Food Chemistry Advances 2 (2023) 100241

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Food Chemistry Advances

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

Preservation of high pressure pasteurised milk by hyperbaric storage at

room temperature versus refrigeration – Effect on natural microbiota and
physicochemical properties
Álvaro T. Lemos, José A. Lopes-da-Silva, Ivonne Delgadillo, Jorge A. Saraiva∗
LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

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

Keywords: Compared to refrigeration (RF), hyperbaric storage (HS) has been applied as a preservation methodology to
Hyperbaric storage increase foods shelf-life. The aim of this study was to compare HS at naturally variable uncontrolled room tem-
Refrigeration perature (RT) to RF and evaluate its feasibility to preserve high pressure pasteurised milk (HPP milk), using
High pressure pasteurised milk
different pressure levels (50, 75 and 100 MPa), by testing microbial and physicochemical parameters.
Microbiology
While RF merely inhibited microbial growth over 40 days, HS/RT (75/100 MPa) maintained pH and gradually
Preservation
Shelf-life reducing all endogenous microorganisms tested to below the detection limit (< 1.0 log CFU/mL) corresponding
to at least 2.60 log CFU/mL reduction on total aerobic mesophiles. Additionally, total soluble solids and apparent
viscosity could both be kept using HS/RT for at least for 21 days. For 40 days, the total colour difference showed
a small but discernible change for RF and HS/RT conditions, being also observed an increase on soluble protein
for HS/RT samples, which may have resulted by pressure leading to aggregation of micelle caseins and whey
proteins.
In conclusion, the present work showed the potential of HS to preserve HPP milk with longer microbial shelf-
life compared to RF and with a minor impact on physicochemical properties, being more environmentally friendly
and sustainable than conventional RF.

1. Introduction
Raw milk drinking has been linked to a public health risk
(David, 2012), since it can support a rich microflora that can be eas-
ily introduced into milk, at any stage from the production to con-
sumption, including collection, processing, handling, distribution and
post-harvest storage (Lee, Barbano & Drake, 2016; Porcellato et al.,
2018). Governments, regulatory authorities, and public health practi-
tioners recommend or even mandate conventional heat treatments such
as pasteurisation and sterilization to prevent milk spoilage and ensure
safe products, (Alegbeleye, Guimarães, Cruz & Sant’Ana, 2018; Codina-
Torrella, Guamis, Zamora, Quevedo & Trujillo, 2018). However, these
treatments can result in undesirable losses of the most appreciated milk
sensory characteristics, particularly in the case of sterilization (Lee et al.,
2016). Hence, the consumers, in many countries, prefer consuming pas-
teurised milk because of its perceived better flavour, even with its in-
herent shorter shelf-life under refrigeration (RF). Moreover, consumers
demand for fresher-like tasting products has driven research and inno-
vation for alternative preservation methods, such as is the case of non-
thermal high pressure processing (HPP) pasteurisation.
Indeed, HPP has been so far extensively studied to pasteurise many
food products with minimal impact on their natural nutritional and
sensory characteristics, while also ensuring at the same time micro-
bial safety, as thermal pasteurisation does (Codina-Torrella et al., 2018;
Machado et al., 2019). HPP was first reported precisely to preserve raw
milk by Hite (1899) who applied 680 MPa for 10 min at room tem-
perature and achieved a 5‐6 log reduction in the number of bacteria in
milk. Nowadays, HPP pasteurisation is well established commercially,
applying pressures from 400 to 600 MPa for some minutes to pasteurise
diverse products like fruit juices, vegetables, seafood and meat products
(Duarte et al., 2014). Several authors have investigated the use of HPP
to pasteurise raw milk, not only due to its ability to inactivate spoilage
and pathogenic bacteria, but also to maintain the quality characteris-
tics of dairy products, while minimising the loss of vitamins and the
production of off-flavours (Machado et al., 2019; Yang et al., 2012).
Low acidity pasteurised food products must be refrigerated, re-
gardless of the pasteurisation process used, for its preservation
(Fidalgo, Lemos, Delgadillo & Saraiva, 2018). However, while RF
increases food shelf-life the energetic cost of RF is significant in
the food industry, with 40 % of all food requiring refrigeration

∗
Corresponding author.
E-mail address: jorgesaraiva@ua.pt (J.A. Saraiva).

https://doi.org/10.1016/j.focha.2023.100241
Received 22 September 2022; Received in revised form 14 February 2023; Accepted 13 March 2023
2772-753X/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
Á.T. Lemos, J.A. Lopes-da-Silva, I. Delgadillo et al.
and RF accounting for 15 % of the electricity consumed globally
(James & James, 2010).
Hyperbaric storage (HS), which typically ranges between 25 and
100 MPa, is a new concept of food preservation, relying on constant
pressure application to store foods as a hurdle to microbial growth as
RF (Lemos, Ribeiro, Fidalgo, Delgadillo & Saraiva, 2017). In last years,
HS has received a lot of attention for its ability to extend the microbial
shelf-life of diverse highly perishable foods compared to RF, such as fruit
juices (Lemos et al., 2017), fish (Fidalgo et al., 2018), raw bovine meat
(Fernandes et al., 2019; Freitas et al., 2016), and ready-to-eat meals
(Moreira et al., 2015). Generally, HS at room temperature (RT), allows
for lower energy consumption and carbon footprint, because energy is
only needed for compression to generate the pressure and not to main-
tain pressure during storage, which is another significant advantage over
RF (Bermejo-Prada, Colmant, Otero & Guignon, 2017).
Thus, the purpose of this study was to compare HS feasibility to RF
as a preservation methodology for high pressure pasteurised milk (HPP
milk), at three different pressure levels (50 MPa, 75 MPa and 100 MPa),
at naturally variable uncontrolled room temperature (RT ≈ 20 °C). For
that, microbiological (Enterobacteriaceae, coliform bacteria, total aero-
bic mesophiles, psychrotrophic bacteria, lactic acid bacteria, and yeasts
and moulds) and physicochemical analyses (pH, total soluble solids,
colour, apparent viscosity and soluble protein content) were carried out
to perceive possible differences between HS at RT and conventional RF,
over a long storage period (40 and 60 days, respectively for microbial
and chemical analyses). Prior to the storage study, effect of high pres-
sure pasteurisation on raw milk was also briefly investigated. As far as
the authors are aware, this is the first study concerning HS of a highly
perishable pasteurised food product.
2. Materials and methods
2.1. Raw bovine milk collection
Raw bovine milk was obtained from a dairy products company in
Portugal and transported refrigerated to the laboratory, where it was
packaged in 250 mL plastic bottles (PET, EspaçoPlás, Leiria, Portugal)
and high pressure pasteurised immediately.
2.2. High pressure pasteurisation of raw bovine milk
The bottles with raw milk, at an initial temperature of ≈ 4 °C, were
pasteurised by high pressure processing using as pressurization fluid
potable water at ≈ 20 °C, with a first cycle of 600 MPa/90 s, followed
by a second cycle of 600 MPa/120 s (Cornell, 2017). Pressure process-
ing was performed in a pilot-industrial scale high pressure press (Model
55, Hyperbaric, Burgos, Spain), with a pressure vessel of 200 mm in-
ner diameter and 2000 mm length, with a capacity of 55 L and a maxi-
mum operating pressure of 600 MPa. The compression time was approx-
imately 30 s/100 MPa and decompression time was only a few seconds.
After pasteurisation, the bottles were kept at ≈ 4 °C until being trans-
ferred (no more than 2 h) to previously UV sterilised (BioSafety Cabinet
Telstar Bio II Advance, Terrassa, Spain) low permeability polyamide-
polyethylene bags (PA/PE-90, Albipack - Packaging Solutions, Portu-
gal), being the bags then manually heat sealed with the amount of air
inside the bags being minimised, followed by storage under the different
conditions studied, as detailed below.
2.3. Hyperbaric storage experiments
Storage experiments were carried out at naturally variable room tem-
perature (RT ≈ 20 °C) in a laboratorial SFP FPG13900 hydrostatic press
(Stansted Fluid Power, Stansted, United Kingdom) at 50 MPa, 75 MPa
and 100 MPa. This system has three pressure vessels, each one with
30 mm diameter and 500 mm height (volume of 400 mL), using a mix-
ture of propylene glycol and water (40:60, v/v) as pressurization fluid.
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Food Chemistry Advances 2 (2023) 100241
Two control samples were kept at atmospheric pressure (0.1 MPa) un-
der: room temperature (AP/RT) and refrigeration (RF ≈ 4 °C) at the
same time. Except for pressure, both control samples were immersed in
the same pressurization fluid and kept in the dark, for the same amount
of time, to mimic the same samples experiment conditions stored under
HS.
2.4. Natural microbiota of milk
From each sample, 1.0 mL was obtained aseptically and ho-
mogenised with 9.0 mL of ringer’s solution. Further, decimal dilutions
were made with the same diluent and duplicates of dilutions were plated
on the appropriate media according to the microorganisms analysed: En-
terobacteriaceae counts were quantified in violet red bile dextrose agar
(VRBDA), being incubated at 37 ± 1 °C for 24 ± 1 h (ISO 21,528:2017);
coliform bacteria counts were plated and counted in chromocult col-
iform agar (CCA) after incubation at 37 ± 1 °C for 24 ± 1 h (ISO 4832:
2007); total aerobic mesophiles (TAM) and psychrotrophic bacteria (PB)
counts were determined in plate count agar (PCA), by incubation at
30 ± 1 °C for 72 ± 3 h and at 20 ± 1 °C for 120 ± 3 h, respectively (ISO
4833:2013); lactic acid bacteria (LAB) counts were determined in pour
plate using de man, rogosa and sharpe (MRS) agar, by incubation during
120 ± 3 h days at 30 ± 1 °C (ISO 11,133:2014); yeasts and moulds (YM)
counts were enumerated on rose-bengal chloramphenicol agar (RBCA),
being incubated at 25 ± 1 °C for 120 ± 3 h (ISO 21,527:2008). In all
cases, petri dishes containing 15 – 300 colony forming units (CFU) were
selected for counting and the results expressed as decimal logarithm of
colony forming units per millilitre of milk (log CFU/mL). The maximum
quantifiable load in this study was 6.48 log CFU/mL (dilution 10−4 ),
while the detection and quantification limits were < 1.0 log CFU/mL
and < 2.0 log CFU/mL, respectively.
2.5. pH, total soluble solids and colour
The pH value of milk was measured with a pH electrode at 20 °C
(Testo 205, Testo, Inc., New Jersey, USA) properly calibrated and the
total soluble solids (TSS) content was determined by measuring °Brix
using a handheld refractometer (Atago, ATC1E) at 20 °C. The colour
analysis was performed using the CIELab space at 20 °C, by determina-
tion of the parameters: L∗ - lightness (0, dark; 100, light), a∗ - redness
(+, red; −, green) and b∗ - yellowness (+, yellow; −, blue), using a Kon-
ica Minolta CM 2300d spectrophotometer (Tokyo, Japan). The CIELab
parameters was determined and directly computed through the origi-
nal SpectraMagicTM NX software (Konica Minolta, Tokyo, Japan), ac-
cording to regulations of the International Commission on Illumination
(ISO 11,664–2:2007). The standard illuminant D65 and a standard ob-
server 10° were used. The total colour difference (∆E∗ ) was calculated
by Eq. (1):
[( )2 ( )2 ( )2 ]1∕2
Δ𝐸 ∗ = 𝐿∗ − 𝐿∗0 + 𝑎∗ − 𝑎∗0 + 𝑏∗ − 𝑏∗0 (1)
where ∆E∗ is the total colour difference between the sample and the
control, L∗ and L0 ∗ are the lightness of sample and control, respectively;
a∗ and a0 ∗ are the redness of sample and control, respectively; and b∗
and b0 ∗ are the yellowness of sample and control, respectively.
2.6. Viscosity analysis
The apparent viscosity of milk was measured using a controlled-
stress rheometer (AR-1000, TA Instruments, New Castle, USA), equipped
with a system of cone and plate geometry (acrylic cone, 6 cm diameter
and 2° angle). Control of temperature was achieved by a circulating ther-
mostatic bath (Circulating Bath 1156D, VWR International, Carnaxide,
Portugal), connected to the bottom plate of the rheometer. Flow curves
were obtained by applying an increasing linear shear stress ramp, from 0
to 2 Pa, for 3 min, at 20 °C. Apparent viscosity was calculated at a shear
Á.T. Lemos, J.A. Lopes-da-Silva, I. Delgadillo et al.
rate of 300 s- 1 and for each measurement for comparison amongst sam-
ples. Samples were visually free from foam/bubbles and measurements
were carried out for raw milk, HPP pasteurised milk before storage (day
0) and after 3, 7, 10, 14, 21, 40 and 60 days at the different storage
conditions. Measurements were made in duplicate for each one of the
triplicate samples.
2.7. Soluble protein content
The Bradford method was used to determine the soluble protein con-
tent of HPP pasteurised milk (Bradford, 1976). Briefly, 0.10 mL of milk
samples were diluted in 9.90 mL of distilled water and then, were cen-
trifuged at 6000 g for 15 min at 4 °C (Heraeus Biofuge Stratos, Thermo
Electron Corporation, Massachusetts, USA). Then, 50 μL of supernatant
was added to 200 μL of Bradford reagent, and shaked for 40 s, followed
absorbence measurement at 595 nm after 20 min of reaction (Microplate
Spectrophotometer Multiskan GO, Thermo Scientific, Waltham, Mas-
sachusetts, USA) (for the blank, the sample was replaced by distilled wa-
ter). Measurements were taken in triplicate for each sample triplicate.
The standard curve was created using bovine serum albumin (Sigma-
Aldrich, Lisbon, Portugal), with 0.02–0.40 g/L and the soluble protein
content was expressed as mg albumin/mL of milk.
2.8. Statistical analyses
Storage experiments were carried out in triplicate of sample and du-
plicate of analyses for the majority of raw and HPP pasteurised milk.
Statistical data analysis of the results was performed using the Analysis
of Variance (ANOVA) and Tukey’s honestly significant difference test,
at a 5 % level of significance (p < 0.05).
3. Results and discussion
3.1. Effect of high-pressure pasteurisation on raw milk
3.1.1. Initial microbial load
Fig. 1 depicts the effect of HPP pasteurisation on natural spoilage mi-
crobiota is raw milk. As expected, the samples subjected to HPP showed
a decrease in all microorganisms tested. Regarding Enterobacteriaceae,
coliform bacteria and yeast and moulds (YM) was observed a signif-
icant inactivation (p < 0.05) to below the detection limit (< 1.0 log
CFU/mL), of at least 1.40, 1.45 and 1.99 log units, respectively. Con-
cerning total aerobic mesophiles (TAM), lactic acid bacteria (LAB) and
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Food Chemistry Advances 2 (2023) 100241
psychrotrophic bacteria (PB), were significantly reduced (p < 0.05) in
1.33, 1.44 and 1.17 log units, respectively. So, results proved that the
application of HPP pasteurisation was able to significantly reduce all
microorganisms tested. Bacterial counts found on raw milk ranged be-
tween 4.08 and 4.82 log CFU/mL in the literature, and coliform bacte-
ria ranged between approximately 2.40 and 3.40 log CFU/mL (Fromm
& Boor, 2004). Wang, Fritsch and Moraru (2019) found an average of
vegetative bacteria count for skim raw milk of about 4.11 log CFU/mL
and Lee et al. (2016) found counts of psychrotrophic bacteria on raw
milk of 3.62 log CFU/mL, which is cose comparable our results. Fur-
thermore, studies in literature concerning HPP milk pasteurisation at
600 MPa/3 min, reported that the initial load of Enterobacteriaceae on
milk was reduced by approximately 1.7 log CFU/ml to below the detec-
tion limit, while for TAM and LAB was observed a reduction of 3.95 and
3.26 log CFU/mL, respectively which is nearly 2-fold of that verified
in our work (Stratakos et al., 2019). However, the initial loads of the
latter microorganisms in that study were approximately 6.0 and 4.3 log
CFU/ml respectively, which was higher than the initial load in our case
(4.9 and 3.6 log CFU/ml, respectively), which could explain the some-
what different results. Other works reported HPP pasteurisation at 400/
600 MPa for 10 min caused a reduction on TAM counts in raw milk of
about 2.1 and 5.1 log CFU/ml, respectively (Erkmen & Dogan, 2004).
HPP pasteurisation reduces the number of vegetative microorganisms
in foods due to a variety of injuries associated in several components,
including the cell namely cell membrane, nucleoid, ribosomes, protein
synthesis, and enzymes which are responsible for the nutrient and res-
piration transport mechanisms of the cell (Abe, 2015; Daryaei & Bala-
subramaniam, 2012). As a result, the level of microbial inactivation is
determined on the level pressure applied, number of pressure cycles,
duration of treatment (holding time), temperature, environment, and
number of microorganisms.
TAM counts should not exceed 5.00 log CFU/mL according to the
microbiological criteria for raw milk (Melini, Melini, Luziatelli & Ruzzi,
2017), while in pasteurised milk, counts of TAM, coliform bacteria and
Enterobacteriaceae should not exceed 4.30, 1.00 and 1.00 log CFU/mL,
respectively (FDA, 2019).
In this work, the microbial loads after HPP pasteurisation (HPP milk)
were 3.60 ± 0.06, 2.13 ± 0.11 and 2.89 ± 0.05 log CFU/mL, for TAM,
LAB and PB, respectively, while Enterobacteriaceae, coliform bacteria
and YM counts, were below the detection limit (< 1.0 log CFU/mL),
which is consistent with all microbiology criteria mentioned above for
pasteurised milk.
Fig. 1. Microbial loads for Enterobacteriaceae,
coliform bacteria, total aerobic mesophiles, lac-
tic acid bacteria, psychrotrophic bacteria, and
yeasts and moulds for raw milk ( ) and high
pressure pasteurised milk (HPP milk) ( ). Un-
filled broken line bars (∗ ) indicate that micro-
bial counts were below the detection limit (<
1.0 log CFU/mL). Different letters (a-b) denote
significant differences (p < 0.05) between raw
and HPP pasteurised milk for each microorgan-
ism.
Á.T. Lemos, J.A. Lopes-da-Silva, I. Delgadillo et al.
Table 1
Physicochemical parameters of bovine milk before (raw milk) and after
high pressure pasteurisation (HPP milk). Different letters (a-b) denote sig-
nificant differences (p < 0.05) between raw and HPP milk for each param-
eter analysed.
Raw Milk HPP Milk
Parameter analysed Mean ± Standard Deviation
pH 6.74 ± 0.01a 6.73 ± 0.00a
Total soluble solids (°Brix) 10.2 ± 0.1a 10.2 ± 0.1a
Apparent viscosity (mPa.s) 2.64 ± 0.01b 2.95 ± 0.06a
Soluble protein content (mg/mL) 27.55 ± 1.45a 24.60 ± 1.22a
Colour
L∗ 50.27 ± 0.04b 52.82 ± 0.34a
a∗ -0.60 ± 0.01b -0.81 ± 0.03a
b∗ 0.95 ± 0.03b 3.38 ± 0.14a
𝚫E∗ 3.53 ± 0.33
3.1.2. Physicochemical properties
Raw milk was tested for pH, total soluble solids (TSS), apparent vis-
cosity, soluble protein content and colour before (raw milk) and imme-
diately after high pressure pasteurisation (HPP milk) and the results are
presented in Table 1. The pH and TSS of raw milk were 6.74 ± 0.01
and 10.2 ± 0.1 respectively, which is very similar to the pH values re-
ported on the literature (Broyard & Gaucheron, 2015; Harte, Luedecke,
Swanson & Barbosa-Cánovas, 2003). In line with previous reports, these
values did not show significant (p ≥ 0.05) differences after HPP pasteuri-
sation (Harte et al., 2003).
Concerning apparent viscosity and colour, significant changes (p <
0.05) occurred due to HPP pasteurisation. The apparent viscosity of
milk increased from 2.64 ± 0.01 mPa.s to 2.95 ± 0.06 mPa.s. The val-
ues of viscosity obtained by Adapa, Schmidt and Toledo (1997) were
2.3 ± 0.00 mPa.s for raw milk and raised to 2.8 ± 0.00 mPa.s with
application of HPP at 310 MPa/1 min. The increase in viscosity of
skimmed bovine milk with the increment of pressure was also reported
by Huppertz, Fox and Kelly (2003), pointing to effects of HPP on ca-
sein micelles, as the responsible for the viscosity changes, since in raw
milk casein micelles are roughly spherical, but in HPP milk, irregularly
shaped casein particles have been observed, mainly at pressures above
300 MPa, due to considerable disruption of micelles and denaturation of
whey proteins (Huppertz, Fox & Kelly, 2004b). Furthermore, HPP was
reported to cause liberation of colloidal calcium phosphate resulting in
individual caseins concentration in the serum in which submicelles are
suspended, which may explain the viscosity increase (Harte et al., 2003).
After HPP pasteurisation was found an increase on L∗ and (Table 1),
which corresponds to the increase on bright as well as an increase
of milk yellowness, respectively. Moreover, as the total colour differ-
ence (ΔE∗ =3.53 ± 0.33) between raw and HPP milk was slightly higher
than 3.0, at naked eye was observed a slight raise on yellow hue of
HPP pasteurised milk (Fig. S1). The same way, the decrease of a∗
with HPP means an increase on green colour of the milk, although ei-
ther a∗ or b∗ were close to the neutral colour (grey values when a∗
and b∗ were equal to 0). These results corroborate those reported by
Stratakos et al. (2019) that also observed an increase on greenness (de-
crease of a∗ ) and an increase on yellowness (increase of b∗ ) when the raw
milk was pasteurised by HPP. The HPP pasteurised milk lost its typical
white colour to a yellowish colour, looking more like milk whey, due to
the decrease of casein micelles and fat globules size promoted by HPP,
causing this light scattering and turning milk translucent (Harte et al.,
2003). HPP caused no significant effects on soluble protein content.
3.2. Hyperbaric storage (HS) of high pressure pasteurised milk (HPP milk)
3.2.1. Effect on natural microbiota
HPP milk was preserved at different HS conditions, ranging between
50 and 100 MPa, at uncontrolled variable room temperature (RT ≈ 20
°C) and compared to refrigeration storage at 4 °C (RF) up to 40 days
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Food Chemistry Advances 2 (2023) 100241
(a control at atmospheric pressure and room temperature, AP/RT, was
also studied), as shown in Fig. 2.
In the case of samples stored at AP/RT, the samples showed a quickly
and significant (p < 0.05) increase in all microbials counts, reaching 6.0
log CFU/mL just after 3 days of storage, even when the initial loads were
under the detection limit (< 1.0 log CFU/mL). These samples presented
a typical undesirable odour and off-flavour of spoilage milk, and the
sample packages were swollen, due to CO2 production, showing also
small aggregates.
According to Fig. 2, the microorganisms that were under the detec-
tion limit initially, displayed the same behaviour when HPP milk was
stored at RF or under HS (50 MPa, 75 MPa, and 100 MPa), with the val-
ues remaining below the detection limit throughout the storage time.
Concerning the quantifiable microorganisms after pasteurisation, TAM,
LAB and PB, differed between RF and HS conditions. Under RF, mi-
crobial growth inhibition occurred during 40, 21 and 21 days for LAB,
TAM and PB, respectively. After the 40th day, a significant increase (p <
0.05) was verified for TAM and PB to 3.93 ± 0.28 and 3.89 ± 0.34 log
CFU/mL, respectively despite the samples remaining below the maxi-
mum limit values for pasteurised milk consumption (4.30 log CFU/mL)
(FDA, 2019). The microbial behaviour under HS was different depend-
ing on the different levels of pressure applied. Regarding 50 MPa, was
verified LAB growth inhibition during the first 10 days, but after that
these bacteria grew reaching values of 4.77 ± 0.37 at the 40th day of
storage. For TAM and PB, although a slight decrease occurred in the first
10 days, after that microbial growth increased significantly (p < 0.05)
to values of 4.68 ± 0.33 and 4.78 ± 0.28 log CFU/mL, respectively, at
the end of storage, being already above of the consumption level. This
is consistent with findings in the literature, which show that with HS at
lower pressure (50 MPa), initially inhibits microbial development but
with the passage of time, microbial growth is observed (Lemos et al.,
2017; Pinto et al., 2017).
In contrast, at higher pressures (75 MPa and 100 MPa) the inacti-
vation effect obtained with the application of HPP pasteurisation on
raw milk was enhanced, being verified a significant (p < 0.05) pro-
gressive decrease of microbial loads throughout HS. For 75 MPa, both
TAM or PB, decreased significantly just after 3 days to 2.19 ± 0.07 and
2.21 ± 0.12 log CFU/mL, respectively, reaching after the quantification
limit (< 2.0 log CFU/mL) on 7th day for TAM, and detection limit (<
1.0 log CFU/mL) on 10th and 3rd day for TAM and PB, respectively. The
LAB counts were reduced first to below the quantification level, and
then to below the detection level on 3rd and 7th days respectively. HS at
100 MPa showed a faster microbial inactivation profile, achieving the
detection level just in 3 days for LAB and PB. TAM counts were reduced
to below the quantification limit in the third day of storage and reached
the detection level on 10th day.
These results with HS (75 MPa and 100 MPa) at RT are very promis-
ing, because the inactivation effect observed for all microorganisms
studied during storage, point to a lower microbial load of HPP milk
stored by HS, compared to RF along storage, where was verified only
microbial growth inhibition. This lower microbial load observed for HS
at 75 MPa and 100 MPa enhances HPP milk microbial safety, indicat-
ing also a longer shelf-life, being these two important advantages of
HS compared to RF. Nonetheless, the maximum acceptable values for
consumption for TAM (4.3 log CFU/mL), Enterobacteriaceae (1.0 log
CFU/mL) and coliform bacteria (1.0 log CFU/mL) were met for up to 40
days using either RF or HS/RT (75 MPa and 100 MPa). However, it is
also clearly that with 50 MPa and RF, TAM, LAB and PB exhibited the
capacity to grow at the later stages of storage, which not happen under
HS at 75 MPa and 100 MPa. Additionally to the advantages indicated
above, HS was performed at natural variable RT, thus being quasi ener-
getically costless, differently than RF that needs constant energy supply
to maintain the low temperature (Bermejo-Prada et al., 2017). In fact,
Bermejo-Prada et al. (2017) showed that HS reduces very considerable
the energetic costs with food production (0.026 €/kg to RF instead of
0.001 €/kg to HS), achieving as well as the lowest carbon emissions
Á.T. Lemos, J.A. Lopes-da-Silva, I. Delgadillo et al. Food Chemistry Advances 2 (2023) 100241

Fig. 2. Microbial growth of Enterobacteriaceae, coliform bacteria, total aerobic mesophiles, lactic acid bacteria, psychrotrophic bacteria, and yeasts and moulds for
high pressure pasteurised milk up to 40 days of storage under: atmospheric pressure at ≈ 20 °C (AP/RT), refrigeration at 4 °C (RF) and HS/RT at ≈ 20 °C (50 MPa,
75 MPa and 100 MPa). Unfilled symbols on graphics mean that microbial counts were ≥ 6.0 log CFU/mL, below the quantification limit (< 2.0 log CFU/mL) or
below the detection limit (< 1.0 log CFU/mL), which are represented on tables by +, # and ∗ , respectively. Different letters denote significant differences (p < 0.05)
between storage days for each storage condition (A-D) and between storage conditions for each storage day (a-d).

5
Á.T. Lemos, J.A. Lopes-da-Silva, I. Delgadillo et al.
Fig. 3. pH and total difference colour (ΔE∗ ) for high pressure pasteurised milk (HPP milk) stored up to 40 days under: atmospheric pressure at ≈ 20 °C (AP/RT),
refrigeration at 4 °C (RF) and HS at ≈ 20 °C (50 MPa/RT, 75 MPa/RT and 100 MPa/RT). Different letters denote significant differences (p < 0.05) between storage
days for each storage condition (A-E) and between storage conditions for each storage day (a-b).
(0.1085 kg CO2 /kg to RF against 0.0042 kg CO2 /kg for HS), represent-
ing an HS carbon footprint approximately 26 times lower than RF.
As was highlighted in previous studies concerning HS of dairy prod-
ucts, HS at 100 MPa for 4 h of whey cheese, allowed to maintain micro-
bial levels similarly to RF (about 3 log CFU/g for TAM, Enterobacteri-
aceae, and LAB), whereas at 150 MPa for 8 h, the microbial load were
reduced to undetectable levels (< 1.0 log CFU/mL), except for TAM (re-
duction of approximately 1.0 log CFU/g) (Duarte et al., 2014). When the
study was prolonged to 10 days, TAM were also reduced to below the
detection limit (Duarte et al., 2017). The best case reported in literature
so far, for extending the shelf-life of a highly perishable food product by
HS was for raw watermelon juice at 75 MPa/RT, which demonstrated
microbial counts below the detection limit for up at least one year (the
longest period studied) (Lemos, Ribeiro, Delgadillo & Saraiva, 2020).
3.2.2. Effect on physicochemical parameters
Because the microbial results clearly indicated a shelf-life greater
than the 40 days studied, physicochemical parameters were examined
up to 60 days, except for pH and colour, because these two parameters
remained unchanged after 40 days, excluding AP/RT samples (but these
samples presented off-flavours and gas formation due to its high spoilage
state after 7 days).
3.2.2.1. pH. Fig. 3 shows the pH value of HPP milk along HS. AP/RT
samples presented a decrease (p < 0.05) on pH from 6.73 ± 0.00 (initial
value) to 3.64 ± 0.02 (40th day). This pH decrease is probably caused by
the accumulation of lactic acid produced by LAB (Fidalgo et al., 2019).
On the other hand, the pH changes (varied between 6.68 and 6.75) were
not statistically significant (p ≥ 0.05) for RF and HS conditions dur-
ing 40 days of storage. The first study on literature concerning HS at
100 MPa of a dairy product (whey cheese) reported pH retention for 8 h
(Duarte et al., 2014). Later, Duarte et al. (2017) observed that pH of
whey cheese preserved by HS remained stable for 10 days.
3.2.2.2. Total soluble solids (°Brix). The initial TSS of HPP milk was
10.2 ± 0.1 °Brix (Table 2), a slight lower value than reported on lit-
erature (11.69 ± 0.34) (Hernández-Falcón et al., 2018). For samples
stored at AP/RT, a significant and continuous TSS decrease (p < 0.05)
was observed reaching 7.5 ± 0.2 °Brix at the 7th day.
6
Food Chemistry Advances 2 (2023) 100241
Differently, no considerable changes (p ≥ 0.05) in TSS values were
detected for samples kept at RF over 60 days, being similar to the ini-
tial value. Regarding samples under HS at 50 MPa, it was observed a
decrease (p < 0.05) reaching the lowest value at 60th day (8.0 ± 0.5
°Brix), explained in part due to the microbial growth observed from 21
days onwards. The TSS reduction (p < 0.05) was also observed at 75 MPa
and 100 MPa at 40th and 21st days, respectively. However, at 75 MPa
and 100 MPa, the decrease on TSS cannot be explained by microbial
spoilage since with these levels of pressure the microbial loads decrease
along storage. In fact, some authors reported that pressure induce the
unfolding of 𝛽-lactoglobulin and its interaction with 𝜅-casein in stable
complexes, meaning the formation of casein micelle and 𝛽-lactoglobulin
aggregates (Bogahawaththa, Buckow, Chandrapala & Vasiljevic, 2018).
Nevertheless, the first 21 days of all HS conditions presented a TSS value
close to the initial one. On literature, TSS of watermelon juice stored
under RF remained stable for 10 days (Pinto et al., 2017). Meanwhile,
TSS of watermelon juice at 50 MPa showed a decrease relatively to the
initial value, with a slight variation verified at 75 MPa and 100 MPa
(Lemos et al., 2020; Pinto et al., 2017).
3.2.2.3. Apparent viscosity. Firstly, flow curves of all milk samples ex-
hibited newtonian behaviour. The apparent viscosity of AP/RT, in-
creased considerably from 2.95 ± 0.06 mPa.s to values of 14.56 ± 1.10
mPa.s, just in 7 days (Table 2), possibly due to microbial spoilage. Con-
cerning RF samples, the apparent viscosity values were close to the ini-
tial ones, with values of 2.92 ± 0.02 mPa.s at the 60th day. Under HS con-
ditions, the apparent viscosity was maintained (p ≥ 0.05) for 21 days. An
increase of apparent viscosity to 13.01 ± 2.22 mPa.s and 14.31 ± 3.47
mPa.s was observed at 60th day for 50 MPa and 75 MPa, respectively.
At 100 MPa, the apparent viscosity reached values of 8.24 ± 1.49 mPa.s
at 60th day. Thus, from 21st day onwards, viscosity increased, possibly
due to the formation of small casein micelle aggregates (Fig. S2), as pre-
viously explained, resulting in greater heterogenicity and higher vari-
ation of the experimental values for HS samples. Although an increase
in viscosity of bovine milk after HPP pasteurisation was reported before
(Huppertz et al., 2003, 2004b), the bibliography concerning the effect
of HS on the rheological properties of foods is still largely lacking. Thus,
because HS requires the constant application of a mild pressure during
the whole storage period (in our case 50 MPa, 75 MPa, and 100 MPa
Á.T. Lemos, J.A. Lopes-da-Silva, I. Delgadillo et al. Food Chemistry Advances 2 (2023) 100241

Table 2
Physicochemical properties for high pressure pasteurised milk (HPP milk) stored up to 60 days under: atmospheric pressure at ≈ 20 °C (AP/RT),
refrigeration at 4 °C (RF) and HS at ≈ 20 °C (50 MPa/RT, 75 MPa/RT and 100 MPa/RT). Results are presented as mean ± standard deviation.
Different letters (a-f) denote statistical differences (p < 0.05) between storage conditions for each parameter analysed.

Storage time Total soluble Apparent Colour Parameters

Storage Condition (days) solids (°Brix) viscosity (mPa.s)
L∗ a∗ b∗
a c b abc
HPP milk 0 10.2 ± 0.1 2.95 ± 0.06 52.82 ± 0.34 -0.81 ± 0.03 3.38 ± 0.14a
AP/RT 3 7.5 ± 0.2d 15.25 ± 0.60a 53.96 ± 0.22ab -0.82 ± 0.03abc 3.15 ± 0.18abc
7 7.5 ± 0.1d 14.56 ± 1.10a 53.83 ± 0.18ab -0.85 ± 0.04abcd 3.00 ± 0.13abcd
RF 3 10.2 ± 0.1a 2.92 ± 0.02c 52.91 ± 0.56ab -0.80 ± 0.05ab 3.15 ± 0.17abc
7 10.2 ± 0.2a 2.93 ± 0.06c 53.33 ± 0.24ab -0.85 ± 0.04abcd 3.26 ± 0.21ab
10 10.2 ± 0.1a 2.94 ± 0.03c 53.04 ± 0.37ab -0.84 ± 0.04abcd 2.83 ± 0.09bcde
14 10.1 ± 0.1a 2.93 ± 0.04c na (1) na (1) na (1)
21 10.1 ± 0.1a 2.89 ± 0.04c 53.44 ± 0.44ab -0.83 ± 0.04abcd 2.63 ± 0.12de
40 10.1 ± 0.1a 2.84 ± 0.05c 53.46 ± 0.51ab -0.80 ± 0.03a 2.56 ± 0.15de
60 10.1 ± 0.1a 2.92 ± 0.02c na (1) na (1) na (1)
50 MPa/RT 3 10.2 ± 0.1a 2.69 ± 0.04c 53.10 ± 0.15ab -0.92 ± 0.06bcd 2.58 ± 0.15de
7 10.2 ± 0.1a 2.67 ± 0.04c 53.61 ± 0.29ab -0.92 ± 0.04abcd 2.63 ± 0.38de
10 10.2 ± 0.1a 2.68 ± 0.08c 53.77 ± 0.36ab -0.93 ± 0.05cd 2.61 ± 0.10de
14 10.1 ± 0.0a 2.73 ± 0.02c na (1) na (1) na (1)
21 10.1 ± 0.1a 3.22 ± 0.18c 53.76 ± 0.32ab -0.91 ± 0.03abcd 2.47 ± 0.23e
40 8.8 ± 0.4b 8.78 ± 2.98b 53.97 ± 0.36a -0.94 ± 0.02d 2.70 ± 0.11cde
60 8.0 ± 0.5c 13.01 ± 2.22a na (1) na (1) na (1)
75 MPa/RT 3 10.2 ± 0.1a 2.68 ± 0.07c 53.51 ± 0.35ab -0.88 ± 0.04abcd 2.63 ± 0.15de
7 10.2 ± 0.1a 2.65 ± 0.08c 53.72 ± 0.33ab -0.95 ± 0.01d 2.44 ± 0.17e
10 10.2 ± 0.1a 2.73 ± 0.11c 53.60 ± 0.45ab -0.89 ± 0.03abcd 2.46 ± 0.17e
14 10.1 ± 0.1a 2.77 ± 0.01c na (1) na (1) na (1)
21 10.1 ± 0.1a 3.16 ± 0.06c 53.71 ± 0.44ab -0.86 ± 0.03abcd 2.52 ± 0.12de
40 9.1 ± 0.1b 5.39 ± 2.64bc 53.72 ± 0.32ab -0.89 ± 0.04abcd 2.77 ± 0.12bcde
60 9.1 ± 0.1b 14.31 ± 3.47a na (1) na (1) na (1)
100 MPa/RT 3 10.2 ± 0.1a 2.74 ± 0.03c 53.19 ± 0.14ab -0.90 ± 0.04abcd 2.67 ± 0.05cde
7 10.2 ± 0.1a 2.69 ± 0.10c 53.66 ± 0.37ab -0.89 ± 0.03abcd 2.58 ± 0.03de
10 10.2 ± 0.1a 2.74 ± 0.04c 53.73 ± 0.59ab -0.87 ± 0.06abcd 2.53 ± 0.17de
14 10.1 ± 0.0a 3.04 ± 0.01c na (1) na (1) na (1)
21 9.1 ± 0.1b 7.76 ± 0.53b 53.91 ± 0.25ab -0.88 ± 0.03abcd 2.77 ± 0.03bcde
40 9.1 ± 0.1b 6.98 ± 1.09b 53.95 ± 0.24ab -0.85 ± 0.06abcd 2.73 ± 0.09cde
60 9.1 ± 0.0b 8.24 ± 1.49b na (1) na (1) na (1)

(1) na - not analysed.

up to 60 days), it may promote various changes that alter the fluidity of
milk, in ways that require further investigation.
3.2.2.4. Colour evolution. Table 2 shows the CIELab colour parameters
for HPP milk under HS are shown in. Initial L∗ , a∗ , and b∗ values of
HPP milk observed were 52.82 ± 0.34, -0.81 ± 0.03 and 3.38 ± 0.14,
respectively and no significant differences (p ≥ 0.05) in L∗ value were
observed under all conditions.
When a∗ and b∗ values were considered, different results were ob-
tained. Under RF, the a∗ value at 40th day (-0.80 ± 0.03) is similar
(p ≥ 0.05) to the initial one. Nonetheless, on HS samples was observed a
slight variation on the a∗ values, even though not statistically different
(p ≥ 0.05), in the majority of the cases. The HS at 50 MPa showed a lower
a∗ values, presenting a final storage value (p < 0.05) of -0.94 ± 0.02.
Generally, at 75 MPa and 100 MPa, the a∗ values ranged between -0.86
and 0.90. Overall, for all HS conditions tested, the b∗ value had a ten-
dency to decrease during the storage, but, once again, almost all of them
were similar (p ≥ 0.05). Under RF, the a∗ value at 40th day (-0.80 ± 0.03)
is similar (p ≥ 0.05) to the initial one. Nonetheless, on HS samples was
observed a slight variation on the a∗ values, even though not statisti-
cally different (p ≥ 0.05), in the majority of the cases. The HS at 50 MPa
showed a lower a∗ values, presenting a final storage value (p < 0.05) of
-0.94 ± 0.02. Generally, at 75 MPa and 100 MPa, the a∗ values ranged
between -0.86 and -0.90. Overall, regarding b∗ value, it was observed
a tendency to decrease during the storage for all HS conditions tested.
However, once again, almost all of them were similar (p ≥ 0.05).
One way to understand better the colour changes is by analysing
the total colour difference (ΔE∗ ) that is presented in Fig. 3. With RF,
it was observed a significant increase (p < 0.05), although minor, of
ΔE∗ value at the 21st and 40th days of 1.03 ± 0.22 and 1.12 ± 0.17,
respectively, mainly due to the decrease on b∗ values. Regarding HS,
all storage pressures used (50 MPa, 75 MPa and 100 MPa) presented a
similar behaviour (p ≥ 0.05), with a significant ΔE∗ value increase (p <
0.05), although minor, at the 3rd day (0.81 - 1.06), remaining unchanged
until the 40th day (1.11 - 1.36), which may be justified by decrease on
a∗ and b∗ parameters over storage time (Table 2). Furthermore, ΔE∗
can be classified as not noticeable (0 - 0.5), slightly noticeable (0.5 -
1.5), noticeable (1.5 - 3.0), well visible (3.0 - 6.0) and great (6.0 - 12.0)
to indicate the degree of colour difference between treated/untreated
samples or before/after storage (Stratakos et al., 2019). On the 40th
day of storage, only minor differences in RF and HS were observed,
according to this classification. Overall, it is reported in the literature
that HS allows for a preservation of colour or causes minor changes
when compared to RF for liquid foods (Santos et al., 2020).
3.2.2.5. Soluble protein content. After 7 days at AP/RT, the soluble pro-
tein content decreased significantly (p < 0.05) from 21.99 ± 2.72 to
5.53 ± 1.48 (Fig. 4). As previously stated, these results are possibly could
be attributed to an increase of the microbial loads, as explained before.
Differently, with RF the soluble protein values were constant (p ≥ 0.05)
along the whole storage being observed a value of 21.97 ± 2.78 at the
60th day.
Regarding HS conditions, soluble protein content increased at the
7th day, being then maintained till the end of storage, except at 50 MPa,
where was observed a decrease after 21 days of storage, possible due
to the microbial grow observed from 21st onwards, reaching the low-
est value of 8.14 ± 3.23 at the 60th day. In contrast, at 75 MPa, solu-
ble protein showed a value of 37.06 ± 3.69 at 7th day with no signif-
icant changes at the 60th day (41.68 ± 1.77). Similarly, 100 MPa was
able to maintain the soluble protein content (p ≥ 0.05) from the 7th
day (37.86 ± 2.33) till the end of the storage (39.11 ± 2.23 at 60th
day). Furthermore, HS at 75 MPa and 100 MPa showed no significant

7
Á.T. Lemos, J.A. Lopes-da-Silva, I. Delgadillo et al.
Fig. 4. Soluble protein content (mg of albumin/mL of milk) for high pressure pasteurised milk (HPP milk) stored up to 60 days under: atmospheric pressure at ≈
20 °C (AP/RT), refrigeration at 4 °C (RF) and HS at ≈ 20 °C (50 MPa/RT, 75 MPa/RT and 100 MPa/RT). Different letters denote significant differences (p < 0.05)
between storage days for each storage condition (A-D) and between storage conditions for each storage day (a-c).
difference (p ≥ 0.05), indicating that the values found at the end of
storage similar to that of the raw milk (Table 1). Milk proteins (whey
proteins and micellar caseins) can be denatured by high pressure pro-
cessing, resulting in protein structure changes depending of pressure
levels, treatment time and temperature, and milk pH (Baier, Schmitt
& Knorr, 2015; Bogahawaththa et al., 2018; Huppertz, Fox, de Kruif &
Kelly, 2006). As previously stated, not only caseins can form aggregates
(flocs) but also the solubility can increase by the formation of smaller
micelles (Baier et al., 2015). Baier et al. (2015) also pointed out that the
disruption of casein micelles promoted by pressure is accompanied by
a decrease in particle size and an increase in the amount of caseins in
the serum phase. For example, HPP at 100–600 MPa for 30 min, lead to
a considerable increase in the levels of caseins in the soluble phase of
milk (Huppertz, Fox & Kelly, 2004a).
4. Conclusions
High-pressure processing (HPP) was effective in pasteurising raw
milk, reducing the microbial counts while maintaining the main physic-
ochemical parameters and causing minor changes on apparent viscosity
and colour and a reduction of soluble protein.
Furthermore, unlike refrigeration (RF), hyperbaric storage at room
temperature (HS at RT) of HPP milk not only inhibited microbial de-
velopment (at 50 MPa), but also inactivated all microorganisms tested
to below the detection limit (at 75 MPa and 100 MPa), extending the
microbial shelf-life of milk for at least up to 40 days (the longest period
studied). RF, on the other hand, could only slow down microbial growth
for 40 days.
When comparing HS to RF, physicochemical parameters such as pH
and colour were equal to better maintained by HS at RT during the 60
days of storage at 75 MPa and 100 MPa, when compared to RF, while
apparent viscosity and protein soluble content increased.
The results of this study clearly show that HS at RT was capable
of considerable extending the microbial shelf-life of HPP pasteurised
milk compared to RF, with minimal effects on the main physicochemical
characteristics. As a result, more research is needed on quality param-
eters such as volatiles compounds, fatty acids composition and others,
8
Food Chemistry Advances 2 (2023) 100241
like sensorial properties, to gain a better understanding of the overall
effect of HS on HPP milk, to better assess its overall effectiveness.
Declaration of Competing Interest
The authors declared that they have no conflict of interest.
CRediT authorship contribution statement
Álvaro T. Lemos: Investigation, Formal analysis, Visualization,
Writing – original draft. José A. Lopes-da-Silva: Methodology, Writing
– review & editing. Ivonne Delgadillo: Writing – review & editing, Su-
pervision. Jorge A. Saraiva: Conceptualization, Methodology, Writing
– review & editing, Supervision.
Data availability
Data will be made available on request.
Acknowledgements
Thanks are due to the University of Aveiro and FCT/MCT for the fi-
nancial support for LAQV-REQUIMTE (UIDB/50006/2020) through na-
tional founds and, where applicable, co-financed by the FEDER, within
the PT2020 Partnership Agreement and for supporting the PhD grant of
Álvaro T. Lemos (SFRH/BD/129848/2017).
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.focha.2023.100241.
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