Effect of Combined High-Pressure Homogenization and Biotechnological Processes on Chitin, Protein, and Antioxidant Activity of Cricket Powder-Based Ingredients.

The main objective of this work was to evaluate the combined effect of a biotechnology process, based on selected yeast strains, and a high-pressure homogenization (HPH) treatment on the microbiological quality, structural organization of proteins, chitin content, and antioxidant activity of a mixture of cricket powder (Acheta domesticus) and water. Compared to untreated samples, the cricket matrix treated with HPH four times at 180 MPa promoted the growth of the inoculated Yarrowia lipolytica and Debaryomyces hansenii strains. HPH did not affect the concentration of chitin; however, the combination with microorganisms tended to reduce the content. Although the antioxidant activity increased from 0.52 to 0.68 TAC mM/TE after a 48 h incubation in the control, it was further improved by the combination of HPH and D. hansenii metabolism, reaching a value of 0.77 TAC mM/TE. The combination of the two approaches also promoted a reduction in the intensity of bands with molecular weights between 31 and 21.5 kDa in favor of bands with a lower molecular weight. In addition, HPH treatment reduced the number of accessible thiols, suggesting protein structure changes that may further impact the technological properties of cricket powder.

Essential Oils and Their Combination with Lactic Acid Bacteria and Bacteriocins to Improve the Safety and Shelf Life of Foods: A Review.

The use of plant extracts (e.g., essential oils and their active compounds) represents an interesting alternative to chemical additives and preservatives applied to delay the alteration and oxidation of foods during their storage. Essential oils (EO) are nowadays considered valuable sources of food preservatives as they provide a healthier alternative to synthetic chemicals while serving the same purpose without affecting food quality parameters. The natural antimicrobial molecules found in medicinal plants represent a possible solution against drug-resistant bacteria, which represent a global health problem, especially for foodborne infections. Several solutions related to their application on food have been described, such as incorporation in active packaging or edible film and direct encapsulation. However, the use of bioactive concentrations of plant derivatives may negatively impact the sensorial characteristics of the final product, and to solve this problem, their application has been proposed in combination with other hurdles, including biocontrol agents. Biocontrol agents are microbial cultures capable of producing natural antimicrobials, including bacteriocins, organic acids, volatile organic compounds, and hydrolytic enzymes. The major effect of bacteriocins or bacteriocin-producing LAB (lactic acid bacteria) on food is obtained when their use is combined with other preservation methods. The combined use of EOs and biocontrol agents in fruit and vegetables, meat, and dairy products is becoming more and more important due to growing concerns about potentially dangerous and toxic synthetic additives. The combination of these two hurdles can improve the safety and shelf life (inactivation of spoilage or pathogenic microorganisms) of the final products while maintaining or stabilizing their sensory and nutritional quality. This review critically describes and collects the most updated works regarding the application of EOs in different food sectors and their combination with biocontrol agents and bacteriocins.

Effect of High Hydrostatic Pressure on the Metabolite Profile of Striped Prawn (Melicertus kerathurus) during Chilled Storage.

A variety of metabolites contribute to the freshness and taste characteristics of seafood. This study investigated the effects of high hydrostatic pressure (HHP; 400, 500, and 600 MPa) for 10 min) on the metabolome of striped prawn during chilled storage, in relation to microorganisms' development. All treated samples showed lower viable counts throughout storage compared to the untreated counterparts. The limit of acceptability from a microbiological point of view was extended from 9 to as many as 35 days by 600 MPa treatment. Metabolites were quantified by 1H-NMR through a targeted-untargeted metabolomic approach. Molecules linked to nucleotides' degradation and amines' anabolism suggested an overall freshness improvement granted by HHP. Notably, putrescine and cadaverine were detected only in untreated prawn samples, suggesting the inactivation of degradative enzymes by HHP. The concentration of molecules that influence umami perception was significantly elevated by HHP, while in untreated samples, the concentration of molecules contributing to a sour taste gradually increased during storage. As metabolomics was applied in its untargeted form, it allowed us to follow the overall set of metabolites related to HHP processing and storage, thus providing novel insights into the freshness and taste quality of striped prawn as affected by high hydrostatic pressure.

Sublethal HPH treatment is a sustainable tool that induces autolytic-like processes in the early gene expression of Saccharomyces cerevisiae.

Sublethal HPH treatments have been demonstrated to impact the technological properties and functions of treated microorganisms by inducing specific enzymes/genes or modulating membrane structures and inducing autolysis. In this work, the early effects of a 100 MPa HPH treatment on the winery starter Saccharomyces cerevisiae ALEAFERM AROM grown in synthetic must were assessed. While there were no differences in cell cultivability during the first 48 h between treated and untreated cells, a reduction in volatile metabolites released by HPH-treated cells during the first 2 h was observed. This reduction was only temporary since after 48 h, volatile molecules reached similar or even higher concentrations compared with the control. Moreover, the gene expression response of HPH-treated cells was evaluated after 1 h of incubation and compared with that of untreated cells. A massive rearrangement of gene expression was observed with the identification of 1220 differentially expressed genes (DEGs). Most of the genes related to energetic metabolic pathways and ribosome structure were downregulated, while genes involved in ribosome maturation, transcription, DNA repair, response to stimuli and stress were upregulated. These findings suggest that HPH induces or promotes an autolytic-like behaviour that can be exploited in winemaking.

Role of Yeasts on the Sensory Component of Wines.

The aromatic complexity of a wine is mainly influenced by the interaction between grapes and fermentation agents. This interaction is very complex and affected by numerous factors, such as cultivars, degree of grape ripeness, climate, mashing techniques, must chemical−physical characteristics, yeasts used in the fermentation process and their interactions with the grape endogenous microbiota, process parameters (including new non-thermal technologies), malolactic fermentation (when desired), and phenomena occurring during aging. However, the role of yeasts in the formation of aroma compounds has been universally recognized. In fact, yeasts (as starters or naturally occurring microbiota) can contribute both with the formation of compounds deriving from the primary metabolism, with the synthesis of specific metabolites, and with the modification of molecules present in the must. Among secondary metabolites, key roles are recognized for esters, higher alcohols, volatile phenols, sulfur molecules, and carbonyl compounds. Moreover, some specific enzymatic activities of yeasts, linked above all to non-Saccharomyces species, can contribute to increasing the sensory profile of the wine thanks to the release of volatile terpenes or other molecules. Therefore, this review will highlight the main aroma compounds produced by Saccharomyces cerevisiae and other yeasts of oenological interest in relation to process conditions, new non-thermal technologies, and microbial interactions.

Isolation and Identification of Wild Yeast from Malaysian Grapevine and Evaluation of Their Potential Antimicrobial Activity against Grapevine Fungal Pathogens.

Pathogenic fungi belonging to the genera Botrytis, Phaeomoniella, Fusarium, Alternaria and Aspergillus are responsible for vines diseases that affect the growth, grapevine yield and organoleptic quality. Among innovative strategies for in-field plant disease control, one of the most promising is represented by biocontrol agents, including wild epiphytic yeast strains of grapevine berries. Twenty wild yeast, isolated and molecularly identified from three different Malaysian regions (Perlis, Perak and Pahang), were evaluated in a preliminary screening test on agar to select isolates with inhibition against Botrytis cinerea. On the basis of the results, nine yeasts belonging to genera Hanseniaspora, Starmerella, Metschnikowia, Candida were selected and then tested against five grape berry pathogens: Aspergillus carbonarius, Aspergillus ochraceus, Fusarium oxysporum, Alternaria alternata and Phaeomoniella chlamydospora.Starmerella bacillaris FE08.05 and Metschnikowia pulcherrima GP8 and Hanseniaspora uvarum GM19 showed the highest effect on inhibiting mycelial growth, which ranged between 15.1 and 4.3 mm for the inhibition ring. The quantitative analysis of the volatile organic compound profiles highlighted the presence of isoamyl and phenylethyl alcohols and an overall higher presence of low-chain fatty acids and volatile ethyl esters. The results of this study suggest that antagonist yeasts, potentially effective for the biological control of pathogenic moulds, can be found among the epiphytic microbiota associated with grape berries.

High-Pressure Homogenization and Biocontrol Agent as Innovative Approaches Increase Shelf Life and Functionality of Carrot Juice.

Recently, application of high-pressure homogenization (HPH) treatments has been widely studied to improve shelf life and rheological and functional properties of vegetable and fruit juices. Another approach that has drawn the attention of researchers is the use of biocontrol cultures. Nevertheless, no data on their possible combined effect on fruit juices shelf life and functionality have been published yet. In this work, the microbial, organoleptic, and technological stability of extremely perishable carrot juice and its functionality were monitored for 12 and 7 days (stored at 4 and 10 °C, respectively) upon HPH treatment alone or in combination with a fermentation step using the biocontrol agent L. lactis LBG2. HPH treatment at 150 MPa for three passes followed by fermentation with L. lactis LBG2 extended the microbiological shelf life of the products of at least three and seven days when stored at 10 °C and 4 °C, respectively, compared to untreated or only HPH-treated samples. Moreover, the combined treatments determined a higher stability of pH and color values, and a better retention of ß-carotene and lutein throughout the shelf-life period when compared to unfermented samples. Eventually, use of combined HPH and LBG2 resulted in the production of compounds having positive sensory impact on carrot juice.

Effects of Sub-Lethal High Pressure Homogenization Treatment on the Adhesion Mechanisms and Stress Response Genes in Lactobacillus acidophilus 08.

Cell surface hydrophobicity (CSH) and adhesion are very important phenotypical traits for probiotics that confer them a competitive advantage for the resilience in the human gastrointestinal tract. This study was aimed to understand the effects over time of a 50 MPa hyperbaric treatment on the surface properties of Lactobacillus acidophilus 08 including CSH, autoaggregation, and in vitro adhesion (mucin layer and Caco-2 cells). Moreover, a link between the hurdle applied and the expression of genes involved in the general stress response (groEL and clpP) and adhesion processes (efTu and slpA) was evaluated. High pressure homogenization (HPH) at 50 MPa significantly increased the CSH percentage (H%), autoaggregation and in vitro adhesion on mucin of L. acidophilus 08 cells compared with the untreated cells. Moreover, the hyperbaric hurdle induced an upregulation of the stress response genes groEL and ef-TU together with a down regulation of the clpP and S-layer slpA genes. Looking at the protein profile, HPH-treatment showed an increase in the number or intensity of protein bands at high and low molecular weights.

Lactobacillus paracasei A13 and High-Pressure Homogenization Stress Response.

Sub-lethal high-pressure homogenization treatments applied to Lactobacillus paracasei A13 demonstrated to be a useful strategy to enhance technological and functional properties without detrimental effects on the viability of this strain. Modification of membrane fatty acid composition is reported to be the main regulatory mechanisms adopted by probiotic lactobacilli to counteract high-pressure stress. This work is aimed to clarify and understand the relationship between the modification of membrane fatty acid composition and the expression of genes involved in fatty acid biosynthesis in Lactobacillus paracasei A13, before and after the application of different sub-lethal hyperbaric treatments. Our results showed that Lactobacillus paracasei A13 activated a series of reactions aimed to control and stabilize membrane fluidity in response to high-pressure homogenization treatments. In fact, the production of cyclic fatty acids was counterbalanced by the unsaturation and elongation of fatty acids. The gene expression data indicate an up-regulation of the genes accA, accC, fabD, fabH and fabZ after high-pressure homogenization treatment at 150 and 200 MPa, and of fabK and fabZ after a treatment at 200 MPa suggesting this regulation of the genes involved in fatty acids biosynthesis as an immediate response mechanism adopted by Lactobacillus paracasei A13 to high-pressure homogenization treatments to balance the membrane fluidity. Although further studies should be performed to clarify the modulation of phospholipids and glycoproteins biosynthesis since they play a crucial role in the functional properties of the probiotic strains, this study represents an important step towards understanding the response mechanisms of Lactobacillus paracasei A13 to sub-lethal high-pressure homogenization treatments.

How Listeria monocytogenes Shapes Its Proteome in Response to Natural Antimicrobial Compounds.

The goal of this study was to investigate the adaptation of L. monocytogenes Scott A cells to treatments with sublethal doses of antimicrobials (ethanol, citral, carvacrol, E-2-hexenal and thyme essential oil). The survival of L. monocytogenes cells was not affected by the antimicrobials at the concentrations assayed, with the exception of ethanol (1% v/v) and thyme essential oil (100 mg/L), which decreased cell viability from 8.53 ± 0.36 to 7.20 ± 0.22 log CFU/mL (P = 0.04). We subsequently evaluated how L. monocytogenes regulates and shapes its proteome in response to antimicrobial compounds. Compared to the control cells grown under optimal conditions, L. monocytogenes treated for 1 h with the antimicrobial compounds showed increased or decreased (≥ or ≤2-fold, respectively, P < 0.05) levels of protein synthesis for 223 protein spots. As shown multivariate clustering analysis, the proteome profiles differed between treatments. Adaptation and shaping of proteomes mainly concerned cell cycle control, cell division, chromosome, motility and regulatory related proteins, carbohydrate, pyruvate, nucleotide and nitrogen metabolism, cofactors and vitamins and stress response with contrasting responses for different stresses. Ethanol, citral (85 mg/l) or (E)-2-hexenal (150 mg/L) adapted cells increased survival during acid stress imposed under model (BHI) and food-like systems.

Flow Cytometric Assessment of the Morphological and Physiological Changes of Listeria monocytogenes and Escherichia coli in Response to Natural Antimicrobial Exposure.

Essential oils (EOs) or their components represent one of the most promising natural, safe, and feasible alternatives to prevent the growth of food-borne pathogens like Listeria monocytogenes and Escherichia coli in food matrices. Although antimicrobial properties of EOs and their components are well-documented, limited and fragmented information is available on the changes induced by these compounds, even at sub-lethal concentrations, in the physiological properties of microbial cells. The aim of this study was to explore the morpho-physiological changes of L. monocytogenes Scott A and E. coli MG 1655 induced after 1 h exposure to different sub-lethal and lethal concentrations of citral, carvacrol, (E)-2-hexenal, and thyme EO. For this purpose, different cell viability parameters such as membrane integrity, esterase activity, and cytoplasmic cell membrane potential were measured by flow cytometry. Flow cytometric data revealed specific response patterns in relation to the strain, the natural antimicrobial and its concentrations. Both the target microbial strains showed an increased cell membrane permeabilization without a loss of esterase activity and cell membrane potential with increasing citral, carvacrol and thyme EO concentrations. By contrast, (E)-2-hexenal did not significantly affect the measured physiological properties of L. monocytogenes Scott A and E. coli MG 1655. The used approach allowed identifying the most effective natural antimicrobials in relation to the microbial target.

Gene expression responses of Listeria monocytogenes Scott A exposed to sub-lethal concentrations of natural antimicrobials.

One of the emerging strategies proposed to prevent the presence and the growth of Listeria monocytogenes in food products is the use of natural antimicrobial compounds like essential oils (EOs) or their components alone or in combination with other mild hurdles. The aim of this study was to explore the gene expression mechanisms of L. monocytogenes Scott A exposed for 1 h to different sub-lethal antimicrobial concentrations of (E)-2-hexenal, citral, carvarcol and thyme EO in order to understand the impact of these molecules on the main metabolic pathways of this pathogenic strain. RT-qPCR has been performed on L. monocytogenes cells exposed to the target antimicrobials. The presence of the antimicrobials induced a clear unbalance in the catabolic processes, suggesting a shift from oxidation to fermentation metabolism (pdhD, pgm). Moreover, the results highlighted how antimicrobials belonged to the same chemical class induced different stress response mechanisms on L. monocytogenes Scott A. The information about the cell responses to the exposure to the natural antimicrobials selected is crucial to understand which cell target(s) can be affected, and consequently how the inhibition of pathogens survival can be further enhanced.

Microencapsulation of functional strains by high pressure homogenization for a potential use in fermented milk.

This study was aimed to evaluate the potential of high pressure homogenization for the microencapsulation of two probiotic lactic acid bacteria, Lactobacillus paracasei A13 and Lactobacillus salivarius subsp. salivarius CET 4063 to produce functional fermented milks. Microcapsules of the considered functional microorganisms were obtained by HPH treatments at 50MPa in the presence of sodium alginate and vegetable oil. The microencapsulated microorganisms were then inoculated as adjuncts to produce fermented milks. As controls were used fermented milks in which the two probiotic lactobacilli were inoculated without encapsulation. The viability of the strains was monitored during almost 2months of refrigerated storage. The survival of lactic acid bacteria after the gastric-duodenal simulated test was determined. Fermented milk texture parameters, the presence of exo-polysaccharides and the production of volatile molecules were also evaluated over storage. The microcapsules, for both the considered probiotic strains, were homogeneous and with a size<100µM and therefore did not adversely affect the sensory properties of the fermented milks. The encapsulation decreased the hyperacidity phenomena generally related to the inclusion of probiotic microorganisms in fermented milks. The lower acidity of the products due to the microencapsulation was fundamental for the improvement of the viability of the starter culture and the sensory characteristics of the products. The microencapsulation conditions increased the resistance to the simulated digestion processes, although the strain Lb. paracasei A13 generally showed a higher resistance to the gastric barrier respect to Lb. salivarius CECT 4063. By contrast, the data obtained showed a reduction of EPS production by the microencapsulation. The volatile profiles showed specific profiles in relation to the probiotic strain used and microencapsulation process. In conclusion, the results of this study underlined the applicative potential of HPH microencapsulation of probiotic microorganisms to produce fermented milk with improved functionality and with enhanced sensory properties.