- Email: [email protected]
Effect of high isostatic pressure on the peptidase activity and viability of Pseudomonas fragi isolated from a dairy processing plant
Accepted Manuscript Effect of high isostatic pressure on the peptidase activity and viability of Pseudomonas fragi isolated from a dairy processing plant Wilson R. Pinto Júnior, Leandro O. Joaquim, Patricia R. Pereira, Marcelo Cristianinni, Eduardo M. Del Aguila, Vânia M. Flosi Paschoalin PII:
S0958-6946(17)30157-7
DOI:
10.1016/j.idairyj.2017.07.007
Reference:
INDA 4204
To appear in:
International Dairy Journal
Received Date: 15 December 2016 Revised Date:
12 July 2017
Accepted Date: 12 July 2017
Please cite this article as: Pinto Júnior, W.R., Joaquim, L.O., Pereira, P.R., Cristianinni, M., Del Aguila, E.M., Flosi Paschoalin, V.M., Effect of high isostatic pressure on the peptidase activity and viability of Pseudomonas fragi isolated from a dairy processing plant, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2017.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT Effect of high isostatic pressure on the peptidase activity and viability of
RI PT
Pseudomonas fragi isolated from a dairy processing plant
Wilson R. Pinto Júniora, Leandro O. Joaquimb, Patricia R. Pereiraa, Marcelo
a
M AN U
SC
Cristianinnib, Eduardo M. Del Aguilaa, Vânia M. Flosi Paschoalina*
Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira
Ramos, 149 - Bloco A - sala 545, Cidade Universitária – 21941-909. Rio de Janeiro-RJ. Brazil
Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Av.
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b
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EP
Monteiro Lobato, 80 - sala 6121- 13083-862. Campinas-SP. Brazil
*Corresponding author. Tel.: +55 21 39387362 E-mail address: [email protected] (V. M. F. Paschoalin)
1
ACCEPTED MANUSCRIPT ______________________________________________________________________ ABSTRACT
To produce safe and high quality processed milk, high pressure (HP) technology was
RI PT
tested to inactivate undesired microorganisms and their caseinolytic activity. A
Pseudomonas fragi strain, isolated from the inner surface of a cheese-making machine from a dairy plant, was shown to harbour the aprX gene and cause casein proteolysis.
SC
Single-cycle HP processing of P. fragi-spiked milk at 450 MPa and 25 °C for 20 min decreased bacteria viability to lower levels and reduced peptidase activity by 14%.
M AN U
However when HP processing was performed at 50 °C, a synergistic effect on peptidase was observed, reaching 40% inactivation. Multiple HP treatment cycles at 450 MPa and 25 °C were less effective and reduced peptidase activity by only 23%. HP treatment could aid in the challenge to reduce AprX peptidase activity produced by microbial
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contaminants, but partial inactivation of peptidase was not effective in preventing UHT milk coagulation during storage at room temperature.
AC C
EP
_____________________________________________________________________________
2
ACCEPTED MANUSCRIPT 1.
Introduction
Industrial losses are an unwelcome problem that must be prevented or minimised, especially when dealing with perishable foodstuffs such as milk. Control of
RI PT
foodborne pathogens and their undesired metabolites is essential to extend food shelflife and preserve food quality.
Milk spoilage after a successful UHT treatment has been attributed to the
SC
original microbiota of raw milk, where bacteria can secrete the alkaline zinc metallic
metalloprotease encoded by the aprX gene (Martins, Pinto, Riedel, & Vanetti, 2015).
M AN U
This peptidase preferentially hydrolyses κ-casein, followed by β- and α-casein (Zhang et al., 2015), The increases in casein-derived peptides and decreases of particle hydration and zeta potential trigger physicochemical changes, characterised by gel and sediment formation and off-flavours, which are easily perceived by consumers (Gaucher, Tanguy,
TE D
Fauquant, Madec, & Gaucheron, 2011).
Conventional thermal methods, such as pasteurisation or UHT treatment, have been successfully applied to eliminate spoilage bacteria to guarantee the high quality
EP
and microbiological safety of dairy products for human consumption. However these treatments are not able to inactivate the already secreted thermostable peptidases. High-
AC C
pressure (HP) processing (Mújica-Paz, Valdez-Fragoso, Samson, Welti-Chanes, & Torres, 2011) is a non-thermal technology that could be used to inactivate thermostable peptidases to prolong the shelf-life of dairy products (Savadkoohi, Bannikova, Van, & Kasapis, 2014). In this study, the effectiveness of single and multiple-cycle HP applied to UHT milk artificially contaminated by a Pseudomonas fragi strain, isolated from a dairy plant
3
ACCEPTED MANUSCRIPT and able to secrete caseinolytic activity, was evaluated as an alternative technology to
2.
Material and methods
2.1.
Pseudomonas fragi isolation and characterization
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avoid milk spoilage.
SC
Conventional bacteriology methods were used to isolate a P. fragi strain at the cheese-making equipment from a dairy industrial plant in North-eastern Brazil. DNA
M AN U
preparation, quantification and sequencing, and identification of bacteria were performed as described previously (Nunes, Signori, Souza, Del Aguila, Paschoalin, 2016). PCR assay targeting for the aprx gene sequence was performed as described by
2.2.
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Marchand et al. (2009).
Cell growth and peptidase activity
EP
The P. fragi strain (105 cfu mL-1) was inoculated on UHT milk and cell growth at 7 °C was estimated by plating on Oxoid nutrient media. Cells were harvested by
AC C
centrifugation and the cell-free supernatant was used for enzyme assays. Protein content was determined by using the Qubit® Protein Assay Kit (Applied Biosystems/Life Technologies Co).
Peptidase activity was performed in triplicate as described previously (Christen & Marshall, 1984). One arbitrary unit (AU) of peptidase activity was defined as the minimum amount of protein required to enhance the absorbance by 0.01 AU (λ= 345 nm) in 60 min at 35.5 °C (Thys, Lucas, Riffel, Heeb, & Brandelli, 2004). Peptidase 4
ACCEPTED MANUSCRIPT activity was expressed as a percentage of the initial activity (100%) estimated before HP and/or thermal treatment.
2.3.
HP treatments
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A P. fragi inoculum at 109 cfu mL-1 was used to artificially contaminate 4 mL samples of UHT milk, vacuum-packaged in LDPE-Nylon bags. HP treatments were
performed in duplicate and on different days using an HP batch model QFP 2L–700
SC
(Avure Technologies Inc., KY, USA). Single-cycle treatments were performed at 450 or 550 MPa for 10, 15 or 20 min. Multiple-cycle treatments consisting of three 10-min
M AN U
cycles at 450 MPa. The water temperatures used as the pressure-transmitting fluid were 25 and 50 °C. Final pressures were reached in 2 min and depressurisation in 3 s.
Milk coagulation assay
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2.4.
Cell-free supernatants exhibiting 0.6 AU mL-1 h-1 or 0.36 AU mL-1 h-1 were combined with UHT milk in sterile glass tubes. Mixtures were maintained at 7 °C or 25
EP
°C for 30 days and milk coagulation was monitored by visual inspection (Nörnberg,
AC C
Friedrich, Weiss, Tondo, & Brandelli, 2010).
2.5.
Statistical analyses
ANOVA was used to compare the effects of pressures on peptidase activity. Tukey’s test was used to evaluate differences between samples at a 95% confidence level. Statistical analyses were performed using the STATISTICA 7.0 software package (StatiSoft Inc., OK, USA) and the results were expressed as means ± standard deviation, 5
ACCEPTED MANUSCRIPT where means followed by distinct lowercase (same processing × different time) or uppercase (different processing × same time) letters indicate significant difference (p <
Results and discussion
3.1.
Identification and characterisation of spoilage bacteria
SC
3.
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0.05) after the post-hoc test.
The P. fragi strain harbours the aprx gene in its genome, as shown by
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amplification of the 900 bp fragment by PCR assay (Fig. 1A). The strain was able to secrete peptidases from the mid-exponential growth phase in UHT milk at 7 °C (Fig. 1B), which is a typical milk storage temperature, according to the current Brazilian legislation (Brasil, 2011 http://www.apcbrh.com.br/files/IN62.pdf), while the milk is
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still in the dairy farm. After 5 days, P. fragi growth reached 3.4 log cycles, displaying a peptidase activity of 2.0 ± 0.2 AU mL-1 h -1, similar to the reference strain (p < 0.05). The peptidase showed thermostability when exposed to 50 °C for up 20 min (Fig
EP
1C), confirming that P. fragi was able to express the aprX gene detected in its genome by PCR assay secreting the AprX metalloprotease, the major contributor to casein
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proteolysis (von Neubeck et al., 2015). Contamination of post-pasteurised milk during the processing in the cheese
making apparatus could favour the rapid proliferation of P. fragi, since competition for nutrients decreases with the reduction of biodiversity, leading to severe milk spoilage (FDA, 2011).
3.3.
Effects of single- and multiple-cycle HP treatments on peptidase activity 6
ACCEPTED MANUSCRIPT
HP treatments at 450 or 550 MPa reduced the viability of P. fragi to < 1 cfu mL1
(Fig. 2A). However, peptidases secreted to UHT milk artificially contaminated by P.
fragi showed low susceptibility to HP, since a maximum of 14% peptidase inactivation
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was reached after treatment at 550 MPa for 15 min or at 450 MPa for 20 min, at 25 °C. No increment in peptidase inactivation was seen when HP treatment time at 550 MPa was extended to 20 min (Fig 2B).
SC
When HP was combined to 50 °C, peptidase inactivation reached 40% following pressurisation at 450 MPa for 20 min (Fig. 2C). No improvement in peptidase
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inactivation was observed at 550 MPa. Although peptidase activity was reduced by 32% at 550 MPa and 50 °C, treatment at 450 MPa and 50 °C was more effective in reducing peptidase activity (Fig. 2C). Combination of HP treatments at 50 °C showed synergistic effects on UHT milk preservation, demonstrated by the decrease of peptidase activity
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(compare Figs. 2C and 1C). Peptidase seems to be reversibly unfolded at 450 MPa, whereas at 550 MPa a temperature-dependent reversible association may occur, similar to that already described for β-casein (Balny & Masson, 1993).
EP
UHT milk treatment by HP in multiple 10-minute cycles at 450 MPa and 25 °C was more effective than the single 10-min cycle treatment (Fig. 2B and 2D). The
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multiple-cycle processing caused a 23% reduction in peptidase activity compared with 14% in the single-cycle treatment at the same temperature (Fig. 2B and 2D), corroborating previous studies that have already described greater efficiency of multiple-cycle HP (Buzrul, 2015). The effect of HP on peptidases varies according to their conformational features, resulting in folding and unfolding domains (Bilbao-Sainz, Younce, Rasco, & Clark, 2009). The β-domain on the secondary structure of proteins was found to be more stable 7
ACCEPTED MANUSCRIPT than the α-domain against pressure-induced unfolding. Indeed, the β-domain is essentially uncompressible, and increments in pressure do not necessarily increase enzyme inactivation (Gross & Jaenicke, 1994). Increasing pressure from 450 to 550 MPa caused no additional peptidase inactivation, indicating that P. fragi secretes
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peptidases that may be enriched in β-domain secondary structures.
In addition, the HP resistance of a peptidase depends on the matrix
characteristics. The industrial homogenisation process of raw milk reduces the size of
SC
fat globules (Michalski & Januel, 2006) and smaller fat globules coated by a protein
layer can form a stable emulsion, protecting peptidase against HP (Bilbao-Sainz et al.,
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2009). Also, the protein composition of the food matrix might protect the threedimensional conformation of the peptidase during compression, also preserving enzymatic activity (Claeys, Indrawati, & Hendrickx, 2003).
Spoilage of UHT milk inoculated with P. fragi peptidase extract
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3.5.
To investigate whether the reduction in peptidase activity following HP
EP
treatment would be enough to prevent UHT milk coagulation at storage conditions, curdling was monitored during storage at 25 °C and 7 °C in UHT milk containing full
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(0.6 AU mL-1 h-1) or 60% peptidase activity (0.36 AU mL-1 h-1). Curdling was not prevented by reducing the peptidase activity to 60%, since
after 12 days at 25 °C the coagulation signs became visually evident (Fig. 3, tube 60%, bottom panel). However, some curd reduction and change in curd consistency were observed, indicating that partial peptidase inactivation achieved after HP treatment at 450 MPa and 50 °C for 20 min (Fig 2C). The milk matrix offers optimal conditions for peptidase activity, while temperature (25 °C), high casein concentration and neutral pH 8
ACCEPTED MANUSCRIPT favors UHT milk spoilage, with the development of a bitter flavour, clearing, or coagulation (Adams et al, 1975). Since there is a direct correlation between milk shelf life and peptidase activity (Datta & Deeth, 2001; Stoeckel et al., 2016), HP treatment should decrease the levels of
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peptidase activity in a range enough to hinder clot formation. A threshold for peptidase activity and its ability to spoil UHT milk was estimated and peptidase activity ≤ 0.1 AU
Conclusions
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4.
SC
mL-1 can ensure UHT milk shelf life for 1 year (Adams, Barach, & Speck, 1975).
In UHT milk, P. fragi can reach the end of the exponential growth phase and release a thermostable peptidase promoting the spoilage of UHT milk and could affect the quality of cheese and processed cheese in dairy plants. The peptidase also exhibited
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resistance to HP, possibly due to the stabilisation induced by high pressure on the secondary structure of enzymes and/or the complex environment of the milk matrix. The thermo- and baro-resistance of the peptidase can promote curdling when UHT milk
EP
is stored at room temperature. Further investigation is required to determine the optimal combinations of HP treatment, time and temperature to reduce and prevent peptidase
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stabilisation. Understanding the mechanisms of peptidase resistance could be the way to overcome this issue.
Acknowledgments
The authors acknowledge financial support from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ; grant numbers: E9
ACCEPTED MANUSCRIPT 26/010.001968/2014, E-26 010.002864/2014, E-26/102.876/2012, E-26/202.861/2016), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant number: 142033/2012-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível
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Superior (CAPES; grant number: 8271-13-5).
References
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Adams, D. M., Barach, J. T., & Speck, M. L. (1975). Heat resistant proteases produced
828–835.
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in milk by psychrotrophic bacteria of dairy origin. Journal Dairy Science, 58,
Balny, C., & Masson, P. (1993). Effect of high pressure on proteins. Food Reviews International, 9, 611–628.
Bilbao-Sáinz, C., Younce, F. L., Rasco, B., & Clark, S. (2009). Protease stability in
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bovine milk under combined thermal-high hydrostatic pressure treatment. Innovative Food Science Emerging Technologies, 10, 314–320. Buzrul, S. (2015). Multi-pulsed high hydrostatic pressure treatment of foods. Foods, 4,
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173–183.
Christen, G. L., & Marshall, R. T. (1984). Selected properties of lipase and protease of
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Pseudomonas fluorescens 27 produced in four media. Journal of Dairy Science,
97, 1680–1687.
Claeys, W. L., Indrawati, A. M., & Hendrickx, M. E. (2003). Review: Are intrinsic TTIs for thermally processed milk applicable for high-pressure processing assessment? Innovative Food Science and Emerging Technologies, 4, 1–14. Datta, N., & Deeth, H. C. (2001). Age gelation of UHT milk - a review. Food and Bioproducts Processing, 79, 197–210. 10
ACCEPTED MANUSCRIPT FDA. (2011). Introduction of pathogenic bacteria after pasteurization and specialized cooking processes. In US Food and Drug Administration, Lactation: Fish and fishery products hazards and controls guidance (4th Edn., pp. 345–354). Washington, DC, USA: Center for Food Safety and Applied Nutrition. Office of
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Seafood.
Gaucher, I., Tanguy, G., Fauquant, J., Madec, M. -N., & Gaucheron, F. (2011). Proteolysis of casein micelles by Pseudomonas fluorescens CNRZ 798
SC
contributes to the destabilization of UHT milk during its storage. Dairy Science Technology, 91, 413–429.
Biochemistry, 221, 617–630.
M AN U
Gross, M., & Jaenicke, R. (1994). Proteins under pressure. European Journal of
Marchand, S., Vandriesche, G., Coorevits, A., Coudijzer, K., de Jonghe, V., Dewettinck, K., et al. (2009). Heterogeneity of heat-resistant proteases from
68–77.
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milk Pseudomonas species. International Journal of Food Microbiology, 133,
Martins, M. L., Pinto, U. M., Riedel, K., & Vanetti, M. C. (2015). Milk-deteriorating
EP
exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk. Brazilian Journal of Microbiology, 46, 207–217.
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Michalski, M. -C., & Januel, C. (2006). Does homogenization affect the human health properties of cow’s milk? Trends in Food Science and Technology, 17, 423–437.
Mújica-Paz, H., Valdez-Fragoso, A., Samson, C. T., Welti-Chanes, J., & Torres, A. (2011). High-pressure processing technologies for the pasteurization and sterilization of foods. Food Bioprocess Technology, 4, 969–985.
11
ACCEPTED MANUSCRIPT Nörnberg, M. F. B. L., Friedrich, R. S. C., Weiss, R. D. N., Tondo, E. C., & Brandelli, A. (2010). Proteolytic activity among psychrotrophic bacteria isolated from refrigerated raw milk. International Journal of Dairy Technology, 63, 41–46. Nunes, R. S. C. N., Signori, K., Souza, C. P., Del Aguila, E. M., & Paschoalin, V. M. F.
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(2016). Identification and molecular phylogeny of coagulase-negative
staphylococci isolates from Minas Frescal cheese in southeastern Brazil:
Superantigenic toxin production and antibiotic resistance. Journal of Dairy
SC
Science, 99, 2641–2653.
Savadkoohi, S., Bannikova, A., Van, T. T. H., & Kasapis, S. (2014). Inactivation of
M AN U
bacterial proteases and foodborne pathogens in condensed globular proteins following application of high pressure. Food Hydrocolloids, 42, 244–250. Stoeckel, M., Lidolt, M., Achberger, V., Glück, C., Krewinkel, M., Stressler, T., et al. (2016). Growth of Pseudomonas weihenstephanensis, Pseudomonas proteolytica
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and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life. International Dairy Journal, 59, 20–28.
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Thys, R. C. S., Lucas, F. S., Riffel, A., Heeb, P., & Brandelli, A. (2004). Characterization of a protease of a feather-degrading Microbacterium species.
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Letters in Applied Microbiology, 39, 181–186.
von Neubeck, M., Baur, C., Krewinkel, M., Stoeckel, M., Kranz, B., Stressler, T., et al. (2015). Biodiversity of refrigerated raw milk microbiota and their enzymatic spoilage potential. International Journal of Food Microbiology, 211, 57–65. Zhang, S., Li, H., Uluko, H., Liu, L., Pang, X. & Lv, J. (2015). Investigation of protease production by Pseudomonas fluorescens BJ‐10 and degradation on milk proteins. Journal of Food Processing and Preservation, 39, 2466–2472. 12
ACCEPTED MANUSCRIPT Figure legends
Fig. 1. Peptidase release during bacterial growth (A) PCR screening for aprX amplicons (900 bp) on a 1% agarose gel, stained with GelRedTM. Lane P: DNA molecular weight
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marker (GeneRulerTM 1kb DNA ladder); lane (C+): P. fluorescens ATCC 13525; lane
(C-): S. aureus ATCC 25923 (reference strains); lane 3: P. fragi. Panel B: growth of P. fragi () and peptidase activity () in UHT milk inoculated with 105 cfu mL-1 at 7 °C.
SC
Growth was expressed as log (N/N0), where N0 and N correspond to initial and final cell concentrations. Panel C: thermostability of peptidase activity in milk inoculated
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with P. fragi, at 25 °C (black bars) or 50 °C (grey bars).
Fig. 2. Effect of HP single- and multiple-cycles on peptidase activity and cell viability. Panel A: viability of P. fragi (109 cfu mL-1) before (white bars) and after HP processing
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at 450 MPa (black bars) and 550 MPa (grey bars) and 25 °C for 10, 15 and 20 min. Panels B, C and D: residual peptidase activity in UHT seeded milk after HP single-cycle treatment at 450 (black bars) or 550 MPa (grey bars) for 10, 15 or 20 min and 25 °C (B)
EP
or 50 °C (C), or after multiple 10 min-cycle HP treatments at 450 MPa and 25 °C (D).
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Fig. 3. Spoilage of UHT milk inoculated with P. fragi during storage at 7 °C or 25 °C. Clot formation by the remaining peptidase after HP was monitored during storage at 7 °C (upper panel) or at 25 °C (lower panel). Control tubes have no peptidase; 100% tubes had full added peptidase content of 0.6 AU mL-1 h-1 and 60% tubes contained 0.36 AU mL-1 h-1 corresponding to the remaining peptidase activity after HP at 450 MPa and 50 °C for 15 min. Tube contents were drained and are in the up-side-down position to facilitate milk-clotting visualisation (right-hand set of three, both panels).
ACCEPTED MANUSCRIPT Figure 1
P
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3
C+ 1
2
AC C
A
EP
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M AN U
C -
SC
900 pb
B
ACCEPTED MANUSCRIPT
A
M AN U
C
SC
(cf u m L1 )
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Figure 2
AC C
EP
TE D
B
C
AC C
D
EP TE D M AN U SC
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12 days of storage at 25 Ԩ
ACCEPTED MANUSCRIPT
Figure 3
S0958-6946(17)30157-7
DOI:
10.1016/j.idairyj.2017.07.007
Reference:
INDA 4204
To appear in:
International Dairy Journal
Received Date: 15 December 2016 Revised Date:
12 July 2017
Accepted Date: 12 July 2017
Please cite this article as: Pinto Júnior, W.R., Joaquim, L.O., Pereira, P.R., Cristianinni, M., Del Aguila, E.M., Flosi Paschoalin, V.M., Effect of high isostatic pressure on the peptidase activity and viability of Pseudomonas fragi isolated from a dairy processing plant, International Dairy Journal (2017), doi: 10.1016/j.idairyj.2017.07.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
ACCEPTED MANUSCRIPT Effect of high isostatic pressure on the peptidase activity and viability of
RI PT
Pseudomonas fragi isolated from a dairy processing plant
Wilson R. Pinto Júniora, Leandro O. Joaquimb, Patricia R. Pereiraa, Marcelo
a
M AN U
SC
Cristianinnib, Eduardo M. Del Aguilaa, Vânia M. Flosi Paschoalina*
Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira
Ramos, 149 - Bloco A - sala 545, Cidade Universitária – 21941-909. Rio de Janeiro-RJ. Brazil
Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Av.
TE D
b
AC C
EP
Monteiro Lobato, 80 - sala 6121- 13083-862. Campinas-SP. Brazil
*Corresponding author. Tel.: +55 21 39387362 E-mail address: [email protected] (V. M. F. Paschoalin)
1
ACCEPTED MANUSCRIPT ______________________________________________________________________ ABSTRACT
To produce safe and high quality processed milk, high pressure (HP) technology was
RI PT
tested to inactivate undesired microorganisms and their caseinolytic activity. A
Pseudomonas fragi strain, isolated from the inner surface of a cheese-making machine from a dairy plant, was shown to harbour the aprX gene and cause casein proteolysis.
SC
Single-cycle HP processing of P. fragi-spiked milk at 450 MPa and 25 °C for 20 min decreased bacteria viability to lower levels and reduced peptidase activity by 14%.
M AN U
However when HP processing was performed at 50 °C, a synergistic effect on peptidase was observed, reaching 40% inactivation. Multiple HP treatment cycles at 450 MPa and 25 °C were less effective and reduced peptidase activity by only 23%. HP treatment could aid in the challenge to reduce AprX peptidase activity produced by microbial
TE D
contaminants, but partial inactivation of peptidase was not effective in preventing UHT milk coagulation during storage at room temperature.
AC C
EP
_____________________________________________________________________________
2
ACCEPTED MANUSCRIPT 1.
Introduction
Industrial losses are an unwelcome problem that must be prevented or minimised, especially when dealing with perishable foodstuffs such as milk. Control of
RI PT
foodborne pathogens and their undesired metabolites is essential to extend food shelflife and preserve food quality.
Milk spoilage after a successful UHT treatment has been attributed to the
SC
original microbiota of raw milk, where bacteria can secrete the alkaline zinc metallic
metalloprotease encoded by the aprX gene (Martins, Pinto, Riedel, & Vanetti, 2015).
M AN U
This peptidase preferentially hydrolyses κ-casein, followed by β- and α-casein (Zhang et al., 2015), The increases in casein-derived peptides and decreases of particle hydration and zeta potential trigger physicochemical changes, characterised by gel and sediment formation and off-flavours, which are easily perceived by consumers (Gaucher, Tanguy,
TE D
Fauquant, Madec, & Gaucheron, 2011).
Conventional thermal methods, such as pasteurisation or UHT treatment, have been successfully applied to eliminate spoilage bacteria to guarantee the high quality
EP
and microbiological safety of dairy products for human consumption. However these treatments are not able to inactivate the already secreted thermostable peptidases. High-
AC C
pressure (HP) processing (Mújica-Paz, Valdez-Fragoso, Samson, Welti-Chanes, & Torres, 2011) is a non-thermal technology that could be used to inactivate thermostable peptidases to prolong the shelf-life of dairy products (Savadkoohi, Bannikova, Van, & Kasapis, 2014). In this study, the effectiveness of single and multiple-cycle HP applied to UHT milk artificially contaminated by a Pseudomonas fragi strain, isolated from a dairy plant
3
ACCEPTED MANUSCRIPT and able to secrete caseinolytic activity, was evaluated as an alternative technology to
2.
Material and methods
2.1.
Pseudomonas fragi isolation and characterization
RI PT
avoid milk spoilage.
SC
Conventional bacteriology methods were used to isolate a P. fragi strain at the cheese-making equipment from a dairy industrial plant in North-eastern Brazil. DNA
M AN U
preparation, quantification and sequencing, and identification of bacteria were performed as described previously (Nunes, Signori, Souza, Del Aguila, Paschoalin, 2016). PCR assay targeting for the aprx gene sequence was performed as described by
2.2.
TE D
Marchand et al. (2009).
Cell growth and peptidase activity
EP
The P. fragi strain (105 cfu mL-1) was inoculated on UHT milk and cell growth at 7 °C was estimated by plating on Oxoid nutrient media. Cells were harvested by
AC C
centrifugation and the cell-free supernatant was used for enzyme assays. Protein content was determined by using the Qubit® Protein Assay Kit (Applied Biosystems/Life Technologies Co).
Peptidase activity was performed in triplicate as described previously (Christen & Marshall, 1984). One arbitrary unit (AU) of peptidase activity was defined as the minimum amount of protein required to enhance the absorbance by 0.01 AU (λ= 345 nm) in 60 min at 35.5 °C (Thys, Lucas, Riffel, Heeb, & Brandelli, 2004). Peptidase 4
ACCEPTED MANUSCRIPT activity was expressed as a percentage of the initial activity (100%) estimated before HP and/or thermal treatment.
2.3.
HP treatments
RI PT
A P. fragi inoculum at 109 cfu mL-1 was used to artificially contaminate 4 mL samples of UHT milk, vacuum-packaged in LDPE-Nylon bags. HP treatments were
performed in duplicate and on different days using an HP batch model QFP 2L–700
SC
(Avure Technologies Inc., KY, USA). Single-cycle treatments were performed at 450 or 550 MPa for 10, 15 or 20 min. Multiple-cycle treatments consisting of three 10-min
M AN U
cycles at 450 MPa. The water temperatures used as the pressure-transmitting fluid were 25 and 50 °C. Final pressures were reached in 2 min and depressurisation in 3 s.
Milk coagulation assay
TE D
2.4.
Cell-free supernatants exhibiting 0.6 AU mL-1 h-1 or 0.36 AU mL-1 h-1 were combined with UHT milk in sterile glass tubes. Mixtures were maintained at 7 °C or 25
EP
°C for 30 days and milk coagulation was monitored by visual inspection (Nörnberg,
AC C
Friedrich, Weiss, Tondo, & Brandelli, 2010).
2.5.
Statistical analyses
ANOVA was used to compare the effects of pressures on peptidase activity. Tukey’s test was used to evaluate differences between samples at a 95% confidence level. Statistical analyses were performed using the STATISTICA 7.0 software package (StatiSoft Inc., OK, USA) and the results were expressed as means ± standard deviation, 5
ACCEPTED MANUSCRIPT where means followed by distinct lowercase (same processing × different time) or uppercase (different processing × same time) letters indicate significant difference (p <
Results and discussion
3.1.
Identification and characterisation of spoilage bacteria
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3.
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0.05) after the post-hoc test.
The P. fragi strain harbours the aprx gene in its genome, as shown by
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amplification of the 900 bp fragment by PCR assay (Fig. 1A). The strain was able to secrete peptidases from the mid-exponential growth phase in UHT milk at 7 °C (Fig. 1B), which is a typical milk storage temperature, according to the current Brazilian legislation (Brasil, 2011 http://www.apcbrh.com.br/files/IN62.pdf), while the milk is
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still in the dairy farm. After 5 days, P. fragi growth reached 3.4 log cycles, displaying a peptidase activity of 2.0 ± 0.2 AU mL-1 h -1, similar to the reference strain (p < 0.05). The peptidase showed thermostability when exposed to 50 °C for up 20 min (Fig
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1C), confirming that P. fragi was able to express the aprX gene detected in its genome by PCR assay secreting the AprX metalloprotease, the major contributor to casein
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proteolysis (von Neubeck et al., 2015). Contamination of post-pasteurised milk during the processing in the cheese
making apparatus could favour the rapid proliferation of P. fragi, since competition for nutrients decreases with the reduction of biodiversity, leading to severe milk spoilage (FDA, 2011).
3.3.
Effects of single- and multiple-cycle HP treatments on peptidase activity 6
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HP treatments at 450 or 550 MPa reduced the viability of P. fragi to < 1 cfu mL1
(Fig. 2A). However, peptidases secreted to UHT milk artificially contaminated by P.
fragi showed low susceptibility to HP, since a maximum of 14% peptidase inactivation
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was reached after treatment at 550 MPa for 15 min or at 450 MPa for 20 min, at 25 °C. No increment in peptidase inactivation was seen when HP treatment time at 550 MPa was extended to 20 min (Fig 2B).
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When HP was combined to 50 °C, peptidase inactivation reached 40% following pressurisation at 450 MPa for 20 min (Fig. 2C). No improvement in peptidase
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inactivation was observed at 550 MPa. Although peptidase activity was reduced by 32% at 550 MPa and 50 °C, treatment at 450 MPa and 50 °C was more effective in reducing peptidase activity (Fig. 2C). Combination of HP treatments at 50 °C showed synergistic effects on UHT milk preservation, demonstrated by the decrease of peptidase activity
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(compare Figs. 2C and 1C). Peptidase seems to be reversibly unfolded at 450 MPa, whereas at 550 MPa a temperature-dependent reversible association may occur, similar to that already described for β-casein (Balny & Masson, 1993).
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UHT milk treatment by HP in multiple 10-minute cycles at 450 MPa and 25 °C was more effective than the single 10-min cycle treatment (Fig. 2B and 2D). The
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multiple-cycle processing caused a 23% reduction in peptidase activity compared with 14% in the single-cycle treatment at the same temperature (Fig. 2B and 2D), corroborating previous studies that have already described greater efficiency of multiple-cycle HP (Buzrul, 2015). The effect of HP on peptidases varies according to their conformational features, resulting in folding and unfolding domains (Bilbao-Sainz, Younce, Rasco, & Clark, 2009). The β-domain on the secondary structure of proteins was found to be more stable 7
ACCEPTED MANUSCRIPT than the α-domain against pressure-induced unfolding. Indeed, the β-domain is essentially uncompressible, and increments in pressure do not necessarily increase enzyme inactivation (Gross & Jaenicke, 1994). Increasing pressure from 450 to 550 MPa caused no additional peptidase inactivation, indicating that P. fragi secretes
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peptidases that may be enriched in β-domain secondary structures.
In addition, the HP resistance of a peptidase depends on the matrix
characteristics. The industrial homogenisation process of raw milk reduces the size of
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fat globules (Michalski & Januel, 2006) and smaller fat globules coated by a protein
layer can form a stable emulsion, protecting peptidase against HP (Bilbao-Sainz et al.,
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2009). Also, the protein composition of the food matrix might protect the threedimensional conformation of the peptidase during compression, also preserving enzymatic activity (Claeys, Indrawati, & Hendrickx, 2003).
Spoilage of UHT milk inoculated with P. fragi peptidase extract
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3.5.
To investigate whether the reduction in peptidase activity following HP
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treatment would be enough to prevent UHT milk coagulation at storage conditions, curdling was monitored during storage at 25 °C and 7 °C in UHT milk containing full
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(0.6 AU mL-1 h-1) or 60% peptidase activity (0.36 AU mL-1 h-1). Curdling was not prevented by reducing the peptidase activity to 60%, since
after 12 days at 25 °C the coagulation signs became visually evident (Fig. 3, tube 60%, bottom panel). However, some curd reduction and change in curd consistency were observed, indicating that partial peptidase inactivation achieved after HP treatment at 450 MPa and 50 °C for 20 min (Fig 2C). The milk matrix offers optimal conditions for peptidase activity, while temperature (25 °C), high casein concentration and neutral pH 8
ACCEPTED MANUSCRIPT favors UHT milk spoilage, with the development of a bitter flavour, clearing, or coagulation (Adams et al, 1975). Since there is a direct correlation between milk shelf life and peptidase activity (Datta & Deeth, 2001; Stoeckel et al., 2016), HP treatment should decrease the levels of
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peptidase activity in a range enough to hinder clot formation. A threshold for peptidase activity and its ability to spoil UHT milk was estimated and peptidase activity ≤ 0.1 AU
Conclusions
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4.
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mL-1 can ensure UHT milk shelf life for 1 year (Adams, Barach, & Speck, 1975).
In UHT milk, P. fragi can reach the end of the exponential growth phase and release a thermostable peptidase promoting the spoilage of UHT milk and could affect the quality of cheese and processed cheese in dairy plants. The peptidase also exhibited
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resistance to HP, possibly due to the stabilisation induced by high pressure on the secondary structure of enzymes and/or the complex environment of the milk matrix. The thermo- and baro-resistance of the peptidase can promote curdling when UHT milk
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is stored at room temperature. Further investigation is required to determine the optimal combinations of HP treatment, time and temperature to reduce and prevent peptidase
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stabilisation. Understanding the mechanisms of peptidase resistance could be the way to overcome this issue.
Acknowledgments
The authors acknowledge financial support from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ; grant numbers: E9
ACCEPTED MANUSCRIPT 26/010.001968/2014, E-26 010.002864/2014, E-26/102.876/2012, E-26/202.861/2016), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant number: 142033/2012-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível
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Superior (CAPES; grant number: 8271-13-5).
References
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Adams, D. M., Barach, J. T., & Speck, M. L. (1975). Heat resistant proteases produced
828–835.
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in milk by psychrotrophic bacteria of dairy origin. Journal Dairy Science, 58,
Balny, C., & Masson, P. (1993). Effect of high pressure on proteins. Food Reviews International, 9, 611–628.
Bilbao-Sáinz, C., Younce, F. L., Rasco, B., & Clark, S. (2009). Protease stability in
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bovine milk under combined thermal-high hydrostatic pressure treatment. Innovative Food Science Emerging Technologies, 10, 314–320. Buzrul, S. (2015). Multi-pulsed high hydrostatic pressure treatment of foods. Foods, 4,
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173–183.
Christen, G. L., & Marshall, R. T. (1984). Selected properties of lipase and protease of
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Pseudomonas fluorescens 27 produced in four media. Journal of Dairy Science,
97, 1680–1687.
Claeys, W. L., Indrawati, A. M., & Hendrickx, M. E. (2003). Review: Are intrinsic TTIs for thermally processed milk applicable for high-pressure processing assessment? Innovative Food Science and Emerging Technologies, 4, 1–14. Datta, N., & Deeth, H. C. (2001). Age gelation of UHT milk - a review. Food and Bioproducts Processing, 79, 197–210. 10
ACCEPTED MANUSCRIPT FDA. (2011). Introduction of pathogenic bacteria after pasteurization and specialized cooking processes. In US Food and Drug Administration, Lactation: Fish and fishery products hazards and controls guidance (4th Edn., pp. 345–354). Washington, DC, USA: Center for Food Safety and Applied Nutrition. Office of
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Seafood.
Gaucher, I., Tanguy, G., Fauquant, J., Madec, M. -N., & Gaucheron, F. (2011). Proteolysis of casein micelles by Pseudomonas fluorescens CNRZ 798
SC
contributes to the destabilization of UHT milk during its storage. Dairy Science Technology, 91, 413–429.
Biochemistry, 221, 617–630.
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Gross, M., & Jaenicke, R. (1994). Proteins under pressure. European Journal of
Marchand, S., Vandriesche, G., Coorevits, A., Coudijzer, K., de Jonghe, V., Dewettinck, K., et al. (2009). Heterogeneity of heat-resistant proteases from
68–77.
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milk Pseudomonas species. International Journal of Food Microbiology, 133,
Martins, M. L., Pinto, U. M., Riedel, K., & Vanetti, M. C. (2015). Milk-deteriorating
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exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk. Brazilian Journal of Microbiology, 46, 207–217.
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Michalski, M. -C., & Januel, C. (2006). Does homogenization affect the human health properties of cow’s milk? Trends in Food Science and Technology, 17, 423–437.
Mújica-Paz, H., Valdez-Fragoso, A., Samson, C. T., Welti-Chanes, J., & Torres, A. (2011). High-pressure processing technologies for the pasteurization and sterilization of foods. Food Bioprocess Technology, 4, 969–985.
11
ACCEPTED MANUSCRIPT Nörnberg, M. F. B. L., Friedrich, R. S. C., Weiss, R. D. N., Tondo, E. C., & Brandelli, A. (2010). Proteolytic activity among psychrotrophic bacteria isolated from refrigerated raw milk. International Journal of Dairy Technology, 63, 41–46. Nunes, R. S. C. N., Signori, K., Souza, C. P., Del Aguila, E. M., & Paschoalin, V. M. F.
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(2016). Identification and molecular phylogeny of coagulase-negative
staphylococci isolates from Minas Frescal cheese in southeastern Brazil:
Superantigenic toxin production and antibiotic resistance. Journal of Dairy
SC
Science, 99, 2641–2653.
Savadkoohi, S., Bannikova, A., Van, T. T. H., & Kasapis, S. (2014). Inactivation of
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bacterial proteases and foodborne pathogens in condensed globular proteins following application of high pressure. Food Hydrocolloids, 42, 244–250. Stoeckel, M., Lidolt, M., Achberger, V., Glück, C., Krewinkel, M., Stressler, T., et al. (2016). Growth of Pseudomonas weihenstephanensis, Pseudomonas proteolytica
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and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life. International Dairy Journal, 59, 20–28.
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Thys, R. C. S., Lucas, F. S., Riffel, A., Heeb, P., & Brandelli, A. (2004). Characterization of a protease of a feather-degrading Microbacterium species.
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Letters in Applied Microbiology, 39, 181–186.
von Neubeck, M., Baur, C., Krewinkel, M., Stoeckel, M., Kranz, B., Stressler, T., et al. (2015). Biodiversity of refrigerated raw milk microbiota and their enzymatic spoilage potential. International Journal of Food Microbiology, 211, 57–65. Zhang, S., Li, H., Uluko, H., Liu, L., Pang, X. & Lv, J. (2015). Investigation of protease production by Pseudomonas fluorescens BJ‐10 and degradation on milk proteins. Journal of Food Processing and Preservation, 39, 2466–2472. 12
ACCEPTED MANUSCRIPT Figure legends
Fig. 1. Peptidase release during bacterial growth (A) PCR screening for aprX amplicons (900 bp) on a 1% agarose gel, stained with GelRedTM. Lane P: DNA molecular weight
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marker (GeneRulerTM 1kb DNA ladder); lane (C+): P. fluorescens ATCC 13525; lane
(C-): S. aureus ATCC 25923 (reference strains); lane 3: P. fragi. Panel B: growth of P. fragi () and peptidase activity () in UHT milk inoculated with 105 cfu mL-1 at 7 °C.
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Growth was expressed as log (N/N0), where N0 and N correspond to initial and final cell concentrations. Panel C: thermostability of peptidase activity in milk inoculated
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with P. fragi, at 25 °C (black bars) or 50 °C (grey bars).
Fig. 2. Effect of HP single- and multiple-cycles on peptidase activity and cell viability. Panel A: viability of P. fragi (109 cfu mL-1) before (white bars) and after HP processing
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at 450 MPa (black bars) and 550 MPa (grey bars) and 25 °C for 10, 15 and 20 min. Panels B, C and D: residual peptidase activity in UHT seeded milk after HP single-cycle treatment at 450 (black bars) or 550 MPa (grey bars) for 10, 15 or 20 min and 25 °C (B)
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or 50 °C (C), or after multiple 10 min-cycle HP treatments at 450 MPa and 25 °C (D).
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Fig. 3. Spoilage of UHT milk inoculated with P. fragi during storage at 7 °C or 25 °C. Clot formation by the remaining peptidase after HP was monitored during storage at 7 °C (upper panel) or at 25 °C (lower panel). Control tubes have no peptidase; 100% tubes had full added peptidase content of 0.6 AU mL-1 h-1 and 60% tubes contained 0.36 AU mL-1 h-1 corresponding to the remaining peptidase activity after HP at 450 MPa and 50 °C for 15 min. Tube contents were drained and are in the up-side-down position to facilitate milk-clotting visualisation (right-hand set of three, both panels).
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Figure 2
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12 days of storage at 25 Ԩ
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Figure 3