Porosira glacialis as a possible source of lipids for human consumption and aquaculture feed

Det er et stort behov for marine langkjedede omega-3 fettsyrer både til humant konsum og til fiskefôr. De langkjedede omega-3 fettsyrene EPA og DHA har gunstige helseeffekter for både mennesker og fisk. Mengden villfanget fisk har de siste 40 årene nådd maksimal utnyttelse mens volumet av oppdrettsfisk har økt betraktelig. Mangelen på marint råstoff har ført til en økt andel terrestrisk råstoff i fiskefôret, noe som påvirker næringssammensetningen i oppdrettsfisken. Dyrking av marine mikroalger kan være med på å løse utfordringene knyttet til mangel på marine langkjedede omega-3 fettsyrer, da de er havets primærprodusenter av EPA og DHA. Mikroalger dyrkes i vann og trenger CO2, lys og uorganiske næringsstoffer for å vokse. Mikroalger kan også være med på å redusere direkte utslipp av CO2 fra store utslippspunkter ved å benytte denne gassen til slik dyrking. I dette prosjektet ble den kaldtvannstilpassede kiselalgen Porosira glacialis dyrket for å undersøke om lipidfraksjonen er egnet som tilskudd i fiskefôr og/eller til menneskelig konsum. De spesifikke delmålene var i) å undersøke lagringsstabiliteten til lipidene i våt biomasse, ii) sammenligne in vitro fordøyelighet av lipidene i P. glacialis med vanlige kilder til langkjedede omega-3 fettsyrer, iii) undersøke utbytte av lipider med ulike fettekstraksjonsmetoder sammenlignet med andre massedyrkede mikroalger. P. glacialis inneholder store mengder flerumettet fett, hvorav fettsyren EPA utgjør en stor andel. Sammenlignet med lipidfraksjonen i de andre artene og oljene som ble undersøkt, er omega-3-innholdet høyt. Størstedelen av lipidklassene i P. glacialis er polare, blant annet galaktolipider og fosfolipider. Disse lipidklassene skiller seg ut fra vanlige fiskeoljeprodukter som tran og omega-3-konsentrater. Lipidene i P. glacialis er svært utsatt for hydrolyse under lagring, noe som fører til økte mengder frie fettsyrer samt at oljen er mer utsatt for oksidasjon. Våre resultater viser at lagringsstabiliteten til lipidene i våt biomasse kan økes betraktelig ved en enkel varmebehandling før lagring ved 4 °C. Det ser også ut til at lipidene i P. glacialis er godt fordøyelige sammenlignet med vanlige omega-3 kosttilskudd. Den eneste oljen som ble hydrolysert til en høyere grad under in vitro fordøyelse var tran, som i hovedsak inneholder triacylglycerol. Ekstraksjon av lipider fra P. glacialis krever ingen forbehandling. Men som andre arter med høyt innhold av polare lipidklasser trengs et delvis polart løsemiddel for ekstraksjon. Den har god vekst på CO2 fra røykgassen til et smelteverk og kan derfor være med på å begrense direkte utslipp av drivhusgasser. Det vil være spennende å undersøke om denne kiselalgen kan benyttes direkte til fiskefôr eller humant konsum.The demand for marine long-chain omega-3 fatty acids for both human consumption and fish feed is large. The long-chain omega-3 fatty acids, EPA and DHA, have several health benefits for both humans and fish. The amount of wild caught fish has reached maximum exploitation over the last 40 years, while the production from aquaculture has increased drastically. The shortage of marine raw material has led to an increased inclusion of terrestrial raw material in the aquaculture feed, which has affected the nutritional composition of the farmed fish. Cultivation of marine microalgae may be part of the solution to these challenges, since these are the ocean’s primary producers of EPA and DHA. Microalgae can be cultivated in water using only CO2, light, and inorganic nutrients. Microalgae can also reduce the amount of direct CO2 emissions from large industry by utilizing the gas for cultivation. In this project, the cold-water adapted diatom Porosira glacialis was cultivated to investigate the lipid fraction's suitability for fish feed and/or human consumption. The specific aims were to i) investigate the storage stability of lipid in wet biomass, ii) compare the in vitro digestibility of lipids from P. glacialis with common sources of long-chain omega-3 fatty acids, iii) investigate the extractability of lipids from P. glacialis compared to other mass-cultivated microalgae. P. glacialis contains large amounts of polyunsaturated fatty acids, of which EPA makes up a substantial part. Compared to the lipid fraction of the other oils that were investigated, the omega-3 content is high. The main fraction of lipid classes in P. glacialis is the polar galactolipids and phospholipids. These lipid classes differ from the usual fish oil products, like cod-liver oil and omega-3 concentrates. The lipids in P. glacialis are exposed to hydrolysis during storage, which results in increased amounts of free fatty acids that are more exposed to oxidation. Our results indicate that the storage stability of lipids in the wet biomass can be enhanced by a simple heat treatment and storage at 4 °C. The lipids in P. glacialis appear to be digested well compared to common omega-3 supplements. The only other oil with a higher degree of hydrolysis was cod-liver oil, which contain mostly triacylglycerol. The extraction of lipids from P. glacialis does not require any pre-treatment. However, like other species with large amounts of polar lipid classes, a partially polar solvent is needed for complete extraction. P. glacialis grows well using CO2 from the flue gas of a smelting plant and could therefore help limit the emission of greenhouse gases. The prospect of investigating this diatom directly for fish feed or human consumption is interesting

In vitro intestinal digestion of lipids from the marine diatom Porosira glacialis compared to commercial LC n-3 PUFA products

Marine sources of long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) are in high demand for use in health supplements. Mass cultivated marine microalgae is a promising and sustainable source of LC n-3 PUFA, which relieves pressure on natural fish stocks. The lipid class profile from cultivated photosynthetic algae differ from the marine organisms currently used for the production of LC n-3 PUFA. The objective of this study was to compare in vitro intestinal digestion of oil extracted from the cold-adapted marine diatom Porosira glacialis with commercially available LC n-3 PUFA supplements; cod liver oil, krill oil, ethyl ester concentrate, and oil from the copepod Calanus finmarchicus (Calanus® oil). The changes in the free fatty acids and neutral and polar lipids during the enzymatic hydrolysis were characterized by liquid and gas chromatography. In Calanus® oil and the Ethyl ester concentrate, the free fatty acids increased very little (4.0 and 4.6%, respectively) during digestion. In comparison, free fatty acids in Krill oil and P. glacialis oil increased by 14.7 and 17.0%, respectively. Cod liver oil had the highest increase (28.2%) in free fatty acids during the digestion. Monounsaturated and saturated fatty acids were more easily released than polyunsaturated fatty acids in all five oils

Microbial Community Dynamics during a Harmful Chrysochromulina leadbeateri Bloom in Northern Norway

A harmful algal bloom occurred in late spring 2019 across multiple, interconnected fjords and bays in northern Norway. The event was caused by the haptophyte Chrysochromulina leadbeateri and led to severe fish mortality at several salmon aquaculture facilities. This study reports on the spatial and temporal succession dynamics of the holistic marine microbiome associated with this bloom by relating all detectable 18S and 16S rRNA gene amplicon sequence variants to the relative abundance of the C. leadbeateri focal taxon. A k-medoid clustering enabled inferences on how the causative focal taxon cobloomed with diverse groups of bacteria and microeukaryotes. These coblooming patterns showed high temporal variability and were distinct between two geographically separated time series stations during the regional harmful algal bloom. The distinct blooming patterns observed with respect to each station were poorly connected to environmental conditions, suggesting that other factors, such as biological interactions, may be at least as important in shaping the dynamics of this type of harmful algal bloom. A deeper understanding of microbiome succession patterns during these rare but destructive events will help guide future efforts to forecast deviations from the natural bloom cycles of the northern Norwegian coastal marine ecosystems that are home to intensive aquaculture activities

Porosira glacialis as a possible source of lipids for human consumption and aquaculture feed

Det er et stort behov for marine langkjedede omega-3 fettsyrer både til humant konsum og til fiskefôr. De langkjedede omega-3 fettsyrene EPA og DHA har gunstige helseeffekter for både mennesker og fisk. Mengden villfanget fisk har de siste 40 årene nådd maksimal utnyttelse mens volumet av oppdrettsfisk har økt betraktelig. Mangelen på marint råstoff har ført til en økt andel terrestrisk råstoff i fiskefôret, noe som påvirker næringssammensetningen i oppdrettsfisken. Dyrking av marine mikroalger kan være med på å løse utfordringene knyttet til mangel på marine langkjedede omega-3 fettsyrer, da de er havets primærprodusenter av EPA og DHA. Mikroalger dyrkes i vann og trenger CO2, lys og uorganiske næringsstoffer for å vokse. Mikroalger kan også være med på å redusere direkte utslipp av CO2 fra store utslippspunkter ved å benytte denne gassen til slik dyrking. I dette prosjektet ble den kaldtvannstilpassede kiselalgen Porosira glacialis dyrket for å undersøke om lipidfraksjonen er egnet som tilskudd i fiskefôr og/eller til menneskelig konsum. De spesifikke delmålene var i) å undersøke lagringsstabiliteten til lipidene i våt biomasse, ii) sammenligne in vitro fordøyelighet av lipidene i P. glacialis med vanlige kilder til langkjedede omega-3 fettsyrer, iii) undersøke utbytte av lipider med ulike fettekstraksjonsmetoder sammenlignet med andre massedyrkede mikroalger. P. glacialis inneholder store mengder flerumettet fett, hvorav fettsyren EPA utgjør en stor andel. Sammenlignet med lipidfraksjonen i de andre artene og oljene som ble undersøkt, er omega-3-innholdet høyt. Størstedelen av lipidklassene i P. glacialis er polare, blant annet galaktolipider og fosfolipider. Disse lipidklassene skiller seg ut fra vanlige fiskeoljeprodukter som tran og omega-3-konsentrater. Lipidene i P. glacialis er svært utsatt for hydrolyse under lagring, noe som fører til økte mengder frie fettsyrer samt at oljen er mer utsatt for oksidasjon. Våre resultater viser at lagringsstabiliteten til lipidene i våt biomasse kan økes betraktelig ved en enkel varmebehandling før lagring ved 4 °C. Det ser også ut til at lipidene i P. glacialis er godt fordøyelige sammenlignet med vanlige omega-3 kosttilskudd. Den eneste oljen som ble hydrolysert til en høyere grad under in vitro fordøyelse var tran, som i hovedsak inneholder triacylglycerol. Ekstraksjon av lipider fra P. glacialis krever ingen forbehandling. Men som andre arter med høyt innhold av polare lipidklasser trengs et delvis polart løsemiddel for ekstraksjon. Den har god vekst på CO2 fra røykgassen til et smelteverk og kan derfor være med på å begrense direkte utslipp av drivhusgasser. Det vil være spennende å undersøke om denne kiselalgen kan benyttes direkte til fiskefôr eller humant konsum

In vitro digestion of lipids from the marine diatom Porosira glacialis compared to commercially available marine oils

The digestibility of lipids from P. glacialis were investigated using an in vitro digestion mode

Trans isomers of EPA and DHA in refining and concentration of fish oils.

The omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are strongly recommended to be part of a normal diet due to the many positive health effects in humans. Health authorities in many countries recommend an average daily intake of 0.25 – 0.5 grams of EPA and DHA by eating fish at least twice a week or alternatively, consume fish oil supplements. The unsaturated fatty acids in native marine and vegetable lipids are all present as cis-isomers while trans-isomers are found in industrially hydrogenated oils and in minor amounts in fat from ruminants. The intake of large amounts of trans-fatty acids is considered to have serious negative health effects and the advices are to reduce the consumption as much as possible. Several processing steps are necessary to produce high quality fish oil supplements, including concentrated forms of EPA and DHA. These steps include high temperature processes that might induce transformation of cis-double bonds to trans-double bonds in the unsaturated fatty acids. The objectives of this thesis was determine if the processing conditions used at Nordic Pharma Inc. resulted in the formation of trans fatty acids in both natural “1812” fish oils (18 % EPA, 12 % DHA) and EPA/DHA concentrates and to investigate how time and temperature used in the processing steps affected the formation of trans fatty acids in fish oil concentrates. Initially, a method for the analysis of trans fatty acids in fish oils was established. Methylated standards of trans EPA and DHA were produced using p-toluenesulfinic acid as catalyst and separating the different isomers on high performance thin layer chromatography (HPTLC) plates impregnated with silver nitrate. A 100 meter SLB-IL111, the most polar gas chromatography (GC) column commercially available, was used to analyze the samples. The preparation and the separation of the trans standards on HPTLC plates was successful and gave sufficient amounts to optimize a temperature program for GC separation. The results from samples of processed fish oil showed that only minor amounts of trans fatty acids were formed during the processing conditions applied at Nordic Pharma Inc. The content of trans fatty acids was far below the amount allowed for such products. The SLB-IL111 column worked well as a tool for analysis of trans fatty acids in fish oil, but some further investigations are needed for this analysis system to be optimized. The temperature experiment showed that 200 °C for more than an hour was needed for the formation of larger amounts of trans LC-PUFA to occur. Keywords: Fish oil, industrial processing, trans fatty acids, EPA, DHA, SLB-IL111, silver ion TL

Temperature dependent growth rate, lipid content and fatty acid composition of the marine cold-water diatom Porosira glacialis

In this study, the northern cold-water marine diatom Porosira glacialis was cultivated in a pilot-scale mass cultivation system at 5 different temperatures (−2 to 12 °C), in order to evaluate temperature-dependent growth rate (in vitro Chl a), lipid content (Folch's method) and fatty acid (FA) composition (GC–MS) in the exponential growth phase. We found that P. glacialis has a wide temperature range, with maximum growth at 12 °C and positive growth even at sub-zero water temperatures. The lipid content was inversely correlated with temperature, peaking at 33.4 ± 4.0% at 2 °C, and was highly desaturated independently of temperature; PUFA content varied from 71.50 ± 0.88% at 12 °C to 82 ± 0.64% at −2 °C. EPA was the main FA at all temperatures (31.0 ± 0.7–40.4 ± 1.2% of total FAs)

Stability of lipids during wet storage of the marine diatom Porosira glacialis under semi-preserved conditions at 4 and 20 °C

Cultivation of diatoms may help alleviate the pressure on wild fish stocks for marine nutrient availability in aquaculture feed and for human consumption. However, the lipids in microalgae biomass are easily deteriorated, both trough lipolysis and degradation of polyunsaturated fatty acids (PUFA). Proper storage conditions are therefore necessary to maintain the lipid quality. Additionally, the storage conditions must have a low cost and facilitate further processing of the biomass. In this study, we investigated the formation of free fatty acids, changes in lipid classes, and fatty acid composition of the psychrophilic marine diatom Porosira glacialis under storage. The wet biomass was stored for 14 days at 4 and 20 °C with either heat treatment, formic acid, or benzoic acid addition, and a control sample. Heat-treated and formic acid samples had the lowest rate of free fatty acid formation during storage. Mainly, polar lipids were hydrolyzed to free fatty acids and this occurred fastest at 20 °C. The fatty acid composition remained stable in heat-treated samples during storage, whereas a loss of PUFA was observed in the other treatments. The lack of effect from benzoic acid indicates that the loss of lipid quality stems from endogenous enzymes rather than exogenous organisms. Heat treatment and formic acid appeared to effectively reduce lipase activity, and potentially lipoxygenase and similar enzymes that affect the fatty acids. The low pH of the formic acid samples seems to have a negative effect on the PUFA content, in particular at 20 °C

Lipid yield from the diatom Porosira glacialis is determined by solvent choice and number of extractions, independent of cell disruption

Cell wall disruption is necessary to maximize lipid extraction yields in conventional species of mass-cultivated microalgae. This study investigated the effect of sonication, solvent choice and number of extractions on the lipid yield, lipid class composition and fatty acid composition of the diatom Porosira glacialis. For comparison, the diatom Odontella aurita and green alga Chlorella vulgaris were included in the study. Sonication effectively disrupted P. glacialis cells, but did not increase the total lipid yield compared to physical stirring (mixing). In all three microalgae, the content of membrane-associated glyco- and phosopholipids in the extracted lipids was strongly dependent on the solvent polarity. A second extraction resulted in higher yields from the microalgae only when polar solvents were used. In conclusion, choice of solvent and number of extractions were the main factors that determined lipid yield and lipid class composition in P. glacialis

Protein determination - Method matters

The reported protein content of foods depends on the analytical method used for determination, making a direct comparison between studies difficult. The aim of this study was to examine and compare protein analytical methods. Some of these methods require extraction preceding analysis. The efficacy of protein extraction differs depending on food matrices and thus extraction yield was determined. Overall, most analytical methods overestimated the protein contents. The inaccuracies were linked to indirect measurements, i.e. nitrogen determination and subsequent conversion to protein, or interference from other chemical substances. Amino acid analysis is the only protein analysis method where interfering substances do not affect the results. Although there is potential for improvement in regards to the hydrolysis method, we recommend that this method should be the preferred for food protein determination