Journal of Food Engineering 128 (2014) 19

Contents lists available at ScienceDirect

Journal of Food Engineering

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

Combination of microltration and heat treatment for ESL milk

production: Impact on shelf life
L. Fernndez Garca, F.A. Riera Rodrguez
Chemical Engineering and Environmental Technology Department, Faculty of Chemistry, University of Oviedo, Asturias, Spain

a r t i c l e

i n f o

Article history:
Received 14 August 2013
Received in revised form 11 October 2013
Accepted 24 November 2013
Available online 4 December 2013
Keywords:
ESL
Microltration
Heat treatments

a b s t r a c t
Thermized defatted cow milk was submitted to different heat treatments (between 73 and 130 C, 2 and
15 s) and combined with a microltration step (1.4 lm cut-off ceramic membrane) to study the inuence
of these treatments on milk shelf life. Thirty thousand colony forming units/mL was selected as the limit
parameter for extended shelf life. The logarithmic reduction in bacteria was estimated for each treatment
and the total bacteria count was measured during the storage of milk at 46 C and at room temperature.
Microorganism growth kinetic data during storage were also estimated. A maximum extended shelf life
of 74 days was found for milk after the combination of microltration and direct heat treatment at
125130 C and storage at room temperature. An extended shelf life of 33 days was obtained after microltration followed by pasteurization at 90 C and storage at 46 C.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
The shelf life of milk is an important concept that denes the
ability to widen the distribution chain of the product. As milk
provides a favorable medium for spoilage microorganisms, pretreatment as well as temperature/time conditions must be chosen
in order to control microbial growth. Heat treatments are the most
widely used processes for lowering the bacterial content of milk
and milk products (Olesen and Jensen, 1989). Currently, pasteurization and ultra-high temperature (UHT) processing are common
heat treatments used in the dairy industry. Pasteurization, however, cannot totally prevent the survival of all bacteria, some of
which may affect the storage qualities of milk and milk products.
One signicant barrier to extending the shelf life of dairy products is the difculty in balancing the removal or destruction of
spoilage micro-organisms and spores present in raw milk while
limiting product color changes, vitamin destruction and milk protein denaturation. Extended shelf life (ESL) milk provides the possibility of extending the shelf life of a range of products that can
stay under refrigerated conditions beyond the traditional limits
of conservation (Goff and Grifths, 2006).
Some of the possible ESL technologies are bactofugation (Giffel
and van der Horst, 2004), pulsed electric elds (Barbosa-Cnovas
et al., 1999), high pressure processing (Trujillo et al., 2002), high
heat treatment (Fredsted et al., 1996) and microltration (MF).
Cross-ow MF for bacteria removal provides a low-temperature
Corresponding author. Address: C/Julin Clavera, 8, 33008 Asturias, Spain.
Tel.: +34 985103436.
E-mail address: far@uniovi.es (F.A. Riera Rodrguez).
0260-8774/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2013.11.021

approach for the control of microbial growth and is one of the

ESL techniques employed at the industrial scale for this application
(Skrzypek and Burger, 2010).
The effectiveness of the MF separation process in reducing bacterial levels in milk was conrmed by Olesen and Jensen (1989).
MF led to a logarithmic bacteria reduction (LBR) of 4 for total bacteria and 2.33.7 for spores. Experimental results obtained under
various operating conditions have been reported in a number of
publications and reviews (Saboya and Maubois, 2000; Brans
et al., 2004; Fernandez et al., 2013). MF membranes with a pore
size of about 1.4 lm can achieve the right balance between rejection of bacteria and long-term ux, with little or no rejection of
other milk components such as protein, lactose and ash. However,
most fat globules in milk are similar in size to bacteria; this results
in very rapid fouling of the membrane due to the deposition of a fat
layer on the membrane surface and the constriction of pores,
which consequently affect MF performance. MF for microbial removal is only applied to skimmed milk on an industrial scale
(Guerra et al., 1997).
Although very efcient regarding the removal of bacteria and
spores, MF cannot guarantee 100% removal of pathogenic bacteria,
as required for milk pasteurization. After milk treatment and during storage, surviving spores and microorganisms can germinate
and grow and thus limit the milk shelf life. For this reason, heat
treatment is needed after the MF process. MF prior to heat treatment can remove some microorganisms and reduce enzyme activity during storage that can degrade lactose, protein and fat. Some
authors have studied different combinations of MF and mild heat
treatments. Elwell and Barbano (2006) studied the shelf life of pasteurized (72 C, 15 s) skimmed milk with and without MF (1.4 and

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19

Nomenclature
Symbols
A
CFU
CWF
DHHT
ESL
HT
IHHT
LBR

membrane area
colony forming units
membrane clean water ux
direct high heat treatment
extended shelf life
heat treatment
indirect high heat treatment
logarithmic bacteria reduction

0.8 lm) at different storage temperatures (0.16.1 C), and obtained a maximum bacterial log reduction of 5.63 and 92 days of
milk shelf life. Tomasula et al. (2011) microltered (0.8 lm)
previously pasteurized milk (72 C, 18.2 s) in order to study Bacillus
anthracis spore removal, and found a maximum log reduction of
about 6.
Information on the combination of MF and pasteurization treatments can be found in Schmidt et al. (2012) and Elwell and
Barbano (2006), but treatment data at higher temperatures are
scarce. The use of MF could reduce the temperature of traditional
ultra-high temperature processes, giving products with organoleptic properties similar to those of pasteurized milk. The method proposed in this work could obtain a premium milk type with an ESL
greater than that provided by the new ultrapasteurization products
that are currently available.
In this study, several combinations of MF and temperature heat
treatments (indirect and direct, between 73 and 130 C) with and
without MF were studied in order to evaluate the effect of all treatments on the milk shelf life maintained at refrigeration and room
temperatures. Organoleptic and proteolytic aspects were not
studied.
2. Materials and methods
2.1. Milk
The raw milk used in all experiments was submitted to a mild
heat treatment at 50 C and then centrifuged (GEA Westfalia,
Germany) by a dairy company (CAPSA, Asturias, Spain). The
average milk properties and composition were: pH 6.79 0.04, fat
content 0.03 0.01%, mean total protein 3.4 1%, 4.7 1% lactose,
and 8.4 1% non-fat solids. Initial milk bacterial counts varied between 50,000 and 200,000 CFU mL 1.
2.2. MF rig and membranes
MF experiments were conducted using the pilot-scale unit
(Orelis Rhodia, France) shown in Fig. 1. The capacity of the feeding
tank (E-1) was 50 L and was designed with automatic monitoring
of the liquid level (KROHNE, Romans CEDEX, France). The temperature was controlled by means of a tank jacket with automatic regulation of ows of cooling and heating uids to adjust to the set
point (V-1). The tangential ow rate in membrane channels was
ensured by a ow rate frequency-regulated vertical multistage
centrifugal pump (Grundfos, St. Quentin-Fallavier, France) (E-2).
The pump provided a maximum ow rate of 8 m3 h 1. The crossow velocity was obtained by adjusting the ow rate of the feeding
pump.
Experimental data measurements were performed using
electronic volume ow meters for the permeate and retentate
(Endress-Hausser Promass 60, Weil am Rhein, Germany) (M-1,

LHT
MF
N
N0
Qf
TMP
TBC
RD

low temperature treatments

microltration
number of microorganisms a time t
number of microorganisms a t = 0
membrane permeate ow rate (with water)
transmembrane pressure
total bacterial count
reduction degree
viscosity

M-2); platinum resistance in a mineral-insulated cable process

thermometer was used for the feeding solution (Endress-Hausser,
Weil am Rhein, Germany) (M-3) and differential pressure transducers (Endress-Hausser, Weil am Rhein, Germany) were used
for the inlet and outlet transmembrane pressure (M-4, M-5,
M-6). Additionally, three manometers (WIKA, Barcelona, Spain)
were placed closer to the membrane inlet and outlet to ensure
the correct measurement of the transmembrane pressure (Pi and
Po, respectively) and the pressure on the permeate side (Pp). Transmembrane pressure (TMP) was calculated using the following
equation, TMP = [(Pi + Po)/2] Pp. There were two needle valves
at the retentate and permeate outlet to control the pressure of
the system (V-2, V-3). The experimental rig had an automatic
control system to operate at a constant permeate ux. A pneumatic
control valve (SAMSON, Vaulx en Velin CEDEX, France) (V-4)
allowed variation of the pressure on the permeate side.
The membrane used in the MF trials was an Isoux ceramic
membrane (Tami, France). The Isoux membrane was designed
to compensate for the pressure drop by a thickness gradient on
the top layer along the membrane length (Grangeon et al., 2000).
Such a membrane design produces a constant ux along the length
of the membrane element. This feature leads to improved performance of the membrane in terms of long-term ux rates required
with industrial feeds and allows for more effective cleaning since
membrane fouling is similar along the full length of the membrane
element (Saboya and Maubois, 2000).
The Isoux membrane chosen for this purpose had 23 channels with an internal diameter of 3.5 mm, a length of 1178 mm, a
ltering area of 0.35 m2 and a pore size of 1.4 lm (measured by
the porosity method, as reported by the manufacturer).
2.3. Membrane cleaning procedure
The membrane was chemically cleaned after each run using
Alkaline P-3 Ultrasil 25 1% (v/v) (Henkel-Ecolab SNC, Issy les Moulineaux, France) at 75 C, for 15 min without permeation (permeate
valve closed) and 15 min with permeate ux followed by nitric
acid (HNO3, 58% purity) at 1% (v/v) (Brenntag, Sevilla, Spain) at
50 C. Finally, the system was rinsed completely with tap water.
2.4. Heat treatment equipment
Two different equipments were used for the heat treatments.
2.4.1. Indirect heat treatment
A tubular heat exchanger was used for indirect heat treatment
(IHHT) and for the pasteurization step. The system (OMVE
HT220, Netherlands) consisted of a feed tank, pump, heat exchanger, temperature and pressure sensors, boiler and computer. The
product was pumped from the reservoir to the heat exchanger
where the product was rst preheated and then directed to the

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19
M-5
PT
Dps
V-1

V-1

V-1

V-1

E-3

M-2
FIC

V-1

FT

V-1

M-6

Dpp

PT

M
V-1

V-1
V-16

FV

E-1
E-5

Dpe

E-1

LSL

P
M-1
FIC
SCM
FT

V-1

M-3

M-4

TT

PT

E-2

Fig. 1. Ceramic MF pilot plant piping and instrumentation diagram.

main heating area. After the treatment, the product was cooled.
The system was designed to work with a product ow rate of
20 L h 1. The maintenance tube length was selected to x the pasteurization step to 15 s and to 6 s for the indirect high heat
treatments.

Finally, for both heat treatments studied, the product was collected in a laminar air ow cabinet. Samples were taken in 100
mL sterile containers in a sterile atmosphere. There was no significant contamination of the circuit after UHT and its connection
with the laminar air ow cabinet.

2.4.2. Direct heat treatment

In this case was used an UHT pilot plant (APV, United Kingdom)
consisted of a feed tank with a capacity of 200 L (AISI 316L), a positive pump which provided a variable ow from 100 to 1000 L h 1
and pressures ranging from 2 to 10 bar, a ow meter and a plate
heat exchanger for previous heating (at 80 C). This plate heat exchanger had two bodies. The milk was rst heated with hot water.
Preheating was carried out at 80 C, and a controller was used to
adjust the temperature to the desired value. In this case, only the
rst body of the heat exchanger was used.
In addition to the elements described above, the equipment had
several temperature and pressure sensors placed at the inlet and
the outlet of the exchangers, steam injection, circuits of feed and
product, etc. The recirculation of the product was also possible
and was useful for the cleaning steps. The steam temperature could
be adjusted by a needle valve which regulated the steam injection.
The product remained at this temperature for 6 s. This could vary
slightly depending on the ow provided by the positive pump. At
the ash cooler, the product was cooled to 80 C (the same temperature it was before steam injection in order not to dilute or concentrate the product). The steam was extracted from the expansion
chamber through a vacuum pump and the product was drained
with a centrifugal pump.
The pilot plant had a homogenizer connected after the heating
process. It is important that the homogenizer is placed here because heating can destabilize the mixture and separate the fat
again. Subsequently, the homogenized product was cooled in a
plate heat exchanger with tap water.

2.5. Operating procedure

Water from the industry tap network was used to warm the
previous membrane rig to 45 C.
The cleaning protocol was performed before each MF run with
the alkaline cleaner and after the water rinse, and the acid cleaner
was circulated. Finally, a water rinse was performed until neutralization. Prior to the experiments with milk, membrane clean water
ux (CWF) was measured to ensure that it was the same from run to
run, by performing a MF trial using water at 20 C and 6 m s 1 linear
velocity. The CWF was then calculated using the following equation:
CWF = Qf  l/(TMP  A), where Qf is the permeate water ow rate
in L h 1, l is the water viscosity (1.0 cP at 20 C), and A is the membrane ltration area (0.35 m2). The CWF trial was performed in
duplicate or until the original CWF was obtained. If not, the cleaning
procedure was repeated. After each run, the milk was drained from
the feed tank and water was added to initiate a water rinse cycle for
about 20 min before the cleaning protocol was applied. If the CWF
was less than 10% of the CWF before the experiments with milk,
the cleaning cycle was repeated, and the CWF and pH of the permeate and retentate streams were measured again.
After each cleaning cycle, the unit was emptied and lled with
150 L of raw skimmed milk. Before the operating parameters were
set, 15 L of milk (corresponding to the volume of the retentate
compartment) were used to ush the loop of the remaining water.
The unit almost instantly reached 80% of the desired pressure and
ow velocity and needed roughly 3 min to attain the desired values. Around 200 L were used for each experiment.

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19

When the processing run was initiated, the retentate was returned to the feed tank and the permeate was collected in order
to perform the thermal treatment afterwards. The experimental
conditions for the MF stage were xed at: v = 6 m s 1, TMP = 0.55
bar and T = 45 C. Experiments showed no membrane fouling after
4 h and the permeate ow rate varied between 450 and 500 l h 1
m 2 in all cases. Total protein retention at the aforementioned conditions was lower than 1.5% (Fernandez et al., 2013).
For the combination of MF and heat treatment trials, the process began with the collection of 200 L of skimmed milk at 50 C
right after skimming (0.3% fat content). The milk was pumped to
the MF equipment and, after bacteria removal, was directed to
the heat treatment apparatus (either direct or indirect). After heat
treatment, samples were collected in a laminar ow cabinet in
sterile containers.
2.6. Samples and analyses
Samples of the feeding solution, permeates and nal product
after heat treatments were aseptically withdrawn from all experiments. The nal product samples were kept under refrigerated
conditions (4 C) in the case of heat treatment at 7390 C and at
room temperature for the 115130 C treatments.
2.6.1. Physico-chemical analyses
Total acidity was determined by titration with 0.1 M NaOH
according to the procedure given in IDF 220 (ISO 29981) (2010).
A Foss Milko-Scan 50 apparatus (short-wave near-infrared [NIR]
spectroscopic analysis) was used to determine total solids and proteins, lactose and fat content. The pH was measured using a pH
meter (Crison Instruments SA, Barcelona, Spain).

2.6.3. Storage/shelf life studies

Samples of the permeate (80 mL) and nal product were
drawn after the MF and the heat treatment units at various times
in all experiments in sterile specimen cups and kept at 46 C or
room temperature (1820 C) (depending on the heat treatment)
for the shelf life study. The main parameters considered for the
milk quality evaluation were pH, titratable acidity and TBC. These
parameters were analyzed at different time intervals depending on
the type of study. In the case of commercial milk, with a shorter
shelf life, parameters were monitored three times weekly in the
initial stage of refrigerated storage and then daily when a decline
in the quality parameters was observed. For the rest of the trials,
the frequency of the analysis increased when any of the parameters started to show a relevant variation. From that point on, samples were measured more frequently.
Acidity, pH, and TBC parameters were selected to follow milk
shelf life. Limit values considered for acceptable milk life were:
pH: between 6.6 and 6.8.
Acidity < 18 Dornic.
TBC < 30,000 CFU mL 1.
When the samples did not reach one of these values, the milk
was considered not suitable for consumption. Simultaneously,
organoleptic properties such as odor, color and appearance were
taken into account, but organoleptic statistical analysis was not
performed.

3. Experimental results
First, two commercial milks were maintained at refrigerated
conditions (46 C) following pH, titratable acidity and TBC assessments during storage, to determine milk shelf life and changes in
each parameter with storage. The results of this study are shown
in Fig. 2.
Milk 1 was a conventionally pasteurized skimmed milk (75 C,
15 s) and Milk 2 was a commercial milk treated by an ultrapasteurization infusion process (135140 C; 0.21 s). Both were stored

45000

20

40000

18

35000

16

pH and Acidity (Dornic)

TBC (CFU/m L)

2.6.2. Bacterial analyses

Indigenous microora were monitored during MF. To enumerate the microora in raw milk, skimmed milk, permeate and nal
heat treated product, total plate count agar medium was used for
the determination of total bacterial count (TBC). Suitable dilutions
of the samples were plated in duplicate and the plates were incubated for 24 h at 37 C. The TBC was determined after the incubation time using the direct count method (IDF RM 204) (2012),
whereby colonies were counted and reported as colony forming
units per mL (CFU mL 1), with the limit of detection of 1 log10
CFU mL 1. Reported counts are the average number of colonies
from duplicate plates.
Analyses to determine the removal of somatic cells and microorganisms commonly found in raw milk were not performed in
this study because previous studies have conrmed their removal

from skim milk by MF using a 1.4 lm membrane (Pafylias et al.,

1996; Elwell and Barbano, 2006; Fritsch and Moraru, 2008).
In order to obtain information about the sterility of the samples
prior to the 48 h incubation period for the TBC analysis, bioluminescence was measured for the experiments carried out with a
high heat treatment process (data nor shown). Contaminated samples were rejected from the study.

14

30000
25000
20000
15000

Milk 2: TBC

10

Milk 1: pH

Milk 2: pH

10000

5000

Milk 1: TBC

12

Milk 1: Acidity
Milk 2: Acidity

0
1

10 11 12 13 14 15 16 17

Shelf life (days)

Fig. 2. Titrable acidity, pH and total bacteria count (TBC) of two commercial milks during milk life. Milk 1 is a pasteurized milk (75 C, 15 s) and Milk 2 is a ultrapasteurized
milk (130140 C, 0.21 s). Both milks were stored at 46 C.

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19

2006) have selected similar values (20,000 CFU mL 1) according

to the requirements for pasteurized milk.
The shelf life of different milk samples was estimated after different heat treatments (LHT: low heat treatment/pasteurization at
90, 80, 75 and 73 C, 15 s; IHHT: indirect heat treatment at 130,

between 4 and 6 C inside their original closed containers. Samples

for analysis were obtained through a septum using a syringe.
From a bacteriological point of view, the selected end of shelf
life was a TBC greater than 30,000 CFU mL 1, as this is the value
used in the dairy industry. Other authors (Elwell and Barbano,
40000
35000

MF+LHT, 90C

TBC (CFU/mL)
MF+LHT, 80C

30000

MF+LHT, 75C
25000
MF+LHT, 73C
20000

LHT, 90C
LHT, 80C

15000

LHT, 75C

10000

LHT, 73C
5000
0
0

10

15

20

25

30

35

Shelf life (days)

Fig. 3. Microorganisms growth after low temperature treatments (LHT) and combined Microltration + low temperature treatment (LHT + MF). Heat treatment: 15 s. Storage
temperature between 4 and 6 C.

40000
35000
IHHT, 130C

TBC
30000
(CFU/mL)

IHHT, 125C
25000
MF+IHHT, 120C
20000

MF+IHHT, 115C

15000

MF+IHHT, 130C
MF+IHT 125C

10000

MF+IHT 120C
5000
MF+IHT 115C
0
0
10

20

30

40

50

60

70

Shelf life (days)

Fig. 4. Microorganisms growth after indirect high temperature treatments (IHHT) and combined Microltration + indirect high temperature treatment (IHHT + MF). Heat
treatment: 2 s Storage temperature between 20 and 22 C.
40000
35000
DDHT, 130C

TBC
(CFU/mL) 30000

DDHT, 125C
DDHT, 120C

25000

DDHT, 115C

20000

MF+DDHT, 130C
15000

MF+DDHT, 125C
MF+DDHT, 120C

10000

MF+DDHT, 115C

5000
0
0

10

20

30

40

50

60

70

80

Shelf life (days)

Fig. 5. Microorganisms growth after direct high temperature treatments (DHHT: T, 6 s) and combined Microltration + direct high temperature treatment (DHHT + MF). Heat
treatment: 6 s Storage temperature between 20 and 22 C.

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19
5.00
MF+LHT, 90C - R2=0.984

4.50
4.00

MF+LHT, 80C- R2=0.980

3.50

MF+LHT, 75C-R2=0.986

Log (TCB)
3.00

MF+LHT, 73C - R2=0.992

2.50
2.00

LHT, 90C - R2=0.996

1.50

LHT, 80C - R2=0.998

1.00

LHT, 75C - R2=0.990

0.50
LHT, 73C - R2=0.994

0.00
0

10

15

20

25

30

35

Shelf life (days)

Fig. 6. Linealization of the rst order equation dN/dt = kN for low heat treatments (LHT) and microltration follow by low heat treatment (LHT + MF). Heat treatment: 15 s.
Storage temperature of 46 C.

5.00
IHHT, 130C - R2=0.989

4.50
Log (TBC)

4.00

IHHT, 125C - R2=0.986

3.50

IHHT, 120C - R2=0.987

3.00

IHHT, 115C - R2=0.982

2.50
MF+IHHT, 130C -R2=0.993

2.00
MF+IHHT, 125C - R2=0.986

1.50
1.00

MF+IHHT, 120C - R2=0.988

0.50

MF+IHHT, 115C - R2=0.992

0.00
0

20

40

60

80

Shelf life (days)

Fig. 7. Linealization of the rst order equation dN/dt = kN for indirect heat treatments (IHHT) and microltration follow by indirect heat treatment (IHHT + MF). Heat
treatment: 2 s Storage temperature: 2022 C.

125, 120 and 115 C, 2 s; DHHT: direct heat treatment at 130, 125,
120 and 115 C, 6 s) and the combination of MF + heat treatment at
the same temperatures and treatment periods mentioned above.
Microorganism growth of all these treatments are shown in Figs. 3
5. The dotted line represents the limit for the end of milk shelf life
according to the TBC analysis.
The microorganism growth followed rst order kinetics, as can
be seen in Figs. 68, in which Log (N/N0) is represented against
(t t0) to obtain the kinetic constant, k (days 1). N0 is the initial
TBC before treatment and N is the TBC value after each treatment.
(t t0) corresponds to the milk shelf life. As can be seen in the gures, the R2 values are greater than 0.98 in all experiments.
The slope of the straight lines in Figs. 68, that represent the
growth kinetic constant during storage, are shown in Table 1.
The logarithmic bacteria reduction (LRD), presented in Table 2,
was estimated as the ratio between the initial bacteria count of
each milk sample and the nal bacteria count after complete
treatment.
Finally, the values of milk shelf life for all the treatments studied are represented in Fig. 9. The values were obtained as the intersection between microorganism growth and established the TBC
limit (30,000 CFU mL 1). In the gure, the temperature in parentheses is the storage temperature.

4. Discussion
In Fig. 2, it can be seen that Milk 1 had a shelf life between 7 and
8 days according to the TBC stablished limits. However, changes in
pH and titratable acidity were very small (constant values around
6.4 and 15, respectively) throughout this period of time. Milk 2 can
be considered an ultrapasteurized milk (treated at 135140 C for
0.21 s), and could duplicate the shelf life of pasteurized milk life,
reaching between 16 and 17 days with a TBC lower than the limit,
but, in this case, the titratable acidity increased slightly from 14 to
17 at the end of its shelf life. Titratable acidity and pH do not seem
to be adequate parameters to follow the state of milk. Some experiments (not included in this work) showed that milk samples with
TBC higher than 30,000 CFU mL 1 maintained normal values of pH
and acidity, so the TBC limits selected in this work are conservative
and real shelf life probably is longer. The shelf life for these two
milk samples used as reference were within the range established
by manufacturers. In the case of Milk 2, the apparently short life, in
spite of the temperature treatment (135140 C) was due to the
short duration of treatment. For the following experiments, TBC
was selected as the parameter to estimate the milk shelf life.
Figs. 35 show the effect of combination of MF + heat treatment
(HT) on the shelf life as well as the inuence of different heat treat-

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19
5.00

DHHT, 130C - R2=0.992

4.50

Log (TCB) 4.00

DHHT, 125C - R2=0.987

3.50

DHHT, 120C - R2=0.994

3.00

DHHT, 115C - R2=0.988

2.50

MF+DHHT, 130C - R2=0.996

2.00
1.50

MF+DHHT, 125C - R2=0.995

1.00

MF+DHHT, 120C - R2=0.986

0.50

MF+DHHT, 115C - R2=0.996

0.00
0

20

40

60

80

Shelf Life (days)

Fig. 8. Linealization of the rst order equation dN/dt = kN for direct heat treatments (DHHT) and microltration follow by direct heat treatment (DHHT + MF). Heat
treatment: 6 s. Storage temperature: 2022 C.

Table 1
Kinetic constants (day

a
b

) of each treatment at different temperatures.

Treatments

90 C

80 C

75 C

73 C

LHTa
MFa + LHT
IHHTb
MFb + IHHT
DHHTb
MFb + DHHT

0.5628
0.2943

0.3985
0.2864

0.4266
0.3195

0.4006
0.3032

130 C

125 C

120 C

115 C

0.3240
0.1609
0.2593
0.2613

0.2035
0.1600
0.1941
0.1806

0.2118
0.1516
0.2078
0.2037

0.1560
0.1775
0.1749
0.1371

Storage temperature between 4 and 6 C.

Storage temperature between 20 and 22 C.

Table 2
Logarithmic bacteria reduction for all the treatments studied.
Treatment

Logarithmic bacteria reduction (LBR)

LHT (90 C)
LHT (80 C)
LHT (75 C)
LHT (73 C)
MF + LHT (90 C)
MF + LHT (80 C)
MF + LHT (75 C)
MF + LHT (73 C)
IHHT (130 C)
IHHT (125 C)
IHHT (120 C)
IHHT (115 C)
MF + IHHT (130 C)
MF + IHHT (125 C)
MF + IHHT (120 C)
MF + IHHT (115 C)
DHHT (130 C)
DHHT (125 C)
DHHT (120 C)
DHHT (115 C)
MF + DHHT (130 C)
MF + DHHT (125 C)
MF + DHHT (120 C)
MF + DHHT (115 C)

2.7
2.0
1.8
1.6
5.1
4.8
4.3
4.1
6.5
5.2
4.6
3.3
6.4
5.3
4.9
4.8
7.8
5.6
5.3
4.0
9.1
6.5
6.3
4.6

ments alone. At low temperatures (LHT and MF + LHT) (Fig. 3), the
differences in shelf life were considerable (69 days vs. 26
33 days), and the growth curves of both groups of treatments were
well different. Schmidt et al. (2012) studied combination of
MF + pasteurization (77 C, 30 s), obtaining a milk shelf life between 18 and 22 days (considering a TBC limit of about
30,000 CFU mL 1), depending on the storage temperature (4 and

8 C), and using raw milk with similar initial microorganism counts
as in this work. Elwell and Barbano (2006) published shelf life
values between 16 days (storage at 6.1 C) and 68 days (with storage at 0.1 C). In this work, the effect of heat treatment on shelf life
was clear between 75 and 80 C and differences between 80 and
90 C were almost negligible (as well as between 75 and 73 C, as
expected). This behavior was observed with and without MF. The
main difference in shelf life was related to the lag time, i.e. the time
after treatment at which bacteriological growth is detected. The lag
time for MF + LHT was greater than 10 days for the MF + LHT treatment at 73 and 75 C and greater than 18 days for treatment at 80
and 90 C. On the other hand, TBC values after the LHT treatment
were non-zero and microbiological growth started immediately
after treatment (day 1).
When compared, the IHHT and MF + IHHT (Fig. 4) curve shapes
were similar, with a longer lag time (around 35 days for MF + IHHT
at 130 C) than in previous experiments. In this case, the differences between treatments with and without MF were not as clear
as before, but MF + IHHT samples showed a microorganisms
growth slightly slower. The shelf life of milk after MF + IHHT treatment during the 45 rst days at the lowest temperature studied
(115 C) was similar to those obtained with IHHT at the highest
temperature studied (130 C). After this time, the growth curves
were similar. Combined methods provided a longer shelf life (between 42 and 50 days against 5865 with IHHT alone) than the
LHT and MF + LHT treatments, even taking into account that storage of the IHHT and MF + IHHT samples (as well as DHHT and
MF + DHHT samples later on) took place at higher temperatures
(2022 C). Milk shelf life is strongly affected by storage temperature, as it was stated by other authors (Schmidt et al., 2012;
Tomasula et al., 2011; Ranieri et al., 2009) and changes of a few de-

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19

80

LHT (73C)
LHT (75C)
LHT (80C)

70

LHT (90C)
MF+LHT (73C)
MF+LHT (75C)

Shelf Life
(days)

60

MF+LHT (80C)
MF+LHT (90C)
IHHT (115C)

50

IHHT (120C)
IHHT (125C)
IHHT (130C)

40

MF+IHHT (115C)
MF+IHHT (120C)

30

MF+IHHT (125C)
MF+IHHT (130C)
DHHT (115C)

20

DHHT (120C)
DHHT (125C)
DHHT (130C)

10

MF+DHHT (115C)
MF+DHHT (120C)
MF+DHHT (125C)

0
Treatments

MF+DHHT (130C)

Fig. 9. Milk shelf life for all the treatments studied. Storage temperatures between brackets.

grees in the storage temperature lead to important differences in

shelf life, so a comparison with published data must be done
carefully.
Fig. 5 shows a microorganism growth similar for direct high
heat treatment (DHHT) and combination with MF. From these
experiments, it can be observed that shelf life values after DHHT
treatments were very much dependent on the temperature treatments (see Fig. 9 later on). Around 20 days of extra life can be obtained by increasing the treatment temperature by 15 C. The
maximum milk shelf life obtained in this work was about 74 days
with TBC lower than 30,000 CFU mL 1. Even though statistical
organoleptic analysis was not performed, chemical analysis and
the general aspect of the milk was good, without odd colors or
odors. The microltration stage allows for reducing the temperature common in UHT processes by more than 20 C in (around
150 C in most of the milk industry), which leads to better milk
quality.
Figs. 68 show lineal behavior when plotting log TBC vs. shelf
life, what demonstrates that microorganisms growth follow a rst
order kinetic, being the R2 values of all experiments higher than
0.98.
Kinetic constants (k) values presented in Table 1 do not show
important differences. For LHT and MF + LHT did not depend on
the heat treatment, as expected (see Fig. 6). Note that the temperature does not refer to the value at which microorganisms were
grown but to the intensity of the previous heat treatment. The k
values for MF + LHT and for MF + IHHT were slightly lower when
compared to LHT and IHHT alone. However these differences between MF + DHHT and the corresponding DHHT are negligible.
Fig. 6 shows almost parallel lines with a different cut-off on the
y-axis (which means that the TBC at t = 0 days was clearly different); this is related to the lag time for each pasteurization treatment. As Ranieri et al. (2009) demonstrated, the pasteurization
process does not preferentially affect the microorganism population, and the parallel lines observed in this gure conrm this

statement. However, lower k values obtained for MF + LHT and

for MF + IHHT demonstrate that the removal of some of the bacteria present in raw milk leads to a lower microorganism growth rate
(lower k values). The higher k values in the LHT and IHHT experiments mean that the rate of microorganism growth during storage
was higher. In the case of MF + LHT and MF + IHHT treatments, previous removal of microorganisms probably included psychrotrophs, so growth was reduced in samples stored at 46 C after
MF + LHT. On the contrary the differences in k values for DHHT
and MF + DHHT treatments are very small. Time treatment in direct heating was 6 s (2 s in the rest of treatments) and psychrotrophs probably are destroyed in these treatments. k values
during milk storage at low and room temperatures are difcult
to nd in the literature.
Heat treatments without MF at temperatures higher than
120 C deactivated most of the psychrotolerant bacteria and microorganism growth during storage and did not depend on the microltration step, except that the lag time was longer after combined
treatments.
The shelf life was longer for the DHHT and MF + DHHT treatments due to the longer duration of treatment (6 s vs. 2 s in the
case of indirect HT). The maximum shelf life obtained was 74 days
in the case of the MF + DHHT treatment between 120 and 130 C.
Table 2 shows the logarithmic bacteria reduction (LBR) values
for each treatment. Pasteurization treatments (75 C, 15 s) gave
an LBR value of 1.8, and higher pasteurization temperature increased the LBR to 2.7 (at 90 C). Combined MF + LHT increased
the LBR value (between 4.1 and 5.1). Additionally, microltration
before heat treatment noticeably increased the LBR. Some published data gave LBR values between 2 and 4 for a 1.4 lm microltration membrane without heat treatment (Malmberg and Holm,
1988; Elwell and Barbano, 2006; Giffel and van der Horst, 2004),
depending on the membrane operation conditions and the microorganisms studied (TBC, Bacillus cereus, spores, etc.). However,
other published results (Schmidt et al., 2012) published higher val-

L. Fernndez Garca, F.A. Riera Rodrguez / Journal of Food Engineering 128 (2014) 19

ues (between 5 and 6) after combined MF + pasteurization. Differences between these results are due to the difculty in performing
experiments under equivalent conditions, particularly regarding
the initial milk bacteria count and microltration conditions (especially the membrane cut-off and temperature). Table 2 shows,
however, reasonable LBR values when different treatments are
compared. MF treatment always increased the milk ESL. The
MF + IHHT treatment led to a higher LBR value, especially at lower
temperatures when compared with treatments without MF (4.8 at
115 C vs. 3.3 without the MF step). In the case of direct heat treatment, the LBR values were higher due to the longer duration of
treatment.
Finally, Fig. 9 shows the ESL of all the studied treatments.
Important increases in shelf life were observed. Major effects of
MF were found with low heat treatment (the shelf life increased
by a factor of two or three at temperatures between 73 and
90 C). Note that the results shown in this gure are not fully comparable because, in some cases, the milk was stored at 20 C (treatments at high temperature) but, in spite of this, important
increases in shelf life were found. The maximum milk shelf life
was found for MF + DHHT (at 125130 C) with a treatment duration of 6 s. The results shown in this gure provide a new way of
combining ultrapasteurization processes with a microltration
step to obtain defatted milk with more than two months of shelf
life at room temperature. Extra efforts must be made to statistically evaluate the organoleptic properties of the obtained products
as well as to follow proteolysis over time. These aspects were not
the objective of the present study.
5. Conclusions
MF has proven to be an adequate tool for the removal of bacteria in milk. The combination of this technology and a subsequent
pasteurization treatment (73 C for 15 s) has enabled the production of ESL milk with a lifetime close to 30 days (70% longer than
regular pasteurized milk). A shelf life longer than 21 days allows
the distribution of this ESL milk together with other fresh dairy
products such as yogurt and facilitates its arrival to markets. Treatment at higher temperature (always lower than 130 C for 6 s) allow extending the shelf life to more than 70 days, even when
maintaining the product at ambient temperature. The kinetics of
microorganism growth show that the growth rate was similar
regardless of the selected treatment, and that the microorganism
growth lag time is the main reason for the increased shelf life.
Acknowledgements
The authors acknowledge the nancial support from the Spanish Ministry of Science and Innovation (Project AGL2007-63998/
ALI). We would also like to thank FICYT (Fundacin para el

Fomento en Asturias de la Investigacin Cientca Aplicada y la

Tecnologa) for the grant of PhD studies of Leticia Fernndez (BP
08-050), and C.A.P.S.A. (Corporacin Alimentaria Peasanta S.A.)
for its technical support for the experiments.
References
Barbosa-Cnovas, G.V., Palou, E., Pothekemury, U., Swamson, B.G., 1999.
Conservacin no trmica de alimentos. Acribia, Zaragoza, Spain.
Brans, G., Schren, C.G.P.H., van der Sman, R.G.M., Boom, R.M., 2004. Membrane
fractionation of milk: state of the art and challenges. J. Membr. Sci. 243, 263
272.
Elwell, M.W., Barbano, D.M., 2006. Use of microltration to improve uid milk
quality. J. Dairy Sci. 89, 2030.
Fernandez, L., lvarez, S., Riera, F.A., 2013. Microltration applied to dairy streams:
removal of bacteria. J. Food Sci. Agric. 93, 187196.
Fredsted, L.B., Rysstad, G., Eie, T., 1996. Pure-Lac: the new milk with protected
freshness and extended shelf life. In: Proceedings of the IDF Symposium 1995:
Heat Treatments and Alternative Methods, pp. 104125. Brussels, Belgium.
Fritsch, J., Moraru, C.I., 2008. Development and optimization of carbon dioxideaided cold microltration process for the physical removal of microorganisms
and somatic cells from skim milk. J. Dairy Sci. 91, 37443760.
Giffel, M.C., van der Horst, H.C., 2004. Comparison between bactofugation and
microltration regarding efciency of somatic cell and bacteria removal. Bull.
Int. Dairy Fed. 389, 4953.
Goff, H.D., Grifths, M.W., 2006. Major advances in fresh milk and milk products:
uid milk products and frozen desserts. J. Dairy Sci. 89, 11631173.
Grangeon, A., Lescoche, P., Millares, M., 2000. Isoux membrane: the
microltration mastering. In: Proc. 6th Int. Conf. on Inorganic Membranes
(ICIM-6). Montpellier, France.
Guerra, A., Jonsson, G., Rasmussen, A., Waagner Nielsen, E., Edelsten, D., 1997. Low
cross-ow velocity microltration of skim milk for removal of bacterial spores.
Int. Dairy J. 7, 849861.
International Dairy Federation (IDF 220), 2010. (ISO 29981:2010) Milk productscolony count technique at 37 C.
International Dairy federation (IDF RM 204), 2012. Milk and milk productsdetermination of the titratable acidity of milk fat.
Malmberg, M., Holm, S., 1988. Producing low bacteria skim milk by microltration.
J. Membr. Sci. 274, 7578.
Olesen, N., Jensen, F., 1989. Microltration. The inuence of operation parameters
on the process. Milchwissenshaft 44, 476479.
Pafylias, I., Cheryan, M., Mehaia, M.A., Saglam, N., 1996. Microltration of milk with
ceramic membranes. Food Res. Int. 29, 141146.
Ranieri, M.L., Huck, J.R., Sonnen, M., Barbano, D.M., Boor, K.J., 2009. High
temperature, short time pasteurisation temperatures inversely affect bacterial
numbers during refrigerated storage pasteurised uid milk. J. Dairy Sci. 92,
48234832.
Saboya, L.V., Maubois, J.-L., 2000. Current developments of microltration
technology in the dairy industry. Lait 80, 541553.
Schmidt, V.S.J., Kaufmann, V., Kulozik, U., Scherer, S., Wenning, M., 2012. Microbial
biodiversity, quality and shelf life of microltered and pasteurised extended
shelf life (ESL) milk from Germany, Australia and Switzerland. Int. J. Food
Microbiol. 154, 19.
Skrzypek, M., Burger, M., 2010. Isoux ceramic membranes practical experiences
in dairy industry. Desalination 250, 10951100.
Tomasula, P.M., Mukhopadhyay, S., Datta, N., Porto-Fett, A., Call, J.E., Luchansky, J.B.,
Renye, J., 2011. Pilot-scale crossow-microltration and pasteurization to
remove spores of Bacillus anthracis (Sterne) from milk. J. Dairy Sci. 94, 4277
4291.
Trujillo, A.J., Capellas, M., Saldo, J., Gervilla, R., Guamis, B., 2002. Applications of
high-hydrostatic pressure on milk and dairy products: a review. Innovative
Food Sci. Emer. Technol. 3, 295307.