Food ingredients from cultivated seaweeds-Improving storage stability and protein recovery

There is a global demand for new vegetarian protein sources, and seaweed have for multiple reasons been identified as a promising candidate. The overall aim of this thesis was to evaluate the potential of three different Swedish seaweed species as food protein sources, but also as sources of unsaturated fatty acids, vitamins and minerals. To accomplish this, the goals were to i) obtain a seaweed biomass high in protein and lipids, ii) find strategies to maintain the quality of the seaweed biomass during drying and storage and iii) develop an efficient method for recovering seaweed proteins. The protein and lipid content in seaweed is related, e.g. to the nutrient access and physical parameters of the surrounding environment. By applying alterative cultivation, the protein and fatty acid content in U. lactuca were increased 3.4 respective 1.5 times by nitrate addition. Furthermore, the protein content was increased by cultivation at low temperature (12 \ub0C) and light (50 μmol photons m-2 s-1), and the fatty acid content was increased by low light and high temperature (18\ub0C).The polyunsaturated fatty acids (PUFAs) in seaweed could make the dried biomass susceptible to lipid oxidation, with co-oxidation of pigments and vitamins. However, during long-term storage (≤520 days) of oven- and freeze-dried P. umbilicalis and U. lactuca, there was only a moderate development of the lipid oxidation-derived aldehydes, malondialdehyde, 4-hydroxy-trans-2-hexenal and 4-hydryoxy-trans-2-nonenal, while there was a great loss of unsaturated fatty acids and ascorbic acid. Light stimulated the fatty acid loss as well as bleaching of chlorophyll. Several advantages are foreseen from concentrating the seaweed proteins. This calls for food grade and scalable fractionation methods. The pH-shift process, using alkaline protein solubilisation followed by isoelectric precipitation, was in this work adapted and improved for P. umbilicalis, U. lactuca and S. latissima, e.g. by including freeze-thawing-stimulated protein precipitation. When then comparing the new pH-shift process to two other fractionation methods, the pH-shift method resulted in extracts with the highest protein content: 71%, 51% and 41% per dry weight for P. umbilicalis, U. lactuca and S. latissima, respectively. The protein contained 37-41% essential amino acids. The highest achieved protein yields using the pH-shift method were 23%, 6% and 25%, respectively, for the listed species. For U. lactuca, the yield was further raised to 29% by incorporating a pre-incubation step at pH 8.5 prior to further protein solubilisation at pH 12. The pH-shift process was also successfully used as a first step in a sequential recovery of proteins, carrageenan, pectin and cellulose from P. umbilicalis, showing potential as a biorefinery tool. Throughout the work, the effect of different protein analysis methods on the achieved concentrations of protein in seaweed and seaweed protein extracts was evaluated. This revealed that the analytical choice has a profound impact on the results, especially for the extracts.To summarise, it was possible to increase the level of proteins and lipids in seaweed through alterative cultivation protocols, and the seaweed proteins could then be further concentrated using the pH-shift process. These results strengthen the possibility that seaweed biomass can be a valuable complement to terrestrial vegetarian food protein sources. If considering the seaweed PUFAs as an added value to recover along with proteins, caution must, however, be taken when it comes to pre-processing storage of the seaweed biomass; these nutrients easily degrade during storage of dried seaweeds, especially in the presence of light

Production of protein extracts from Swedish red, green, and brown seaweeds, Porphyra umbilicalis Kützing, Ulva lactuca Linnaeus, and Saccharina latissima (Linnaeus) J. V. Lamouroux using three different methods

peer-reviewedThe demand for vegetable proteins increases globally and seaweeds are considered novel and promising protein sources. However, the tough polysaccharide-rich cell walls and the abundance of polyphenols reduce the extractability and digestibility of seaweed proteins. Therefore, food grade, scalable, and environmentally friendly protein extraction techniques are required. To date, little work has been carried out on developing such methods taking into consideration the structural differences between seaweed species. In this work, three different protein extraction methods were applied to three Swedish seaweeds (Porphyra umbilicalis, Ulva lactuca, and Saccharina latissima). These methods included (I) a traditional method using sonication in water and subsequent ammonium sulfate-induced protein precipitation, (II) the pH-shift protein extraction method using alkaline protein solubilization followed by isoelectric precipitation, and (III) the accelerated solvent extraction (ASE®) method where proteins are extracted after pre-removal of lipids and phlorotannins. The highest protein yields were achieved using the pH-shift method applied to P. umbilicalis (22.6 ± 7.3%) and S. latissima (25.1 ± 0.9%). The traditional method resulted in the greatest protein yield when applied to U. lactuca (19.6 ± 0.8%). However, the protein concentration in the produced extracts was highest for all three species using the pH-shift method (71.0 ± 3.7%, 51.2 ± 2.1%, and 40.7 ± 0.5% for P. umbilicalis, U. lactuca, and S. latissima, respectively). In addition, the pH-shift method was found to concentrate the fatty acids in U. lactuca and S. latissima by 2.2 and 1.6 times, respectively. The pH-shift method can therefore be considered a promising strategy for producing seaweed protein ingredients for use in food and feed

A trout (Oncorhynchus mykiss) perfusion model approach to elucidate the role of blood removal for lipid oxidation and colour changes in ice-stored fish muscle

Whole body saline-perfused rainbow trout (Oncorhynchus mykiss) was ice-stored for 4\ua0weeks and compared with unwashed/washed minces from unbled and bled trout in terms of rancid odour, peroxide value (PV), thiobarbituric acid reactive substances (TBARS) and redness loss. Muscle from saline-perfused fish, which had 72% less total haem, was deficient in rancid odour during the whole storage, while bled (54% less haem) and unbled samples developed rancid odour already after ~4 and 2\ua0days; higher intensity without bleeding. PV/TBARS also developed in the order unbled\ua0>\ua0bled\ua0>\ua0perfused samples; however, PV/TBARS were not as completely prevented as rancid odour after perfusion. Saline washing (3\ua0 7\ua03 volumes) of unbled mince removed 84% haem and yielded the second most stable sample while saline washing (1\ua0 7\ua01 volumes) destabilised unbled mince, despite 64% haem removal. Concurrent antioxidant removal during washing of minces obviously counteracted the effect of blood removal and washing fish mince with small volumes of solution should be used with great care

Formation of malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) in fish and fish oil during dynamic gastrointestinal in vitro digestion

Marine lipids contain a high proportion of polyunsaturated fatty acids (PUFA), including the characteristic long chain (LC) n-3 PUFA. Upon peroxidation these lipids generate reactive products, such as malondialdehyde (MDA), 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE), which can form covalent adducts with biomolecules and thus are regarded as genotoxic and cytotoxic. PUFA peroxidation can occur both before and after ingestion. The aim of this study was to determine what levels of MDA, HHE and HNE can evolve in the gastric and intestinal lumen after ingesting meals containing fish or fish oil using a dynamic gastrointestinal (GI) model (TIM). The impact of the fish muscle matrix, lipid content, fish species, and oven baking on GI oxidation was evaluated. MDA and HHE concentrations in gastric lumen increased for all meals during digestion, with the highest level found with herring mince; similar to 25 mu M MDA and similar to 850 nM HHE. Aldehyde concentrations reached in intestinal lumen during digestion of fish containing meals were generally lower than in gastric lumen, while isolated herring oils (bulk and emulsified) generated higher MDA and HHE values in intestinal lumen compared to gastric lumen. Based on aldehyde levels in gastric lumen, meals containing herring lipids were ranked: raw herring (17% lipid) = baked herring (4% lipid) > raw herring (4% lipid) >> herring oil emulsion > herring oil. Herring developed higher concentrations of MDA and HHE during gastric digestion compared to salmon, which initially contained lower levels of oxidation products. Cooked salmon generated higher MDA concentrations during digestion than raw salmon. Low levels of HNE were observed during digestion of all test meals, in accordance with the low content of n-6 PUFA in fish lipids

A Strategy for the Sequential Recovery of Biomacromolecules from Red Macroalgae Porphyra umbilicalis K\ufctzing

A nondestructive, multicomponent fractionation strategy has been developed to extract proteins and polysaccharides from the red macroalgae Porphyra umbilicalis collected along the west coast of Sweden and cultivated indoors under controlled conditions. First, a protein-rich fraction was extracted in an ice-cold alkaline solution. The overall protein content in Porphyra umbilicalis was estimated to be 30.6% of the dry weight, and out of that, 15.0% could be recovered. Water-soluble polysaccharides were then extracted from the insoluble residual fraction using sequential alkaline and acidic treatments at 90 \ub0C for 4 h. Spectroscopic and chromatographic analyses of the polysaccharide fractions show that high-molecular-weight carrageenans were obtained from the alkaline extraction and a galactose-rich pectin substance was obtained from the acidic extraction. The insoluble fraction remaining after all extraction steps was rich in cellulose. An elemental analysis of Porphyra umbilicalis via scanning electron microscopy with energy-dispersive X-ray spectrometry (SEM-EDS) showed the presence of C, O, Na, Ca, Mg, Al, Cl, and S. However, no heavy metals or other toxic elements, such as Pb, Hg, and As, were found

Effect of storage conditions on lipid oxidation, nutrient loss and colour of dried seaweeds, Porphyra umbilicalis and Ulva fenestrata, subjected to different pretreatments

Here we evaluated the levels of lipid oxidation products, fatty acids, ascorbic acid and colour of Porphyra and Ulva after oven-drying at 40 \ub0C, and during subsequent storage for ≥370 days under light, semi-light and dark conditions. Part of the seaweed was pre-soaked in freshwater or pre-coated with a whey protein mixture. Controls consisted of freeze-dried seaweeds. Throughout storage there was a moderate development of the lipid oxidation-derived aldehydes, malondialdehyde, 4-hydroxy-trans-2-hexenal and 4-hydroxy-trans-2-nonenal, while there was a great loss of unsaturated fatty acids and ascorbic acid. Light storage and freeze-drying stimulated the fatty acid loss as well as pigment bleaching, seen as increased a*-values. For Ulva, the coating reduced malondialdehyde, 4-hydroxy-trans-2-hexenal and 4-hydroxy-trans-2-nonenal formation during drying and slightly prevented loss of polyunsaturated fatty acids during light storage. Pre-soaking in freshwater had no effect on the seaweed stability, although it reduced the ash content and thereby increased the relative content of ascorbic acid and fatty acids of the biomasses

In vitro digestibility and Caco-2 cell bioavailability of sea lettuce (Ulva fenestrata) proteins extracted using pH-shift processing

Seaweed is a promising sustainable source of vegan protein as its farming does not require arable land, pesticides/insecticides, nor freshwater supply. However, to be explored as a novel protein source the content and nutritional quality of protein in seaweed need to be improved. We assessed the influence of pH-shift processing on protein degree of hydrolysis (%DH), protein/peptide size distribution, accessibility, and cell bioavailability of Ulva fenestrata proteins after in vitro gastrointestinal digestion. pH-shift processing of Ulva, which concentrated its proteins 3.5-times, significantly improved the %DH from 27.7\ub12.6% to 35.7\ub12.1% and the amino acid accessibility from 56.9\ub14.1% to 72.7\ub10.6%. Due to the higher amino acid accessibility, the amount of most amino acids transported across the cell monolayers was higher in the protein extracts. Regarding bioavailability, both Ulva and protein extracts were as bioavailable as casein. The protein/peptide molecular size distribution after digestion did not disclose a clear association with bioavailability

Effects of irradiance, temperature, nutrients, and pCO2 on the growth and biochemical composition of cultivated Ulva fenestrata

Ulva fenestrata is an economically and ecologically important green algal species with a large potential in seaweed aquaculture due to its high productivity, wide environmental tolerance, as well as interesting functional and nutritional properties. Here, we performed a series of manipulative cultivation experiments in order to investigate the effects of irradiance (50, 100, and 160\ua0μmol photons m−2\ua0s−1), temperature (13 and 18\ua0\ub0C), nitrate (< 5, 150, and 500\ua0μM), phosphate (< 1 and 50\ua0μM), and pCO2 (200, 400, and 2500\ua0ppm) on the relative growth rate and biochemical composition (fatty acid, protein, phenolic, ash, and biochar content) in indoor tank cultivation of Swedish U. fenestrata. High irradiance and low temperature were optimal for the growth of this northern hemisphere U. fenestrata strain, but addition of nutrients or changes in pCO2 levels were not necessary to increase growth. Low irradiance resulted in the highest fatty acid, protein, and phenolic content, while low temperature had a negative effect on the fatty acid content but a positive effect on the protein content. Addition of nutrients (especially nitrate) increased the fatty acid, protein, and phenolic content. High nitrate levels decreased the total ash content of the seaweeds. The char content of the seaweeds did not change in response to any of the manipulated factors, and the only significant effect of changes in pCO2 was a negative relationship with phenolic content. We conclude that the optimal cultivation conditions for Swedish U. fenestrata are dependent on the desired biomass traits (biomass yield or biochemical composition)

Cultivation conditions affect the monosaccharide composition in Ulva fenestrata

In recent years, the interest in using seaweed for the sustainable production of commodities has been increasing as seaweeds contain many potentially worthwhile compounds. Thus, the extraction and refining processes of interesting compounds from seaweeds is a hot research topic but has been found to have problems with profitability for novel applications. To increase the economic potential of refining seaweed biomass, the content of the compounds of interest should be maximized, which can potentially be achieved through optimization of cultivation conditions. In this study, we studied how the monosaccharide composition of the green seaweed species Ulva fenestrata is influenced by the abiotic factors; irradiance, temperature, nitrate, phosphate, and pCO2. It was evident that lower nitrate concentration and cultivation at elevated temperature increased monosaccharide contents. A 70% increase in iduronic acid and a 26% increase in rhamnose content were seen under elevated irradiance and temperature conditions, though the absolute differences in monosaccharide concentration were small. Irradiance and nitrate impacted the ratio between iduronic and rhamnose, which is an indicator of the ulvan structure. These results could potentially be utilized to coax the ulvan towards specific bioactivities, and thus have a considerable impact on a potential biorefinery centered around Ulva.\ua0\ua9 2020, The Author(s)