Shotgun-Based Mass Spectrometry Analysis of Phospholipid and Triacylglycerol Molecular Species and Eicosanoids in Salmon Muscle Tissue on Feeding Microbial Oil.

The continuous growth of aquaculture places a growing demand on alternative sources of fish oil (FO). Certain microorganisms provide a sustainable replacement for FO due to their content of EPA and DHA, which are essential for fish health. Appreciable evidence shows that changes in feeding sources may alter the nutritional components of salmon; however, the influence of diets on lipid species remains unclear. In this study, the identification and semi-quantification of lipid molecular species in salmon muscle during feeding with a microbial oil (MO) were carried out by focusing on triacylglycerol (TAG) and diacyl-phospholipid using shotgun-based mass spectrometry analysis. DHA in the MO diet was efficiently incorporated into phospholipid structures on feeding, followed by accumulation in salmon muscle. The MO diet elevated the level of certain EPA-containing TAGs, such as TAG C52:5 (16:0_16:0_20:5) and TAG C54:6 (16:0_18:1_20:5), indicating that the MO diet may be an excellent source for enhancement of the abundance of ω3 lipids. Further, prostaglandins (PGs) PGE2 and PGF3α were identified and quantified for the first time in salmonid tissue.

Shotgun Lipidomics for the Determination of Phospholipid and Eicosanoid Profiles in Atlantic Salmon (Salmo salar L.) Muscle Tissue Using Electrospray Ionization (ESI)-MS/MS Spectrometric Analysis.

Shotgun lipidomics was applied to identify and quantify phospholipids (PLs) in salmon muscle tissue by focusing on the distribution of ω-3 fatty acids (e.g., docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) in the form of phospholipids, as well as to identify and quantify eicosanoids, which has not yet been attempted in Atlantic salmon muscle. Shotgun lipidomics enabled the identification of 43 PL species belonging to four different classes: phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylserines (PSs), and phosphatidylinositols (PIs). Among others, 16:0-22:6 PtdCho m/z [M + Na]+ at 828.4 was the predominant PL species in salmon muscle tissue. The present study provided the quantification of individual phospholipid species, which has not been performed for salmon muscle tissue so far. In addition, two eicosanoids-prostaglandin E2 (PGE2) and prostaglandin F3α (PGF3α)-were identified for the first time in salmon muscle. Thus, the rapid and high-throughput shotgun lipidomics approach should shed new light on phospholipids and eicosanoids in salmon muscle tissue.

Gut Microbiota Extracellular Vesicles as Signaling Molecules Mediating Host-Microbiota Communications.

Over the past decade, gut microbiota dysbiosis has been linked to many health disorders; however, the detailed mechanism of this correlation remains unclear. Gut microbiota can communicate with the host through immunological or metabolic signalling. Recently, microbiota-released extracellular vesicles (MEVs) have emerged as significant mediators in the intercellular signalling mechanism that could be an integral part of microbiota-host communications. MEVs are small membrane-bound vesicles that encase a broad spectrum of biologically active compounds (i.e., proteins, mRNA, miRNA, DNA, carbohydrates, and lipids), thus mediating the horizontal transfer of their cargo across intra- and intercellular space. In this study, we provide a comprehensive and in-depth discussion of the biogenesis of microbial-derived EVs, their classification and routes of production, as well as their role in inter-bacterial and inter-kingdom signaling.

Bioactivities of Phenolics by Focusing on Suppression of Chronic Diseases: A Review.

Phenolics, which are secondary metabolites of plants, exhibit remarkable bioactivities. In this contribution, we have focused on their protective effect against chronic diseases rather than their antioxidant activities, which have been widely discussed in the literature. A large body of epidemiological studies has proven the bioactivities of phenolics in both standard compounds and natural extracts: namely, anticancer, anti-inflammatory, and antibacterial activities as well as reducing diabetes, cardiovascular disease, and neurodegenerative disease. Phenolics also display anti-analgesic, anti-allergic, and anti-Alzheimer's properties. Thus, this review provides crucial information for better understanding the bioactivities of phenolics in foods and fills a gap in the existing collective and overall knowledge in the field.

Insoluble-Bound Phenolics in Food.

This contribution provides a review of the topic of insoluble-bound phenolics, especially their localization, synthesis, transfer and formation in plant cells, as well as their metabolism in the human digestive system and corresponding bioactivities. In addition, their release from the food matrix during food processing and extraction methods are discussed. The synthesis of phenolics takes place mainly at the endoplasmic reticulum and they are then transferred to each organ through transport proteins such as the ATP-binding cassette (ABC) and multidrug and toxic compound extrusion (MATE) transporter at the organ's compartment membrane or via transport vesicles such as cytoplasmic and Golgi vesicles, leading to the formation of soluble and insoluble-bound phenolics at the vacuole and cell wall matrix, respectively. This part has not been adequately discussed in the food science literature, especially regarding the synthesis site and their transfer at the cellular level, thus this contribution provides valuable information to the involved scientists. The bound phenolics cannot be absorbed at the small intestine as the soluble phenolics do (5%-10%), thus passing into the large intestine and undergoing fermentation by a number of microorganisms, partially released from cell wall matrix of foods. Bound phenolics such as phenolic acids and flavonoids display strong bioactivities such as anticancer, anti-inflammation and cardiovascular disease ameliorating effects. They can be extracted by several methods such as acid, alkali and enzymatic hydrolysis to quantify their contents in foods. In addition, they can also be released from the cell wall matrix during food processing procedures such as fermentation, germination, roasting, extrusion cooking and boiling. This review provides critical information for better understanding the insoluble-bound phenolics in food and fills an existing gap in the literature.

Food-Derived Extracellular Vesicles as Multi-Bioactive Complex and Their Versatile Health Effects.

Extracellular vesicles (EVs) are membrane-bound organelles that are generally released by eukaryotic cells and enclose various cellular metabolic information, such as RNA, meta-proteins, and versatile metabolites. The physiological properties and diverse functions of food-derived EVs have been extensively elucidated, along with a recent explosive upsurge in EV research. Therefore, a concise review of the health effects of food-derived EVs is necessary. This review summarizes the structural stability and uptake pathways of food-derived EVs to target cells and their health benefits, including antioxidant, anti-inflammatory, and anticarcinogenic effects, gut microbiome modulation, and intestinal barrier enhancement.

Influence of food-derived bioactives on gut microbiota compositions and their metabolites by focusing on neurotransmitters.

The behavior of gut microbiota is closely involved in sustaining balanced immune and metabolic homeostasis, and the dysbiosis of gut microbiota can lead to severe disease. Foods and dietary patterns are the primary drivers in shaping/designing gut microbiota compositions and their metabolites across the lifetime. This indicates the importance of functional molecules present in the food matrix in the life of gut microbiota and their influence on the host's biological system. In this contribution, the effects of different dietary choices and bioactive compounds (i.e., phenolics, vitamins, carotenoids) on gut microbiome compositions and their metabolites are comprehensively discussed by focusing on neurotransmitters. This study may provide useful information that fills a gap in understanding the role of the gut microbiota and its alterations as affected by foods and food-derived bioactives.

Neuromicrobiology, an emerging neurometabolic facet of the gut microbiome?

The concept of the gut microbiome is emerging as a metabolic interactome influenced by diet, xenobiotics, genetics, and other environmental factors that affect the host's absorption of nutrients, metabolism, and immune system. Beyond nutrient digestion and production, the gut microbiome also functions as personalized polypharmacy, where bioactive metabolites that our microbes excrete or conjugate may reach systemic circulation and impact all organs, including the brain. Appreciable evidence shows that gut microbiota produce diverse neuroactive metabolites, particularly neurotransmitters (and their precursors), stimulating the local nervous system (i.e., enteric and vagus nerves) and affecting brain function and cognition. Several studies have demonstrated correlations between the gut microbiome and the central nervous system sparking an exciting new research field, neuromicrobiology. Microbiome-targeted interventions are seen as promising adjunctive treatments (pre-, pro-, post-, and synbiotics), but the mechanisms underlying host-microbiome interactions have yet to be established, thus preventing informed evidence-based therapeutic applications. In this paper, we review the current state of knowledge for each of the major classes of microbial neuroactive metabolites, emphasizing their biological effects on the microbiome, gut environment, and brain. Also, we discuss the biosynthesis, absorption, and transport of gut microbiota-derived neuroactive metabolites to the brain and their implication in mental disorders.

A Critical Overview of HPLC-MS-Based Lipidomics in Determining Triacylglycerol and Phospholipid in Foods.

With the current advancement in mass spectrometry (MS)-based lipidomics, the knowledge of lipidomes and their diverse roles has greatly increased, enabling a deeper understanding of the action of bioactive lipid molecules in plant- and animal-based foods. This review provides in-depth information on the practical use of MS techniques in lipidomics, including lipid extraction, adduct formation, MS analysis, data processing, statistical analysis, and bioinformatics. Moreover, this contribution demonstrates the effectiveness of MS-based lipidomics for identifying and quantifying diverse lipid species, especially triacylglycerols and phospholipids, in foods. Further, it summarizes the wide applications of MS-based lipidomics in food science, such as for assessing food processing methods, detecting food adulteration, and measuring lipid oxidation in foods. Thus, MS-based lipidomics may be a useful method for identifying the action of individual lipid species in foods.

Phenolic Compounds and Antioxidant Capacity of Sea Cucumber (Cucumaria frondosa) Processing Discards as Affected by High-Pressure Processing (HPP).

Sea cucumber processing discards, which include mainly internal organs, represent up to 50% of the sea cucumber biomass, and are a rich source of bioactive compounds, including phenolics. This work aimed to extract free, esterified, and insoluble-bound phenolics from the internal organs of the Atlantic sea cucumber (C. frondosa) using high-pressure processing (HPP) pre-treatment. The sea cucumber internal organs were subjected to HPP (6000 bar for 10 min), followed by the extraction and characterization of phenolics. Samples were evaluated for their total contents of phenolics and flavonoids, as well as several in vitro methods of antioxidant activities, namely, free radical scavenging and metal chelation activities. Moreover, anti-tyrosinase and antiglycation properties, as well as inhibitory activities against LDL cholesterol oxidation and DNA damage, were examined. The results demonstrated that HPP pre-treatment had a significant effect on the extraction of phenolics, antioxidant properties, and other bioactivities. The phenolics in sea cucumber internal organs existed mainly in the free form, followed by the insoluble-bound and esterified fractions. Additionally, UHPLC-QTOF-MS/MS analysis identified and quantified 23 phenolic compounds from HPP-treated samples, mostly phenolic acids and flavonoids. Hence, this investigation provides fundamental information that helps to design the full utilization of the Atlantic sea cucumber species and the production of a multitude of value-added products.

Riboflavin-Sensitized Photooxidation of Low-Density-Lipoprotein (LDL) Cholesterol: A Culprit in the Development of Cardiovascular Diseases (CVDs).

The oxidation of human low-density-lipoprotein (LDL) particles is responsible for the development of cardiovascular diseases (CVDs). In the present study, the occurrence of riboflavin-sensitized photooxidation of LDL particles was examined in an in vitro system. The presence of light, oxygen, and photosensitizer (50 µM riboflavin) caused the riboflavin-sensitized photooxidation of human LDL particles thereby increasing in the conjugated dienes (CDs) by 32.5 ± 4.8% (p < 0.05), indicating that this could serve as a major culprit in the development of CVDs. A 1 h radiation caused a 63.6 ± 0.3% degradation of the riboflavin content, and this indicates the extremely fast reaction of the riboflavin-sensitized photooxidation. The singlet oxygen quenching capacity of ß-carotene was determined at three different concentrations (10, 50, and 100 µM), exhibiting both antioxidant and prooxidant effects, depending on the concentrations used. In addition, ascorbic acid displayed a high incorporation rate into the LDL particles, implying its potential in preventing riboflavin photosensitization of LDL particles. To the best of our knowledge, this is the first report on the riboflavin-sensitized photooxidation of LDL particles in an in vitro system, proposing a new possible mechanism in the development of CVDs.

Liberation of insoluble-bound phenolics from lentil hull matrices as affected by Rhizopus oryzae fermentation: Alteration in phenolic profiles and their inhibitory capacities against low-density lipoprotein (LDL) and DNA oxidation.

Fermentation is an effective non-thermal food processing operation used for enhancing the nutritional and functional properties of food. HPLC-ESI-MS/MS analysis and inhibitory capacity of the soluble- and insoluble-bound phenolics in lentil hulls in retarding the oxidation of LDL and DNA strand scission were determined following fermentation. In HPLC-ESI-MS/MS analysis, most insoluble-bound phenolics in lentil hulls were significantly decreased, indicating their liberation from the cell wall matrix upon fermentation. However, the released insoluble-bound phenolics did not show an efficient conversion into the bioavailable soluble phenolics as reflected in the inhibitory capacity against oxidation of LDL and DNA strands. The low efficiency in bioconversion from insoluble-bound to soluble phenolics might be due to the loss of the released bound phenolics during the fermentation process. Following the alterations of individual insoluble-bound phenolics in legumes upon fermentation in this work may fill the existing gap in the related areas.

Determination of soluble and insoluble-bound phenolic compounds in dehulled, whole, and hulls of green and black lentils using electrospray ionization (ESI)-MS/MS and their inhibition in DNA strand scission.

The soluble and insoluble-bound phenolic fractions of hull, whole, and dehulled black and green lentil extracts were identified and quantified using electrospray ionization (ESI)-MS/MS. Several in vitro antioxidant tests and inhibition of DNA strand scission were conducted to assess different pathways of activity. The most abundant phenolics in the soluble fractions were caffeic acid (412.2 µg/g), quercetin, (486.5 µg/g) quercetin glucoside (633.6 µg/g) luteolin glucoside (239.1 µg/g) and formononetin (920 µg/g), while myricetin (534.1 µg/g) and catechin (653.4 µg/g) were the predominant phenolics in the insoluble bound fraction. Hulls of both lentil cultivars had the highest phenolic content and the strongest antioxidant activity followed by whole and dehulled samples. Thus, lentil hulls would serve as an excellent source for the production of functional foods. Moreover, ESI-MS/MS (direct infusion) analysis was the rapid and high-throughput approach for the determination of bioactives in lentils by reducing the analysis time.

Identification and quantification of soluble and insoluble-bound phenolics in lentil hulls using HPLC-ESI-MS/MS and their antioxidant potential.

The identification and quantification of soluble- and insoluble-bound phenolics in lentil hulls were studied using HPLC-DAD-ESI-MSn and their antioxidant potential determined using DPPH radical scavenging ability (DRSA), reducing power (RP), and hydroxyl radical scavenging ability (HRSA) assays to test their electron and hydrogen donating abilities. A number of soluble phenolics such as phenolic acids, flavonoids, and proanthocyanidins were found, which lead to the remarkable antioxidant potential as reflected in DRSA, RP, and HRSA. Meanwhile, insoluble-bound phenolics displayed a relatively lower number of peaks and contents than their corresponding soluble phenolics, leading to a lower antioxidant potential than that of soluble phenolics. Moreover, dihydrokaempferol dimer and carboxylated kaempferol diglucoside were identified for the first time in the insoluble-bound form in lentils. This study offers important data for the identification of phenolic compounds derived from lentils and their antioxidant potential.

Evaluation of triacylglycerol (TAG) profiles and their contents in salmon muscle tissue using ESI-MS/MS spectrometry with multiple neutral loss scans.

Identification and quantification of triacylglycerols (TAGs) in salmon muscle tissue were conducted using electrospray ionization (ESI)-MS/MS in a triple quadrupole mass spectrometer. The confirmation of three fatty acid moieties of individual TAGs was determined using the multiple neutral loss (NL) scanning mode. A total of 98 TAGs were identified, and the predominant TAG species were 16:0-18:0-20:5 (10.4%), 18:1-18:2-22:6 (9.0%), and 18:0-18:1-22:6 (16.4%) in salmon muscle tissue. NL scanning was an effective means to confirm the three fatty acid moieties of the TAGs, leading to the rapid and accurate identification of individual TAGs. To the best of our knowledge, this is the first application of multiple neutral loss scanning to identify TAGs in salmonoid tissue, and many TAG species have been newly identified (i.e., 18:1-18:2-22:6, 16:0-18:2-20:5, 18:1-18:2-20:5, etc.). This study showed that the shotgun lipidomic approach along with NL scans is a useful means for studying TAG metabolism in fish.

Critical Re-Evaluation of DPPH assay: Presence of Pigments Affects the Results.

A limitation of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability (DRSA) due to the presence of pigments and colors in the extracts of plant-based foods was addressed. The pigments present in the test samples absorb in the same wavelength region as the DPPH radicals; this interferes with the elaborate absorbance readings of the DPPH radicals. In this contribution, electron paramagnetic resonance (EPR) spectroscopy for DPPH assay is proposed in order to avoid this limitation. In the EPR-DPPH assay, the underestimation of the radical scavenging abilities of four different pigment-containing extracts was corrected and this reflected up to a 16.1% change compared to the original values. Thus, the EPR-DPPH assay eliminated interference from the pigments coexisting in the reaction medium and improved the accuracy of the DPPH radical scavenging potential of such extracts. The principle of the proposed solution might also be employed in other assays which suffer from pigment interference.

Critical evaluation of changes in the ratio of insoluble bound to soluble phenolics on antioxidant activity of lentils during germination.

A new indicator, the ratio of insoluble bound phenolics (IBPs) to soluble phenolics (SPs), is suggested as an effective means to monitor changes in the antioxidant activity of lentils during germination. This indicator may be used to monitor other process-induced changes in antioxidant potential of food phenolics in other foods. The antioxidant activity of SPs, IBPs, and total value, the sum of both free and esterified phenolics, of germinated CDC Richlea lentil variety was evaluated for 4 days. Total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging ability were employed to record antioxidant activities. An incremental increase in IBPs was found in TPC, TFC, DPPH, and ABTS radical cation scavenging ability, whereas SPs showed a declining trend in TFC, DPPH, and ABTS, except TPC during 4 days of germination. The ratio of IBPs to SPs increased using most methods, and this may be possibly due to the changes of phenolic compound formation from soluble into insoluble bound form during germination process. The ratio can be used as a novel method for monitoring process-induced changes in the antioxidant activity of foods.

Determination of the degree of oxidation in highly-oxidised lipids using profile changes of fatty acids.

The degree of highly oxidised lipids was determined by a modified method using profile changes of fatty acids in lard and soybean oil heated at 180°C. The usefulness of the modified method was compared through conjugated dienoic acid (CDA) and/or p-anisidine value (p-AV) methods. Absolute values, which were expressed as equivalent to an internal standard (C11:0), of both unsaturated fatty acids (UFAs) and saturated fatty acids (SFAs) decreased significantly during thermal oxidation (p<0.05) while relative percentage of SFA increased and those of UFA decreased significantly (p<0.05). The content of caprylic acid (C8:0) increased significantly (p<0.05) as thermal oxidation time increased. The ratio of total saturated over total unsaturated fatty acids (SFAs/UFAs) or caprylic acid (C8:0) over UFAs could be useful markers to determine the degree of oxidation. Antioxidant capacity of sesamol, a free radical scavenger, was determined using the ratio of fatty acids. The modified method can be applied to determine the quality control of fried foods containing highly oxidised and abused oils, which may not be measured correctly using CDA and p-AV.

Application of solid phase-microextraction (SPME) and electronic nose techniques to differentiate volatiles of sesame oils prepared with diverse roasting conditions.

Headspace volatiles of sesame oil (SO) from sesame seeds roasted at 9 different conditions were analyzed by a combination of solid phase microextraction (SPME)-gas chromatography/mass spectrometry (GC/MS), electronic nose/metal oxide sensors (MOS), and electronic nose/MS. As roasting temperature increased from 213 to 247 °C, total headspace volatiles and pyrazines increased significantly (P < 0.05). Pyrazines were major volatiles in SO and furans, thiazoles, aldehydes, and alcohols were also detected. Roasting temperature was more discrimination factor than roasting time for the volatiles in SO through the principal component analysis (PCA) of SPME-GC/MS, electronic nose/MOS, and electronic nose/MS. Electronic nose/MS showed that ion fragment 52, 76, 53, and 51 amu played important roles in discriminating volatiles in SO from roasted sesame seeds, which are the major ion fragments from pyrazines, furans, and furfurals. SO roasted at 213, 230, and 247 °C were clearly differentiated from each other on the base of volatile distribution by SPME-GC/MS, electronic nose/MOS, and electronic nose/MS analyses. Practical Application: The results of this study are ready to apply for the discriminating samples using a combinational analysis of volatiles. Not only vegetable oils prepared from roasting process but also any food sample possessing volatiles could be targets for the SPME-GC/MS and electronic nose assays. Contents and types of pyrazines in sesame seed oil could be used as markers to track down the degree of roasting and oxidation during oil preparation.

Effects of riboflavin photosensitization on daidzein and its photosensitized derivatives.

Photosensitized compounds from daidzein were studied in a riboflavin model system under visible light irradiation by high-performance liquid chromatography (HPLC). As the period of light irradiation increased, concentration of daidzein decreased significantly (P < 0.05) and new peaks of daidzein derivatives were observed and changed during photosensitization. Three new peaks from photosensitized daidzein were tentatively identified as 7-, 3', 4'-trihydroxyisoflavone (or 3'-hydroxydaidzein) and 2 dimmers of daidzein by a combination of HPLC-mass spectrometry (MS) and retention times of standard compounds by HPLC. Addition of sodium azide and removal of headspace oxygen treatment affected the formation of newly formed peaks. The type I pathway of riboflavin photosensitization played more important roles than type II pathways on the formation of daidzein derivatives. Practical Application: Isoflavones are important phytochemicals found in soy foods. Generally, many foods containing soy ingredients are displayed under visible light irradiation. Also, riboflavin can be found in many foods containing vegetables. The results of this study can be used to understand the stability and changes of isoflavone aglycones in soy and soy-based foods under visible light irradiation.