Cephalopods as Challenging and Promising Blue Foods: Structure, Taste, and Culinary Highlights and Applications.

Foods are complex systems due to their biological origin. Biological materials are soft matter hierarchically structured on all scales from molecules to tissues. The structure reflects the biological constraints of the organism and the function of the tissue. The structural properties influence the texture and hence the mouthfeel of foods prepared from the tissue, and the presence of flavour compounds is similarly determined by biological function. Cephalopods, such as squid, cuttlefish, and octopuses, are notoriously known for having challenging texture due to their muscles being muscular hydrostats with highly cross-linked collagen. Similar with other marine animals such as fish and crustaceans, cephalopods are rich in certain compounds such as free amino acids and free 5'-ribonucleotides that together elicit umami taste. Scientific investigations of culinary applications of cephalopods as foods must therefore involve mechanical studies (texture analysis), physicochemical measurements of thermodynamic properties (protein denaturation), as well as chemical analysis (taste and aroma compounds). The combination of such basic science investigations of food as a soft material along with an exploration of the gastronomic potential has been termed gastrophysics. In this review paper, we reviewed available gastrophysical studies of cephalopod structure, texture, and taste both as raw, soft material and in certain preparations.

Gastrophysical and chemical characterization of structural changes in cooked squid mantle.

Squid (Loligo forbesii and Loligo vulgaris) mantles were cooked by sous vide cooking using different temperatures (46°C, 55°C, 77°C) and times (30 s, 2 min, 15 min, 1 h, 5 h, 24 h), including samples of raw tissue. Macroscopic textural properties were characterized by texture analysis (TA) conducted with Meullenet-Owens razor shear blade and compared to analysis results from differential scanning calorimetry. The collagen content of raw tissues of squid was quantified as amount of total hydroxyproline using ultra-high-performance liquid chromatography. Structural changes were monitored by Raman spectroscopy and small-angle X-ray scattering and visualized by second harmonic generation microscopy. Collagen in the squid tissue was found to be highest in arms (4.3% of total protein), then fins (3.0%), and lowest in the mantle (1.5%), the content of the mantle being very low compared to that of other species of squid. Collagen was found to be the major protein responsible for cooking loss, whereas both collagen and actin were found to be key to mechanical textural changes. A significant decreased amount of cooking loss was obtained using a lower cooking temperature of 55°C compared to 77°C, without yielding significant textural changes in most TA parameters, except for TA hardness which was significantly less reduced. An optimized sous vide cooking time and temperature around 55-77°C and 0.5-5 h deserves further investigation, preferably coupled to sensory consumer evaluation. PRACTICAL APPLICATION: The study provides knowledge about structural changes during sous vide cooking of squid mantle. The results may be translated into gastronomic use, promoting the use of an underutilized resource of delicious and nutritious protein (Loligo vulgaris and Loligo forbesii).

Umami potential of fermented beverages: Sake, wine, champagne, and beer.

The free amino acid (FAA) contents of a special selection of fermented beverages have been measured by ultra-high performance liquid chromatography (UHPLC). The selection, which includes 8 sakes, 9 white, rosé, and sparkling wines, 9 genuine champagnes, as well as 5 types of beer, was made to uncover the umami potential of different types of fermented beverages, in particular whether long yeast contact and ageing may influence the contents of free glutamate that is known to elicit umami sensation. The data show that in particular sakes as well as some beers, wines and champagnes with long yeast contact contain appreciable amounts of free glutamate. The results are discussed in the context of food pairing where umami synergy can be achieved by combining fermented beverages with long yeast contact with food rich in free nucleotides.

Saved by seaweeds: phyconomic contributions in times of crises.

Seaweeds (macroalgae) are, together with microalgae, main contributors to the Earth's production of organic matter and atmospheric oxygen as well as fixation of carbon dioxide. In addition, they contain a bounty of fibres and minerals, as well as macro- and micronutrients that can serve both technical and medicinal purposes, as well as be a healthy and nutritious food for humans and animals. It is therefore natural that seaweeds and humans have had a myriad of interwoven relationships both on evolutionary timescales as well as in recent millennia and centuries all the way into the Anthropocene. It is no wonder that seaweeds have also entered and served as a saviour for humankind around the globe in many periods of severe needs and crises. Indeed, they have sometimes been the last resort, be it during times of famine, warfare, outbreak of diseases, nuclear accidents, or as components of securing the fabric of social stability. The present topical review presents testimony from the history of human interaction with seaweeds to the way humankind has, over and over again, been 'saved by seaweeds'. It remains a historical fact that in extreme conditions, such as shortage and wars, humans have turned to seaweeds in times of 'needs must' and created new opportunities for their uses in order to mitigate disasters. Lessons to be learned from this history can be used as reminders and inspiration, and as a guide as how to turn to seaweeds in current and inevitable, future times of crises, not least for the present needs of how to deal with changing climates and the pressing challenges of sustainable and healthy eating.

Umami synergy as the scientific principle behind taste-pairing champagne and oysters.

Food and flavour pairing are commonly used as an empirically based phenomenology by chefs and food innovators for creating delicious dishes. However, there is little if any science behind the pairing systems used, and it appears that pairing is determined by food culture and tradition rather than by chemical food composition. In contrast, the pairing implied by the synergy in the umami taste, elicited by free glutamate and free nucleotides, is scientifically founded on an allosteric action at the umami receptor, rendering eggs-bacon and cheese-ham delicious companions. Based on measurement of umami compounds in champagnes and oysters we suggest that a reason why champagne and oysters are considered good companions may be the presence of free glutamate in champagne, and free glutamate and 5'-nucleotides in oysters. By calculations of the effective umami potential we reveal which combinations of oysters and champagnes lead to the strongest umami taste. We also show that glutamate levels and total amount of free amino acids are higher in aged champagnes with long yeast contact, and that the European oyster (Ostrea edulis) has higher free glutamate and nucleotide content than the Pacific oyster (Crassostrea gigas) and is thus a better candidate to elicit synergistic umami taste.

Effects of seaweed sterols fucosterol and desmosterol on lipid membranes.

Higher sterols are universally present in large amounts (20-30%) in the plasma membranes of all eukaryotes whereas they are universally absent in prokaryotes. It is remarkable that each kingdom of the eukaryotes has chosen, during the course of evolution, its preferred sterol: cholesterol in animals, ergosterol in fungi and yeast, phytosterols in higher plants, and e.g., fucosterol and desmosterol in algae. The question arises as to which specific properties do sterols impart to membranes and to which extent do these properties differ among the different sterols. Using a range of biophysical techniques, including calorimetry, fluorescence microscopy, vesicle-fluctuation analysis, and atomic force microscopy, we have found that fucosterol and desmosterol, found in red and brown macroalgae (seaweeds), similar to cholesterol support liquid-ordered membrane phases and induce coexistence between liquid-ordered and liquid-disordered domains in lipid bilayers. Fucosterol and desmosterol induce acyl-chain order in liquid membranes, but less effectively than cholesterol and ergosterol in the order: cholesterol>ergosterol>desmosterol>fucosterol, possibly reflecting the different molecular structure of the sterols at the hydrocarbon tail.

Data for the size of cholesterol-fat micelles as a function of bile salt concentration and the physico-chemical properties of six liquid experimental pine-derived phytosterol formulations in a cholesterol-containing artificial intestine fluid.

The data in this paper are additional information to the research article entiltled "Inhibition of cholesterol transport in an intestine cell model by pine-derived phytosterols" (Yi et al.,2016) [1]. The data derived from the measurement on six liquid formulations of commercial pine-derived phytosterol (CPP) by dynamic light scattering. The data cover micelle size and the zeta-potential for formulations with cholesterol including monoglyceride, oleic acid, and bile salt. The data demonstrate the critical effect of the bile salt concentration on the size of cholesterol-digested fat micelles.

Contents of capsaicinoids in chillies grown in Denmark.

We have studied 15 different cultivars of chillies (Capsicum var.) grown in temperate climate Denmark and determined the contents of the four major capsaicinoids: capsaicin, dihydrocapsaicin, nordihydrocapsaicin, and homocapsaicin. From these contents we have, as commonly done for chillies, calculated the so-called pungency in Scoville heat units in order to compare with previous studies from other climatical zones. For three of the investigated cultivars, Serrano, Habanero and BIH Jolokia, for which reliable pungencies has previously been reported, we have found pungencies of 34,000±1400, 247,000±24,000 and 665,000±4000, respectively, which are all in the same ranges as found earlier for chillies grown in more traditional chilli growing areas. Furthermore we have found that the relative distribution of the four capsaicinoids in the 15 different cultivars is highly variable, with the content of capsaicin ranging from 31% to 71% of the total capsaicinoid content.

Effect of fatty acids on the permeability barrier of model and biological membranes.

Because of the amphipathicity and conical molecular shape of fatty acids, they can efficiently incorporate into lipid membranes and disturb membrane integrity, chain packing, and lateral pressure profile. These phenomena affect both model membranes as well as biological membranes. We investigated the feasibility of exploiting fatty acids as permeability enhancers in drug delivery systems for enhancing drug release from liposomal carriers and drug uptake by target cells. Saturated fatty acids, with acyl chain length from C8 to C20, were tested using model drug delivery liposomes of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the breast cancer MCF-7 cell line as a model cell. A calcein release assay demonstrated reduction in the membrane permeability barrier of the DPPC liposomes, proportionally to the length of the fatty acid. Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) experiments revealed that C12 to C20 fatty acids can stabilize DPPC liposomal bilayers and induce the formation of large structures, probably due to liposome aggregation and bilayer morphological changes. On the other hand, the short fatty acids C8 and C10 tend to destabilize the bilayers and only moderately cause the formation of large structures. The effect of fatty acids on DPPC liposomes was not completely transferrable to the MCF-7 cell line. Using cytotoxicity assays, the cells were found to be relatively insensitive to the fatty acids at apoptotic sub-millimolar concentrations. Increasing the fatty acid concentration to few millimolar substantially reduced the viability of the cells, most likely via the induction of necrosis and cell lysis. A bioluminescence living-cell-based luciferase assay showed that saturated fatty acids in sub-cytotoxic concentrations cannot reduce the permeability barrier of cell membranes. Our results confirm that the membrane perturbing effect of fatty acids on model membranes cannot simply be carried over to biological membranes of live cells.

Inhibition of cholesterol transport in an intestine cell model by pine-derived phytosterols.

We have quantified the inhibition of intestinal cholesterol transport by pine-derived phytosterols using an HT29-MTX intestine cell model that forms a mucus layer similar to that in the intestine. An artificial intestinal fluid consisting of digested fat, bile salt, cholesterol, and phytosterols was formulated in order to mimic the conditions in the intestine. The apparent permeability coefficient (Papp) of the positive control, i.e., 0.1mM of cholesterol solubilized in the artificial intestine fluid, was found to be 0.33 (±0.17)×10-6cm/s. When 0.1mM ß-sitosterol was solubilized alongside, Papp was effectively zero, corresponding to a total inhibition of cholesterol transport. A similar strong inhibition was found when commercial pine-derived phytosterols, PinVita™ FSP DuPont, were co-solubilized with cholesterol in the dietary model micelles, leading to Papp=0.06 (±0.06)×10-6cm/s, i.e., 5.5 times lower than the cholesterol positive control. Additionally, the effect of potential oral administration formulations generated by the pine-derived phytosterols was also characterized. The formulations were produced as a liquid formulation of the cholesterol-containing artificial intestine fluid. Six liquid formulations were tested of which four displayed a Papp in the range of 0-0.09×10-6cm/s. The remaining two formulations did not show any inhibition effect on cholesterol transport and even enhanced cholesterol transport. It was furthermore observed that the phytosterols were found in the collected intestine cells but not transported to the basolateral region in the intestinal cell model system.

Optimization and modeling of the remote loading of luciferin into liposomes.

We carried out a mechanistic study to characterize and optimize the remote loading of luciferin into preformed liposomes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPC/DPPG) 7:3 mixtures. The influence of the loading agent (acetate, propionate, butyrate), the metal counterion (Na(+), K(+), Ca(+2), Mg(+2)), and the initial extra-liposomal amount of luciferin (nL(add)) on the luciferin Loading Efficiency (LE%) and luciferin-to-lipid weight ratio, i.e., Loading Capacity (LC), in the final formulation was determined. In addition, the effect of the loading process on the colloidal stability and phase behavior of the liposomes was monitored. Based on our experimental results, a theoretical model was developed to describe the course of luciferin remote loading. It was found that the highest luciferin loading was obtained with magnesium acetate. The use of longer aliphatic carboxylates or inorganic proton donors pronouncedly reduced luciferin loading, whereas the effect of the counterion was modest. The remote-loading process barely affected the colloidal stability and drug retention of the liposomes, albeit with moderate luciferin-induced membrane perturbations. The correlation between luciferin loading, expressed as LE% and LC, and nL(add) was established, and under our conditions the maximum LC was attained using an nL(add) of around 2.6µmol. Higher amounts of luciferin tend to pronouncedly perturb the liposome stability and luciferin retention. Our theoretical model furnishes a fair quantitative description of the correlation between nL(add) and luciferin loading, and a membrane permeability coefficient for uncharged luciferin of 1×10(-8)cm/s could be determined. We believe that our study will prove very useful to optimize the remote-loading strategies of moderately polar carboxylic acid drugs in general.

Spatial distribution and activity of Na(+)/K(+)-ATPase in lipid bilayer membranes with phase boundaries.

We have reconstituted functional Na(+)/K(+)-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at lo/ld interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to softening of the membrane.

Gastrophysics of the Oral Cavity.

BACKGROUND: Gastrophysics is the science that pertains to the physical and physico-chemical description of the empirical world of gastronomy, with focus on sensory perception in the oral cavity and how it is related to the materials properties of food and cooking processes. Flavor (taste and smell), mouthfeel, chemesthesis, and astringency are all related to the chemical properties and the texture of the food and how the food is transformed in the oral cavity. METHODS: The present topical review will primarily focus attention on the somatosensory perception of food (mouthfeel or texture) and how it interacts with basic tastes (sour, bitter, sweet, salty, and umami) and chemesthetic action. RESULTS: Issues regarding diet, nutrition, and health will be put into an evolutionary perspective, and some mention will be made of umami and its importance for (oral) health.

Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures.

Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biological membranes involving heterogeneities in lipid composition, shape, mechanical properties, and chemical properties. We have devised a method that makes it possible to prepare a uniform sample of ternary GUVs of a prescribed composition and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixture of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compositions. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liquid ordered-liquid disordered phases in giant vesicles and is found to be comparable to that in GUVs of the prescribed composition produced from hydration of dried lipids mixed in organic solvent. Our method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiological buffer and temperature conditions. The new protocol will permit quantitative biophysical studies of a whole new class of well-defined model membrane systems of a complexity that resembles biological membranes with rafts.

Enzymatic action of phospholipase A2 on liposomal drug delivery systems.

The overexpression of secretory phospholipase A2 (sPLA2) in tumors has opened new avenues for enzyme-triggered active unloading of liposomal antitumor drug carriers selectively at the target tumor. However, the effects of the liposome composition, drug encapsulation, and tumor microenvironment on the activity of sPLA2 are still not well understood. We carried out a physico-chemical study to characterize the sPLA2-assisted breakdown of liposomes using dye-release assays in the context of drug delivery and under physiologically relevant conditions. The influence of temperature, lipid concentration, enzyme concentration, and drug loading on the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, Tm=42°C) liposomes with snake venom sPLA2 was investigated. The sensitivity of human sPLA2 to the liposome composition was checked using binary lipid mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) phospholipids with C14 and C16 acyl chains. Increasing temperature (36-41°C) was found to mainly shorten the enzyme lag-time, whereas the effect on lipid hydrolysis rate was modest. The enzyme lag-time was also found to be inversely dependent on the lipid-to-enzyme ratio. Drug encapsulation can alter the hydrolysis profile of the carrier liposomes. The activity of human sPLA2 was highly sensitive to the phospholipid acyl-chain length and negative surface charge density of the liposomes. We believe our work will prove useful for the optimization of sPLA2-susceptible liposomal formulations as well as will provide a solid ground for predicting the hydrolysis profile of the liposomes in vivo at the target site.

Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release.

The feasibility of exploiting secretory phospholipase A2 (sPLA2) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.

Lipid domains in model membranes: a brief historical perspective.

All biological membranes consist of a complex composite of macromolecules and macromolecular assemblies, of which the fluid lipid-bilayer component is a core element with regard to cell encapsulation and barrier properties. The fluid lipid bilayer also supports the functional machinery of receptors, channels and pumps that are associated with the membrane. This bilayer is stabilized by weak physical and colloidal forces, and its nature is that of a self-assembled system of amphiphiles in water. Being only approximately 5 nm in thickness and still encapsulating a cell that is three orders of magnitude larger in diameter, the lipid bilayer as a material has very unusual physical properties, both in terms of structure and dynamics. Although the lipid bilayer is a fluid, it has a distinct and structured trans-bilayer profile, and in the plane of the bilayer the various molecular components, viz different lipid species and membrane proteins, have the capacity to organize laterally in terms of differentiated domains on different length and time scales. These elements of small-scale structure and order are crucial for the functioning of the membrane. It has turned out to be difficult to quantitatively study the small-scale structure of biological membranes. A major part of the insight into membrane micro- and nano-domains and the concepts used to describe them have hence come from studies of simple lipid bilayers as models of membranes, by use of a wide range of theoretical, experimental and simulational approaches. Many questions remain to be answered as to which extent the result from model studies can carry over to real biological membranes.