The Efect Of High Pressure Homogenisation On Raw Bovine Milk Properties.

(1)

THE EFECT OF HIGH PRESSURE HOMOGENISATION ON RAW

BOVINE MILK PROPERTIES

YANA CAHYANA, STP.DEA

DEPARTEMENT OF FOOD INDUSTRIAL TECHNOLOGY FACULTY OF AGRICULTURAL INDUSTRIAL TECHNOLOGY

PADJADJARAN UNIVERSITY

DIPRESENTASIKAN PADA SEMINAR ILMIAH DIES NATALIS UNPAD 2008

(2)

PREFACE

This paper dealing with one of the emerging technologies in food science technology was presented in scientific seminar in dies natalis of Padjadjaran University (UNPAD) in 2008. The results and discussion provided in the paper were taken from the research the author conducted during his master’s degree.

Obviously, there are some excellent reference works in food science technology. However, the scarcity of references on emerging technology especially on high pressure homogenisation and its application in food displayed in the library of UNPAD is still apparent. The author hopes the paper will contribute as a valuable resource for either students or lecturers attracted in the field of the application of high pressure homogenisation in food technology.

Some parts of the paper were deliberately changed, especially regarding the format of the paper, and consequently have been slightly different from the original paper presented in the seminar. The editing was inevitable in order to be in line with the rule of DIKTI as a prerequisite to be assessed as a reliable paper.

The author would like to thank to Prof J.C Cheftel and Dr Eliane Dumay at Université Montpellier 2, France for the supervision and discussion they devoted during the author’s research.

Jatinangor, October 2008

(3)

ABSTRACT

The treatment on raw bovine milk by high pressure homogenisation (HPH) at 100-300 MPa was performed. The mean diameter of milk fat globules lowered when the homogenisation pressure increased. At 100 MPa of pressure, the size of milk fat globules reduced to a great extent, 6 times smaller (d4,3 = 0,61 µm) than that of unhomogenised milk. Furthermore, homogenisation at 300 MPa resulted in the smallest size of milk fat globules (0,19 µm).

For the pressure at 100 and 200 MPa, the use of secondary valve at a pressure equal to 10% of pressure at primary valve decreased significantly the average diameter of fat globules compared to that solely treated by primary valve. Running the milk through homogeniser for 2-3 times of pass (recycling) conducted at 200 MPa (primary valve) and 20 MPa (secondary valve) lowered more significantly fat globule size.

The possibility of alkaline phosphatase as an indice of adequate HPH treatment was investigated as well. The enzyme was potential as the indice of adequate HPH treatment since it was entirely inactivated by HPH treatment at an inlet temperature about 24°C.

(4)

INTRODUCTION

Heat treatment is one of the most commonly used preservation treatment for food and other perishable products, but it cannot be used on heat-labile compounds or heat labile dispersion. Therefore, over the last 10 years, considerable research efforts have been directed towards the development of novel nonthermal process for preservation, such as the use of high hydrostatic pressure, pulsed electric field, ultraviolet light, pulsed light and high pressure homogenisation (Diels et al, 2003). The latter, high pressure homogenisation (HPH) at 100-500MPa, has been considered to be one of the most promising methods for the food treatment and preservation at room temperature and of the great concern since the nutritional and sensory qualities of foods such as nutrient retention, flavour and colour are generally not adversely affected by the process.

Homogenisation was first presented by Auguste Gaulin at the Paris World Fair in 1990. Since then, homogenisation has been introduced to the food, pharmaceutical and cosmetic industries to disperse non-miscible phases, to stabilise emulsions and/or to prepare products with appropriate rheological properties. Homogenisation is of special interest in the dairy industry to reduce the size of fat globules in milk and cream (from 1-8µm in raw milk to 0.3-0.8µm in homogenised milk) and to prevent creaming and coalescence during long shelf storage (Walstra& Jeness, 1984).

Over the years, homogenisation technology has been evolved; the demand for longer shelf life and products with better stability has led to new developments. For products such as aseptic UHT milk and cream or baby food formulas requiring a shelf life up to 6-12 months, higher pressures or double homogenisations have been successful in stabilizing these emulsion systems. However, these homogenisation processes employ pressures of less than 50 MPa (Paquin, 1999).

In classical homogenisation processes, milk heated at 60°-70°C is forced under moderate pressure (20-50MPa) and high velocity through a narrow opening (homogenisation valve). Turbulence, shear and cavitation are the main physical causes for fat globule disruption. Pressure generated-energy transmitted to the fluid is mainly converted into heat as it leaves the valve, raising the milk temperature by 2.5°C per 10 MPa in the case of moderate pressure homogenisers (Walstra& Jeness, 1984).

More recently high pressure homogenisation (HPH) has been developed, which is based on the same design principle as conventional/or moderate pressure homogenisation but works at significantly higher pressures (100-500MPa). High pressure homogenisation (also

(5)

known as dynamic high pressure) is different from high hydrostatic pressure. In high pressure homogenisation, the exposure time is in the order of seconds or less, while in high hydrostatic pressure treatment it is in the order of minutes or more. Furthermore, high pressure homogenisation treated-products are exposed to hydrodynamic cavitations, impingement against static surfaces, high turbulence and fluid shear. These are not the case in high hydrostatic pressure treatment.

High pressure homogenisation (HPH) is used in the pharmaceutical, cosmetic, chemical and food industries for the preparation or stabilization of emulsions and suspensions, or for creating physical changes, such as viscosity changes in product (Wuytack, 2002; Popper & Knorr, 1990). Another application is cell disruption of yeasts or bacteria in order to release intracellular products such as recombinant proteins (Middelberg, 1995). The fact that HPH is capable to disrupt cell has led to various discoveries reporting that HPH could inactivate mikroorganism, indicating a potential application of HPH in food preservation after being pasteurised using HPH.

This potential application of HPH necessities an indicator showing that HPH treated- products are safe to consume. The indicator in question must be easy, rapid and simple to carry out.

The aim of the present study was to investigate the feasibility of the use of a milk indigenous enzyme (alkaline phosphatase) as indice of adequate HPH and to elucidate the change of milk properties especially milk fat globule diameter.

(6)

MATERIAL AND METHODS

2.1. Milk Supply

Fresh raw whole bovine milk was purchased in a local dairy farm. Milk containers were transported from the farm to the laboratory in less than 1h in refrigerated conditions (milk temperature at arrival < 10°C), and were then stored at 4°C for maximum of 24h before HPH. Milk pH ranged between 6.6 and 6.7. The milk was equilibrated at 24°C for 40-50 min before HPH.

2.2. High Pressure Homogenisation

Homogenisation of whole raw milk was performed with Stansted HPH (model FPG7400 H, Stansted Fluid Power Ltd., Essex, UK) shown in Fig.1.

Fig. 1. Schematic representation of Stansted high pressure homogenisation: Tin, milk inlet temperature;

T1/P1, temperature and pressure probes before the HP valpe; T2/P2, temperature and pressure probes at the HP valve outlet; T3 and T4, temperature probes before and after the spiral heat exchanger

This machine comprises a High Pressure (HP) valve made of HP resistant ceramics, and is able to support up to 350 MPa of pressure. The HP system consists of a single intensifier (80 mL useful volume), driven by a hydraulic pump. The operating cycle comprises 2 steps. The first one (9s) corresponds to the filling of the intensifier. The other (10 s) corresponds to the pressure build-up and to the discharge of the intensifier. The pulsed flow rate depends on the homogenisation pressure and ranges from 16.0 Lh-1 at 100 MPa to 13.6 Lh-1 at 300 MPa. To avoid a loss of homogenisation performance due to a temperature increase in the first stage valve, the second stage valve is cooled by circulating water at 18°C in an external jacket built

Spiral heat Exchanger T4

T1/P1 T2 / P2

HP Valve (1er_effect)

Tin

Intensifier

HP Pump

HP Valve (2nd effect) Feed Tank

(7)

aroud the valve. The pressures before the HP valve (P1) and after the HP valve(P2) were measured using two pressure gauges. The milk inlet temperature (Tin) measured with a thermistance (Checktemp, Hanna Instruments, Tanneries, France, ± 0.2°C accuracy) in the feeding tank were 24.0 ± 0.2°C. The fluid temperatures before the HP valve (T1), immediately after the HP valve (T2), as well as the temperature before the cooling device (T3) was measured with T-thermocouples of low inertia (1.5 mm diam.; ± 1°C accuracy). Temperature and pressure were displayed on digital indicators. The homogenised milk was rapidly cooled to 14.7 ± 1.7°C (T4) by passing through a spiral type heat exchanger located after the HP valve and cooled to 10°C using an external cryostat. Milk samples were stored at 4°C for 18 h before fat globule size determination. Each HP experiment was repeated three times.

2.3 Determination of fat globule size

The size distribution of fat globules in untreated (control) and processsed milk samples was determined with a Mastersizer 2000 laser diffractometer (Malvern instruments, Malvern, UK) equipped with He-Ne laser (λ =633). All milk samples were first diluted to 1/10 (v/v) in deionised water or in two different dissociation solution for 1 h to disrupt clusters of fat and/or casein micelles (Robin & Paquin, 1991; Strawbridged, Ray, Hallet, Tosh, & Dalgleish, 1995): solution 1 contained 5 gL-1 sodium dodecyl sulphate (L-4509, sigma), and solution 2 contained 8 M urea plus 50mM EDTA, pH 7.0. The diluted samples were then diluted again to 1/1000 (v/v) in deionised water in the diffractometer cell under moderate stirring at 20°C. The refractive index of the fat globule and of the dispersant (water) at 25°C was 1.452 and 1.33, respectively. The absorbance of fat particles was taken as 0.01. It was checked that absorbance values between 0.001 and 0.01 did not significantly influence the determination of fat globule size. It was also checked that the presence of 8M urea did not change the refractive index of the dispersant when diluted to 1/1000.

The size of milk fat globule was characterised by diameter of d4,3, defined as

nidi4/nidi3 where ni is the number of fat globules of diameter di.

2.4. Alkaline phosphatase (ALP) inactivation

Proper pasteurisation is a critical step in the manufacture of safe and high-quality dairy products. Thus, the presence of an indicator showing that a pasteurisation has been properly carried out needs to be developed. As an indigenous enzyme in milk, Bovine alkaline phosphatase (ALP) has a slightly higher resistance to heat than non spore-forming pathogens. Reportedly, several time/temperature relationships for processing, based upon ALP

(8)

inactivation, ensure pathogen-free milk. Lack of ALP activity is the critical determinant of pasteurisation adequacy (Angelino, et al, 1999). In this context, it is interesting to investigate the potentiality of using ALP as high pressure homogenisation (HPH) adequacy. One of the most widely used methods to quantify residual ALP activity is flourometric method.

2.4.1. Principle

ALP activity in fluid dairy products is measured by continous fluorometric direct kinetic assay. A nonfluoresent aromatic monophosphoric ester substrate called Fluorophos® undergoes hydrolysis of its phosphate radical and is converted to a highly fluorescent product, Fluoroyellow® (Rocco, 1990). ALP is measured at 38°C during 3 minutes read time.

2.4.2. Assessment of alkaline phosphatase (ALP) inactivation

Two mL of substrat (Fluorophos) was dispensed into glass cuvettes and pre-incubated at 38°C for 5 min. A 0.075mL milk sample was added to pre-warmed substrat, mixed by inversion, and placed into the fluorometer (fluorimeter Cary Eclipse, Murgrave, Australie). The kinetic increase in fluorescence was monitored for 2 to 3 min and the average increase in fluorescence per min was obtained. Results were reported in intensity of fluorescence (% IF).

2.4.3 Heat treatment

The laboratory heat treatments were performed using a stainless steel coil (2 mm x 183 mm). Raw milk samples were pumped through the coil at such rate that the residence time of milk was 4 or 15 seconds. The coil was immersed in a water bath equilibrated at different temperature (15-87°C)

(9)

RESULTS AND DISCUSSION

Milk fat globule size

Fat globule size of milk samples treated by HPH is shown in Table 1. The data of milk fat diameter prepared in the table can be divided into 4 groups namely fat globule diameter of control milk, that of homogenised milk with neither recycling nor secondary valve, that of homogenised milk using both primary and secondary valve but no recycling, and that of homogenised milk using both primary and secondary valve as well as recycling. As expected, it can be immediately noticed that the increase in the pressure of homogenisation led to a decrease in the mean diameter of milk fat globules. Milk homogenised at 100 MPa (with neither recycling nor secondary valve) resulted in average diameters of milk fat globules about 0,612 ± 0,004 µm, 6 folds smaller than those of control milk (untreated milk).

Furthermore, homogenisation at 300 MPa of pressure (with neither recycling nor secondary valve) lowered fat globules size to smallest one compared to others. Tested by t student showed that all fat globule diameters of homogenised milk were significantly smaller than those of untreated milk (control milk) and significantly different among them (P<0,05).

Further study on the effect of secondary valve on fat globule diameter reveals that except for milk treated at 300MPa, the use of secondary valve (low pressure valve) having a pressure value equal to 10% of the pressure at primary valve (high pressure valve) resulted in reduction of globule sizes compared to those of milk treated by only primary valve. The increase in homogenisation pressure also reduced the milk fat diameter. However, at pressure of 300 MPa, the fat globule diameter of milk homogenised by only primary valve showed significantly smaller than that homogenised by both primary and secondary valve, hence the ineffectiveness of the treatment at 300 MPa in reduction of fat globule size using secondary valve. For this reason, the further study on the effect of recycling on fat globules diameters was limited at pressure of 200 MPa.

The treatment of recycling milk reveals that the recycling for two-stage or three –stage led to a smaller fat globule size compared to that treated by single-stage at the same pressure. This fact demonstrates that the recycling was an effective way to reduce fat globules diameter. The reduction in the fat globule diameter by recycling seemingly stemmed from a decrease in re-coalescence of a number of broken uncoated fat globules.

(10)

Table 1. Effect of homogenisation pressure on the size of fat globules in raw bovine milk

Pression (MPa)

Traitement _{Primary Valve} _{Secondary Valve} d4,3 (µm)

Control 3,836 (0,001)a

100 0,612 (0,004)b

150 0,410 (0,001)c

200 0,251 (0,001)d

250 0,216 (0,001)e

Homogenisation without recycling (Primary Valve)

300

~ 1-3

0,194 (0,000)f

100 10 0,515 (0,002)

200 20 0,236 (0,001)

Homogenisation without recycling (Primary and

secondary Valve) 300 30 0,198 (0,001)

Single stage

200 20 0,233 (0,001)x

Two-stage

200 20 0,170 (0,000)y

Three-stage Homogenisation

with recycling (Primary and secondary Valve)

200 20 0,157 (0,000)z

a-f

:significantly different values among the data for P<0,05

x,y,z

(11)

Effect of HPH on alkaline phosphatase inactivati

Inactivation of raw bovine milk alkaline phosphatase by heat treatment is presented at Fig 2. The result showed that the enzyme commenced to be inactivated at the temperatures more than 60°C and was entirely inactivated by heat treatment at 75°C for 15 seconds, while the heat treatment of 4 seconds required a temperature of at least 80°C in order to totally inactivate alkaline phosphatase. The enzyme also lost its bioactivity when the temperature reached about 70°C. This result demonstrated that the higher the temperature, the shorter the time required to completely inactivate the enzyme.

Fig.2. Alkaline phosphatase activity reported as relatif intensity of fluorescence (IF,%) as a function of temperature of heat treatment, for holding time of 4 (•) or 15 (∆) seconds.

Fig 3 depicted alkaline phosphatase inactivation by HPH. The result exhibited that HPH commenced to inactivate alkaline phosphatase at pressures > 200 MPa, corresponding to temperatures > 60°C. Bear in mind that HHP treatment causes a temperature increase at primary valve (high pressure valve). The temperature increase correlates well to the pressure increase. For milk inlet temperature (T1) about 24°C, it is known that T2 = 0,18P1 + 24,2 where r 2 = 0,9942, T2 = outlet temperature at primary valve (°C), P1= pression (MPa). The Fig 3 allows demonstrating the alkaline phosphatase inactivation as a function of HPH temperature and pression. From the equation, we are therefore capable to predict the required homogenisation pressure to achieve the temperature about 75-80°C. These temperatures correspond to pressures about 282-310 MPa. Theses pressures are believed to be capable to inactivate alkaline phosphatase by assuming that the only temperature plays a major role in alkaline phosphatase inactivation. In fact, as shown fig 2, inactivation of alkaline phosphatase was apparent at about 60°C, and at about 75-80°C for entire inactivation. The results suggest

0 20 40 60 80 100 120

25 35 45 55 65 75 85 95

Température (°C)

IF (%

)

15 s 4 s

(12)

that the temperature play a pivotal role in inactivating alkaline phosphatase by HPH treatment compared to the effect of shear and cavitation.

Fig 3. Inactivation of alkaline phosphatase as a function of homogenisation.

The result lead to a conclusion that HPH treatment at about 300 MPa inactivate entirely alkaline phosphatase on condition that the milk residence time in HPH be about 4 secondes. Furthermore, the result suggests that the use of alkaline phosphatase as indice of adequate HPH is potential. However, this conclusion requires another research on pathogenic microbial inactivation by HPH.

T2 = 0,18 P1+ 24,2

r2_{= 0,9942}

0 20 40 60 80 100 120

0 50 100 150 200 250 300 350

Pression (MPa) 0 10 20 30 40 50 60 70 80 90 Tem péra ture T 2 ( °C) IF (% ) Temper ature T2 (°C)

(13)

CONCLUSION

HHP treatment on raw bovine milk resulted in reduction of milk fat globule size. The use of secondary valve as well as recycling treatment influences the reduction of milk fat globule size. HPH at 300 MPa inactivate alkaline phosphatase of milk at inlet temperature about 24°C. The use of alkaline phosphatase as indice of adequate HPH is potential on condition that we could demonstrate that milk pathogenic microorganism is also inactivated by the same type HPH at the same pressure.

For the future research, it would be interesting to determine the possible effect of shear force and of cavitation on enzyme inactivation compared to the effect of temperature generated by the process.

(14)

REFERENCES

Angelino, P.D., Christen, G.L., Penfield, M.P., Beattie, S. 1999. Residual alkaline phosphatase activity in pasteurized milk heated at various temperatures-measurement with the fluorophos and scharer rapid phosphatase tests. Journal of food protection. 62 (1), 81-85 Cahyana, Y. 2004. Homogéneisation haut pression: a recherche d’indicateurs de traitement. Master’s thesis. Université Montpellier 2. France

Diels, AM.J., Wuytack, A.Y., Michiels C.W. 2003. Modelling inactivation of Staphylococcus aureus and Yersinia enterocolitica by high-pressure homogenisation at different temperatures. International journal of food microbiology. 87, 55-62

Middelberg, A. 1995. Process-scale disruption of microorganisms. Biotechnology Advances, 13 (3), 491-551.

Paquin, P. 1999. Technological properties of high pressure homogenizers: the effect of fat globules, milk proteins, and polysaccharides. International dairy journal. 9, 329-335.

Popper, L., Knorr, D., 1990. Application of high-pressure homogenization for food preservation. Food Technol. 44,84-89

Rocco, R.M. 1990. Fluorometric analysis of alkaline phosphatse in fluid dairy milk products. Journal of food protection. 53 (7), 588-591.

Walstra, P., & Jenness, R. 1984. Milk fat globules . In P Walstra & R Jenness (Eds), Dairy chemistry & physics (pp 254-278).New York: Wiley

Wuytack, E.Y., Diels, A.M.J., Michiels, A.W. 2002. Bacterial inactivation by high-pressure homogenisation and high hydrostatic pressure. International journal of food microbiology. 77, 205-212.

(1)

RESULTS AND DISCUSSION

Milk fat globule size

(2)

Table 1. Effect of homogenisation pressure on the size of fat globules in raw bovine milk Pression (MPa)

Traitement _{Primary Valve} _{Secondary Valve} d4,3 (µm)

Control 3,836 (0,001)a

100 0,612 (0,004)b

150 0,410 (0,001)c

200 0,251 (0,001)d

250 0,216 (0,001)e

Homogenisation without recycling (Primary Valve)

300

~ 1-3

0,194 (0,000)f

100 10 0,515 (0,002)

200 20 0,236 (0,001)

Homogenisation without recycling (Primary and

secondary Valve) 300 30 0,198 (0,001)

Single stage

200 20 0,233 (0,001)x

Two-stage

200 20 0,170 (0,000)y

Three-stage Homogenisation

with recycling (Primary and secondary Valve)

200 20 0,157 (0,000)z

a-f

:significantly different values among the data for P<0,05 x,y,z

(3)

Effect of HPH on alkaline phosphatase inactivati

Fig.2. Alkaline phosphatase activity reported as relatif intensity of fluorescence (IF,%) as a function of temperature of heat treatment, for holding time of 4 (•) or 15 (∆) seconds.

0 20 40 60 80 100 120

25 35 45 55 65 75 85 95

Température (°C)

IF (%

)

15 s 4 s

(4)

that the temperature play a pivotal role in inactivating alkaline phosphatase by HPH treatment compared to the effect of shear and cavitation.

Fig 3. Inactivation of alkaline phosphatase as a function of homogenisation.

T2 = 0,18 P1+ 24,2

r2_{= 0,9942}

0 20 40 60 80 100 120

0 50 100 150 200 250 300 350

Pression (MPa)

0 10 20 30 40 50 60 70 80 90

Tem

péra

ture

(

°C)

IF (%

)

Temper

ature

(5)

CONCLUSION

(6)

REFERENCES

Cahyana, Y. 2004. Homogéneisation haut pression: a recherche d’indicateurs de traitement. Master’s thesis. Université Montpellier 2. France

Middelberg, A. 1995. Process-scale disruption of microorganisms. Biotechnology Advances, 13 (3), 491-551.

Paquin, P. 1999. Technological properties of high pressure homogenizers: the effect of fat globules, milk proteins, and polysaccharides. International dairy journal. 9, 329-335.

Popper, L., Knorr, D., 1990. Application of high-pressure homogenization for food preservation. Food Technol. 44,84-89

Rocco, R.M. 1990. Fluorometric analysis of alkaline phosphatse in fluid dairy milk products. Journal of food protection. 53 (7), 588-591.

Walstra, P., & Jenness, R. 1984. Milk fat globules . In P Walstra & R Jenness (Eds), Dairy chemistry & physics (pp 254-278).New York: Wiley

Wuytack, E.Y., Diels, A.M.J., Michiels, A.W. 2002. Bacterial inactivation by high-pressure homogenisation and high hydrostatic pressure. International journal of food microbiology. 77, 205-212.

The Efect Of High Pressure Homogenisation On Raw Bovine Milk Properties.