Visualisation of microalgal lipid bodies through electron microscopy.

In this study, transmission electron microscopy (TEM) and cryo-scanning electron microscopy (cryo-SEM) were evaluated for their ability to detect lipid bodies in microalgae. To do so, Phaeodactylum tricornutum and Nannochloropsis oculata cells were harvested in both the mid-exponential and early stationary growth phase. Two different cryo-SEM cutting methods were compared: cryo-planing and freeze-fracturing. The results showed that, despite the longer preparation time, TEM visualisation preceded by cryo-immobilisation allows a clear detection of lipid bodies and is preferable to cryo-SEM. Using freeze-fracturing, lipid bodies were rarely detected. This was only feasible if crystalline layers in the internal structure, most likely related to sterol esters or di-saturated triacylglycerols, were revealed. Furthermore, lipid bodies could not be detected using cryo-planing. Cryo-SEM is also not the preferred technique to recognise other organelles besides lipid bodies, yet it did reveal chloroplasts in both species and filament-containing organelles in cryo-planed Nannochloropsis oculata samples.

Stabilization of Vitamin A by Cereal Bran: The Importance of the Balance between Antioxidants, Pro-oxidants, and Oxidation-Sensitive Components.

This study investigated the contribution of bran antioxidants and lipids to the stabilizing effect of cereal bran on vitamin A during accelerated storage. Hereto, wheat and rice bran samples subjected to a sequential extraction process were used. Vitamin A stabilization was more pronounced for wheat compared to rice bran. This was attributed to the higher antioxidant capacity and lower degree of lipid oxidation of wheat compared to rice bran. Removal of the chloroform/methanol-extractable fraction resulted in a substantial decrease in vitamin A retention from 78 to 26% for wheat bran and from 30 to 0% for rice bran after 2 weeks of accelerated storage. However, the vitamin A-stabilizing effect could not be attributed to specific components. The ability of cereal bran to stabilize vitamin A is therefore believed to be determined by the balance of antioxidants, pro-oxidants, and oxidation-sensitive components in the system.

Ultrasound-Assisted Extraction of Applewood Polyphenols at Lab and Pilot Scales.

This study focused on the extraction of polyphenols from applewood using ultrasonic-assisted extraction (UAE). First, the influence of solvent composition and mass-volume (m:v) ratio on the extraction yield was studied at a lab scale (200 mL). Overall, a ratio of 1:33 (m:v) resulted in a higher yield of polyphenols. Furthermore, both a higher yield of polyphenols and higher antioxidant capacity were detected in the extracts produced in the presence of a 30 v/v % ethanol mixture compared to pure water; a further increase in ethanol did not improve the extraction yield. Second, under the optimal conditions (30 v/v % ethanol-water; 1:33 and 1:20 (m:v)), the UAE technique was applied at a pilot scale (100 L). At 1:33 (m:v), the polyphenol yield was lower at the pilot scale compared to the lab scale; by contrast, at 1:20 (m:v), production at the pilot scale resulted in a higher yield compared to the lab scale. To identify and quantify individual polyphenols, HPLC-PDA analyses were performed. Phloridzin appears to be the major identified compound. Finally, the UAE process was compared to a conventional solid-liquid extraction technique, showing that a significantly higher yield could be obtained with UAE.

Oxidative stability differences of aqueous model systems of photoautotrophic n-3 LC-PUFA rich microalgae: The antioxidative role of endogenous carotenoids.

Microalgae rich in omega-3 long-chain polyunsaturated fatty acids (n-3LC-PUFA) have already shown their potential for developing functional food rich in these healthy fatty acids. Not only could they offer a more sustainable alternative for the fish stock that is currently relied upon but is unable to keep up with the demand, enrichment with certain microalgae also leads to oxidatively stable products. Although the reason for this stability has been attributed to the presence of endogenous carotenoids, further insight into their antioxidative role is missing and would be clarifying for selecting the proper microalgae for food enrichment. In trying to further accomplish this, a storage experiment (4 weeks, 37 °C) was set up with the parallel analysis of both oxidation products (primary and secondary) and carotenoids of two aqueous model systems of different (promising) microalgae (Nannochloropsis and Phaeodactylum). The results showed a clear difference in oxidative stability despite both microalgae containing endogenous carotenoids: Nannochloropsis led to oxidatively unstable and Phaeodactylum to oxidatively stable products. This was clearly confirmed by the analysis of n-3LC-PUFA throughout storage which showed a breakdown of half of the n-3LC-PUFA for Nannochloropsis. All carotenoids (violaxanthin, zeaxanthin and ß-carotene for Nannochloropsis, and fucoxanthin and ß-carotene for Phaeodactylum) acted as an antioxidant as shown by their degradation throughout storage, but the difference in oxidative stability pointed out an impact of carotenoid content and (possibly) type. The presence of a sufficient amount of carotenoids seems to be an important factor for perceiving oxidative stability. Phaeodactylum has shown to be more potent for food enrichment.

Stabilisation of vitamin A by wheat bran is affected by wheat bran antioxidants, bound lipids and endogenous lipase activity.

Food fortification is an efficient strategy to combat vitamin A deficiency. However, the stability of vitamin A during storage is low. Cereal bran can be used as a natural and affordable stabilising agent, but the mechanism behind this stabilisation remains unclear. To unravel this mechanism, vitamin A stabilisation was studied during an accelerated storage experiment (60 °C, 70% relative humidity) using a set of 30 in-house modified wheat bran samples. The characteristics of these samples were linked to vitamin A stabilisation during storage using forward regression modelling. While all wheat bran samples could stabilise vitamin A to a significant extent, the stabilising effect was more pronounced for samples with a high antioxidant capacity, high bound lipid content and low lipase activity. The main effect of lipase activity was more than thrice as large as the main effects of antioxidant capacity and bound lipid content. These results suggest that wheat bran antioxidants and bound lipids protect vitamin A from degradation during storage, while endogenous lipase activity counteracts the stabilising effect. Based on these findings, modified wheat bran mixed with vitamin A can be a cost-effective and healthy aid in food fortification by providing high vitamin A stability.

A simple method for analysis of vitamin A palmitate in fortified cereal products using direct solvent extraction followed by reversed-phase HPLC with UV detection.

Vitamin A is generally analysed using a time-consuming and possibly detrimental saponification step, followed by extraction and HPLC analysis. We here developed a new method to analyse retinyl palmitate (RP) (also known as vitamin A palmitate) without the need for saponification and validated it in model systems consisting of RP, soy oil and wheat bran, and in RP-fortified cereal products. Two direct solvent extraction protocols using acetone/methanol (7/3, v/v) or chloroform/methanol (1/1, v/v) were tested. After extraction, RP was quantified by reversed-phase HPLC with UV detection. The HPLC method had low limits of detection (0.01 µg/mL) and quantification (0.03 µg/mL). Both extraction protocols showed a good recovery (88-105 %) and intra-and inter-day precision (<5%) for RP extraction from the model systems. The obtained results corresponded to results obtained using a golden standard approach. For complex food matrices like bread and cookies, the chloroform/methanol extraction protocol showed the best performance characteristics.

Antioxidative capacity of microalgal carotenoids for stabilizing n-3LC-PUFA rich oil: Initial quantity is key.

The health-beneficial long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFA) are easily affected by the undesired process of lipid oxidation in fish oil, while being stable in the lipid extracts of photoautotrophic microalgae. The current research investigates the role of carotenoids by evaluating the oxidative stability of mixtures of fish oil with total lipid extracts of two different microalgae (Phaeodactylum and Isochrysis) throughout an accelerated storage experiment of 4 weeks at 37 °C. A clear separation between oxidatively stable and oxidatively unstable mixtures was observed for which the initial amount of carotenoids relative to the amount of n-3LC-PUFA was a good indicator. The lipid class composition, clearly differing between the two algae, was probably of minor influence. The antioxidative role of fucoxanthin, and diatoxanthin and ß-carotene as minor carotenoids, was illustrated by their gradual degradation throughout storage. However, when their initial contents were too low, this role could not be exerted leading to thorough lipid oxidation.

Conversion of Retinyl Palmitate to Retinol by Wheat Bran Endogenous Lipase Reduces Vitamin A Stability.

Wheat bran can be used as a cost-effective food ingredient to stabilise vitamin A. However, wheat bran endogenous enzymes have been shown to reduce vitamin A stability. In this study, we elucidated the mechanism for this negative effect in an accelerated storage experiment with model systems consisting of native or toasted wheat bran, soy oil and retinyl palmitate (RP). Both native and toasted wheat bran substantially stabilised RP. While RP was entirely degraded after ten days of storage in the absence of wheat bran, the RP retention after ten days was 22 ± 2% and 75 ± 5% in the presence of native and toasted bran, respectively. The significantly stronger stabilising effect of toasted bran was attributed to the absence of bran endogenous enzymes. In contrast to toasted bran systems, noticeable free fatty acid production was observed for native bran systems. However, this did not result in a pronounced lipid oxidation. Next to lipid hydrolysis, wheat bran lipase was shown to hydrolyse retinyl esters to the less stable retinol and fatty acids. This reaction could explain the major part, about 66 ± 5%, of the difference in RP stabilisation between native and toasted wheat bran.

The Potential of Phaeodactylum as a Natural Source of Antioxidants for Fish Oil Stabilization.

Worldwide, fish oil is an important and rich source of the health-beneficial omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). It is, however, troubled by its high susceptibility towards lipid oxidation. This can be prevented by the addition of (preferably natural) antioxidants. The current research investigates the potential of Phaeodactylum carotenoids in this regard. The oxidative stability of fish oil and fish oil with Phaeodactylum addition is evaluated by analyzing both primary (PV) and secondary (volatiles) oxidation products in an accelerated storage experiment (37 °C). A first experimental set-up shows that the addition of 2.5% (w/w) Phaeodactylum biomass is not capable of inhibiting oxidation. Although carotenoids from the Phaeodactylum biomass are measured in the fish oil phase, their presence does not suffice. In a second, more elucidating experimental set-up, fish oil is mixed in different proportions with a Phaeodactylum total lipid extract, and oxidative stability is again evaluated. It was shown that the amount of carotenoids relative to the n-3 LC-PUFA content determined oxidative stability. Systems with a fucoxanthin/n-3 LC-PUFA ratio ≥ 0.101 shows extreme oxidative stability, while systems with a fucoxanthin/n-3 LC-PUFA ratio ≤ 0.0078 are extremely oxidatively unstable. This explains why the Phaeodactylum biomass addition did not induce oxidative stability.

Photo-Oxidative Stability of Aqueous Model Systems Enriched with Omega-3 Long-Chain Polyunsaturated Fatty Acid-Rich Microalgae as Compared to Autoxidative Stability.

Several species of microalgae are promising as an alternative source of omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Photoautotrophic species show the greatest potential, since incorporating them into food products leads to oxidatively stable products; however, the presence of photosensitizers could reduce the shelf-life due to the appearance of photo-oxidation on exposure to light. This study investigated the oxidative impact of illumination for aqueous model suspensions enriched with Phaeodactylum (phototrophic microalgae─containing potential photosensitizers) and Schizochytrium (heterotrophic microalgae─lacking photosensitizers) during storage for 28 days at 37 °C. Primary (peroxide value) and secondary (volatiles with gas chromatography (GC)-mass spectrometry) oxidation products, n-3 LC-PUFA content (GC), and pigments (high-pressure liquid chromatography) were assessed. The results showed that photo-oxidation did not cause oxidative instability for Phaeodactylum samples compared with strong autoxidation in Schizochytrium samples. For the Phaeodactylum-enriched suspensions, only minimal photo-oxidation could be detected and the n-3 LC-PUFA content remained stable throughout storage regardless of illumination.

Normal-Phase HPLC-ELSD to Compare Lipid Profiles of Different Wheat Flours.

Normal-phase high-performance liquid chromatography (HPLC) is widely used in combination with evaporative light scattering detection (ELSD) for separating and detecting lipids in various food samples. ELSD responses of different lipids were evaluated to elucidate the possibilities and challenges associated with quantification by means of HPLC-ELSD. Not only the number and type of polar functional groups but also the chain length and degree of unsaturation of (free or esterified) fatty acids (FAs) had a significant effect on ELSD responses. Tripalmitin and trilinolein yielded notably different ELSD responses, even if their constituting free FAs produced identical responses. How FA structure impacts ELSD responses of free FAs is thus not predictive for those of triacylglycerols and presumably other lipids containing esterified FAs. Because ELSD responses of lipids depend on the identity of the (esterified) FA(s) which they contain, fully accurate lipid quantification with HPLC-ELSD is challenging and time-consuming. Nonetheless, HPLC-ELSD is a good and fast technique to semi-quantitatively compare the levels of different lipid classes between samples of comparable FA composition. In this way, lipid profiles of different flours from near-isogenic wheat lines could be compared.

Phase Behavior and Polymorphism of Saturated and Unsaturated Phytosterol Esters.

This study investigated how the physicochemical characteristics of phytosterol esters are influenced by the chain length and degree of unsaturation of the fatty acid ester moiety. Saturated and unsaturated phytosterol esters (PEs) were synthesized by the esterification of different types of fatty acids (stearic, palmitic, lauric, oleic, and linoleic acid) to ß-sitosterol. The non-isothermal crystallization and melting behavior of the pure PEs were analyzed. It was proven by X-ray diffraction that saturated ß-sitosteryl esters and ß-sitosteryl oleate formed a bilayer crystal structure. The lamellar spacings of the bilayer structure decreased with decreasing fatty acid chain length and with an increasing degree in unsaturation. The degree of unsaturation of the fatty acid chain of the ß-sitosteryl esters also influenced the type of subcell packing of the fatty acid moieties in the bilayer structure, whether or not a metastable or stable liquid crystalline phase was formed during cooling. Furthermore, it was found that the melting temperature and enthalpy of the ß-sitosteryl esters increased with an increasing fatty acid chain length while they decreased with an increasing degree of unsaturation. The microscopic analyses demonstrated that ß-sitosteryl oleate formed much smaller spherulites than their saturated ß-sitosteryl analogues.

The potential of microalgae and their biopolymers as structuring ingredients in food: A review.

Microalgae are considered promising functional food ingredients due to their balanced composition, containing multiple nutritional and health-beneficial components. However, their functionality in food products is not limited to health aspects, since microalgae can also play a structuring role in food, for instance as a texturizing ingredient. Photoautotrophic microalgae are actually rich in structural biopolymers such as proteins, storage polysaccharides, and cell wall related polysaccharides, and their presence might possibly alter the rheological properties of the enriched food product. A first approach to benefit from these structural biopolymers consists of isolating the cell wall related polysaccharides for use as food hydrocolloids. The potential of extracted cell wall polysaccharides as food hydrocolloids has only been shown for a few microalgae species, mainly due to an enormous diversity in molecular structure and composition. Nevertheless, with intrinsic viscosities comparable or higher than those of commercial thickening agents, extracellular polysaccharides of red microalgae and cyanobacteria could be a promising source of novel food hydrocolloids. A more sustainable approach would be to incorporate the whole microalgal biomass into food products, to combine health benefits with potential structuring benefits, i.e. providing desired rheological properties of the enriched food product. If microalgal biomass would act as a thickening agent, this would actually reduce the need for additional texturizing ingredients. Even though only limitedly studied so far, food processing operations have been proven successful in establishing desired microstructural and rheological properties. In fact, the use of cell disruption techniques allows the release of intracellular compounds, which become available to create strong particle aggregates resulting in an improved viscosity and network structure. Food processing operations might not only be favorable in terms of rheological properties, but also for enhancing the bioaccessibility of several bioactive compounds. However, this research area is only very scarcely explored, and there is a demand for more standardized research studies to draw conclusions on the effect of processing on the nutritional quality of food products enriched with microalgae. Even though considered as promising food ingredients, some major scientific challenges have been pointed out throughout this review paper for the successful design of microalgal based food products.

Impact of Nannochloropsis sp. dosage form on the oxidative stability of n-3 LC-PUFA enriched tomato purees.

Microalgae are a sustainable alternative source of n-3 LC-PUFA that can be incorporated into the food chain either via the incorporation of the (intact or disrupted) biomass or by the incorporation of the oil extracted from the biomass. However, the impact of the dosage form on the enrichment of food products with n-3 LC-PUFA and their oxidative stability has never been described before. This study aims to contribute more insight on the impact of the dosage form of the photoautotrophic microalga Nannochloropsis in enriched tomato puree. Three different dosage forms of Nannochloropsis were compared to commercial fish oil and analyzed for their amount of n-3 LC-PUFA, lipid oxidation products, antioxidants and free fatty acids. Tomato purees supplemented with dosage forms derived from Nannochloropsis showed higher oxidative stability than those supplemented with commercial fish oil. The highest oxidative stability was observed for purees supplemented with Nannochloropsis biomass irrespective of whether it was pre-disrupted.

Impact of processing on n-3 LC-PUFA in model systems enriched with microalgae.

Microalgae have already shown their potential as an alternative source of n-3 LC-PUFA. In this study, 5 different microalgal species (Isochrysis, Nannochloropsis, Phaeodactylum, Porphyridium and Schizochytrium) were added to an acidic model system and screened on their potential use in acidic food matrices. The impact of mechanical and thermal processing on the model systems was studied by analyzing the amount of n-3 LC-PUFA, free fatty acids, carotenoids, lipid polymers and the oxidative stability. A (limited) reduction of n-3 LC-PUFA was observed. Thermal alterations combined with the presence of free fatty acids seemed to be the causing factor for this decrease. Furthermore, the oxidative stability of model systems enriched with photoautotrophic microalgae was significantly higher than of those enriched with heterotrophic microalgae. It can therefore be concluded that photoautotrophic microalgae low in initial free fatty acid content are a promising source of n-3 LC-PUFA in thermally processed acidic food systems.

Effect of Meat Type, Animal Fatty Acid Composition, and Isothermal Temperature on the Viscoelastic Properties of Meat Batters.

The aim of this research was to simultaneously study the effect of meat type (chicken breast and leg meat), animal fatty acid composition (selected pork backfats having a low and high degree of saturation, respectively), and isothermal temperature (50, 60, 70, and 80 °C) on the viscoelastic properties of meat batters during and after application of different time-temperature profiles. Gelation of meat proteins contributed most to the viscoelastic properties of meat batters during heating, whereas crystallization of the lipids especially contributed to the viscoelastic properties during the cooling phase. Although the meat type had little effect on the final viscoelastic properties of the meat product, the fatty acid composition had a clear impact on the melting peak area (and therefore solid fat content) of lard, and subsequently on the final viscoelastic properties of meat batters prepared with different types of fats, with higher G' (elastic modulus) values for the most saturated animal fat. The crystallization of the fat clearly transcended the effect of the meat type with regard to G' at the end of the process. With increasing (isothermal) temperature, G' of meat batters increased. Therefore, it could be concluded that the structural properties of heated meat batters mainly depend on the heating temperature and the fatty acid composition, rather than the meat type. PRACTICAL APPLICATION: Quality characteristics of cooked sausages depend on multiple factors such as the meat and fat type, non-meat ingredients and processing conditions. From this study it could be concluded that the structural properties of cooked sausage batters mainly depend on the heating temperature and the fatty acid composition, rather than the meat type. Because the fatty acid composition of different animal fats differs widely, these results may be a concern for all manufactures of cooked sausages products with regard to the product structure and final texture, keeping in mind that rendered fat was used in this study, which is not common in sausage making.

Molecular and rheological characterization of different cell wall fractions of Porphyridium cruentum.

Cell wall related polysaccharides of the red microalga Porphyridium sp. were shown to be a promising source of new sustainable thickening agents. Isolated extracellular polysaccharides (EPS) consisted of high molecular weight polymers, showing a higher intrinsic viscosity compared to several commercially used hydrocolloids. Aqueous solutions of EPS (2% w/w) were characterized by substantial viscosities and weak gel behavior. Even though the extracted water soluble cell wall polysaccharides exhibited the same monosaccharide profile as EPS (composed of galactose, glucose, xylose and glucuronic acid), a lower molecular weight and intrinsic viscosity was observed for this fraction, resulting in poor rheological properties. Therefore, it was hypothesized that the physicochemical properties were related with a different molecular structural organization of these monosaccharides and sulfate groups. The main challenge for commercialization of extracellular polysaccharides of Porphyridium sp. remains the purification of these fractions to obtain polysaccharide extracts with low protein and salt contents.

Impact of different sequences of mechanical and thermal processing on the rheological properties of Porphyridium cruentum and Chlorella vulgaris as functional food ingredients.

Microalgae are a promising and sustainable source for enhancing the nutritional value of food products. Moreover, incorporation of the total biomass might contribute to the structural properties of the enriched food product. Our previous study demonstrated the potential of Porphyridium cruentum and Chlorella vulgaris as multifunctional food ingredients, as they displayed interesting rheological properties after applying a specific combination of mechanical and thermal processing. The aim of the current study was to investigate the impact of a different sequence of high pressure homogenization (HPH) and thermal processing on the thickening and gelling potential of these microalgal biomasses in aqueous suspensions. Thermal processing largely increased the gel strength and viscosity of both microalgae, which was ascribed to larger and stronger aggregates as a result of partial solubilization of polymers, while subsequent HPH generally reduced the rheological properties. Interestingly, large amounts of intact cells were still observed for both microalgae when HPH was performed after a thermal treatment, irrespective of the applied homogenization pressure, implying that cell disruption was hindered by the preceding thermal treatment. Although thermal processing was regarded as the most effective processing technique to obtain increased rheological properties, the combination with a preceding HPH treatment should still be considered when cell disruption is desired, for instance to increase the bioavailability of intracellular components. Finally, biomass of P. cruentum showed the largest potential for use as a structuring agent, as the gel strength and viscosity in thermally treated suspensions of this microalga were about 10 times higher than for C. vulgaris.

Isothermal Crystallization Kinetics of Palm Oil as Influenced by Addition of a Commercial Phytosterol Ester Mixture.

In literature there is good agreement on the health-promoting effects of phytosterols. However, addition of phytosterol esters (PEs) to lipid (containing food products) may influence its crystallization behavior. This study investigated the crystallization kinetics of palm oil (PO) after addition of PEs in high concentrations (≥10%). The isothermal crystallization of the PE-PO blends was analyzed at a temperature of 20 °C and at a supercooling of 18.7 °C using differential scanning calorimetry and time-resolved synchrotron X-ray diffraction. At increasing PE concentrations, PO crystallization at an isothermal temperature of 20 °C started later and was slower and a smaller amount of crystals were formed. Furthermore, a delay in polymorphic transition from α to ß' was observed. When the blends were isothermally crystallized at a supercooling of 18.7 °C, only two of these effects remained: the delay in polymorphic transition and the decrease in crystalline content.

The effect of adding a commercial phytosterol ester mixture on the phase behavior of palm oil.

The objective of this study was to investigate in depth the non-isothermal crystallization and melting behavior of binary blends of palm oil (PO) with a commercial, multi-component phytosterol ester (PE) mixture. DSC and time-resolved synchrotron X-ray diffraction (XRD) experiments were conducted on blends with a PE concentration from 0 to 100% at intervals of 10% for DSC and 20% for XRD. Based on XRD, two different ordered structures were identified in pure the PEs. The structure designated as PEx was truly crystalline and needed a very high degree of supercooling for its nucleation from the melt. The structure designated PEy formed without supercooling and showed long-range order with multiple reflections at small angles but only one broad reflection at high angle, typical of liquid crystalline samples. Furthermore, PEy had a high tolerance for molecules of different chemical nature. In the PE-PO blends no other ordered structures were formed other than the ones observed in the pure PEs and PO. The peaks in the DSC runs of the PE-PO blends were linked to transitions of the different polymorphic forms. All structural information of the binary blends as a function of concentration and temperature was collected in morphology maps. The binary blends exhibited eutectic characteristics visualized in the morphology maps with a eutectic point at 40% PEs.