Introduction
Yeasts can convert organic side streams into high value protein and have become one of the most promising alternative feed ingredients for aquaculture and specifically salmonid fish
. The range of side streams which may be used for yeast cultivation is broad and different yeast strains thrive in different culture conditions. Saccharomyces cerevisiae and most other terrestrial yeast do not tolerate high salt concentrations, rendering marine side streams unsuitable for cultivation of these yeasts. Candida sake , on the other hand, is a cold water marine yeast adapted to high salt concentrations and thus can utilize processing side streams of marine biomass. Similar to other yeast used as additives in feed, low levels of C. sake have been shown to have immune stimulating effects in the Indian prawn.
The aim of the present study was therefore to cultivate C. sake on herring brine (processing water of the herring industry) and to evaluate C. sake as a nutrient source and immunostimulant in feed for rainbow trout (Oncorhynchus mykiss).
Material and Methods
The cultivation substrate was comprised of 50% pre-salting brine from North Sea herring and 10 % molasses. After freeze drying, a sub-sample of the product was exposed to an additional heat treatment (105°C). The experimental diets were formulated to substitute a fish meal-based control (C) diet with 20% C. sake (Y20), 20% heat-treated C. sake (YE20) or 3% C. sake (Y3). All diets were extruded, and fish were fed twice daily for 10 weeks. Measurements of weight and length were taken monthly. After finalizing the feeding trial, 61 fish from C and Y3 diet, were intraperitoneally injected with either LPS or PBS. Fish were sampled for tissue both at the end of the feeding trial and 12- and 24-hour post injection. The tissue samples included: blood, liver, head kidney, spleen, and intestinal mucosa. Additionally, samples of both proximal and distal intestine were used to assess intestinal nutrient uptake and barrier functions in vitro.
Results
The C. sake product contained 54% protein and 13% fat including 1% DHA and 1.1% EPA. The results of the feeding trial revealed no effect on specific growth rate, feed conversion, condition factor and fat deposition in any of the diets. An increase in lamina propria width was observed in the distal intestine of fish fed the Y3 diet (Fig 1. left). Other parameters of intestinal health including other morphological measures and electrophysiology were unaffected by C. sake inclusion. Heat treating C. sake improved the apparent digestibility coefficient (ADC) of dry matter by 7% and gross energy (GE) by 2.7%, but reduced the overall amino acid ADC by ca. 3%. The ADC of C. sake for crude protein, crude fat, amino acids and GE was generally above 80%. LPS simulation had no effect on mRNA concentrations of selected immune genes, but increased levels of TGFβ in the 3% diet compared to the control fish were observed (Fig 1. right).
Discussion
The present study demonstrated the viability of cultivating C. sake on herring brine. Additional heat treatment of the yeast biomass increased dry matter and gross energy digestibility of the feed, possibly do to the rupture of long, for salmonids indigestible, polysaccharides (Agboola et al., 2021). The amino acids content, and ADC however decreased after heat treatment indicating endothermic breakdown of amino acids. However, the heat treatment of 105°C is rather mild considering extrusion temperatures of generally above 100°C. Thus, other mechanisms causing the lower ADC may be involved. C. sake inclusion of 3% resulted in an increase in lamina propria width indicating immune stimulation and possibly infiltration of lymphocytes (Gisbert et al., 2013). While lamina propria width is a common histopathological indicator of intestinal inflammation (enteritis), no effect was observed in fish fed either diet with 20% yeast inclusion. This suggest that the increased lamina propria width most likely is not due to enteritis but rather a stimulation of the gut associated immune tissue. The 3% C. sake inclusion also increased transcription of TGFβ in the proximal intestine further suggesting an immune stimulating effect. This effect was LPS independent, and no other immune genes were affected.
Conclusion
The production of Candida sake using herring brine was successful and yielded a product containing over 50% protein and 3% omega-3 fatty acids. C. sake inclusion in feeds for rainbow trout did not affect growth and feed utilization. Due its nutrient profile and the sustainable production using industry process water, C. sake has great potential as an aquafeed ingredient. Downstream processing techniques increase the bioavailability of complex carbohydrate but alternative techniques, such as autolysis, may be more suitable than heat. Three percent yeast inclusion may alter gene expression and intestinal morphology. However, additional research is needed to understand the implications of these mild effects on innate immune response and disease resistance in rainbow trout.
References
Agboola, J.O., Øverland, M., Skrede, A., Hansen, J.Ø., 2021. Yeast as major protein-rich ingredient in aquafeeds: a review of the implications for aquaculture production. Rev. Aquac. 13, 949–970. https://doi.org/10.1111/raq.12507
Gisbert, E., Castillo, M., Skalli, A., Andree, K.B., Badiola, I., 2013. Bacillus cereus var. toyoi promotes growth, affects the histological organization and microbiota of the intestinal mucosa in rainbow trout fingerlings. J. Anim. Sci. 91, 2766–2774. https://doi.org/10.2527/jas2012-5414