2nd Meet Hpressure

�

Process Optimisation andMinimal Processing of Foods

Proceedings of the second main meeting

European CommissionCOPERNICUS PROGRAMME

Concerted action CIPA-CT94-0195

Warsaw Agricultural University, Warsaw, Poland, December 1996

Editors : Jorge C. Oliveira and Dietrich KnorrProject Coordinator : Fernanda A. R. Oliveira Area leader : Dietrich Knorr

Volume 4: High Pressure

Proceedings of the second project workshop

The proceedings of the second workshop organised by the COPERNICUS concerted action Process

Optimisation and Minimal Processing of Foods in December 1996 at the Agricultural University of

Warsaw, Poland, consist of five booklets, one for each project area:

¥ Thermal Processing

¥ Freezing

¥ Drying

¥ High Pressure

¥ Minimal and Combined Processes

Each booklet includes all communications that were presented at the meeting, either orally or in

poster, and later forwarded by the authors as written text, plus the questions and answers that

were recorded. As with the set of booklets related to the first meeting, some editorial effort was

put into trying to harmonise the written style and improve the readability of the texts, but no

scientific reviewing was performed.

The third and final project meeting will lead to a third set of booklets. A book including selected

papers is also being prepared, to be published by a professional scientific publisher.

We would like to thank all participants that have generously contributed to making the project

meetings very successful and lively, and particularly to those that have taken the effort to present

oral or poster communications with accompanying written texts. This effort has allowed the

project to gather a very good set of disseminating materials and we hope that in this way the

project results can be of better use to all partners and to a wider audience.

Porto, October 5th, 1997Fernanda A. R. Oliveira

Jorge C. OliveiraDietrich Knorr

i

Foreword

High PressureProceedings of the second project workshop

iii

Table of Contents

S. Denys, A. Van Loey, S. De Cordt, M. Hendrickx and P. Tobback 1

Modeling Heat Transfer During High Pressure Freezing and Thawing

L. Ludikhuyze, C. Weemaes, I. Van den Broeck, S. De Cordt, M. Hendrickx and P. Tobback 5

Modelling Thermal and Pressure-Temperature Inactivation

of Bacillus Subtilis α-Amylase

C. Weemaes, L. Ludikhuyze, I. Van den Broeck, M. Hendrickx and P. Tobback 10

Inactivation of Mushroom PPO by Pressure and/or Temperature:

Influence of pH and Inhibitors

V. Heinz and D. Knorr 15

High Pressure Inactivation and Germination Kinetics of Spore-Forming Bacteria

D. Brùna, M. Voldrich, M. Marek and J. Kamarád 19

Effect of High Pressure Treatment on the Patulin Content of Apple Concentrate

P. Butz, A. Fernandez, H. Fister and B. Tauscher 23

Influence of High Hydrostatic Pressure on Dipeptides

G. van Almsick, R. Eiche and H. Ludwig 26

High Pressure Inactivation of Hyphae, Condiospores and Ascospores

of the Fungus Eurotium Repens

A. Hernández and M. Pilar Cano 32

High Pressure and Temperature Effects on Enzyme Inactivation in Tomato Puree

J. Szczawinski, M. Szczawinska, B. Stanczak, 42

M. Fonberg-Broczek, J. Arabas, J. Szczepek and S. Porowski

Comparison of the Influence of High Pressure Treatment on the Survival

of Listeria Monocytogenes in Minced Meat, Sliced, Cured Ham and Ripened, Sliced Cheeses

J. Szczepek, J. Arabas, M. Fonberg-Broczek, T. Strzelecki, J. Zurkowska-Beta and S. Porowski 48

The Laboratory of High Pressure Processing of Food Products

of the High Pressure Research Center UNIPRESS, Warsaw

P.D. Sanz, N. Zaritzky, L. Otero and M. Martino 54

Effect of High-Pressure-Assisted Freezing and Air-Blast Freezing

on the Microstructure of Pork Meat

High PressureDenys, Van Loey, De Cordt, Hendrickx & Tobback

1

Modeling Heat Transfer During High Pressure Freezing and Thawing

S. Denys, A. Van Loey, S. De Cordt, M. Hendrickx and P. Tobback

Faculty of Agricultural and Applied Biological Sciences, Department of Food and Microbial

Technology, Laboratory of Food Technology

Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium

Abstract

An existing theoretical model for predicting heat transfer during freezing and thawing was

extended for modeling high pressure freezing and thawing processes. To deal with the two-

dimensional problem (temperature and pressure), a simplifying approach is suggested, consisting

of ÔshiftingÕ the known thermal property data (1 atm) on the temperature scale according to the

prevalent pressure. Good agreement of experimental and predicted profiles were obtained and

considering its simplicity, the method is very promising.

1. Introduction

Effects of high pressure on the liquid-solid behaviour of water (Bridgman, 1912) open the way

to new applications in food technology, including High Pressure Freezing and High Pressure Thawing.

High pressure freezing or Ôpressure-shift freezingÕ (Kanda and Aoki, 1993) or Ôhigh pressure

assisted freezingÕ (Kalichevsky et al., 1995) involves cooling an unfrozen sample under pressure

(usually up to 200 MPa), to -20¡C or above (without freezing) and suddenly releasing the pressure,

thus causing a high degree of supercooling. As supercooling increases the rate of ice-nucleation,

and because pressure is transmitted uniformly and instantaneously in the food, uniform and rapid

ice nucleation and growth result throughout the sample, if the heat of fusion can be removed.

Analogously, high pressure thawing involves forcing a frozen sample to the liquid area of the

phase diagram, thus increasing the thawing rate, if the appropriate heat of fusion is provided from

a heater (Kalichevsky et al., 1995; Deuchi and Hayashi, 1992). This comminication presents a first

attempt to model heat transfer during high pressure freezing and thawing.

2. Heat Transfer Model and Approach Followed

A computer program was written for numerically solving the heat transfer differential

equations in a cylindrical shape, using an explicit finite difference scheme. An infinite heat transfer

Process Optimisation and Minimal Processing of Foods Process Optimisation

2

coefficient at the surface of the cylinder (inner wall of the high pressure vessel) was assumed.

To deal with the problem of phase change over a range of temperatures, temperature

dependent apparent specific heat and thermal conductivity were considered. Specific heat and

thermal conductivity data given by Cleland and Earle (1984) for Tylose were used. To preserve

a correct heat balance and to avoid the possibility of missing peak values, an enthalpy

correction method was used.

The approach followed for dealing with the two-dimensional situation (temperature and

pressure) basically consists of ÔshiftingÕ the thermal property data (1 atm) on the temperature

scale, according to the prevalent pressure. Using the thermal property data obtained in the

computer model, the phase change (heat capacity peak) occurs at a lower temperature. A Ônew

initial sample temperatureÕ after pressure release or pressure build-up in case of a high pressure

freezing or thawing process, respectively, is calculated by use of an energy balance. This is a

simplifying approach, assuming a Ôvolumetric latent heatÕ independent of the applied pressure. In

reality, latent heat of water shows a slight pressure dependency.

3. Experimental Setup

Experiments were carried out in a warm isostatic press (SO. 5-7422-0, National Forge, Belgium)

with an operating pressure of 600 MPa and a volume of 590 ml (fl 50 mm, height 300 mm). A food

analogue material known as Tylose, a 23% hydroxymethylcellulose gel, was used because the

thermal properties of this substance are well known (5, 6). Tylose samples were placed in 50 ml

syringes, allowing axial volume change upon pressurizing and depressurizing. Thermocouples

were placed in the center of the sample.

High pressure freezing and thawing processes were performed and temperatures as well as

pressures were measured every 30 seconds.

4. Results and Discussion

4.1. High Pressure Freezing

Experimental temperature profiles for freezing Tylose samples are shown in figure 1. The

positive influence of applying high pressure on the time required to release the latent heat is

visible. The contribution of uniform ice nucleation, at a higher rate, on pressure release is visible

when comparing the ÔconventionalÕ process and the high pressure process with similar

temperature of the medium (-16.65¡C): only half of the time is required to release the latent heat

of melting. It is expected that crystallization occurs more homogeneously and that a better

product is obtained in terms of texture. Investigation of this was not the aim of the work.

High PressureDenys, Van Loey, De Cordt, Hendrickx & Tobback

3

Using the appropriate boundary conditions, a temperature profile for the process at 1 atm was

generated and is shown in figure 1. The best fit was obtained when the thermal conductivity of

the high pressure medium was estimated to be – 0.20 W/m¡C. The slight deviation from the

known value of 0.16 W/m¡C (provided by the distributor), creates the assumption of convective

currents in the high pressure medium, rather than heating by pure conduction. Using this thermal

conductivity value for the high pressure medium and the appropriate boundary conditions,

temperature profiles for the corresponding high pressure freezing processes were generated with

the model, and are shown in figure 1. Good predictions were obtained.

4.2. High Pressure Thawing

Figure 2 shows the experimental temperature profiles of five thawing processes. Here also, a

reduction of thawing time, using high pressure, can be observed. Temperature profiles of the

ÔconventionalÕ thawing process and of the four high pressure thawing processes were generated

with the model and are plotted in figure 2. The best result was obtained when estimating the

thermal conductivity of the high pressure medium to be –0.33 W/m¡C. This was assumed to be a

consequence of the higher temperature gradient during the thawing experiments (vessel at room

temperature) compared to the freezing experiments (vessel ranging from -11 to -18 ¡C), causing an

increase of convective currents and thus a higher ÔapparentÕ thermal conductivity. Considering the

simplification, the model predicts the temperature profiles well.

Figure 1 - Experimental and simulated temperature profiles for classical and high pressure freezingof Tylose

Time (Min)

Tem

erat

ure

(ºC

)


4

Figure 2 - Experimental and simulated temperature profiles for classical and high pressure thawing of Tylose

5. Conclusions

The method seems promising for the evaluation of high pressure freezing and thawing

processes. The main criterion determining the accuracy of the prediction was the determination of

an ÔapparentÕ thermal conductivity value of the high pressure medium. This value is dependent on

temperature gradients within the high pressure medium and should be incorporated in the model.

Experiments with different products should be performed in order to further test the proposed

method for the evaluation of high pressure freezing and thawing processes.

References

Bridgman, P.W. (1912) Water, in the liquid and five solid forms, under pressure. Proc. Am. Acad. Arts

Sci. XLVII (13), 439.

Kanda, Y. and Aoki, M. (1993). Development of pressure-shift freezing. Part I. Observtion of ice

crystals of frozen tofu. in High Pressure Bioscience and Food Science (R. Hayashi ed.), 33.

Kalichevsky, M.T., Knorr, D. and Lillford, P.J. (1995). Potential food applications of high-pressure

effects on ice-water transitions. Trends in Food Science & Technology, 6, 253-259.

Deuchi, T. and Hayashi, R. (1992). High pressure treatments at subzero temperature: application to

preservation, rapid freezing and rapid thawing of foods. High Pressure and Biotechnology (C. Balny,

R. Hayashi, K. Heremans & P. Masson eds.) Colloque INSERM, 224, 353-355.

Cleland, A. and Earle, R.L. (1984). Assessment of freezing time prediction methods. J. Food Sci., 49, 1034.

Riedel, L. (1960). A test substance for freezing experiments. Kaltetechnik 12, 222.

Time (Min)

Tem

erat

ure

(ºC

)

High PressureLudikhuyze, Weemaes, V. d. Broeck, D. Cordt, Hendrickx & Tobback

5

Modelling Thermal and Pressure-Temperature Inactivation

of Bacillus Subtilis α-Amylase

L. Ludikhuyze, C. Weemaes, I. Van den Broeck, S. De Cordt, M. Hendrickx and P. Tobback

Department of Food and Microbial Technology. Katholieke Universiteit Leuven. Kardinaal

Mercierlaan 92, B-3001 Leuven, Belgium

Abstract

Isobaric-isothermal inactivation of Bacillus subtilis α-amylase (BSA) could be accurately described

by a first order kinetic model. At ambient pressure an activation energy (Ea-value) of 219 kJ/mole and

an inactivation rate constant at a reference temperature (Tref) of 50¡C (kref-value) of 1.2x10-4 min-1 were

calculated. At a reference pressure of 500 MPa, the kref-value determined was 7.9x10-2 min-1. A

kinetic model to describe isobaric-isothermal inactivation of BSA was fit to these data. Subsequently,

non-isobaric/non-isothermal experiments were carried out. Assuming first order kinetics and constant

pressure during the entire treatment, Ea-values and kref-values were determined at different constant

pressure levels. Finally, taking the actual pressure-temperature profiles into account, the proposed kinetic

model was validated for dynamic conditions and the kinetic parameters were re-estimated.

1. Introduction

Recently, high pressure has gained renewed interest in the food industry and research as a

promising non-thermal preservation and processing method (Mozhaev et al., 1994). High pressure

has been shown to inactivate spoilage micro-organisms and enzymes, while leaving most quality

carriers, such as flavour, taste, colour and nutrient and vitamin content, intact (Hoover, 1993;

Hayakawa et al., 1994). Despite the advantages of high pressure processing, pressure treated food

products, such as fruit juices, jams and yoghurts, are until now only available on the Japanese

market. American and European industries are currently investigating the possibilities of high

pressure as a preservation technique and are exploring high pressure processing to determine

ideal parameters for a variety of foods (Mertens and Deplace, 1993). To develop such ideal processes,

the exact behaviour of micro-organisms, proteins, enzymes and other food components under

high pressure has to be known. Unfortunately, the mechanisms and certainly the kinetics of pressure

inactivation of enzymes are much less documented than those of thermal inactivation.

In this paper, the kinetic behaviour of an enzymatic model system, Bacillus subtilis α-amylase,

during pressure-temperature inactivation was investigated. This study will contribute to the

development of a concept to design, evaluate and optimise high pressure processes.


6

2. Materials and Methods

2.1. The enzyme and activity measurement

α-Amylase from Bacillus subtilis (BSA, Fluka) was purchased as a dry powder. The enzyme was

dissolved in a 0.01 M Tris HCl buffer at pH 8.6 in a concentration of 15mg/ml. The activity of BSA

was measured spectrophotometrically at 405nm according to procedure no. 577 of Sigma

Diagnostics. The temperature of the activity measurement was kept constant at 30¡C. The activity,

expressed as the change in optical density per minute, was calculated by linear regression of the

absorption as a function of time.

2.2. Thermal and pressure-temperature inactivation

Isothermal inactivation treatments were performed in a water bath with temperature control.

The samples were enclosed in capillary tubes to ensure isothermal heating. After withdrawal from

the waterbath, the samples were immediately cooled down in ice water.

Isobaric-isothermal inactivation treatments were performed in a multivessel high pressure

equipment (Roden, Holland). This equipment allows pressure up to 1000 MPa to be combined with

temperatures between 0 and 100¡C.

Non-isobaric/non-isothermal inactivation treatments were performed in a laboratory pilot scale

warm isostatic press (Engineered Pressure Systems International, Belgium). Using this equipment,

pressure (1 to 600 MPa) can be combined with temperatures between -30 and 100¡C. An output

card for pressure registration and four thermocouples type K, connected to a datalogger, allow the

pressure-temperature profiles to be recorded. These profiles are characterised by an initial phase

of pressure overshoot, accompanied by a temperature increase due to adiabatic heating.

2.3. Data analysis

Based on the linear curves in the semi-logarithmic plots of the activity retention as a function

of time, isothermal as well as isobaric-isothermal inactivation of BSA was assumed to follow first

order kinetics (1).

(1)

The activation energy at constant pressure was determined using the Arrhenius equation (2).

(2)k kE

R T Trefa

ref= × − × −

exp

1 1

lnA

Ak tt

0

= − ×


7

Using the data from isobaric-isothermal inactivation of BSA, a pressure-temperature kinetic

diagram, indicating combinations of pressure and temperature resulting in the same inactivation

rate constant was constructed. A kinetic model was fit to these data (3).

(3)

Based on literature data and by analogy with isobaric-isothermal inactivation, non-

isobaric/non-isothermal inactivation of BSA was assumed to follow first order kinetics. Firstly,

the pressure was considered constant during the entire experiment, which means that the

building up of pressure and the initial pressure overshoot were neglected. The integral effect

of a non-isothermal inactivation process at constant elevated pressure is given by equation

(4).

(4)

Applying a non-linear regression method to fit equation (3) to the data, the kinetic parameters

at one specific pressure were determined. Secondly, the proposed kinetic model (4) was validated

for dynamic conditions. Hereto, the pressure was no longer assumed constant, but the actual

pressure-temperature profiles were taken into account.


Isobaric-isothermal inactivation of BSA could be accurately described by a first order

kinetic model. At ambient pressure, the enzyme was inactivated in the temperature range 72-

82¡C. The kref-value (Tref=50¡C) was 1.2x10-4 min-1 and the Ea-value 219 kJ/mole.

Furthermore, k-values for about 50 combinations of constant pressure (100-800 MPa) and

temperature (25-70¡C) were determined (1). At a reference pressure of 500 MPa, the kref-value

was 7.9x10-2 min-1. The proposed kinetic model to describe isobaric-isothermal inactivation

of BSA is given in equation 4.

The kinetic parameters at a reference temperature of 50¡C and a reference pressure of 500

MPa determined were: a krefPT-value of 0.086 min-1, a B-value of 9031 and a C-value of

-0.000676.

Firstly, the kinetic parameters for non-isobaric/non-isothermal inactivation of BSA were

determined by a non-linear regression method applied to a first order kinetic model (3), assuming

constant pressure during the entire treatment (table 1).

k k BT T

C P PrefPTref

ref= × − × −

× − × −exp exp( ( ))

1 1

ln expA

Ak

E

R T Tdtref

a

ref

t

0 0

1 1

= − ×

−

× −

×∫


8

Table 1Kinetic parameters for non-isobaric/non-isothermal inactivation

of BSA according to equation (3).(Tref=50¡C)

Pressure BSA in Tris HCl

kref-value Standard error Ea-value Standard error(min-1) (kJ/mole,K)

250 MPa 8.8x10-5 1.1x10-5 130 12.2

350 MPa 5.2x10-4 1.7x10-5 112 3.8

400 MPa 9.9x10-4 1.3x10-5 85 2.3

450 MPa 1.4x10-3 1.0x10-5 77 1.9

500 MPa 1.9x10-3 3.2x10-5 79 2.4

550 MPa 2.7x10-3 3.5x10-5 76 1.3

From table 1 it could be concluded that kref-values increased and Ea-values decreased with

increasing pressure, i.e. the inactivation occurred more rapidly at higher pressure and the

temperature sensitivity of the inactivation rate constant was lowered at higher pressure.

Secondly, the proposed kinetic model was validated for non-isothermal/non-isobaric

conditions. Equation (5) was used as the model to fit the data from non-isobaric/non-isothermal

experiments. The pressure was no longer assumed to be constant, but the actual pressure and

temperature profiles, recorded with the datalogger, were taken into account.

(5)

Applying a non-linear regression method to this model, the kinetic parameters were re-

estimated under dynamic conditions. The re-estimated parameters calculated were: a krefPT-value

of 0.109 min-1, a B-value of 10022 and a C-value of -0.00086. According to the 95% confidence

intervals of the individual parameters, only the parameters kref and C were significantly influenced

by re-estimation.

4. Conclusions

Isothermal as well as isobaric-isothermal inactivation of BSA could be accurately described by

first order kinetics.

A model was formulated to describe the dependence of the inactivation rate constant of

BSA on pressure and temperature. This model was able to describe sufficiently accurately

isobaric-isothermal inactivation as well as pressure-temperature inactivation under dynamic

conditions.

ln exp exp( ( ))A

Ak B

T TC P P dtrefPT

refref

t

0 0

1 1

= − × − × −

× − × −

×∫


9

List of Symbols

At : enzyme activity at time t

B : T-dependence of the k-value

C : P-dependence of the k-value

Ea : activation energy (kJ/mole,K)

k : inactivation rate constant (min-1)

kref : k-value at reference T (min-1)

kTPref: k-value at reference T and P (min-1)

P : pressure (MPa)

R : gas constant (8.314 J/mole,K)

T : absolute temperature (K)

t : time (min)

Acknowledgements

This research was supported by the National Fund for Scientific Research (project G.0189.95),

the KULeuven Research Council (project OT/94/19) and the European Commission (project AIR1-

CT92-0296).

References

Mozhaev, V.V., Heremans, K., Frank, J., Masson, P. and Balny, C. (1994). Exploiting the effects of

high hydrostatic pressure in biotechnological application. Trends in Biotechnology., 12, 493-500.

Hayakawa, I., Kanno, T., Tomito, M. and Fujio, Y. (1994). Application of high pressure for spore

inactivation and protein denaturation, Journal of Food Science, 59(1), 159-163.

Hoover, D.G. (1993). Pressure effects on biological systems. Food Technology, 47(6), 150-155.

Mertens, B. and Deplace, G. (1993). Engineering aspects of high pressure technology in the food

industry. Food Technology, 47(6), 164-169.


10

Inactivation of Mushroom PPO by Pressure and/or Temperature: Influence of pH and Inhibitors

C. Weemaes, L. Ludikhuyze, I. Van den Broeck, M. Hendrickx and P. Tobback

Department of Food and Microbial Technology, Katholieke Universiteit Leuven,

Kard. Mercierlaan 92, B-3001 Leuven, Belgium

Abstract

Mushroom polyphenoloxidase is a thermosensitive but pressure resistant enzyme. The

enzyme resistance to pressure and temperature can be altered by intrinsic factors (pH, additives).

However, the intrinsic factors can affect the pressure and/or temperature resistance in a different

way. A remarkable sensitising effect of glutathione on the pressure and temperature stability was

noted.

1. Introduction

Polyphenoloxidase (PPO) is responsible for enzymatic browning of damaged fruits and

vegetables. In the presence of oxygen, PPO oxidises o-diphenolic compounds to the

corresponding o-quinones. Subsequently, the o-quinones polymerise with other o-quinones,

proteins, amino acids, etc. resulting in the formation of brown complexes (1,2,3). Enzymatic

browning is highly undesirable in the food industry, as it causes deleterious changes in colour,

flavour and nutritional quality of fruits and vegetables (1,2).

Prevention of enzymatic browning is often accomplished by the use of anti-browning agents,

such as complexing agents, substrate structure analogues, reducing agents and acidulants.

Complexing agents chelate the active site copper, required for enzyme activity. Structure

analogues of the substrate can take place at the active site and can, therefore, reduce the amount

of active sites involved in enzymatic browning. Reducing agents reduce the o-quinones formed

back to the corresponding o-diphenols. Acidulants lower the pH to a value well below the pH for

optimal activity (1,2). Another method to prevent enzymatic browning is to inactivate the enzyme

e.g. by thermal or pressure treatment (2,3).

In this study it is investigated whether the pressure and/or temperature stability can be

reduced by the addition of anti-browning agents (complexing agents, substrate structure

analogues, reducing agents and acidulants).

High PressureWeemaes, Ludikhuyze, V. d. Broeck, Hendrickx & Tobback

11


2.1. Materials

2.1.1. Enzyme

Mushroom PPO was purchased from Sigma (T7755) as a dry powder and was dissolved in 0.1

M phosphate buffer (ÒPBÓ, pH 8-6.5) or in McIlvaine buffer (ÒMBÓ, pH 6.5-4). To study the effect of

EDTA, benzoic acid or glutathione, the respective substance was added to the enzyme system in

phosphate buffer (pH 6.5, 0.1 M).

2.2. Methods

2.2.1. Enzyme assay

The PPO activity (A) was measured polarographically or spectrophotometrically. These activity

measurements are based, respectively, on the oxygen consuming oxidation of catechol to o-

benzoquinone and its subsequent polymerisation to brown pigments. The method of

Thannhauser et al. (4) was used to determine the presence of disulphide bonds.

2.2.2. Pressure and thermal treatments

To facilitate the kinetic analysis of the results, the applied thermal and/or pressure

treatments were designed to be isothermal and/or isobaric. At atmospheric pressure, capillary

tubes containing the enzyme solution were heated during pre-set times (t) in a temperature

controlled water bath. Isobaric treatments were carried out in a thermostatic multi-vessel

high pressure apparatus (HPIU 10.000, Resato, The Netherlands). Micro tubes, containing

enzyme sample, were pressurised at 8 kbar during pre-set times (t). At pH 4, however, a

pressure of 7.5 kbar was used. After pressure and/or temperature treatment, the samples were

transferred to and stored on ice water.

2.2.3. Data analysis

The results of the isobaric/isothermal treatments were processed into kinetic parameter

values, i.e. rate constants (k), activation energies (EA) and reaction orders (n). For the

estimation of k and n (if n≠1), equations (1) and (2) were used. To estimate EA the Arrhenius

equation was used.

n = 1 ln(A) = ln(A0) - k * t (1)

n ≠ 1 A = ( A1-n0 + (n - 1) * k * t)

11 - n

(2)


12

Table 1Reaction order (n) of the decay process of mushroom PPO

caused by temperature and/or pressure

P/T condition lot 24H9542 lot 112H9580 lot 73H9524

temperature (T) 2.0 1.6 1.0

pressure (P) 1.0 1.0 1.0

P/T 1.0 1.0 1.0


3.1. Study of the inactivation kinetics by spectrophotometry and polarography

The thermal inactivation of mushroom PPO (lot 73H9524) was followed by means of

spectrophotometry and polarography. Both activity measurements showed that the thermo-

inactivation followed first-order kinetics, i.e. the enzyme activity decreased log-linearly as a

function of heating time. However, the estimated k-values did not coincide for the two assay

methods. The k-values derived by polarography were about twice as high as those determined

by spectrophotometry. The temperature dependence of the k-values, as indicated by the

activation energy, was about the same for both activity measurements. The activation energy

was determined as 309 and 319 kJ/mole according to the polarographic and

spectrophotometric measurement, respectively. Because of the discrepancy between the

activity assays, one method was selected for further use. The spectrophotometric measurement

was chosen because it was fast and accurate.

3.2. Thermal and/or pressure inactivation kinetics

Three lots of mushroom PPO were mutually compared with respect to thermal and pressure

stability. Thermal inactivation was obtained upon heating at temperatures exceeding 50¡C. The

reaction order of the thermal inactivation was, however, different for the three lots studied, as

indicated in table 1. For pressure inactivation at room temperature, pressures of about 8 kbar

were needed. The high pressure resistance could be reduced by applying mild heating (up to

45¡C) along with the high pressure treatment. At all temperatures studied, the pressure

inactivation followed first-order kinetics (see table 1). At 8 kbar, the EA-values were all about

43 kJ/mole, which is much lower than the one determined for atmospheric pressure. It seems

that the temperature dependence of the k-value is much lower at higher pressure.

Lot 24H9542 was selected to study the effect of intrinsic factors on the pressure and/or

temperature stability of mushroom PPO.

High PressureWeemaes, Ludikhuyze, V. d. Broeck, Hendrickx & Tobback

13

Table 2 Effect of pH on the P and/or T resistance of mushroom PPO

P/T condition ranking according to increasing stability

temperature (T) pH 4 << pH 5 << pH 8 < pH 6.5 (MB) < pH 6.5 (PB)

pressure (P) pH 4 << pH 5 <pH 6.5 (MB + PB) < pH 8

P/T T < 37 ¡C : pH 4 << pH 5 < pH 6.5 (MB + PB) » pH 8

T > 37 ¡C : pH 4 << pH 5 » pH 6.5 (MB + PB) » pH 8

Table 3Effect of additives on the P and/or T resistance of mushroom PPO

P/T condition EDTA benzoic acid glutathione

5 mM 5(a) ; 50(b) mM 5 mM

temperature (T) stabilising a + b : stabilising sensitising

pressure (P) no effect a : no effect sensitising

b : sensitising

P/T no effect a : no effect sensitising

b : sensitising

3.3. Effect of intrinsic factors on the thermal and/or pressure stability

The effect of pH on the pressure and/or temperature stability of mushroom PPO was studied in

the pH range 4-8. The results of the inactivation experiments are summarised in table 2.

It appears that the enzyme at pH 4 is less stable in all conditions studied. Besides, three additives

were evaluated for their ability to modify the pressure and/or temperature stability: EDTA,

benzoic acid and glutathione. These additives belong to the complexing agents, the structure

analogues of the substrate and the reducing agents, respectively. The sensitising/stabilising ability

of the additives is presented in table 3. From this table it appears that glutathione sensitises the

enzyme in all conditions studied. Using the method of Thanhauser et al. (4), it was found that

disulphide bonds become exposed to the surrounding medium upon heating and pressurising. It

is, therefore, possible that glutathione interacts with the disulphide bonds, resulting in a

decreased stability.

4. Conclusion

Mushroom PPO is a thermo-sensitive enzyme that can be inactivated by temperatures

exceeding 50¡C. On the contrary, the enzyme is very pressure resistant. Pressures as high as 8

kbar are needed to cause enzyme inactivation. Both pressure and temperature inactivations were

most pronounced at low pH. Glutathione in a concentration of 5 mM decreased significantly the

pressure and temperature sensitivity of the enzyme. The sensitising effect of glutatione was

thought to be due to an interaction with the disulphide bonds of the enzyme.


14

Acknowledgements

This research has been supported by the Flemish Institute for the promotion of scientific-

technological research in industry (IWT), KULeuven Research Council (project OT/94/19) and

National Fund for Scientific Research (project G.0189.95).

References

V�mos-Vigy�z� L. (1981). Polyphenol oxidase and peroxidase in fruits and vegetables. CRC Critical

Reviews in Food Science and Nutrition, 15 (5), 49-127.

McEvily A.J., Iyengar R. and Otwell W.S. (1992). Inhibition of enzymatic browning in foods and

beverages. CRC Critical reviews in Food Science and Nutrition, 32 (3), 253-273.

Golan-Goldhirsh A., Whitaker J.R. and Kahn V. (1984). Relation between structure of polyphenol

oxidase and prevention of browning. Journal of Agricultural and Food Chemistry, 177, 437-456.

Thannhauser T.W., Konishi Y. and Sheraga H.A. (1984). Sensitive quantitative analysis of disulfide

bonds in polypeptides and proteins. Analytical Biochemistry, 138, 181-188.

High PressureHeinz & Knorr

15

High Pressure Inactivation and Germination Kinetics of Spore-Forming Bacteria

V. Heinz and D. Knorr

Department of Food Technology, Berlin University, K�nigin-Luise-Str. 22,

D-14 195, Berlin, Germany

Abstract

The pressure effect on the interaction of germination and inactivation of spore forming bacteria

was studied for Bacillus subtilis between 10-70oC and up to 600 MPa. The death kinetics of

vegetative cells were well described by a kinetic model that assumed a two-step mechanism: the

transition of the population from the vital into a postulated intermediate state where all

homeostatic activity has stopped, and a subsequent first-order death kinetics. The time of

transition of the first step was described by a probability density function. Germination could not

be induced without inactivation at higher temperatures, but at lower temperatures (ideally

38oC). It is recommended to develop a pressure-temperature profile with prolonged pressure build-

up to maximise germination prior to inactivation, thus optimising the integral lethal effect on

the microbial load reduction.

1. Introduction

Pasteurisation or sterilisation by high hydrostatic pressure is one of the most promising novel

techniques of food processing. It provides the advantage of less severe damage of structural and

nutritional properties of the product and low operating energy input. To minimise the microbiological

hazards during storage, a high level of microbial reduction during the treatment is necessary. In the

last decade, a huge amount of high pressure inactivation data was presented. It shows that

vegetative organisms are generally assessable by the lethal action of high hydrostatic pressure. Spores

are more resistant than vegetative cells. The general behaviour of spores during pressure treatment

is in fact complex, and the application of higher pressure does not necessarily produce an increasingly

large effect. On the other side, the inactivation of bacterial spores by hydrostatic pressure is

supposed to occur in two stages: first pressure causes germination of the spores and then inactivates

the germinated forms.

Therefore, the main purpose of the research work during the run of this project was to

identify the pressure effect on the interaction of germination and inactivation of spore-

forming bacteria. The organism tested was Bacillus subtilis ATCC 9372. High pressure

inactivation kinetics were investigated separately with vegetative organisms and spores.


16

The maximum pressure of the available high pressure equipment was 600 MPa.

Temperature variation was possible in a range between 10 and 70oC. Germination was tracked

by different characteristic physical and physiological indicators. The use of a pressure cell with

sapphire windows allowed in situ observation of the pressure induced germination.

Interpretation of the results was performed by kinetic modelling taking into account the

high degree of variability within a microbial population. The validation and the applicability

of the models on non-isobaric treatments was checked. Consequences to high pressure

processing of foods are presented.

2. Vegetative Cells

Vegetative cells of B. subtillis were cultured with shaking in nutrient-broth for 24 hours.

Inoculation of the test medium yielded an initial concentration of approximately 107cfu/mL.

The samples were sealed in polyethylene-bags and stored in ice for no longer than 2 h

before treatment. The determination of the surviving fraction was performed immediately

after decompression. Experiments were carried out in Ringer solution or in carrot-puree.

The semi-logarithmic plot of the survival data versus treatment time showed typical

sigmoidal shaped curves, independently of treatment pressure or temperature. Extensive

shoulder-formation and tailing occurred especially during low-intensity treatments (<300

MPa). A portion of the surviving population showed a delayed or inhibited recovery when

incubated at non-optimal conditions. This behaviour indicated that homeostatic mechanisms

were still active within the more resistant survivors. Due to this pressure induced segregation

of organisms, the direct application of first-order kinetics would be improper. Obviously, the

basic assumption of the mechanistic concept, that all organisms in a population are totally

indistinguishable, was not valid.

As a way out, a first-order model of inactivation was extended with an exclusively time-

dependent transition of the population from the vital into a postulated intermediate state.

The main property of this metastable state is the complete absence of homeostatic activity.

All organisms which have fallen into this state are supposed to have regained homogeneity

concerning the lethal action of high pressure. Inactivation is defined and can only occur as an

irreversible step from the metastable to a completely unrecoverable state of death.

Introducing this two-step-mechanism, it was possible to combine the populationÕs

diversity of resistance phenomena and the mechanistic single-hit/single-target assumption.

Mathematically, the time of transition was described by a continuous distribution function and

connected to a first-order reaction supposed to govern the second irreversible step to the

finally inactivated cells.

Fitting the derived model-equation to the experimental inactivation data yielded good results

without exception. Moreover, the characteristic model-parameters showed logarithmic-linear

High PressureHeinz & Knorr

17

behaviour when plotted versus treatment pressure. The possibility of applying the model to

non-isobaric treatments was experimentally checked and confirmed during linear pressure

build-up periods.

3. Spores

Kinetic measurements of the pressure-induced germination were performed in a pressure

and temperature range between 0.1 - 400 MPa and 10 - 70oC. In situ scanning of the change

of a spore solutionÕs optical density was used as an indicator of the progressive degradation

of the cortex. Clear pressure-temperature optima with an increased rate of absorbency-

change were observed, suggesting that germination presents a complex system of

corresponding reactions, partly affected by changing environmental conditions. Above 75

MPa and 38oC, no further improvement could be obtained. The detection of the release of

dipicolinic acid (DPA) from the core into the medium indicated no basics to e.g. L-alanin

induced germination. The main characteristic of this germination pathway is the activation of

specific cortex-immobilised enzymes by L-alanin which irreversibly triggers the degradative

events leading to the loss of typical spore properties. This behaviour could also be identified

during pressure treatment. Heat-resistance of a spore population as well as phase-contrast

refractivity of single spores was found to be reduced in parallel to the PD-release. Moreover,

it was observed that germination continued to a certain extent, even if the pressure had

already been relieved.

It seems likely that the role of high hydrostatic pressure during the induction of

germination is restricted to an activation of specific enzymes, only occurring in a narrow

pressure range between 50 and 250 MPa. More intensive treatments may lead to the direct

inactivation of the germination system. Inactivation at 150MPa and 70oC (6 log-cycles in 30

min) was found to occur prior to the release of DPA.

Similarly to vegetative cells, the high degree of variability within a spore population

governed the macroscopic kinetic behaviour. Passive homeostatic mechanisms led to

statistical distributed times of germination triggering. Additionally, the detectable events like

DPA-release were always interfered by a certain delay which may be due to diffusional

limitation in the outer parts of the spore. Modelling this simulation was performed by

combining a distribution function on describing the triggering-times with a concentration

dependent exponential decay of the remaining DPA-content of the core. Especially at 150 MPa

the analysis of the experimental data yielded further information: from 10 to 38oC average

triggering-times were reduced from 2h to 2min. Higher temperatures did not improve this

value. Concerning the DPA-transfer, a strong discontinuity at 38oC characterised the

temperature dependence. Obviously, the onset of the inactivation time of the germination of

specific enzymes limited the DPA-release by a delayed degradation of the cortex.


18

4. Conclusion

Vegetative forms of B. subtilis are easily inactivated at non-thermal conditions (20-40oC) at

a pressure level of approximately 400 MPa. The killing of spores requires the application of

more severe conditions. Though extreme pressure (>1GPa) cannot improve the situation

dramatically, a temperature rise of 70oC at pressure as low as 150 MPa is very effective.

Germination cannot be induced without inactivation at the same temperature, but at lower

temperatures (ideally 38oC).

Usual process concepts favour the application of relatively high pressure (800 MPa),

reached in a short time. However, spore inactivation would be more effective with pressure

and increased temperature. As a compromise, more prolonged phases of pressure build-up and

decompression are recommended. The pressure-temperature-profile of such a treatment

should include conditions of optimal induction of germination. Models of inactivation or

germination can serve for process development by estimating the integral lethal effect of

those non-isobaric treatments.

High PressureBr�na, Voldrich, Marek & Kamar�d

19

Effect of High Pressure Treatment on the Patulin Content of Apple Concentrate

D. Br�na1, M. Voldrich1, M. Marek1 and J. Kamar�d2

1 Department of Food Preservation and Meat Technology, Institute of Chemical Technology,

Technick� 5, 16628 Praha 6, Czech Republic

2 Institute of Physics, Academy of Sciences of Czech Republic, Cukrovarnick� 10, Praha 6,

Czech Republic

Abstract

Patulin, a toxic mould metabolite, is frequently found in apple juices and concentrates. The

effect of high pressure treatment on the patulin content in the apple concentrate and the model

apple juice (both at the initial concentration level of 155 mg/kg) was investigated. One hour

treatment of the samples at room temperature under pressures of 300, 500 and 800 MPa caused

a decrease of the patulin content of 16, 20 and 23% of the initial value in the apple concentrate

and of 42, 53 and 62% in the juice, respectively. The high pressure pasteurisation can significantly

reduce the mycotoxin content and improve the quality of products with retention of the

nutritional and sensorial components of fruit juices.

1. Introduction

The ultra high pressure pasteurisation in combination with aseptic filling systems seems to be

one of the most promissing applications of high pressure technology in food processing (Knorr,

1995, Ifuku et al., 1993). The high pressure treatment for 5 - 10 min at 300 - 600 MPa and 20 -

50¡C allows the reduction of vegetative microbial cells by 4 - 5 log cycles, although enzymes,

especially pectinesterase in citrus juices, are more pressure resistant and their inactivation needs

additional approaches (Knorr, 1995, Ifuku et al., 1993, Takahashi et al., 1993). The main advantage

of high pressure pasteurisation is that the products retain the flavour and nutrition level of fresh

fruits (Knorr, 1995, Cheftel, 1992). However, high pressure processing affects chemical reactions in

the food material in both positive and negative ways. Nonenzymatic browning (Maillard reaction)

as well as hydrolysis of sucrose are inhibited by pressure. The degradation of colour and slight

changes of flavour due to the higher content of dissolved oxygen in products are mentioned as

examples of negative pressure effects (Knorr, 1995, Cheftel, 1992). The possible effect of high

pressure treatment on the chemical reactions in foods could be interesting also from the point of

view of degradation of toxic constituents of food.

Patulin (4-hydroxy-4H-furo(3,2-c)pyran-2(6H)-one) (1), a toxic metabolite produced by several

species of Aspergillus, Penicillium and Byssochlamys is frequently found in fruit products especially in


20

apple juices and concentrates as a consequence of processing of fruits decayed by these moulds. In

large scale harvest and industrial processing of fruit it is almost impossible to obtain raw materials

absolutely free of such contamination, although the level in products is regulated. In several European

countries the limit for soft drinks is 50 µg/kg, in the Czech Republic it is 10 µg/kg (Tarter, 1991, van

Egmond, 1989). Heating has only very low effect on patulin content, that decreases very slowly

during storage. The mechanisms of its degradation in complex food matrixes is not sufficiently clear.

Patulin can react with thiol compounds to form additional products, similar reactions are known also

for the SO2 (bisulfite ion) (Pohland and Allen, 1970). The addition of ascorbic acid also accelerates

the degradation of patulin. It is suggested that degradation products of patulin are less toxic than

the original molecule (Pohland and Allen, 1970, Aytac and Acar, 1994, Woller and Majerus, 1982).

(1)

The aim of this communication is to evaluate the effect of high pressure treatment on the

degradation of patulin content in apple juice and concentrate.

2. Materials and methods

2.1. Material

Patulin free (patulin content less than 3 µg/kg) apple concentrate (pH 3.4, 70 ¡Brix) was obtained

from Fruiko Prus�nky. Concentrate was fortified with patulin (Aldrich Chem. Co.) to the level of 155

µg/kg (A) or with patulin and sodium metabisulfite (Lachema Neratovice) (100 mg/kg) (B). Both model

samples were pressurised also in diluted forms with 37¡Brix and 22¡Brix. Distilled water was used.

2.2. Methods

2.2.1. Pressure treatment

The samples (0.5 - 1g) were closed into PE capsules 2 x 2 cm and jointly treated (A and B) in an

hydrostatic pressure cell to guarantee identical pressure, temperature and time conditions. The

steel pressure cell (with i.d. 22 mm) enables treatment under pressures up to 1GPa in the

temperature range from 0 to 100¡C. A mixture of mineral oils was used as a pressure transmission

medium, no contamination of samples was detected. The samples were pressurised to 300, 500

and 800 MPa at 20 and 50¡C during 20, 60 and 150 min. Both pressure and temperature were

measured inside the pressure cell by a manganin sensor and a thermocouple, respectively.

O

O

OH

O

21

2.2.2. Determination of patulin

Patulin was determined (Tarter and Scott, 1991, Adensam et al., 1989) in pressurised samples as

well as nontreated reference ones. Patulin was extracted with ethyl acetate, extract purified using SPE

(Tessek Praha) on silica gel, evaporated to dryness, derivatized using N,O-Bis (trimethylsilyl)-

trifluoroacetamid (BSTFA, Fluka Chemical Co.) and analysed by gas chromatography on DB 5 capillary

column (J&W Scientific) with ECD detection. Internal standard p,p«-DDT (Sigma Chemical Co.) was used.


The rate of patulin degradation in fortified apple

concentrate (155 µg/kg) with and without addition

of sodium metabisulfite at 500 MPa and 20¡C is

given in figure 1. It is obvious that high pressure

accelerates the degradation, which is faster in the

case of the sulphited sample probably due to the

higher concentration of possible reaction partners

of patulin. As an usual pasteurisation has almost

negligible effect on patulin content in fruit

products, any pressure treatment seems to be a

useful procedure to reach a higher reduction of

patulin content.

The course of patulin degradation is pressure

dependent (figure 2). Higher pressure causes higher

decrease of patulin content. In spite of a limited

number of data, a D-p degradation curve at 20¡C

was drawn (figure 3). The D-p degradation curve

means the dependence of the exposition time

needed to reduce the concentration of patulin of

one log cycle on pressure, at constant temperature.

One point of the D-p curve at 50¡C is significantly

below the D-p curve at 20¡C in figure 3. It reflects

the more pronounced effect of temperature on log

D in comparison with the effect of pressure. At

higher temperatures (about 50¡C), the reduction of

patulin will be remarkable for the short treatment

time used.

In addition to the composition of juice or

concentrate (content of thiol, ascorbic acid, sulphites,

High PressureBr�na, Voldrich, Marek & Kamar�d

0

50

100

150

200

0 50 100 150

time (min)patulin content (

µ( g/kg)AB

0

50

100

150

200

0 200 400 600 800

pressure (MPa)

patu

lin c

onte

nt (µ

g/kg

) A

B

50°C

1

1,5

2

2,5

3

0 500 1000

pressure (MPa)

log

D (

min

)

Figure 1 - The effect of time of pressuretreatment (500 MPa, 20°C) on therate of patulin decrease. A - fortifiedapple concentrate, B - fortified appleconcentrate with addition of sodiummetabisulfite.

Figure 2 - The effect of pressure on patulincontent reduction at 20°C.

Figure 3 - Effect of Pressure on patulindegradation at 20°C.

22


etc.), the course of patulin degradation is also

affected by osmotic pressure. The dependence of

relative patulin content reduction on the refractive

index is given in figure 4. A similar protective effect

of osmotic pressure was observed. The degradation

of patulin in concentrate was lower than in juice. In

practice, juice is usually treated before the aseptic

filling, therefore the reduction of patulin content

can be expected to be more significant.

4. Conclusions

High pressure accelerates the degradation reactions of patulin in apple concentrates and juices.

The reduction depends on the conditions used; the increase of pressure or temperature causes a

more significant reduction of patulin content. The effect is higher at lower osmotic pressure.

The effect of high pressure treatment on the degradation of patulin frequently present in fruit

products is the other positive factor supporting a wide use of high pressure in food processing.

References

L. Adensam, M. Lebedov�, J. Pavlosek and B. Turek. (1989). Pr�mysl potravin, 40, 127.

S.A. Aytac and J. Acar. (1994). Ern�hrung/Nutrition, 18, 15.

J.C. Cheftel. (1992). Effect of high hydrostartic pressure on food constituents: an overview. In:

Proceedings of High Pressure and Biotechnology, C. Balny, R. Hayashi, K. Heremans, P. Masson

(Eds.), Colloque INSERM/John Libbey Eurotext, 195.

H.P. van Egmond. (1989). Food Addit. Contam., 6, 139.

Y. Ifuku, Y. Takahashi and S. Yamasaki. (1993). International Markets, Fruit Processing, 3(1), 19.

D. Knorr. (1995). High pressure effect on plant derived foods. In: High Pressure Processing of

Foods, D.A. Leward, D.E. Johnston, R.G. Earnshaw, A.P.M. Hasting (eds.), Nottingham University

Press, 123.

A.E. Pohland and R. Allen. (1970). J. AOAC, 53, 688.

A. Pr�bela and T. Sinkov�. (1995). Mycotoxins in Natural toxic compounds of foods, J. Dav�dek (ed.),

CRC Press, 170.

Y. Takahashi, H. Ohta, H. Yonei and Y. Ifuku. (1993). Int. J. Food Sci. and Technol., 28 (1), 95.

E.J. Tarter and P.M. Scott. (1991). J. Chromatogr. 538, 441.

R. Woller, P. Majerus. (1982). Fl�ssiges Obst

0

10

20

30

40

50

0 20 40 60 80

refractive index (°Brix)

rela

tive

patu

lin c

onte

ntre

duct

ion

(%)

A

B

Figure 4 - Dependence of the relative patulincontent reduction on the refractiveindex of apple concentrate - juice(500 MPa, 60 min, 20°C).

High PressureButz, Fernandez, Fister & Tauscher

23

Influence of High Hydrostatic Pressure on Dipeptides

P. Butz, A. Fernandez, H. Fister and B. Tauscher

Institute of Chemistry and Biology, Federal Research Centre for Nutrition.

Karlsruhe. Germany

Abstract

The pressure stability of the synthetic dipeptides and derivatives in solution was investigated.

1-2 mmol/l solutions of L-aspartyl-L-phenylalanine, cis-3,6-dioxo-5-(phenylmethyl)-2S-piperazine

acetic acid (=diketopiperazine, DKP), and L-aspartyl-L-phenylalanine methylester (= Aspartame,

Nutrasweet) in 0.05 mol/l Tris/HCI buffer, pH 7, were treated at 700 MPa for 3, 10 and 30 minutes.

Aspartame was very unstable under the given conditions (neutral pH is essential). Degradation

products are diketopiperazine and aspartyl-phenylalanine. Diketopiperazine and aspartyl-

phenylalanine themselves did not change under the same conditions.

1. Introduction

High pressure is a thermodynamic factor which is known to influence chemical equilibria and

reaction rates according to the Le Chatelier principle. Pressure allows covalent bond ruptures in

reactions associated with reductions in volume. Therefore the pressure stability of food ingredients

should be studied. It has to be confirmed that no undesirable degradation products form, as e.g.

peptides which may become biologically active. In this work the stability of three dipeptides under

conditions of commercial pressure treatment was investigated.


2.1. Materials

Three synthetic dipeptides were used for the experiments: L-aspartyl-L-phenylalanine (Bachem

AG, Switzerland), cis-3,6-dioxo-5-(phenylmethyl)-2S-piperazine acetic acid (=diketopiperazine,

DKP) and L-aspartyl-L-phenylalanine methylester (=Aspartame) (a kind gift from Nutrasweet AG,

Zug, Switzerland).

24


2.1.1 High pressure apparatus

The high pressure device consisted of a series of thermostated micro-autoclaves (ID 16 mm, ca.

l 0 ml) connected by valves (Butz et al. 1994). Pressure was generated manually by a hand pump in

combination with a pressure intensifier. The pressure transmision medium was water.

2.1.2 HPLC analysis

The three dipeptides and degradation products, were analyzed according to Langguth et al.

(1991) with minor modifications. A high-performance liquid chromatograph SPD-IOAD/LC-IOAD

(Shimazdu. D�sseldorf Germany) with UV-detection was used with a reversed-phase HD-Sil-18-5s-

80 column (Orpegen, Heidelberg, Germany). Detection was at 200 nm, flow rate was 1.0 ml/min.

The mobile phase was a 80:20 mixture of 0.5 mol/l NaH2PO4 (Merck 106346) adjusted to pH 2.1 by

H3PO4 and methanol (Merck 106007), injection volume was 20 111, pressure was 26-28 MPa. Peak

areas were measured by integration software (PC Integration Pack, Kontron).

2.2. Methods

Aspartame, diketopiperazine and L-aspartyl-L-phenylalanine were dissolved in 0.05 mol/l

Tris/HCI buffer, pH 7, to yield a concentration of about 1-2 mmol/ml. Teflon tubes (inner/outer

diameter 6/8 mm; 1-2 ml) with silicon stoppers were filled for the treatment under pressure.

Temperature controls were identically packaged samples in pressureless microautoclaves of the

same device. Pressure treatment was 3, 10 and 30 minutes at 700 MPa and 60¡C .


Aspartame is very unstable under the given conditions; degradation products are

diketopiperazine and aspartyl-phenylalanine (figure 1). The same products are also formed

during thermal treatment and storage of Aspartame. The stability of Aspartame in solution

depends strongly on pH and temperature with greater stability in acid media. At neutral pH,

pressure of 700 MPa shows an additional detrimental effect on Aspartame compared to thermal

treatment only (upper line in figure 1).

While the dipeptide-methylester Aspartame under pressure quickly forms the cyclization

product diketopiperazine, the analog dipeptide aspartyl-phenylalanine fails to do so (figure 2).

This could, at least in part, be due to the cleavage of methanol which is accompanied by a

negative volume effect. The cyclic dipeptide diketopiperazine does not change either under the

given conditions (figure 2); however, there is a significant change at different pH and longer

treatment (data not shown).

25

High PressureButz, Fernandez, Fister & Tauscher

References

Butz, P.; Koller, W.-D.; Tauscher, B.; Wolf, S. Ultra high pressure processing of onions: chemical and

sensory changes. Lebensm.-Wiss. u. Technol. 1994, 27, 463-467.

Langguth, P.; Alder R; Merkle, H.P. Studies on the stability of aspartame (I): specific and reproducible

HPLC assay for aspartame and its potential degradation products and applications to acid hydrolysis

of aspartame. Pharmazie. 1991. 46. 188-192.

Figure 1 - Pressure treatment of L-aspartyl-L-phenylal-anylmethylester (Aspartame) in 0.05 mol/lTris/HCI buffer, pH 7, at 700 MPa and 60°C.■ Aspartame, ◆ Aspartame control at 60°Cand normal pressure, ● DKP(diketopiperazine), ▲ aspartylphenylalanine.

Figure 2 - Pressure treatment of dipeptide solutions in0.05 mol/l Tris/HCI buffer, pH 7, at 700 MPa and60°C, ● diketo-piperazine, ▲ aspartyl-phenylalanine.

26


Abstract

The mould Eurotium repens can cause spoilage of products with high sugar content. In order to

analyse the potential of high pressure to inactivate this mould, the pressure resistance of its three

different forms was studied. It was found that the minimum pressure required for an effective

inactivation varies from 150 MPa for the vegetative mycelium to 450 MPa for the more resistant

ascospores, with conidiospores requiring a minimum of 250 MPa. Concentrated solutions of sugars

or salts showed a stabilising effect of conidiospores against pressure.

1. Introduction

The mould Eurotium repens is able to grow even on media which contain high amounts of sugars,

e.g. jams and may cause spoilage of such products. As a perfect fungus it forms three different kinds

of cells — vegetative hyphae, asexually derived conidiospores and sexually derived ascospores.

A method to separate these species by density gradient centrifugation is demonstrated, and for

each of them the characteristics of high pressure inactivation are shown.

The vegetative hyphae are most pressure sensitive and ascospores are most resistant to pressure.

Conidiospores, less pressure resistant than ascospores, are highly stabilized against pressure in

concentrated salt- and sugar-solutions (van Almsick et al., 1995).


Eurotium repens DSM 62631 was purchased from the Deutsche Sammlung von Mikroorganismen,

Braunschweig. It was grown at 24¡C on the medium recommended for osmophilic fungi (DSM). For

each experimental run cells were freshly prepared. Vegetative cells were harvested after 24 to 48h

before any formation of spores started. Conidiospores were taken after 5 to 7 days. A large inoculum

stimulated the formation of ascospores which were harvested after at least 14 days. To break asci

and get free ascospores the raw material was shaken with glass beads. To avoid aggregation of spores

the suspensions contained 0.1% of Polysorbate 80.

High Pressure Inactivation of Hyphae, Conidiospores and Ascosporesof the Fungus Eurotium Repens

G. van Almsick, R. Eiche and H. Ludwig

Universit�t Heidelberg, Institut f�r Pharmazeutische Technologie und Biopharmazie,

69120 Heidelberg, Germany

27

High PressureVan Almsick, Eicher & Ludwig

Separation of ascospores from vegetative cells and conidiospores was performed by density

gradient centrifugation using two CsCl-density gradients. The first 2 ml of a spore suspension

were loaded onto 50 ml of a gradient of 22 to 34% CsCl and centrifuged for 15 min at 1100g. The

pellet, containing ascospores and still some conidiospores, was loaded onto a gradient of 34 to

44% CsCl and centrifuged for 20 min at 1100g. The well separated white band in the middle of the

tube contained more than 90% of single ascospores. These could be distinguished from

conidiospores by phase contrast microscopy. The shape of ascospores occurs brighter and sharper,

thus indicating a lower content of water (figure 1 and 2).

The high pressure device consisted of ten small pressure vessels . These were filled with 1 to

2.5 ml samples, enclosed in polyethylene tubes, pressurized simultaneously and thermostated.

The maximum pressure was 700 MPa, the pressure medium was water. The single vessels could be

opened at different times in order to measure the kinetics of inactivation (Butz et al., 1990).

The number of surviving organisms was determined by counting colony forming units per ml

(cfu) on agar plates. The results given in the figures are from single experimental runs. All

experiments were replicated at least once.

Figure 1 - Phase contrast microscopy of conidiospores,1000 times magnified

Figure 2 - Phase contrast microscopy of ascospores,1000 times magnified

28



As expected, the vegetative hyphae are the least barotolerant cells formed by Eurotium repens.

Figure 3 shows the inactivation of hyphae at 200 MPa and 25¡C. Under these conditions viable

counts of hyphae are reduced with a D-value of 2 min, whereas conidiospores have a D-value of 8

min when inactivated at 300 MPa and 25¡C. In contrast to the inactivation of vegetative cells,

reduction of conidiospores is not linear in the semilogarithmic plot over the whole range of cells.

This is caused by very small amounts of ascospores which cannot completely be removed from the

preparations (figure 4).

Figure 3 - ● Inactivation of vegetative cells of Eurotium repensat 200MPa and 25ºC; open symbol is control

Figure 4 - ● Inactivation of conidiospores at 300 MPa and25ºC; open symbol is control

29


Figure 5 gives the inactivation of separated ascospores at different pressures. Substantial

reductions in viable counts occur at pressures above 400 MPa. Therefore ascospores are the most

barotolerant cells of the fungus. In kinetic studies (figure 6) a D-value of 30 min at a pressure of 500

MPa was ascertained. For non-purified ascospores the inactivation is faster initially. This is due to

the amount of vegetative cells and conidiospores. Separated ascospores do not show this phenomenon.

The remarkable barotolerance of ascospores found here is in agreement with results of Butz from

investigations on Byssochlamys nivea (Butz et al., 1996). For the yeast Rhodotorula rubra it has been

Figure 5 - ● Inactivation of separated ascospores, 30 min at25ºC and different pressures

Figure 6 - Inactivation of ● non-purified and ■purified ascosporesat 500 MPa and 25ºC, open symbols are controls


30

reported that high concentrations of different sugars and NaCl led to a decrease in pressure sensitivity

(Oxen et al., 1993). Therefore, conidiospores of Eurotium repens were suspended in solutions of sucrose,

glucose, NaCl and KCl. Figure 8 exemplifies the results for sucrose and NaCl. Both, concentrated solutions

of sugars and salts stabilize conidiospores of Eurotium repens against high hydrostatic pressure. At 300MPa

in 60% sucrose and in 25% NaCl no reduction in viable counts could be determined after 30min of pressure

treatment. For sucrose this stabilization could be overcome by elevated pressures as demonstrated in figure

8. All germs were inactivated when the spore suspension was pressurized to 550MPa for 30 min.

Figure 7 - Inactivation of E. repens (conidiospores) at 25ºCfor 30 min, ● suspended in sucrose-solutions, ■suspended in NaCI-solutions, ■■-■ indicates theinitial number of germs

Figure 8 - ■ Inactivation of conidiospores in 60% sucrosesolutions, 30 minat 25ºC and different pressures,■ initial number of germs

31


4. Conclusions

The barotolerance of Eurotium repens depends on the developmental form in which the mould

is present. The minimum pressure needed for an effective inactivation varies from 150 MPa for the

vegetative mycelium and 250 MPa for the conidiospores to 450 MPa for the ascospores.

For preparations consisting of only one kind of cells the kinetics of inactivation follows a first

order reaction over several log cycles.

The various pressure sensitivities of conidio- and ascospores may be caused by different water

activities inside the specimens. Concentrated solutions of sugars or salts stabilize conidiospores

against pressure and therefore, in high osmolalic foods, conidiospores may also be problematical

to inactivate.

Acknowledgements

This work was supported by the EU, Project. No. AIR11-CT92-0296 and FAIR-CT96-1175

References

G. van Almsick, Ch. Schreck, H. Ludwig, Basic and Applied High Pressure Biology IV. 1995, 5, 69

P. Butz, S. Funtenberger, T. Haberditzl, B. Tauscher, Lebensmittel-Wissenschaft und -Technologie

1996, 29, 404

P. Butz, J. Ries, u. Traugott, H. Weber, H. Ludwig, Pharm. Ind. 1990, 52, 487

P. Oxen, D. Knorr, Lebensmittel-Wissenschaft und -Technologie 1993, 26, 220

Questions and Answers

Is there any logarithmic relation between the velocity of killing microorganisms

(vegetative and spores) and pressure value in MPa, like the dependence between Dt and

temperature and Dt and intensity of irradiation.

Stoyan Tantchev

There is a relation similar to irradiation (minimal dose) with a minimal pressure needed

for inactivation. Above that minimal pressure there exists a nearly linear relationship

between log D and pressure, but only until an upper pressure limit is reached where the rate does

not further increase substantially.

A

Q

32


Abstract

The effect of high hydrostatic pressure treatment (50-500 MPa) combined with heat treatment

(20-60…C) on peroxidase (POD), polyphenol oxidase (PPO) and pectin methylesterase (PME)

activities in tomato puree were studied. Assays were carried out with fresh made tomato puree

and a 15 minutes treatment time was prefixed. Pressurization/depressurization treatments caused

a continous denaturation of soluble proteins at room temperature (20…C). Also, UHP/mild heat

treatments produced a significant reduction (32.5%) of PME activity when a combination of 150

MPa/30…C treatment were employed, while some activation was observed for treatments carried

out at 335-500 MPa, at different temperatures. A fair reduction of POD activity (25%) was obtained

in tomato purees treated at 350 MPa/20…C, but combination of higher pressures and mild

temperatures (30-60…C) produced an enhancement of this activity. PPO activity did not experiment

any significant changes due to UHP/mild temperature treatments in the tomato product. Only a

combination of 200 MPa/20…C seemed to produced a significant loss (10%) in PPO activity.

1. Introduction

High hydrostatic pressure treatment reduces microbial counts and enzyme activity and affects

product functionality (Farr, 1990; Hoover et al., 1989; Cheftel, 1991). This provides a good

potential basis for development of new processes for food preservation or product modifications

(Mertens and Knorr, 1992). The first commercial products made using high pressure treatments

have been almost exclusively plants or products containing plants (Knorr, 1995).

Effects of high pressure treatments on enzymes may be related to reversible or irreversible

changes in protein structure (Cheftel, 1992). However, loss of catalytic activity can differ

depending on the enzyme, the nature of the substrates, the temperature and the length of

processing (Cheftel, 1992; Kunugi, 1992; Cano et al., 1996).

The demand for minimally processed tomato products of rich flavour and high consistency has

risen markedly in these last years. In this way, some authors (Porreta et al., 1995) reported the effects

of ultra-high hydrostatic pressure treatments on the quality of tomato juice. This product (juice)

High Pressure and Temperature Effects on Enzyme Inactivation in Tomato Puree

A. Hern�ndez and M. Pilar Cano

Department of Plant Foods Science and Technology, Instituto del Fr�o (C.S.I.C.),

28040-Madrid, Spain

33

High PressureHern�ndez & Pilar Cano

was prepared using a pH adjustement and no combinations of UHP/temperature were employed.

Also, no enzymatic inactivation studies were made.

The objective of the present work was to determine the effects of high pressure treatments up to

500 MPa combined with mild heat treatments up to 60…C on peroxidase (POD; EC 1.11.1.7), polyphenol

oxidase (PPO, EC 1.10.3.1) and pectin methylesterase (PME, EC 3.1.1.11) activities in tomato puree.


2.1. Materials

2.1.1. Plant material

Full ripe tomatoes (Lycopersicum esculentum, var. Pera) from Valencia (Spain) were obtained from

commercial sources. Fruits for processing were selected attending to their maturity and disease

free. The characteristics of the tomato puree are shown in table 1.

2.1.2. UHP equipment

A high pressure unit GEC ALSTHOM ACB 900 HP, type ACIP No 665 (Nantes, France), with

2,350 ml capacity was used.

2.2. Methods

2.2.1. Combined UHP/Temperature Treatments

UHP treatments (50-500 MPa) were employed. The time of the treatments was constant at 15

min and the temperature of the immersion medium (initial sample at atmospheric pressure: 20…C)

was varied between 20…C and 60…C. Samples were placed in polyethylene bottles (250 ml)

Table 1Physicochemical and biochemical characteristics

of tomato puree before pressurization

Characteristics Tomato puree

Titratable acidity (g citric acid/100g f.w.) 0.38 – 0.02

pH 4.08 – 0.05

Soluble solids (¡ Brix at 20¡C) 5.6 – 0.1

Total solids (mg/100 g f.w.) 5.69 – 0.17

Moisture content (%) 94.3 – 0.18

POD activity 40.41 – 1.33

PPO activity 0.79 – 0.03

PME activity 5.32 – 0.08

Protein content (mg/g f.w.) 0.31 – 0.05

Color L (luminosity) 23.42 – 0.08

aL 12.39 – 0.05

bL 9.95 – 0.04

Values are average – standard deviation of three independent determinations.


34

and then introduced in the pressure unit filled with pressure medium (water). Pressure was

increased and released at 2.5 MPa/s. After pressure treatments samples were immediatelly

analyzed or stored at -80…C for later enzyme activity determination.

2.2.2. Quality determinations

Soluble solids of fresh made tomato puree were determined using a digital refractrometer

(ATAGO, Tokyo, Japan). Results were reported as Brix at 20…C. For titratable acidity, the puree was

macerated and 10 g samples were accurately weighed into beakers. Distilled water (40 ml) was

added to each sample. The resulting mixture was titrated with 0.1N Na OH to pH 8.1, monitored

with a pH meter (Microph 2000, Crison, spain). The results were expressed as g citric acid/100g

sample. The pH of the samples was determined before titration. For moisture content, the AOAC

(1984) vacuum oven method was modified, using a microwave oven operating at 200W for 20-25

min, as described by Cano et al. (1990).

The colour of the tomato puree was measured in a cylindrical sample cup, 5 cm diam X 2 cm

high, filled to the top, using a colourimeter model 25-9 (Hunter associates Laboratory mod. 25-9,

Reston, VA, USA). A standard colour plate No. C2-19952 with reflectance values L=77.65, a=-1.51,

b=21.41 was used as reference.

2.2.3. Biochemical analysis

The enzyme extracts for the determination of polyphenol oxidase (PPO) and peroxidase (POD)

were made by homogenization of 10g of each sample with 10ml of 0.2M sodium phosphate buffer

(pH 6.5) (containing 1% (w/v) insoluble polyvinylpolypirrolidone (PVPP)) or for pectin

methylesterase determination with 10 ml of 0.2M sodium phosphate buffer (pH 7.5) in an

ultrahomogenizer (Omnimixer, mod. ES-207, Omni International, Inc., Gainsville, VA, USA) with

external cooling, for 3 min with stop intervals each 30 sec.

Peroxidase activity was assayed spectrophotometrically using aliquots (0.025ml) of extract and

a reaction mixture composed by 2.7ml 0.05M sodium phosphate buffer (pH 6.5) with 0.2 ml 1%

(w/v) p-phenylenediamine as H-donor and 0.1 ml 1.5% (w/v) hydrogen peroxide as oxidant. The

oxidation of p-phenylenediamine was measured using a double beam spectrophotometer (Perkin-

Elmer, mod. Lambda 15, Bodenseewerk, FRG) at 485 nm and 25…C.

Polyphenol oxidase activity was assayed using aliquots (0.1 ml) of extract and 3.0ml of a

solution of 0.15M cathecol in 0.05M sodium phosphate buffer (pH 6.5). The reaction was

measured with the spectrophotometer at 420 nm and 25…C.

Pectin methylesterase activity was assayed using aliquots (0.05 ml) of extract, adjusted to pH

7.5 if necessary, and a reaction mixture composed by 2.8ml 0.5% (w/v) citrus pectin (pH 7.5), 0.2 ml

0.01% (w/v) bromothymol blue in 0.003M potassium phosphate buffer (pH 7.5). The reaction was

measured spectrophotometrically at 620 nm and 25…C.

All enzyme activities were determined by measuring the slope of the reaction. The enzyme

activity unit was defined as the change in absorbance/min/g fresh weight of sample.

35


Soluble proteins were analyzed according to the Bradford (1976) method, measuring optical

density (OD) at 595 nm, with bovine albumin as a standard.

2.3. Experimental design

High pressure treatments were carried out in triplicate. Values from chemical and biochemical

analysis are averages of three independent determinations. A STATGRAPHICS (Statistical Graphics

system, vers. 7.0, USA) software program was employed for statistical data analysis and graphycal

presentation.

Response surface methodology (RSM) was used to study the simultaneous effect of two processing

variables. The experiments were designed according to a central composite rotatable design (Cochran

and Cox, 1957). The variables studied were pressure and temperature. Five levels of each variable

were chosen in accordance with the

principles of central composite design

(table 2). Thirteen combinations of two

variables were performed following the

designs of Cochran and Cox (1957).

Assessment of error was derived from

replication of treatment combination as

suggested in the design.

For each factor assessed a second-order polynomial equation was fitted as follows:

y = bo + ∑ bixi + ∑ biixi2 + ∑ bijxixj

Where y is the estimated response, bo, bi, bii, bij are equation parameters to be estimated

(constant, bo; coefficients of the linear terms, bi ;for quadratic terms, bii; for interaction terms, bij),

xi, xj are levels of factors and k the number of factors. For each factor the variance was partitioned

into linear, quadratic and interactive components in order to assess the adequacy of the second

order polynomial function and the relative importance of the components. The significance of the

equation parameters for each response variable was assessed by a t-test.


Initial enzyme activities in freshly prepared tomato puree (table 1) showed a very high peroxidase

activity (40.41 ∆OD/min/g f.w.), a relatively low polyphenol oxidase activity (0.79 ∆OD/min/g f.w.),

a fair pectin methylesterase (5.32 ∆OD/min/g f.w.) activity and a protein content of 0.31 mg/g f.w.

These are the control values employed for UHP/temperature inactivation studies. Tomato puree

Table 2Levels of variables in tomato puree UHP processing

according to the experimental design

Pressure (MPa) Temperature (¡C)

50 20.0

115.8 25.0

275.0 40.0

434.1 54.1

500.0 60.0


36

proteins suffered a continuous denaturation when high pressure increased to 500 MPa at

room temperature (20…C), as shown in figure 1. However, when a combined treatment

UHP/temperature was employed, the effects on proteins were different. In general, UHP/mild

temperature treatments caused a continuous denaturation of soluble proteins, but this

denaturation increased with pressure. Better results, in terms of protein modification,

were obtained at temperatures of 20/30…C and pressures between 100-300 MPa.

Peroxidase activity in tomato puree suffered an activation when combined treatments were carried

out at pressures below 350 MPa at room temperature (20…C), while a significant inactivation of this

enzyme can be obtained using treatments at pressures above 350 MPa, as shown in figure 2.

Figure 1 - Effect of P and T on protein content. Untreatedsample (control): 0.311 mg/g

Table 3Regression model for protein extracted from tomato pureea

RC SE SL

Constant 0.190 0.010 0.000

Linear

P -0.090 0.008 0.000

T 0.004 0.008 0.590

Quadratic

P x P 0.020 0.008 0.040

T x T -0.040 0.008 0.002

Interaction

P x T 0.030 0.011 0.017

a P = Pressure (MPa); T = Temperature (¡C); RC = Regression Coefficient; SE = Standard Error;

SL = Significance Level


37

However, combinations of higher pressures (400-500 MPa) and mild temperatures (30-

60…C) produced an increase of this activity. The observed effects of UHP/temperature

treatments in tomato POD activity were opposite of those reported by Cano et al. (1996) for

POD inactivation in strawberry puree and orange juice. In this work, strawberry POD can be

successfully inactivated using combinations of pressures up to 280 MPa and temperatures up

to 45…C. In the same way, these authors obtained good POD inactivation (50%) in orange juice

employing a combination of 400 MPa and 32…C. In all studies, a constant treatment time (15

min) was employed.

Figure 2 - Effect of P and T on POD activity. Untreated sample(control): 40.41 DO/min/g

Table 4Regression model fitted for peroxidase (POD) inactivation in tomato pureea

b RC SE SL

Constant 69.48 2.41 0.00

Linear

P -1.86 1.91 0.36

T 0.89 1.91 0.65

Quadratic

P x P -11.46 2.05 0.00

T x T -9.81 2.05 0.00

Interaction

P x T 6.85 2.70 0.03



38


Similar results were observed for tomato puree polyphenol oxidase (PPO) activity, as shown in

figure 3. The effect of different pressurization/depressurization treatments on this enzyme showed

that tomato puree PPO suffered an increase in activity when a pressure below 200 MPa was

employed, while a significant activity loss was observed using pressures from 200 up to 500 MPa,

working at room temperature (20…C). The better result in terms of PPO inactivation was obtained

using a combination of 200 MPa/20…C (15 min).

Pectin methylesterase activity in fresh tomato puree was 5.32 (∆OD/min/g f.w.). This initial activity

was reduced to 35% using a combined treatment of 150 MPa/30…C during 15 minutes, figure 4. This

combination was the most efficient in terms of PME inactivation. In general, PME is the enzyme most

affected in tomato products by UHP treatments. This higher efficiency of low pressure/mild temperature

treatments on tomato puree PME was also reported in pressurized orange juice (Cano et al., 1996).

Figure 3 - Effect of P and T on PPO activity. Untreated sample(control): 0.792 DO/min/g

Table 5Regression model for polyphenol oxidase (PPO) inactivation in tomato pureea

b RC SE SL

Constant 2.010 0.05 0.00

Linear

P 0.012 0.04 0.76

T 0.044 0.04 0.31

Quadratic

P x P -0.490 0.04 0.00

T x T -0.470 0.04 0.00

Interaction

P x T 0.050 0.05 0.42



39


The activation effects, observed in some cases, as consequence of combined UHP/temperature

treatments could be attributed to reversible configuration and/or conformation changes of the

enzyme and/or substrate molecules (Ogawa et al., 1990; Anese et al., 1995). The pH dependence of

such activation effects seemed to confirm this hypothesis. Tomato puree showed a relatively high

pH value (4.08) and also a fairly low soluble solids content (5.6 Brix at 20…C) compared to other

studies of UHP/temperature effects on other fruit-derived products, such as strawberry puree or

orange juice.

Anese et al. (1995) reported that the pH of enzymatic crude extracts from juices seemed to strongly

affect the extent of enzyme inactivation, which reached a maximum at pH 6.0.

Figure 4 - Effect on P and T on PME activity. Untreated sample(control): 5.32 DO/min/g

Table 6Regression model fitted for pectin methylesterase (PME) inactivation in tomato pureea

b RC SE SL

Constant 7.42 0.20 0.00

Linear

P 0.04 0.15 0.78

T 0.20 0.15 0.23

Quadratic

P x P -0.95 0.17 0.00

T x T -1.40 0.17 0.00

Interaction

P x T -0.58 0.22 0.03

a P = Pressure (Mpa); T = Temperature (¡C); RC = Regression Coefficient; SE = Standard Error;



40

In addition, the soluble solids content of the sample also influences the inactivation of

enzymes (Ogawa et al., 1990). Increased soluble solids protect PME against pressure as well as heat

inactivation. However, in the present study tomato PME suffered a significantly greater

inactivation using UHP/temperature combination than orange juice (Cano et al., 1996), in spite of

this tomato product having a higher pH and lower soluble solids than the orange juice. In this way,

other factors in addition to pH and soluble solids must contribute to the effectiveness or not of

combined UHP/temperature treatments in enzyme inactivation of fruit-derived products.

Acknowledgements

This work was supported by the Spanish project no. ALI94-0786, Comisi�n Interministerial de

Ciencia y Tecnolog�a .

References

Anese, M.; Nicoli, M.C.; Dall,aglio G. And Lerici, C.R. (1995). Effect of high pressure treatments on

peroxidase and polyphenoloxidase activities. Journal Food Biochem., 18, 285-293

AOAC (1984). Official Methods of Analysis, 14th de. Association of Official Analytical Chemists,

Washington, D.C.

Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of

protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254

Cano, M.P.; Mar�n, M.A. and Fuster, C. (1990). Freezing of banana slices. Influence of maturity level

and thermal treatment prior to freezing. Journal of Food Science, 55 (4), 1070-1073

Cano, M.P.; Hern�ndez, A. and De Ancos, B. (1996). High Pressure and Temperature Effects on

Enzyme Inactivation in Strawberry and Orange Products. Journal of Food Science, in press.

Cheftel, J.C. (1991). Applications des hautes pressions en technologie alimentaire. Actualit� des

Industries Alimentaires et Agro-alimentaires, 108 (3), 141-153

Cheftel, J.C. (1992). Effects of high hydrostatic pressure on food constituents: An overview. In High

Pressure and Biotechnology, C. Balny. R. Hayashi, K. Heremans and P. Masson. Editions John Libbey

Euro-text, Montrouge, 195-209

Cochran, W.G. and Cox, G. M. (1957). Experimental Design, 2nd de. Wiley International, New York

Farr, D. (1990). High pressure technology in the food industry. Trends Food Sci. Technol., 1, 14-16

Hoover, D.G.; Metrick, C.; Papineau, A.M.; Farkas, D.F. and Knorr, D. (1989). Biological effects of

high hydrostatic pressure on food microorganisms. Food Technology 43 (3), 99-107

Knorr, D. (1995). High pressure effects on plant derived products. In High Pressure Processing of

Foods, D.A. Ledward, D. E. Johnston, R.G., Earnshaw and A.P.H. Hasting (Ed), Nottinghan University

Press, 123-135


41

Kunugi, S. (1992). Effect of pressure on activity and specificity of some hydrolytic enzymes. In High

Pressure and Biotechnology, C. Balny. R. Hayashi, K. Heremans and P. Masson. Editions John Libbey

Euro-text, Montrouge, 129-137

Mertens, B. and Knorr, D. (1992). Development of nonthermal processes for food preservation.

Food Technology, 46 (5), 124-133

Ogawa, H.; Fukuhisa, K.; Kubo, Y. and Fukumoto, H. (1990). Pressure inactivation of yeast, molds

and pectinesterase in Satsuma mandarin juice: effect of juice concentration, pH and organic acids

and comparison with heat sanitation. Agric. Biol. Chem., 54 (5), 1219-1225

Porreta, S.; Birzi, A.; Ghizzoni, C. and Vicini, E. (1995). Effects of ultra-high Hydrostatic pressure

treatments on the quality of tomato juice. Food Chemistry, 52, 35-41


You observed POD and OPP (re-)generation during frozen storage. Are you sure that it is an

artifact related to the sensitivity/specificity of your assays, or is there enough enzyme

acivity at freezing temperature to drive the (re-)generation process?

Leon Gorris

Yes, we concluded that it is a regeneration, because we did not study these enzymes only

from the point of view of quantification of enzyme activity. We also characterised the

enzymes by biochemical techniques, and the enzyme activity during frozen storage (in the right

analytical conditions of assay for each tissue) was enough to establish this conclusion. This work

was published in 1994.

A

Q

42


Abstract

The samples of ripened, sliced cheeses, minced beef and cured, sliced ham were placed in

commercially used polyamide-polyethylene bags, inoculated with L. monocytogenes and vacuum

sealed. The samples were exposed to hydrostatic pressures ranging from 100 to 500 MPa for 5, 10

and 15 min. The numbers of L. monocytogenes and Aerobic Plate Counts in high pressure treated

and in control samples were determined using the decimal dilution technique. The 6D-values, i.e.

time required for reduction of bacteria by 6 log cycles for different pressure levels, were

calculated. The uninoculated samples treated with high pressure were used in sensory studies. It

was found that L. monocytogenes subjected to high pressure in ripened cheeses is more resistant

than in minced beef and cured, sliced ham. Generally, the results confirm the usefulness of high

pressure technology for inactivation of L. monocytogenes and reduction of inherent microflora in

vacuum-packaged food products.

1. Introduction

During industrial processing of food, particularly during slicing and packaging, secondary

contamination is practically unavoidable (Berends et al., 1995). Microbial contamination consists of

various microorganisms including pathogenic bacteria: Staphylococcus aureus, Salmonella spp.,

Listeria spp. (Berends et al., 1995; Kwiatek, 1991, 1992, 1993). Contamination with L. monocytogenes

seems to be particularly dangerous. Contrary to the majority of pathogens, L. monocytogenes

(classified as a psychotrophic micro-organism) has the ability to multiply in vacuum-packaged

food products, stored in a refrigerator (Grau and Vanderlinde, 1990). Listeria monocytogenes is

responsible for severe food poisonings and may cause death in sensitive persons (Zar�ba and

Borowski, 1994). Foodborne listeriosis outbreaks have been linked to the consumption of dairy

products including raw and pasteurized milk, ice cream and a variety of cheeses, meat and meat

products, as well as chicken and fish (McLauchlin, 1992).

Various control measures have been applied to reduce the incidence of listeria in food

including an improvement of GMP by introduction of the HACCP concept, however a complete

Comparison of the Influence of High Pressure Treatment on the Survival of ListeriaMonocytogenes in Minced Meat, Sliced, Cured Ham and Ripened, Sliced Cheeses

J. Szczawinski1, M. Szczawinska1, B. Stanczak1, M. Fonberg-Broczek2,3,

J. Arabas2, J. Szczepek2 and S. Porowski2

1Warsaw Agricultural University, Faculty of Veterinary Medicine,

Department of Food Hygiene, Warszawa, Poland2Polish Academy of Sciences, High Pressure Research Center, Warszawa, Poland

3National Institute of Hygiene, Warszawa, Poland

43

High PressureSzczawinski, Szczawinska, Stanczak, Fonberg-Broczek

Arabas, Szczepek & Porowski

elimination of the pathogen by those means seems to be impossible (Tompkin, 1990). Properly

conducted heat treatment during preparation of food at home as well as an industrial pasteurization

or sterilization should cause complete inactivation of L. monocytogenes (Sta�czak and Szczawi�ski,

1996), however most vacuum-packaged food products cannot be subjected to heat treatment.

Ionizing radiation has been successful in removing non-spore-forming pathogens, like Listeria and

Salmonella, from vacuum-packaged food products (Huhtanen et al., 1989; Szczawi�ska, 1994;

Szczawi�ski et al., 1996b), but so far radiation treatment has not been well accepted by consumers.

The aims of this study were: (a) to compare the effect of high pressure on the survival of L.

monocytogenes in minced beef, cured ham and in three different ripened, hard cheeses (Edamski,

Gouda, Podlaski - the most common cheeses in Poland), sliced and vacuum-packaged in

polyamide-polyethylene bags; (b) to compare the effect of high pressure on the sensory

properties of the treated samples.


Lean beef meat was minced, divided into 10-gram portions and placed in polyamide-polyethylene

bags (Multiseven 78 TOP, Wipak, Finland). In order to restrict the growth of saprophytic microflora

(which might have affected the determination of L. monocytogenes), the samples were decontaminated

by gamma irradiation at a dose of 10 kGy. Cured, sliced, pasteurized pork ham and hard, sliced cheeses

(Edamski, Gouda, Podlaski), were divided into 10 g portions. Every portion, made of two adjoining

slices, was placed in a commercially used polyamide-polyethylene bag. These samples were not

exposed to ionizing radiation. All samples were inoculated with L. monocytogenes (a mixture of

strains isolated from milk, obtained from the Polish National Veterinary Institute, incubated on BHI

at 37oC for 24 h) and vacuum-packaged (Henkovac 1000).

The samples were exposed to high pressure treatment in the High Pressure Research Center of

the Polish Academy of Sciences, where a special stand for food testing was constructed. The

process of high pressure treatment requires the following steps: a high pressure chamber, closed

from the bottom with a special cover, is filled with distilled water. After placing vacuum-packaged

food products in the chamber, the top cover is closed. Pressure is generated in 10-60 seconds by

a special plunger, descending into the chamber. The measurement of hydrostatic pressure inside

the chamber is done indirectly with the help of a manometer, indicating the pressure under the piston

rod. After the high pressure treatment is finished, the pressure in the chamber is relieved,

approximately twice slower than it has been generated.

The samples of minced beef and cured ham were subjected to 100, 150, 200, 300 and 400 MPa

of hydrostatic pressure, whereas the samples of cheeses were treated with 200, 350 and 500 MPa.

All samples were exposed for 5, 10 and 15 min. After treatment, the samples were stored at 4oC for

18 hours. The number of surviving listeria in each sample per gram was determined by the ten fold

dilution method and plating onto Oxford Agar (Oxoid). The plates were incubated for 72 hours at 30oC.

44


The bacterial counts were transformed into logarithms and statistically analyzed.

Additionally, some control (uninoculated) samples for sensory studies were treated with high

pressure. The appearance, colour, smell and taste of the samples were evaluated by a trained panel

(6 persons) using 9-point quality scores.


In previous studies (Szczawi�ski et al., 1995a, 1995b, 1996a) it was found that mathematical

parameters applied in thermobacteriology, i.e. D-value (time required for decimal reduction of L.

monocytogenes at given pressure) and z-value (coefficient of resistance of L. monocytogenes to high

pressure), can be also useful for predicting the effects of high pressure treatment on microflora

present in food. Because it is generally accepted that reduction of L. monocytogenes by 6 log units

in slightly contaminated food is large enough for consumer safety, 6D values (time required for

reduction of bacteria by 6 log cycles at given pressure) were calculated on the basis of those

previously obtained results.

The results presented in figure 1 demonstrate that the resistance of L. monocytogenes to high

pressure is different in various foods.

Figure 1 - Comparison of 6D-values for L. monocytogenes in different products at various pressure levels

45



In hard cheeses bacteria are much more resistant to pressure than in minced meat and

cured ham. This could be explained by the lower water activity of the cheeses tested compared

to beef and ham (Johnston, 1995). It is more difficult to find a reason for the large differences

in 6D-values found in the three kinds of cheeses, because all of them had similar basic

chemical compositions (content of fat, water and protein), pH and physical structure. Those

differences are particularly visible at the lowest (100 MPa) pressure (6D value ranged from 264

to 2460 min). The 6D-values for various products tend to level off together with the increase

of pressure. Therefore, it seems that the effect of high pressure treatment on microflora can be

predicted better at higher levels of pressure. At 500 MPa the 6D values for the specific food

products ranged from only 6 to 17 min.

Raw meat samples which were subjected to high pressure treatment revealed undesirable

organoleptic changes, especially visible when pressures of 300 and 400 MPa were employed.

Even before opening the experimental bags, changes in colour and consistency and leakage

were observed. Organoleptic changes of raw meat subjected to high pressure were reported

by other researchers (Johnston, 1995; Lewicki, 1992).

Samples of cured, sliced and pasteurized pork ham subjected to the pressure of 400 MPa

for 15 min did not differ statistically from untreated samples in general appearance, smell and

taste. High pressure (400 MPa for 15 min) had a slight impact only on the colour of cured ham.

High pressure treatment (up to 500 MPa) did not have any influence on appearance, smell

and taste of the ripened, hard cheeses. Only once, in the case of Gouda cheese, a positive

effect of high pressure on the colour of the samples was found (statistically significant

difference at P < 0.05).

4. Conclusions

The application of high pressure technology to the processing of vacuum-packaged cured,

pasteurized pork ham and hard cheeses can cause reduction in L. monocytogenes contamination

by 6 logarithmic cycles, with practically unchanged organoleptic product quality. The results

obtained seem to be promising and confirm the need for further investigations in this field.

Acknowledgements

This work was founded by National Committee of Scientific Research, Poland, Grant Nr PB

112/P06G 9610 1996/97 and High Pressure Research Center of Polish Academy of Sciences,

Warsaw, Poland

46


References

Berends, B., Burt S., Snijders J. (1995). Critical points in relation to breaking Salmonella and

Listeria cycles in pork production. New challenges in meat hygiene: specific problems in cleaning

and disinfection. Ed. Sara A. Burt and Friedrich Bauer. Utrecht ECCEAMST Foundation, The

Netherlands, 9-20

Johnston, D.E. (1995). High pressure effects on milk and meat. High Pressure Processing of

Foods. Ed. Ledward D.A., Johnston D.E., Earnshaw R.G., Hasting A.P.M., Nottingham University

Press, 99-121

Grau, F. H., Vanderlinde, P.B. (1990). Growth of Listeria monocytogenes on vacuum-packaged beef.

J. Food Prot. 53, 739-741

Huhtanen, C. N., Jenkins, R. K. and Thayer, D. W. (1989). Gamma radiation sensitivity of Listeria

monocytogenes. J. Food Prot. 52, 610-613

Kwiatek, K. (1991). Wystepowanie Listeria monocytogenes w wolowinie, wieprzowinie i miesie

drobiowym. Medycyna Weterynaryjna, 42 (12), 69-71

Kwiatek, K., Wojton, B., Rola, J., R�zanska, H. (1992). The incidence of Listeria monocytogenes and

other Listeria spp. in meat, poultry and raw milk. Bulletin of Veterinary Institute in Pulawy, 35, 7-11

Kwiatek, K. (1993). Wystepowanie L. monocytogenes w miesie oraz w produktach miesnych. Zycie

Weterynaryjne, (12), 304-06

Lewicki, P.P. (1992). Zastosowanie wysokich cisnien w technologii zywnoˇci. Przemysl Spozywczy,

46 (11), 280-282

Mc Lauchlin, J. (1992). Listeriosis. Declining but may return. Brit. Med. J. 304, 1583-1584

Rönner, U. (1994). Food preservation by ultrahigh pressure. Food Preservation by Combined

Processes. Ed. Leistner L., Gorris L.G.M., Final Report FLAIR Concerted Action No. 7, Subgroup B,

EUR 15776 EN, 1994, 31-35

Stanczak, B.J., Szczawinski, J. (1996). Cieploopornosc Listeria monocytogenes w mleku i smietance

o r�znej zawartosci tluszczu. Medycyna Weterynaryjna, 52 (6), 389-391

Szczawinska, M.E. (1994). Prospects for elimination of salmonellae from poultry by irradiation in

Poland. IAEA-TECDOC-754, 77-96

Szczawinski, J., Peconek, J., Fonberg-Broczek, M., Arabas, J., Szczawinska, M. (1995a). Mozliwosc

zastosowania wysokich cisnien do inaktywacji L. monocytogenes w miesie i przetworach miesnych.

Magazyn Weterynaryjny 4 (6), 516-519

Szczawi�ski, J., P�conek, J., Szczawi�ska, M., Porowski, S., Fonberg-Broczek, M., Arabas, J. (1995b).

High pressure inactivation of Listeria monocytogenes in meat and meat products. Proceedings of

the first main meeting Process Optimization and Minimal Processing of Foods. Copernicus Programme.

Contract CIPA - CT94-0195, Porto, November 12th, 1995. In press.

Szczawinski, J., Szczawinska, M., Stanczak, B., Fonberg-Broczek, M., Arabas, J., Szczepek, J. (1996a).

Effect of high pressure on survival of Listeria monocytogenes in ripened, sliced cheeses at ambient

temperature. Proceedings of XXXIV Meeting High Pressure Bio-science and Bio-technology joint

47



Meeting with Japanese and European Seminar on High Pressure Bioscience and biotechnology.

Katholieke Universiteit Leuven, Belgium, September 1-5, 1996. In press.

Szczawinski, J., Szczawinska, M. E., Stanczak B. (1996b). Radioresistance of Listeria monocytogenes

in meat and cheese. Acta Alimentaria, 25 (4), 397-401

Tompkin, R. B. (1990). The use of HACCP in the production of meat and poultry products. J. Food

Prot. 53, 795-803

Zar�ba, M.L., Borowski, J. (1994). Podstawy mikrobiologii lekarskiej. Ed. Wydawnictwo Lekarskie

PZWL, Warszawa


Did you use a simple strain in a mixture of Listeria monocytogenes, because it is well known

that there are big differences between strains on even within a population?

Leon Gorris

A mixture of five (5) Listeria monocytogenes strains isolated from milk was used in the

experiments.A

Q

48


Abstract

This communication describes a new equipment for high pressure food processing on

laboratory scale, developed and installed in the High Pressure Research Center of the Polish

Academy of Sciences (Warsaw) in 1996. The main part of the processor is a multilayer vessel with

inner diameter of 110 mm, total working volume of 1.5 litre and pressure up to 700 MPa. The

vessel is pressurised by means of a high-capacity hydraulic press 10 MN equipped with special

tools for operating the vessel during food processing. Using the equipment, about 250 kg of

various fruit jams and low-sweetened fruit desserts were produced during the last 6 months,

packed in commercially available plastic packages. The most common Polish fruits were used for

jam production: strawberries and apples. Low-sweetened fruit desserts were manufactured from

raspberry, blueberry, strawberry, cherry, apricot and pears. As a result, high pressure fruit products

were obtained which maintained very good sensory and microbiological properties during 5

month-storage at temperatures +4…C to +6…C.

1. Introduction

The high pressure method of food preservation was introduced in the early 1990Õs, on limited

scale, after suppressing a range of technical problems and technological barriers. In this method,

food products are placed in suitable high pressure containers and subjected to high pressure

treatment at pressures up to 800 MPa. This treatment leads to inactivation of micro-organisms and

preserves food products without harmful influence on organoleptic properties, quality and taste of

the pressurised products.

Food products preserved using high pressure, often referred to as Ultra High Pressure (UHP)

products, were first introduced in Japan, on a limited scale. Although the prices of UHP products

are higher compared to those of conventional products, the new UHP products looked

competitive and requested, especially by more wealthy consumers.

The High Pressure Research Center (HPRC) of the Polish Academy of Sciences in Warsaw (Poland)

is involved in high pressure research since the early 1970Õs, mainly in physics and material sciences.

The Laboratory of High Pressure Processing of Food Products of the High Pressure Research Center UNIPRESS, Warsaw

J. Szczepek1, J. Arabas1, M. Fonberg-Broczek1,2, T. Strzelecki1,

J. Zurkowska-Beta1 and S. Porowski1

1 High Pressure Research Center, Polish Academy of Sciences, Warsaw, Poland2 National Institute of Hygiene, Warsaw, Poland

49

High PressureSzczepek, Arabas, Fonberg-Broczek. Strzelecki,

Zurkowska-Beta &Porowski

High pressure research in HPRC is strongly accompanied by the development and construction of

laboratory equipment. This high pressure equipment is in use in many research laboratories all over

the world.

An the HPRC, research works are carried out on application of high pressure in the

preservation of food products since the early 1990Õs . Up to now, two research programmes in this

field were conducted, sponsored by the Polish Committee of Sciences.

In the first programme, Application of high pressure in processing of fruits and vegetables (1993-94),

the pressure-induced inactivation of various micro-organisms in fruit and vegetable products was

studied. Thanks to this programme, the first UHP fruit jams were manufactured. The experiments

were conducted in a rather small pressure vessel with a diameter of 50 mm and the samples were

packed in plastic bags.

The second research programme, Investigation of biologically-active substances and inactivation of micro-

organisms in jellied fruit products preserved by high pressure (1996-97) is being currently conducted.

The results of these investigations are indicated in the References.

This communication describes a new laboratory stand for high pressure food processing on

laboratory scale, developed and installed in the HPRC in 1996. Using this stand, the production of

UHP fruit products was undertaken. During the last 6 months about 250 g of various fruit jams and

low-sweetened fruit desserts were produced, packed in commercially available plastic packages.

2. Laboratory Equipment for Processing UHP Products

Taking advantage of the significant

experience in the design and manufacturing of

high pressure laboratory equipment during the

last 25 years, and in particular thanks to the

experience collected during the exploitation of

the first laboratory stand for investigation of

food samples with capacity of 0.5 litre, a new

high pressure laboratory food processor with

1.5 litre volume and working pressure up to

700 MPa at temperatures from -20 …C to

+90…C was installed in 1995-96.

The vessel is pressurised by means of a

high capacity two-drive hydraulic press 10 MN.

The ÒheartÓ of the processor is a piston-

cylinder-type multilayer vessel (figure 1., 1)

with inner diameter of 110 mm and volume

of about 1.5 litre.

Figure 1 - Scheme of the equipment for laboratory highpressure processing of food products in theHigh Pressure Research Centre, Warsaw 1 -high pressure vessel, volume 1.5 litre, diameter110 mm, max. pressure 700 MPa, 2 - spiralheat exchanger, 3 - bottom closure, 4 - upperclosure, 5 - moveable plunger, 6 - manometer

50


The vessel has the form of a compound cylinder, incorporating an external thick-wall body made of

high quality steel and internal thin layers made of stainless steel. According to the general requirements

for food processing, water is used as the pressure transmitting medium in the processor. The vessel

is supplied with a spiral heat exchanger situated between the inner layers (figure 1, item 2).

The vessel is closed from the bottom with a steady plug with diameter of 110 mm (figure 1,

item 3). The plug contains a threaded socket for a connecting pressure gauge. The upper closure

of the vessel forms a plug (figure 1, item 4) with a diameter of 110 mm. The plug incorporates a

moveable plunger (figure 1, item 5) with a diameter of 80 mm, which generates pressure when

pushed by the press into the vessel. The plugs and the plunger are sealed with multi-use seals. The

upper plug with the plunger is integrated with the upper piston of the two-drive hydraulic press.

For easy operation of the vessel, the press is equipped with a rail transport system. Thanks to

it, the vessel can be easily loaded/unloaded outside the press. After loading the vessel with the

products and placing the vessel inside the press, the lower piston of the press lifts the vessel and

closes the upper closure. Afterwards, the upper piston of the press moves down, and pushing the

plunger into the vessel, generates pressure.

The pressure in the vessel is measured with a Bourdon gauge (figure 1, item 6).

Figure 2 shows the vessel placed in the two-drive hydraulic press 10 MN.

3. Preparation and High Pressure Processing of Fruit Products

Fresh fruits - strawberry, apple, cherry, blueberry, raspberry, apricot, blackberry and pear were used

for the production of high pressure fruit products. All fruits were of very good quality, without any

mechanical (bruise, crushing) nor biological damage (mouldiness, wounds caused by insects or birds).

Figure 2 - Photograph of the equipment forlaboratory high pressure processingof food products

51



Fruits were carefully sorted out and stoned, and other useless particles (peduncles) were also

removed. Next, the fruits were crumbled, depending on the type of fruit by means of cutting

(apple, pear) or mixing (strawberry), or left as a whole (blueberry, raspberry). The prepared fruits

were then placed in a special container suitable for heating and afterwards pectin (gelling substance)

with citric acid (acidity regulator) were added - about 40 g per 1 kg of fruit (preparation Zelfix). After

thorough mixing, the contents were heated up to the moment when pectin dissolved totally in the

fruit juice. The process lasted usually 2-3 minutes and the temperature reached 40…-50…C.

Subsequently, an amount of 50-100 g of sugar per 1 kg of fruit was added (5-10%;), depending

on the type of fruit. The mixture was heated again for the next 2-3 minutes until the sugar

dissolved totally. At the end of this process the temperature reached about 60…C. Warm fruit

dessert was then placed in commercially produced polystyrene packages. The packages were then

closed with thin metal foil cover, sealed by means of a heat-sealer.

The closed packages were then placed in a high pressure vessel and exposed to a pressure of

400 MPa, during 10 minutes at ambient temperature (22…C). This combination of the operating

variables was developed during previous model studies and accepted as optimal for laboratory

experiments.

The parameters of the pressurisation process applied to the UHP fruit jams and low-sweetened

products are summarised in table I.

4. Results

After high pressure treatment, the UHP fruit products were stored in a refrigerator at

temperatures from +3…C to +5…C for a period of 5 months. After this period, the UHP products

underwent a series of microbiological investigations, conducted in the Sanitary - Epidemiological

Station in Warsaw.

Table IParameters of the pressurisation process applied to our UHP

fruit jams and low-sweetened products

Total production of UHP fruit products: 250 kg

Fruits used: strawberry, apple, cherry, blueberry, raspberry,

apricot, blackberry, pear

kind of products: fruit jams

low-sweetened fruit desserts

Description of the process 1. Preliminary thermal processing:

temperature 40… to 60… C.

time 2 -3 minutes

2. High pressure processing: pressure 400 MPa

temperature: ambient

time: 10 minutes

52


The products were examined for content of moulds and yeasts. These investigations confirmed

that the UHP fruit products are characterised by lightly gelatinous consistency of fruit jelly, natural colour

and fresh fruit aroma. They are significantly more tasteful than products prepared using traditional

food processing technology. The low content of sugar of the UHP fruit products should be noted again.

Currently, studies conducted are devoted to the evaluation of the influence of high pressure on

enzyme activity in fruits treated with UHP.

5. Conclusions

The equipment for laboratory high pressure processing of food products developed and

installed recently in the High Pressure Research Center in Warsaw was subjected to about 500 high

pressure cycles. Both initial tests as well as pressure cycles during pressurisation of fruit products

proved that the equipment is a very good and efficient food processor for pilot scale food

processing under high pressure.

Using this apparatus a significant amount of UHP fruit products has been produced during the

last six months: fruit jams and low sweetened fruit desserts. The UHP products were then stored

during 5 months and afterwards a series of biological investigations were conducted. The samples

proved to be microbiologically pure, without any signs of spoilage after a 5-month storage period.

Acknowledgements

This research was partially sponsored by the Polish Committee of Sciences (grant Nr

5P06G00510

References

Arabas J., Fonberg-Broczek M. (1995). High pressure food processing acivities in the High Pressure

Research Center - Warsaw, Poland. 1st Main Meeting ÒProcess Optimization and Minimal

Processing of FoodsÓ COPERNICUS PROGRAMME, CONTRACT CIPA - CT94-0195, Porto, December

6-8 1995, in press.

Fonberg-Broczek M., Windyga B., Sciezynska H., Grochowska A., Gorecka K., Salanski P., Arabas J.,

Szczepek J., Podlasin S., Porowski S. (1995) Effect of high hydrostatic pressure on microorganisms.

Symposium: High pressure effects on foods, 9th World Congress of Food Science and Technology,

Budapest, Hungary, 07.30-8.04,1995, Abstracts Vol. II. P207, p. 74.

Fonberg-Broczek M., Windyga B., Sciezynska H., Gorecka K., Grochowska A., Napiorkowska B.,

Karlowski K., Arabas J., Jurczak J., Podlasin S., Porowski S., Salanski P., Szczepek J. (1995) The effect



53

of high hydrostatic pressure on vegetative bacteria and spores of Aspergillus flavus and Bacillus

cereus. Joint XV AIRAPT & XXXIII EHPRG International Conference ÒHigh Pressure Science &

TechnologyÓ, Warsaw, Poland, September 11-15, 1995, Conference Proceedings ÒHigh Pressure

Science & TechnologyÓ, ed. W. Trzeciakowski, World Sci. Publ. 892-894

Fonberg-Broczek M., Arabas J., Porowski S. (1995) The Effect of High Hydrostatic Pressure in

Vegetative Microorganisms and Spores of Chosen Moulds. 1st Main Meeting ÒProcess

Optimization and Minimal Processing of FoodsÓ COPERNICUS PROGRAMME, CONTRACT CIPA -

CT94-0195, Porto, December 6-8 1995, in press

Fonberg-Broczek M., Arabas J., Porowski S., Windyga B. (1995). The Effect of High Hydrostatic

Pressure in Vegetative Microorganisms and Spores of Chosen Moulds. 1st Main Meeting ÒProcess

Optimization and Minimal Processing of FoodsÓ COPERNICUS PROGRAMME, CONTRACT CIPA -

CT94-0195, Porto, December 6,-8 1995, in press

Kolakowski P., Reps A., Babuchowski A., Zmudzian L, Podlasin S., Porowski S. (1995) Influence of

high pressure on changes of cheeses characteristics. Joint XV AIRAPT & XXXIII EHPRG

International Conference ÒHigh Pressure Science & TechnologyÓ, Warsaw, Poland, September 11-

15, 1995, Conference Proceedings ÒHigh Pressure Science & TechnologyÓ, ed. W. Trzeciakowski,

World Sci. Publ., 898-901

Sa3a�ski J., Jurczak J., Fonberg-Broczek M., Szczepek J. (1996). Influence of Ultra High Pressure on

chosen natural plant pigments. European High Pressure Research Group, XXXIV Meeting High

Pressure Bio-Science and Bio-Technology, Leuven, Belgium, Sept. 1-5, 1996.

Szczawinski J, Peconek J., Szczawinska M., Porowski S., Fonberg-Broczek M., Arabas J. (1995) The

Effect of High Hydrostatic Pressure on Listeria monocytogenes in Minced Meat and Sliced Ham in

Ambient Temperature. 1st Main Meeting ÒProcess Optimization and Minimal Processing of FoodsÓ

COPERNICUS PROGRAMME, CONTRACT CIPA - CT94-0195, Porto, December 6-8 1995, in press

Szczawi�ski J., P�conek J., Szczawi�ska M., Fonberg-Broczek M., Arabas J. (1995). High pressure

inactivation of Listeria monocytogenes in meat and meat products, Process Optimization and

Minimal Processing of Foods. Contract CIPA CT 94-0195 COPERNICUS PROGRAMME, I Main

Meeting, Dec,6-8, 1995 , PORTO, in press

Szczawi�ski J., Szczawi�ska M., Sta�czak B., Fonberg-Broczek M., Arabas J. Szczepek J. (1996).

Influence of Ultra High Pressure on survival of Listeria monocytogenes in riplned, sliced cheeses in

ambient temperature. European High Pressure Research Group, XXXIV Meeting, High Pressure

Bio-Science and Bio-Technology, euven, Belgium, Sept. 1-5, 1996

Szczepek J., Arabas J. (1996). Experimental techniqe for high pressure ivestigationin chemistry,

biology and food scienxce. European High Pressure Research Group, XXXIV MeetingHigh Pressure

Bio-Science and Bio-Technology, Leuven, Belgium, Sep. 1-5, 1996.

Windyga B., Fonberg-Broczek M., Sciezynska H., Gorecka K., Grochowska A., Arabas J., Szczepek

J., Podlasin S., Porowski S. (1994) Effect of high hydrostatic pressure on yeast Candida albicans. 7th

International Congress of Bacteriology and Applied Microbiology Division, 7th International Congress

of Mycology Division, Prague, Czech Republic, July 3rd - 8th, 1994, Abstract book, PC-6/52, p. 272

54


Abstract

Air-blast and high-pressure-assisted freezing systems were used to freeze Longissimus dorsi

muscle of pork meat. Samples were taken from both the centre and the surface region and

processed by freeze substitution. They were fixed at the final freezing temperature (-20…C) with

Carnoy fluid. Finally, the samples were micrographed by light microscopy. When air-blast

freezing was used, large extracellular crystals located both at the surface and at the centre

region were observed. With high-pressure-assisted freezing, small intracellular crystals were

obtained both at the surface and at the centre regions. Thus, freezing with the high-pressure-

assisted method shows a uniform and homogeneous nucleation that produces a large amount of

small crystals located in the whole volume of the sample, avoiding the cellular damage

produced by big ice crystals.

1. Introduction

Water, the main constituent of foods, can be located inside and outside the cells. This water

can be divided into two types: bound water and bulk water. Bound water is defined as the water

in the vicinity of macromolecules whose properties differ detectably from those of pure water; i.e.

it does not freeze at low temperatures, shows less mobility and a lower water vapour pressure

than pure water (Kuntz and Kauzman, 1974). The bulk water is the part of water in foods which

has properties that are similar to those of pure water and in the freezing process, bulk water of

foods is the fraction that changes its state from liquid to solid.

Ice, with a larger volume than liquid water, is the main agent that produces cellular damage

and affects the final quality of the frozen food obtained. Damage can be more or less important

depending on the size and location of ice crystals. These two factors are a function of the

freezing rate.

The higher the freezing rate, the smaller the ice crystals. Therefore, with low freezing rates

and higher temperature gradients, big ice crystals are produced and with high freezing rates and

lower temperature gradients, many very small crystals are formed (Bevilacqua et al., 1979).

Effect of High-Pressure-Assisted Freezing and Air-Blast Freezing on the Microstructure of Pork Meat

P.D. Sanz1, N. Zaritzky2, L. Otero1 and M. Martino2

1 Instituto del Fr�o (C.S.I.C.), Ciudad Universitaria, 28040-Madrid, Spain2 Centro de Investigaci�n y Desarrollo en Criotecnolog�a de Alimentos,

47 y 116, 1900-La Plata, Argentina

55

High PressureSanz, Zaritzky, Otero & Martino

At the beginning, the crystallisation is always extracellular. The first water that is frozen is

water at the meatus since the freezing point is higher because of the more diluted fluid here than

inside the cell. Afterwards, with low freezing rates water leaves the cells because of the vapour

pressure difference between water inside the cell and water at the meatus. Ice at the extracellular

space grows, distorting and desaggregating the fibres. As a result of the dehydration, protoplasm

shrinks and an irreversible plasmolisis is produced because of the denaturation of proteins that

lose its water retention capacity. With very high freezing rates, crystallisation occurs inside and

outside the cell simultaneously and there is no translocation of water (Menegalli and Calvelo,

1979).

The objective of this work was to compare the different size and location of ice crystals

formed in pork meat frozen by air-blast and by high-pressure-assisted freezing methods.


2.1. Materials

Cylinders of Longissimus dorsi post-rigor pork muscles of approximately 5 cm in height, 7.5 cm

in length and 11 cm in width were cut with the principal axis parallel to the muscle fibres. Each

sample was isolated with a poliethilene bag.

2.2. Methods

2.2.1 Freezing methods

Two different freezing methods were studied: air-blast and high-pressure-assisted freezing. Air-

blast freezing was chosen to represent the most usual freezing method in the industries when large

pieces of meat are frozen and it was compared with the novel high-pressure-assisted freezing

method. In each experiment the thermal history of the meat was registered during freezing with two

thermocouples of copper-constantan

inserted at two predetermined points in

the samples: one at the surface and the

other at the centre.

Air-blast freezing was performed in a

Lab Freezer device (FRIGOSCANDIA, S.A.;

Paris, France) at a fixed temperature of -

40…C. Air speed was about 5.5 m/s. A

thermocouple was connected to register

the thermal history at the surrounding

medium (figure 1).Figure 1 - Air-blast freezing device


56

High-pressure-assisted freezing was performed in an experimental equipment (ACB GEC

ALSTHON, Nantes, France) that was complemented with a thermally isolated thermostatic circuit

and with a pressure and temperature computerised measuring system (figure 2). Two

thermocouples were placed at the beginning and at the exit of the thermostatic circuit,

respectively, to register the thermal history (Sanz et al. 1997).

2.2.2. Histological method

Immediately after freezing, the pieces of meat were introduced in a chamber at -20…C. Inside

the chamber, small cylindrical samples of about 5mm in diameter and 5mm in height were taken

for subsequent histological examination. Two samples were taken from each piece of meat: one

located at the surface and the other at the centre.

For the microscopic analysis of the frozen tissue, an indirect technique was used based on the

observations of the holes left by the ice in the tissue. The technique used was a modification of

the classical freeze-substitution method (Cerrella

and Zaritzky, 1975) in which the samples were

fixed at the final freezing temperature with

Carnoy fluid which has a low freezing point and

diffuses rapidly through the tissue (Martino and

Zaritzky, 1986). Sections of the samples were

stained with hematoxylin-eosin for histological

examination and in each experiment a sample

of fresh meat was also processed at the same

time to act as a control (figure 3). Figure 3 - Fresh pork meat

Figure 2 - High-pressure assisted freezing device

57

High PressureSanz, Zaritzky, Otero & Martino


The micrographs obtained show the size and location of the ice crystals in pork meat frozen

by air-blast and by high-pressure-assisted freezing methods.

When meat is frozen by air-blast (low freezing rate) only extracellular ice crystals appear

(figures 4 and 5). There are few and big crystals. These crystals desaggregate the fibres causing

important damages and cells are dehydrated and distorted. When micrographs from the surface

and from the centre of the sample are studied, differences are found between them. Crystals at

the centre of the sample are bigger than those at the surface because of thermal gradients

established between surface and centre, which means that the freezing rate at the centre of the

sample is lower that at the surface.

On the other side, micrographs of meat frozen with high pressure show a lot of very small

crystals inside and outside the cells (figures 6 and 7) causing minimal damages in the

microstructure. These crystals must be formed instantaneously in the whole volume of the

meat because of the supercooling reached in the sample when this freezing method is

performed (Otero et al., 1996). No differences are shown between the centre and the surface

of the sample.

Figure 4 - Air-blast frozen meat (surface region)

Figure 6 - High-pressure-assisted frozen meat(surface region)

Figure 7 - High-pressure-assisted frozen meat(central region)

Figure 5 - Air-blast frozen meat (central region)

58


4. Conclusions

From the results obtained it can be concluded that high pressure assisted freezing is an

interesting freezing method because of the small and numerous ice crystals located uniformly

along the sample. This freezing method can be specially useful to freeze foods with large

dimensions in which significant thermal gradients are established when classical freezing

methods are used.

References

Bevilacqua, A.; Zaritzky, N.E. and Calvelo, A. (1979).Histological measurements of ice in frozen

beef. J. Fd. Technology, 14, 237-251.

Cerrella, E.G. and Zaritzky, N. E. (1975). CIDCA. Pub. Int. n… 9.

Kuntz, I.D. and Kauzman, W. (1974). Hydratation of Proteins and Polypeptides. Adv. Protein Chem.

vol. 38. 239-245.

Martino, M., Zaritzky, N.(1986). Fixing conditions in the freeze substitution technique for light

microscopy observation of frozen beef tissue. Food Microstruct. 5, 19-24.

Menegalli, F. C. and Calvelo, A. (1979). Dendritic growth of ice crystals during the freezing of beef.

Meat science, vol. 3, 179-198.

Otero, L., Sanz, P.D., de Elvira, Carrasco, J.A. (1996). Modelling thermodynamic properties of water

in the high-pressure-assisted freezing process. XXXIV Meeting High Pressure Bio-Science and Bio-

Technology. Kat. Uni. Leuven, B�lgica, 1-5 Sep.

Sanz, P.D., Otero, L., de Elvira, L., Carrasco, J.A. ÒFreezing processes in high-pressure domainsÓ

International Journal of Refrigeration. To be published.

2nd Meet Hpressure

Documents

phase contrast microscopy

blast frozen meat

van den broeck

assisted frozen meat

simulated temperature profiles

kc sh slce

le se li1s

high pressure treatment