Nannochloropsis oceanica, a novel natural source of rumen-protected eicosapentaenoic acid (EPA) for ruminants

Sci Rep. 2018; 8: 10269. Published online 2018 Jul 6. doi: 10.1038/s41598-018-28576-7 PMCID: PMC6035222 PMID: 29980726 Susana P. Alves,corresponding author1 Sofia H. Mendonça,2 Joana L. Silva,2 and Rui J. B. Bessa1 1CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Av. da Universidade Técnica, 1300-477 Lisboa, Portugal 2ALLMICROALGAE, Av. Eng. Duarte Pacheco 19, 9° piso, 1070-100 Lisboa, Portugal Susana P. Alves, Email: tp.aobsilu.vmf@sevlaanasus. corresponding authorCorresponding author. Author information ▼ Article notes ► Copyright and License information ► Disclaimer Go to: Abstract We hypothesize that whole microalga biomass is a natural rumen-protected source of eicosapentaenoic acid (EPA, 20:5n-3) for ruminants. To test our hypothesis, we studied the ruminal biohydrogenation of EPA from two microalgae, Nannochloropsis oceanica and Phaeodactylum tricornutum using in vitro incubations with rumen fluid. A total mixed ration was incubated with: no EPA (control), EPA as free-fatty acid, N. oceanica spray-dried (SD), N. oceanica freeze-dried (FD), or P. tricornutum FD. The kinetics of EPA disappearance and of products formed during the 24 hours of incubation were evaluated, and complemented by deuterated-EPA incubation. Results showed that EPA metabolism from the N. oceanica was remarkably reduced compared with the P. tricornutum and free-EPA, and this reduction was even more effective with the N. oceanica FD. Our data also indicates that neither feed dry matter disappearance nor rumen microbial markers (branched-chain fatty acids and dimethyl acetals) were affected by EPA-sources. We reported for the first time the kinetics of EPA biohydrogenation class products and the unequivocal formation of 20:0 from EPA. Overall, N. oceanica shows a strong potential to be used as a natural dietary source of EPA to ruminants, nevertheless further studies are needed to verify its protection in vivo. Go to: Introduction Public health nutrition guidelines recommend population-wide decreased saturated fatty acid (FA) and trans FA and increased polyunsaturated fatty acid (PUFA) consumption to lower the incidence of clinical metabolic diseases1,2. Indeed, the n-3 long-chain PUFA (n-3 LC-PUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and the docosahexaenoic acid (DHA, 22:6 n-3) have been recommended due to their beneficial effects for humans, which includes anti-atherogenic, anti-thrombotic and anti-inflammatory properties3. Meat from land animals can be a source of n-3 LC-PUFA, particularly for populations where the consumption of fish and seafood is very low4. Nevertheless, in ruminants the content of n-3 LC-PUFA in meat is low (about 2 to 40 mg/100 g meat)5. Thus, there is a need to develop strategies to increase n-3 LC-PUFA in meat and milk5,6. The most efficacious approach is to feed animals with dietary n-3 LC-PUFA, like fish oil or more recently microalgae6. These strategies might have high success with monogastrics, however in ruminants most of the n-3 LC-PUFA included in the diet are hydrogenated in the rumen, producing saturated FA and a range of other FA intermediates5,7. This is due to the microbial processes that occur in the rumen, i.e. lipolysis of dietary esterified lipids followed by biohydrogenation of the released PUFA. The microbial biohydrogenation is the process responsible for the isomerization and hydrogenation of the FA unsaturated double bonds resulting in extensive metabolism of PUFA into saturated end-products limiting the escape of PUFA from the rumen. For that reason and considering the importance of consumption of ruminant derived products, several companies and research groups are trying to develop methods to protect n-3 LC-PUFA from the ruminal microbial population to increase the amount of PUFA available for deposition, elongation and desaturation in muscle and adipose tissue8,9. Lipid rumen-protection approaches such as calcium salts, fatty acyl amides, aldehyde treatment, non-enzymatic browning, lipid composite gels, as well as novel interfacial cross-linking emulsions have been researched to resist to the ruminal microbial attack as was recently reviewed8. These approaches involve blocking the free FA carboxyl-end by forming calcium salts or fatty acyl amides, or involve encapsulation the lipid or FA inside a microbial-resistant shell. However, none of these methods were successfully applied in practice, either due to high cost, use of harmful products (e.g. formaldehyde encapsulation), or lack of consistency regarding rumen protection efficiency8. Thus, efficient rumen lipid bypass approaches are still needed. There has been a growing interested in microalgae as a natural resource for biofuel, food and nutraceutical applications, including animal feed due to the nutritional and energetic value of microalgae constituents, particular n-3 LC-PUFA10,11. Microalgae are unicellular organisms in which the plasma membrane is protected by a complex cell wall that might display great diversity among species, strain and growing conditions. However, the most common constituents of microalgae cell walls are polysaccharides, including cellulose, lipids and proteins although some microalgae are also protected by an inorganic rigid wall composed of silica frustule of diatoms or calcium carbonate12. Mechanical, chemical and biological methods have been studied for cell wall disruption in order to extract the valuable cell components such as lipid and pigments12. From research of methods based on microbial enzymatic cell wall lysis, it became evident that several microalgae cell walls are somehow resistant to microbial attack13. Thus, we hypothesize that microalgae cell walls could partially resist ruminal microbial attack and protect PUFA from biohydrogenation. Although inclusion of fish oil or marine algae oils in ruminant diets have been shown to result in marginal PUFA enrichment in ruminant derived foods14, there are limited studies on the use of PUFA-rich microalgae biomass in ruminant diets. Therefore, this work aims to study if EPA-rich microalgae biomass has potential to be used as a natural source of rumen-protected n-3 LC-PUFA for ruminants. To this end, we evaluated in vitro the ruminal metabolism of EPA from two microalgae sources, the Nannochloropsis oceanica and the Phaeodactylum tricornutum, and compared that with the metabolism of free-EPA. However, because microalgae dehydration can cause damage to cells walls, the N. oceanica biomass was dehydrated by centrifugal spray-drying (SD) or freeze-drying (FD) processes. The effect of microalgae biomass on the ruminal biohydrogenation of C18 FA was also evaluated. In addition, the pattern of EPA biohydrogenation products formed from both free-EPA and microalgae were accessed and to confirm the origin of those products we also incubated EPA labeled with deuterium atoms. Go to: Results Influence of EPA-source on the ruminal metabolism and EPA disappearance Dry matter (DM) disappearance in tubes containing about 0.05 mg of EPA per mL of incubation medium added in the form of free-FA or microalgae biomass did not differ (P > 0.05) from control tubes (without EPA addition) during 0 to 24 hours of incubation (Fig. 1). However, considering the EPA-source we verified that EPA was metabolized at different rates in rumen fluid (Fig. 2). At 2 hours of incubation, the disappearance of EPA in tubes with the addition of free-EPA was already 51% while in tubes with P. tricornutum FD, N. oceanica SD and FD the EPA disappearance was only 29, 18 and 25%, respectively. At 24 hours of incubation the highest disappearance of EPA was observed in tubes with the addition of free-EPA or P. tricornutum, reaching 88 and 83%, respectively. The lowest disappearance was observed in tubes containing N. oceanica FD, reaching only 44% of EPA disappearance at 24 hours of incubation and the N. oceanica SD showed intermediate values, i.e. 69% of EPA disappearance.