Factors impacting the microbial production of eicosapentaenoic acid.

The increasing applications for eicosapentaenoic acid (EPA) and the potential shortfall in supply due to sustainability and contamination issues related with its conventional sources (i.e., fish oils; seafood) led to an extensive search for alternative and sustainable sources, as well as production processes. The present mini-review covers all the steps involved in the production of EPA from microorganisms, with a deeper focus on microalgae. From production systems to downstream processing, the most important achievements within each area are briefly highlighted. Comparative tables of methodologies are also provided, as well as additional references of recent reviews, so that readers may deepen their knowledge in the different issues addressed. KEY POINTS: • Microorganisms are more sustainable alternative sources of EPA than fish. • Due to the costly separation from DHA, species that produce only EPA are preferable. • EPA production can be optimised using non-genetic and genetic tailoring engineering.

Combining high pressure and electric fields towards Nannochloropsis oculata eicosapentaenoic acid-rich extracts.

Nannochloropsis oculata is naturally rich in eicosapentaenoic acid (EPA). To turn this microalga into an economically viable source for commercial applications, extraction efficiency must be achieved. Pursuing this goal, emerging technologies such as high hydrostatic pressure (HHP) and moderate electric fields (MEF) were tested, aiming to increase EPA accessibility and subsequent extraction yields. The innovative approach used in this study combined these technologies and associated tailored, less hazardous different solvent mixtures (SM) with distinct polarity indexes. Although the classical Folch SM with chloroform: methanol (PI 4.4) provided the highest yield concerning total lipids (166.4 mglipid/gbiomass), diethyl ether: ethanol (PI 3.6) presented statistically higher values in terms of EPA per biomass, corresponding to 1.3-fold increase. When SM were used in HHP and MEF, neither technology independently improved EPA extraction yields, although the sequential combination of technologies did result in 62% increment in EPA extraction. Overall, the SM and extraction methodologies tested (HHP-200 MPa, 21 °C, 15 min, followed by MEF processing at 40 °C, 15 min) enabled increased EPA extraction yields from wet N. oculata biomass. These findings are of high relevance for the food and pharmaceutical industries, providing viable alternatives to the "classical" extraction methodologies and solvents, with increased yields and lower environmental impact. KEY POINTS: • Et2O: EtOH is a less toxic and more efficient alternative to Folch solvent mixture • HHP or MEF per se was not able to significantly increase EPA extraction yield • Combinations of HHP and MEF technologies increased both lipids and EPA yields.

Modulated stress to balance Nannochloropsis oculata growth and eicosapentaenoic acid production.

Two environmental parameters, temperature and light intensity, were independently used as stress modulators to enhance eicosapentaenoic acid (EPA) production by the microalga Nannochloropsis oculata, without hindering biomass production. A sinusoidal approach was used, as environmental conditions were alternated between optimum and stress status in multi-day cycles. Low temperatures (5 and 10 °C) and light intensities (30 and 50 µmol photons/m2/s) were tested. Results revealed that the modulated stress approach used was able to avoid decreases in biomass production. Temperature stress (10 °C) presented the highest impact, increasing EPA content to 12.8 mgEPA/L, 158% more than the amount obtained in optimum (non-modulated) growth conditions at that point in time, while the lower light intensity stress was able to increase to 126% more. It is important to point out that in both cases increases in EPA amounts resulted from increased content in each individual cell and not just from increased biomass contents. KEY POINTS: • Temperature stress (10 °C) presented the highest impact increasing EPA content 158% • Lower light intensity stress was able to increase EPA to 126% more • EPA increased in individual cell contents simultaneous with biomass increase.

Can Growth of Nannochloropsis oculata under Modulated Stress Enhance Its Lipid-Associated Biological Properties?

Nannochloropsis oculata is well-recognized as a potential microalgal source of valuable compounds such as polyunsaturated fatty acids, particularly, eicosapentaenoic acid (EPA). The content and profile of these lipids is highly dependent on the growth conditions and can, therefore, be tailored through modulation of the growth parameters, specifically, temperature. Moreover, biological activities are composition dependent. In the present work, lipid extracts obtained from N. oculata, grown under constant temperature and under modulated temperature stress (to increase EPA content; Str) were characterized by GC-FID and several bioactivities were evaluated, namely, antioxidant (L-ORACFL), cytotoxic (MTT), adipolytic, anti-hepatic lipid accumulation (steatosis), and anti-inflammatory properties. Both extracts exhibited antioxidant activity (c.a. 49 µmol Troloxequivalent/mgextract) and the absence of toxicity (up to 800 µg/mL) toward colon and hepatic cells, adipocytes, and macrophages. They also induced adipolysis and the inhibition of triglycerides hepatic accumulation, with a higher impact from Str. In addition, anti-inflammatory activity was observed in the lipopolysaccharide-induced inflammation of macrophages in the presence of either extract, since lower levels of pro-inflammatory interleukin-6 and interferon-ß were obtained, specifically by Str. The results presented herein revealed that modulated temperature stress may enhance the health effects of N. oculata lipid extracts, which may be safely utilized to formulate novel food products.

Pomegranate Oil's Potential as an Anti-Obesity Ingredient.

In recent years, pomegranate oil has obtained more attention due to its content of conjugated linolenic acids and possible application in the prevention of many diseases. The purpose of this work was to evaluate the potential ability of pomegranate oil to modulate obesity-related metabolism and immune response using in vitro models. In this regard, pomegranate oil was characterized in terms of fatty acids profile, tocopherols and phytosterols, and antioxidant capacity. After evaluation of the safety profile, pomegranate oil's capacity to modulate obesity-related metabolism was evaluated through adipolysis and adipokines secretion quantification in 3T3-L1 differentiated adipocytes and hepatic lipid accumulation assay in Hep G2 hepatocytes. The immunomodulatory activity was evaluated in Caco-2 cells by quantification of pro-inflammatory cytokines IL-6, IL-8, and TNF-α. This oil showed high antioxidant capacity and was mainly composed of conjugated fatty acid, namely punicic acid. Its chemical composition was responsible for its capacity to reduce the lipid accumulation in Hep G2 cells and 3T3-L1 differentiated adipocytes. In short, pomegranate oil shows great potential for the development of functional foods and nutraceuticals targeting obesity.

Assessment of the efficacy of the utilisation of conventional and electric toothbrushes by the older adults.

OBJECTIVE: To evaluate the efficacy of electric and conventional toothbrushes for a group of elderly individuals. BACKGROUND: Although the electric toothbrush has been recommended for elderly individuals, there had previously never been a study regarding its efficacy. MATERIAL AND METHODS: Sixty independent elders of both genders with different oral conditions from the Center Adult Day Vitória, Espírito Santo, Brazil, were randomly divided into two groups of 30 individuals. One group received the Oral B CrossAction Power electric toothbrush, whereas the other received a conventional Bitufo Class 32 soft toothbrush to perform oral hygiene. The bacterial plaque index (O'Leary Plaque Index) and DMFT index were assessed as a measure of oral hygiene and oral health. The data were analysed using the Shapiro-Wilk, Mann-Whitney and Wilcoxon tests. RESULTS: The results of the efficacy of the Oral B Cross Action Power electric toothbrush demonstrated that on the 7th and 15th days, the bacterial plaque indexes were 24.91 ± 12.81 and 22.11 ± 14.46, respectively, which corresponds to a 50.24% removal of bacterial plaque on the 7th and 55.83% on the 15th days. Although the electric toothbrush removed more bacterial plaque than the conventional toothbrush, the difference was not statistically significant. CONCLUSION: Both the conventional and the electric toothbrushes were effective in removing bacterial plaque within the elderly group. More studies are necessary to test the efficacy of electric toothbrushes in relation to conventional toothbrushes for elderly patients.

Sargassum muticum and Osmundea pinnatifida Enzymatic Extracts: Chemical, Structural, and Cytotoxic Characterization.

Seaweeds, which have been widely used for human consumption, are considered a potential source of biological compounds, where enzyme-assisted extraction can be an efficient method to obtain multifunctional extracts. Chemical characterization of Sargassum muticum and Osmundea pinnatifida extracts obtained by Alcalase and Viscozyme assisted extraction, respectively, showed an increment of macro/micro elements in comparison to the corresponding dry seaweeds, while the ratio of Na/K decreased in both extracts. Galactose, mannose, xylose, fucose, and glucuronic acid were the main monosaccharides (3.2-27.3 mg/glyophilized extract) present in variable molar ratios, whereas low free amino acids content and diversity (1.4-2.7 g/100gprotein) characterized both extracts. FTIR-ATR and 1H NMR spectra confirmed the presence of important polysaccharide structures in the extracts, namely fucoidans from S. muticum or agarans as sulfated polysaccharides from O. pinnatifida. No cytotoxicity against normal mammalian cells was observed from 0 to 4 mglyophilized extract/mL for both extracts. The comprehensive characterization of the composition and safety of these two extracts fulfils an important step towards their authorized application for nutritional and/or nutraceutical purposes.

Application of High Pressure with Homogenization, Temperature, Carbon Dioxide, and Cold Plasma for the Inactivation of Bacterial Spores: A Review.

Formation of highly resistant spores is a concern for the safety of low-acid foods as they are a perfect vehicle for food spoilage and/or human infection. For spore inactivation, the strategy usually applied in the food industry is the intensification of traditional preservation methods to sterilization levels, which is often accompanied by decreases of nutritional and sensory properties. In order to overcome these unwanted side effects in food products, novel and emerging sterilization technologies are being developed, such as pressure-assisted thermal sterilization, high-pressure carbon dioxide, high-pressure homogenization, and cold plasma. In this review, the application of these emergent technologies is discussed, in order to understand the effects on bacterial spores and their inactivation and thus ensure food safety of low-acid foods. In general, the application of these novel technologies for inactivating spores is showing promising results. However, it is important to note that each technique has specific features that can be more suitable for a particular type of product. Thus, the most appropriate sterilization method for each product (and target microorganisms) should be assessed and carefully selected.

Evidences and perspectives in the utilization of CLNA isomers as bioactive compounds in foods.

Conjugated alpha linolenic acid (CLNA) isomers are promising lipids owing to their similarities with conjugated linoleic acid (CLA) but exerting their bioactivity at lower doses; some isomers also belong to omega 3 family. This review aims to summarize the state of the art about the utilization of CLNA as a functional ingredient. Indeed, in vitro and in vivo studies reported that CLNA exerted anticancer, anti-inflammatory, anti-obese, and antioxidant activities. However, CLNA has not been tested in humans. These compounds are naturally present in meat and milk fat from ruminants but the highest concentrations are found in vegetable oils. Their incorporation in foodstuffs is one of the most effective strategies to elaborate CLNA-enriched products together with the microbiological production. Lactobacilli, propionibacteria, and bifidobacteria strains have been assayed to produce CLNA isomers but at the current moment there are not high CLNA concentration products elaborated using these strains. Furthermore, it is known that CLNA isomers are highly prone to oxidation when compared with linoleic acid and CLA, but the possible effects of elaboration and storage on high CLNA productsare unknown.The utilization of CLNA as a functional compound still remains a challenge and requires more research to address all of its technological and bioactivity aspects.

Valorization of By-Products from Commercial Fish Species: Extraction and Chemical Properties of Skin Gelatins.

Fish skins constitute an important fraction of the enormous amount of wastes produced by the fish processing industry, part of which may be valorized through the extraction of gelatins. This research exploited the extraction and characterization of gelatins from the skin of three seawater fish species, namely yellowfin tuna (Thunnus albacares), blue shark (Prionace glauca), and greenland halibut (Reinhardtius hippoglossoides). Characterization included chemical composition, rheology, structure, texture, and molecular weight, whereas extraction studies intended to reduce costly steps during extraction process (reagents concentration, water consumption, and time of processing), while maintaining extraction efficiency. Chemical and physical characterization of the obtained gelatins revealed that the species from which the gelatin was extracted, as well as the heat treatment used, were key parameters in order to obtain a final product with specific properties. Therefore, the extraction conditions selected during gelatin production will drive its utilization into markets with well-defined specifications, where the necessity of unique products is being claimed. Such achievements are of utmost importance to the food industry, by paving the way to the introduction in the market of gelatins with distinct rheological and textural properties, which enables them to enlarge their range of applications.

Carotenoids and Intestinal Harmony: Exploring the Link for Health.

Carotenoids, prominent lipid-soluble phytochemicals in the human diet, are responsible for vibrant colours in nature and play crucial roles in human health. While they are extensively studied for their antioxidant properties and contributions to vitamin A synthesis, their interactions with the intestinal microbiota (IM) remain poorly understood. In this study, beta (ß)-carotene, lutein, lycopene, a mixture of these three pigments, and the alga Osmundea pinnatifida were submitted to simulated gastrointestinal digestion (GID) and evaluated on human faecal samples. The results showed varying effects on IM metabolic dynamics, organic acid production, and microbial composition. Carotenoid exposure influenced glucose metabolism and induced the production of organic acids, notably succinic and acetic acids, compared with the control. Microbial composition analysis revealed shifts in phyla abundance, particularly increased Pseudomonadota. The α-diversity indices demonstrated higher diversity in ß-carotene and the pigments' mixture samples, while the ß-diversity analysis indicated significant dissimilarity between the control and the carotenoid sample groups. UPLC-qTOF MS analysis suggested dynamic changes in carotenoid compounds during simulated fermentation, with lutein exhibiting distinct mass ion fragmentation patterns. This comprehensive research enhances our understanding of carotenoid-IM interactions, shedding light on potential health implications and the need for tailored interventions for optimal outcomes.

More than a meat- or synthetic nitrogen fertiliser-substitute: a review of legume phytochemicals as drivers of 'One Health' via their influence on the functional diversity of soil- and gut-microbes.

Legumes are essential to healthy agroecosystems, with a rich phytochemical content that impacts overall human and animal well-being and environmental sustainability. While these phytochemicals can have both positive and negative effects, legumes have traditionally been bred to produce genotypes with lower levels of certain plant phytochemicals, specifically those commonly termed as 'antifeedants' including phenolic compounds, saponins, alkaloids, tannins, and raffinose family oligosaccharides (RFOs). However, when incorporated into a balanced diet, such legume phytochemicals can offer health benefits for both humans and animals. They can positively influence the human gut microbiome by promoting the growth of beneficial bacteria, contributing to gut health, and demonstrating anti-inflammatory and antioxidant properties. Beyond their nutritional value, legume phytochemicals also play a vital role in soil health. The phytochemical containing residues from their shoots and roots usually remain in-field to positively affect soil nutrient status and microbiome diversity, so enhancing soil functions and benefiting performance and yield of following crops. This review explores the role of legume phytochemicals from a 'one health' perspective, examining their on soil- and gut-microbial ecology, bridging the gap between human nutrition and agroecological science.

Metabolic Evaluation of Lupin-Enriched Yogurt by Nuclear Magnetic Resonance Metabolomics.

Untargeted nuclear magnetic resonance (NMR) metabolomics was used to evaluate compositional changes during yogurt fermentation upon lupin enrichment compared to traditional conditions. Lupin significantly changed the sample metabolic profile and its time course dynamics, seemingly delaying microbial action. The levels of organic and amino acids were significantly altered, along with those of some sugars, nucleotides, and choline compounds. Lupin seemed to favor acetate and formate synthesis, compared to that of citrate and fumarate; a higher formate levels may suggest increased levels of Streptococcus thermophilus action, compared toLactobacillus bulgaricus. Lupin-yogurt was poorer in hippurate, lactose (and hence lactate), galactose, glucose-1-phosphate, and galactose-1-phosphate, containing higher orotate levels (possibly related to increased uridine derivatives), among other differences. Trigonelline was confirmed as a lupin marker, possibly together with glutamate and histidine. Other metabolite trajectories remained unchanged upon lupin addition, unveiling unaffected underlying processes. These results demonstrate the usefulness of untargeted NMR metabolomics to understand/develop new foodstuffs and their production processes, highlighting the identity of a variety of bioactive metabolites with importance for human health.

Bioactivity of probiotic whey cheese: characterization of the content of peptides and organic acids.

BACKGROUND: Probiotic whey cheeses have been produced for several years. It is recognized that several bacterium-mediated metabolic activities contribute differently to the final sensory and nutritional profiles of dairy products. Hence the metabolic activity of probiotic strains in a whey cheese and their contribution to the bioactivity of such matrices were investigated here, including in particular Bifidobacterium animalis, Lactobacillus acidophilus and Lactobacillus casei. RESULTS: Both L. casei and B. animalis produce lactic and acetic acids, whereas L. acidophilus produce mainly lactic acid; these metabolites may be considered bioprotection factors. Water-soluble extracts (WSE) obtained from these cheese matrices were subjected to ultrafiltration through a 3 kDa cut-off membrane, and the eluted peptides were resolved by high-performance liquid chromatography. Different qualitative and quantitative profiles were obtained, depending on the strain. WSE were further assayed for their ability to inhibit angiotensin-converting enzyme; the <3 kDa fraction exhibited higher activities in the case of L. casei and B. animalis than the control and L. acidophilus. CONCLUSION: Whey cheeses with higher nutritional value were those inoculated with L. casei.

Development of probiotic tablets using microparticles: viability studies and stability studies.

Alternative vectors to deliver viable cells of probiotics, to those conferring limited resistance to gastrointestinal conditions, still need to be sought. Therefore the main goal of the study was to develop tablets able to protect entrapped probiotic bacteria from gastric acidity, thus providing an easily manufacturing scale-up dosage form to deliver probiotics to the vicinity of the human colon. Whey protein concentrate microparticles with Lactobacillus paracasei L26 were produced by spray-drying and incorporated in tablets with cellulose acetate phthalate and sodium croscarmellose. The viability of L. paracasei L.26 throughout tableting as well as its gastric resistance and release from the tablets were evaluated. Storage stability of L. paracasei L26 tablets was also performed by evaluation of viable cells throughout 60 days at 23°C and 33% relative humidity. A decrease of approximately one logarithmic cycle was observed after the acid stage and the release of L. paracasei L26 from the tablets occurred only after 4 h in the conditions tested. Microencapsulated L. paracasei L26 in tablets revealed some susceptibility to the storage conditions tested since the number of viable cells decreased 2 log cycles after 60 days of storage. However, the viability of L. paracasei L26 after 45 days of storage did not reveal significant susceptibility upon exposure to simulated gastrointestinal conditions. The developed probiotic tablets revealed to be potential vectors for delivering viable cells of L. paracasei L26 and probably other probiotics to persons/patients who might benefit from probiotic therapy.

Carotenoids Diet: Digestion, Gut Microbiota Modulation, and Inflammatory Diseases.

Several epidemiologic studies have found that consuming fruits and vegetables lowers the risk of getting a variety of chronic illnesses, including several types of cancers, cardiovascular diseases (CVDs), and bowel diseases. Although there is still debate over the bioactive components, various secondary plant metabolites have been linked to these positive health benefits. Many of these features have recently been connected to carotenoids and their metabolites' effects on intracellular signalling cascades, which influence gene expression and protein translation. Carotenoids are the most prevalent lipid-soluble phytochemicals in the human diet, are found in micromolar amounts in human serum, and are very susceptible to multiple oxidation and isomerisation reactions. The gastrointestinal delivery system, digestion processes, stability, and functionality of carotenoids, as well as their impact on the gut microbiota and how carotenoids may be effective modulators of oxidative stress and inflammatory pathways, are still lacking research advances. Although several pathways involved in carotenoids' bioactivity have been identified, future studies should focus on the carotenoids' relationships, related metabolites, and their effects on transcription factors and metabolism.

Improving fresh cheese shelf-life through hyperbaric storage at variable room temperature.

The changes in microbiological, physiochemical, and textural properties in fresh cheeses made from either cow or goat milk were observed under hyperbaric storage (HS, 50-100 MPa) at room temperature (RT) and compared with refrigerated storage under normal atmospheric pressure for 60 days. An initial microbial growth inhibition was observed for both cheese types, as well as a considerable inactivation of all endogenous microbiota under HS/RT (75-100 MPa/RT). This contributed to a higher stability of pH and color values, especially at the higher pressure at room temperature (100 MPa/RT) throughout 60 days storage. A compression effect occurred during HS/RT, resulting in higher whey loss, reduction in moisture content, and textural changes. Such changes tended to decrease over time, to values closer to the initial ones, with hardness values at the 60th day of storage at 75/RT similar to those observed for refrigeration on the 7th day and 1.4-fold higher than those observed at 100/RT. Overall, HS/RT reduced the microbial populations load during storage (≥5 log units in some cases), with minimal effects on most of the evaluated quality parameters. These results point to a considerable shelf-life extension of HS fresh cheeses, without temperature control, pinpointing HS as a more sustainable preservation strategy than refrigeration, with great potential for industrial application. PRACTICAL APPLICATION: The results presented in this study point to increased microbial stability of fresh cheeses when stored under hyperbaric storage without temperature control, leading possibly to an increased shelf-life, of up to 60 days. This kind of new food preservation strategy may be suitable for longer transportation of foods, where energy may not be handily and widely available, while additionally contributing to increased shelf-life and safety. Also, hyperbaric storage could be applied throughout the food storage, improving shelf-life with a lower carbon footprint than refrigeration.

Comparing Different Packaging Conditions on Quality Stability of High-Pressure Treated Serra da Estrela Cheeses during Cold Storage.

Serra da Estrela cheese with a Protected Designation of Origin (PDO) is a traditional cheese that is wrapped in paper without vacuum. High-pressure processing (HPP), which requires vacuum packaging of the cheese, has been used for its cold pasteurization to overcome safety issues. In this study, two packaging systems were studied: non-vacuum greaseproof paper wrapping package and vacuum packaging in plastic film. Lactococci, lactobacilli, enterococci, and total mesophiles reached ca. 8 log cfu g-1 and 4-6 log cfu g-1 in control (unpasteurized) and HPP-treated cheeses, respectively, with no significant differences between packaging systems. Spoilage microorganisms' viable cell numbers were reduced to <3 log cfu g-1 (quantification limit) in HPP-treated cheeses, independently of the packaging system. Yeasts and molds reached >5 log cfu g-1 in non-vacuum paper-wrapped cheeses. A vacuum-packaging system enabled better control of cheese proteolysis, which was revealed to be closer to that of the original control cheese values at the end of the 10-month storage period. In addition, cheese stored under vacuum film packaging became harder than non-vacuum paper-wrapped cheeses at each time point. Overall, conventional non-vacuum paper wrapping is adequate for short storage periods (<3 months), but for long periods vacuum packaging in plastic film is preferable.

A chemical study of yoghurt produced under isostatic pressure during storage.

Yoghurt fermented under sub-lethal high pressure (10, 20, 30 and 40 MPa at 43 °C), and afterward placed under refrigeration (4 °C for 23 days) was studied and compared with yoghurt fermented at atmospheric pressure (0.1 MPa). For a deeper analysis, metabolite fingerprinting by nuclear magnetic resonance (NMR), sugars and organic acids assessment by high performance liquid chromatography (HPLC), total fatty acids (TFA) determination and quantification by gas chromatography with a flame ionization detector (GC-FID) were performed. Metabolomic analyses revealed that only 2,3-butanediol, acetoin, diacetyl and formate vary with the increase of pressure and probable relation with pressure influenced diacetyl reductase, acetoin reductase and acetolactate decarboxylase. Yoghurts fermented at 40 MPa had the lowest content in lactose (39.7 % of total sugar reduction) and the less content in TFA (56.1 %). Further research is of interest to understand more about fermentation processes under sub-lethal high pressure.

Optimization of Raw Ewes' Milk High-Pressure Pre-Treatment for Improved Production of Raw Milk Cheese.

Serra da Estrela protected designation of origin (PDO) cheese is manufactured with raw milk from Bordaleira and/or Churra Mondegueira da Serra da Estrela sheep breeds. Several socio-environmental shortcomings have reduced production capacity; hence, treatments that may contribute to its efficient transformation into cheese are welcome. High-pressure processing (HPP) milk pre-treatment may contribute to a cheese yield increment, yet optimization of processing conditions is warranted. An initial wide-scope screening experiment allowed for pinpointing pressure intensity, holding time under pressure and time after HPP as the most important factors influencing curd yield. Based on this, a more targeted screening experiment allowed for selecting the range of experimental conditions to be used for an experimental design study that revealed an HPP treatment at 121 MPa for 30 min as the optimum for milk processing to improve curd yield (>9%) and effectively maintain the beneficial cheese microbiota; the optimum was validated in a final experimental framework.