Can fish grow on trees? Nutritional and functional properties of yeasts in diets for Atlantic salmon (Salmo salar)

Abstract

Yeasts are gaining attention as alternative ingredients in fish feeds. The nutritional and health potentials of non-saccharomyces yeasts in fish are scarce in literature. Three non-saccharomyces yeasts; Cyberlindnera jadinii (CJ), Blastobotrys adeninivorans (BA) and Wickerhamomyces anomalus (WA) are the focus of this thesis. The objective of the current thesis was to investigate the nutritional values and health effects of the three selected yeasts in the diets of Atlantic salmon (Salmo salar) with focus on growth performance, intestinal health, gut microbiota, and immune responses of fish. The three yeasts were produced in-house using a growth medium containing a blend of enzymatic hydrolysates of pre-treated spruce wood (Picea abies) and chicken by-products. After harvesting, the selected yeasts were processed by direct heat-inactivation with spray-drying (ICJ, IBA and IWA) or autolyzed at 50 ºC for 16 h, followed by spray-drying (ACJ, ABA and AWA). The present thesis comprises of seven papers. Paper I used a desk study approach to review the state-of-the art on the use of yeasts in fish feeds and identified gaps in literature regarding the use of yeasts as aquafeed ingredients. Yeasts are efficient converter of low-value non-food biomass into high-value resources. Yeasts showed comparatively similar amino acids with fishmeal (FM) and soybean meal (SBM), except for methionine, lysine, arginine, and phenylalanine which need to be supplemented when used in fish feeds. Genetic modification and/or nutrient digestibility through exogenous enzyme supplementation and the use of cost-effective down-stream processing (DSP) are possible strategies to increase the nutritive values of yeasts in fish. Additional investment in large-scale production at competitive price is needed for yeasts to be considered as feasible replacement for FM and SBM in fish feeds. Paper II investigated the impacts of yeast species and processing on performance, immune response and gut health of Atlantic salmon fry fed SBM-based diet in freshwater. In a 37-day feeding experiment, the fish were fed one of the nine experimental diets: a FM-based diet, a challenging diet with 40% SBM and six other diets containing 40% SBM and 5% each of ICJ, ACJ, IBA, ABA, IWA and AWA yeast products. An additional control containing 40% SBM and 5% of a reference inactivated C. jadinii (ICU), known for its ability to counteract SBM-induced enteritis (SBMIE) was used in this experiment. C. jadinii and W. anomalus yeasts showed the most promising effects on gut health based on widening of lamina propria and immune response parameters. The AWA was effective in ameliorating SBMIE in fish, while only limited effects were observed for other yeasts products. The ability of yeasts to counteract SBMIE is linked to the activation of immune responses in fish. The results also revealed that the amounts, length, adhesion, and accessibility of cell wall components could be important for the ameliorating effects of yeasts on SBMIE in fish. Paper III assessed the effects of yeasts species and processing on systemic immune response of Atlantic salmon fry fed SBM-based diet in freshwater and demonstrated whether spleen can be used as a target organ to characterize immunomodulatory effects of functional ingredients in fish. The production of yeasts, experiment diets and fish experimental protocol were fully described in Paper II. Four experimental diets (FM, SBM, ICJ, and ACJ) were used in Paper III. The immunomodulatory effects of the diets were analyzed in the spleen of fish after 37 days of feeding, using a transcriptomic evaluation by RNA sequencing and protein expression of specific immunological markers through indirect ELISA. The results showed that SBM induced a down-regulation of pathways associated with ion binding and transport, along with an increase at the protein levels of pro-inflammatory cytokines TNFα and IFNγ. The inclusion of ACJ in the diet was able to control the inflammatory profile caused by SBM through activation of biological pathways related to endocytosis, along with increased protein expression of IL-10 and decreased level of TNFα. The functionality of yeasts in improving gut health of fish is dependent on the yeast species and DSP used after harvesting the yeasts. The results also showed that spleen was a good target organ to characterize the immunomodulatory effects of functional ingredients in Atlantic salmon. Paper IV investigated the effects of yeast species and processing on nutrient digestibility of yeasts in Atlantic salmon. The production and processing of yeasts used in Paper IV were as described in Paper II. Seven experimental diets were used in paper. The control feeds consisted of 100% reference diet (REF) and six other diets comprising of 70% REF diet and 30% each of the yeast products (ICJ, ACJ, IBA, ABA, IWA and AWA). The protein and amino acids of the three yeast species were moderately digested in Atlantic salmon. Autolysis slightly increased protein digestibility of C. jadinii and W. anomalus in Atlantic salmon, but not B. adeninivorans. The results revealed that cell wall porosity as demonstrated by nitrogen solubility had larger impact on nutrient digestibility of yeasts than cell wall thickness. The nutrient digestibility of yeasts in Atlantic salmon is dependent on the yeast species and DSP used after harvesting the yeasts. Based on the results of Papers II, III and IV, a second batch of C. jadinii and W. anomalus yeast were produced to understand the response of Atlantic salmon reared in seawater to dietary yeasts. Therefore, Paper V evaluated the effects of yeasts species and processing on intestinal health and transcriptomic profile from DI and spleen tissue of Atlantic salmon fed SBM-based diet in seawater. The yeasts were produced and processed following the procedure described in Paper II. The ICJ, ACJ, IWA and AWA yeasts products were used in this paper. Six diets were formulated, one based on FM, a challenging diet containing 30% SBM and four other diets containing 30% SBM and 10% each of the yeast products (ICJ, ACJ, IWA and AWA). The inclusion of ICJ and ACJ yeasts reduced the loss of enterocyte supranuclear vacuolization and reduced the population of CD8α positive cells in the lamina propria of fish fed SBM diets. The ICJ and ACJ yeasts controlled the inflammatory profile through upregulation of pathways connected to wound healing and taurine metabolism. The IWA and AWA yeasts controlled the inflammatory profile in fish fed SBM through down-regulation of pathways associated with toll-like receptor signaling, C-lectin receptor and signal transduction. This paper strengthened our earlier observations (Papers II and III) that C. jadinii and W. anomalus are promising novel ingredients with health beneficial effects in terms of controlling distal intestine inflammation associated by feeding plant based diets to Atlantic salmon. Paper VI investigated the effects of yeast species and processing on gut microbiota of fish. The yeast production and processing, experimental diets and fish experimental protocol were as described in Paper V. After 42 days of feeding, six fish from each tank were randomly selected to collect digesta samples from the DI for 16S rRNA sequencing. Water samples (from the source and rearing tanks) and feed samples were also collected for the sequencing analysis. The microbiota of fish fed SBM diet differed from those fed FM diet. The microbiota composition, richness and diversity were similar in fish fed ICJ, IWA and SBM diets. Fish fed ACJ increased relative abundance of Pediococcus, and mucin O-glycan degradation pathway, while fish fed AWA diet increased relative abundance of Bacillaceae compared with fish fed the other diets. Despite the significant modulation of intestinal microbiota of fish fed the autolyzed yeasts (ACJ and AWA), the histological and transcriptomic results revealed that the autolyzed yeasts did not improve gut health of fish beyond the level observed for the inactivated yeasts (ICJ and IWA) (Paper V). These results suggest that the ameliorating effects of yeasts on SBMIE is connected to their ability to stimulate immune responses in Atlantic salmon (Papers II, III and V), rather than through modulation of intestinal microbiota (Paper VI).