International Dairy Journal 75 (2017) 51e55

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International Dairy Journal

journal homepage: www.elsevier.com/locate/idairyj

Short communication

Effect of high isostatic pressure on the peptidase activity and viability

of Pseudomonas fragi isolated from a dairy processing plant
Wilson R. Pinto Júnior a, Leandro O. Joaquim b, Patricia R. Pereira a, Marcelo Cristianini b,
Eduardo M. Del Aguila a, Va ^nia M. Flosi Paschoalin a, *
a ria, 21941-909, Rio
Instituto de Química, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149 e Bloco A e sala 545, Cidade Universita
de Janeiro, RJ, Brazil
b
Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Av. Monteiro Lobato, 80 e sala 6121, 13083-862, Campinas, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: To produce safe and high quality processed milk, high pressure (HP) technology was tested to inactivate
Received 15 December 2016 undesired microorganisms and their caseinolytic activity. A Pseudomonas fragi strain, isolated from the
Received in revised form inner surface of a cheese-making machine from a dairy plant, was shown to harbour the aprX gene and
12 July 2017
cause casein proteolysis. Single-cycle HP processing of P. fragi-spiked milk at 450 MPa and 25
C for
Accepted 12 July 2017
Available online 1 August 2017
20 min decreased bacteria viability to lower levels and reduced peptidase activity by 14%. 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 contaminants, but partial inactivation of peptidase was not effective in preventing
UHT milk coagulation during storage at room temperature.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction bacteria to guarantee the high quality and microbiological safety of

Industrial losses are an unwelcome problem that must be pre-
vented or minimised, especially when dealing with perishable
foodstuffs such as milk. Control of foodborne pathogens and their
undesired metabolites is essential to extend food shelf-life and
preserve food quality.
Milk spoilage after a successful UHT treatment has been
attributed to the original microbiota of raw milk, where bacteria
can secrete the alkaline zinc metallic metalloprotease encoded by
the aprX gene (Martins, Pinto, Riedel, & Vanetti, 2015). This pepti-
dase preferentially hydrolyses k-casein, followed by b- and a-casein
(Zhang et al., 2015), The increases in casein-derived peptides and
decreases of particle hydration and zeta potential trigger physico-
chemical changes, characterised by gel and sediment formation and
off-flavours, which are easily perceived by consumers (Gaucher,
Tanguy, Fauquant, Madec, & Gaucheron, 2011).
Conventional thermal methods, such as pasteurisation or UHT
treatment, have been successfully applied to eliminate spoilage
* Corresponding author. Tel.: þ55 21 39387362.
E-mail address: paschv@iq.ufrj.br (V.M.F. Paschoalin).
dairy products for human consumption. However these treatments
are not able to inactivate the already secreted thermostable pep-
tidases. High pressure (HP) processing (Mújica-Paz, Valdez-
Fragoso, Samson, Welti-Chanes, & Torres, 2011) is a non-thermal
technology that could be used to inactivate thermostable pepti-
dases 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 and able to secrete casein-
olytic activity, was evaluated as an alternative technology to avoid
milk spoilage.
2. Material and methods
2.1. P. fragi isolation and characterisation
Conventional bacteriology methods were used to isolate a
P. fragi strain at the cheese-making equipment from a dairy in-
dustrial plant in North-eastern Brazil. DNA preparation, quantifi-
cation and sequencing, and identification of bacteria were
performed as described previously (Nunes, Signori, Souza, Del

http://dx.doi.org/10.1016/j.idairyj.2017.07.007
0958-6946/© 2017 Elsevier Ltd. All rights reserved.
52 W.R. Pinto Júnior et al. / International Dairy Journal 75 (2017) 51e55

Aguila, & Paschoalin, 2016). PCR assay targeting for the aprx gene
sequence was performed as described by Marchand et al. (2009).
2.2. Cell growth and peptidase activity
The P. fragi strain (105 cfu mL1) was inoculated on UHT milk
and cell growth at 7
C was estimated by plating on Oxoid nutrient
media. Cells were harvested by centrifugation and the cell-free
supernatant was used for enzyme assays. Protein content was
determined by using the Qubit® Protein Assay Kit (Applied Bio-
systems/Life Technologies Co, Foster City, California, USA).
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 (l ¼ 345 nm) in
60 min at 35.5
C (Thys, Lucas, Riffel, Heeb, & Brandelli, 2004).
Peptidase activity was expressed as a percentage of the initial ac-
tivity (100%) estimated before HP and/or thermal treatment.
2.3. HP treatments
A P. fragi inoculum at 109 cfu mL1 was used to artificially
contaminate 4 mL samples of UHT milk, vacuum-packaged in low-
density polyethylene (LDPE)-Nylon bags. HP treatments were per-
formed in duplicate and on different days using an HP batch model
QFP 2Le700 (Avure Technologies Inc., KY, USA). Single-cycle treat-
ments were performed at 450 or 550 MPa for 10, 15 or 20 min.
Multiple-cycle treatments consisting of three 10-min 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.
differences between samples at a 95% confidence level. Statistical
analyses were performed using the STATISTICA 7.0 software pack-
age (StatiSoft Inc., OK, USA) and the results were expressed as
means ± standard deviation, where means followed by distinct
lowercase (same processing  different time) or uppercase
(different processing  same time) letters indicate significant dif-
ference (p < 0.05) after the post-hoc test.
3. Results and discussion
3.1. Identification and characterisation of spoilage bacteria
The P. fragi strain harbours the aprx gene in its genome, as
shown by 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 Brazil-
ian legislation (Brasil, 2011 http://www.apcbrh.com.br/files/IN62.
pdf), while the milk is still in the dairy farm. After 5 days,
P. fragi growth reached 3.4 log cycles, displaying a peptidase ac-
tivity of 2.0 ± 0.2 AU mL1 h1, similar to the reference strain
(p < 0.05).
The peptidase showed thermostability when exposed to 50
C
for up 20 min (Fig. 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 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).

2.4. Milk coagulation assay
Cell-free supernatants exhibiting 0.6 AU mL1 h1 or
0.36 AU mL1 h1 were combined with UHT milk in sterile glass
tubes. Mixtures were maintained at 7
C or 25
C for 30 days and
milk coagulation was monitored by visual inspection (No € rnberg,
Friedrich, Weiss, Tondo, & Brandelli, 2010).
2.5. Statistical analyses
Analysis of variance (ANOVA) was used to compare the effects of
pressures on peptidase activity. Tukey's test was used to evaluate
3.2. Effects of single- and multiple-cycle HP treatments on
peptidase activity
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 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 treat-
ment time at 550 MPa was extended to 20 min (Fig. 2B).
When HP was combined to 50
C, peptidase inactivation
reached 40% following pressurisation at 450 MPa for 20 min

Fig. 1. Peptidase release during bacterial growth (A) PCR screening for aprX amplicons (900 bp) on a 1% agarose gel, stained with GelRed™. Lane P: DNA molecular weight marker
(GeneRuler™ 1 kb DNA ladder); lane (Cþ): 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 mL1 at 7
C. 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 with P. fragi, at 25
C (black bars) or 50
C (grey bars).
 W.R. Pinto Júnior et al. / International Dairy Journal 75 (2017) 51e55 53

(Fig. 2C). No improvement in peptidase 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
(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 b-casein (Balny & Masson, 1993).
UHT milk treatment by HP in multiple 10-min cycles at 450 MPa
and 25
C was more effective than the single 10-min cycle treat-
ment (Fig. 2B and D). The 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 D), corrobo-
rating previous studies that have already described greater effi-
ciency of multiple-cycle HP (Buzrul, 2015).
The effect of HP on peptidases varies according to their
conformational features, resulting in folding and unfolding do-
mains (Bilbao-Sa inz, Younce, Rasco, & Clark, 2009). The b-domain
on the secondary structure of proteins was found to be more stable
than the a-domain against pressure-induced unfolding. Indeed,
the b-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 se-
cretes peptidases that may be enriched in b-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 fat globules (Michalski & Januel, 2006)
and smaller fat globules coated by a protein layer can form a stable
emulsion, protecting peptidase against HP (Bilbao-Sa inz et al.,
2009). Also, the protein composition of the food matrix might
protect the three-dimensional conformation of the peptidase dur-
ing compression, also preserving enzymatic activity (Claeys,
Indrawati, & Hendrickx, 2003).
3.3. Spoilage of UHT milk inoculated with P. fragi peptidase extract
To investigate whether the reduction in peptidase activity
following HP treatment would be enough to prevent UHT milk
coagulation at storage conditions, curdling was monitored dur-
ing storage at 25
C and 7
C in UHT milk containing full
(0.6 AU mL1 h1) or 60% peptidase activity (0.36 AU mL1 h1).
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 tem-
perature (25
C), high casein concentration and neutral pH favours
UHT milk spoilage, with the development of a bitter flavour,
clearing, or coagulation (Adams, Barach, & Speck, 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 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 pepti-
dase activity 0.1 AU mL1 can ensure UHT milk shelf life for 1 year
(Adams et al., 1975).

Fig. 2. Effect of HP single- and multiple-cycles on peptidase activity and cell viability. Panel A: viability of P. fragi (109 cfu mL1) before (white bars) and after HP processing 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) or 50
C (C), or after multiple 10 min-cycle HP treatments at 450 MPa and 25
C (D).
54 W.R. Pinto Júnior et al. / International Dairy Journal 75 (2017) 51e55

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 mL1 h1 and 60% tubes contained 0.36 AU mL1 h1
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).

4. Conclusions numbers: E-26/010.001968/2014, E-26 010.002864/2014, E-26/

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 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 pepti-
dase can promote curdling when UHT milk is stored at room tem-
perature. Further investigation is required to determine the optimal
combinations of HP treatment, time and temperature to reduce and
prevent peptidase stabilisation. Understanding the mechanisms of
peptidase resistance could be the way to overcome this issue.
Acknowledgements
The authors acknowledge financial support from Fundaç~ao de
 Pesquisa do Estado do Rio de Janeiro (FAPERJ; grant
Amparo a
102.876/2012, E-26/202.861/2016), Conselho Nacional de Desen-
 gico (CNPq; grant number: 142033/
volvimento Científico e Tecnolo
2012-0) and Coordenaç~ ao de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES; grant number: 8271-13-5).
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