- Email: [email protected]
Stability of listerial bacteriophage A511 in bovine milk fat globules
Journal Pre-proof Stability of listerial bacteriophage A511 in bovine milk fat globules Mayra C. García-Anaya, David R. Sepúlveda, Claudio Ríos-Velasco, Paul B. Zamudio-Flores, Alejandro Romo-Chacón, Carlos H. Acosta-Muñiz PII:
S0958-6946(19)30264-X
DOI:
https://doi.org/10.1016/j.idairyj.2019.104627
Reference:
INDA 104627
To appear in:
International Dairy Journal
Received Date: 14 September 2019 Revised Date:
6 December 2019
Accepted Date: 8 December 2019
Please cite this article as: García-Anaya, M.C., Sepúlveda, D.R., Ríos-Velasco, C., Zamudio-Flores, P.B., Romo-Chacón, A., Acosta-Muñiz, C.H., Stability of listerial bacteriophage A511 in bovine milk fat globules, International Dairy Journal, https://doi.org/10.1016/j.idairyj.2019.104627. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
1
Stability of listerial bacteriophage A511 in bovine milk fat globules
2 3 4 5 6
Mayra C. García-Anaya, David R. Sepúlveda, Claudio Ríos-Velasco, Paul B. Zamudio-Flores,
7
Alejandro Romo-Chacón, Carlos H. Acosta-Muñiz*
8 9 10 11 12 13
Centro de Investigación en Alimentación y Desarrollo, A. C. Departamento de Microbiología y
14
Biología Molecular. Av. Río Conchos S/N Parque Industrial. Z.C. 31570. Cd. Cuauhtémoc,
15
Chihuahua, México.
16 17 18 19 20 21
*Corresponding author. Tel.: +52 625 581 2921
22
E-mail address: [email protected] (C. H. Acosta-Muñiz)
23
1
24
_______________________________________________________________________________
25
ABSTRACT
26 27
Raw milk and milk fractions were analysed for their activity against the bacteriophage A511. The
28
whole milk was separated into fat, casein and whey. Fat was fractionated further into butter serum,
29
butter oil, butter milk and milk fat globule membrane. Bacteriophage was inoculated and was
30
enumerated at different time periods (0, 24, 48, 72 and 96 h). Results (plaque-forming units, PFU)
31
showed that raw milk reduced phage counts 2.4 log10 PFU mL . Among milk fractions, fat and whey
32
were the most active fractions, reducing the phage titre 2.6 and 2.1 log10 PFU mL , respectively,
33
compared with 0.8 log10 PFU mL in casein. Fat fractions also influenced phage stability. The main
34
effect was attributed to the milk fat globule membrane (2.1 log10 PFU mL reduction) followed by
35
butter milk and butter serum (1.0 log10 PFU mL reduction). Butter oil showed no effect on phage
36
stability.
37
_______________________________________________________________________________
-1
-1
-1
-1
-1
2
38
1.
Introduction
39 40
The literature about the interference of food matrix on phage activity is steadily growing,
41
focusing on dairy products (García-Anaya et al., 2020). The effect of milk on the reduction in the
42
phage titre has been reported in two studies. For phage A511, a reduction of 43% of the total
43
phages was observed after being exposed to raw milk (García-Anaya, Sepulveda, Rios-Velasco,
44
Zamudio-Flores, & Acosta-Muñiz, 2019). Similar observations were made by Garcia, Madera,
45
Martinez, Rodriguez, and Suarez (2009), who found a reduction of 90% for a cocktail of two
46
staphylococcal phages (φ5 and φ72) after their exposure in raw milk at 37 °C for 1 1 h. The mechanism by which reduction in phage titre occurs in milk is still unclear. To date,
47 48
only one study has made suggestions about the influence of milk proteins; in this study, Das and
49
Marshall (1967) reported an inhibition of a staphylococcal phage in skim milk. The effect was
50
attributed to casein micelles, since their experiments showed that the change in pH of the skim milk
51
(4.0 to 9.0) modified phage recovery. Although the information in this area has dealt with proteins,
52
no study has addressed the complexity of milk fat.
53
To date, there is no study that has been focused on the possible effects of pure fractions,
54
either of milk (fat, whey and casein) or fat [butter oil (BO), butter milk (BM), butter serum (BS) and
55
membrane fat globule (MFGM)] on phage stability. However, it is well known that all of these
56
fractions have different compositions, chemical structures, and activities against viruses (Hamosh,
57
et al., 1999; Korhonen, 2009; Parrón et al., 2018). If so, this could be relevant as all milk fractions
58
would represent potential elements to inhibit phages. Thus, the aim of the study was to evaluate the
59
effect of milk fractions as well as fatty fractions on stability of phage A511.
60 61
2.
Materials and methods
2.1.
Milk sample collection
62 63 64
3
Bulk tank milk samples of different cows were obtained from a dairy farm and maintained at
65 66
4 °C prior to their use within 1 to 3 h.
67 68
2.2.
Phage inoculation in SM buffer and milk
69 Phage A511 was purchased from American Type Culture Collection (ATCC, Manassas, VA,
70 71
USA) and propagated with the bacterial host Listeria ivanovii (ATCC, 19119) since it does not give
72
rise to mutants resistant to phage A511 (Loessner, Goeppl, & Busse, 1991; Loessner, Rees,
73
Stewart, & Scherer, 1996). Phage propagation was performed in brain heart infusion broth (BHI) at
74
30 °C for 16 h, as described by Guenther, Huwyler, Richard, and Loessner (2009). Aliquots of the
75
purified phage suspensions were prepared at concentrations of 1 × 10 plaque-forming units per mL
76
(PFU mL ) and stored at 4 °C. Aliquots of SM buffer and raw milk (900 µL) were inoculated with the
77
purified phage at a final concentration of 3 × 10 PFU mL . Samples were vortexed to ensure a
78
complete dispersion of phages before titration.
9
-1
7
-1
79 80
2.3.
Obtaining milk fractions
81 82
Both milk and fat fractions were obtained using the procedure illustrated in Fig 1. The
83
separation of fat, whey and caseins was carried out according to García-Anaya et al. (2019) with
84
some modifications (Fig. 1, procedure 1-A). Three replicates of each treatment were performed, and
85
for each replicate 10 L of raw milk was used to obtain a considerable quantity of MFGM. The milk
86
fat was separated from milk by a cream separator (Kamdhenu Cream Separator KD60-E, Delhi,
87
India). Then, the fat was washed twice with two volumes of washing buffer (2.5 mM Na2HPO4, 2mM
88
KH2PO4, 0.14 M NaCl, 3 mM KCl, 1 mM EDTA, pH 7.2) and centrifuged at 2800 × g for 15 min at 4
89
°C, followed by solidification at −20 °C for 5 min. The upper layer (fat) was transferred into a new
90
tube. Then, an aliquot of fat (2 g) was separated for analysis of phage stability and the rest was
91
used for fat fractionation. Skim milk was used to obtain the protein fraction: casein micelles
92
(precipitate) and whey proteins (supernatant). For this, skim milk was ultracentrifuged (Beckman
4
93
Coulter ultracentrifuge, Optima XPN-100, Brea, CA, USA) using a Beckman SW 60Ti rotor at
94
98,699 × g (k factor 170.1) for 60 min at 4 °C, with 1 min ac celeration and 1 min deceleration times.
95
The supernatant was separated and the precipitate was washed twice with sterile distilled water
96
and stirred with a spatula. Casein micelles and whey were transferred into a new tube separately for
97
phage inoculation. The fat fractionation was carried out according to Parrón et al. (2018) with some
98 99
modifications (Fig. 1. Procedure 2-A). The fat obtained previously in the 1-A procedure was stored
100
at 4 °C overnight. Then, fat was churned into butte r using a Sunbeam Mixmaster (Model 03371,
101
Sunbeam Appliance Co., Mexico). The BM released in the process was filtered through several
102
layers of cheesecloth. Butter was melted at 50 ºC for 10 min and centrifuged at 4000 × g for 10 min
103
to obtain BS and BO. The MFGM was obtained through ultracentrifugation as above. All fractions of
104
interest were immediately inoculated with phage A511 and subjected to titration.
105 106
2.4.
Phage inoculation and phage enumeration
107 108
To determine phage stability, aliquots of each sample (900 µL) were inoculated with the
109
purified phage at a final concentration of 3 × 107 PFU mL-1. Samples were vortexed to ensure a
110
complete dispersion and were stored at 4 °C. Phages were enumerated by plaque assay on tryptic
111
casein soy agar (TSA) plates using antibiotics (ceftazidime 20 mg L , nalidixic acid 20 mg L and
112
cycloheximide 10 mg L ) and L. monocytogenes Scott A (ATCC 15313) as host organism, as
113
described by García-Anaya et al. (2019). Phage enumeration was carried out at 0, 24, 48, 72 and
114
96 h of storage.
-1
-1
-1
115 116
2.5.
Statistical analysis
117 118 119
Phage numbers (PFU) obtained for treated samples (control and milk fractions) were log10 transformed to obtain homogeneity of variance in the data. Data were analysed using a mixed
5
120
model for repeated measures using SAS software version 9.2 (SAS Institute Inc., 2002 Cary, NC,
121
USA).
122 123
3.
Results and discussion
3.1.
Effect of raw milk on the stability of phage A511
124 125 126 Fig 2 shows the reduction of phage A511 by raw milk. A reduction of 2.4 log10 PFU mL-1 of
127 128
phage was observed after 96 h of exposure in raw milk. Similar reductions have been reported by
129
other authors. For example, Garcia et al. (2009) studied the effect of skimmed and whole-fat raw
130
milk samples on the stability of two staphylococcal phages (φH5 and φA72) and found reductions of
131
1 log10 PFU mL after 8 h at 37 °C. Also, García-Anaya et al. (201 9) reported a reduction of 1 log10
132
PFU mL for the phage A511 in raw milk samples.
-1
-1
133 134
3.2.
Effect of milk fractions on phage stability
135 Milk fractions had a different effect on the stability of phage A511 (Fig 3). When phage was
136
-1
137
treated with fat, whey, and casein the phage count decreased 2.6, 2.1 and 0.8 log10 PFU mL ,
138
respectively. The mechanism for which milk fractions reduced the phage titre is unknown. Despite
139
knowledge of the antiviral properties of milk fat (Hamosh et al., 1999; Thormar, 2011), antiphage
140
properties only have been investigated in whey and casein fractions. In whey fraction, the antiphage
141
activity (against phage M13) has been related to the presence of α-lactalbumin (α-LA) and β-
142
lactoglobulin (β-LG) (Sitohy, Chobert, Karwowska, Gozdzicka-Jozefiak, & Haertlé, 2006). The
143
reduction in the phage titre by caseins has been related to electrostatic interactions causing the
144
emergence of casein-phage complexes (Das & Marshall, 1967).
145 146
3.3.
Phage stability in fat fractions
147
6
The stability of phage A511 in fat fractions is shown in Fig 4. The MFGM was the most
148
-1
149
active fraction against phage (2.2 log10 PFU mL ), followed by BM and BS that showed the same
150
reduction (1 log10 PFU mL ). On the contrary, in presence of BO, there was no reduction of phage.
151
To our knowledge, the effect of fat fractions on phage stability has not been evaluated with other
152
phages, but some studies give an insight into the possible factors that could reduce the phage titre.
153
For example, an study also has showed the stability of another viruses in BO samples, whereas
154
another has reported the effect of unsaturated fatty acids against phage T5 (5.9 to 97.6 % of
155
inactivation) (Blackwell, 1978; Hirotani, Ohigashi, Kobayashi, Koshimizu, & Takahashi, 1991).
156
Therefore, the instability of phage in fat could be due to differences in their physical properties (e.g.,
157
chain length, number of double bonds, availability of active groups) (Hamosh et al., 1999).
-1
158
Phage instability could be related to the presence of bioactive molecules with antiviral
159
activity or to the emergence of phage-molecule complexes that hinder the interaction with the
160
bacterial host (Civra et al., 2015; Fuller, Kuhlenschmidt, Kuhlenschmidt, Jiménez-Flores, &
161
Donovan, 2013; Parrón et al., 2018). However, it is not yet known which features of milk
162
compounds (binding interactions and/or active groups) have the effect on phage reduction.
163 164
4.
Conclusions
165 166
The present study showed the effect of raw milk on the stability of phage A511. The results
167
indicated that milk fat had the major effect in phage stability followed by whey and casein micelles.
168
Fat fractions also showed different levels of stability for phage A511. The BM and BS fractions
169
showed the same effect in the reduction of phage titre. In butter oil, phage A511 was completely
170
stable, whereas MFGM showed a great influence on phage stability. The mechanism for phage
171
reduction is unknown, but could be related to several factors including the physical structure of the
172
molecule (lipid and protein), binding interactions (electrostatic and hydrophobic) or the presence of
173
bioactive molecules. More studies are needed to determine which milk compounds cause the
174
reduction in the phage titre, as well as their mechanism of action.
175
7
176
Acknowledgements
177 178
Garcia-Anaya gratefully acknowledges the National Council for Science and Technology
179
(CONACyT) to finance her doctoral studies. This research did not receive any specific grant from
180
funding agencies in the public, commercial, or not -for-profit sectors.
181 182
References
183 184 185 186
Blackwell, J. H. (1978). Persistence of foot-and-mouth disease virus in butter and butter oil. Journal of Dairy Research, 45, 283–285. Civra, A., Giuffrida, M. G., Donalisio, M., Napolitano, L., Takada, Y., Coulson, B. S., et al. (2015).
187
Identification of equine lactadherin-derived peptides that inhibit rotavirus infection via
188
integrin receptor competition. Journal of Biological Chemistry, 290, 12403–12414.
189 190 191
Das, N., & Marshall, R. (1967). Adsorption of staphylococcal bacteriophage by milk proteins. Applied Microbiology, 15, 1095–1098. Fuller, K., Kuhlenschmidt, T., Kuhlenschmidt, M. S., Jiménez-Flores, R., & Donovan, S. M. (2013).
192
Milk fat globule membrane isolated from buttermilk or whey cream and their lipid
193
components inhibit infectivity of rotavirus in vitro. Journal of Dairy Science, 96, 3488–3497.
194
García-Anaya, M. C., Sepulveda, D. R., Rios-Velasco, C., Zamudio-Flores, P. B., & Acosta-Muñiz,
195
C. H. (2019). Effect of homogenization on binding-affinity of bacteriophage A511 in bovine
196
milk fractions. Journal of Food Engineering, 244, 73–79.
197
García-Anaya, M. C., Sepulveda, D. R., Sáenz-Mendoza, A. I., Rios-Velasco, C., Zamudio-Flores,
198
P. B., & Acosta-Muñiz, C. H. (2020). Phages as biocontrol agents in dairy products. Trends
199
in Food Science & Technology, 95, 10–20.
200
Garcia, P., Madera, C., Martinez, B., Rodriguez, A., & Suarez, J. E. (2009). Prevalence of
201
bacteriophages infecting Staphylococcus aureus in dairy samples and their potential as
202
biocontrol agents. Journal of Dairy Science, 92, 3019–3026.
8
203
Guenther, S., Huwyler, D., Richard, S., & Loessner, M. J. (2009). Virulent bacteriophage for efficient
204
biocontrol of Listeria monocytogenes in ready-to-eat foods. Applied and Environmental
205
Microbiology, 75, 93–100.
206
Hamosh, M., Peterson, J. A., Henderson, T. R., Scallan, C. D., Kiwan, R., Ceriani, R. L., et al.
207
(1999). Protective function of human milk: the milk fat globule. Seminars in Perinatology,
208
23, 242–249.
209
Hirotani, H., Ohigashi, H., Kobayashi, M., Koshimizu, K., & Takahashi, E. (1991). Inactivation of T5
210
phage by cis‐vaccenic acid, an antivirus substance from Rhodopseudomonas capsulata,
211
and by unsaturated fatty acids and related alcohols. FEMS Microbiology Letters, 77, 13–18.
212
Korhonen, H. J. (2009). Bioactive components in bovine milk. In Y. P. Park (Ed.), Bioactive
213
components in milk and dairy products (pp. 15–42). Chichester, UK: Wiley.
214
Loessner, M., Goeppl, S., & Busse, M. (1991). The phagovar variability of Listeria strains under the
215
influence of virulent and temperate bacteriophages. Letters in Applied Microbiology, 12,
216
192–195.
217
Loessner, M. J., Rees, C., Stewart, G., & Scherer, S. (1996). Construction of luciferase reporter
218
bacteriophage A511::luxAB for rapid and sensitive detection of viable listeria cells. Applied
219
and Environmental Microbiology, 62, 1133–1140.
220
Parrón, J. A., Ripollés, D., Sánchez, A. C., Pérez, M. D., Calvo, M., López, S., et al. (2018).
221
Antirotaviral activity of bovine milk components: Extending the list of inhibitory proteins and
222
seeking a better understanding of their neutralization mechanism. Journal of Functional
223
Foods, 44, 103–111.
224
Sitohy, M., Chobert, J.-M., Karwowska, U., Gozdzicka-Jozefiak, A., & Haertlé, T. (2006). Inhibition
225
of bacteriophage M13 replication with esterified milk proteins. Journal of Agricultural and
226
Food Chemistry, 54, 3800–3806.
227
Thormar, H. (2011). Antimicrobial lipids and innate immunity. In H. Thomar (Ed.), Lipids and
228
essential oils as antimicrobial agents (pp. 123–150). Chichester, UK: Wiley.
9
1
Figure legends
2 3
Fig. 1. Experimental isolation procedure of both milk and fat fractions. Fractions of interest are
4
indicated with bold font. Approximate yield of pure fractions in basis of 10 L of raw milk was 3.5–
5
4.0% fat; 7–10% casein; 80–85% whey, 0.6–0.7% butter milk; 2.6–2.8% butter oil; 0.4–0.5% butter
6
serum and 0.005% MFGM.
7 8
Fig. 2. Effect of raw milk on phage stability (, control; , raw milk). Error bars represent the
9
relative error from the mean of three independent experiments.
10 11
Fig. 3. Effect of milk fractions on phage stability: () milk fat, () whey and () and caseins
12
compared with the control (). Error bars represent the relative error from the mean of three
13
independent experiments.
14 15
Fig. 4. Effect of fat fractions on phage stability: () butter milk, () butter serum, () butter oil and
16
() milk fat globule membrane compared with the control (). Error bars represent the relative error
17
from the mean of three independent experiments.
18 19
1
Figure 1
Figure 2
Figure 3
Figure 4
Author Contributions
M.C. Mayra C. García-Anaya The author contributed to the conceptualization and development of experiments, as well as the writing, review and edition of the manuscript as part of her doctoral studies. Dr. David Sepúlveda The author contributed to the conceptualization of the project. Dr. Claudio Rios Velasco The author contributed to the conceptualization of the project. Dr. Paul B. Zamudio-Flores The author contributed to the conceptualization of the project. M.C. Alejandro Romo-Chacón The author contributed to the statistical analysis of data Dr. Carlos H. Acosta-Muñiz The author contributed to the conceptualization, visualization, supervision, founding acquisition and administration of the project.
S0958-6946(19)30264-X
DOI:
https://doi.org/10.1016/j.idairyj.2019.104627
Reference:
INDA 104627
To appear in:
International Dairy Journal
Received Date: 14 September 2019 Revised Date:
6 December 2019
Accepted Date: 8 December 2019
Please cite this article as: García-Anaya, M.C., Sepúlveda, D.R., Ríos-Velasco, C., Zamudio-Flores, P.B., Romo-Chacón, A., Acosta-Muñiz, C.H., Stability of listerial bacteriophage A511 in bovine milk fat globules, International Dairy Journal, https://doi.org/10.1016/j.idairyj.2019.104627. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Ltd. All rights reserved.
1
Stability of listerial bacteriophage A511 in bovine milk fat globules
2 3 4 5 6
Mayra C. García-Anaya, David R. Sepúlveda, Claudio Ríos-Velasco, Paul B. Zamudio-Flores,
7
Alejandro Romo-Chacón, Carlos H. Acosta-Muñiz*
8 9 10 11 12 13
Centro de Investigación en Alimentación y Desarrollo, A. C. Departamento de Microbiología y
14
Biología Molecular. Av. Río Conchos S/N Parque Industrial. Z.C. 31570. Cd. Cuauhtémoc,
15
Chihuahua, México.
16 17 18 19 20 21
*Corresponding author. Tel.: +52 625 581 2921
22
E-mail address: [email protected] (C. H. Acosta-Muñiz)
23
1
24
_______________________________________________________________________________
25
ABSTRACT
26 27
Raw milk and milk fractions were analysed for their activity against the bacteriophage A511. The
28
whole milk was separated into fat, casein and whey. Fat was fractionated further into butter serum,
29
butter oil, butter milk and milk fat globule membrane. Bacteriophage was inoculated and was
30
enumerated at different time periods (0, 24, 48, 72 and 96 h). Results (plaque-forming units, PFU)
31
showed that raw milk reduced phage counts 2.4 log10 PFU mL . Among milk fractions, fat and whey
32
were the most active fractions, reducing the phage titre 2.6 and 2.1 log10 PFU mL , respectively,
33
compared with 0.8 log10 PFU mL in casein. Fat fractions also influenced phage stability. The main
34
effect was attributed to the milk fat globule membrane (2.1 log10 PFU mL reduction) followed by
35
butter milk and butter serum (1.0 log10 PFU mL reduction). Butter oil showed no effect on phage
36
stability.
37
_______________________________________________________________________________
-1
-1
-1
-1
-1
2
38
1.
Introduction
39 40
The literature about the interference of food matrix on phage activity is steadily growing,
41
focusing on dairy products (García-Anaya et al., 2020). The effect of milk on the reduction in the
42
phage titre has been reported in two studies. For phage A511, a reduction of 43% of the total
43
phages was observed after being exposed to raw milk (García-Anaya, Sepulveda, Rios-Velasco,
44
Zamudio-Flores, & Acosta-Muñiz, 2019). Similar observations were made by Garcia, Madera,
45
Martinez, Rodriguez, and Suarez (2009), who found a reduction of 90% for a cocktail of two
46
staphylococcal phages (φ5 and φ72) after their exposure in raw milk at 37 °C for 1 1 h. The mechanism by which reduction in phage titre occurs in milk is still unclear. To date,
47 48
only one study has made suggestions about the influence of milk proteins; in this study, Das and
49
Marshall (1967) reported an inhibition of a staphylococcal phage in skim milk. The effect was
50
attributed to casein micelles, since their experiments showed that the change in pH of the skim milk
51
(4.0 to 9.0) modified phage recovery. Although the information in this area has dealt with proteins,
52
no study has addressed the complexity of milk fat.
53
To date, there is no study that has been focused on the possible effects of pure fractions,
54
either of milk (fat, whey and casein) or fat [butter oil (BO), butter milk (BM), butter serum (BS) and
55
membrane fat globule (MFGM)] on phage stability. However, it is well known that all of these
56
fractions have different compositions, chemical structures, and activities against viruses (Hamosh,
57
et al., 1999; Korhonen, 2009; Parrón et al., 2018). If so, this could be relevant as all milk fractions
58
would represent potential elements to inhibit phages. Thus, the aim of the study was to evaluate the
59
effect of milk fractions as well as fatty fractions on stability of phage A511.
60 61
2.
Materials and methods
2.1.
Milk sample collection
62 63 64
3
Bulk tank milk samples of different cows were obtained from a dairy farm and maintained at
65 66
4 °C prior to their use within 1 to 3 h.
67 68
2.2.
Phage inoculation in SM buffer and milk
69 Phage A511 was purchased from American Type Culture Collection (ATCC, Manassas, VA,
70 71
USA) and propagated with the bacterial host Listeria ivanovii (ATCC, 19119) since it does not give
72
rise to mutants resistant to phage A511 (Loessner, Goeppl, & Busse, 1991; Loessner, Rees,
73
Stewart, & Scherer, 1996). Phage propagation was performed in brain heart infusion broth (BHI) at
74
30 °C for 16 h, as described by Guenther, Huwyler, Richard, and Loessner (2009). Aliquots of the
75
purified phage suspensions were prepared at concentrations of 1 × 10 plaque-forming units per mL
76
(PFU mL ) and stored at 4 °C. Aliquots of SM buffer and raw milk (900 µL) were inoculated with the
77
purified phage at a final concentration of 3 × 10 PFU mL . Samples were vortexed to ensure a
78
complete dispersion of phages before titration.
9
-1
7
-1
79 80
2.3.
Obtaining milk fractions
81 82
Both milk and fat fractions were obtained using the procedure illustrated in Fig 1. The
83
separation of fat, whey and caseins was carried out according to García-Anaya et al. (2019) with
84
some modifications (Fig. 1, procedure 1-A). Three replicates of each treatment were performed, and
85
for each replicate 10 L of raw milk was used to obtain a considerable quantity of MFGM. The milk
86
fat was separated from milk by a cream separator (Kamdhenu Cream Separator KD60-E, Delhi,
87
India). Then, the fat was washed twice with two volumes of washing buffer (2.5 mM Na2HPO4, 2mM
88
KH2PO4, 0.14 M NaCl, 3 mM KCl, 1 mM EDTA, pH 7.2) and centrifuged at 2800 × g for 15 min at 4
89
°C, followed by solidification at −20 °C for 5 min. The upper layer (fat) was transferred into a new
90
tube. Then, an aliquot of fat (2 g) was separated for analysis of phage stability and the rest was
91
used for fat fractionation. Skim milk was used to obtain the protein fraction: casein micelles
92
(precipitate) and whey proteins (supernatant). For this, skim milk was ultracentrifuged (Beckman
4
93
Coulter ultracentrifuge, Optima XPN-100, Brea, CA, USA) using a Beckman SW 60Ti rotor at
94
98,699 × g (k factor 170.1) for 60 min at 4 °C, with 1 min ac celeration and 1 min deceleration times.
95
The supernatant was separated and the precipitate was washed twice with sterile distilled water
96
and stirred with a spatula. Casein micelles and whey were transferred into a new tube separately for
97
phage inoculation. The fat fractionation was carried out according to Parrón et al. (2018) with some
98 99
modifications (Fig. 1. Procedure 2-A). The fat obtained previously in the 1-A procedure was stored
100
at 4 °C overnight. Then, fat was churned into butte r using a Sunbeam Mixmaster (Model 03371,
101
Sunbeam Appliance Co., Mexico). The BM released in the process was filtered through several
102
layers of cheesecloth. Butter was melted at 50 ºC for 10 min and centrifuged at 4000 × g for 10 min
103
to obtain BS and BO. The MFGM was obtained through ultracentrifugation as above. All fractions of
104
interest were immediately inoculated with phage A511 and subjected to titration.
105 106
2.4.
Phage inoculation and phage enumeration
107 108
To determine phage stability, aliquots of each sample (900 µL) were inoculated with the
109
purified phage at a final concentration of 3 × 107 PFU mL-1. Samples were vortexed to ensure a
110
complete dispersion and were stored at 4 °C. Phages were enumerated by plaque assay on tryptic
111
casein soy agar (TSA) plates using antibiotics (ceftazidime 20 mg L , nalidixic acid 20 mg L and
112
cycloheximide 10 mg L ) and L. monocytogenes Scott A (ATCC 15313) as host organism, as
113
described by García-Anaya et al. (2019). Phage enumeration was carried out at 0, 24, 48, 72 and
114
96 h of storage.
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-1
-1
115 116
2.5.
Statistical analysis
117 118 119
Phage numbers (PFU) obtained for treated samples (control and milk fractions) were log10 transformed to obtain homogeneity of variance in the data. Data were analysed using a mixed
5
120
model for repeated measures using SAS software version 9.2 (SAS Institute Inc., 2002 Cary, NC,
121
USA).
122 123
3.
Results and discussion
3.1.
Effect of raw milk on the stability of phage A511
124 125 126 Fig 2 shows the reduction of phage A511 by raw milk. A reduction of 2.4 log10 PFU mL-1 of
127 128
phage was observed after 96 h of exposure in raw milk. Similar reductions have been reported by
129
other authors. For example, Garcia et al. (2009) studied the effect of skimmed and whole-fat raw
130
milk samples on the stability of two staphylococcal phages (φH5 and φA72) and found reductions of
131
1 log10 PFU mL after 8 h at 37 °C. Also, García-Anaya et al. (201 9) reported a reduction of 1 log10
132
PFU mL for the phage A511 in raw milk samples.
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-1
133 134
3.2.
Effect of milk fractions on phage stability
135 Milk fractions had a different effect on the stability of phage A511 (Fig 3). When phage was
136
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137
treated with fat, whey, and casein the phage count decreased 2.6, 2.1 and 0.8 log10 PFU mL ,
138
respectively. The mechanism for which milk fractions reduced the phage titre is unknown. Despite
139
knowledge of the antiviral properties of milk fat (Hamosh et al., 1999; Thormar, 2011), antiphage
140
properties only have been investigated in whey and casein fractions. In whey fraction, the antiphage
141
activity (against phage M13) has been related to the presence of α-lactalbumin (α-LA) and β-
142
lactoglobulin (β-LG) (Sitohy, Chobert, Karwowska, Gozdzicka-Jozefiak, & Haertlé, 2006). The
143
reduction in the phage titre by caseins has been related to electrostatic interactions causing the
144
emergence of casein-phage complexes (Das & Marshall, 1967).
145 146
3.3.
Phage stability in fat fractions
147
6
The stability of phage A511 in fat fractions is shown in Fig 4. The MFGM was the most
148
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149
active fraction against phage (2.2 log10 PFU mL ), followed by BM and BS that showed the same
150
reduction (1 log10 PFU mL ). On the contrary, in presence of BO, there was no reduction of phage.
151
To our knowledge, the effect of fat fractions on phage stability has not been evaluated with other
152
phages, but some studies give an insight into the possible factors that could reduce the phage titre.
153
For example, an study also has showed the stability of another viruses in BO samples, whereas
154
another has reported the effect of unsaturated fatty acids against phage T5 (5.9 to 97.6 % of
155
inactivation) (Blackwell, 1978; Hirotani, Ohigashi, Kobayashi, Koshimizu, & Takahashi, 1991).
156
Therefore, the instability of phage in fat could be due to differences in their physical properties (e.g.,
157
chain length, number of double bonds, availability of active groups) (Hamosh et al., 1999).
-1
158
Phage instability could be related to the presence of bioactive molecules with antiviral
159
activity or to the emergence of phage-molecule complexes that hinder the interaction with the
160
bacterial host (Civra et al., 2015; Fuller, Kuhlenschmidt, Kuhlenschmidt, Jiménez-Flores, &
161
Donovan, 2013; Parrón et al., 2018). However, it is not yet known which features of milk
162
compounds (binding interactions and/or active groups) have the effect on phage reduction.
163 164
4.
Conclusions
165 166
The present study showed the effect of raw milk on the stability of phage A511. The results
167
indicated that milk fat had the major effect in phage stability followed by whey and casein micelles.
168
Fat fractions also showed different levels of stability for phage A511. The BM and BS fractions
169
showed the same effect in the reduction of phage titre. In butter oil, phage A511 was completely
170
stable, whereas MFGM showed a great influence on phage stability. The mechanism for phage
171
reduction is unknown, but could be related to several factors including the physical structure of the
172
molecule (lipid and protein), binding interactions (electrostatic and hydrophobic) or the presence of
173
bioactive molecules. More studies are needed to determine which milk compounds cause the
174
reduction in the phage titre, as well as their mechanism of action.
175
7
176
Acknowledgements
177 178
Garcia-Anaya gratefully acknowledges the National Council for Science and Technology
179
(CONACyT) to finance her doctoral studies. This research did not receive any specific grant from
180
funding agencies in the public, commercial, or not -for-profit sectors.
181 182
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1
Figure legends
2 3
Fig. 1. Experimental isolation procedure of both milk and fat fractions. Fractions of interest are
4
indicated with bold font. Approximate yield of pure fractions in basis of 10 L of raw milk was 3.5–
5
4.0% fat; 7–10% casein; 80–85% whey, 0.6–0.7% butter milk; 2.6–2.8% butter oil; 0.4–0.5% butter
6
serum and 0.005% MFGM.
7 8
Fig. 2. Effect of raw milk on phage stability (, control; , raw milk). Error bars represent the
9
relative error from the mean of three independent experiments.
10 11
Fig. 3. Effect of milk fractions on phage stability: () milk fat, () whey and () and caseins
12
compared with the control (). Error bars represent the relative error from the mean of three
13
independent experiments.
14 15
Fig. 4. Effect of fat fractions on phage stability: () butter milk, () butter serum, () butter oil and
16
() milk fat globule membrane compared with the control (). Error bars represent the relative error
17
from the mean of three independent experiments.
18 19
1
Figure 1
Figure 2
Figure 3
Figure 4
Author Contributions
M.C. Mayra C. García-Anaya The author contributed to the conceptualization and development of experiments, as well as the writing, review and edition of the manuscript as part of her doctoral studies. Dr. David Sepúlveda The author contributed to the conceptualization of the project. Dr. Claudio Rios Velasco The author contributed to the conceptualization of the project. Dr. Paul B. Zamudio-Flores The author contributed to the conceptualization of the project. M.C. Alejandro Romo-Chacón The author contributed to the statistical analysis of data Dr. Carlos H. Acosta-Muñiz The author contributed to the conceptualization, visualization, supervision, founding acquisition and administration of the project.