Blue Whiting (Micromesistius poutassou) Protein Hydrolysates Increase GLP-1 Secretion and Proglucagon Production in STC-1 Cells Whilst Maintaining Caco-2/HT29-MTX Co-Culture Integrity.

Inducing the feeling of fullness via the regulation of satiety hormones presents an effective method for reducing excess energy intake and, in turn, preventing the development of obesity. In this study, the ability of blue whiting soluble protein hydrolysates (BWSPHs) and simulated gastrointestinal digested (SGID) BWSPHs, to modulate the secretion and/or production of satiety hormones, such as glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK) and peptide YY (PYY), was assessed in murine enteroendocrine STC-1 cells. All BWSPHs (BW-SPH-A to BW-SPH-F) (1.0% w/v dw) increased active GLP-1 secretion and proglucagon production in STC-1 cells compared to the basal control (Krebs-Ringer buffer) (p < 0.05). The signaling pathway activated for GLP-1 secretion was also assessed. A significant increase in intracellular calcium levels was observed after incubation with all BWSPHs (p < 0.05) compared with the control, although none of the BWSPHs altered intracellular cyclic adenosine monophosphate (cAMP) concentrations. The secretagogue effect of the leading hydrolysate was diminished after SGID. Neither pre- nor post-SGID hydrolysates affected epithelial barrier integrity or stimulated interleukin (IL)-6 secretion in differentiated Caco-2/HT-29MTX co-cultured cells. These results suggest a role for BWSPH-derived peptides in satiety activity; however, these peptides may need to be protected by some means to avoid loss of activity during gastrointestinal transit.

A comprehensive review on carotenoids in foods and feeds: status quo, applications, patents, and research needs.

Carotenoids are isoprenoids widely distributed in foods that have been always part of the diet of humans. Unlike the other so-called food bioactives, some carotenoids can be converted into retinoids exhibiting vitamin A activity, which is essential for humans. Furthermore, they are much more versatile as they are relevant in foods not only as sources of vitamin A, but also as natural pigments, antioxidants, and health-promoting compounds. Lately, they are also attracting interest in the context of nutricosmetics, as they have been shown to provide cosmetic benefits when ingested in appropriate amounts. In this work, resulting from the collaborative work of participants of the COST Action European network to advance carotenoid research and applications in agro-food and health (EUROCAROTEN, www.eurocaroten.eu, https://www.cost.eu/actions/CA15136/#tabs|Name:overview) research on carotenoids in foods and feeds is thoroughly reviewed covering aspects such as analysis, carotenoid food sources, carotenoid databases, effect of processing and storage conditions, new trends in carotenoid extraction, daily intakes, use as human, and feed additives are addressed. Furthermore, classical and recent patents regarding the obtaining and formulation of carotenoids for several purposes are pinpointed and briefly discussed. Lastly, emerging research lines as well as research needs are highlighted.

High-Pressure Processing on Whole and Peeled Potatoes: Influence on Polyphenol Oxidase, Antioxidants, and Glycaemic Indices.

Polyphenol oxidase (PPO) inactivation in five whole and peeled Irish potato cultivars was investigated using high-pressure processing (HPP) at 400 MPa and 600 MPa for 3 min. PPO activity was significantly lower in most of the HPP-treated samples, while the highest PPO inactivation was observed after HPP at 600 MPa. No significant (p > 0.05) changes were observed on the total phenolic content and antioxidant activity of all the HPP-treated potatoes. Regarding individual phenolic acids, chlorogenic acid was decreased significantly (p < 0.05) in all studied varieties with a concomitant increase (p < 0.05) in caffeic and quinic acid. Similarly, ferulic acid was also increased (p < 0.05) in all studied varieties after the HPP treatment, while there was a variation in rutin and 4-coumaric acid levels depending on the cultivar and the sample type. Anthocyanins in the coloured whole potato varieties (i.e., Kerr's Pink and Rooster), tentatively identified as pelargonidin-O-ferulorylrutinoside-O-hexoside and pelargonidin-O-rutinoside-O-hexoside, also exhibited significantly (p < 0.05) higher levels in the HPP-treated samples as opposed to those untreated. Glycaemic indices of the potatoes treated with HPP did not differ with the corresponding untreated cultivars.

The impact of thermal processing on the simulated infant gastrointestinal digestion, bactericidal and anti-inflammatory activity of bovine lactoferrin - An in vitro study.

Lactoferrin (LF) is a multifunctional glycoprotein which, when thermally processed, undergoes significant physicochemical changes. The link between such changes and the bioactivity of LF is not well characterised and requires much research. In this work, bovine LF solutions (1%, w/v, protein, pH 7) were thermally processed using high temperature short time conditions (72, 80, 85 or 95 °C with 15 s holding times). Following this, it was shown that LF and heat induced LF aggregates were largely resistant to simulated infant gastric, but not intestinal, digestion. Also, the efficacy of LF bactericidal activity, and inhibition of lipopolysaccharide-induced NF-κB activation were negatively impacted by thermal processing. This study confirmed that the efficacy of LF bio-functionalities was affected by the extent of heat-induced changes in protein structure whereby processing conditions of least severity (i.e. pasteurisation) had the least impact on bioactivity.

Assessment of the biological activity of fish muscle protein hydrolysates using in vitro model systems.

The generation of biologically active fish protein hydrolysates (FPH) is a useful technique to produce value-added products with potential application in the functional food and nutraceutical industries. Fish muscle is an attractive substrate for the production of protein hydrolysates due to its rich protein content, containing 15-25% of total fish protein. This paper reviews the production of protein hydrolysates from fish muscle, most commonly via enzymatic hydrolysis, and their subsequent bioactivities including anti-obesity, immunomodulatory, antioxidant, angiotensin I-converting enzyme (ACE)-inhibitory, anti-microbial, and anti-cancer activities as measured by in vitro testing methods. Disease prevention with FPH potentially offers a safe and natural alternative to synthetic drugs. Small molecular weight (MW) FPHs generally exhibit favourable bioactivity than large MW fractions via enhanced absorption through the gastrointestinal tract. This review also discusses the relationship between amino acid (AA) composition and AA sequence of FPH and peptides and their exhibited in vitro bioactivity.

Carotenoids: Considerations for Their Use in Functional Foods, Nutraceuticals, Nutricosmetics, Supplements, Botanicals, and Novel Foods in the Context of Sustainability, Circular Economy, and Climate Change.

Carotenoids are versatile isoprenoids that are important in food quality and health promotion. There is a need to establish recommended dietary intakes/nutritional reference values for carotenoids. Research on carotenoids in agro-food and health is being propelled by the two multidisciplinary international networks, the Ibero-American Network for the Study of Carotenoids as Functional Foods Ingredients (IBERCAROT; http://www.cyted.org) and the European Network to Advance Carotenoid Research and Applications in Agro-Food and Health (EUROCAROTEN; http://www.eurocaroten.eu). In this review, considerations for their safe and sustainable use in products mostly intended for health promotion are provided. Specifically, information about sources, intakes, and factors affecting bioavailability is summarized. Furthermore, their health-promoting actions and importance in public health in relation to the contribution of reducing the risk of diverse ailments are synthesized. Definitions and regulatory and safety information for carotenoid-containing products are provided. Lastly, recent trends in research in the context of sustainable healthy diets are summarized.

A Marine-Derived, Multi-mineral Supplement Influences Bacterial Fermentation and Short Chain Fatty Acid Profile In Vitro.

Aquamin is a calcium-rich multi-mineral supplement derived from the red marine algae, Lithothamnion species. Calcium supplementation has been shown to exert a prebiotic-like effect on the gut microbiota and has been associated with distinct changes in lactate and short chain fatty acid (SCFA) production. Irritable bowel syndrome (IBS) subtype is associated with changes in SCFA levels compared with healthy controls. Using an ex vivo simulation model, and a fecal inoculum from a patient diagnosed with IBS, we evaluated the effects of Aquamin (at 6 and 30 mg/mL) on SCFAs and lactate production, pH and gas production, and human microbiota composition. Our results demonstrate that Aquamin increased SCFA production (acetate and propionate by 8% and 24%, respectively, at 30 mg/mL dose), significantly decreased lactate production (30 mg/mL), and increased colonic fluid pH without inducing changes in colonic gas production or gastrointestinal (GI) microbiota composition. These results indicate that Aquamin may play a role in optimizing GI microbial function in an ex vivo setting.

Whey for Sarcopenia; Can Whey Peptides, Hydrolysates or Proteins Play a Beneficial Role?

As the human body ages, skeletal muscle loses its mass and strength. It is estimated that in 10% of individuals over the age of 60, this muscle frailty has progressed to sarcopenia. Biomarkers of sarcopenia include increases in inflammatory markers and oxidative stress markers and decreases in muscle anabolic markers. Whey is a high-quality, easily digested dairy protein which is widely used in the sports industry. This review explores the evidence that whey protein, hydrolysates or peptides may have beneficial effects on sarcopenic biomarkers in myoblast cell lines, in aged rodents and in human dietary intervention trials with the older consumer. A daily dietary supplementation of 35 g of whey is likely to improve sarcopenic biomarkers in frail or sarcopenia individuals. Whey supplementation, consumed by an older, healthy adult certainly improves muscle mTOR signaling, but exercise appears to have the greatest benefit to older muscle. In vitro cellular assays are central for bioactive and bioavailable peptide identification and to determine their mechanism of action on ageing muscle.

Development of a dehydrated fortified food base from fermented milk and parboiled wheat, and comparison of its composition and reconstitution behavior with those of commercial dried dairy-cereal blends.

Dehydrated blends of milk and cereal are reconstituted and consumed as a nutritious soup or porridge in many regions; the composition and reconstitution behavior of the blends are likely to impact on nutritional quality and consumer acceptability of the soup/porridge. Experimental samples of dried fermented milk-bulgur wheat blend (FMBW) and commercial samples of dried dairy-cereal blends, namely kishk, tarhana, and super cereal plus corn-soy blend (SCpCSB) were compared for composition, color, water sorption, and reconstitution characteristics. FMBW blends had higher contents of protein, Ca, lactose and lactic acid, lower levels of salt (NaCl) and Fe, and a lighter, more-yellow color (higher L* and b*-color co-ordinates) than tarhana or kishk. Compared with SCpCSB, FMBW had numerically higher levels of protein, lactose, and lactic acid, lower levels of Ca, Fe, Zn, and Mg, and lower pH. Tarhana had highest mean levels of starch, and on reconstitution (133 g/kg) had highest water holding capacity, viscosity during pasting and cooling, yield stress (σ 0), consistency coefficient (K), and viscosity on shearing from 20 to 120 s-1 at 60°C. Reconstituted FMBW, kishk, and SCpCSB had similar pasting and flow behavior properties. Overall, the composition (starch, protein, Ca, Mg), pasting and flow behavior characteristics of FMBW were closer to those SCpCSB and kishk than to tarhana. The results suggest that the FMBW powder, on appropriate supplementation with Ca, Fe, Zn and Mg, could be used for the development of customized fortified blended foods for specific groups.

The Effect of High Pressure Processing on Polyphenol Oxidase Activity, Phytochemicals and Proximate Composition of Irish Potato Cultivars.

Polyphenol oxidase (PPO) activity, proximate composition, and phytochemicals were determined in four common Irish potato cultivars following a high pressure processing (HPP) at 600 MPa for 3 min. PPO activity was significantly (p < 0.05) lower in all HPP treated samples, while the overall proximate composition was not affected. The total phenolic content was significantly higher in the HPP treated samples. Chlorogenic acid levels significantly decreased with simultaneous increase of caffeic acid and p-coumaric acid levels upon HPP treatment. No significant changes were observed in rutin and ferulic acid levels, although their levels varied, depending on the potato cultivars, while the levels of cytotoxic glycoalkaloids (α-solanine and α-chaconine) remained unaltered.

Fortified Blended Food Base: Effect of Co-Fermentation Time on Composition, Phytic Acid Content and Reconstitution Properties.

Dehydrated blends of dairy-cereal combine the functional and nutritional properties of two major food groups. Fortified blended food base (FBFB) was prepared by blending fermented milk with parboiled wheat, co-fermenting the blend at 35 °C, shelf-drying and milling. Increasing co-fermentation time from 0 to 72 h resulted in powder with lower lactose, phytic acid and pH, and higher contents of lactic acid and galactose. Simultaneously, the pasting viscosity of the reconstituted base (16.7%, w/w, total solids) and its yield stress (σ0), consistency index (K) and viscosity on shearing decreased significantly. The changes in some characteristics (pH, phytic acid, η120) were essentially complete after 24 h co-fermentation while others (lactose, galactose and lactic acid, pasting viscosities, flowability) proceeded more gradually over 72 h. The reduction in phytic acid varied from 40 to 58% depending on the pH of the fermented milk prior to blending with the parboiled cereal. The reduction in phytic acid content of milk (fermented milk)-cereal blends with co-fermentation time is nutritionally desirable as it is conducive to an enhanced bioavailability of elements, such as Ca, Mg, Fe and Zn in milk-cereal blends, and is especially important where such blends serve as a base for fortified-blended foods supplied to food-insecure regions.

Characterisation of the in vitro bioactive properties of alkaline and enzyme extracted brewers' spent grain protein hydrolysates.

Brewer's spent grain (BSG) is a co-product of the brewing industry that has been shown to contain a range of bioactive peptides encrypted within its protein sequences. Two methods were evaluated herein to generate bioactive peptides; (i) an alkaline extracted BSG protein rich fraction (BSG-PI) was hydrolysed using different combinations of proteolytic enzymes and (ii) BSG was pre-treated with carbohydrases followed by direct hydrolysis using proteolytic enzymes (BSG-DH). BSG-DH with Alcalase/Flavourzyme resulted in significantly higher (p < .05) protein yield when compared to BSG-PI (63.09 ±â€¯0.27 and 58.90 ±â€¯1.45%, respectively). The antioxidant activities (ORAC, FRAP and ABTS) of the BSG-PI and -DH hydrolysates differed depending on the assay and proteolytic enzyme combination preparations used for hydrolysis. Inhibition of DPP-IV by the BSG-PI hydrolysates ranged from 87.01 ±â€¯0.15 to 89.61 ±â€¯0.12% while inhibition by the BSG-DH hydrolysates ranged from 35.71 ±â€¯0.72 to 85.06 ±â€¯0.17%. A significant reduction in the release of interleukin-6 in lipopolysaccharide-stimulated RAW 264.7 cells was observed following treatment with BSG-PI hydrolysates generated with Prolyve/Protease P (58.30 ±â€¯13.76%) and Corolase PP/Flavourzyme (48.02 ±â€¯10.82%) when compared to untreated LPS stimulated control cells (100%). BSG-DH hydrolysates were subjected to in vitro simulated gastrointestinal digestion (SGID) which resulted in a reduction in antioxidant activity, an increase in DPP-IV inhibition and no change in the immunomodulatory activity. Ultrafiltration of selected BSG-DH hydrolysates (through 30 and 10 kDa membranes) gave some permeates with enhanced bioactivities. The results demonstrate that direct enzymatic hydrolysis of BSG is a feasible approach for the generation of bioactive peptides without the prior use of an alkali protein extraction step.

Biotin attenuation of oxidative stress, mitochondrial dysfunction, lipid metabolism alteration and 7ß-hydroxycholesterol-induced cell death in 158N murine oligodendrocytes.

Mitochondrial dysfunction and oxidative stress are involved in neurodegenerative diseases associated with an enhancement of lipid peroxidation products such as 7ß-hydroxycholesterol (7ß-OHC). It is, therefore, important to study the ability of 7ß-OHC to trigger mitochondrial defects, oxidative stress, metabolic dysfunctions and cell death, which are hallmarks of neurodegeneration, and to identify cytoprotective molecules. The effects of biotin were evaluated on 158N murine oligodendrocytes, which are myelin synthesizing cells, exposed to 7ß-OHC (50 µM) with or without biotin (10 and 100 nM) or α-tocopherol (positive control of cytoprotection). The effects of biotin on 7ß-OHC activities were determined using different criteria: cell adhesion; plasma membrane integrity; redox status. The impact on mitochondria was characterized by the measurement of transmembrane mitochondrial potential (ΔΨm), reactive oxygen species (ROS) overproduction, mitochondrial mass, quantification of cardiolipins and organic acids. Sterols and fatty acids were also quantified. Cell death (apoptosis, autophagy) was characterized by the enumeration of apoptotic cells, caspase-3 activation, identification of autophagic vesicles, and activation of LC3-I into LC3-II. Biotin attenuates 7ß-OHC-induced cytotoxicity: loss of cell adhesion was reduced; antioxidant activities were normalized. ROS overproduction, protein and lipid oxidation products were decreased. Biotin partially restores mitochondrial functions: attenuation of the loss of ΔΨm; reduced levels of mitochondrial O2•- overproduction; normalization of cardiolipins and organic acid levels. Biotin also normalizes cholesterol and fatty acid synthesis, and prevents apoptosis and autophagy (oxiapoptophagy). Our data support that biotin, which prevents oligodendrocytes damages, could be useful in the treatment of neurodegeneration and demyelination.

Antifungal activity of a de novo synthetic peptide and derivatives against fungal food contaminants.

The development of novel solutions to fight microbial food contaminants rests upon two pillars, which are the development of resistant strains and consumers' desire for a reduced consumption of synthetic drugs. Natural antimicrobial peptides possess the qualities to overcome these issues. De novo synthesis of novel antifungal compounds is a major progress that has been facilitated by the identification of parameters involved in the antimicrobial activity. A 14-residue peptide named KK14, with the sequence KKFFRAWWAPRFLK-NH2 , was designed and inhibited conidial germination and fungal growth of food contaminants within the range 6.25 to 50 µg/ml and 6.25 to 100 µg/ml, respectively. The study of three analogues of the peptide highlighted the role of some residues in the structural conformation of the peptide and its antifungal activity. The substitution of a Pro residue with Arg increased the helical content of the peptide not only its antifungal activity but also its cytotoxicity. The insertion of an unnatural bulky residue ß-diphenylalanine or a full d-enantiomerization overall increased the antifungal potency. The four peptides showed similar behaviour towards salt increase, heat treatment, and pH decrease. Interestingly, the denantiomer remained the most active at high pH and after proteolytic digestion. The four peptides did not present haemolytic activity up to 200 µg/ml but had different behaviours of cytotoxicity. These differences could be crucial for potential application as pharmaceutical or food preservatives.

Cereal type significantly affects the composition and reconstitution characteristics of dried fermented milk-cereal composites.

BACKGROUND: Dairy and cereal products are frequently combined to create composites with enhanced nutritional benefits. Commercially available dried dairy-cereal composites are typically reconstituted and cooked to produce porridge or soup. RESULTS: Dried fermented milk-cereal composites (FMCC) with ∼193 g kg-1 protein were prepared by blending fermented milk with parboiled oats (FMCCo), wheat (FMCCw), or barley (FMCCb), incubating the blend, drying, and milling. Cereal type significantly affected the composition of the FMCC and the properties of the reconstituted, cooked FMCC (R-FMCC). The FMCCo had a higher starch and fat content and lower levels of lactose, lactic acid, and amylose than FMCCb. The R-FMCCo had higher viscosity during cooking at 95 °C and cooling to 35 °C, and higher values of yield stress (σ0 ), consistency index (K) and viscosity on shearing from 20 to 120 s-1 at 60 °C than R-FMCCb. The FMCCw had lower levels of fat and ß-glucan than FMCCo or FMCCb, but was otherwise closer to FMMCb with respect to composition, cooking properties and flow behavior. CONCLUSION: Differences in composition and consistency associated with cereal type are likely to affect the nutritional value of the FMCC. © 2018 Society of Chemical Industry.

Comparison of the nutritional composition of experimental fermented milk:wheat bulgur blends and commercially available kishk and tarhana products.

Dried, fermented blends of dairy products and cereals, such as kishk and tarhana, are foodstuffs traditionally consumed in many regions as they possess good nutritional qualities and extended storage stability. This study examined the nutritional composition of kishk or tarhana type products and compared with experimental blends of fermented milk and wheat bulgur containing 60-80% milk. The blends with higher milk contents had levels of protein (18.9%) and fat (5.8%) at the concentrations specified in fortified blended foods as outlined by the World Food Program. Higher milk contents were also associated with higher contents of calcium (323.2 mg/100 g), phosphorus (335.3 mg/100 g), vitamin A (486.7 µg/100 g) and α-tocopherol (174.5 µg/100 g). The nutritional content of the experimental fermented milk:wheat bulgur blends compared favourably with that of the commercial samples. These blends may be suitable as base products, to be fortified with micronutrients, for the development of fortified blended foods (FBFs) for humanitarian distribution.

The Proportion of Fermented Milk in Dehydrated Fermented Milk⁻Parboiled Wheat Composites Significantly Affects Their Composition, Pasting Behaviour, and Flow Properties on Reconstitution.

Dairy and cereal are frequently combined to create composite foods with enhanced nutritional benefits. Dehydrated fermented milk⁻wheat composites (FMWC) were prepared by blending fermented milk (FM) and parboiled wheat (W), incubating at 35 °C for 24 h, drying at 46 °C for 48 h, and milling to 1 mm. Increasing the weight ratio of FM to W from 1.5 to 4.0 resulted in reductions in total solids (from 96 to 92%) and starch (from 52 to 39%), and increases in protein (15.2⁻18.9%), fat (3.7⁻5.9%), lactose (6.4⁻11.4%), and lactic acid (2.7⁻4.2%). FMWC need to be reconstituted prior to consumption. The water-holding capacity, pasting viscosity, and setback viscosity of the reconstituted FMWC (16.7% total solids) decreased with the ratio of FM to W. The reconstituted FMWC exhibited pseudoplastic flow behaviour on shearing from 18 to 120 s-1. Increasing the FM:W ratio coincided with a lower yield stress, consistency index, and viscosity at 120 s-1. The results demonstrate the critical impact of the FM:W ratio on the composition, pasting behavior, and consistency of the reconstituted FMWC. The difference in consistency associated with varying the FM:W ratio is likely to impact on satiety and nutrient value of the FMWCs.

Bioaccessibility and Bioavailability of a Marine-Derived Multimineral, Aquamin-Magnesium.

Introduction: Magnesium is an essential mineral involved in a range of key biochemical pathways. Several magnesium supplements are present on the market and their degree of bioavailability differs depending on the form of magnesium salt used. Aquamin-Mg is a natural source of magnesium, containing 72 additional trace minerals derived from the clean waters off the Irish coast. However, the in vitro bioaccessibility and bioavailability of Aquamin-Mg in comparison with other supplement sources of magnesium has yet to be tested. Method: Aquamin-Mg, magnesium chloride (MgCl2) and magnesium oxide (MgO) were subjected to gastrointestinal digestion according to the harmonized INFOGEST in vitro digestion method and in vitro bioavailability tested using the Caco-2 cell model. Magnesium concentration was measured by atomic absorption spectrophotometry (AAS). Results: Magnesium recovery from both Aquamin-Mg and MgCl2 was greater than for MgO. Magnesium from all three sources was transported across the epithelial monolayer with Aquamin-Mg displaying a comparable profile to the more bioavailable MgCl2. Conclusions: Our data support that magnesium derived from a marine-derived multimineral product is bioavailable to a significantly greater degree than MgO and displays a similar profile to the more bioavailable MgCl2 and may offer additional health benefits given its multimineral profile.

Concurrent iron and zinc deficiencies in lactating mothers and their children 6-23 months of age in two agro-ecological zones of rural Ethiopia.

PURPOSE: The aim of this study is to examine the co-occurrences of low serum ferritin and zinc and anaemia among mothers and their children in two agro-ecological zones of rural Ethiopia. METHODS: Data were collected from 162 lactating mothers and their breast fed children aged 6-23 months. The data were collected via a structured interview, anthropometric measurements, and blood tests for zinc, ferritin and anaemia. Correlation, Chi-square and multivariable analysis were used to determine the association between nutritional status of mothers and children, and agro-ecological zones. RESULTS: Low serum levels of iron and zinc, anaemia and iron deficiency anaemia were found in 44.4, 72.2, 52.5 and 29.6% of children and 19.8, 67.3, 21.8, 10.5% of mothers, respectively. There was a strong correlation between the micronutrient status of the mothers and the children for ferritin, zinc and anaemia (p < 0.005). Deficiency in both zinc and ferritin and one of the two was observed in 19.1, and 53.7% of the mothers and 32.7 and 46.3%, of their children, respectively. In the 24 h before the survey, 82.1% of mothers and 91.9% of their infants consumed foods that can decrease zinc bioavailability while only 2.5% of mothers and 3.7% of their infants consumed flesh foods. CONCLUSION: This study shows that micronutrient deficiencies were prevalent among lactating mothers and their children, with variation in prevalence across the agro-ecological zones. This finding calls for a need to design effective preventive public health nutrition programs to address both the mothers' and their children's needs.

Optimisation of the antifungal potency of the amidated peptide H-Orn-Orn-Trp-Trp-NH2 against food contaminants.

The design of novel efficient antimicrobial peptides (AMPs) faces several issues, such as cost of synthesis, proteolytic stability or cytotoxicity. The identification of key determinants involved in the activity of AMPs, such as cationicity and amphipathicity, allowed the synthesis of short peptides with optimized properties. An ultrashort peptide made of the sequence H-Orn-Orn-Trp-Trp-NH2 (O3TR) showed antifungal activity against several contaminants from food products. This peptide inhibited the growth of the filamentous fungi Fusarium culmorum, Penicillium expansum and Aspergillus niger within a range of concentration of 12.5-50µg/ml. In addition, O3TR inhibited the growth of the yeast Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii and Kluyveromyces lactis within the range 12.5-50µg/ml. A derivative peptide, called C12O3TR, made by the addition of lauric acid at the N-terminus of O3TR was 2- to 8-fold more active than O3TR against every species. In addition to the inhibition of conidial germination, O3TR and C12O3TR killed F. culmorum hyphae at 100 and 50µg/ml respectively. The MIC of the two peptides against F. culmorum and Z. bailii after heat treatment at 100°C for 60 min and within the pH range 3-10, were not changed. However, the activity of O3TR against F.culmorum and Z. bailii was strongly reduced in salt solutions, whereas the lauric acid peptide kept its antifungal activity and resistance to proteolytic digestion. The conjugation with lauric acid reduced the random coiled structure and increased the α-helical content of O3TR. After conjugation with the dye tetramethylrhodamine (TMR), both peptides entered F. culmorum spores. They also both induced permeabilization of F. culmorum hyphae but only C12O3TR permeabilized Z. bailii membrane. In contrast to the lipopeptide, O3TR did not show haemolytic or cytotoxic activity when applied at the concentrations that exhibited antifungal potency. The two peptides were challenged against a yeast cocktail of S. cerevisiae and Z. bailii, and A. niger in different commercial beverages. After 7 days, O3TR was able to inhibit the yeast cocktail in a commercial lager and carbonated drink. Due to its antifungal potency, high stability and low cytotoxicity, the tetrapeptide could represent a promising starting point of a novel food preservative.