181 Bull Vet Inst Pulawy 54, 181-187, 2010
EFFECT OF BREWERS’ YEAST (SACCHAROMYCES CEREVISIAE) EXTRACT ON SELECTED PARAMETERS OF HUMORAL AND CELLULAR IMMUNITY IN LAMBS ROMAN WÓJCIK Department of Microbiology and Clinical Immunology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-957 Olsztyn, Poland
[email protected] Received for publication September 11, 2009
Abstract The objective of this study was to determine the stimulating effect of the brewers’ yeast Saccharomyces cerevisiae dietary supplement on selected parameters of specific and non-specific humoral and cellular immunity in lambs. The study involved 32 lambs aged 30 ±3 d, divided into two equal groups: control and experimental. Animals in the experimental group were fed a C-J concentrate mixed with a prebiotic, the extract of dried brewers’ yeast containing 10%-15% MOS and 25%-30% β-1,3/1,6-D-glucan in the amount of 3 g/kg of the concentrate. At the beginning of the experiment (day 0) and on the 15th, 30th, and 60th d of the study, blood was sampled from the jugular vein to determine γ-globulin levels, lysozyme and ceruloplasmin activity, proliferative response of blood lymphocytes (MTT) after stimulation with LPS or ConA, metabolic activity (RBA), and potential killing activity (PKA) of phagocytes. As regards humoral immunity parameters, significantly higher γ-globulin levels and higher lysozyme and ceruloplasmin activity were noted in the blood serum of experimental lambs supplemented with the yeast extract, in comparison with control lambs not fed the supplement. No statistically significant differences in serum total protein were found between the control and experimental groups. The analysis of cellular immunity indicators revealed significantly higher levels of RBA and PKA, and higher MTT rates after stimulation with LPS or ConA in the experimental group, in comparison with the control group.
Key words: lambs, prebiotics, Saccharomyces cerevisiae, protein content, humoral immunity, cellular immunity. Owing to its prebiotic properties, Saccharomyces cerevisiae can be widely used as a natural productivity stimulator added to animals’ feed (9). The beneficial effects of yeast in the nutrition of ruminants have been noted in experiments on dairy cows (8) and suckling lambs (22). The above experiments also confirmed a stimulating effect of yeast on the health status of animals. The results of studies involving lambs (22, 35) indicate that β-1,3/1,6-D-glucan, a structural component of the cellular wall of Saccharomyces cerevisiae, plays an important role in this process, in regard to the indicators of both non-specific humoral immunity (19) and cellular immunity (35). The immunomodulating effect of Saccharomyces cerevisiae yeast is ascribed to mannan-oligosaccharides (MOS), which build the yeast's cell wall structure (19). By binding selected pathogens, MOS prevent them from colonising the host's gastrointestinal system and support their elimination by specialised immune cells (7, 12, 16). So far no studies have been conducted concerning the effect of dried yeast and yeast-derivative prebiotics on immunity indicators in lambs. The objective of this study was to determine the effect of the extract of dried brewers’ yeast
Saccharomyces cerevisiae (Biolex-MB40) with an increased β-1,3/1,6-D-glucan and MOS content on selected parameters of humoral and cellular immunity in lambs.
Material and Methods Experimental design. Thirty-two suckling Kamieniec breed lambs from a conservative herd, aged 30 ±3 d, were divided into two equal groups: I – control and II – experimental. Both groups were identical in terms of body weight on the second day of life, gender, birth type, as well as the age of the ewes, to eliminate possible differences in milk yield. Uniform feeding standards were applied in both groups in line with lamb nutrient requirements. Suckling lambs were fed haysilage of grass and legumes, and C-J concentrate. The quantity of administered feed and leftovers was monitored throughout the experiment. Experimental lambs were fed the Biolex–MB40 (Leiber GmbH) brewer's yeast (Saccharomyces cerevisiae) extract containing 10%-15% MOS and 25%-30% β-1,3/1,6-Dglucan. The extract was mixed with C-J concentrate in
182 the amount of 3 g/kg of the concentrate. C-J concentrate doses, identical for both groups, were increased every 10 d by 0.05 kg/animal/d, starting from 0.15 kg/animal/d. At the beginning of the experiment (day 0) and on the 15th, 30th, and 60th d of the study, blood was sampled from the jugular vein to determine selected parameters of humoral and cellular immunity. Evaluation of non-specific humoral immunity parameters. Lysozyme activity was determined by the turbidimetric method (25) modified by Siwicki and Anderson (29), ceruloplasmin activity – by the method developed by Siwicki and Studnicka (31), total protein content was determined by spectrophotometry as described by Lowry et al. (17) and modified by Siwicki and Anderson (29), and γ-globulin level was determined by the precipitation method modified by Siwicki and Anderson (29). Lysozyme activity. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. The serum was diluted 1:1 with phosphate buffer, and 0.1 ml of the solution was placed in microplate wells. Next, 0.5 ml of Micrococcus lysodeikticus suspension (25 mg bacteria/100 ml of phosphate buffer) (Sigma Chemical Co.) was added. Absorbance was measured directly after the addition of bacteria (E0) and after 1, 2, 3, and 30 min (final E). The final absorbance was subtracted from the initial absorbance (E0) to determine lysozyme activity with the use of a standard curve. The standard curve was plotted based on the optical density values for known lysozyme concentrations. Ceruloplasmin activity. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. The following buffers were prepared: 1) acetate buffer (pH 5.2, containing crystalline acetic acid, sodium acetate trihydrate, and 15 mg of EDTA), 2) buffered substrate solution (0.2% pphenyldiamine (PPD) in acetic buffer), 3) sodium azide solution (0.02% sodium azide solution in deionised water). 0.5 ml of buffered solution was added to each of two 16 x 100 mm test tubes immersed in a water bath at 37ºC. One test tube served as an experimental sample, and the other as control. 50 µl of serum was added to the experimental sample, which was incubated for 15 min at 37ºC. Next, 2 ml of a sodium azide solution was added to the experimental and control samples. 50 µl of serum was added to the control sample, and both samples were mixed. The absorbance of the experimental sample was measured at a wavelength of 540 nm, using the control sample as a blind test. Ceruloplasmin activity was determined with the use of the standard curve. The standard curve was plotted based on the optical density values for known ceruloplasmin concentrations. Total protein level. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. 5 µl of serum was placed in the wells, and 25 µl of reagent A and 200 µl of reagent B were added (Rio-Rad, Hercules, USA). Well contents were gently stirred with a pipette. The microplates were incubated at room temperature for 15 min. Next optical density was measured in a microplate reader at 620 nm. Total protein level was determined using a standard
curve as a reference. The standard curve was plotted based on optical density values for known protein dilutions. γ-globulin level. Whole blood samples were centrifuged for 5 min at 1,000 g to separate blood cells from the serum. The optical density of total protein was determined according to following procedure: 0.1 ml of serum was placed in the microplate wells, and 0.1 ml of 12% polyethylene glycol (10,000 kD) (Sigma Chemical Co.) suspended in deionised water was added. The microplates were incubated at room temperature for 2 h, and well contents were stirred continuously. The microplates were centrifuged for 10 min at 5,000 g to separate the γ-globulin fraction bound by polyethylene glycol (plate sediment) from the remaining total protein fraction, which constituted the supernatant. The optical density of supernatant was measured in a microplate reader at 620 nm. The optical density of supernatant was subtracted from the optical density of total protein. γglobulin content was determined using a standard curve (plotted earlier for total protein) as a reference, based on the ability of γ-globulins to bind with polyethylene glycol and precipitate. Evaluation of non-specific cellular immunity parameters. The metabolic activity of blood phagocytic cells was determined based on the intracellular measurements. Respiratory burst activity (RBA) after stimulation with PMA (Phorobol Myristate Acetate, Sigma), was determined according to the method described by Chung and Secombes (6) and adapted for dogs by Siwicki et al. (30). The isolated cells were resuspended in RPMI-1640 medium (Sigma) at 106 cells/mL. On 96-well U-shaped microplates, 100 µl of the isolated blood leukocytes was mixed with 100 µl of a 0.2% nitro blue tetrazolium (NBT, Sigma) solution in 0.2 M phosphate buffer (pH 7.2), and 1 µl of PMA at a concentration of 1 mg/mL in ethanol was added. After 30 min of incubation at 370C, the supernatant was removed from each well. The cell pellet was washed with absolute ethanol, and three times with 70% ethanol, and then was dried at room temperature. The amount of extracted reduced NBT after incubation with 2M KOH and DMSO (dimethylsulfoxide, Sigma) was measured colorimetrically at 620 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values. Potential killing activity (PKA) of mononuclear (MN) phagocytes and polymorphonuclear (PMN) phagocytes was determined in isolated blood leukocytes stimulated with killed microorganisms, according to the method presented by Rook et al. (26) and adapted for dogs by Siwicki et al. (30). On 96-well U-shaped microplates, 100 µl of leukocytes was mixed with 100 µl of 0.2 % NBT in phosphate buffer (pH 7.2), and 10 µl of killed Staphylococcus aureus strain 209P (containing 106 bacteria) was added. The mixture was incubated for 1 h at 370C and the supernatant was removed. The cell pellet was washed with absolute ethanol and three times with 70% ethanol, and then was dried at room temperature. This was followed by the
183 differences between groups was verified by the Student's t-test with the use of GraphPad Prism 5 software.
addition of 2 M KOH and DMSO to each well. The amount of extracted reduced NBT was measured at 620 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values. Evaluation of specific cellular immunity parameters. Lymphocyte proliferation rates after stimulation with mitogens, concanavalin A (ConA), and lipopolysaccharide (LPS), were determined by MTT spectrophotometry (OD 570 nm) using (3-[4,5dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide – 3-[4,5-dimethyl-2-thiazol]-2,5-diphenyl-2H-tetrazolium bromide), as described by Mosmann (23). MTT (Sigma) was dissolved in PBS at a concentration of 5 mg/ml and filtered. On 96-well culture plates (Sarstetd, USA), 100 µl of blood lymphocytes in RPMI - 1640 containing 10% FCS, 2 mM L-glutamine, 0.02 mM 2-mercaptoethanol, 1% hepes buffer, and penicillin/streptomycin (100 U/100 µg/ml) was mixed with 100 µl of RPMI - 1640 containing mitogens ConA (5 µg/ml), PHA (10µg/ml) or LPS (20 µg/ml). After 72 h incubation at 370C in a 5% carbon dioxide atmosphere (Memmert Incubator), 50 µl of MTT solution was added into each well, and plates were incubated for 4 h at 370C. After incubation the plates were centrifuged (1,400 g, 150C, 5 min). Supernatants were removed and 100 µl of DMSO (Sigma) were added into each well and incubated for 15 min at room temperature. After incubation, the solubilised reduced MTT was measured colorimetrically at 570 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values. The final results are presented as the reactivity index (RI). Statistical analysis. The obtained results were processed statistically by a one-factorial analysis of variance in an orthogonal design. The significance of
Results The obtained results indicate a significant effect of Biolex-MB40 on the analysed parameters of humoral and cellular immunity in lambs. An analysis of humoral immunity indicators– lysozyme and ceruloplasmin activities (Table 1) – revealed significantly (P≤0.01) higher values in the experimental group fed a diet with the addition of dried brewers’ yeast Saccharomyces cerevisiae than in the control group administered feed without yeast supplementation, over the entire experimental period. As regards the serum levels of γ-globulins (Table 1), their significant increase in the experimental group was reported only on the 30th (P≤0.05) and 60th (P≤0.01) d of the experiment in comparison with control group. In comparison with day 0 a significant increase in both ceruloplasmin and lysozyme activities (P≤0.01) was noted only in the experimental group on the successive days (15, 30, 60) of the experiment. Serum total protein content showed no significant differences between the experimental and control group (Table 1). In regard to the investigated indicators of nonspecific cellular immunity – RBA and PKA of phagocytes, and of specific cellular immunity - MTT stimulated with LPS and ConA (Table 2) – a statistically significant (P≤0.01 or P≤0.05) increase in their values was observed in the experimental group in comparison with the control group. The RBA in the group II was not marked by a significant increase in comparison with the group I only on the 60th d of the experiment. The RBA and MTT stimulated with LPS and ConA (Table 2) showed a significant increase (P≤0.01) only in the group I on successive days (15, 30, 60) of the experiment in comparison with day 0.
Table 1 Effect of Biolex-MB40 on the parameters of non-specific humoral immunity in lambs Parameter
Group
0
x Lysozyme activity (mg/L) Ceruloplasmin activity (mg/L) γ-globulin level (g/L) Total protein content (g/L)
I II I II I II I II
0.79 0.79 31.75 31.20 30.22 34.11 57.18 57.10
15 SD 0.08 0.06 0.61 0.77 5.41 6.81 4.07 2.85
x B
0.79 1.09 A** 31.28 B 37.18 A** 33.17 36.44 62.28 61.67
Experimental day 30 SD x 0.09 0.77 B 0.09 1.14 A** 1.15 31.60 B 0.98 37.52 A** 2.83 31.61 b 5.82 35.44 a 3.88 57.30 2.24 56.17
60 SD 0.08 0.06 0.49 0.7 1.27 3.87 2.26 1.53
a, b - P≤0.05; A, B - P≤0.01; SD - standard deviation; I – experimental group; II – control group. * P≤0.05 in comparison with experimental day 0, ** P≤0.01 in comparison with experimental day 0.
x B
0.81 1.17 A** 31.22 B 37.48 A** 30.67 B 39.44 A 58.15 56.25
SD 0.06 0.04 0.61 1.38 3.13 3.83 3.22 3.75
184 Table 2 Effect of Biolex-MB40 on the parameters of specific and non-specific cellular immunity in lambs Experimental day Parameter
0
Group
x I
RBA (OD 620 nm)
II I
PKA (OD 620 nm)
II I
MTT-ConA (RI)
II I
MTT-LPS (RI)
II
0.51 0.51 0.47 0.50 1.15 1.20 1.10 1.12
15 SD 0.06 0.02 0.04 0.04 0.08 0.15 0.05 0.11
x
SD
0.51 0.58
0.56
B
A*
1.30 1.55
b
a**
0.45
b
a**
1.02 1.36
30
B
A**
0.05 0.04 0.02 0.04 0.05 0.21 0.08 0.07
x 0.49 0.57
A**
0.45 0.54
B
A**
0.90 1.32
B
A
1.34 1.61
B
B
A**
60 SD
x
0.05
0.52
0.02 0.03 0.03 0.08 0.19 0.06 0.09
SD 0.02
B
0.02
A
0.03
b
0.12
a**
0.03
B
0.06
A**
0.07
0.56
0.43 0.54
1.35 1.53
1.03 1.33
0.05
**
Symbols as in Table 1.
Discussion The conducted study was the first ever attempt to determine the stimulating effect of a natural immunostimulator – Biolex-MB40 containing increased levels of β-1,3/1,6-D-glucan and MOS – on selected indicators of specific and non-specific humoral and cellular immunity in lambs. Glucans are a group of compounds known as glucose homopolymers. They are isolated from fungi, yeast, and plants, including oat and barley. Glucans derived from yeast and fungi have 1-3 bonds, and may occasionally feature additional 1-6 branches, whose number varies subject to glucan type. Glucans isolated from barley and oat are mostly linear compounds comprising regions with 1-4 bonds that separate smaller structures with 1-3 bonds (1). The most frequently described glucans that have been proven to have a stimulating effect on the immune system are: β-(1,3)(1,6)-glucan extracted from Saccharomyces cerevisiae, scleroglucan produced by Sclerotium glucanicum, grifolan (GRN) isolated from Grifola frondosa, SSG found in Sclerotinia sclerotiorum, and laminarin extracted from Laminaria digitata (2). The biological activity of β-glucan is determined by its origin, occurrence frequency, isolation method, size (molecular weight), physicochemical properties (such as solubility), primary structure, shape, degree of branching, and polymer charge (36). High molecular weight β-glucans (e.g. zymosan) may directly stimulate leukocytes, enhancing their phagocytic, cytotoxic, and antibacterial activity, including the production of reactive oxygen species and indirect nitrogen compounds. Low and medium molecular weight β-glucans (e.g. phosphate glucan) have a weaker effect on immune system cells. Short β-glucans molecules (e.g. laminarin with molecular weight of
