The object of this study was to investigate antimicrobial potential of raw Domestic Balcan donkey milk against L. monocytogenes and S. aureus. The content of LYZ as well as fatty acid composition with an emphasis on the fatty acids with known antimicrobial activity were also examined.
Milk samples were obtained from Domestic Balcan donkey breed, from “Zasavica” Special Nature Reserve, Serbia. Eight clinically healthy donkeys from different lactation periods (65 – 210 days post-partum (pp.) were included in the trial. Samples collected after morning hand-milking, were immediately cooled at 4 °C and transported to the laboratory where the samples were frozen at – 20 °C. The samples marked as A, B, C were collected in the early stage (65-90 days pp.), samples F, G in the middle stage (140-160 days pp.) and samples D, E, H in the late stage of lactation (190-210 days pp.).
Sample preparation was carried out according to Tidona et al. (2011) with some modifications. Milk samples were diluted in 1:1.5 (v/v), sample:buffer (0.125 M TriseHCl, 4% SDS, 2% glycerol, 2% b-mercaptoethanol, pH 6.8) and heated at 100 °C for 5 min. The chip-based separations were performed on the Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) in combination with the Protein 80 Plus LabChip kit and the dedicated Protein 80 software assay on 2100 expert software. Chips were prepared according to the protocol provided by the Protein 80 LabChip kit. Bovine serum albumin was used as the standard for quantitation of the milk proteins. All samples were analyzed in triplicate.
The antibacterial assay was performed on each individual milk sample using Listeria monocytogenes ATCC 19111 and Staphylococcus aureus ATCC 25923. Cultures were stored on nutrient agar slants in a refrigerator at 4 °C and subcultured on fresh slants weekly. After overnight incubation on nutrient agar at 37 °C, well-isolated colonies of each test microorganisms were selected and transferred with an inoculating loop to a tube of sterile saline and vortexed thoroughly. The density of the bacterial suspension was adjusted to the 0.5 McFarland standard using DEN-1 densitometer (Biosan, Riga, Latvia). Further decimal dilutions in sterile saline were prepared from initial suspensions. DM samples were artificially contaminated with these bacterial strains at the level of contamination of 102 cfu/mL. 25 mL of each artificially contaminated sample were placed into a sterile vessel and kept in water bath (Raypa, Spain) at 38 ± 0.5 °C during 8 h. Changes in the number of tested bacteria were monitored every hour according to the international standards (EN ISO, 2004; ISO, 2003). The results were expressed as log10 cfu/mL. Non inoculated DM was used as negative control, while artificially contaminated nutrient broth (Himedia, India) was used as positive control. All samples were analyzed in triplicate.
Results were expressed as mean along with standard deviations, of triplicate analyses for all measurements. Analysis of variance was followed by Duncan's multiple comparison tests using STATISTICA version 10 (StatSoft Inc., Tulsa, OK, USA).
incubation (h) |
Donkey milk samples |
||||||||
A |
B |
C |
D |
E |
F |
G |
H |
NB |
|
0 |
2.30(0.05)de,A |
2.31(0.04)c,A |
2.32(0.03)d,A |
2.30(0.04)d,A |
2.33(0.02)c,A |
2.30(0.02)f,A |
2.34(0.05)d,A |
2.30(0.03)e,A |
2.33(0.04)a,A |
1 |
1.94(0.03)a,DE |
1.78(0.00)a,A |
1.85(0.04)a,B |
1.90(0.05)a,BCD |
1.99(0.04) a,E |
1.87(0.03) a,BC |
1.85(0.04) a,B |
1.92(0.02)a,CD |
2.39(0.02)b,F |
2 |
2.08(0.04)b,A |
2.04(0.18)b,A |
2.07(0.06)b,A |
2.10(0.08)c,A |
2.00(0.05) ab,A |
2.11(0.04) b,A |
2.02(0.07) b,A |
2.00(0.05)b,A |
2.38(0.02)ab,B |
3 |
1.93(0.08)a,A |
2.03(0.02)b,B |
2.06(0.04)b,BC |
2.09(0.03)c,BC |
2.07(0.08) ab,BC |
2.13(0.04) cde,C |
2.01(0.05) b,AB |
2.08(0.04)c,BC |
2.85(0.03)c,D |
4 |
2.25(0.07)d,B |
1.97(0.12)b,A |
2.06(0.09)b,A |
2.07(0.05)bc,A |
2.10(0.09) b,A |
2.07(0.06) bcd,A |
2.10(0.08) bc,A |
2.11(0.06)c,A |
3.37(0.01)d,C |
5 |
2.10(0.05)bc,CD |
1.95(0.00)b,A |
2.02(0.04)b,ABC |
2.03(0.05)bc,ABC |
2.05(0.06) ab,ABCD |
2.15(0.08) de,D |
1.99(0.10) b,AB |
2.09(0.00)c,BCD |
3.80(0.00)e,E |
6 |
2.25(0.07)d,D |
1.94(0.03)b,A |
2.04(0.05)b,BC |
1.98(0.06)a,AB |
2.04(0.04) ab,BC |
2.05(0.05) bc,BC |
2.06(0.04) bc,BC |
2.12(0.03)c,C |
4.11(0.07)f,E |
7 |
2.20(0.05)cd,AB |
2.25(0.01)c,A |
2.18(0.03)c,AB |
2.22(0.04)d,AB |
2.25(0.06)c,A |
2.21(0.03)e,AB |
2.15(0.07)c,B |
2.19(0.05)d,AB |
4.54(0.02)g,C |
8 |
2.37(0.10)e,B |
2.27(0.08)c,AB |
2.30(0.03)d,AB |
2.28(0.06)d,AB |
2.33(0.04)c,AB |
2.32(0.07)f,AB |
2.35(0.03)d,AB |
2.26(0.04)de,A |
5.00(0.01)h,C |
Each value is the mean of three replicates; Standard deviation values are given in parentheses; Means presented in the same column followed by different lowercase letters are statistically different (P<0.05); Means in the same row followed by different capital letters are statistically different (P<0.05); NB - nutrient broth
Incubation (h) |
Donkey milk samples |
||||||||||
A |
B |
C |
D |
E |
F |
G |
H |
NB |
|||
0 |
2.32(0.05) |
2.30(0.05) |
2.35(0.05) |
2.34(0.05) |
2.33(0.05) |
2.32(0.04) |
2.34(0.05) |
2.33(0.05) |
2.30(0.05) |
||
1 |
2.43(0.04) |
2.43(0.01) |
2.34(0.09) |
2.22(0.06) |
2.46(0.10) |
2.27(0.04) |
2.33(0.07) |
2.26(0.01) |
2.25(0.04) |
||
2 |
2.45(0.04) |
2.44(0.00) |
2.32(0.01) |
2.18(0.03) |
2.27(0.05) |
2.25(0.07) |
2.28(0.02) |
2.27(0.09) |
2.26(0.06) |
||
3 |
2.48(0.02) |
2.44(0.02) |
2.57(0.03) |
2.28(0.06) |
2.35(0.03) |
2.41(0.03) |
2.28(0.06) |
2.35(0.04) |
2.30(0.00) |
||
4 |
2.52(0.04) |
2.56(0.02) |
2.60(0.12) |
2.49(0.06) |
2.40(0.13) |
2.38(0.05) |
2.71(0.01) |
2.27(0.06) |
2.70(0.00) |
||
5 |
2.55(0.03) |
2.52(0.10) |
3.21(0.06) |
2.53(0.19) |
2.75(0.05) |
2.70(0.01) |
2.71(0.21) |
2.26(0.24) |
3.71(0.02) |
||
6 |
2.57(0.02) |
2.68(0.05) |
3.64(0.03) |
2.85(0.00) |
3.07(0.10) |
3.00(0.04) |
2.87(0.09) |
2.28(0.20) |
4.39(0.09) |
||
7 |
3.06(0.02) |
3.45(0.02) |
3.92(0.03) |
3.14(0.01) |
3.37(0.02) |
3.33(0.03) |
3.57(0.11) |
2.48(0.00) |
5.39(0.09) |
||
8 |
3.29(0.11) |
3.85(0.02) |
3.92(0.03) |
3.30(0.05) |
3.83(0.03) |
3.70(0.04) |
3.90(0.09) |
3.00(0.01) |
6.84(0.06) |
Each value is the mean of three replicates; Standard deviation values are given in parentheses; Means presented in the same column followed by different lowercase letters are statistically different (P<0.05); Means in the same row followed by different capital letters are statistically different (P<0.05); NB - nutrient broth
The count of S. aureus in most of the tested samples slowly increased or remained at almost constant level during the first 5 h. After 8 h of incubation, count of S. aureus was in range from 3.00 to 3.92, whereas this number in positive control was much higher (6.84 log cfu/mL). The tested bacterial strains did not detect in negative controls. It was obvious that the antibacterial activity of DM toward S. aureus could be characterized as growth inhibitory in terms of the extension of its lag phase and growth slowing. Similar tendency was observed in the experiment with L. monocytogenes, where lag phase practically lasted 8 h. DM exibited stronger antibacterial effect toward L. monocytogenes under the experimental conditions used. Itwas especially clearly visible after the first hour of incubation, when its count was significantly reduced in all samples.
Tidona et al. (2011) reported inhibitory activity of undigested and digested DM against L. monocytogenes in a dose dependent manner, while Nazzaro et al. (2010) showed antibacterial activity of hydrolyzed DM toward S. aureus. The antimicrobial activity of DM is explained by its high concentration of lysozyme (Coppola et al., 2002; Vincenzetti et al., 2008; Zhang et al., 2008). This protein exibits strong antibacterial activity, particularly toward Gram positive bacteria. LYZ belongs to a family of enzymes which cleave the b-1,4 linkages between N-acetylmuramic acid and 2-acetylamino-2-deoxy-D-glucose residues in mucopolysaccharide or mucopeptide components in cell walls (Floris et al., 2003). Gram negative bacteria are less susceptible to its activity owing to their outer layer which does not allow the entry of LYZ moleculs to the target places in peptidoglycan structure (Floris et al., 2003). The obtained different values of LYZ content in tested milk samples (Table 3) can be due to different lactation periods (65-210 days pp.) of the donkeys and to different analytical methods applied (Salimei & Fantuz, 2012). Therefore, literature data about the LYZ content in DM ranges from 1 g/L (Vincenzetti et al., 2008) to 4 g/L (Coppola et al., 2002). The relationship between LYZ content and antibacterial activity of DM samples toward tested bacteria was not established in this investigation, since DM samples with LYZ content ranging from 0.67 to 3.54 g/L showed similar effect against L. monocytogenes, while sample H (1.24 g/L of LYZ) had stronger activity against S. aureus compared to other samples with higher LYZ content. This probably indicates that LYZ is not the only antimicrobial agent in DM which can act against tested Gram positive bacteria, since it is a complex medium with numerous different compounds which could contribute to the overal antimicrobial potential. Among those compounds are definitely certain fatty acids, which composition in the tested DM samples is presented in Table 3.
Main determinated fatty acids with well known antibacterial activity toward Gram positive bacteria (Galbraith et al., 1971; Galbraith & Miller, 1973) were linoleic acid (C18:2), lauric acid (C12:0) and oleic acid (C18:1) (Table 3), which account from 40.3 to 54.7% of the total fatty acids content in analysed milk samples. These long chain fatty acids (C18:2, C18:1) stimulated oxygen uptake by Gram positive bacteria at bactericidal and protoplast lytic concentrations and produced inhibition at higher levels (Galbraith & Miller, 1973). Linolenic acid caused an increase in lag phase duration of Gram positive bacteria (Galbraith et al., 1971). The palmitic acid (C16:0) was also detected in high contentration in all tested milk samples, but there is no data on its antimicrobial action against L. monocytogenes or S. aureus. Although fatty acids probably play a supporting role in the total antimicrobial activity of DM it would be useful to investigate their individual contribution to this activity, especially if it is known that calcium and magnesium ions, also present in DM, reverse their activities.
Since this experiment was performed at 38 °C which is the donkeys’ body temperature (Etana et al., 2011), antimicrobial agents will not allow quick growth of L. monocytogenes and S. aureus in milk, if it is not cooled imidiately. However, DM at applied level of contamination did not possess any bactericidal activity against tested bacteria. Therefore, their presence and number in raw DMwill depend on the health of the mammary gland and hygienic milking procedures applied. DM could be effective in the treatment of listeriosis and staphylococcosis, since Tidona et al. (2011) reported high resistance of DM LYZ to the degradation by gastrointestinal enzymes, while Nazzaro et al. (2010) detected the presence of compounds in hydrolyzed DM, different from LYZ, capable of inhibiting the growth of S. aureus.
Fatty acid (% m/m) |
Donkey milk samples |
|||||||||
|
|
A |
B |
C |
D |
E |
F |
G |
H |
|
8:0 |
2.2(0.12) |
2.8(0.19) |
4.2(0.13) |
1.5(0.14) |
2.6(0.21) |
2.1(0.24) |
4.0(0.10) |
3.7(0.34) |
||
10:0 |
5.3(0.37) |
12.8(1.28)d |
11.3(0.59)c |
4.8(0.34) |
9.3(0.33) |
5.4(0.25) |
11.0(0.18)c |
13.8(0.99)d |
||
11:0 |
0.9(0.04) |
2.1(0.19) |
1.1(0.15) |
0.8(0.05) |
1.9(0.12) |
1.1(0.12) |
2.1(0.16) |
2.2(0.17) |
||
12:0 |
6.1(0.42) |
15.7(0.98)e |
10.6(0.20)c |
4.8(0.15) |
13.3(1.19)d |
6.2(0.39) |
11.8(0.72)c |
14.2(0.12)d |
||
14:0 |
6.3(0.42) |
13.5(0.94)f |
8.6(0.31) |
5.1(0.41) |
13.3(0.81)f |
6.6(0.36) |
10.7(0.86)e |
12.2(0.86)e |
||
14:1 |
0.2(0.03) |
0.9(0.04) |
n.d. |
0.5(0.01) |
1.1(0.05) |
0.4(0.03) |
0.2(0.03) |
n.d. |
||
15:0 |
1.0(0.08) |
0.5(0.03) |
0.8(0.09) |
0.6(0.03) |
0.8(0.06) |
0.8(0.09) |
n.d. |
n.d. |
||
16:0 |
19.4(0.95)d |
17.8(0.09)b |
16.0(0.58)a |
18.1(0.14)bc |
18.5(0.64)bcd |
19.2(0.46)cd |
18.3(1.14)bcd |
18.8(0.50)cd |
||
16:1 |
6.6(0.46) |
4.1(0.31)ab |
6.3(0.59) |
6.1(0.14) |
6.6(0.66) |
5.2(0.15) |
3.5(0.04) |
4.3(0.42) |
||
18:0 |
1.3(0.15) |
1.9(0.28) |
1.6(0.01) |
1.4(0.13)bc |
1.5(0.05) |
1.5(0.06) |
1.0(0.10) |
1.6(0.21) |
||
18:1 |
20.1(0.90)d |
10.9(1.13)a |
13.1(0.16)b |
20.9(0.08)d |
11.8(1.04)ab |
20.3(1.35)d |
15.0(0.55)c |
12.3(1.22) |
||
18:2n6 |
24.3(0.52)e |
14.4(1.37)b |
18.9(0.47)c |
28.7(0.49)f |
15.8(0.42)b |
21.9(0.90)d |
18.1(0.30)c |
12.6(1.54)a |
||
18:3n3 |
6.3(0.11) |
2.6(0.07) |
7.6(0.62) |
6.5(0.15) |
3.4(0.38) |
8.6(0.48) |
4.1(0.34) |
4.4(0.21) |
||
20:2 |
n.d. |
n.d. |
n.d. |
n.d. |
n.d. |
0.8(0.08) |
n.d. |
n.d. |
||
LYZ |
3.52 |
3.54 |
3.42 |
1.3 |
0.67 |
2.52 |
1.74 |
1.24 |
Each value is the mean of three replicates. Standard deviation values are given in parentheses. Means presented in the same line followed by different lower case are statistically different (P<0.05)