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

Accepted Manuscript Combination of microfiltration and heat treatment for ESL milk production: Impact on shelf life L. Fernández García, F.A. Riera Rodríguez PII: DOI: Reference:

S0260-8774(13)00597-9 http://dx.doi.org/10.1016/j.jfoodeng.2013.11.021 JFOE 7643

To appear in:

Journal of Food Engineering

Received Date: Revised Date: Accepted Date:

14 August 2013 11 October 2013 24 November 2013

Please cite this article as: Fernández García, L., Riera Rodríguez, F.A., Combination of microfiltration and heat treatment for ESL milk production: Impact on shelf life, Journal of Food Engineering (2013), doi: http://dx.doi.org/ 10.1016/j.jfoodeng.2013.11.021

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Combination of microfiltration and heat treatment for ESL milk production: Impact on shelf life L. Fernández García and F. A. Riera* Rodríguez Chemical Engineering and Environmental Technology Department, Faculty of Chemistry, University of Oviedo, Asturias, Spain * Corresponding author: C/Julián Clavería, 8, 33008. [email protected] Abstract Thermized defatted cow milk was submitted to different heat treatments (between 73 and 130ºC, 2 and 15 s) and combined with a microfiltration step (1.4 μm cut-off ceramic membrane) to study the influence 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 4-6ºC 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 microfiltration and direct heat treatment at 125-130ºC and storage at room temperature. An extended shelf life of 33 days was obtained after microfiltration followed by pasteurization at 90ºC and storage at 4-6ºC. Keywords: ESL, microfiltration, heat treatments List of symbols A: membrane area CFU: colony forming units CWF: membrane clean water flux DHHT: direct high heat treatment ESL: extended shelf life HT: Heat Treatment IHHT: indirect high heat treatment LBR: logarithmic bacteria reduction LHT: low temperature treatments MF: microfiltration N: Number of microorganisms a time t N0: Number of microorganisms a t=0 Qf: Membrane permeate flow rate (with water) TMP: transmembrane pressure TBC: total bacterial count RD: reduction degree μ: viscosity

1

1. Introduction The shelf life of milk is an important concept that defines the ability to widen the distribution chain of the product. As milk provides a favorable medium for spoilage microorganisms, pre-treatment 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 significant barrier to extending the shelf life of dairy products is the difficulty 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 Griffiths, 2006). Some of the possible ESL technologies are bactofugation (Giffel and van der Horst, 2004), pulsed electric fields (Barbosa-Cánovas et al., 1999), high pressure processing (Trujillo et al., 2002), high heat 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 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 confirmed by Olesen and Jensen (1989). MF led to a logarithmic bacteria reduction (LBR) of 4 for total bacteria and 2.3–3.7 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; Fernández et al., 2013). MF membranes with a pore size of about 1.4 µm can achieve the right balance between rejection of bacteria and longterm flux, 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 efficient 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 (72ºC, 15 s) skimmed milk with and without MF (1.4 and 0.8

2

μm) at different storage temperatures (0.1-6.1ºC), and obtained a maximum bacterial log reduction of 5.63 and 92 days of milk shelf life. Tomasula et al. (2011) microfiltered (0.8 μm) previously pasteurized milk (72ºC, 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 ultrahigh 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 130ºC) 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 50ºC 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 Figure 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 flows of cooling and heating fluids to adjust to the set point (V-1). The tangential flow rate in membrane channels was ensured by a flow rate frequency-regulated vertical multistage centrifugal 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 feeding pump.

3

Figure 1: Ceramic MF pilot plant piping and instrumentation diagram

Experimental data measurements were performed using electronic 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 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 flux. 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 Isoflux® ceramic membrane (Tami, France). The Isoflux® 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 flux along the length of the membrane element. This feature leads to improved performance of the membrane in terms of long-term flux 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 & Maubois, 2000).

4

The Isoflux® membrane chosen for this purpose had 23 channels with an internal diameter of 3.5 mm, a length of 1178 mm, a filtering area of 0.35 m2 and a pore size of 1.4 µm (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 75ºC, for 15 min without permeation (permeate valve closed) and 15 min with permeate flux followed by nitric acid (HNO3, 58% purity) at 1% (v/v) (Brenntag, Sevilla, Spain) at 50ºC. 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 first preheated and then directed to the main heating area. After the treatment, the product was cooled. The system was designed to work with a product flow rate of 20 L.h-1. The maintenance tube length was selected to fix the pasteurization step to 15 s and to 6 s for the indirect high heat treatments. 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 flow from 100 to 1,000 L.h-1 and pressures ranging from 2 to 10 bar, a flow meter and a plate heat exchanger for previous heating (at 80ºC). This plate heat exchanger had two bodies. The milk was first 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 first 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 flow provided by the positive pump. At the flash cooler, the product was cooled to 80ºC (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.

5

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. Finally, for both heat treatments studied, the product was collected in a laminar air flow 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 flow cabinet. 2.5. Operating procedure Water from the industry tap network was used to warm the previous membrane rig to 45ºC. 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 flux (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 × µ/(TMP × A), where Qf is the permeate water flow rate in L.h-1, µ is the water viscosity (1.0 cP at 20°C), and A is the membrane filtration 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 filled 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 flush the loop of the remaining water. The unit almost instantly reached 80% of the desired pressure and flow velocity and needed roughly 3 min to attain the desired values. Around 200 L were used for each experiment. 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 fixed at: v= 6m.s-1, TMP=0.55 bar and T=45ºC. Experiments showed no membrane fouling after 4 hours and the permeate flow 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% (Fernández, 2013). For the combination of MF and heat treatment trials, the process began with the collection of 200 L of skimmed milk at 50ºC 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 flow cabinet in sterile containers. 2. 6. Samples and analyses

6

Samples of the feeding solution, permeates and final product after heat treatments were aseptically withdrawn from all experiments. The final product samples were kept under refrigerated conditions (4ºC) in the case of heat treatment at 73-90ºC and at room temperature for the 115-130ºC 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 nearinfrared [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). 2.6.2. Bacterial analyses Indigenous microflora were monitored during MF. To enumerate the microflora in raw milk, skimmed milk, permeate and final 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 37ºC. 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 confirmed their removal from skim milk by MF using a 1.4 µm 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 hour 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. 2.6.3. Storage/shelf life studies Samples of the permeate (~80 mL) and final product were drawn after the MF and the heat treatment units at various times in all experiments in sterile specimen cups and kept at 46ºC or room temperature (18-20ºC) (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:

7

pH: between 6.6 and 6.8 Acidity < 18º Dornic TBC < 30000 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 (4-6ºC) 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 Figure 2. Milk 1 was a conventionally pasteurized skimmed milk (75ºC, 15 s) and Milk 2 was a commercial milk treated by an ultrapasteurization infusion process (135-140ºC; 0.2-1 s). Both were stored between 4 and 6ºC inside their original closed containers. Samples for analysis were obtained through a septum using a syringe.

45000

20

40000

18

35000

16 14

30000

12

25000

10

20000

8

15000

6

10000

4

5000

2

0

pH and Acidity (ºDornic)

TBC (CFU/m L)

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, 2006) have selected similar values (20,000 CFU.mL-1) according to the requirements for pasteurized milk.

Milk 1: TBC Milk 2: TBC Milk 1: pH Milk 2: pH Milk 1: Acidity Milk 2: Acidity

0 1

2

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17

Shelf life (days)

Figure 2. Titratable acidity, pH and total bacteria count (TBC) of two commercial milks during milk shelf life evaluations. Milk 1: Pasteurized milk (75ºC, 15 s). Milk 2: Ultrapasteurized milk (130-140ºC, 0.2-1 s). Both milks were stored at 4-6ºC.

The shelf life of different milk samples was estimated after different heat treatments (LHT: low heat treatment/pasteurization at 90, 80, 75 and 73ºC, 15 s; IHHT: indirect heat treatment at 130, 125, 120 and 115ºC, 2 s; DHHT: direct heat treatment at 130, 125, 120 8

and 115ºC, 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 Figures 3, 4 and 5. The dotted line represents the limit for the end of milk shelf life according to the TBC analysis.

40000 35000

MF+LHT, 90ºC

TBC (CFU/mL) MF+LHT, 80ºC

30000

MF+LHT, 75ºC 25000 MF+LHT, 73ºC 20000

LHT, 90ºC LHT, 80ºC

15000

LHT, 75ºC

10000

LHT, 73ºC 5000 0 0

5

10

15

20

25

30

35

Shelf life (days)

Figure 3. Microorganism growth after low temperature treatment (LHT) and combined microfiltration + low temperature treatment (MF+LHT). Heat treatment: 15 s. Storage temperature between 46ºC

40000 35000 TBC (CFU/mL)

IHHT, 130ºC

30000

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

MF+IHHT, 115ºC

15000

MF+IHHT, 130ºC MF+IHT 125ºC

10000

MF+IHT 120ºC 5000 MF+IHT 115ºC 0 0 10

20

30

40

50

60

70

Shelf life (days)

9

Figure 4. Microorganism growth after indirect high temperature treatment (IHHT) and combined microfiltration + indirect high temperature treatment (MF+IHHT). Heat treatment: 2 s. Storage temperature between 20-22ºC. 40000 35000 DDHT, 130ºC

TBC (CFU/mL) 30000

DDHT, 125ºC DDHT, 120ºC

25000

DDHT, 115ºC

20000

MF+DDHT, 130ºC 15000

MF+DDHT, 125ºC MF+DDHT, 120ºC

10000

MF+DDHT, 115ºC

5000 0 0

10

20

30

40

50

60

70

80

Shelf life (days)

Figure 5. Microorganism growth after direct high temperature treatments (DHHT) and combined microfiltration + direct high temperature treatment (MF+DDHT). Heat treatment: 6 s. Storage temperature between 20-22ºC

The microorganism growth followed first order kinetics, as can be seen in Figures 6, 7 and 8, in which Log (N / N 0 ) is represented against (t − t 0 ) 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 figures, the R2 values are greater than 0.98 in all experiments.

10

5.00 MF+LHT, 90ºC - R2=0.984

4.50 Log (TCB)

4.00

MF+LHT, 80ºC- R2=0.980

3.50

MF+LHT, 75ºC-R2=0.986

3.00

MF+LHT, 73ºC - R2=0.992

2.50 LHT, 90ºC - R2=0.996

2.00 1.50

LHT, 80ºC - R2=0.998

1.00

LHT, 75ºC - R2=0.990

0.50

LHT, 73ºC - R2=0.994

0.00 0

5

10

15

20

25

30

35

Shelf life (days)

Figure 6. Linearization of the first order equation dN/dt =kN for low heat treatment (LHT) and microfiltration follow by low heat treatment (MF+LHT). Heat treatment: 15 s. Storage temperature of 4-6 ºC. 5.00 IHHT, 130ºC - R2=0.989

4.50 Log (TBC)

4.00

IHHT, 125ºC - R2=0.986

3.50

IHHT, 120ºC - R2=0.987

3.00

IHHT, 115ºC - R2=0.982

2.50 MF+IHHT, 130ºC -R2=0.993

2.00

MF+IHHT, 125ºC - R2=0.986

1.50 1.00

MF+IHHT, 120ºC - R2=0.988

0.50

MF+IHHT, 115ºC - R2=0.992

0.00 0

20

40

60

80

Shelf life (days)

Figure 7. Linearization of the first order equation dN/dt =kN for indirect heat treatment (IHHT) and microfiltration follow by indirect heat treatment (MF+IHHT). Heat treatment: 2 s. Storage temperature: 20-22 ºC.

11

5.00

DHHT, 130ºC - R2=0.992

4.50

Log (TCB) 4.00

DHHT, 125ºC - R2=0.987

3.50

DHHT, 120ºC - R2=0.994

3.00

DHHT, 115ºC - R2=0.988

2.50

MF+DHHT, 130ºC - R2=0.996

2.00 1.50

MF+DHHT, 125ºC - R2=0.995

1.00

MF+DHHT, 120ºC - R2=0.986

0.50

MF+DHHT, 115ºC - R2=0.996

0.00 0

20

40

60

80

Shelf Life (days)

Figure 8. Linearization of the first order equation dN/dt =kN for direct heat treatment (DHHT) and microfiltration follow by direct heat treatment (MF+DHHT). Heat treatment: 6 s. Storage temperature: 20-22 ºC.

The slope of the straight lines in Figures 6, 7 and 8, that represent the growth kinetic constant during storage, are shown in Table 1. Treatments LHT* MF*+LHT IHHT** MF**+IHHT DHHT** MF**+DHHT

90ºC 0.5628 0.2943

80ºC 0.3985 0.2864

75ºC 0.4266 0.3195

73ºC 0.4006 0.3032

130ºC

125ºC

120ºC

115ºC

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

Table 1. Kinetic constants (day-1) of each treatment at different temperatures * Storage temperature between 4-6 ºC ** Storage temperature between 20-22ºC

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 final bacteria count after complete treatment. Treatment

LHT (90ºC) LHT (80ºC) LHT (75ºC) LHT (73ºC)

Logarithmic Bacteria reduction (LBR)

2.7 2.0 1.8 1.6

12

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

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

Table 2. Logarithmic bacteria reduction for all the treatments studied

Finally, the values of milk shelf life for all the treatments studied are represented in Figure 9. The values were obtained as the intersection between microorganism growth and established the TBC limit (30,000 CFU.mL-1). In the figure, the temperature in parentheses is the storage temperature.

13

80

LHT (73ºC) DHHT+MF (20ºC)

70

Shelf Life (days)

MF+LHT (73ºC) DHHT (20ºC)

IHHT (20ºC)

50

LHT (80ºC) LHT (90ºC)

IHHT+MF (20ºC)

60

LHT (75ºC)

MF+LHT (75ºC) MF+LHT (80ºC) MF+LHT (90ºC) IHHT (115ºC) IHHT (120ºC) IHHT (125ºC)

40

IHHT (130ºC)

MF+LHT (4-6ºC)

MF+IHHT (115ºC) MF+IHHT (120ºC)

30

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

20

DHHT (120ºC) DHHT (125ºC) LHT (4-6ºC)

DHHT (130ºC)

10

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

0 Treatments

MF+DHHT (130ºC)

Figure 9. Milk shelf life for all the treatments studied. Storage temperatures are in parentheses.

4. Discussion In Figure 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 135-140ºC for 0.2-1 s), and could duplicate the shelf life of pasteurized milk life, reaching between 16-17 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

14

temperature treatment (135-140ºC) was due to the short duration of treatment. For the following experiments, TBC was selected as the parameter to estimate the milk shelf life. Figures 3, 4 and 5 show the effect of combination of MF + heat treatment (HT) on the shelf life as well as the influence of different heat treatments alone. At low temperatures (LHT and MF+LHT) (Figure 3), the differences in shelf life were considerable (6-9 days vs. 2633 days), and the growth curves of both groups of treatments were well different. Schmidt et al. (2012) studied combination of MF + pasteurization (77ºC, 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 8ºC), and using raw milk with similar initial microorganism counts as in this work. Elwell & Barbano (2006) published shelf life values between 16 days (storage at 6.1ºC) and 68 days (with storage at 0.1ºC). In this work, the effect of heat treatment on shelf life was clear between 75 and 80ºC and differences between 80 and 90ºC were almost negligible (as well as between 75 and 73ºC, 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 75ºC and greater than 18 days for treatment at 80 and 90ºC. 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 (Figure 4) curve shapes were similar, with a longer lag time (around 35 days for MF+IHHT at 130ºC) 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 first days at the lowest temperature studied (115ºC) was similar to those obtained with IHHT at the highest temperature studied (130ºC). After this time, the growth curves were similar. Combined methods provided a longer shelf life (between 42 and 50 days against 58-65 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 (20-22ºC). Milk shelf life is strongly affected by storage temperature, as it was stated by other authors (Schmidt et al. 2012; Tomasula, et al., 2011; Raneri, et al., 2009) and changes of a few degrees in the storage temperature lead to important differences in shelf life, so a comparison with published data must be done carefully. Figure 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 Figure 9 later on). Around 20 days of extra life can be obtained by increasing the treatment temperature by 15ºC. 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 microfiltration stage allows for reducing the temperature common in UHT processes by more than 20ºC in (around 150ºC in most of the milk industry), which leads to better milk quality.

15

Figures 6, 7 and 8 show lineal behaviour when plotting log TBC vs. shelf life, what demonstrates that microorganisms growth follow a first 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 Figure 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. Figure 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 figure confirm 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 4-6ºC 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 seconds (2 seconds in the rest of treatments) and psychrotrophs probably are destroyed in these treatments. k values during milk storage at low and room temperatures are difficult to find in the literature. Heat treatments without MF at temperatures higher than 120ºC deactivated most of the psychrotolerant bacteria and microorganism growth during storage and did not depend on the microfiltration 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 130ºC. Table 2 shows the logarithmic bacteria reduction (LBR) values for each treatment. Pasteurization treatments (75ºC, 15 s) gave an LBR value of 1.8, and higher pasteurization temperature increased the LBR to 2.7 (at 90ºC). Combined MF+LHT increased the LBR value (between 4.1 and 5.1). Additionally, microfiltration before heat treatment noticeably increased the LBR. Some published data gave LBR values between 2 and 4 for a 1.4 μm microfiltration 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 values (between 5 and 6) after combined MF + pasteurization. Differences between these results are due to the difficulty in performing experiments under equivalent conditions, particularly regarding the initial milk bacteria count and microfiltration conditions (especially the membrane cut-off and temperature). Table 2 shows, however, reasonable LBR values when different

16

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 115ºC 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, Figure 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 90ºC). Note that the results shown in this figure are not fully comparable because, in some cases, the milk was stored at 20ºC (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 125130ºC) with a treatment duration of 6 s. The results shown in this figure provide a new way of combining ultrapasteurization processes with a microfiltration 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. 4. 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 (73ºC 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 130ºC 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 financial support from the Spanish 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 la Tecnología) for the grant of PhD studies of Leticia Fernández (BP 08-050), and C.A.P.S.A. (Corporación Alimentaria Peñasanta S. A.) for its technical support for the experiments. References Barbosa-Cánovas, G.V., Palou, E., Pothekemury, U., & Swamson, B.G. (1999). Conservación no térmica de alimentos. Acribia. Zaragoza. Spain. Brans, G., Schröen, C.G.P.H., van der Sman, R.G.M. & Boom, R.M. (2004). Membrane fractionation of milk: state of the art and challenges. Journal of Membrane Science, 243, 263–272. 17

Elwell, M.W., & Barbano, D.M. (2006). Use of microfiltration to improve fluid milk quality. Journal of Dairy Science, 89, 20-30. Fernandez, L., Álvarez, S. & Riera, F.A. (2013). Microfiltration applied to dairy streams: removal of bacteria. Journal of Food Science and Agriculture, 93,187-196. Fredsted, L.B., Rysstad, G. & Eie, T. (1996). Pure-Lac™: the new milk with protected freshness and extended shelf life. Proceedings of the IDF Symposium 1995: Heat Treatments and Alternative Methods, pp. 104–125. Brussels, Belgium. Fritsch, J. & Moraru, C.I. (2008). Development and optimization of carbon dioxide-aided cold microfiltration process for the physical removal of microorganisms and somatic cells from skim milk. Journal of Dairy Science, 91, 3744-3760. Giffel, M.C. & van der Horst, H.C. (2004). Comparison between bactofugation and microfiltration regarding efficiency of somatic cell and bacteria removal. Bulletin of the International Dairy Federation 389, 49–53. Goff, H.D. & Griffiths, M.W. (2006). Major advances in fresh milk and milk products: Fluid Milk Products and Frozen Desserts. J. Dairy Science, 89, 1163-1173. Grangeon, A., Lescoche, P. & Millares, M. (2000). Isoflux® membrane: the microfiltration mastering. 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-flow velocity microfiltration of skim milk for removal of bacterial spores. International Dairy Journal, 7, 849–861. International Dairy Federation (IDF 220) (2010). (ISO 29981:2010) Milk products-colony count technique at 37ºC. 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 microfiltration. J. Membrane Science, 274, 75-78 Olesen, N. & Jensen, F. (1989). Microfiltration. The influence of operation parameters on the process. Milchwissenshaft, 44, 476–479. Pafylias, I., Cheryan, M., Mehaia, M.A. & Saglam, N. (1996). Microfiltration of milk with ceramic membranes. Food Research international, 29,141-146. 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 fluid milk. J. Dairy Sci., 92, 4823-4832. 18

Saboya, L.V. & Maubois, J-L. (2000). Current developments of microfiltration technology in the dairy industry. Lait, 80, 541–553. Schmidt, V.S.J., Kaufmann, V., Kulozik, U., Scherer, S., & Wenning, M. (2012). Microbial biodiversity, quality and shelf life of microfiltered and pasteurised extended shelf life (ESL) milk from Germany, Australia and Switzerland. Int. J. of Food Microbiology, 154, 19. Skrzypek, M. & Burger, M. (2010). Isoflux® ceramic membranes – practical experiences in dairy industry. Desalination, 250,1095–1100. Tomasula, P. M., Mukhopadhyay, S. Datta, N, Porto-Fett, A, Call, J.E., Luchansky, J.B., Renye, J. (2011). Pilot-scale crossflow-microfiltration 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 Science and Emerging Technologies, 3, 295-307. List of captions Figure 1: Ceramic MF pilot plant piping and instrumentation diagram Figure 2. Titrable acidity, pH and total bacteria count (TBC) of two commercial milks during milk life. Milk 1 is a pasteurised milk (75ºC, 15 s) and Milk 2 is a ultrapasteurised milk (130-140ºC, 0.2-1 s). Both milks were stored at 4-6ºC. Figure 3. Microorganisms growth after low temperature treatments (LHT) and combined Microfiltration + low temperature treatment (LHT+MF). Heat treatment: 15 seconds. Storage temperature between 4-6ºC. Figure 4. Microorganisms growth after indirect high temperature treatments (IHHT) and combined Microfiltration + indirect high temperature treatment (IHHT+MF). Heat treatment: 2 seconds Storage temperature between 20-22ºC. Figure 5. Microorganisms growth after direct high temperature treatments (DHHT: T, 6 seconds) and combined Microfiltration + direct high temperature treatment (DHHT+MF). Heat treatment: 6 seconds Storage temperature between 20-22ºC. Figure 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 seconds. Storage temperature of 4-6 ºC. Figure 7. Linealization of the first order equation dN/dt = kN for indirect heat treatments (IHHT) and microfiltration follow by indirect heat treatment (IHHT+MF). Heat treatment: 2 seconds Storage temperature: 20-22 ºC. 19

Figure 8. Linealization of the first order equation dN/dt = kN for direct heat treatments (DHHT) and microfiltration follow by direct heat treatment (DHHT+MF). Heat treatment: 6 seconds. Storage temperature: 20-22 ºC. Figure 9. Milk shelf life for all the treatments studied. Storage temperatures between brackets.

Highlights Raw defatted milk was treated by combination of microfiltration (1.4µm) and heat treatments. Maximum ESL of 74 days (CFU/ml <30 000) was found after microfiltration and heat treatment of 125-130 ºC, 6 seconds. Estimated kinetic constants do not depend on the previous low heat treatments.

20