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Evaluation of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae yeasts as protein sources in diets for Atlantic salmon (Salmo salar)
Aquaculture 402–403 (2013) 1–7
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Evaluation of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae yeasts as protein sources in diets for Atlantic salmon (Salmo salar) Margareth Øverland ⁎, Anders Karlsson, Liv Torunn Mydland, Odd Helge Romarheim, Anders Skrede Aquaculture Protein Centre, CoE, Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, NO-1432 Ås, Norway
a r t i c l e
i n f o
Article history: Received 10 January 2013 Received in revised form 14 March 2013 Accepted 19 March 2013 Available online 26 March 2013 Keywords: Atlantic salmon Candida utilis Kluyveromyces marxianus Saccharomyces cerevisiae Single cell protein Nutrient utilization
a b s t r a c t The use of the yeasts Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae as protein sources in diets for Atlantic salmon (Salmo salar) pre-smolts was investigated. An 89 day study was conducted using triplicate groups of Atlantic salmon with 28 g initial weight kept in fresh water. The dietary treatments consisted of a control diet based on high-quality fish meal (FM diet) and three experimental diets with C. utilis (CU diet), K. marxianus (KM diet) or S. cerevisiae (SC diet) yeast, substituting 40% of the crude protein (CP) from FM. There was no difference in final weight or specific growth rate (SGR) of fish fed the CU diet and the FM diet. The SC diet resulted in lower final weight and SGR, and higher average daily feed intake (ADFI) and feed conversion ratio (FCR) than the FM diet, and higher ADFI and FCR than the KM and CU diets. Fish fed the SC diet had decreased retention of nitrogen and energy, while fish fed the CU diet had increased nitrogen retention compared with those fed the FM diet. In general, feeding the SC diets gave lower digestibility of CP, amino acids, and energy compared with the FM, KM and CU diets, whereas there was no difference in nutrient or amino acid digestibility between the KM and CU diet and the FM diet. Feeding KM and SC gave higher total gut weight and higher distal intestinal weight compared with the FM diet. Plasma cholesterol level was lower for the KM and SC diets while plasma glutathione peroxidase levels were higher for the KM diet compared with the FM diet. In conclusion, feeding moderate levels of S. cerevisiae reduced growth performance and nutrient utilization, while C. utilis and K. marxianus were shown to be promising protein sources in diets for Atlantic salmon, capable of replacing 40% of the protein from high-quality fishmeal without adversely affecting growth performance, digestibility or nutrient retention. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Atlantic salmon (Salmo salar) have traditionally been fed a diet based on marine ingredients such as fish meal (FM). However, marine feed ingredients are limited resources as the world's fish stocks are already either fully exploited or depleted (Tacon and Metian, 2008). Moreover, the aquaculture industry is undergoing rapid growth (FAO, 2012), and as a consequence there is increased demand for aquafeeds. Securing ingredient supply by increased use of new sustainable feed ingredients is therefore crucial. Microbial products like yeast represent potential sustainable ingredients in aquafeeds due to their ability to convert low-value biomass from forestry and agricultural industry into high-value feed ingredients and with limited dependence on agricultural land, water, and changing climatic conditions. Yeast, depending on the strains and species, can thus be produced
⁎ Corresponding author. Tel.: +47 64965206; fax: +47 64965101. E-mail address: [email protected] (M. Øverland). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.03.016
from fermentation of sugar-rich feed stock such as sugar cane or from lignocellulosic biomass from forestry or agriculture industry. Recent advances in fermentation technology have resulted in more efficient and less costly yeast production, making these ingredients more feasible as nutrient sources for use in aquafeeds (Kim et al., 1998; Omar et al., 2012). Whole cell yeast or products containing different yeast cell wall fractions (β-glucan and mannan oligosaccharide) and nucleic acids are commonly used as immunostimulants in fish diets (Gatesoupe, 2007; Gopalakannan and Arul, 2010; Li and Gatlin, 2006; Oliva-Teles and Goncalves, 2001; Refstie et al., 2010). Low levels (1–4%) of inactive brewer's yeast have been shown to improve growth performance and modulate intestinal microbiota in different fish species (Abdel-Tawwab et al., 2008, 2010; Hoseinifar et al., 2011; Li and Gatlin, 2003). Yeasts are also used as live feed in aquaculture (Abdel-Tawwab et al., 2008; Brown et al., 1996). Less interest has been devoted to the potential of dried yeast as a major protein source in fish diets. Thus, there is a scarcity of literature on alternative yeasts as a replacement for fish meal in salmon diets. As an easily available source, brewer's yeast (Saccharomyces cerevisiae) have been applied in a number of studies with different fish
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species, whereas there are few reports on Candida utilis, and to the author's knowledge no studies on Kluyveromyces marxianus (formerly K. fragilis) as a protein source for fish have been published. The objective of the present experiment was to investigate the effect of moderate levels of C. utilis, K. marxianus and S. cerevisiae yeasts as a partial replacement of FM in diets for Atlantic salmon on growth performance, nutrient digestibility, retention of nitrogen and energy, organ weights and blood chemistry. 2. Materials and methods 2.1. Fish and facilities The Atlantic salmon (S. salar) pre-smolts used in this experiment were acquired as eyed eggs from Aqua Gen AS in the spring of 2011. The fish were fed a commercial salmon feed prior to the start of the 89-day growth experiment at the fish nutrition laboratory at the Norwegian University of Life Sciences, Ås, Norway. After a 48 h fasting period, a total of 720 salmon (average start weight of 27.7 g) were randomly distributed into 12 fiberglass tanks with 60 fish per tank, each with a 300-l capacity. The fish were kept under continuous light in recirculated fresh water with a water supply of 6–7 l min −1 and with temperatures ranging from 8.8 to 14.5 °C. Dissolved oxygen levels were measured daily and kept above 6.0 mg l −1 in the outlet water. 2.2. Diets and feeding The dietary treatments consisted of a control diet based on highquality FM (FM diet) and three experimental diets containing either C. utilis, K. marxianus or S. cerevisiae yeast, all spray dried and inactivated, replacing 40% of the CP from the FM. The diets were formulated to be isonitrogenous and isolipidic based on the analyzed chemical content of the ingredients. All diets contained 0.1 g kg−1 yttrium oxide (Y2O3) as an indigestible marker for determination of nutrient digestibility (Austreng et al., 2000). The chemical composition of FM, C. utilis, K. marxianus and S. cerevisiae is given in Table 1, while the formulation and composition of the diets are given in Table 2. The diets were processed at the feed laboratory of the Norwegian University of Life Sciences, Ås, Norway. All dry ingredients, excluding gelatin, were mixed in a Moretti Foreni kneading machine (Spiry 25, Mondolfo, Italy). The fish oil was then added to the dry ingredients and mixed thoroughly. The gelatin was dissolved in hot water (70–80 °C) and mixed with the mash, resulting in a firm dough that was pelleted using an Italgi pasta extruder (P35A, Carasco, Italy) equipped with a 3 mm die. The pellets were then cooled to room temperature and later dried at 45–60 °C to about 95% dry matter (DM) and stored at 0–4 °C in zip-lock bags until feeding. The four experimental diets were fed to triplicate groups of fish over a period of 5 h (07:00 to 12:00) daily by automatic belt feeders. Fish were fed 20% in excess, based on recorded feed intake over the previous 3-day period. Uneaten feed was collected from feed collectors in the tank outlets directly after each feeding period. Feed intake was adjusted for uneaten feed and calculated as: Feed fed DM (g) − (uneaten feed DM (g) / recovery). Recovery was estimated according to Helland et al. (1996). 2.3. Sampling Before the start of the experiment, 10 fish from the holding tank were euthanized with an overdose of MS-222 (Metacaine, Norsk Medisinaldepot AS, Norway), and stored at − 20 °C for whole body analysis. Fish were anesthetized with MS-222 and batched weighted at the start of the experiment (Day 0) and individually weighted at the end (Day 89) of the experiment. At the termination of the experiment, eight fish were randomly sampled from each tank (24 fish per diet), anesthetized, and blood was collected from the caudal vessel
Table 1 Chemical composition of fish meal (FM), Candida utilis (CU), Kluyveromyces marxianus (KM) and Saccharomyces cerevisiae (SC) used in the dietsa (g kg−1).
Dry matter Crude protein Nucleic acids Crude lipid Starch Ash Ca P Se, mg kg−1 Gross energy, MJ kg−1
FMb
CUc
KMc
SCc
918 735 13 114 10 139 26 20 2.7 22.3
937 560 93 3 37 54 2.7 15 b0.04 21.4
939 511 102 8 8 76 0.5 15 b0.04 20.5
968 460 58 2 11 64 0.7 11 0.12 19.6
4.67 1.96 4.22 6.94 6.95 1.36 4.16 4.77 4.51 1.08
4.20 1.71 3.90 6.05 6.51 1.54 3.71 4.47 4.04 0.98
4.07 1.82 3.41 5.44 6.19 1.23 3.29 4.23 3.96 1.08
5.22 9.29 3.52 10.99 0.82 3.59 3.28 4.76
5.92 8.55 3.260 11.90 0.91 3.15 2.90 4.67
4.71 8.87 2.98 13.09 1.02 2.95 2.85 4.26
Essential amino acids, g 16 g N−1,d Arginine 5.23 Histidine 1.97 Isoleucine 3.35 Leucine 6.25 Lysine 6.79 Methionine 2.50 Phenylalanine 3.26 Threonine 3.97 Valine 3.93 Tryptophan 0.84 Non-essential amino acids, g 16 g N−1,d Alanine 5.00 Aspartic acid 8.24 Glycine 4.40 Glutamic acid 11.92 Cysteine 0.84 Tyrosine 2.64 Proline 3.26 Serine 3.95
a Proximate analysis and starch are means of duplicate analyses, minerals are means of triplicate analysis, while nucleic acids are means of duplicate analysis of the fish meal and triplicate analysis of the yeasts. b NorsECO-LT, Egersund Sildoljefabrikk AS, Egersund, Norway. c Borregaard ASA, Sarpsborg, Norway. d Water corrected amino acids.
with heparinized syringes. The blood was divided into three aliquots before further processing and analysis of blood chemistry, glutathione peroxidase (GPx) and plasma cholesterol. One aliquot of whole blood was immediately analyzed for standard blood chemistry on a portable blood analyzer (i-Stat, Medinor ASA, Norway) and the results were later temperature corrected following the procedures specified by the i-Stat manufacturer. One aliquot of whole blood for GPx analysis was immediately transferred to Eppendorf tubes, frozen on dry ice and kept at − 80 °C until analysis. The remaining blood samples were centrifuged at 2000 ×g for 3 min and the plasma was kept frozen at − 20 °C until analysis of cholesterol. Another 10 fish from each tank were randomly selected, anesthetized with MS-222 and euthanized with a blow to the head, dissected and eviscerated. Surface fat and connective tissue were carefully removed before weighing of the gastrointestinal tract (from the esophageal sphincter to the anus) and the individual sections: stomach (ST), mid-intestine (MI, from distal side of the pyloric sphincter to distal intestine), and distal intestine (DI). The liver was also removed from the same fish, weighted and used for calculation of liver index. The fish and all organs were then placed in a plastic zip lock bag and frozen at −20 °C for whole body analysis. For whole body analysis, all fish from each tank were ground twice through a meat grinder (first with 8 and then 5 mm die holes) and thoroughly mixed before freeze drying and analysis. Feces were stripped in two rounds according to the method described by Austreng (1978). Feces from all fish in each tank were pooled and immediately frozen at − 20 °C and freeze dried prior to analysis.
M. Øverland et al. / Aquaculture 402–403 (2013) 1–7 Table 2 Formulation and chemical composition (as is) of diets. FM diet
CU diet
KM diet
SC diet
Formulation, g kg−1 Fish meal Candida utilis Kluyveromyces marxianus Saccharomyces cerevisiae Gelatinized potato starcha Fish oilb Gelatinc Cellulosed Premixe Y2O3f
579 0 0 0 126 137 80 67 9.9 0.1
347 283 0 0 118 162 80 0 9.9 0.1
347 0 302 0 100 161 80 0 9.9 0.1
347 0 0 345 56 161 80 0 9.9 0.1
Analyzed content, kg−1 Dry matter, g Crude protein, g Total amino acids, g Crude lipid, g Starch, g Ash, g Gross energy, MJ
968 491 382 195 126 79 22.6
943 470 374 192 124 63 22.5
946 466 346 201 105 74 22.5
955 475 379 202 71 73 22.6
Essential amino acids, g kg−1 Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Tryptophan
2.61 0.88 1.48 2.76 3.05 1.07 1.45 1.72 1.83 0.86
2.25 0.76 1.43 2.46 2.73 0.85 1.37 1.62 1.69 0.86
2.46 0.87 1.53 2.64 2.98 0.87 1.48 1.78 1.87 0.88
2.46 0.87 1.58 2.73 2.97 0.86 1.53 1.79 1.88 0.87
Non-essential amino acids, g kg−1 Alanine Aspartic acid Glycine Glutamic acid Cysteine Tyrosine Proline Serine
2.55 3.68 3.15 5.53 0.33 1.11 2.20 1.81
2.44 3.36 2.77 5.00 0.30 1.06 1.97 1.73
2.54 3.76 2.97 5.73 0.35 1.18 2.15 1.87
2.51 3.61 2.99 5.17 0.31 1.20 2.17 1.86
a
Lygel F 60, Lyckeby Culinar AB, Fjälkinge, Sweden. b Skretting Norway, Stavanger, Norway. c Rousselot™ 250 PS, Rousselot SAS, Courbevoie Cedex, France. d Cellulose powder, AWL Kjemi AS, Kolbotn, Norway. e Provided the following amount per kg of diet: Ca 1.07 g, Mn2SO4 14.7 mg, ZnSO4 117 mg, CuSO4 4.90 mg, CoSO4 980 μg, Ca(IO3)2 2.94 mg, retinol 2450 IU, cholecalciferol 1470 IU, tocopherol 196 mg, menadione 9.80 mg, thiamine 14.7 mg, riboflavin 24.5 mg, pyridoxine 14.7 mg, cobalamine 19.6 μg, pantothenic acid 29.4 mg, folic acid 4.90 mg, niacin 73.5 mg, biotin 245 μg, vitamin C 1.75 (Rovimix® Stay-C® 35, DSM Nutritional Products, Basel, Switzerland) ascorbic acid 123 mg, AS Norsk Mineralnæring, Hønefoss, Norway. f Di-yttrium tri-oxide (Y2O3), Rare Earth Metal Limited, Shenzhen, Guangdong, China.
3
Ultimate 3000 system fitted with an YMC-Pack ODS-A column) after perchloric acid hydrolysis. The total nucleic acid content of the ingredients was calculated assuming that the molar fraction of each nucleotide is equal to that of its respective nucleobase. Gross energy in diets, feces and fish was measured by bomb calorimetry (Parr 1271 Bomb calorimeter, Parr, Moline, Il, USA). Fish were also analyzed for CP using Kjeldahl nitrogen (Commission dir. 93/28/EEC) × 6.25. For digestibility calculations, DM in feces was determined by freeze drying, and diets and feces were analyzed for CP using the Dumas method (Kirsten and Hesselius, 1983) on an EA 1108 element analyzer (Fison, Waltham, USA) and yttrium by ICP-AES analysis (NS-EN ISO 11885). Whole blood GPx was analyzed according to the method described by Paglia and Valentine (1967). Plasma cholesterol was analyzed using a Maxmat PL multianalyzer and a commercially available kit (Cholesterol CP A11A01634, Bergman Diagnostica AS, Norway). 2.5. Calculations and statistical analysis Fish growth was calculated as specific growth rate (SGR) according to the following equation: SGR ¼ 100 ðlnðfinal body weight ðBWÞÞ−lnðinitial BWÞ=ΔtÞ; where Δt is the number of experimental days: Feed conversion ratio (FCR) was calculated as: FCR = Feed DM ingested (g) × fish weight gain (g)−1. Daily feed intake is expressed as % of the current BW: Current BW (BWn) = BWn − 1 + (current daily DM feed intake / FCR). Apparent digestibility was calculated as: 100 ð1−ðYd Yf−1 Nf Nd−1ÞÞ; where Yd and Yf represent the concentration of yttrium in the diet and feces, and Nd and Nf represent the concentration of individual nutrients or energy in the diet and feces, respectively. Nitrogen and energy retentions were calculated as: 1) Of ingested = nutrient retained in the body/nutrient ingested × 100. 2) Of digested = nutrient retained in the body / (nutrient ingested × digestibility) × 100. The data were analyzed in Statistical Analysis Software (SAS) v. 9.2, using a general linear model (GLM procedure) with diet as fixed effect. The tank mean for each time point was used as a single observation in the statistical analysis. Effect of diet was tested on all parameters using an F-test. Comparisons of diet means were performed using a t-test with Tukey–Kramer correction for multiple comparisons. Pvalues b 0.05 were considered statistically significant. Unless stated otherwise, all numbers are presented as diet means ± standard error of the mean.
2.4. Chemical analysis
3. Results
Ingredients, diets and feces were ground with a pestle and mortar prior to analysis. Diets, ingredients and fish were analyzed for DM by drying to constant weight at 104 °C (Commission dir. 71/393/EEC). Diets, ingredients and feces were analyzed for CP using Kjeldahl nitrogen (Commission dir. 93/28/EEC) × 6.25, crude lipid (CL) by HCl hydrolysis followed by diethyl ether extraction (Commission dir. 98/64/EC), ash (Commission dir. 71/250/EEC), amino acids (Commission dir. 98/64/EC) and tryptophan (Commission dir. 2000/45/EC). Tryptophan in feces was not analyzed due to limited material available. Starch in diets and ingredient was analyzed using the AOAC enzymatic method 996.11. Nucleic acids in the ingredients were analyzed according to methods described by Mydland et al. (2008). In brief, the individual nucleobases were analyzed by HPLC (Dionex
The daily relative feed intake of fish during the experimental period is given in Fig. 1. The feed intake increased gradually during the first two weeks of feeding, followed by a decrease in parallel with decreasing water temperature until the end of the experiment. Growth performance and fecal dry matter are given in Table 3. There were no differences among the diets for initial weights. By comparing fish fed the CU diet with those fed the FM diet there was no difference in final weight or SGR. Salmon fed the FM diet had higher final weights than those fed the KM and SC diets and higher SGR than those fed the SC diet. The fish fed the SC diet also had higher average daily feed intake (ADFI) and FCR than those fed the control and the KM and CU diets. The FM diet and the SC diet gave higher fecal DM than the KM and CU diets.
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M. Øverland et al. / Aquaculture 402–403 (2013) 1–7
15
FM 2.0 %
CU
14
KM
13
Temperature 1.5 %
12
1.0 %
11
Water temperature (°C)
Feed intake (% of body weight)
SC
10 0.5 % 9
0.0 %
8 0
7
14
21
28
35
42
49
56
63
70
77
84
Experiment duration (day) Fig. 1. Feed intake of Atlantic salmon pre-smolts fed a fish meal control diet (FM diet) or one of three diets containing spray-dried yeast as fish meal substitution (40% of CP) in an 89-day growth trial. The CU diet contained Candida utilis, the KM diet contained Kluyveromyces marxianus and the SC diet contained Saccharomyces cerevisiae yeast. Each data point is the diet mean of three tanks for a single day. The gray line is water temperature recorded daily from the inlet water source.
The apparent digestibility of CP and energy is shown in Table 4. There was no difference in digestibility parameters between salmon fed the CU diet and those fed the FM diet. The SC diet gave a lower digestibility of CP and energy compared with the FM diet and the KM and CU diets. There were significant effects of adding yeast to diets on the retention of nitrogen (Table 4). Feeding SC decreased nitrogen and energy retention as a percentage of intake compared with the FM diet. Nitrogen retention of fish fed the KM diet was unaffected, while the CU diet caused an increase in nitrogen retention as a percentage of ingested and digested nitrogen compared with the FM control. The digestibility of individual amino acids was significantly lower for the SC diet compared with all other diets, except for methionine, aspartic acid and cysteine (Table 5). Compared with the FM diet, the KM diet had a lower digestibility of cysteine, while the KM and SC diets gave a lower digestibility of methionine and the SC diet had a lower digestibility of aspartic acid. There was no difference in digestibility of total amino acids among fish fed the FM, CU and KM diets. The relative weights of liver and gastrointestinal sections, and blood metabolites, GPx and plasma cholesterol of fish fed the experimental diets are given in Table 6. Feeding KM and SC gave higher total gut weight, while liver weight was higher in the fish fed the CU and KM diets compared with those fed the FM control diet. The KM and SC diets gave a higher stomach weight than the CU diet, but not compared
with those fed the FM control. Fish fed the SC diet had a higher MI weight compared with the FM control and the other yeast-containing diets, while fish fed the SC and KM diets had higher DI weights than fish fed the FM diet. The blood ions, pH and partial pressure of CO2 were not significantly affected by the dietary treatments. Blood glucose level was lower for the SC diet than for the CU diet, but did not differ from the FM control, while plasma cholesterol level was lower for the KM and SC diets compared with the FM control. Also, fish fed the KM diet had higher blood GPx levels than those fed the FM diet, while feeding CU and the SC did not affect blood GPx levels of the fish compared with the FM diet. 4. Discussion The recent development toward a supply shortage of FM for use in diets for salmonid fish has provoked an increasing search for sustainable protein substitutes. The present study revealed that C. utilis and K. marxianus could be fed at about 30% of the diet to Atlantic salmon, replacing FM protein, without significant negative effects on feed intake, SGR or FCR, whereas feeding of S. cerevisiae had negative effects on these parameters. The high feed intake and poor FCR of fish fed the SC diet may be interpreted as an indirect effect of lower digestibility of CP, amino acids, and energy. Presumably, the high feed
Table 3 Growth performance and fecal dry matter in salmon fed the experimental diets. FM diet Start weight, g1 Final weight, g Specific growth rate, % Daily feed intake, % of BW Feed conversion ratio Fecal dry matter, % 1
27.8 92.4 1.36 0.95 0.70 16.0
± ± ± ± ± ±
CU diet 0.2 2.8a 0.03a 0.02a 0.01a 0.6a
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
27.7 88.6 1.32 0.91 0.70 12.5
± ± ± ± ± ±
0.0 1.1a,b 0.01a,b 0.01a 0.01a 0.3b
KM diet
SC diet
27.7 81.3 1.22 0.89 0.73 12.3
27.6 78.8 1.19 1.07 0.90 18.3
± ± ± ± ± ±
0.0 3.1b 0.04a,b 0.03a 0.00a 0.4b
± ± ± ± ± ±
P-value 0.1 2.1b 0.03b 0.03b 0.01b 1.0a
0.38 0.01 0.01 0.002 b0.001 0.001
M. Øverland et al. / Aquaculture 402–403 (2013) 1–7 Table 4 Apparent digestibility (%) of CP and energy and retention of nitrogen and energy in salmon fed experimental diets1.
Digestibility Crude protein Energy
FM diet
CU diet
88.4 ± 0.7a 85.5 ± 0.8a
88.0 ± 0.5a 86.1 ± 0.7 84.3 ± 0.4a 82.2 ± 1.4
Nutrient retention Nitrogen % of ingested 52.9 % of digested 59.9 Energy % of ingested 53.7 % of digested 62.8 1
± 0.6a 57.2 ± 0.4c ± 0.6ab 65.0 ± 0.4c
KM diet
SC diet a a
53.2 ± 0.3a 61.8 ± 0.3b
P-value
72.6 ± 2.3 67.8 ± 2.5
b b
b0.001 b0.001
42.9 ± 0.5b 59.1 ± 0.7a
b0.001 0.001
± 0.9a 55.9 ± 0.4a 52.2 ± 0.5a 40.4 ± 1.4b ± 1.1ab 66.3 ± 0.5b 63.5 ± 0.6ab 59.6 ± 2.1a
b0.001 0.03
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
intake could not compensate for the reduced digestibility as the SGR was lower than in fish fed the FM diet, and retention of digested nitrogen and energy was lower in fish fed the SC diet than for any of the other diets. The proximal composition of yeast is dependent on strain, growth media and growing conditions; the CP content varies on average between 40 and 55% of the cell dry matter, including a nucleic acid content of 6–12% (Halasz and Lasztity, 1991). The dried yeasts used in the present study were all commercially available products, and contained 56.0% (C. utilis), 51.1% (K. marxianus) and 46.0% (S. cerevisiae) CP, respectively. As opposed to S. cerevisiae, C. utilis and K. marxianus have a broad substrate spectrum and are able to utilize pentoses such as xylose, the second most abundant carbohydrate in lignocellulosic biomass (Nasseri et al., 2011; Rodrussamee et al., 2011). Furthermore, both C. utilis and K. marxianus have obtained the generally-regarded-as-safe (GRAS) status. The diets used were about isonitrogenous on a DM basis, but amino acid composition was slightly different among diets. It is well known that yeast contains lower levels of sulfur-containing amino acids than FM (Kuhad et al., 1997), and all yeasts used in our study contained less methionine than the FM. Accordingly, the yeast-containing diets had slightly lower levels of methionine than the FM diet, and the CU diet also had lower levels of threonine and valine. Comparing with data from Kaushik and Seiliez (2010), it is possible that especially the poorly digestible SC diet may have been suboptimal in digestible methionine. Thus, the possibility that this may have Table 5 Apparent digestibility (%) of amino acids in salmon fed experimental diets1. FM diet Essential amino Arginine Isoleucine Histidine Leucine Lysine Methionine Threonine Phenylalanine Tyrosine Valine
1
acids 89.4 84.8 80.0 88.0 87.0 86.4 81.2 83.0 83.9 84.5
CU diet
KM diet
SC diet
P-value
0.2a 0.4a 0.7a 0.3a 0.4a 0.4a 0.7a 0.3a 0.4a 0.5a
89.5 82.6 79.6 85.6 85.9 82.9 77.8 81.4 82.8 82.8
± ± ± ± ± ± ± ± ± ±
0.4a 0.3a 0.4a 0.3a 0.3a 0.6ab 0.6a 0.3a 0.4a 0.3a
86.8 77.7 74.8 81.3 84.0 79.5 72.4 75.3 77.8 78.5
± ± ± ± ± ± ± ± ± ±
0.3a 1.0a 1.4a 0.9a 1.0a 1.1bc 1.8a 1.2a 1.3a 1.0a
75.3 61.2 61.3 66.8 68.4 73.9 57.7 61.5 58.8 62.2
± ± ± ± ± ± ± ± ± ±
2.7b 4.2b 3.6b 3.6b 3.6b 2.4c 3.7b 3.5b 4.0b 3.9b
b0.001 b0.001 b0.001 b0.001 b0.001 0.001 b0.001 b0.001 b0.001 b0.001
Non-essential amino acids Alanine 87.4 ± 0.4a Aspartic acid 70.6 ± 1.1a Glutamic acid 87.4 ± 0.5a Glycine 81.6 ± 0.8 Proline 84.1 ± 0.9a Serine 80.2 ± 0.6a Cysteine 57.7 ± 1.2a Total AA 83.5 ± 0.6a
86.6 70.7 86.2 81.7 84.4 79.1 51.9 82.4
± ± ± ± ± ± ± ±
0.3a 0.8a 0.2a 0.5 0.4a 0.4a 0.9ab 0.3a
84.1 69.3 84.3 79.7 81.3 75.1 44.5 79.4
± ± ± ± ± ± ± ±
0.8a 2.6ab 0.9a 1.4 1.4a 1.3a 3.9b 1.3a
75.0 59.5 75.6 77.4 74.6 61.5 51.0 68.8
± ± ± ± ± ± ± ±
2.6b 3.3b 2.5b 2.0 2.4b 3.3b 2.6ab 3.0b
b0.001 0.02 0.001 0.14 0.005 b0.001 0.03 b0.001
± ± ± ± ± ± ± ± ± ±
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
5
contributed to poor growth performance, in spite of high feed intake, cannot be excluded. In studies with sea bass (Dicentrarchus labrax) it was concluded that brewer's yeast can replace 50% of FM protein with no negative effects on fish performance (Oliva-Teles and Goncalves, 2001). In their study, inclusion of up to about 30% brewer's yeast had no effects on protein digestibility, and feed conversion and nitrogen retention were improved compared with the FM diet. Rumsey et al. (1991a) reported that feeding 25% brewer's yeast, replacing casein protein, improved growth and feed conversion in rainbow trout (Oncorhynchus mykiss), whereas diets with 50% of brewer's yeast appeared to be unpalatable. Working with pacu (Piaractus mesopotamicus, Holmberg), Ozòrio et al. (2010) observed that replacing 50% of the FM with brewer's yeast had no effect on protein digestibility, and growth performance and feed conversion were improved. Similar results were obtained by feeding 20% inactive baker's yeast (S. cerevisiae) as a FM substitute to Gilthead sea bream (Sparus aurata) (Salnur et al., 2009). A recent study by Ozòrio et al. (2012) showed that feeding 15% brewer's yeast to Nile tilapia (Oreochromis niloticus) promoted growth and efficient nutrient utilization, while higher levels gave a negative dose dependent effect on growth performance and nutrient utilization. Although our study was done with a different species of fish, it may be speculated that the divergent results with S. cerevisiae may partially be due to different characteristics of the brewer's yeast used in the experiments. It has been suggested that the tough cell wall is a major constrain in industrial processing of yeast protein (Kim et al., 1998), and different processing may influence digestibility in salmonids (Murray and Marchant, 1986). Accordingly, Rumsey et al. (1991b) showed that digestibility of CP and energy in intact brewer's yeast was lower than that of disrupted brewer yeast cells in rainbow trout. Various methods can be used for cell wall destruction with the purpose of increasing digestibility of yeast nutrients (reviewed by Nasseri et al., 2011). The poor digestibility of energy and amino acids in the SC diet used in our study indicates that other methods than spray drying should be applied for S. cerevisiae intended as a protein source for salmonids. Matty and Smith (1978) showed that Candida yeast (C. lipolytica) was well utilized as a protein source for rainbow trout. Among the dried yeasts used in our study, C. utilis (also known as Torula yeast) supported the best growth and digestibility, although not significantly higher than K. marxianus, and the CU diet resulted in higher retention of ingested nitrogen than the FM diet. The increased nitrogen retention of salmon fed the CU diet compared with the FM control diet indicates that the absorbed nutrients from C. utilis conformed well with the requirements of the salmon. Previously, Olvera-Novoa et al. (2002) observed that C. utilis could be used as a primary protein source for tilapia, replacing anchovy meal, and Kim et al. (1998) indicated that K. marxianus can be developed as a protein source for aquaculture. The satisfactory growth performance obtained with these yeast strains in our experiment with Atlantic salmon is thus supporting the views of C. utilis and K. marxianus as promising protein sources for use in aquaculture. Rumsey et al. (1991a) observed in studies with high levels of brewer's yeast that rainbow trout appeared not to be adversely affected physiologically by high levels of nucleic acids. In later studies, Andersen et al. (2006) showed that the urolytic pathway of Atlantic salmon is well regulated to handle high dietary purine levels. It has also been suggested that nucleic acids from yeast can be utilized by rainbow trout for N retention and growth (Rumsey et al., 1992) and may in fact increase non-specific immune responses and disease resistance in fish (Li and Gatlin, 2004, 2006; Lin et al., 2009). Our study showed that the KM and SC diets resulted in higher weight of total gut, stomach and DI. The SC diet also increased weight of MI. The effects of yeast on intestinal weights may be caused by nucleotides derived from nucleic acids as nucleotides have been shown to increase the height of intestinal villa in Atlantic salmon (Burrels et al., 2001). This effect of nucleotides has been associated with improved growth of intestinal epithelial cells (Li and Gatlin, 2006).
6
M. Øverland et al. / Aquaculture 402–403 (2013) 1–7
Table 6 Relative weight of the liver and different sections of the gastrointestinal tract, blood metabolites and glutathione peroxidase, and plasma cholesterol of salmon fed the experimental diets1. FM diet
1
CU diet
KM diet
SC diet
P-value
Organ weight, g kg−1 body weight Total gut Liver Stomach Mid intestine Distal intestine
73.8 11.4 44.1 1.91 3.74
± ± ± ± ±
1.7a 0.5a 0.3ab 0.04a 0.04a
76.0 13.3 41.7 1.79 4.11
± ± ± ± ±
0.2ab 0.4b 0.7a 0.01a 0.02ab
82.9 14.1 48.5 1.97 4.45
± ± ± ± ±
3.2b 0.5b 2.3b 0.05a 0.17b
82.3 11.1 48.9 2.76 5.04
± ± ± ± ±
0.2b 0.2a 1.2b 0.21b 0.17c
0.02 0.002 0.01 b0.001 b0.001
Blood parameters pH CO2 partial pressure, kPa Bicarbonate, mmol l−1 Sodium, mmol l−1. Chloride, mmol l−1 Potassium, mmol l−1 Glucose, mmol l−1 Glutathione peroxidase, U l−1 Plasma cholesterol, mmol l−1
7.20 1.42 4.2 149 133 3.4 5.8 482 13.9
± ± ± ± ± ± ± ± ±
0.05 0.04 0.5 0 0 0.2 0.5ab 25a 0.6a
7.21 1.54 4.7 149 133 3.4 6.5 502 11.6
± ± ± ± ± ± ± ± ±
0.02 0.08 0.2 0 1 0.3 0.1a 6ab 0.56ab
7.16 1.38 3.8 148 133 3.2 6.1 752 9.9
± ± ± ± ± ± ± ± ±
0.02 0.04 0.3 1 1 0.3 0.7ab 114bc 0.6bc
7.26 1.41 4.9 150 131 3.4 4.3 483 7.1
± ± ± ± ± ± ± ± ±
0.05 0.11 0.7 1 1 0.4 0.3b 25ab 0.5c
0.37 0.49 0.35 0.22 0.21 0.45 0.04 0.03 b0.001
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
Also, bacterial meal derived from natural gas fermentation increases the weight of the MI and DI in Atlantic salmon (Romarheim et al., 2011). All yeasts used in the present study contained higher level of nucleic acids than the FM, but the SC diet caused the greatest increase in the weight of DI, notwithstanding that the nucleic acid levels in KM and CU were higher than in SC. This may suggest that different proportional levels or digestibilities of individual nucleobases (Mydland et al., 2008), or other mechanisms may have contributed to differences in intestinal growth of salmon fed different yeasts. The significant decrease in plasma cholesterol following feeding with K. marxianus and S. cerevisiae, and a tendency in the same direction with C. utilis, may be related to the yeast cell wall components. Yoshida et al. (2004) compared the hypocholesterolemic effects of a 10% dietary addition of S. cerevisiae, C. utilis, or K. marxianus in rats. They showed that the effect varied depending on yeast species or strains; the K. marxianus strain was most efficient whereas C. utilis and S. cerevisiae had no significant effect. Other studies have shown that also S. cerevisiae and C. utilis may lower plasma cholesterol in rats, apparently due to active cell wall compounds such as β-glucan (Waszkiewicz-Robak and Bartnikowska, 2009). The reason why S. cerevisiae was more efficient than C. utilis in lowering plasma cholesterol in our study remains unknown, but may be related to the observed differences in energy digestibility and body growth. The selenium-dependent GPx is an important component of the defense system against lipid peroxidation, and GPx activity in blood is considered a useful index of selenium status and antioxidative capacity in fish (Bell et al., 1986). In rainbow trout, the selenium requirement for maximum plasma GPx activity has been estimated to between 0.15 and 0.38 mg per kg diet (Hilton et al., 1980). All yeasts used in our study contained very low levels of selenium compared with FM (Table 1). However, because FM is such a rich selenium source, all diets used in the present study contained selenium levels well above the requirement of salmonid fish. Hence, although selenium in FM may have relatively low availability (Bell and Cowey, 1989), it seems unlikely that any of the diets were suboptimal in selenium content. This explains that the CU and SC diets supported similar GPx levels as the FM control diet. The high GPx level following feeding with K. marxianus may indicate that K. marxianus holds antioxidant properties that increase blood GPx levels. For instance, aerobic microorganisms contain ubiquinone and ubiquinol in their membranes and it has been reported that ubiquinol-10 is an important physiological lipid-soluble antioxidant (Frei et al., 1990).
5. Conclusions The study showed that two of the investigated spray-dried yeasts, C. utilis and K. marxianus, can be used as suitable protein sources in diets for Atlantic salmon, whereas S. cerevisiae appeared to be less promising. However, more research is needed to ascertain the effects of different yeast products as protein sources for salmonids.
Acknowledgments The authors would like to acknowledge Frank Sundby for assisting during feed manufacturing and in the fish laboratory, and the staff of the fish laboratory at the Norwegian University of Life Sciences for excellent support during the course of the experiment. The research was supported by Borregaard ASA and co-financed by the Research Council of Norway, grant no. 417091.
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Evaluation of Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae yeasts as protein sources in diets for Atlantic salmon (Salmo salar) Margareth Øverland ⁎, Anders Karlsson, Liv Torunn Mydland, Odd Helge Romarheim, Anders Skrede Aquaculture Protein Centre, CoE, Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P.O. Box 5003, NO-1432 Ås, Norway
a r t i c l e
i n f o
Article history: Received 10 January 2013 Received in revised form 14 March 2013 Accepted 19 March 2013 Available online 26 March 2013 Keywords: Atlantic salmon Candida utilis Kluyveromyces marxianus Saccharomyces cerevisiae Single cell protein Nutrient utilization
a b s t r a c t The use of the yeasts Candida utilis, Kluyveromyces marxianus and Saccharomyces cerevisiae as protein sources in diets for Atlantic salmon (Salmo salar) pre-smolts was investigated. An 89 day study was conducted using triplicate groups of Atlantic salmon with 28 g initial weight kept in fresh water. The dietary treatments consisted of a control diet based on high-quality fish meal (FM diet) and three experimental diets with C. utilis (CU diet), K. marxianus (KM diet) or S. cerevisiae (SC diet) yeast, substituting 40% of the crude protein (CP) from FM. There was no difference in final weight or specific growth rate (SGR) of fish fed the CU diet and the FM diet. The SC diet resulted in lower final weight and SGR, and higher average daily feed intake (ADFI) and feed conversion ratio (FCR) than the FM diet, and higher ADFI and FCR than the KM and CU diets. Fish fed the SC diet had decreased retention of nitrogen and energy, while fish fed the CU diet had increased nitrogen retention compared with those fed the FM diet. In general, feeding the SC diets gave lower digestibility of CP, amino acids, and energy compared with the FM, KM and CU diets, whereas there was no difference in nutrient or amino acid digestibility between the KM and CU diet and the FM diet. Feeding KM and SC gave higher total gut weight and higher distal intestinal weight compared with the FM diet. Plasma cholesterol level was lower for the KM and SC diets while plasma glutathione peroxidase levels were higher for the KM diet compared with the FM diet. In conclusion, feeding moderate levels of S. cerevisiae reduced growth performance and nutrient utilization, while C. utilis and K. marxianus were shown to be promising protein sources in diets for Atlantic salmon, capable of replacing 40% of the protein from high-quality fishmeal without adversely affecting growth performance, digestibility or nutrient retention. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Atlantic salmon (Salmo salar) have traditionally been fed a diet based on marine ingredients such as fish meal (FM). However, marine feed ingredients are limited resources as the world's fish stocks are already either fully exploited or depleted (Tacon and Metian, 2008). Moreover, the aquaculture industry is undergoing rapid growth (FAO, 2012), and as a consequence there is increased demand for aquafeeds. Securing ingredient supply by increased use of new sustainable feed ingredients is therefore crucial. Microbial products like yeast represent potential sustainable ingredients in aquafeeds due to their ability to convert low-value biomass from forestry and agricultural industry into high-value feed ingredients and with limited dependence on agricultural land, water, and changing climatic conditions. Yeast, depending on the strains and species, can thus be produced
⁎ Corresponding author. Tel.: +47 64965206; fax: +47 64965101. E-mail address: [email protected] (M. Øverland). 0044-8486/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.03.016
from fermentation of sugar-rich feed stock such as sugar cane or from lignocellulosic biomass from forestry or agriculture industry. Recent advances in fermentation technology have resulted in more efficient and less costly yeast production, making these ingredients more feasible as nutrient sources for use in aquafeeds (Kim et al., 1998; Omar et al., 2012). Whole cell yeast or products containing different yeast cell wall fractions (β-glucan and mannan oligosaccharide) and nucleic acids are commonly used as immunostimulants in fish diets (Gatesoupe, 2007; Gopalakannan and Arul, 2010; Li and Gatlin, 2006; Oliva-Teles and Goncalves, 2001; Refstie et al., 2010). Low levels (1–4%) of inactive brewer's yeast have been shown to improve growth performance and modulate intestinal microbiota in different fish species (Abdel-Tawwab et al., 2008, 2010; Hoseinifar et al., 2011; Li and Gatlin, 2003). Yeasts are also used as live feed in aquaculture (Abdel-Tawwab et al., 2008; Brown et al., 1996). Less interest has been devoted to the potential of dried yeast as a major protein source in fish diets. Thus, there is a scarcity of literature on alternative yeasts as a replacement for fish meal in salmon diets. As an easily available source, brewer's yeast (Saccharomyces cerevisiae) have been applied in a number of studies with different fish
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M. Øverland et al. / Aquaculture 402–403 (2013) 1–7
species, whereas there are few reports on Candida utilis, and to the author's knowledge no studies on Kluyveromyces marxianus (formerly K. fragilis) as a protein source for fish have been published. The objective of the present experiment was to investigate the effect of moderate levels of C. utilis, K. marxianus and S. cerevisiae yeasts as a partial replacement of FM in diets for Atlantic salmon on growth performance, nutrient digestibility, retention of nitrogen and energy, organ weights and blood chemistry. 2. Materials and methods 2.1. Fish and facilities The Atlantic salmon (S. salar) pre-smolts used in this experiment were acquired as eyed eggs from Aqua Gen AS in the spring of 2011. The fish were fed a commercial salmon feed prior to the start of the 89-day growth experiment at the fish nutrition laboratory at the Norwegian University of Life Sciences, Ås, Norway. After a 48 h fasting period, a total of 720 salmon (average start weight of 27.7 g) were randomly distributed into 12 fiberglass tanks with 60 fish per tank, each with a 300-l capacity. The fish were kept under continuous light in recirculated fresh water with a water supply of 6–7 l min −1 and with temperatures ranging from 8.8 to 14.5 °C. Dissolved oxygen levels were measured daily and kept above 6.0 mg l −1 in the outlet water. 2.2. Diets and feeding The dietary treatments consisted of a control diet based on highquality FM (FM diet) and three experimental diets containing either C. utilis, K. marxianus or S. cerevisiae yeast, all spray dried and inactivated, replacing 40% of the CP from the FM. The diets were formulated to be isonitrogenous and isolipidic based on the analyzed chemical content of the ingredients. All diets contained 0.1 g kg−1 yttrium oxide (Y2O3) as an indigestible marker for determination of nutrient digestibility (Austreng et al., 2000). The chemical composition of FM, C. utilis, K. marxianus and S. cerevisiae is given in Table 1, while the formulation and composition of the diets are given in Table 2. The diets were processed at the feed laboratory of the Norwegian University of Life Sciences, Ås, Norway. All dry ingredients, excluding gelatin, were mixed in a Moretti Foreni kneading machine (Spiry 25, Mondolfo, Italy). The fish oil was then added to the dry ingredients and mixed thoroughly. The gelatin was dissolved in hot water (70–80 °C) and mixed with the mash, resulting in a firm dough that was pelleted using an Italgi pasta extruder (P35A, Carasco, Italy) equipped with a 3 mm die. The pellets were then cooled to room temperature and later dried at 45–60 °C to about 95% dry matter (DM) and stored at 0–4 °C in zip-lock bags until feeding. The four experimental diets were fed to triplicate groups of fish over a period of 5 h (07:00 to 12:00) daily by automatic belt feeders. Fish were fed 20% in excess, based on recorded feed intake over the previous 3-day period. Uneaten feed was collected from feed collectors in the tank outlets directly after each feeding period. Feed intake was adjusted for uneaten feed and calculated as: Feed fed DM (g) − (uneaten feed DM (g) / recovery). Recovery was estimated according to Helland et al. (1996). 2.3. Sampling Before the start of the experiment, 10 fish from the holding tank were euthanized with an overdose of MS-222 (Metacaine, Norsk Medisinaldepot AS, Norway), and stored at − 20 °C for whole body analysis. Fish were anesthetized with MS-222 and batched weighted at the start of the experiment (Day 0) and individually weighted at the end (Day 89) of the experiment. At the termination of the experiment, eight fish were randomly sampled from each tank (24 fish per diet), anesthetized, and blood was collected from the caudal vessel
Table 1 Chemical composition of fish meal (FM), Candida utilis (CU), Kluyveromyces marxianus (KM) and Saccharomyces cerevisiae (SC) used in the dietsa (g kg−1).
Dry matter Crude protein Nucleic acids Crude lipid Starch Ash Ca P Se, mg kg−1 Gross energy, MJ kg−1
FMb
CUc
KMc
SCc
918 735 13 114 10 139 26 20 2.7 22.3
937 560 93 3 37 54 2.7 15 b0.04 21.4
939 511 102 8 8 76 0.5 15 b0.04 20.5
968 460 58 2 11 64 0.7 11 0.12 19.6
4.67 1.96 4.22 6.94 6.95 1.36 4.16 4.77 4.51 1.08
4.20 1.71 3.90 6.05 6.51 1.54 3.71 4.47 4.04 0.98
4.07 1.82 3.41 5.44 6.19 1.23 3.29 4.23 3.96 1.08
5.22 9.29 3.52 10.99 0.82 3.59 3.28 4.76
5.92 8.55 3.260 11.90 0.91 3.15 2.90 4.67
4.71 8.87 2.98 13.09 1.02 2.95 2.85 4.26
Essential amino acids, g 16 g N−1,d Arginine 5.23 Histidine 1.97 Isoleucine 3.35 Leucine 6.25 Lysine 6.79 Methionine 2.50 Phenylalanine 3.26 Threonine 3.97 Valine 3.93 Tryptophan 0.84 Non-essential amino acids, g 16 g N−1,d Alanine 5.00 Aspartic acid 8.24 Glycine 4.40 Glutamic acid 11.92 Cysteine 0.84 Tyrosine 2.64 Proline 3.26 Serine 3.95
a Proximate analysis and starch are means of duplicate analyses, minerals are means of triplicate analysis, while nucleic acids are means of duplicate analysis of the fish meal and triplicate analysis of the yeasts. b NorsECO-LT, Egersund Sildoljefabrikk AS, Egersund, Norway. c Borregaard ASA, Sarpsborg, Norway. d Water corrected amino acids.
with heparinized syringes. The blood was divided into three aliquots before further processing and analysis of blood chemistry, glutathione peroxidase (GPx) and plasma cholesterol. One aliquot of whole blood was immediately analyzed for standard blood chemistry on a portable blood analyzer (i-Stat, Medinor ASA, Norway) and the results were later temperature corrected following the procedures specified by the i-Stat manufacturer. One aliquot of whole blood for GPx analysis was immediately transferred to Eppendorf tubes, frozen on dry ice and kept at − 80 °C until analysis. The remaining blood samples were centrifuged at 2000 ×g for 3 min and the plasma was kept frozen at − 20 °C until analysis of cholesterol. Another 10 fish from each tank were randomly selected, anesthetized with MS-222 and euthanized with a blow to the head, dissected and eviscerated. Surface fat and connective tissue were carefully removed before weighing of the gastrointestinal tract (from the esophageal sphincter to the anus) and the individual sections: stomach (ST), mid-intestine (MI, from distal side of the pyloric sphincter to distal intestine), and distal intestine (DI). The liver was also removed from the same fish, weighted and used for calculation of liver index. The fish and all organs were then placed in a plastic zip lock bag and frozen at −20 °C for whole body analysis. For whole body analysis, all fish from each tank were ground twice through a meat grinder (first with 8 and then 5 mm die holes) and thoroughly mixed before freeze drying and analysis. Feces were stripped in two rounds according to the method described by Austreng (1978). Feces from all fish in each tank were pooled and immediately frozen at − 20 °C and freeze dried prior to analysis.
M. Øverland et al. / Aquaculture 402–403 (2013) 1–7 Table 2 Formulation and chemical composition (as is) of diets. FM diet
CU diet
KM diet
SC diet
Formulation, g kg−1 Fish meal Candida utilis Kluyveromyces marxianus Saccharomyces cerevisiae Gelatinized potato starcha Fish oilb Gelatinc Cellulosed Premixe Y2O3f
579 0 0 0 126 137 80 67 9.9 0.1
347 283 0 0 118 162 80 0 9.9 0.1
347 0 302 0 100 161 80 0 9.9 0.1
347 0 0 345 56 161 80 0 9.9 0.1
Analyzed content, kg−1 Dry matter, g Crude protein, g Total amino acids, g Crude lipid, g Starch, g Ash, g Gross energy, MJ
968 491 382 195 126 79 22.6
943 470 374 192 124 63 22.5
946 466 346 201 105 74 22.5
955 475 379 202 71 73 22.6
Essential amino acids, g kg−1 Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Valine Tryptophan
2.61 0.88 1.48 2.76 3.05 1.07 1.45 1.72 1.83 0.86
2.25 0.76 1.43 2.46 2.73 0.85 1.37 1.62 1.69 0.86
2.46 0.87 1.53 2.64 2.98 0.87 1.48 1.78 1.87 0.88
2.46 0.87 1.58 2.73 2.97 0.86 1.53 1.79 1.88 0.87
Non-essential amino acids, g kg−1 Alanine Aspartic acid Glycine Glutamic acid Cysteine Tyrosine Proline Serine
2.55 3.68 3.15 5.53 0.33 1.11 2.20 1.81
2.44 3.36 2.77 5.00 0.30 1.06 1.97 1.73
2.54 3.76 2.97 5.73 0.35 1.18 2.15 1.87
2.51 3.61 2.99 5.17 0.31 1.20 2.17 1.86
a
Lygel F 60, Lyckeby Culinar AB, Fjälkinge, Sweden. b Skretting Norway, Stavanger, Norway. c Rousselot™ 250 PS, Rousselot SAS, Courbevoie Cedex, France. d Cellulose powder, AWL Kjemi AS, Kolbotn, Norway. e Provided the following amount per kg of diet: Ca 1.07 g, Mn2SO4 14.7 mg, ZnSO4 117 mg, CuSO4 4.90 mg, CoSO4 980 μg, Ca(IO3)2 2.94 mg, retinol 2450 IU, cholecalciferol 1470 IU, tocopherol 196 mg, menadione 9.80 mg, thiamine 14.7 mg, riboflavin 24.5 mg, pyridoxine 14.7 mg, cobalamine 19.6 μg, pantothenic acid 29.4 mg, folic acid 4.90 mg, niacin 73.5 mg, biotin 245 μg, vitamin C 1.75 (Rovimix® Stay-C® 35, DSM Nutritional Products, Basel, Switzerland) ascorbic acid 123 mg, AS Norsk Mineralnæring, Hønefoss, Norway. f Di-yttrium tri-oxide (Y2O3), Rare Earth Metal Limited, Shenzhen, Guangdong, China.
3
Ultimate 3000 system fitted with an YMC-Pack ODS-A column) after perchloric acid hydrolysis. The total nucleic acid content of the ingredients was calculated assuming that the molar fraction of each nucleotide is equal to that of its respective nucleobase. Gross energy in diets, feces and fish was measured by bomb calorimetry (Parr 1271 Bomb calorimeter, Parr, Moline, Il, USA). Fish were also analyzed for CP using Kjeldahl nitrogen (Commission dir. 93/28/EEC) × 6.25. For digestibility calculations, DM in feces was determined by freeze drying, and diets and feces were analyzed for CP using the Dumas method (Kirsten and Hesselius, 1983) on an EA 1108 element analyzer (Fison, Waltham, USA) and yttrium by ICP-AES analysis (NS-EN ISO 11885). Whole blood GPx was analyzed according to the method described by Paglia and Valentine (1967). Plasma cholesterol was analyzed using a Maxmat PL multianalyzer and a commercially available kit (Cholesterol CP A11A01634, Bergman Diagnostica AS, Norway). 2.5. Calculations and statistical analysis Fish growth was calculated as specific growth rate (SGR) according to the following equation: SGR ¼ 100 ðlnðfinal body weight ðBWÞÞ−lnðinitial BWÞ=ΔtÞ; where Δt is the number of experimental days: Feed conversion ratio (FCR) was calculated as: FCR = Feed DM ingested (g) × fish weight gain (g)−1. Daily feed intake is expressed as % of the current BW: Current BW (BWn) = BWn − 1 + (current daily DM feed intake / FCR). Apparent digestibility was calculated as: 100 ð1−ðYd Yf−1 Nf Nd−1ÞÞ; where Yd and Yf represent the concentration of yttrium in the diet and feces, and Nd and Nf represent the concentration of individual nutrients or energy in the diet and feces, respectively. Nitrogen and energy retentions were calculated as: 1) Of ingested = nutrient retained in the body/nutrient ingested × 100. 2) Of digested = nutrient retained in the body / (nutrient ingested × digestibility) × 100. The data were analyzed in Statistical Analysis Software (SAS) v. 9.2, using a general linear model (GLM procedure) with diet as fixed effect. The tank mean for each time point was used as a single observation in the statistical analysis. Effect of diet was tested on all parameters using an F-test. Comparisons of diet means were performed using a t-test with Tukey–Kramer correction for multiple comparisons. Pvalues b 0.05 were considered statistically significant. Unless stated otherwise, all numbers are presented as diet means ± standard error of the mean.
2.4. Chemical analysis
3. Results
Ingredients, diets and feces were ground with a pestle and mortar prior to analysis. Diets, ingredients and fish were analyzed for DM by drying to constant weight at 104 °C (Commission dir. 71/393/EEC). Diets, ingredients and feces were analyzed for CP using Kjeldahl nitrogen (Commission dir. 93/28/EEC) × 6.25, crude lipid (CL) by HCl hydrolysis followed by diethyl ether extraction (Commission dir. 98/64/EC), ash (Commission dir. 71/250/EEC), amino acids (Commission dir. 98/64/EC) and tryptophan (Commission dir. 2000/45/EC). Tryptophan in feces was not analyzed due to limited material available. Starch in diets and ingredient was analyzed using the AOAC enzymatic method 996.11. Nucleic acids in the ingredients were analyzed according to methods described by Mydland et al. (2008). In brief, the individual nucleobases were analyzed by HPLC (Dionex
The daily relative feed intake of fish during the experimental period is given in Fig. 1. The feed intake increased gradually during the first two weeks of feeding, followed by a decrease in parallel with decreasing water temperature until the end of the experiment. Growth performance and fecal dry matter are given in Table 3. There were no differences among the diets for initial weights. By comparing fish fed the CU diet with those fed the FM diet there was no difference in final weight or SGR. Salmon fed the FM diet had higher final weights than those fed the KM and SC diets and higher SGR than those fed the SC diet. The fish fed the SC diet also had higher average daily feed intake (ADFI) and FCR than those fed the control and the KM and CU diets. The FM diet and the SC diet gave higher fecal DM than the KM and CU diets.
4
M. Øverland et al. / Aquaculture 402–403 (2013) 1–7
15
FM 2.0 %
CU
14
KM
13
Temperature 1.5 %
12
1.0 %
11
Water temperature (°C)
Feed intake (% of body weight)
SC
10 0.5 % 9
0.0 %
8 0
7
14
21
28
35
42
49
56
63
70
77
84
Experiment duration (day) Fig. 1. Feed intake of Atlantic salmon pre-smolts fed a fish meal control diet (FM diet) or one of three diets containing spray-dried yeast as fish meal substitution (40% of CP) in an 89-day growth trial. The CU diet contained Candida utilis, the KM diet contained Kluyveromyces marxianus and the SC diet contained Saccharomyces cerevisiae yeast. Each data point is the diet mean of three tanks for a single day. The gray line is water temperature recorded daily from the inlet water source.
The apparent digestibility of CP and energy is shown in Table 4. There was no difference in digestibility parameters between salmon fed the CU diet and those fed the FM diet. The SC diet gave a lower digestibility of CP and energy compared with the FM diet and the KM and CU diets. There were significant effects of adding yeast to diets on the retention of nitrogen (Table 4). Feeding SC decreased nitrogen and energy retention as a percentage of intake compared with the FM diet. Nitrogen retention of fish fed the KM diet was unaffected, while the CU diet caused an increase in nitrogen retention as a percentage of ingested and digested nitrogen compared with the FM control. The digestibility of individual amino acids was significantly lower for the SC diet compared with all other diets, except for methionine, aspartic acid and cysteine (Table 5). Compared with the FM diet, the KM diet had a lower digestibility of cysteine, while the KM and SC diets gave a lower digestibility of methionine and the SC diet had a lower digestibility of aspartic acid. There was no difference in digestibility of total amino acids among fish fed the FM, CU and KM diets. The relative weights of liver and gastrointestinal sections, and blood metabolites, GPx and plasma cholesterol of fish fed the experimental diets are given in Table 6. Feeding KM and SC gave higher total gut weight, while liver weight was higher in the fish fed the CU and KM diets compared with those fed the FM control diet. The KM and SC diets gave a higher stomach weight than the CU diet, but not compared
with those fed the FM control. Fish fed the SC diet had a higher MI weight compared with the FM control and the other yeast-containing diets, while fish fed the SC and KM diets had higher DI weights than fish fed the FM diet. The blood ions, pH and partial pressure of CO2 were not significantly affected by the dietary treatments. Blood glucose level was lower for the SC diet than for the CU diet, but did not differ from the FM control, while plasma cholesterol level was lower for the KM and SC diets compared with the FM control. Also, fish fed the KM diet had higher blood GPx levels than those fed the FM diet, while feeding CU and the SC did not affect blood GPx levels of the fish compared with the FM diet. 4. Discussion The recent development toward a supply shortage of FM for use in diets for salmonid fish has provoked an increasing search for sustainable protein substitutes. The present study revealed that C. utilis and K. marxianus could be fed at about 30% of the diet to Atlantic salmon, replacing FM protein, without significant negative effects on feed intake, SGR or FCR, whereas feeding of S. cerevisiae had negative effects on these parameters. The high feed intake and poor FCR of fish fed the SC diet may be interpreted as an indirect effect of lower digestibility of CP, amino acids, and energy. Presumably, the high feed
Table 3 Growth performance and fecal dry matter in salmon fed the experimental diets. FM diet Start weight, g1 Final weight, g Specific growth rate, % Daily feed intake, % of BW Feed conversion ratio Fecal dry matter, % 1
27.8 92.4 1.36 0.95 0.70 16.0
± ± ± ± ± ±
CU diet 0.2 2.8a 0.03a 0.02a 0.01a 0.6a
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
27.7 88.6 1.32 0.91 0.70 12.5
± ± ± ± ± ±
0.0 1.1a,b 0.01a,b 0.01a 0.01a 0.3b
KM diet
SC diet
27.7 81.3 1.22 0.89 0.73 12.3
27.6 78.8 1.19 1.07 0.90 18.3
± ± ± ± ± ±
0.0 3.1b 0.04a,b 0.03a 0.00a 0.4b
± ± ± ± ± ±
P-value 0.1 2.1b 0.03b 0.03b 0.01b 1.0a
0.38 0.01 0.01 0.002 b0.001 0.001
M. Øverland et al. / Aquaculture 402–403 (2013) 1–7 Table 4 Apparent digestibility (%) of CP and energy and retention of nitrogen and energy in salmon fed experimental diets1.
Digestibility Crude protein Energy
FM diet
CU diet
88.4 ± 0.7a 85.5 ± 0.8a
88.0 ± 0.5a 86.1 ± 0.7 84.3 ± 0.4a 82.2 ± 1.4
Nutrient retention Nitrogen % of ingested 52.9 % of digested 59.9 Energy % of ingested 53.7 % of digested 62.8 1
± 0.6a 57.2 ± 0.4c ± 0.6ab 65.0 ± 0.4c
KM diet
SC diet a a
53.2 ± 0.3a 61.8 ± 0.3b
P-value
72.6 ± 2.3 67.8 ± 2.5
b b
b0.001 b0.001
42.9 ± 0.5b 59.1 ± 0.7a
b0.001 0.001
± 0.9a 55.9 ± 0.4a 52.2 ± 0.5a 40.4 ± 1.4b ± 1.1ab 66.3 ± 0.5b 63.5 ± 0.6ab 59.6 ± 2.1a
b0.001 0.03
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
intake could not compensate for the reduced digestibility as the SGR was lower than in fish fed the FM diet, and retention of digested nitrogen and energy was lower in fish fed the SC diet than for any of the other diets. The proximal composition of yeast is dependent on strain, growth media and growing conditions; the CP content varies on average between 40 and 55% of the cell dry matter, including a nucleic acid content of 6–12% (Halasz and Lasztity, 1991). The dried yeasts used in the present study were all commercially available products, and contained 56.0% (C. utilis), 51.1% (K. marxianus) and 46.0% (S. cerevisiae) CP, respectively. As opposed to S. cerevisiae, C. utilis and K. marxianus have a broad substrate spectrum and are able to utilize pentoses such as xylose, the second most abundant carbohydrate in lignocellulosic biomass (Nasseri et al., 2011; Rodrussamee et al., 2011). Furthermore, both C. utilis and K. marxianus have obtained the generally-regarded-as-safe (GRAS) status. The diets used were about isonitrogenous on a DM basis, but amino acid composition was slightly different among diets. It is well known that yeast contains lower levels of sulfur-containing amino acids than FM (Kuhad et al., 1997), and all yeasts used in our study contained less methionine than the FM. Accordingly, the yeast-containing diets had slightly lower levels of methionine than the FM diet, and the CU diet also had lower levels of threonine and valine. Comparing with data from Kaushik and Seiliez (2010), it is possible that especially the poorly digestible SC diet may have been suboptimal in digestible methionine. Thus, the possibility that this may have Table 5 Apparent digestibility (%) of amino acids in salmon fed experimental diets1. FM diet Essential amino Arginine Isoleucine Histidine Leucine Lysine Methionine Threonine Phenylalanine Tyrosine Valine
1
acids 89.4 84.8 80.0 88.0 87.0 86.4 81.2 83.0 83.9 84.5
CU diet
KM diet
SC diet
P-value
0.2a 0.4a 0.7a 0.3a 0.4a 0.4a 0.7a 0.3a 0.4a 0.5a
89.5 82.6 79.6 85.6 85.9 82.9 77.8 81.4 82.8 82.8
± ± ± ± ± ± ± ± ± ±
0.4a 0.3a 0.4a 0.3a 0.3a 0.6ab 0.6a 0.3a 0.4a 0.3a
86.8 77.7 74.8 81.3 84.0 79.5 72.4 75.3 77.8 78.5
± ± ± ± ± ± ± ± ± ±
0.3a 1.0a 1.4a 0.9a 1.0a 1.1bc 1.8a 1.2a 1.3a 1.0a
75.3 61.2 61.3 66.8 68.4 73.9 57.7 61.5 58.8 62.2
± ± ± ± ± ± ± ± ± ±
2.7b 4.2b 3.6b 3.6b 3.6b 2.4c 3.7b 3.5b 4.0b 3.9b
b0.001 b0.001 b0.001 b0.001 b0.001 0.001 b0.001 b0.001 b0.001 b0.001
Non-essential amino acids Alanine 87.4 ± 0.4a Aspartic acid 70.6 ± 1.1a Glutamic acid 87.4 ± 0.5a Glycine 81.6 ± 0.8 Proline 84.1 ± 0.9a Serine 80.2 ± 0.6a Cysteine 57.7 ± 1.2a Total AA 83.5 ± 0.6a
86.6 70.7 86.2 81.7 84.4 79.1 51.9 82.4
± ± ± ± ± ± ± ±
0.3a 0.8a 0.2a 0.5 0.4a 0.4a 0.9ab 0.3a
84.1 69.3 84.3 79.7 81.3 75.1 44.5 79.4
± ± ± ± ± ± ± ±
0.8a 2.6ab 0.9a 1.4 1.4a 1.3a 3.9b 1.3a
75.0 59.5 75.6 77.4 74.6 61.5 51.0 68.8
± ± ± ± ± ± ± ±
2.6b 3.3b 2.5b 2.0 2.4b 3.3b 2.6ab 3.0b
b0.001 0.02 0.001 0.14 0.005 b0.001 0.03 b0.001
± ± ± ± ± ± ± ± ± ±
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
5
contributed to poor growth performance, in spite of high feed intake, cannot be excluded. In studies with sea bass (Dicentrarchus labrax) it was concluded that brewer's yeast can replace 50% of FM protein with no negative effects on fish performance (Oliva-Teles and Goncalves, 2001). In their study, inclusion of up to about 30% brewer's yeast had no effects on protein digestibility, and feed conversion and nitrogen retention were improved compared with the FM diet. Rumsey et al. (1991a) reported that feeding 25% brewer's yeast, replacing casein protein, improved growth and feed conversion in rainbow trout (Oncorhynchus mykiss), whereas diets with 50% of brewer's yeast appeared to be unpalatable. Working with pacu (Piaractus mesopotamicus, Holmberg), Ozòrio et al. (2010) observed that replacing 50% of the FM with brewer's yeast had no effect on protein digestibility, and growth performance and feed conversion were improved. Similar results were obtained by feeding 20% inactive baker's yeast (S. cerevisiae) as a FM substitute to Gilthead sea bream (Sparus aurata) (Salnur et al., 2009). A recent study by Ozòrio et al. (2012) showed that feeding 15% brewer's yeast to Nile tilapia (Oreochromis niloticus) promoted growth and efficient nutrient utilization, while higher levels gave a negative dose dependent effect on growth performance and nutrient utilization. Although our study was done with a different species of fish, it may be speculated that the divergent results with S. cerevisiae may partially be due to different characteristics of the brewer's yeast used in the experiments. It has been suggested that the tough cell wall is a major constrain in industrial processing of yeast protein (Kim et al., 1998), and different processing may influence digestibility in salmonids (Murray and Marchant, 1986). Accordingly, Rumsey et al. (1991b) showed that digestibility of CP and energy in intact brewer's yeast was lower than that of disrupted brewer yeast cells in rainbow trout. Various methods can be used for cell wall destruction with the purpose of increasing digestibility of yeast nutrients (reviewed by Nasseri et al., 2011). The poor digestibility of energy and amino acids in the SC diet used in our study indicates that other methods than spray drying should be applied for S. cerevisiae intended as a protein source for salmonids. Matty and Smith (1978) showed that Candida yeast (C. lipolytica) was well utilized as a protein source for rainbow trout. Among the dried yeasts used in our study, C. utilis (also known as Torula yeast) supported the best growth and digestibility, although not significantly higher than K. marxianus, and the CU diet resulted in higher retention of ingested nitrogen than the FM diet. The increased nitrogen retention of salmon fed the CU diet compared with the FM control diet indicates that the absorbed nutrients from C. utilis conformed well with the requirements of the salmon. Previously, Olvera-Novoa et al. (2002) observed that C. utilis could be used as a primary protein source for tilapia, replacing anchovy meal, and Kim et al. (1998) indicated that K. marxianus can be developed as a protein source for aquaculture. The satisfactory growth performance obtained with these yeast strains in our experiment with Atlantic salmon is thus supporting the views of C. utilis and K. marxianus as promising protein sources for use in aquaculture. Rumsey et al. (1991a) observed in studies with high levels of brewer's yeast that rainbow trout appeared not to be adversely affected physiologically by high levels of nucleic acids. In later studies, Andersen et al. (2006) showed that the urolytic pathway of Atlantic salmon is well regulated to handle high dietary purine levels. It has also been suggested that nucleic acids from yeast can be utilized by rainbow trout for N retention and growth (Rumsey et al., 1992) and may in fact increase non-specific immune responses and disease resistance in fish (Li and Gatlin, 2004, 2006; Lin et al., 2009). Our study showed that the KM and SC diets resulted in higher weight of total gut, stomach and DI. The SC diet also increased weight of MI. The effects of yeast on intestinal weights may be caused by nucleotides derived from nucleic acids as nucleotides have been shown to increase the height of intestinal villa in Atlantic salmon (Burrels et al., 2001). This effect of nucleotides has been associated with improved growth of intestinal epithelial cells (Li and Gatlin, 2006).
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M. Øverland et al. / Aquaculture 402–403 (2013) 1–7
Table 6 Relative weight of the liver and different sections of the gastrointestinal tract, blood metabolites and glutathione peroxidase, and plasma cholesterol of salmon fed the experimental diets1. FM diet
1
CU diet
KM diet
SC diet
P-value
Organ weight, g kg−1 body weight Total gut Liver Stomach Mid intestine Distal intestine
73.8 11.4 44.1 1.91 3.74
± ± ± ± ±
1.7a 0.5a 0.3ab 0.04a 0.04a
76.0 13.3 41.7 1.79 4.11
± ± ± ± ±
0.2ab 0.4b 0.7a 0.01a 0.02ab
82.9 14.1 48.5 1.97 4.45
± ± ± ± ±
3.2b 0.5b 2.3b 0.05a 0.17b
82.3 11.1 48.9 2.76 5.04
± ± ± ± ±
0.2b 0.2a 1.2b 0.21b 0.17c
0.02 0.002 0.01 b0.001 b0.001
Blood parameters pH CO2 partial pressure, kPa Bicarbonate, mmol l−1 Sodium, mmol l−1. Chloride, mmol l−1 Potassium, mmol l−1 Glucose, mmol l−1 Glutathione peroxidase, U l−1 Plasma cholesterol, mmol l−1
7.20 1.42 4.2 149 133 3.4 5.8 482 13.9
± ± ± ± ± ± ± ± ±
0.05 0.04 0.5 0 0 0.2 0.5ab 25a 0.6a
7.21 1.54 4.7 149 133 3.4 6.5 502 11.6
± ± ± ± ± ± ± ± ±
0.02 0.08 0.2 0 1 0.3 0.1a 6ab 0.56ab
7.16 1.38 3.8 148 133 3.2 6.1 752 9.9
± ± ± ± ± ± ± ± ±
0.02 0.04 0.3 1 1 0.3 0.7ab 114bc 0.6bc
7.26 1.41 4.9 150 131 3.4 4.3 483 7.1
± ± ± ± ± ± ± ± ±
0.05 0.11 0.7 1 1 0.4 0.3b 25ab 0.5c
0.37 0.49 0.35 0.22 0.21 0.45 0.04 0.03 b0.001
Average ± standard error of the mean. Means in a row with no superscripts in common differ.
a, b, c
Also, bacterial meal derived from natural gas fermentation increases the weight of the MI and DI in Atlantic salmon (Romarheim et al., 2011). All yeasts used in the present study contained higher level of nucleic acids than the FM, but the SC diet caused the greatest increase in the weight of DI, notwithstanding that the nucleic acid levels in KM and CU were higher than in SC. This may suggest that different proportional levels or digestibilities of individual nucleobases (Mydland et al., 2008), or other mechanisms may have contributed to differences in intestinal growth of salmon fed different yeasts. The significant decrease in plasma cholesterol following feeding with K. marxianus and S. cerevisiae, and a tendency in the same direction with C. utilis, may be related to the yeast cell wall components. Yoshida et al. (2004) compared the hypocholesterolemic effects of a 10% dietary addition of S. cerevisiae, C. utilis, or K. marxianus in rats. They showed that the effect varied depending on yeast species or strains; the K. marxianus strain was most efficient whereas C. utilis and S. cerevisiae had no significant effect. Other studies have shown that also S. cerevisiae and C. utilis may lower plasma cholesterol in rats, apparently due to active cell wall compounds such as β-glucan (Waszkiewicz-Robak and Bartnikowska, 2009). The reason why S. cerevisiae was more efficient than C. utilis in lowering plasma cholesterol in our study remains unknown, but may be related to the observed differences in energy digestibility and body growth. The selenium-dependent GPx is an important component of the defense system against lipid peroxidation, and GPx activity in blood is considered a useful index of selenium status and antioxidative capacity in fish (Bell et al., 1986). In rainbow trout, the selenium requirement for maximum plasma GPx activity has been estimated to between 0.15 and 0.38 mg per kg diet (Hilton et al., 1980). All yeasts used in our study contained very low levels of selenium compared with FM (Table 1). However, because FM is such a rich selenium source, all diets used in the present study contained selenium levels well above the requirement of salmonid fish. Hence, although selenium in FM may have relatively low availability (Bell and Cowey, 1989), it seems unlikely that any of the diets were suboptimal in selenium content. This explains that the CU and SC diets supported similar GPx levels as the FM control diet. The high GPx level following feeding with K. marxianus may indicate that K. marxianus holds antioxidant properties that increase blood GPx levels. For instance, aerobic microorganisms contain ubiquinone and ubiquinol in their membranes and it has been reported that ubiquinol-10 is an important physiological lipid-soluble antioxidant (Frei et al., 1990).
5. Conclusions The study showed that two of the investigated spray-dried yeasts, C. utilis and K. marxianus, can be used as suitable protein sources in diets for Atlantic salmon, whereas S. cerevisiae appeared to be less promising. However, more research is needed to ascertain the effects of different yeast products as protein sources for salmonids.
Acknowledgments The authors would like to acknowledge Frank Sundby for assisting during feed manufacturing and in the fish laboratory, and the staff of the fish laboratory at the Norwegian University of Life Sciences for excellent support during the course of the experiment. The research was supported by Borregaard ASA and co-financed by the Research Council of Norway, grant no. 417091.
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