Journal of Food Engineering 128 (2014) 1–9

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Journal of Food Engineering

journal homepage: www.elsevier .com/ locate / j foodeng

Combination of microfiltration and heat treatment for ESL milkproduction: Impact on shelf life

0260-8774/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jfoodeng.2013.11.021

⇑ Corresponding author. Address: C/Julián Clavería, 8, 33008 Asturias, Spain.Tel.: +34 985103436.

E-mail address: far@uniovi.es (F.A. Riera Rodríguez).

L. Fernández García, F.A. Riera Rodríguez ⇑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 2013Received in revised form 11 October 2013Accepted 24 November 2013Available online 4 December 2013

Keywords:ESLMicrofiltrationHeat 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 and15 s) and combined with a microfiltration step (1.4 lm cut-off ceramic membrane) to study the influenceof these treatments on milk shelf life. Thirty thousand colony forming units/mL was selected as the limitparameter for extended shelf life. The logarithmic reduction in bacteria was estimated for each treatmentand the total bacteria count was measured during the storage of milk at 4–6 �C and at room temperature.Microorganism growth kinetic data during storage were also estimated. A maximum extended shelf lifeof 74 days was found for milk after the combination of microfiltration and direct heat treatment at125–130 �C and storage at room temperature. An extended shelf life of 33 days was obtained after micro-filtration followed by pasteurization at 90 �C and storage at 4–6 �C.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The shelf life of milk is an important concept that defines theability to widen the distribution chain of the product. As milkprovides a favorable medium for spoilage microorganisms, pre-treatment as well as temperature/time conditions must be chosenin order to control microbial growth. Heat treatments are the mostwidely used processes for lowering the bacterial content of milkand milk products (Olesen and Jensen, 1989). Currently, pasteuri-zation and ultra-high temperature (UHT) processing are commonheat treatments used in the dairy industry. Pasteurization, how-ever, cannot totally prevent the survival of all bacteria, some ofwhich may affect the storage qualities of milk and milk products.

One significant barrier to extending the shelf life of dairy prod-ucts is the difficulty in balancing the removal or destruction ofspoilage micro-organisms and spores present in raw milk whilelimiting product color changes, vitamin destruction and milk pro-tein denaturation. Extended shelf life (ESL) milk provides the pos-sibility of extending the shelf life of a range of products that canstay under refrigerated conditions beyond the traditional limitsof conservation (Goff and Griffiths, 2006).

Some of the possible ESL technologies are bactofugation (Giffeland van der Horst, 2004), pulsed electric fields (Barbosa-Cánovaset al., 1999), high pressure processing (Trujillo et al., 2002), highheat treatment (Fredsted et al., 1996) and microfiltration (MF).Cross-flow MF for bacteria removal provides a low-temperature

approach for the control of microbial growth and is one of theESL techniques employed at the industrial scale for this application(Skrzypek and Burger, 2010).

The effectiveness of the MF separation process in reducing bac-terial levels in milk was confirmed by Olesen and Jensen (1989).MF led to a logarithmic bacteria reduction (LBR) of 4 for total bac-teria and 2.3–3.7 for spores. Experimental results obtained undervarious operating conditions have been reported in a number ofpublications and reviews (Saboya and Maubois, 2000; Branset al., 2004; Fernandez et al., 2013). MF membranes with a poresize of about 1.4 lm can achieve the right balance between rejec-tion of bacteria and long-term flux, with little or no rejection ofother milk components such as protein, lactose and ash. However,most fat globules in milk are similar in size to bacteria; this resultsin very rapid fouling of the membrane due to the deposition of a fatlayer on the membrane surface and the constriction of pores,which consequently affect MF performance. MF for microbial re-moval is only applied to skimmed milk on an industrial scale(Guerra et al., 1997).

Although very efficient regarding the removal of bacteria andspores, MF cannot guarantee 100% removal of pathogenic bacteria,as required for milk pasteurization. After milk treatment and dur-ing storage, surviving spores and microorganisms can germinateand grow and thus limit the milk shelf life. For this reason, heattreatment is needed after the MF process. MF prior to heat treat-ment can remove some microorganisms and reduce enzyme activ-ity during storage that can degrade lactose, protein and fat. Someauthors have studied different combinations of MF and mild heattreatments. Elwell and Barbano (2006) studied the shelf life of pas-teurized (72 �C, 15 s) skimmed milk with and without MF (1.4 and

Nomenclature

SymbolsA membrane areaCFU colony forming unitsCWF membrane clean water fluxDHHT direct high heat treatmentESL extended shelf lifeHT heat treatmentIHHT indirect high heat treatmentLBR logarithmic bacteria reduction

LHT low temperature treatmentsMF microfiltrationN number of microorganisms a time tN0 number of microorganisms a t = 0Qf membrane permeate flow rate (with water)TMP transmembrane pressureTBC total bacterial countRD reduction degreel viscosity

2 L. Fernández García, F.A. Riera Rodríguez / Journal of Food Engineering 128 (2014) 1–9

0.8 lm) at different storage temperatures (0.1–6.1 �C), and ob-tained a maximum bacterial log reduction of 5.63 and 92 days ofmilk shelf life. Tomasula et al. (2011) microfiltered (0.8 lm)previously pasteurized milk (72 �C, 18.2 s) in order to study Bacillusanthracis spore removal, and found a maximum log reduction ofabout 6.

Information on the combination of MF and pasteurization treat-ments can be found in Schmidt et al. (2012) and Elwell andBarbano (2006), but treatment data at higher temperatures arescarce. The use of MF could reduce the temperature of traditionalultra-high temperature processes, giving products with organolep-tic properties similar to those of pasteurized milk. The method pro-posed in this work could obtain a ‘‘premium’’ milk type with an ESLgreater than that provided by the new ultrapasteurization productsthat are currently available.

In this study, several combinations of MF and temperature heattreatments (indirect and direct, between 73 and 130 �C) with andwithout MF were studied in order to evaluate the effect of all treat-ments on the milk shelf life maintained at refrigeration and roomtemperatures. Organoleptic and proteolytic aspects were notstudied.

2. Materials and methods

2.1. Milk

The raw milk used in all experiments was submitted to a mildheat treatment at 50 �C and then centrifuged (GEA Westfalia,Germany) by a dairy company (CAPSA, Asturias, Spain). Theaverage milk properties and composition were: pH 6.79 ± 0.04, fatcontent 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 be-tween 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 feedingtank (E-1) was 50 L and was designed with automatic monitoringof the liquid level (KROHNE, Romans CEDEX, France). The temper-ature was controlled by means of a tank jacket with automatic reg-ulation of flows of cooling and heating fluids to adjust to the setpoint (V-1). The tangential flow rate in membrane channels wasensured by a flow rate frequency-regulated vertical multistagecentrifugal pump (Grundfos, St. Quentin-Fallavier, France) (E-2).The pump provided a maximum flow rate of 8 m3 h�1. The cross-flow velocity was obtained by adjusting the flow rate of the feedingpump.

Experimental data measurements were performed usingelectronic volume flow meters for the permeate and retentate(Endress-Hausser Promass 60, Weil am Rhein, Germany) (M-1,

M-2); platinum resistance in a mineral-insulated cable processthermometer was used for the feeding solution (Endress-Hausser,Weil am Rhein, Germany) (M-3) and differential pressure trans-ducers (Endress-Hausser, Weil am Rhein, Germany) were usedfor 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 ensurethe correct measurement of the transmembrane pressure (Pi andPo, respectively) and the pressure on the permeate side (Pp). Trans-membrane pressure (TMP) was calculated using the followingequation, TMP = [(Pi + Po)/2] � Pp. There were two needle valvesat the retentate and permeate outlet to control the pressure ofthe system (V-2, V-3). The experimental rig had an automaticcontrol system to operate at a constant permeate flux. A pneumaticcontrol 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 Isoflux� ceramicmembrane (Tami, France). The Isoflux� membrane was designedto compensate for the pressure drop by a thickness gradient onthe top layer along the membrane length (Grangeon et al., 2000).Such a membrane design produces a constant flux along the lengthof the membrane element. This feature leads to improved perfor-mance of the membrane in terms of long-term flux rates requiredwith industrial feeds and allows for more effective cleaning sincemembrane fouling is similar along the full length of the membraneelement (Saboya and Maubois, 2000).

The Isoflux� membrane chosen for this purpose had 23 chan-nels with an internal diameter of 3.5 mm, a length of 1178 mm, afiltering area of 0.35 m2 and a pore size of 1.4 lm (measured bythe porosity method, as reported by the manufacturer).

2.3. Membrane cleaning procedure

The membrane was chemically cleaned after each run usingAlkaline P-3 Ultrasil 25 1% (v/v) (Henkel-Ecolab SNC, Issy les Mou-lineaux, France) at 75 �C, for 15 min without permeation (permeatevalve closed) and 15 min with permeate flux followed by nitricacid (HNO3, 58% purity) at 1% (v/v) (Brenntag, Sevilla, Spain) at50 �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 treatmentA tubular heat exchanger was used for indirect heat treatment

(IHHT) and for the pasteurization step. The system (OMVEHT220, Netherlands) consisted of a feed tank, pump, heat exchan-ger, temperature and pressure sensors, boiler and computer. Theproduct was pumped from the reservoir to the heat exchangerwhere the product was first preheated and then directed to the

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FICSCM

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LSL

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M-6 Dpp

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V-1 V-1

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V-1

V-1

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

L. Fernández García, F.A. Riera Rodríguez / Journal of Food Engineering 128 (2014) 1–9 3

main heating area. After the treatment, the product was cooled.The system was designed to work with a product flow rate of20 L h�1. The maintenance tube length was selected to fix the pas-teurization step to 15 s and to 6 s for the indirect high heattreatments.

2.4.2. Direct heat treatmentIn 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 po-sitive pump which provided a variable flow from 100 to 1000 L h�1

and pressures ranging from 2 to 10 bar, a flow meter and a plateheat exchanger for previous heating (at 80 �C). This plate heat ex-changer had two bodies. The milk was first heated with hot water.Preheating was carried out at 80 �C, and a controller was used toadjust the temperature to the desired value. In this case, only thefirst body of the heat exchanger was used.

In addition to the elements described above, the equipment hadseveral temperature and pressure sensors placed at the inlet andthe outlet of the exchangers, steam injection, circuits of feed andproduct, etc. The recirculation of the product was also possibleand was useful for the cleaning steps. The steam temperature couldbe adjusted by a needle valve which regulated the steam injection.The product remained at this temperature for 6 s. This could varyslightly depending on the flow provided by the positive pump. Atthe flash cooler, the product was cooled to 80 �C (the same temper-ature it was before steam injection in order not to dilute or concen-trate the product). The steam was extracted from the expansionchamber through a vacuum pump and the product was drainedwith a centrifugal pump.

The pilot plant had a homogenizer connected after the heatingprocess. It is important that the homogenizer is placed here be-cause heating can destabilize the mixture and separate the fatagain. Subsequently, the homogenized product was cooled in aplate heat exchanger with tap water.

Finally, for both heat treatments studied, the product was col-lected in a laminar air flow cabinet. Samples were taken in 100mL sterile containers in a sterile atmosphere. There was no signif-icant contamination of the circuit after UHT and its connectionwith the laminar air flow cabinet.

2.5. Operating procedure

Water from the industry tap network was used to warm theprevious membrane rig to 45 �C.

The cleaning protocol was performed before each MF run withthe alkaline cleaner and after the water rinse, and the acid cleanerwas circulated. Finally, a water rinse was performed until neutral-ization. Prior to the experiments with milk, membrane clean waterflux (CWF) was measured to ensure that it was the same from run torun, by performing a MF trial using water at 20 �C and 6 m s�1 linearvelocity. The CWF was then calculated using the following equation:CWF = Qf � l/(TMP � A), where Qf is the permeate water flow ratein L h�1, l is the water viscosity (1.0 cP at 20 �C), and A is the mem-brane filtration area (0.35 m2). The CWF trial was performed induplicate or until the original CWF was obtained. If not, the cleaningprocedure was repeated. After each run, the milk was drained fromthe feed tank and water was added to initiate a water rinse cycle forabout 20 min before the cleaning protocol was applied. If the CWFwas less than 10% of the CWF before the experiments with milk,the cleaning cycle was repeated, and the CWF and pH of the perme-ate and retentate streams were measured again.

After each cleaning cycle, the unit was emptied and filled with150 L of raw skimmed milk. Before the operating parameters wereset, 15 L of milk (corresponding to the volume of the retentatecompartment) were used to flush the loop of the remaining water.The unit almost instantly reached 80% of the desired pressure andflow velocity and needed roughly 3 min to attain the desired val-ues. Around 200 L were used for each experiment.

4 L. Fernández García, F.A. Riera Rodríguez / Journal of Food Engineering 128 (2014) 1–9

When the processing run was initiated, the retentate was re-turned to the feed tank and the permeate was collected in orderto perform the thermal treatment afterwards. The experimentalconditions for the MF stage were fixed at: v = 6 m s�1, TMP = 0.55bar and T = 45 �C. Experiments showed no membrane fouling after4 h and the permeate flow rate varied between 450 and 500 l h�1

m�2 in all cases. Total protein retention at the aforementioned con-ditions was lower than 1.5% (Fernandez et al., 2013).

For the combination of MF and heat treatment trials, the pro-cess began with the collection of 200 L of skimmed milk at 50 �Cright after skimming (0.3% fat content). The milk was pumped tothe MF equipment and, after bacteria removal, was directed tothe heat treatment apparatus (either direct or indirect). After heattreatment, samples were collected in a laminar flow cabinet insterile containers.

2.6. Samples and analyses

Samples of the feeding solution, permeates and final productafter heat treatments were aseptically withdrawn from all experi-ments. The final product samples were kept under refrigeratedconditions (4 �C) in the case of heat treatment at 73–90 �C and atroom temperature for the 115–130 �C treatments.

2.6.1. Physico-chemical analysesTotal 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 pro-teins, lactose and fat content. The pH was measured using a pHmeter (Crison Instruments SA, Barcelona, Spain).

2.6.2. Bacterial analysesIndigenous microflora were monitored during MF. To enumer-

ate the microflora in raw milk, skimmed milk, permeate and finalheat treated product, total plate count agar medium was used forthe determination of total bacterial count (TBC). Suitable dilutionsof the samples were plated in duplicate and the plates were incu-bated for 24 h at 37 �C. The TBC was determined after the incuba-tion time using the direct count method (IDF RM 204) (2012),whereby colonies were counted and reported as colony formingunits per mL (CFU mL�1), with the limit of detection of 1 log10

CFU mL�1. Reported counts are the average number of coloniesfrom duplicate plates.

Analyses to determine the removal of somatic cells and micro-organisms commonly found in raw milk were not performed inthis study because previous studies have confirmed their removal

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/mL)

Fig. 2. Titrable acidity, pH and total bacteria count (TBC) of two commercial milks durinmilk (130–140 �C, 0.2–1 s). Both milks were stored at 4–6 �C.

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 samplesprior to the 48 h incubation period for the TBC analysis, biolumi-nescence was measured for the experiments carried out with ahigh heat treatment process (data nor shown). Contaminated sam-ples were rejected from the study.

2.6.3. Storage/shelf life studiesSamples of the permeate (�80 mL) and final product were

drawn after the MF and the heat treatment units at various timesin all experiments in sterile specimen cups and kept at 4–6 �C orroom temperature (18–20 �C) (depending on the heat treatment)for the shelf life study. The main parameters considered for themilk quality evaluation were pH, titratable acidity and TBC. Theseparameters were analyzed at different time intervals depending onthe type of study. In the case of commercial milk, with a shortershelf life, parameters were monitored three times weekly in theinitial stage of refrigerated storage and then daily when a declinein the quality parameters was observed. For the rest of the trials,the frequency of the analysis increased when any of the parame-ters started to show a relevant variation. From that point on, sam-ples were measured more frequently.

Acidity, pH, and TBC parameters were selected to follow milkshelf 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 milkwas considered not suitable for consumption. Simultaneously,organoleptic properties such as odor, color and appearance weretaken into account, but organoleptic statistical analysis was notperformed.

3. Experimental results

First, two commercial milks were maintained at refrigeratedconditions (4–6 �C) following pH, titratable acidity and TBC assess-ments during storage, to determine milk shelf life and changes ineach parameter with storage. The results of this study are shownin Fig. 2.

Milk 1 was a conventionally pasteurized skimmed milk (75 �C,15 s) and Milk 2 was a commercial milk treated by an ultrapasteur-ization infusion process (135–140 �C; 0.2–1 s). Both were stored

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81012

1416

1820

pH and Acidity (ºDornic)

Milk 1: TBCMilk 2: TBCMilk 1: pHMilk 2: pHMilk 1: AcidityMilk 2: Acidity

g milk life. Milk 1 is a pasteurized milk (75 �C, 15 s) and Milk 2 is a ultrapasteurized

L. Fernández García, F.A. Riera Rodríguez / Journal of Food Engineering 128 (2014) 1–9 5

between 4 and 6 �C inside their original closed containers. Samplesfor analysis were obtained through a septum using a syringe.

From a bacteriological point of view, the selected end of shelflife was a TBC greater than 30,000 CFU mL�1, as this is the valueused in the dairy industry. Other authors (Elwell and Barbano,

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Fig. 3. Microorganisms growth after low temperature treatments (LHT) and combined Mtemperature between 4 and 6 �C.

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TBC (CFU/mL)

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Fig. 4. Microorganisms growth after indirect high temperature treatments (IHHT) andtreatment: 2 s Storage temperature between 20 and 22 �C.

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Fig. 5. Microorganisms growth after direct high temperature treatments (DHHT: T, 6 s) antreatment: 6 s Storage temperature between 20 and 22 �C.

2006) have selected similar values (20,000 CFU mL�1) accordingto the requirements for pasteurized milk.

The shelf life of different milk samples was estimated after dif-ferent heat treatments (LHT: low heat treatment/pasteurization at90, 80, 75 and 73 �C, 15 s; IHHT: indirect heat treatment at 130,

20 25 30 35

(days)

MF+LHT, 90ºC

MF+LHT, 80ºC

MF+LHT, 75ºC

MF+LHT, 73ºC

LHT, 90ºC

LHT, 80ºC

LHT, 75ºC

LHT, 73ºC

icrofiltration + low temperature treatment (LHT + MF). Heat treatment: 15 s. Storage

60 70

fe (days)

IHHT, 130ºC

IHHT, 125ºC

MF+IHHT, 120ºC

MF+IHHT, 115ºC

MF+IHHT, 130ºC

MF+IHT 125ºC

MF+IHT 120ºC

MF+IHT 115ºC

combined Microfiltration + indirect high temperature treatment (IHHT + MF). Heat

)

DDHT, 130ºC

DDHT, 125ºC

DDHT, 120ºC

DDHT, 115ºC

MF+DDHT, 130ºC

MF+DDHT, 125ºC

MF+DDHT, 120ºC

MF+DDHT, 115ºC

50 60 70 80

d combined Microfiltration + direct high temperature treatment (DHHT + MF). Heat

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Shelf life (days)

Log (TCB)

MF+LHT, 90ºC - R2=0.984

MF+LHT, 80ºC- R2=0.980

MF+LHT, 75ºC-R2=0.986

MF+LHT, 73ºC - R2=0.992

LHT, 90ºC - R2=0.996

LHT, 80ºC - R2=0.998

LHT, 75ºC - R2=0.990

LHT, 73ºC - R2=0.994

Fig. 6. Linealization of the first order equation dN/dt = kN for low heat treatments (LHT) and microfiltration follow by low heat treatment (LHT + MF). Heat treatment: 15 s.Storage temperature of 4–6 �C.

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Log (TBC)

IHHT, 130ºC - R2=0.989

IHHT, 125ºC - R2=0.986

IHHT, 120ºC - R2=0.987

IHHT, 115ºC - R2=0.982

MF+IHHT, 130ºC -R2=0.993

MF+IHHT, 125ºC - R2=0.986

MF+IHHT, 120ºC - R2=0.988

MF+IHHT, 115ºC - R2=0.992

Fig. 7. Linealization of the first order equation dN/dt = kN for indirect heat treatments (IHHT) and microfiltration follow by indirect heat treatment (IHHT + MF). Heattreatment: 2 s Storage temperature: 20–22 �C.

6 L. Fernández García, F.A. Riera Rodríguez / Journal of Food Engineering 128 (2014) 1–9

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 atthe 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 lifeaccording to the TBC analysis.

The microorganism growth followed first order kinetics, as canbe seen in Figs. 6–8, in which Log (N/N0) is represented against(t � t0) to obtain the kinetic constant, k (days�1). N0 is the initialTBC 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 fig-ures, the R2 values are greater than 0.98 in all experiments.

The slope of the straight lines in Figs. 6–8, that represent thegrowth 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 ofeach milk sample and the final bacteria count after completetreatment.

Finally, the values of milk shelf life for all the treatments stud-ied are represented in Fig. 9. The values were obtained as the inter-section between microorganism growth and established the TBClimit (30,000 CFU mL�1). In the figure, the temperature in paren-theses is the storage temperature.

4. Discussion

In Fig. 2, it can be seen that Milk 1 had a shelf life between 7 and8 days according to the TBC stablished limits. However, changes inpH and titratable acidity were very small (constant values around6.4 and 15, respectively) throughout this period of time. Milk 2 canbe considered an ultrapasteurized milk (treated at 135–140 �C for0.2–1 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 to17 at the end of its shelf life. Titratable acidity and pH do not seemto be adequate parameters to follow the state of milk. Some exper-iments (not included in this work) showed that milk samples withTBC higher than 30,000 CFU mL�1 maintained normal values of pHand acidity, so the TBC limits selected in this work are conservativeand real shelf life probably is longer. The shelf life for these twomilk samples used as reference were within the range establishedby manufacturers. In the case of Milk 2, the apparently short life, inspite of the temperature treatment (135–140 �C) was due to theshort duration of treatment. For the following experiments, TBCwas selected as the parameter to estimate the milk shelf life.

Figs. 3–5 show the effect of combination of MF + heat treatment(HT) on the shelf life as well as the influence of different heat treat-

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Log (TCB)

DHHT, 130ºC - R2=0.992

DHHT, 125ºC - R2=0.987

DHHT, 120ºC - R2=0.994

DHHT, 115ºC - R2=0.988

MF+DHHT, 130ºC - R2=0.996

MF+DHHT, 125ºC - R2=0.995

MF+DHHT, 120ºC - R2=0.986

MF+DHHT, 115ºC - R2=0.996

Fig. 8. Linealization of the first order equation dN/dt = kN for direct heat treatments (DHHT) and microfiltration follow by direct heat treatment (DHHT + MF). Heattreatment: 6 s. Storage temperature: 20–22 �C.

Table 1Kinetic constants (day�1) of each treatment at different temperatures.

Treatments 90 �C 80 �C 75 �C 73 �C 130 �C 125 �C 120 �C 115 �C

LHTa 0.5628 0.3985 0.4266 0.4006MFa + LHT 0.2943 0.2864 0.3195 0.3032IHHTb 0.3240 0.2035 0.2118 0.1560MFb + IHHT 0.1609 0.1600 0.1516 0.1775DHHTb 0.2593 0.1941 0.2078 0.1749MFb + DHHT 0.2613 0.1806 0.2037 0.1371

a Storage temperature between 4 and 6 �C.b Storage temperature between 20 and 22 �C.

Table 2Logarithmic bacteria reduction for all the treatments studied.

Treatment Logarithmic bacteria reduction (LBR)

LHT (90 �C) 2.7LHT (80 �C) 2.0LHT (75 �C) 1.8LHT (73 �C) 1.6MF + LHT (90 �C) 5.1MF + LHT (80 �C) 4.8MF + LHT (75 �C) 4.3MF + LHT (73 �C) 4.1IHHT (130 �C) 6.5IHHT (125 �C) 5.2IHHT (120 �C) 4.6IHHT (115 �C) 3.3MF + IHHT (130 �C) 6.4MF + IHHT (125 �C) 5.3MF + IHHT (120 �C) 4.9MF + IHHT (115 �C) 4.8DHHT (130 �C) 7.8DHHT (125 �C) 5.6DHHT (120 �C) 5.3DHHT (115 �C) 4.0MF + DHHT (130 �C) 9.1MF + DHHT (125 �C) 6.5MF + DHHT (120 �C) 6.3MF + DHHT (115 �C) 4.6

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ments alone. At low temperatures (LHT and MF + LHT) (Fig. 3), thedifferences in shelf life were considerable (6–9 days vs. 26–33 days), and the growth curves of both groups of treatments werewell different. Schmidt et al. (2012) studied combination ofMF + pasteurization (77 �C, 30 s), obtaining a milk shelf life be-tween 18 and 22 days (considering a TBC limit of about30,000 CFU mL�1), depending on the storage temperature (4 and

8 �C), and using raw milk with similar initial microorganism countsas in this work. Elwell and Barbano (2006) published shelf lifevalues between 16 days (storage at 6.1 �C) and 68 days (with stor-age at 0.1 �C). In this work, the effect of heat treatment on shelf lifewas clear between 75 and 80 �C and differences between 80 and90 �C were almost negligible (as well as between 75 and 73 �C, asexpected). This behavior was observed with and without MF. Themain difference in shelf life was related to the lag time, i.e. the timeafter treatment at which bacteriological growth is detected. The lagtime for MF + LHT was greater than 10 days for the MF + LHT treat-ment at 73 and 75 �C and greater than 18 days for treatment at 80and 90 �C. On the other hand, TBC values after the LHT treatmentwere non-zero and microbiological growth started immediatelyafter treatment (day 1).

When compared, the IHHT and MF + IHHT (Fig. 4) curve shapeswere similar, with a longer lag time (around 35 days for MF + IHHTat 130 �C) than in previous experiments. In this case, the differ-ences between treatments with and without MF were not as clearas before, but MF + IHHT samples showed a microorganismsgrowth slightly slower. The shelf life of milk after MF + IHHT treat-ment during the 45 first days at the lowest temperature studied(115 �C) was similar to those obtained with IHHT at the highesttemperature studied (130 �C). After this time, the growth curveswere similar. Combined methods provided a longer shelf life (be-tween 42 and 50 days against 58–65 with IHHT alone) than theLHT and MF + LHT treatments, even taking into account that stor-age of the IHHT and MF + IHHT samples (as well as DHHT andMF + DHHT samples later on) took place at higher temperatures(20–22 �C). Milk shelf life is strongly affected by storage tempera-ture, 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-

0

10

20

30

40

50

60

70

80

Treatments

LHT (73ºC)

LHT (75ºC)

LHT (80ºC)

LHT (90ºC)

MF+LHT (73ºC)

MF+LHT (75ºC)

MF+LHT (80ºC)

MF+LHT (90ºC)

IHHT (115ºC)

IHHT (120ºC)

IHHT (125ºC)

IHHT (130ºC)

MF+IHHT (115ºC)

MF+IHHT (120ºC)

MF+IHHT (125ºC)

MF+IHHT (130ºC)

DHHT (115ºC)

DHHT (120ºC)

DHHT (125ºC)

DHHT (130ºC)

MF+DHHT (115ºC)

MF+DHHT (120ºC)

MF+DHHT (125ºC)

MF+DHHT (130ºC)

Shelf Life (days)

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

8 L. Fernández García, F.A. Riera Rodríguez / Journal of Food Engineering 128 (2014) 1–9

grees in the storage temperature lead to important differences inshelf life, so a comparison with published data must be donecarefully.

Fig. 5 shows a microorganism growth similar for direct highheat treatment (DHHT) and combination with MF. From theseexperiments, it can be observed that shelf life values after DHHTtreatments were very much dependent on the temperature treat-ments (see Fig. 9 later on). Around 20 days of extra life can be ob-tained by increasing the treatment temperature by 15 �C. Themaximum milk shelf life obtained in this work was about 74 dayswith TBC lower than 30,000 CFU mL�1. Even though statisticalorganoleptic analysis was not performed, chemical analysis andthe general aspect of the milk was good, without odd colors orodors. The microfiltration stage allows for reducing the tempera-ture common in UHT processes by more than 20 �C in (around150 �C in most of the milk industry), which leads to better milkquality.

Figs. 6–8 show lineal behavior when plotting log TBC vs. shelflife, what demonstrates that microorganisms growth follow a firstorder kinetic, being the R2 values of all experiments higher than0.98.

Kinetic constants (k) values presented in Table 1 do not showimportant differences. For LHT and MF + LHT did not depend onthe heat treatment, as expected (see Fig. 6). Note that the temper-ature does not refer to the value at which microorganisms weregrown but to the intensity of the previous heat treatment. The kvalues for MF + LHT and for MF + IHHT were slightly lower whencompared to LHT and IHHT alone. However these differences be-tween MF + DHHT and the corresponding DHHT are negligible.Fig. 6 shows almost parallel lines with a different cut-off on they-axis (which means that the TBC at t = 0 days was clearly differ-ent); this is related to the lag time for each pasteurization treat-ment. As Ranieri et al. (2009) demonstrated, the pasteurizationprocess does not preferentially affect the microorganism popula-tion, and the parallel lines observed in this figure confirm this

statement. However, lower k values obtained for MF + LHT andfor MF + IHHT demonstrate that the removal of some of the bacte-ria present in raw milk leads to a lower microorganism growth rate(lower k values). The higher k values in the LHT and IHHT experi-ments mean that the rate of microorganism growth during storagewas higher. In the case of MF + LHT and MF + IHHT treatments, pre-vious removal of microorganisms probably included psychro-trophs, so growth was reduced in samples stored at 4–6 �C afterMF + LHT. On the contrary the differences in k values for DHHTand MF + DHHT treatments are very small. Time treatment in di-rect heating was 6 s (2 s in the rest of treatments) and psychro-trophs probably are destroyed in these treatments. k valuesduring milk storage at low and room temperatures are difficultto find in the literature.

Heat treatments without MF at temperatures higher than120 �C deactivated most of the psychrotolerant bacteria and micro-organism growth during storage and did not depend on the micro-filtration step, except that the lag time was longer after combinedtreatments.

The shelf life was longer for the DHHT and MF + DHHT treat-ments due to the longer duration of treatment (6 s vs. 2 s in thecase of indirect HT). The maximum shelf life obtained was 74 daysin the case of the MF + DHHT treatment between 120 and 130 �C.

Table 2 shows the logarithmic bacteria reduction (LBR) valuesfor each treatment. Pasteurization treatments (75 �C, 15 s) gavean LBR value of 1.8, and higher pasteurization temperature in-creased the LBR to 2.7 (at 90 �C). Combined MF + LHT increasedthe LBR value (between 4.1 and 5.1). Additionally, microfiltrationbefore heat treatment noticeably increased the LBR. Some pub-lished data gave LBR values between 2 and 4 for a 1.4 lm microfil-tration 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 micro-organisms studied (TBC, Bacillus cereus, spores, etc.). However,other published results (Schmidt et al., 2012) published higher val-

L. Fernández García, F.A. Riera Rodríguez / Journal of Food Engineering 128 (2014) 1–9 9

ues (between 5 and 6) after combined MF + pasteurization. Differ-ences between these results are due to the difficulty in performingexperiments under equivalent conditions, particularly regardingthe initial milk bacteria count and microfiltration conditions (espe-cially the membrane cut-off and temperature). Table 2 shows,however, reasonable LBR values when different treatments arecompared. MF treatment always increased the milk ESL. TheMF + IHHT treatment led to a higher LBR value, especially at lowertemperatures when compared with treatments without MF (4.8 at115 �C vs. 3.3 without the MF step). In the case of direct heat treat-ment, the LBR values were higher due to the longer duration oftreatment.

Finally, Fig. 9 shows the ESL of all the studied treatments.Important increases in shelf life were observed. Major effects ofMF were found with low heat treatment (the shelf life increasedby a factor of two or three at temperatures between 73 and90 �C). Note that the results shown in this figure are not fully com-parable because, in some cases, the milk was stored at 20 �C (treat-ments at high temperature) but, in spite of this, importantincreases in shelf life were found. The maximum milk shelf lifewas found for MF + DHHT (at 125–130 �C) with a treatment dura-tion of 6 s. The results shown in this figure provide a new way ofcombining ultrapasteurization processes with a microfiltrationstep to obtain defatted milk with more than two months of shelflife at room temperature. Extra efforts must be made to statisti-cally evaluate the organoleptic properties of the obtained productsas well as to follow proteolysis over time. These aspects were notthe objective of the present study.

5. Conclusions

MF has proven to be an adequate tool for the removal of bacte-ria in milk. The combination of this technology and a subsequentpasteurization treatment (73 �C for 15 s) has enabled the produc-tion of ESL milk with a lifetime close to 30 days (70% longer thanregular pasteurized milk). A shelf life longer than 21 days allowsthe distribution of this ESL milk together with other fresh dairyproducts such as yogurt and facilitates its arrival to markets. Treat-ment at higher temperature (always lower than 130 �C for 6 s) al-low extending the shelf life to more than 70 days, even whenmaintaining the product at ambient temperature. The kinetics ofmicroorganism growth show that the growth rate was similarregardless of the selected treatment, and that the microorganismgrowth lag time is the main reason for the increased shelf life.

Acknowledgements

The authors acknowledge the financial support from the Span-ish Ministry of Science and Innovation (Project AGL2007-63998/ALI). We would also like to thank FICYT (Fundación para el

Fomento en Asturias de la Investigación Científica Aplicada y laTecnología) for the grant of PhD studies of Leticia Fernández (BP08-050), and C.A.P.S.A. (Corporación Alimentaria Peñasanta S.A.)for its technical support for the experiments.

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