DOI: 10.1111/jpn.12145
ORIGINAL ARTICLE
Brewer’s yeast and sugarcane yeast as protein sources for dogs M. S. Martins1, N. K. Sakomura1, D. F. Souza1, F. O. R. Filho1, M. O. S. Gomes1, R. S. Vasconcellos2 and A. C. Carciofi1 1 Faculdade de Ci^encias Agr arias e Veterinarias, Universidade Estadual Paulista (UNESP), Jaboticabal, Brazil 2 Universidade Estadual de Maringa (UEM), Maringa, Brazil
Summary Brewer’s yeast (BY), autolysed sugarcane yeast (ASCY) and integral sugar cane yeast (ISCY) were studied in two experiments as ingredients for dog diets. In the first experiment, 28 dogs were randomly assigned to four diets; one reference diet and three test diets containing 15% of BY, ASCY or ISCY and 85% of the reference diet (asfed basis). The digestibilities of the yeasts were calculated by the substitution method. In the second experiment, 35 dogs were randomized to five diets with similar chemical composition but different levels of sugarcane yeast inclusion (0%, 7.5% ASCY, 15% ASCY, 7.5% ISCY and 15% ISCY). In both experiments, the coefficient of total tract apparent digestibility (CTTAD) of nutrients was determined through total collection of faeces. During experiment, two additional analyses of food palatability, nitrogen balance and urea postprandial responses were performed. The data were submitted to analysis of variance, and the means were compared by orthogonal or polynomial contrasts or Tukey’s test (p < 0.05). In experiment 1, CTTAD of protein was lower for both sugarcane yeasts than for BY (p = 0.012), as was metabolizable energy content (p = 0.025). In experiment 2, a linear reduction in energy digestibility with ASCY inclusion (p = 0.05) was verified. Furthermore, faecal score and DM content were reduced with ISCY inclusion (p < 0.003). No effect of yeast inclusion on nitrogen balance or postprandial urea response was found. Also, the inclusion of 7.5% of ASCY or ISCY increased diet palatability (p < 0.01). Yeasts present adequate digestibility by dogs, but its effect on faecal formation needs to be considered. No clear advantage for the use of ASCY over ISCY was found. In conclusion, we find that sugarcane yeast is suitable for inclusion in dog food and can enhance the overall palatability of the diet. Keywords canine, digestibility, palatability, Saccharomyces cerevisiae lio de Mesquita Filho (UNESP), Correspondence Dr. A. C. Carciofi, Departamento de Clınica e Cirurgia Veterinaria. Universidade Estadual Paulista Ju Via de Acesso Prof. Paulo Donato Castellane, s/n, Zip code 14884-900, Jaboticabal, SP, Brazil. Tel: +55 16 3209 2626; Fax: +55 16 3203 1226; E-mail: [email protected] Received: 17 March 2013; accepted: 22 October 2013
Introduction Sugarcane yeast and brewer’s yeast (Saccharomyces cerevisiae) are used as feed for many species, including bovine (Pereira et al., 2001), fish (Hisano et al., 2008), swine (Moreira et al., 2002; Castilho, 2004; White et al., 2009) and poultry (Granjeiro et al., 2001). Yeasts contain important characteristics for animal nutrition, such as high levels of B vitamins, nucleotides derived from cellular nucleic acids and mannan oligosaccharides and 1.3/1.6 b-glucan derived from cell membranes, that when consumed together can favourably influence gut and overall animal health (White et al., 2009). They are also considered palatable to many species, which is attributed to their elevated concentrations of glutamic acid and 5′-ribonucleotide which impart a savoury, umami taste (Kurihara and 948
Kashiwayanagi, 2000). In addition to glutamic acid, brewer’s and sugarcane yeasts have high levels of amino acids, and the total protein content varies from approximately 40 to 50% of dry matter. However, yeast has a rigid cell membrane with thicknesses varying from 150 to 400 nm (Osumi, 1998) which could reduce the absorption of the cell proteins and other nutrients. Currently, little is known about the bioavailability of yeast protein (Hisano et al., 2008), and, to our knowledge, no information for dogs is available. The worldwide production of brewer’s yeast is approximately 80 000 tons per year, with an apparently slow growth of 2% per year. Due to the recent increase in the ethanol production from sugarcane in Brazil, sugarcane yeast production is increasing at a rate of 4% per year (Santos, 2009). The potential production of sugarcane yeast in Brazil is around 500 000
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tons per year, but only 15% is being currently utilized for animal feed (Santos, 2009). The production of sugarcane yeast starts with inoculation of fermentation chambers of sugarcane juice. Yeast cell mass is separated from the fermentation liquor by centrifugation, washed and dried, yielding what is called integral sugarcane yeast (ISCY). Additionally, the mass of living yeast cells can be submitted to osmotic stress and enzymatic hydrolysis which promotes cellular autolysis that ruptures the cellular membrane and releases the cell contents. When dried, typically by spray drying this product is called autolysed sugarcane yeast (ASCY). One study using pelleted diets for fish reported better digestibility of ASCY than ISCY (Hisano et al., 2008). Considering the lack of information on the use of yeast as a protein source for dogs, the present study was designed to compare the digestibility of brewer’s yeast (BY) to ASCY and ISCY. Additionally, the effects of different levels of dietary ASCY and ISCY on nutrient digestibility, nitrogen balance, food palatability and urea postprandial response in dogs were evaluated.
Yeast as a protein source for dogs
there were seven replicates (dogs) per treatment (diet). In experiment 2, the CTTAD of nutrients and energy, food palatability and urea postprandial response were evaluated using diets containing different levels of ASCY and ISCY. Five isonutritive diets were used, the standard control diet without yeast and four test diets with 7.5% ASCY, 15% ASCY, 7.5% ISCY or 15% ISCY. Thirty-five dogs were used in a randomized block design, with 20 dogs (four dogs per diet) in the first block and 15 dogs (three dogs per diet) in the second block, totalling seven replicates (dogs) per treatment (diet). Ingredients and diets
The chemical compositions of the BY, ASCY and ISCY ingredients are shown in Table 1 and were determined as follows. The ASCY and ISCY samples were Table 1 Chemical composition of the brewer’s yeast and the autolysed and integral sugarcane yeast used in experiments one and two* Sugarcane yeast
Materials and methods
Item
All experimental procedures were approved by the Ethics and Animal Welfare Committee of the College of Agrarian and Veterinarian Sciences, S~ ao Paulo State University, Jaboticabal, Brazil (protocol 024116-08).
Analysed chemical composition, % (as-fed basis)s Dry matter 93.7 92.1 Crude protein 47.0 39.1 Lysine 2.60 2.27 Threonine 1.82 1.73 Methionine 0.61 0.45 Cystine 0.43 0.25 Alanine 2.62 1.95 Arginine 1.94 1.37 Aspartic acid 3.55 3.09 Glutamic acid 5.36 3.49 Glycine 1.76 1.36 Hystidine 0.84 0.67 Isoleucine 1.66 1.51 Leucine 2.67 2.18 Phenylalanine 1.66 1.38 Serine 1.94 1.84 Tyrosine 1.34 1.11 Valine 2.06 1.76 Acid-hydrolysed fat 2.9 2.5 Neutral detergent fibre 2.2 0.6 Ash 6.0 4.4 Calcium 0.1 0.1 Phosphorous 1.3 0.7 Potassium 1.6 0.9 Magnesium 0.2 0.1 Sodium 0.1 0.4 Chloride 0.0 0.0 Sulphur 0.2 1.2 Gross energy (kJ/g As-fed) 16.1 14.5
Animals and experimental design
A total of 62 healthy adult beagles of average age 6.36 1.31 years and weighing 10.87 3.78 kg were used in the experiments. The dogs belong and were housed in the Laboratory of Research on Nutrition and Nutritional Diseases of Dogs and Cats at the S~ ao Paulo State University in Jaboticabal, Brazil. During the digestibility trials, the dogs were kept in individual stainless steel metabolic cages (100 9 100 9 100 cm), which were equipped with a system to separate faeces and urine for collection. In experiment 1, the substitution method (Matterson et al., 1965) was used to calculate the coefficient of total tract apparent digestibility (CTTAD) of nutrients and metabolizable energy (ME) content of BY, ASCY and ISCY. Twenty-eight dogs were randomly assigned to one of four diets, one reference diet and three test diets composed of 85% reference diet and 15% BY, ASCY or ISCY on as-fed basis. The experiment was conducted in two blocks: the first block included 16 dogs (four dogs per diet) and the second block included 12 dogs (three dogs per diet); thus, Journal of Animal Physiology and Animal Nutrition . © 2013 Blackwell Verlag GmbH
Brewer’s yeast
Autolysed
Integral
92.7 42.2 2.69 1.97 0.58 0.33 2.30 1.63 3.46 3.83 1.55 0.72 1.67 2.46 1.44 2.07 1.14 2.01 2.6 0.7 5.7 0.0 1.3 1.5 0.1 0.0 0.0 0.5 15.8
*Analysed in duplicate with variation coefficient below 5%.
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submitted to electronic microscopy scanning (JEOL JSM 5410 Jeol, Tokyo, Japan). The samples were fixed with double-sided tape to the aluminium supports and coated with a 36–nm-thick palladium gold layer on a vacuum device (Denton vacuum DESK II, Frehold, New Jersey, USA), and the microscope was operated at 15 KV. Micrographs were taken with Sony thermal paper UPP-110 HD and Kodak TMAX ASA 100 film (Sony, Tokyo, Japan) to observe the surface of the yeast cell membranes (Figs 1 and 2). In experiment 1, the reference diet was formulated according to AAFCO (2008) nutritional recommendations for adult dog maintenance. Initially, each of the three test diets was prepared using 70% reference diet
Fig. 1 Scan electronic microscopy picture of the integral sugarcane yeast (Saccharomyces cerevisiae). The cell membranes are intact and well defined (3.5009).
and 30% yeast. However, the dogs presented diarrhoea and soft faeces, so the substitution level was reduced to 85% reference diet and 15% yeast (Sakomura and Rostagno, 2007). The ingredients and chemical composition of the reference diet are shown in Table 2 and of the test diets in Table 3. For experiment 2, the same reference diet was used from experiment 1, and the four other diets were formulated with 7.5% or 15% of ASCY and 7.5% or 15% of ISCY (as-fed basis). The protein in the reference diet was primarily derived from poultry by-product meal. During the formulation of the other diets, yeast protein replaced the protein from the poultry by-product meal to achieve diets with similar protein content. Also, soybean hulls were added to produce diets with similar amounts of neutral detergent fibre (Table 3). All of the diets used in experiments 1 and 2 were mixed and ground in a hammer mill (Model 4, D’Andrea, Limeira, Brazil) fitted with a sieve with 1.0 mm screens before being extruded in a singlescrew extruder (Mab 400S, Extrucenter, Monte Alto, Brazil) and kibbled under identical processing conditions. The manufacturing process was controlled by adjusting kibble density between 400 and 450 g/l (asfed basis) every 15 min to ensure consistent cooking and kibble quality (size and expansion). The extruder preconditioning temperature was kept above 90 °C, and the water, steam, screw speed and ration flux were adjusted according to diet formulation. Extrusion temperatures varied between 125 °C and 135 °C. The extruder die had a single hole of 8 mm, the productivity was approximately 150 kg/h (as-is basis) Table 2 Chemical compositions of the test diets used in the Experiment 1 (DM-basis)*,†
Item
85% Reference diet and 15% of brewer’s yeast
Chemical composition, % (DM-basis) Dry matter 92.0 Ash 6.6 Crude protein 35.6 Acid-hydrolysed 12.8 fat Neutral detergent 8.8 fibre Crude energy (kJ/g) 19.8
85% Reference diet and 15% of autolysed sugarcane yeast
85% Reference diet and 15% of integral sugarcane yeast
92.9 5.4 31.2 11.7
92.1 5.9 31.8 12.4
9.2
8.8
19.6
19.5
Fig. 2 Scan electronic microscopy picture of the autolyzed sugarcane yeast (Saccharomyces cerevisiae). The cell membranes are disrupted and poorly defined (3.5009).
*Analysed in duplicate with variation coefficient below 5%. †The chemical composition of the reference diet used in the Experiment 1 is described on Table 3.
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Table 3 Ingredient and chemical composition, and quality parameters of the experimental diets used in Experiment 1 (reference diet), and Experiment 2 (reference diet and the diets with autolyssed and integral sugarcane yeast)
Item
Reference diet
Autolysed sugarcane yeast
Integral sugarcane yeast
7.5%
7.5%
Ingredients, % (as-fed basis) Maize 30.0 Broken rice 23.2 Poultry by-product meal 26.0 Maize gluten meal 7.0 Sugarcane yeast 0.0 Poultry fat 6.7 Palatant* 2.0 Dicalcium phosphate 0.05 Mineral-vitamin mixture† 0.50 Soybean hulls 2.56 Sodium chloride 0.50 Mould inhibitors‡ 0.10 Dl-Methionine 0.20 L-Lysine 0.21 Antioxidant§ 0.05 Calcium carbonate 0.44 Potassium chloride 0.49 Chemical composition, % (DM-basis)¶ Dry matter 92.9 Crude protein 29.9 Acid-hydrolysed fat 10.6 Neutral detergent fibre 10.5 Ash 6.4 Gross energy (kJ/g, DM-basis) 19.9 Starch gelatinization degree (%) 93.0 Kibble density (g/l) 445 25 430 Contribution to dietary crude protein (% of the total protein of the food) Poultry by-product meal 56.9 Evaluated yeast –
15%
30.0 18.7 21.9 7.0 7.5 7.2 2.0 0.30 0.50 2.90 0.50 0.10 0.20 0.11 0.05 0.62 0.42
30.0 14.3 17.7 7.0 15.0 7.7 2.0 0.60 0.50 3.23 0.50 0.10 0.18 0.01 0.05 0.80 0.34
15%
30.0 19.2 21.7 7.0 7.5 7.2 2.0 0.10 0.50 2.91 0.50 0.10 0.20 0.11 0.05 0.65 0.33
30.0 15.2 17.4 7.0 15.0 7.7 2.0 0.16 0.50 3.25 0.50 0.10 0.18 0.01 0.05 0.79 0.17
92.5 30.2 10.3 10.4 6.7 19.6 85.7 24
91.9 30.4 10.5 10.0 6.9 19.7 86.8 406 15
92.7 30.1 11.2 9.3 6.8 20.0 83.3 414 13
92.4 29.7 11.1 9.5 6.9 19.5 84.8 435 27
47.4 10.5
37.9 20.9
47.0 11.3
38.3 22.9
*Hydrolysed liver. †Added per kilogram of diet: vitamin A, 15 000 IU; vitamin D3, 1300 IU; vitamin E, 200 IU; thiamin, 10 mg; riboflavin, 14 mg; pantothenic acid, 60 mg; niacin, 90 mg; pyridoxine, 9 mg; folic acid, 0.50 mg; vitamin B12, 0.2 mg; iron, 130 mg; copper, 13 mg; magnesium, 13 mg; zinc, 180 mg; iodine, 2 mg; selenium, 0.3 mg. ‡Mould Zap: Ammonium dipropionate, acetic acid, sorbic acid and benzoic acid. Alltech do Brasil Agroindustrial Ltda, Curitiba, Brazil. §Banox: BHA, BHT, propyl gallate and calcium carbonate. Alltech do Brasil Agroindustrial Ltda, Curitiba, Brazil. ¶Analysed in duplicate with variation coefficient below 5%.
and the ratio of the open area to the die food productivity was 300 mm2/Ton-h. The degree of starch gelatinization was measured in all diets to assess extrusion quality using the amyloglucosidase method (Sa et al., 2013).
In each experiment, the dogs were fed with the experimental diets for 10 days, and faeces and urine were collected in total during the last 5 days following the guidelines of Association of American Feed Control
Officials (AAFCO, 2008) to determine food metabolizable energy. The CTTADs of nutrients were also determined with the same protocol. The dogs were fed twice daily (at 0800 and 1600 h), the food ME content was estimated from their chemical composition and the amount supplied calculated by the equation 397 kJ ME per kg body weight 0.75 per day (National Research Council 2006). The dogs were given 15 min to consume their meals after which their bowls were removed and any food remaining was weighed and recorded. The dogs had free access to water. On the first day of collection (day 6), all faeces and urine were removed from the cages and discarded before 0800 h
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and total faecal and urine output for each dog was collected from this point for the next five days. Faeces and urine were collected twice daily, weighed and kept frozen at 15 °C until analysis. Urine was collected in plastic containers containing 1 ml of sulphuric acid 1 Eq/l to avoid loss of nitrogen and bacterial growth. During the collection phase, faecal quality was measured according to the following score system (Carciofi et al., 2008): 0 = watery liquid which can be poured; 1 = soft, unformed; 2 = soft, malformed stool which assumes shape of container; 3 = soft, formed, and moist but retains shape; 4 = well-formed and consistent stool which does not adhere to the floor; and 5 = small, hard, dry pellets. Preparation and analysis of food, urine and faeces
Faeces were dried in a forced air oven at 55 °C for 72 h (320-SE, Fanem, S~ ao Paulo, Brazil). Dried faecal samples and diets were ground in a cutting mill (Mod MA-350, Marconi, Piracicaba, Brazil) fitted with a 1 mm screen. Urine samples (approximately 90 g) were weighed and placed in three Petri dishes (30 g each), dried in a forced air oven at 55 °C for 24 h (320-SE, Fanem, S~ ao Paulo, Brazil), and the dry residue was placed into a silicon capsule for combustion. The yeast ingredients, diets and faeces were tested for dry matter (DM) by oven-drying the sample (method 934.01), ash by muffle furnace incineration (method 942.05), crude protein (CP) by the Kjeldahl method (method 954.01) and acid-hydrolysed fat (AHF) using a Soxhlet apparatus (method 954.02) according to the Association of Official Analytical Chemists (AOAC, 1995). Organic matter (OM) was calculated as DM – ash. Minerals were analysed after nitro perchloric digestion; phosphorus was measured by visible spectrophotometry (Labquest Bio 2000. Labtest Diagn ostica S.A., Lagoa Santa, Brazil), calcium, potassium, magnesium, chloride and sodium by flame atomic absorption spectrophotometry (GBC-932 AA, Scientific Equipment PTY LTD, Melbourne, Australia), and sulphur by the turbidimetric method according to the AOAC (1995). The amino acid contents of the yeast samples were analysed by the performic acid oxidation with acid hydrolysis–sodium metabisulfite method (method 994.12) according to the AOAC (1995). Neutral detergent fibre (Goering and Van Soest, 1970) analysis included a pre-treatment of the sample with heat stable alpha amylase and was expressed exclusive of residual ash (aNDFom; Ud en et al., 2005). Gross energy (GE) content of BY, ASCY, ISCY, diets, faeces and urine was determined in a 952
bomb calorimeter (model 1261, Parr Instrument Company, Moline, IL, USA). Nitrogen content of urine samples was analysed as described previously for diets and faeces (AOAC, 1995). All analyses were carried out in duplicate, and the coefficient of variation was below 5% in all cases. Postprandial urea response
In experiment 2, after the completion of the digestibility assay, the dogs remained in the metabolic cages for three more days to perform a postprandial urea response test according to Carciofi et al. (2009). By this time, the dogs had been conditioned to ingest all of their meal within 15 min; dogs that took longer than this to consume their total amount of food were not tested. The animals were deprived of food for 24 h, and each dog was aseptically catheterized using a peripheral intravenous catheter inserted into the cephalic vein (Angiocath 20 GA 9 1.16 in., Becton, Dickinson, USA). Blood samples were drawn prefeeding (baseline sample, time 0) and 1, 2, 3, 4, 4.5, 5, 6, 7, 8, 9 and 10 h post-feeding (the times were counted from the end of the meal). Meals and blood collections were always performed at the same time, starting at 0800 h. In each collection, a 3-ml blood sample was drawn using a syringe and transferred to a glass tube, and serum was separated within 15 min of collection and stored at 20 °C. Urea concentrations were determined by kinetic ultraviolet assay (Ur eia UV Liquiform, Labtest Diagn ostica S.A., Lagoa Santa, Brazil) within 24 h of collection using a semi-automated analyzer (Labquest model BIO-2000, Labtest Diagn ostica S.A., Lagoa Santa, Brazil). All analyses were carried out in duplicate, and the coefficient of variation was below 5% in all cases. Palatability test
Three of the diets from experiment 2 were used to evaluate the palatability effect of ASCY and ISCY inclusion on formulation. Three comparisons were performed: control diet versus 7.5% ASCY, control diet versus 7.5% ISCY and 7.5% ASCY versus 7.5% ISCY. Dog food palatability was measured using the two-pan method (Griffin, 2003) in which 38 individually housed dogs of different breeds and body weights were tested on two consecutive days. In the morning after a 12-h fast, the dogs received two pans, each containing one of the experimental foods, and were allowed to eat for 30 min. The position of the food pans was alternated at the evening meal. The amount of food offered in each pan surpassed the consumption Journal of Animal Physiology and Animal Nutrition . © 2013 Blackwell Verlag GmbH
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capacity of the animal to ensure there would be leftovers to measure. After 30 min, the pans were removed, the remains weighed and consumption was calculated by taking the difference. Due to the large differences in body weights, the results were calculated as relative consumption of each diet, and the mean intake of the two days for each dog was compared. Consumption rate was calculated by the formula: Relative consumption ð%Þ ¼
Food A consumption 100 Food A consumption + Food B consumption
Coefficient of total tract apparent digestibility, metabolizable energy and nitrogen balance calculations The ME of each experimental diet was calculated according to the quantitative collection of faeces and urine protocol and calculation procedures described by AAFCO (2008). The CTTADs of the nutrients were also calculated by the same procedure, using the analysed chemical composition of the diets and faeces. In experiment 1, the digestibility and ME of BY, ASCY and ISCY were calculated based on the digestibility values of the reference diet and test diets, and the inclusion level of the evaluated yeast corrected to DM-basis (Kawauchi et al., 2011), using the equation proposed by Matterson et al. (1965): ADing ¼ ADrd þ
effects. In experiment 1, when treatment differences were detected by variance analysis, means were compared by Tukey’s test. In experiment 2, polynomial and orthogonal contrasts were used to describe the relationship between ASCY and ISCY inclusion level and the evaluated criteria. Changes in serum urea concentrations were calculated for each postprandial interval. Responses were compared for the average and maximum increase,
ADtd ADrd ðingredientsubstitution; % DM basisÞ
Where ADing is the coefficient of total tract apparent digestibility of the ingredient of interest, ADrd is the CTTAD of the reference diet and ADtd is the CTTAD of the test diet. In experiment 2, nitrogen balance (NB) was determined. Nitrogen was measured in the diets, faeces and urine, and NB was calculated as the difference between ingested nitrogen (Nintake) and nitrogen excreted into the faeces (Nfaecal) and urine (Nurine) according to the following formula:
average and maximum incremental increase (difference between absolute and baseline urea concentrations), and time to peak increase. The integrated area under the curve (AUC) for the postprandial urea response and the AUC for the postprandial incremental urea were calculated by the trapezoidal method, using the software ORIGIN (Microcal Software Version 6.0. Northampton, MA, USA). Repeated measures analysis of variance with two among-animals factors (diet and period) and one within-animals factor (time of sampling) was used to evaluate the effects of diet and time on the dogs’ postprandial urea changes, with seven animals per treatment. Values of p < 0.05 were considered significant. All data were found to comply with the assumptions of ANOVA models. Results were presented as means standard error. Results
For both experiments, the data were analysed as a completely randomized block design using the general linear models procedure of Statistical Analysis Systems Institute (SAS) (1997). Model sums of squares were separated into treatment (diets), block and animal
The chemical composition of BY showed higher protein levels compared with the sugarcane yeasts (Table 1). Between the sugarcane yeasts, ASCY had less protein, potassium and phosphorus but more sodium and sulphur than ISCY. The chemical composition of the diets used in experiment 1 is presented in Table 2. The chemical composition and quality parameters (density and starch gelatinization degree) of the diets of experiment 2 are presented in Table 3. The quality parameters were adequate, and the diets were found to have similar compositions. All dogs adequately consumed the diets in both experiments. In experiment 1, nutrient intake did not differ between diets (p > 0.05; Table 4). The CTTAD of nutrients and energy was similar, except for crude protein which showed less digestibility for the dogs fed the ASCY 15% diet (p = 0.012). Due to this, the calculated protein digestibility of the ASCY was lower than those of ISCY and BY (p < 0.01; Table 5).
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NB (mg/kg BW0.75/d) = Nintake (Nfaecal + Nurine) Statistical analysis
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Table 4 Nutrient intake and coefficient of total tract apparent digestibility of the diets of the Experiment 1
Item
Reference diet (n = 7)
Mean body weight, kg 11.9 Nutrient intake, grams per dog/d Dry matter 148.2 Crude protein 44.3 Acid-hydrolysed fat 15.7 Neutral detergent fibre 15.6 Nutrient digestibility of the diets, % Dry matter 81.9 Organic matter 86.6 Crude protein 84.7a,b Acid-hydrolysed fat 85.8 Neutral detergent fibre 47.4 Gross energy 86.0
85% reference diet and 15% of brewer’s yeast (n = 7)
85% reference diet and 15% of autolysed sugarcane yeast (n = 7)
85% reference diet and 15% of integral sugarcane yeast (n = 7)
SEM*
p value
11.6
10.6
10.5
1.23
0.778
145.8 51.8 19.1 12.9
140.3 43.7 16.5 13.0
156.8 49.4 19.1 13.1
13.59 4.61 1.70 1.21
0.688 0.180 0.340 0.799
0.39 0.52 0.98 0.57 2.74 0.67
0.684 0.469 0.012 0.367 0.865 0.306
82.0 86.0 86.2a 88.3 38.3 84.6
79.9 83.9 80.7b 84.6 34.4 82.7
82.0 86.2 83.2a,b 85.7 35.9 85.7
Means in a row not sharing a common superscript differ (p < 0.05). *SEM = standard error of the mean (n = 7 dogs per diet). a,b
Table 5 Calculated coefficient of total tract apparent digestibility and digestible nutrient contents of extruded brewer’s yeast, and extruded autolysed and integral sugarcane yeast by dogs, obtained by the substitution method in the Experiment 1
Item
Brewer’s yeast
Sugarcane yeast Autolysed
Nutrient digestibility, % Dry matter 82.0 73.2 Crude protein 88.8 a 63.0b Gross energy 84.0 68.4 Digestible nutrients, % (As-fed basis) 34.6b Crude protein 43.4 a a Metabolizable 14.9 10.2b energy content (kJ/g, as-fed basis)
Integral
SEM*
p value
82.1 74.7 a,b 76.0
2.69 9.03 4.14
0.680 0.012 0.301
37.9b 12.3b
3.25 1.12
0.05), in fact none of the parameters evaluated in the urea postprandial response differed (p > 0.05), and due to this, only the AUC response is presented in Table 6. Both yeast promoted diet palatability. Their inclusion at 7.5% in the formula resulted in 70% higher consumption than the reference diet (p < 0.01), as illustrated in Fig. 3. Discussion
The ME (kJ/g of ingredient) contents of both sugarcane yeasts were also lower than in BY. In experiment 2, nutrient intake did not differ between diets (p > 0.05; Table 6). Nutrient digestibility was also similar, except for gross energy digestibility which was lower for the dogs fed the diets with yeast in comparison with the reference diet (p = 0.029). Also, a linear reduction on energy digestibility was verified for ASCY (p = 0.05). Faecal DM content decreased linearly with ISCY inclusion (p < 0.001), leading to a linear decrease in faecal score (p = 0.003). A general effect on faecal DM was also seen where the addition of yeast reduced faecal DM
The differences in the chemical compositions of ISCY and ASCY can be explained by the fact that ISCY is dried, washed yeast that was separated by centrifugation from the fermentation liquor, whereas ASCY is produced from sugarcane yeast that has undergone autolysis and has then been dried by spray drying with the fermentation liquor. No other study on the use of BY or sugarcane yeasts as a food for dogs was found in the literature. Brewer’s yeast and sugarcane yeasts are alternative protein sources that are currently being introduced in animal nutrition. Their high concentrations of protein, nucleotides, vitamins and of the sugars derived of its cell wall, namely mannan oligosaccharides and 1.3/1.6 b-glucan, may be important compounds for nutrition and health. In pigs, it was observed that growth and finishing performance are improved in animals fed yeast extract during the nursery phase (Carlson et al., 2005). We hypothesized that the yeast cell wall would be a barrier that reduced the digestibility of the internal
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Means in a row not sharing a superscript differ (p < 0.05). *SEM = standard error of the mean (n = 7 per diet). a,b
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Table 6 Food consumption, coefficients of total tract apparent digestibility, metabolizable energy content, faecal characteristics, nitrogen balance and postprandial urea response of dogs fed extruded diets with different inclusions of autolysed and integral sugarcane yeast in the Experiment 2 Sugarcane yeast
Item
Reference diet
Mean body weight (kg) 11.9 Nutrient intake, grams per dog/day Dry matter 148.2 Crude protein 44.3 Acid-hydrolysed fat 15.7 Neutral detergent fibre 15.6 Nutrient digestibility of the diets, % Dry matter 81.9 Organic matter 86.6 Crude protein 84.7 Acid-hydrolysed fat 85.8 Neutral detergent fibre 47.4 Gross energy 86.0 Metabolizable energy (kJ/g DM) 15.5 Faecal characteristics Faecal DM (%) 39.7 g faeces/dog/day (wet) 65.3 g faeces/dog/day (dry) 25.8 Faecal score 3.6 Nitrogen balance 210 (mg/kg BW0.75/d) Urea postprandial response test Area under the curve (mg/dl/h) 435
Contrasts
Autolysed
Integral
7.5%
7.5%
15%
Orthogonal
15%
SEM*
Linear
p value
Control x Yeast†
Autolysed x Integral‡
Autolysed
Integral
10.3
10.1
10.4
10.4
1.12
0.921
–
–
–
–
136.7 41.6 14.5 14.8
118.5 35.6 11.4 11.2
134.8 40.8 14.8 12.2
140.2 40.0 15.4 13.3
15.8 6.51 2.33 2.19
0.894 0.992 0.949 0.914
– – – –
– – – –
– – – –
– – – –
82.1 85.8 85.2 85.4 54.2 85.1 15.6
79.1 83.6 81.9 84.1 45.4 82.5 15.3
80.7 85.1 83.3 87.0 46.7 84.8 16.2
80.6 85.1 82.9 85.7 50.9 84.0 15.3
1.21 0.94 0.98 1.35 4.79 1.05 0.48
0.436 0.287 0.130 0.653 0.331 0.050 0.751
– – – – – 0.029 –
– – – – – 0.096 –
– – – – – 0.050 –
– – – – – 0.087 –
35.6 70.5 24.4 3.5 174
34.8 68.6 24.6 3.3 190
37.4 68.6 25.7 3.8 210
33.0 82.1 26.9 2.8 179
1.38 8.88 3.22 0.05 43.9
0.022 0.773 0.997 0.021 0.831
0.028 – – 0.082 –
0.159 – – 0.720 –
0.397 – – 0.330 –
ORIGINAL ARTICLE
Brewer’s yeast and sugarcane yeast as protein sources for dogs M. S. Martins1, N. K. Sakomura1, D. F. Souza1, F. O. R. Filho1, M. O. S. Gomes1, R. S. Vasconcellos2 and A. C. Carciofi1 1 Faculdade de Ci^encias Agr arias e Veterinarias, Universidade Estadual Paulista (UNESP), Jaboticabal, Brazil 2 Universidade Estadual de Maringa (UEM), Maringa, Brazil
Summary Brewer’s yeast (BY), autolysed sugarcane yeast (ASCY) and integral sugar cane yeast (ISCY) were studied in two experiments as ingredients for dog diets. In the first experiment, 28 dogs were randomly assigned to four diets; one reference diet and three test diets containing 15% of BY, ASCY or ISCY and 85% of the reference diet (asfed basis). The digestibilities of the yeasts were calculated by the substitution method. In the second experiment, 35 dogs were randomized to five diets with similar chemical composition but different levels of sugarcane yeast inclusion (0%, 7.5% ASCY, 15% ASCY, 7.5% ISCY and 15% ISCY). In both experiments, the coefficient of total tract apparent digestibility (CTTAD) of nutrients was determined through total collection of faeces. During experiment, two additional analyses of food palatability, nitrogen balance and urea postprandial responses were performed. The data were submitted to analysis of variance, and the means were compared by orthogonal or polynomial contrasts or Tukey’s test (p < 0.05). In experiment 1, CTTAD of protein was lower for both sugarcane yeasts than for BY (p = 0.012), as was metabolizable energy content (p = 0.025). In experiment 2, a linear reduction in energy digestibility with ASCY inclusion (p = 0.05) was verified. Furthermore, faecal score and DM content were reduced with ISCY inclusion (p < 0.003). No effect of yeast inclusion on nitrogen balance or postprandial urea response was found. Also, the inclusion of 7.5% of ASCY or ISCY increased diet palatability (p < 0.01). Yeasts present adequate digestibility by dogs, but its effect on faecal formation needs to be considered. No clear advantage for the use of ASCY over ISCY was found. In conclusion, we find that sugarcane yeast is suitable for inclusion in dog food and can enhance the overall palatability of the diet. Keywords canine, digestibility, palatability, Saccharomyces cerevisiae lio de Mesquita Filho (UNESP), Correspondence Dr. A. C. Carciofi, Departamento de Clınica e Cirurgia Veterinaria. Universidade Estadual Paulista Ju Via de Acesso Prof. Paulo Donato Castellane, s/n, Zip code 14884-900, Jaboticabal, SP, Brazil. Tel: +55 16 3209 2626; Fax: +55 16 3203 1226; E-mail: [email protected] Received: 17 March 2013; accepted: 22 October 2013
Introduction Sugarcane yeast and brewer’s yeast (Saccharomyces cerevisiae) are used as feed for many species, including bovine (Pereira et al., 2001), fish (Hisano et al., 2008), swine (Moreira et al., 2002; Castilho, 2004; White et al., 2009) and poultry (Granjeiro et al., 2001). Yeasts contain important characteristics for animal nutrition, such as high levels of B vitamins, nucleotides derived from cellular nucleic acids and mannan oligosaccharides and 1.3/1.6 b-glucan derived from cell membranes, that when consumed together can favourably influence gut and overall animal health (White et al., 2009). They are also considered palatable to many species, which is attributed to their elevated concentrations of glutamic acid and 5′-ribonucleotide which impart a savoury, umami taste (Kurihara and 948
Kashiwayanagi, 2000). In addition to glutamic acid, brewer’s and sugarcane yeasts have high levels of amino acids, and the total protein content varies from approximately 40 to 50% of dry matter. However, yeast has a rigid cell membrane with thicknesses varying from 150 to 400 nm (Osumi, 1998) which could reduce the absorption of the cell proteins and other nutrients. Currently, little is known about the bioavailability of yeast protein (Hisano et al., 2008), and, to our knowledge, no information for dogs is available. The worldwide production of brewer’s yeast is approximately 80 000 tons per year, with an apparently slow growth of 2% per year. Due to the recent increase in the ethanol production from sugarcane in Brazil, sugarcane yeast production is increasing at a rate of 4% per year (Santos, 2009). The potential production of sugarcane yeast in Brazil is around 500 000
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tons per year, but only 15% is being currently utilized for animal feed (Santos, 2009). The production of sugarcane yeast starts with inoculation of fermentation chambers of sugarcane juice. Yeast cell mass is separated from the fermentation liquor by centrifugation, washed and dried, yielding what is called integral sugarcane yeast (ISCY). Additionally, the mass of living yeast cells can be submitted to osmotic stress and enzymatic hydrolysis which promotes cellular autolysis that ruptures the cellular membrane and releases the cell contents. When dried, typically by spray drying this product is called autolysed sugarcane yeast (ASCY). One study using pelleted diets for fish reported better digestibility of ASCY than ISCY (Hisano et al., 2008). Considering the lack of information on the use of yeast as a protein source for dogs, the present study was designed to compare the digestibility of brewer’s yeast (BY) to ASCY and ISCY. Additionally, the effects of different levels of dietary ASCY and ISCY on nutrient digestibility, nitrogen balance, food palatability and urea postprandial response in dogs were evaluated.
Yeast as a protein source for dogs
there were seven replicates (dogs) per treatment (diet). In experiment 2, the CTTAD of nutrients and energy, food palatability and urea postprandial response were evaluated using diets containing different levels of ASCY and ISCY. Five isonutritive diets were used, the standard control diet without yeast and four test diets with 7.5% ASCY, 15% ASCY, 7.5% ISCY or 15% ISCY. Thirty-five dogs were used in a randomized block design, with 20 dogs (four dogs per diet) in the first block and 15 dogs (three dogs per diet) in the second block, totalling seven replicates (dogs) per treatment (diet). Ingredients and diets
The chemical compositions of the BY, ASCY and ISCY ingredients are shown in Table 1 and were determined as follows. The ASCY and ISCY samples were Table 1 Chemical composition of the brewer’s yeast and the autolysed and integral sugarcane yeast used in experiments one and two* Sugarcane yeast
Materials and methods
Item
All experimental procedures were approved by the Ethics and Animal Welfare Committee of the College of Agrarian and Veterinarian Sciences, S~ ao Paulo State University, Jaboticabal, Brazil (protocol 024116-08).
Analysed chemical composition, % (as-fed basis)s Dry matter 93.7 92.1 Crude protein 47.0 39.1 Lysine 2.60 2.27 Threonine 1.82 1.73 Methionine 0.61 0.45 Cystine 0.43 0.25 Alanine 2.62 1.95 Arginine 1.94 1.37 Aspartic acid 3.55 3.09 Glutamic acid 5.36 3.49 Glycine 1.76 1.36 Hystidine 0.84 0.67 Isoleucine 1.66 1.51 Leucine 2.67 2.18 Phenylalanine 1.66 1.38 Serine 1.94 1.84 Tyrosine 1.34 1.11 Valine 2.06 1.76 Acid-hydrolysed fat 2.9 2.5 Neutral detergent fibre 2.2 0.6 Ash 6.0 4.4 Calcium 0.1 0.1 Phosphorous 1.3 0.7 Potassium 1.6 0.9 Magnesium 0.2 0.1 Sodium 0.1 0.4 Chloride 0.0 0.0 Sulphur 0.2 1.2 Gross energy (kJ/g As-fed) 16.1 14.5
Animals and experimental design
A total of 62 healthy adult beagles of average age 6.36 1.31 years and weighing 10.87 3.78 kg were used in the experiments. The dogs belong and were housed in the Laboratory of Research on Nutrition and Nutritional Diseases of Dogs and Cats at the S~ ao Paulo State University in Jaboticabal, Brazil. During the digestibility trials, the dogs were kept in individual stainless steel metabolic cages (100 9 100 9 100 cm), which were equipped with a system to separate faeces and urine for collection. In experiment 1, the substitution method (Matterson et al., 1965) was used to calculate the coefficient of total tract apparent digestibility (CTTAD) of nutrients and metabolizable energy (ME) content of BY, ASCY and ISCY. Twenty-eight dogs were randomly assigned to one of four diets, one reference diet and three test diets composed of 85% reference diet and 15% BY, ASCY or ISCY on as-fed basis. The experiment was conducted in two blocks: the first block included 16 dogs (four dogs per diet) and the second block included 12 dogs (three dogs per diet); thus, Journal of Animal Physiology and Animal Nutrition . © 2013 Blackwell Verlag GmbH
Brewer’s yeast
Autolysed
Integral
92.7 42.2 2.69 1.97 0.58 0.33 2.30 1.63 3.46 3.83 1.55 0.72 1.67 2.46 1.44 2.07 1.14 2.01 2.6 0.7 5.7 0.0 1.3 1.5 0.1 0.0 0.0 0.5 15.8
*Analysed in duplicate with variation coefficient below 5%.
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submitted to electronic microscopy scanning (JEOL JSM 5410 Jeol, Tokyo, Japan). The samples were fixed with double-sided tape to the aluminium supports and coated with a 36–nm-thick palladium gold layer on a vacuum device (Denton vacuum DESK II, Frehold, New Jersey, USA), and the microscope was operated at 15 KV. Micrographs were taken with Sony thermal paper UPP-110 HD and Kodak TMAX ASA 100 film (Sony, Tokyo, Japan) to observe the surface of the yeast cell membranes (Figs 1 and 2). In experiment 1, the reference diet was formulated according to AAFCO (2008) nutritional recommendations for adult dog maintenance. Initially, each of the three test diets was prepared using 70% reference diet
Fig. 1 Scan electronic microscopy picture of the integral sugarcane yeast (Saccharomyces cerevisiae). The cell membranes are intact and well defined (3.5009).
and 30% yeast. However, the dogs presented diarrhoea and soft faeces, so the substitution level was reduced to 85% reference diet and 15% yeast (Sakomura and Rostagno, 2007). The ingredients and chemical composition of the reference diet are shown in Table 2 and of the test diets in Table 3. For experiment 2, the same reference diet was used from experiment 1, and the four other diets were formulated with 7.5% or 15% of ASCY and 7.5% or 15% of ISCY (as-fed basis). The protein in the reference diet was primarily derived from poultry by-product meal. During the formulation of the other diets, yeast protein replaced the protein from the poultry by-product meal to achieve diets with similar protein content. Also, soybean hulls were added to produce diets with similar amounts of neutral detergent fibre (Table 3). All of the diets used in experiments 1 and 2 were mixed and ground in a hammer mill (Model 4, D’Andrea, Limeira, Brazil) fitted with a sieve with 1.0 mm screens before being extruded in a singlescrew extruder (Mab 400S, Extrucenter, Monte Alto, Brazil) and kibbled under identical processing conditions. The manufacturing process was controlled by adjusting kibble density between 400 and 450 g/l (asfed basis) every 15 min to ensure consistent cooking and kibble quality (size and expansion). The extruder preconditioning temperature was kept above 90 °C, and the water, steam, screw speed and ration flux were adjusted according to diet formulation. Extrusion temperatures varied between 125 °C and 135 °C. The extruder die had a single hole of 8 mm, the productivity was approximately 150 kg/h (as-is basis) Table 2 Chemical compositions of the test diets used in the Experiment 1 (DM-basis)*,†
Item
85% Reference diet and 15% of brewer’s yeast
Chemical composition, % (DM-basis) Dry matter 92.0 Ash 6.6 Crude protein 35.6 Acid-hydrolysed 12.8 fat Neutral detergent 8.8 fibre Crude energy (kJ/g) 19.8
85% Reference diet and 15% of autolysed sugarcane yeast
85% Reference diet and 15% of integral sugarcane yeast
92.9 5.4 31.2 11.7
92.1 5.9 31.8 12.4
9.2
8.8
19.6
19.5
Fig. 2 Scan electronic microscopy picture of the autolyzed sugarcane yeast (Saccharomyces cerevisiae). The cell membranes are disrupted and poorly defined (3.5009).
*Analysed in duplicate with variation coefficient below 5%. †The chemical composition of the reference diet used in the Experiment 1 is described on Table 3.
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Table 3 Ingredient and chemical composition, and quality parameters of the experimental diets used in Experiment 1 (reference diet), and Experiment 2 (reference diet and the diets with autolyssed and integral sugarcane yeast)
Item
Reference diet
Autolysed sugarcane yeast
Integral sugarcane yeast
7.5%
7.5%
Ingredients, % (as-fed basis) Maize 30.0 Broken rice 23.2 Poultry by-product meal 26.0 Maize gluten meal 7.0 Sugarcane yeast 0.0 Poultry fat 6.7 Palatant* 2.0 Dicalcium phosphate 0.05 Mineral-vitamin mixture† 0.50 Soybean hulls 2.56 Sodium chloride 0.50 Mould inhibitors‡ 0.10 Dl-Methionine 0.20 L-Lysine 0.21 Antioxidant§ 0.05 Calcium carbonate 0.44 Potassium chloride 0.49 Chemical composition, % (DM-basis)¶ Dry matter 92.9 Crude protein 29.9 Acid-hydrolysed fat 10.6 Neutral detergent fibre 10.5 Ash 6.4 Gross energy (kJ/g, DM-basis) 19.9 Starch gelatinization degree (%) 93.0 Kibble density (g/l) 445 25 430 Contribution to dietary crude protein (% of the total protein of the food) Poultry by-product meal 56.9 Evaluated yeast –
15%
30.0 18.7 21.9 7.0 7.5 7.2 2.0 0.30 0.50 2.90 0.50 0.10 0.20 0.11 0.05 0.62 0.42
30.0 14.3 17.7 7.0 15.0 7.7 2.0 0.60 0.50 3.23 0.50 0.10 0.18 0.01 0.05 0.80 0.34
15%
30.0 19.2 21.7 7.0 7.5 7.2 2.0 0.10 0.50 2.91 0.50 0.10 0.20 0.11 0.05 0.65 0.33
30.0 15.2 17.4 7.0 15.0 7.7 2.0 0.16 0.50 3.25 0.50 0.10 0.18 0.01 0.05 0.79 0.17
92.5 30.2 10.3 10.4 6.7 19.6 85.7 24
91.9 30.4 10.5 10.0 6.9 19.7 86.8 406 15
92.7 30.1 11.2 9.3 6.8 20.0 83.3 414 13
92.4 29.7 11.1 9.5 6.9 19.5 84.8 435 27
47.4 10.5
37.9 20.9
47.0 11.3
38.3 22.9
*Hydrolysed liver. †Added per kilogram of diet: vitamin A, 15 000 IU; vitamin D3, 1300 IU; vitamin E, 200 IU; thiamin, 10 mg; riboflavin, 14 mg; pantothenic acid, 60 mg; niacin, 90 mg; pyridoxine, 9 mg; folic acid, 0.50 mg; vitamin B12, 0.2 mg; iron, 130 mg; copper, 13 mg; magnesium, 13 mg; zinc, 180 mg; iodine, 2 mg; selenium, 0.3 mg. ‡Mould Zap: Ammonium dipropionate, acetic acid, sorbic acid and benzoic acid. Alltech do Brasil Agroindustrial Ltda, Curitiba, Brazil. §Banox: BHA, BHT, propyl gallate and calcium carbonate. Alltech do Brasil Agroindustrial Ltda, Curitiba, Brazil. ¶Analysed in duplicate with variation coefficient below 5%.
and the ratio of the open area to the die food productivity was 300 mm2/Ton-h. The degree of starch gelatinization was measured in all diets to assess extrusion quality using the amyloglucosidase method (Sa et al., 2013).
In each experiment, the dogs were fed with the experimental diets for 10 days, and faeces and urine were collected in total during the last 5 days following the guidelines of Association of American Feed Control
Officials (AAFCO, 2008) to determine food metabolizable energy. The CTTADs of nutrients were also determined with the same protocol. The dogs were fed twice daily (at 0800 and 1600 h), the food ME content was estimated from their chemical composition and the amount supplied calculated by the equation 397 kJ ME per kg body weight 0.75 per day (National Research Council 2006). The dogs were given 15 min to consume their meals after which their bowls were removed and any food remaining was weighed and recorded. The dogs had free access to water. On the first day of collection (day 6), all faeces and urine were removed from the cages and discarded before 0800 h
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and total faecal and urine output for each dog was collected from this point for the next five days. Faeces and urine were collected twice daily, weighed and kept frozen at 15 °C until analysis. Urine was collected in plastic containers containing 1 ml of sulphuric acid 1 Eq/l to avoid loss of nitrogen and bacterial growth. During the collection phase, faecal quality was measured according to the following score system (Carciofi et al., 2008): 0 = watery liquid which can be poured; 1 = soft, unformed; 2 = soft, malformed stool which assumes shape of container; 3 = soft, formed, and moist but retains shape; 4 = well-formed and consistent stool which does not adhere to the floor; and 5 = small, hard, dry pellets. Preparation and analysis of food, urine and faeces
Faeces were dried in a forced air oven at 55 °C for 72 h (320-SE, Fanem, S~ ao Paulo, Brazil). Dried faecal samples and diets were ground in a cutting mill (Mod MA-350, Marconi, Piracicaba, Brazil) fitted with a 1 mm screen. Urine samples (approximately 90 g) were weighed and placed in three Petri dishes (30 g each), dried in a forced air oven at 55 °C for 24 h (320-SE, Fanem, S~ ao Paulo, Brazil), and the dry residue was placed into a silicon capsule for combustion. The yeast ingredients, diets and faeces were tested for dry matter (DM) by oven-drying the sample (method 934.01), ash by muffle furnace incineration (method 942.05), crude protein (CP) by the Kjeldahl method (method 954.01) and acid-hydrolysed fat (AHF) using a Soxhlet apparatus (method 954.02) according to the Association of Official Analytical Chemists (AOAC, 1995). Organic matter (OM) was calculated as DM – ash. Minerals were analysed after nitro perchloric digestion; phosphorus was measured by visible spectrophotometry (Labquest Bio 2000. Labtest Diagn ostica S.A., Lagoa Santa, Brazil), calcium, potassium, magnesium, chloride and sodium by flame atomic absorption spectrophotometry (GBC-932 AA, Scientific Equipment PTY LTD, Melbourne, Australia), and sulphur by the turbidimetric method according to the AOAC (1995). The amino acid contents of the yeast samples were analysed by the performic acid oxidation with acid hydrolysis–sodium metabisulfite method (method 994.12) according to the AOAC (1995). Neutral detergent fibre (Goering and Van Soest, 1970) analysis included a pre-treatment of the sample with heat stable alpha amylase and was expressed exclusive of residual ash (aNDFom; Ud en et al., 2005). Gross energy (GE) content of BY, ASCY, ISCY, diets, faeces and urine was determined in a 952
bomb calorimeter (model 1261, Parr Instrument Company, Moline, IL, USA). Nitrogen content of urine samples was analysed as described previously for diets and faeces (AOAC, 1995). All analyses were carried out in duplicate, and the coefficient of variation was below 5% in all cases. Postprandial urea response
In experiment 2, after the completion of the digestibility assay, the dogs remained in the metabolic cages for three more days to perform a postprandial urea response test according to Carciofi et al. (2009). By this time, the dogs had been conditioned to ingest all of their meal within 15 min; dogs that took longer than this to consume their total amount of food were not tested. The animals were deprived of food for 24 h, and each dog was aseptically catheterized using a peripheral intravenous catheter inserted into the cephalic vein (Angiocath 20 GA 9 1.16 in., Becton, Dickinson, USA). Blood samples were drawn prefeeding (baseline sample, time 0) and 1, 2, 3, 4, 4.5, 5, 6, 7, 8, 9 and 10 h post-feeding (the times were counted from the end of the meal). Meals and blood collections were always performed at the same time, starting at 0800 h. In each collection, a 3-ml blood sample was drawn using a syringe and transferred to a glass tube, and serum was separated within 15 min of collection and stored at 20 °C. Urea concentrations were determined by kinetic ultraviolet assay (Ur eia UV Liquiform, Labtest Diagn ostica S.A., Lagoa Santa, Brazil) within 24 h of collection using a semi-automated analyzer (Labquest model BIO-2000, Labtest Diagn ostica S.A., Lagoa Santa, Brazil). All analyses were carried out in duplicate, and the coefficient of variation was below 5% in all cases. Palatability test
Three of the diets from experiment 2 were used to evaluate the palatability effect of ASCY and ISCY inclusion on formulation. Three comparisons were performed: control diet versus 7.5% ASCY, control diet versus 7.5% ISCY and 7.5% ASCY versus 7.5% ISCY. Dog food palatability was measured using the two-pan method (Griffin, 2003) in which 38 individually housed dogs of different breeds and body weights were tested on two consecutive days. In the morning after a 12-h fast, the dogs received two pans, each containing one of the experimental foods, and were allowed to eat for 30 min. The position of the food pans was alternated at the evening meal. The amount of food offered in each pan surpassed the consumption Journal of Animal Physiology and Animal Nutrition . © 2013 Blackwell Verlag GmbH
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capacity of the animal to ensure there would be leftovers to measure. After 30 min, the pans were removed, the remains weighed and consumption was calculated by taking the difference. Due to the large differences in body weights, the results were calculated as relative consumption of each diet, and the mean intake of the two days for each dog was compared. Consumption rate was calculated by the formula: Relative consumption ð%Þ ¼
Food A consumption 100 Food A consumption + Food B consumption
Coefficient of total tract apparent digestibility, metabolizable energy and nitrogen balance calculations The ME of each experimental diet was calculated according to the quantitative collection of faeces and urine protocol and calculation procedures described by AAFCO (2008). The CTTADs of the nutrients were also calculated by the same procedure, using the analysed chemical composition of the diets and faeces. In experiment 1, the digestibility and ME of BY, ASCY and ISCY were calculated based on the digestibility values of the reference diet and test diets, and the inclusion level of the evaluated yeast corrected to DM-basis (Kawauchi et al., 2011), using the equation proposed by Matterson et al. (1965): ADing ¼ ADrd þ
effects. In experiment 1, when treatment differences were detected by variance analysis, means were compared by Tukey’s test. In experiment 2, polynomial and orthogonal contrasts were used to describe the relationship between ASCY and ISCY inclusion level and the evaluated criteria. Changes in serum urea concentrations were calculated for each postprandial interval. Responses were compared for the average and maximum increase,
ADtd ADrd ðingredientsubstitution; % DM basisÞ
Where ADing is the coefficient of total tract apparent digestibility of the ingredient of interest, ADrd is the CTTAD of the reference diet and ADtd is the CTTAD of the test diet. In experiment 2, nitrogen balance (NB) was determined. Nitrogen was measured in the diets, faeces and urine, and NB was calculated as the difference between ingested nitrogen (Nintake) and nitrogen excreted into the faeces (Nfaecal) and urine (Nurine) according to the following formula:
average and maximum incremental increase (difference between absolute and baseline urea concentrations), and time to peak increase. The integrated area under the curve (AUC) for the postprandial urea response and the AUC for the postprandial incremental urea were calculated by the trapezoidal method, using the software ORIGIN (Microcal Software Version 6.0. Northampton, MA, USA). Repeated measures analysis of variance with two among-animals factors (diet and period) and one within-animals factor (time of sampling) was used to evaluate the effects of diet and time on the dogs’ postprandial urea changes, with seven animals per treatment. Values of p < 0.05 were considered significant. All data were found to comply with the assumptions of ANOVA models. Results were presented as means standard error. Results
For both experiments, the data were analysed as a completely randomized block design using the general linear models procedure of Statistical Analysis Systems Institute (SAS) (1997). Model sums of squares were separated into treatment (diets), block and animal
The chemical composition of BY showed higher protein levels compared with the sugarcane yeasts (Table 1). Between the sugarcane yeasts, ASCY had less protein, potassium and phosphorus but more sodium and sulphur than ISCY. The chemical composition of the diets used in experiment 1 is presented in Table 2. The chemical composition and quality parameters (density and starch gelatinization degree) of the diets of experiment 2 are presented in Table 3. The quality parameters were adequate, and the diets were found to have similar compositions. All dogs adequately consumed the diets in both experiments. In experiment 1, nutrient intake did not differ between diets (p > 0.05; Table 4). The CTTAD of nutrients and energy was similar, except for crude protein which showed less digestibility for the dogs fed the ASCY 15% diet (p = 0.012). Due to this, the calculated protein digestibility of the ASCY was lower than those of ISCY and BY (p < 0.01; Table 5).
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NB (mg/kg BW0.75/d) = Nintake (Nfaecal + Nurine) Statistical analysis
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Table 4 Nutrient intake and coefficient of total tract apparent digestibility of the diets of the Experiment 1
Item
Reference diet (n = 7)
Mean body weight, kg 11.9 Nutrient intake, grams per dog/d Dry matter 148.2 Crude protein 44.3 Acid-hydrolysed fat 15.7 Neutral detergent fibre 15.6 Nutrient digestibility of the diets, % Dry matter 81.9 Organic matter 86.6 Crude protein 84.7a,b Acid-hydrolysed fat 85.8 Neutral detergent fibre 47.4 Gross energy 86.0
85% reference diet and 15% of brewer’s yeast (n = 7)
85% reference diet and 15% of autolysed sugarcane yeast (n = 7)
85% reference diet and 15% of integral sugarcane yeast (n = 7)
SEM*
p value
11.6
10.6
10.5
1.23
0.778
145.8 51.8 19.1 12.9
140.3 43.7 16.5 13.0
156.8 49.4 19.1 13.1
13.59 4.61 1.70 1.21
0.688 0.180 0.340 0.799
0.39 0.52 0.98 0.57 2.74 0.67
0.684 0.469 0.012 0.367 0.865 0.306
82.0 86.0 86.2a 88.3 38.3 84.6
79.9 83.9 80.7b 84.6 34.4 82.7
82.0 86.2 83.2a,b 85.7 35.9 85.7
Means in a row not sharing a common superscript differ (p < 0.05). *SEM = standard error of the mean (n = 7 dogs per diet). a,b
Table 5 Calculated coefficient of total tract apparent digestibility and digestible nutrient contents of extruded brewer’s yeast, and extruded autolysed and integral sugarcane yeast by dogs, obtained by the substitution method in the Experiment 1
Item
Brewer’s yeast
Sugarcane yeast Autolysed
Nutrient digestibility, % Dry matter 82.0 73.2 Crude protein 88.8 a 63.0b Gross energy 84.0 68.4 Digestible nutrients, % (As-fed basis) 34.6b Crude protein 43.4 a a Metabolizable 14.9 10.2b energy content (kJ/g, as-fed basis)
Integral
SEM*
p value
82.1 74.7 a,b 76.0
2.69 9.03 4.14
0.680 0.012 0.301
37.9b 12.3b
3.25 1.12
0.05), in fact none of the parameters evaluated in the urea postprandial response differed (p > 0.05), and due to this, only the AUC response is presented in Table 6. Both yeast promoted diet palatability. Their inclusion at 7.5% in the formula resulted in 70% higher consumption than the reference diet (p < 0.01), as illustrated in Fig. 3. Discussion
The ME (kJ/g of ingredient) contents of both sugarcane yeasts were also lower than in BY. In experiment 2, nutrient intake did not differ between diets (p > 0.05; Table 6). Nutrient digestibility was also similar, except for gross energy digestibility which was lower for the dogs fed the diets with yeast in comparison with the reference diet (p = 0.029). Also, a linear reduction on energy digestibility was verified for ASCY (p = 0.05). Faecal DM content decreased linearly with ISCY inclusion (p < 0.001), leading to a linear decrease in faecal score (p = 0.003). A general effect on faecal DM was also seen where the addition of yeast reduced faecal DM
The differences in the chemical compositions of ISCY and ASCY can be explained by the fact that ISCY is dried, washed yeast that was separated by centrifugation from the fermentation liquor, whereas ASCY is produced from sugarcane yeast that has undergone autolysis and has then been dried by spray drying with the fermentation liquor. No other study on the use of BY or sugarcane yeasts as a food for dogs was found in the literature. Brewer’s yeast and sugarcane yeasts are alternative protein sources that are currently being introduced in animal nutrition. Their high concentrations of protein, nucleotides, vitamins and of the sugars derived of its cell wall, namely mannan oligosaccharides and 1.3/1.6 b-glucan, may be important compounds for nutrition and health. In pigs, it was observed that growth and finishing performance are improved in animals fed yeast extract during the nursery phase (Carlson et al., 2005). We hypothesized that the yeast cell wall would be a barrier that reduced the digestibility of the internal
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Journal of Animal Physiology and Animal Nutrition . © 2013 Blackwell Verlag GmbH
Means in a row not sharing a superscript differ (p < 0.05). *SEM = standard error of the mean (n = 7 per diet). a,b
M. S. Martins et al.
Yeast as a protein source for dogs
Table 6 Food consumption, coefficients of total tract apparent digestibility, metabolizable energy content, faecal characteristics, nitrogen balance and postprandial urea response of dogs fed extruded diets with different inclusions of autolysed and integral sugarcane yeast in the Experiment 2 Sugarcane yeast
Item
Reference diet
Mean body weight (kg) 11.9 Nutrient intake, grams per dog/day Dry matter 148.2 Crude protein 44.3 Acid-hydrolysed fat 15.7 Neutral detergent fibre 15.6 Nutrient digestibility of the diets, % Dry matter 81.9 Organic matter 86.6 Crude protein 84.7 Acid-hydrolysed fat 85.8 Neutral detergent fibre 47.4 Gross energy 86.0 Metabolizable energy (kJ/g DM) 15.5 Faecal characteristics Faecal DM (%) 39.7 g faeces/dog/day (wet) 65.3 g faeces/dog/day (dry) 25.8 Faecal score 3.6 Nitrogen balance 210 (mg/kg BW0.75/d) Urea postprandial response test Area under the curve (mg/dl/h) 435
Contrasts
Autolysed
Integral
7.5%
7.5%
15%
Orthogonal
15%
SEM*
Linear
p value
Control x Yeast†
Autolysed x Integral‡
Autolysed
Integral
10.3
10.1
10.4
10.4
1.12
0.921
–
–
–
–
136.7 41.6 14.5 14.8
118.5 35.6 11.4 11.2
134.8 40.8 14.8 12.2
140.2 40.0 15.4 13.3
15.8 6.51 2.33 2.19
0.894 0.992 0.949 0.914
– – – –
– – – –
– – – –
– – – –
82.1 85.8 85.2 85.4 54.2 85.1 15.6
79.1 83.6 81.9 84.1 45.4 82.5 15.3
80.7 85.1 83.3 87.0 46.7 84.8 16.2
80.6 85.1 82.9 85.7 50.9 84.0 15.3
1.21 0.94 0.98 1.35 4.79 1.05 0.48
0.436 0.287 0.130 0.653 0.331 0.050 0.751
– – – – – 0.029 –
– – – – – 0.096 –
– – – – – 0.050 –
– – – – – 0.087 –
35.6 70.5 24.4 3.5 174
34.8 68.6 24.6 3.3 190
37.4 68.6 25.7 3.8 210
33.0 82.1 26.9 2.8 179
1.38 8.88 3.22 0.05 43.9
0.022 0.773 0.997 0.021 0.831
0.028 – – 0.082 –
0.159 – – 0.720 –
0.397 – – 0.330 –
