lamb's lettuce leaves were harvested and then packed into perforated bags (control in AIR) or in modified atmosphere packaging (MAP) and stored at 4 °C. RESULTS: At harvest, the yield of lamb's lettuce in the AM and AC systems was higher than that in SB. Phenol and dehydroascorbic acid accumulation was observed in the system with the lowest initial supply of organic amendment (SB). Regarding the effect of packaging, AIR conditions maintained the initial quality attributes for a longer period than MAP, which developed off-odours above the threshold of acceptability at 11 days, irrespective of the production system used. CONCLUSION: In general, the initial differences among the production systems were minimal during the postharvest storage. As for the tested packaging systems, AIR successfully maintained the initial quality attributes for a longer period than the MAP. © 2018 Society of Chemical Industry.

Quality of fresh-cut products as affected by harvest and postharvest operations.

There is a rising demand for fresh-cut convenience products with high quality and nutritional standards that needs to be met by the fresh-cut industry. It is well known that harvest and postharvest handling of fresh produce has a paramount impact on its quality and storage, although most of the existing literature has focused on these impacts related only to fresh produce that is destined for the final consumers. Indeed, current harvest methods and postharvest technologies have improved fruit and vegetable handling and distribution processes by slowing down physiological processes and senescence. Nonetheless, these technologies and methods may influence the quality of fresh produce as raw material for fresh-cut processing as a result of the dynamic responses of fresh produce to handling procedures and treatments. Here, we review the existing literature on the challenges facing the fresh-cut industry, focusing on the impact of harvest, maturity, and handling of fruit and vegetables on the quality of raw materials, as well as the implications for fresh-cut products. The review also highlights areas for further research with the aim of enhancing the sensorial, nutritional and biochemical quality of such products. © 2018 Society of Chemical Industry.

Effect of modified atmosphere packaging and temperature abuse on flavor related volatile compounds of rocket leaves (Diplotaxis tenuifolia L.).

The effect of storage conditions on flavor-related volatile composition of wild rocket (Diplotaxis tenuifolia) was investigated on Modified Atmosphere packed (MAP) leaves stored under isothermal and non-isothermal conditions. In a first experiment the effect of MAP was compared to the storage in air at 5 °C; a second experiment aimed to study the effect of non isothermal conditions, with two temperature abuses (at 13 °C for 24 h) during a 5 °C. Twenty-four volatiles were detected, including C6, C5, isothiocyanate, lipid-derived and sulfur compounds. In the first experiment, MAP-stored rocket showed a slower loss of typical flavour volatiles (thiocyanates and isothiocyanates) and a slower production of off-flavors until 6 days of storage, compared to leaves stored in air. After this time, dimethyl sulfide and acetaldehyde dramatically increased in MAP-stored rocket samples. In the second experiment, samples stored under non-isothermal conditions showed lower O2 and higher CO2 concentrations than samples stored under isothermal conditions. Rocket leaves stored under non-isothermal conditions showed an increased production of volatiles responsible of off-flavors (acetaldehyde and dimethyl sulfide) following temperature abuse comparing to storage in isothermal condition. Thus, dimethyl sulfide and acetaldehyde could be effective markers for tracking the effect of temperature fluctuations on rocket during storage.

The table grape 'Victoria' with a long shaped berry: a potential mutation with attractive characteristics for consumers.

BACKGROUND: Puglia is the most important region in Italy for table grape production. Since consumers look for new products, the number of table grape varieties has greatly increased in recent years. RESULTS: In a survey in the Puglia region, we identified several years ago a potential mutation of the cv. Victoria. We described this accession in comparison with the standard Victoria for some amphelographic traits. All the characteristics were very similar to the standard Victoria except for the berry shape, which was significantly more elongated. Moreover, the berry of the mutated Victoria showed higher firmness, lightness and chroma than the standard one, with a more intense yellow colour of the skin (appreciated by consumers). The molecular characterisation with 25 SSR markers showed that normal and mutant Victoria were genetically identical at all the analysed loci, thus suggesting that the two accessions could be considered as clones with the difference in berry shape probably due to a somatic mutation. CONCLUSIONS: This mutation of the cv. Victoria may have interesting perspective for the market since consumers are always attracted by different shape and colour of the fruits (consumers buy with eyes). This accession can be an alternative clone of the already known standard Victoria. © 2017 Society of Chemical Industry.

Effect of harvest time on table grape quality during on-vine storage.

BACKGROUND: Postponing the harvest of grapes is a common practice in southern Italy, in order to delay harvest up to Christmas and make higher income from their sale. The aim of this work was to evaluate the effect of harvest time (over almost 3 months) on the quality of table grapes (cv. Italia). The experiment was repeated for two years (2010 and 2011). In 2010, grapes were harvested starting from 8 October and after 11, 27 and 48 days. In 2011, five harvest times were compared over a period of 56 days. Respiration rate, firmness, colour, sensory attributes, total soluble solids (TSS), pH, titratable acidity (TA), phenols and antioxidant activity were measured. In addition, in the second year, volatile compounds were evaluated. RESULTS: For both years, harvest time influenced most parameters, which indicated that metabolic changes took place in the plants. In 2010, harvest time influenced respiration rate, cluster and berry appearance scores, colour attributes, crunchiness, pH, TA, total phenol content and antioxidant activity. In 2011, harvest time influenced respiration rate, colour attributes, most sensory attributes, TSS and TA. Generally, late harvested grapes showed higher firmness, berry appearance score, sweetness, fruity taste, overall sensory evaluation score and TSS. Regarding volatile compounds, terpene content decreased during ripening, while C6 compounds showed a nonlinear trend. CONCLUSION: The results showed that table grape sensory quality could be increased by delaying harvest up to a certain time of the season, while excessive delay could reduce final grape quality.

Potential use of microwave treatment on fresh-cut carrots: physical, chemical and microbiological aspects.

BACKGROUND: The effect of microwave treatments (900 and 750 W for 45 and 60 s) on the microbial, physicochemical and sensory properties of fresh-cut carrot slices and the contents of several bioactive compounds was studied. Carrot samples were stored for 7 days at 5 °C. RESULTS: The microwaving of fresh-cut carrots reduced the initial respiration rate (8.6 CO2 mL kg(-1) h(-1)) by 55-74% compared with untreated samples, although the rates then increased during storage. The initial pH (6.7), titratable acidity (0.036%), soluble solid content (8.2 °Brix) and shelf-life of the samples did not differ greatly from those of the untreated samples. Microwaving prevented the incipient whitening and surface dryness during storage. In general, no significant changes in phenylalanine ammonia lyase activity (5.5 µmol t-cinnamic acid kg(-1) h(-1)), total phenolics (TP, 81.3 mg chlorogenic acid equivalent kg(-1) fresh weight (FW)) or total antioxidant capacity (TAC, 74.2 µmol Trolox equivalent kg(-1) FW) were observed on the processing day or over storage. However, the mildest treatment (750 W for 45 s) caused TP and TAC enhancements of 118 and 394% respectively after 7 days of shelf-life. Microwave treatments reduced the initial microbial loads of the samples by up to 1.8 log units, although their microbial growth was greater than that of the untreated samples throughout storage. CONCLUSION: Mild microwave treatments such as 750 W/45 s and 750 W/60 s are a good sustainable alternative to the use of NaOCl; however, combining them with other sanitizing techniques is needed to control microbial growth throughout the shelf-life of fresh-cut carrot slices.

Effect of atmosphere composition on the quality of ready-to-use broccoli raab (Brassica rapa L.).

BACKGROUND: Many leafy brassicas are widely used for ready-to-use salads. Broccoli raab (Brassica rapa L.), also called turnip top, or rapini, is extensively cultivated in southern Italy. The edible portion is made up of the green, immature inflorescences and the stem with its most tender leaves. Recently, interest in this crop has increased among European consumers; moreover, a substantial increase in consumption could come from the ready-to-use product. RESULTS: The effects of four different atmosphere compositions (air, 3% O(2) in nitrogen, 3% O(2) + 10% CO(2) in air, and 10% CO(2) in air) on quality attributes of ready-to-use broccoli raab were studied. Controlled atmosphere affected appearance, composition, respiration rate, weight loss, and presence of off-odours. Storage of broccoli raab florets under low oxygen conditions delayed post-cutting deterioration during storage at 5 degrees C and preserved appearance and typical odour, up to 17 days. Moreover, respiration rate as well as loss of green colour, chlorophyll and vitamin C were also slowed down in this condition. Finally, in this study no effect of controlled atmosphere storage was found on total phenols content and antioxidant activity. CONCLUSION: Results showed that cold storage in 3% O(2) can be beneficial in order to maintain quality of ready-to-use, broccoli raab for up to 17 days.