L'utilizzazione dei campi elettrici pulsati per l ... · Unpasteurized milk or juice Salmonella ,...

Giovanna Ferrari Dipartmento di Ingegneria Industriale ProdAl Scarl – Centro di Competenza Produzioni Agro-Alimentari Università di Salerno – 84084 Fisciano (SA) [email protected] www.prodalricerche.it

L'utilizzazione dei campi elettrici pulsati per

l'inattivazione microbica e l'elettroporazione di

membrane vegetali e della luce pulsata nel

trattamento di alimenti

Milano, 7 novembre 2013

mailto:[email protected]

http://www.prodalricerche.it/

UNISA & PRODAL SCARL

•Schools buildings •Laboratories •Canteen •Dormitories •Sport facilities •Campus transportation system

The Campus is located in Fisciano,12 km from Salerno

UNISA

Primary production

First transformation

Second transformation

… n transformation

Distribution and sale

Consumer

Traceability

Input

Process 1

Process 2

Process … n

Output

Integrated supply chain

Prodal is an Applied Research Centre which is comprised of 7

research institutes (over 400 people) that deals with product

innovation and process innovation in the food industry.

The Centre operates by integrating multidisciplinary skills of the

human resources in order to support agrifood companies

throughout the supply chain.

PRODAL SCARL

At present, ProdAl partners are:

University of Salerno

University of Napoli “Federico II”

Second University of Napoli

University of Napoli “Parthenope”

University of Sannio

National Research Council

Experimental Station SSICA – Angri

PRODAL SCARL

The Board of directors, chairman Professor Giovanna Ferrari.

The Scientific Committee, advising, proposing and addressing the scientific activities of the company, chairman Professor Giorgio Donsì.

PRODAL SCARL

The Mission of ProdAl is to promote the technology transfer,

transforming the experimental development into usable

innovation.

ProdAl is devoted to satisfy the continuously monitored needs of the agri-food productive chain. The offer is divided into 4 Strategic Business Units (SBU):

SBU 1 – Applied research and innovation (core

business)

SBU 2 – Technical consulting

SBU 3 – Strategic and marketing consulting

SBU 4 – Training

PRODAL SCARL

SBU 1 - Applied research and innovation

Primary Production

Transformation

Final Product

Qualifying plant and

animal raw material

Process innovation

Food Packaging

Fingerprinting

regional typical

products

Packing

Product innovation

Recovery and exploitation of waste products

By-products

PRODAL SCARL

Innovative Technologies in use:

• PULSED ELECTRIC FIELDS

• OHMIC HEATING

• HIGH INTENSITY PULSED LIGHT

• HIGH HYDROSTATIC PRESSURE

• HIGH PRESSURE HOMOGENIZATION

• OZONIZATION

PROCESS INNOVATION

FOOD PRESERVATION

Preserving agricultural and animal products for sufficient time after harvesting and/or hunting has been one of the biggest issues since the beginning of human civilization

FOOD PROCESSING

FOOD ALTERATIONS

Biological causes Enzymes

Microorganisms

Chemical-physical causes Radiations

Heat

Change of water content

WHY PROCESSING FOOD?

CONSEQUENCES OF ALTERATION PROCESSES

Occurrence of anomalous organoleptic characteristics (color, odor, taste, texture)

Decay and loss of nutritional value

Toxicity

Reduction of commercial value

WHY PROCESSING FOOD?

UNIT OPERATIONS IN FOOD PRESERVATION

Reduction of the availability of water for microbial growth

Microbial inactivation

FOOD PROCESSING

FOODBORNE DISEASES More than 200 known diseases are transmitted through food. The causes of foodborne illness include viruses, bacteria, parasites, toxins, metals, and prions. The symptoms of foodborne illness range from mild gastroenteritis to life-threatening neurologic (ex. botulism; Clostridium botulinum), hepatic (ex. Hepatitis A), and renal syndromes (ex. E. coli O157:H7). Foodborne diseases cause about 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year.

WHY PROCESSING FOODS?

Known pathogens account for an estimated 14 million illnesses, 60,000 hospitalizations, and 1,800 deaths. Salmonella, Listeria, and Toxoplasma, are responsible for 1,500 deaths each year. More than 75% of these diseases are caused by known pathogens, while unknown agents account for the remaining 62 million illnesses, 265,000 hospitalizations, and 3,200 deaths. Surveillance of foodborne illness is complicated by several factors. They are frequently not reported.


Although foodborne illnesses can be severe or even fatal, milder cases are often not detected through routine surveillance. Second, many pathogens transmitted through food are also spread through water or from person to person, thus obscuring the role of foodborne transmission. Finally, some proportion of foodborne illness is caused by pathogens or agents that have not yet been identified and thus cannot be diagnosed. Many of the pathogens of greatest concern today (e.g., Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Cyclospora cayetanensis) were not recognized as causes of foodborne illness just 20 years ago.


Food Items That Help in the Differential Diagnosis of Foodborne Disease

Item Commonly associated microbes*

Raw seafood Vibrio sp., Hepatitis A virus, Noroviruses (Norwalk-like viruses)

Raw eggs Salmonella

Undercooked meat or

poultry

Salmonella and Campylobacter species, STEC**, Clostridium

perfringens,

Unpasteurized milk or juice Salmonella, Campylobacter, and Yersinia species, STEC

Unpasteurized soft cheeses Salmonella, Campylobacter, Yersinia, and Listeria sp., STEC

Home-made canned goods Clostridium botulinum (botulism)

Raw hot dogs, deli meat Listeria sp.

* Commonly associated organisms, not fully comprehensive.

**STEC = Shiga toxin-producing E coli.


Most Common Microbial Causes of Foodborne Disease*

Disease or agent Estimated total

cases

Percentage

of foodborne

transmission

Noroviruses (Norwalk-like viruses) 23,000,000 40

Campylobacter species 2,500,000 80

Giardia lamblia 2,000,000 10

Salmonella 1,400,000 95

Shigella 450,000 20

Cryptosporidium parvum 300,000 10

Clostridium species 250,000 100

Toxoplasma gondii 225,000 50

Staphylococcus aureus 185,000 100

Shiga toxin-producing Escherichia

coli (STEC)

110,000 85

Yersinia enterocolitica 100,000 90

Bacillus cereus 27,000 100

Cyclospora cayetanensis 16,000 90

Listeria monocytogenes 2,500 99

*Data from Mead et al[1]

Major Foodborne Microbes by Major Presenting Gastrointestinal Symptom*

Major presenting

symptom

Probable Microbes Incubation

Period

Probable food sources

Vomiting Staphylococcus aureus 1-6 hours Prepared food (eg, salads, dairy,

meat)

Bacillus cereus 1-6 hours Rice, meat

Noroviruses (Norwalk-

like viruses)

24-48 hours Shellfish, prepared food, salads,

sandwiches, fruit

Watery diarrhea Clostridium perfringens 8-16 hours Meat, poultry, gravy

Enteric viruses 10-72 hours Feces-contaminated food or water

Enterotoxigenic

Escherichia coli

1-3 days Feces- contaminated food or water

Cyclospora cayetanensis 1-11 days Imported berries, basil.

Clostridium parvum 2-28 days Vegetables, fruit, unpasteurized

milk, water

Inflammatory

diarrhea

Vibrio parahemolyticus 2-48 hours Raw shellfish

Nontyphoidal Salmonella

species

1-3 days Eggs, poultry, meat, unpasteurized

milk or juice, fresh produce

Shigella species 1-3 days Feces-contaminated food and water

Shiga toxin-producing

Escherichia coli

1-8 days Ground beef, unpasteurized milk,

and juice, raw vegetables, water

Campylobacter species 2-5 days Poultry, unpasteurized milk, water

*Modified from Centers for Disease Control.[2]

Major Foodborne Microbes That Usually Have No Gastrointestinal

Manifestations*

Major type of

presenting

symptom

Probable microbes Incubation

period Likely food sources

Neurologic Scromboid 1 minute-3

hours

Bluefin, tuna, skipjack, mackerel, marlin,

mahi mahi

Ciguatara toxin 2-6 hours Large reef fish (eg, grouper, red snapper,

amberjack, barracuda)

Clostridium

botulinum (botulism)

12-72 hours Home-canned foods, fermented fish,

herb-infused oils, bottled garlic, foods

held warm for long periods

Systemic Vibrio vulnificus 1-7 days Shellfish

Listeria

monocytogenes

(listeriosis)

2-6 weeks Deli meats, hot dogs, unpasteurized soft

cheese and milk

Hepatitic Hepatitis A 15-50 days Shellfish, foods prepared by a food-

handler

*Modified from Centers for Disease Control.[2]

UNIT OPERATIONS IN FOOD PRESERVATION

Microbial inactivation Among the preservation methods those based on thermal treatments, carried out at temperatures between 60 and 120 °C with time from few seconds to some hour, are the most traditional and accepted by regulatory authorities.

FOOD PROCESSING: MICROBIAL INACTIVATION

A pasteurization cycle includes three phases: - Heating - Holding - Cooling

Tem

peratu

re (

°C

)

Time (min)

Heating Holding Cooling

FOOD PROCESSING: MICROBIAL INACTIVATION

- Color changes

- Structural changes (loss of consistency)

- Viscosity changes

- Loss of volatiles and thermo-labile compounds (aroma, vitamins)

- Lipids oxidation

- Browning

- Structural changes (shrinkage, case hardening)

- Structure damage due to crystals growth with loss of

constituents

EFFECTS OF PROCESSING ON FOOD PRODUCTS

UNIT OPERATION IN FOOD PROCESSING

-20 0 20 40 60 80 100 10 -1

10 0

10 1

10 2

10 3

t (min)

T (°C)

THERMAL DAMAGE OSMOSIS

CRIOCON-

CENTRA-

TION REVERSE

HIGH

PRESSURE EVAPO-

RATION

HTST

UHTST

OSMOSIS

PEF

NEEDS FOR INNOVATION

In the recent years not only the shelf life of foods but also the quality of processed products became important for the consumers The increasing interests of consumers towards minimal processed foods, which in turn rises the prediction of a rapid and substantial reduction of chemical additives in food processing, stimulated efforts towards the set up of low impact treatments

PROCESS INNOVATION

Development of conceptually new unit operations to perform in a more efficient way and/or

under higher quality requirements a specific stage

in a food production line

Set up new processes based on the integration in a different flow sheet of

conventional unit operations and on the integration and

optimization of their performances

ELECTRO TECHNOLOGIES

Non-thermal technology (PEF)

PULSED ELECTRIC FIELDS IN FOOD PROCESSING

PEF for microbial and enzymatic inactivation: pasteurization

PEF for vegetable tissues permeabilization to improve mass transfer, decrease processing time and increase process throughput: drying, extraction, juice expression

PEF MICROBIAL INACTIVATION

1920 Pasteurization of Milk by Electropure Process Effect: Resistive heating and formation of reactive

radicals and molecules at the electrodes

1949 Plasmolysis of Plant Materials B.L. Flaumenbaum, Odessa

1960 Patent by H. Doevenspeck Existence of a ´non-thermal´ effect on microorganisms

exposed to pulsed electric fields produced by capacitor

discharges

1967 Sale & Hamilton Microbial inactivation depending on: field strength discharge duration

size and shape of microorganisms

1980-1983 Hulsheger et al. Study on the sensitivity of different bacteria to PEF and set-up of a mathematical model (including E and t) to describe the effects of PEF on microorganisms

PEF: PIONEERING WORK

PEF treatment involves application of train of pulses of short duration and high electric field strength to a food product placed between two electrodes

FOOD

+ HV electrode

electrode

E = 5-50 kV/cm

t = 1-10 s

f = 1-1000 Hz

WT = 100-150 kJ/kg

PEF TECHNOLOGY

PEF affects the membrane of biological cells Ec = critical electric field strength

PEF TECHNOLOGY

PEF TECHNOLOGY

cos ERsfU

R

E: External Electric Field

sf: Shape Factor

The effect of PEF is localized at the membrane of biological cells

a = cell membrane with potential V'm b = membrane compression when V>> V'm (*) c = pore formation with reversible breakdown d = large area of the membrane subjected to irreversible breakdown with large pores

(Zimmermann, 1986)

(*) Critical transmembrane potential Vc ≈ 0.7-1 V for most cell membranes

ELECTRICAL FIELD STRENGTH

E= V/d

V= voltage (Volts) d= distance between the electrodes (m)

d

5 kV 0 kV

PEF TECHNOLOGY

PULSE SHAPE AND WIDTH

100%

37 %

Voltage, kV

Pulse duration

Pulse width

t, s

Square wave pulses Exponential decay pulses

Pulse width

Voltage, kV

Pulse duration

100%

t, s

t is defined as the time needed for the decay of voltage to 37% of its maximum value (Zhang et al., 1995a).

t represents the effective time of the high voltage application to the food product

PEF TECHNOLOGY

The pulse repetition frequency is defined as the number of pulses applied per unit of time. In PEF pasteurization applications the frequency interval is between 1-5000 Hz - With increasing the frequency we can reduce the processing time

- With increasing the frequency we can enhance the temperature increase of the product.

- With increasing the frequency, the costs of the power supply unit and of the switch increase

FREQUENCY

PEF TECHNOLOGY

The treatment time is the time interval during which the microorganisms are exposed to the electric field and, therefore, depends on the pulse characteristics and numbers of pulses

t=nt

The calculation of the treatment time is more accurate for square-wave than for exponential decay pulses

TREATMENT TIME

PEF TECHNOLOGY

PEF treatment is based on several charge-discharge cycles during which the energy, stored in the capacitor bank, is delivered to the product in the treatment chamber To calculate the energy Wp (J/ml), depending on pulse shape, it is necessary to consider the effective voltage and current in the treatment chamber.

PULSE ENERGY Wp

PEF TECHNOLOGY

Exponential decay pulses

Umax=voltage applied across the electrodes Imax= current in the food sample v=volume of the food sample in contact with the electrodes

Square wave pulses

u(t)=instantaneous voltage across the electrodes i(t)=instantaneous current in the food sample v=volume of the food sample in contact with the electrodes

t

pulse dttituv

W0

1kpulse RtUtIU

vW /

1 2

maxmaxmax

Effective energy of an exponential decay pulse (a) and of a pseudo square pulse (b) (Gongora-Nieto et al., 2002).

a) b)

PEF TECHNOLOGY

Total specific energy WT:

pulseT WnW

Q

fvWW

pulse

T

In a continuous process:

Q

fvn

TOTAL SPECIFIC ENERGY WT

PEF TECHNOLOGY

Pulsed Electric Field treatment

CONTROL PERMEABILIZED CELL

PULSED ELECTRIC FIELDS AND MEMBRANE PERMEABILIZATION

Microorganism

Cell membrane

Biological cells suspended in electrically conductive medium

PEF TECHNOLOGY: MECHANISM OF MICROBIAL INACTIVATION

The occurrence of sublethal injury depends on:

- microbial strain characteristics, such as cell envelope structure, growth conditions, environmental stress conditions

- process parameters such as E and t

- product parameters such as treatment medium composition

PEF TECHNOLOGY: MECHANISM OF MICROBIAL INACTIVATION

Control &

Monitoring System

High Voltage Pulse

Generator

Treatment Chamber

Treated Product

Raw Product

PEF FOOD PROCESSING UNIT: BASIC COMPONENTS

PEF TECHNOLOGY

136 nF

•Static chamber with parallel electrodes

•Exponential decay pulses

•E = 0-30 kV/cm, n = 1-512, t = 2-9 s, f = 1-5 Hz

BATCH PEF LAB UNIT

Treatment chamber

Area=2.01 cm2,

gap= 0.25 cm

PULSE GENERATOR:

Output voltage: 20 kV

Pulse shape:

exponential decay

Polarity : mono-polar

Pulse width: 1-10 s

Repetition rate: up to

400Hz

TREATMENT CHAMBER:

Geometry: parallel

plate electrodes

Flow rate: 0 to 10 l/h

CONTINUOUS FLOW PEF LAB UNIT

It is a complete system designed for the Pulsed Electric field processing of liquid foods The mains component are: HV pulse generator

Treatment chambers Fluid handling and heating system

Aseptic packaging unit

The pilot plant is provided with a highly flexible control system, allowing independent setting of applied field (0-60 kV/cm), pulse width (2-10 s), pulse repetition rate (200-1000Hz) e flow rate (50-200 l/h)

PEF PILOT PLANT

PULSER

V=50 l

V=49 l

SIMPLIFIED SCHEME

PEF PILOT PLANT

HIGH VOLTAGE

POWER SUPPLY CAPACITORS

HV OUT 220-440

V a.c.

(1.5 - 75 kW)

Transformer/Rectifier

20-60 kV dc

PFN

t

E

t

E

ON Switch (100 kV – 1 MA)

modulator

ON/OFF Switch (~1.2 kV – 1 kA )

PEF FOOD PROCESING UNIT: BASIC COMPONENTS

Pulse forming network (PFN) consists of a special arranged power supply, switch, capacitors, inductors and resistors;

PEF SYSTEM COMPONENTS

HIGH VOLTAGE PULSE GENERATOR

Co-field chamber

- Gap: 0.42 cm

- Electrode diameter: 0.32 cm

- Number of chambers: 4

- Flow rate: 50-200 l/h

TREATMENT CHAMBER


SWITCHES

Switches releases the power stored in the capacitor bank as a high-voltage pulse through a pulse-forming network with defined pulse shape

Because pulse duration can go from few ms up to ms, the switches are designed:

to operate at high frequency, to resist the maximum voltage present across the capacitors, to transfer an electrical current of high intensity resulting primarily from the food samples electric resistivity


The treatment chamber transfer the high voltage pulsed electric field to the food The design of treatment chamber is critical for the utilization of PEF pasteurization technology provided that it should ensure:

- a high intensity electric field

- a uniform distribution of the electric field strength

TREATMENT CHAMBER

TREATMENT CHAMBER


Pulsed Power Treatment of Pumpable Materials Electrodes

PERPENDICULAR CO-AXIAL CO-LINEAR

Product Flow Product Flow Product Flow

FACTORS AFFECTING PEF MICROBIAL INACTIVATION

oSpecies and strain

oSize and morphology

oGrowth conditions

- Growth phase

- Growth temperature

- Growth medium composition

oElectric field strength

oTreatment time

oSpecific energy

oTemperature

oPulse characteristics

oFlow rate

opH

oWater activity

oChemical composition

oConductivity

Process parameters

Food product

characteristics

Microorganisms

characteristics

PROCESS PARAMETERS

0

5

10

15

20

25

0 50 100 150

WT, J/ml

DT

, °C

Q=16.9ml/min Q=33.9ml/min

Q=49ml/min Q=67.9ml/min

Parallel plate continuous chamber (Gap: 2.5 mm; A=2.6 cm2) Electric field strength: 18 kV/cm Pulse width: t=3.5 s Inlet temperature: 25 °C Treatment medium: Trizma HCl buffer (pH 7, k=2 mS/cm)

S. Cerevisiae

TEMPERATURE INCREASE WITH ENERGY INPUT

Treatment conditions: Trizma HCl buffer (pH 7, k=2 mS/cm); f=1Hz; t=3.11 s;

-8

-7

-6

-5

-4

-3

-2

-1

0

0 300 600 900 1200 1500 1800

Treatment time, [s]

Log(

S)

E=3.3 kV/cm E=6.5 kV/cm E=8.4 kV/cmE=10.6 kV/cm E=16.0kV/cm E=22.8 kV/cmE=26.3 kV/cm E=30.9 kV/cm Control

S. cerevisiae

INFLUENCE OF PULSED ELECTRIC FIELD STRENGTH AND TREATMENT TIME

S. cerevisiae E. coli

-8

-7

-6

-5

-4

-3

-2

-1

0

0 50 100 150WT [J/ml]

Lo

g (

S)

E= 13,60 kV/cm E= 18,12 kV/cmE= 22,9 kV/cm E= 27,7 kV/cmControl

-8

-6

-4

-2

0

0 30 60 90 120WT, J/ml

Lo

g(S

)

E= 13,25 kV/cm E= 17,38 kV/cmE= 22,06 kV/cm E= 26,80 kV/cmControl E= 30,38 kV/cm

Parallel plate continuous chamber (Gap: 2.5 mm; A=2.6 cm2); Pulse width: t=3.5 ms , Inlet temperature: 25 °C Flow rate: 2 l/h; Treatment medium: Trizma HCl buffer (pH 7, k=2 mS/cm)

INFLUENCE OF PULSED ELECTRIC FIELD STRENGTH AND ENERGY INPUT

-8

-7

-6

-5

-4

-3

-2

-1

0

0 30 60 90 120

WT, J/ml

Lo

g(S

)

Q=16.9ml/min Q=34ml/minQ=49ml/min Q=67.9ml/minControl Q=100.6ml/min

-8

-7

-6

-5

-4

-3

-2

-1

0

0 30 60 90 120 150

WT, J/ml

Lo

g(S

)

Q=16.2 Q=34.7 ml/minQ=49 ml/min Q=67.9 ml/minControl


Q, [l/h] 1 2 3 4 6

Re, [-] 83 166 250 333 499

Parallel plate continuous chamber (Gap: 2.5 mm; A=2.6 cm2) Electric field strength: 18 kV/cm Pulse width: t=3.5 s Inlet temperature: 25 °C Treatment medium: Trizma HCl buffer (pH 7, k=2 mS/cm)

INFLUENCE OF PRODUCT FLOW RATE

-8-7-6-5-4-3-2-10

0 20 40 60 80 100

Treatment time [s]

Log

(s)

□ t=3.1 s

■ t=6,4 s

--- Control

-8-7-6-5-4-3-2-10

0 50 100 150 200 250 300

Treatment time [s]

Log(

s)

f =1 Hzf =5 HzControl

Batch chamber (Gap: 2.5 mm; A=2 cm2)

Electric field strength: E=22.4 kV/cm

Pulse frequency: 1Hz; Inlet temperature: 25 °C

Treatment medium: TrizmaHCl buffer (pH 7, k=2 mS/cm);

Batch chamber (Gap: 2.5 mm; A=2 cm2)

Electric field strength: E=22.4 kV/cm

Pulse width: t=3.11 s

Inlet temperature: 25 °C

Treatment medium: TrizmaHCl buffer (pH 7, k=2 mS/cm);

S. cerevisiae

INFLUENCE OF PULSE WIDTH AND FREQUENCY

PRODUCT PARAMETERS

INACTIVATION OF S. cerevisiae IN FRUIT JUICES

Treatment conditions : E=22,5 kV/cm; f=1 Hz

Apple juice: Uo=6.9kV, t =3.25 s;

Pineapple juice: Uo=7.1 kV, t =2.53 ms;

Orange juice: Uo=7.5 kV, t=2.42 ms

Medium aw (1)

[-]

pH (1)

[-]

k (1)

[mS/cm]

Apple

juice

0.977 3.65 1.56

Pineapple

juice

0.986 3.74 3.34

Orange

juice

0.990 3.85 3.56

(1) Measured at T=24 °C

-8-7-6-5-4-3-2-10

0 50 100 150 200Treatment time [s]

Log

(S)

Apple JuicePineapple JuiceOrange JuiceControl

At a fixed electric field strength, the lethality of the treatment increases with increasing the treatment time

S. cerevisiae cells are more resistant to PEF treatment when suspended

in apple juice than in orange juice and in pineapple juice

Medium k [mS/cm]

(at 24°C)

W

[J/ml pulse] (at 22.5 kV/cm)

WTOT

[J/ml ] (after 98 s at 22.5

kV/cm)

Apple juice 1.56 2.16 65

Pineapple juice 3.34 3.39 131

Orange juice 3.56 3.78 153

t

pulse dttEkW0

2 )(

Electrical conductivity affects microbial inactivation by PEF

(Jayaram et al, 1993; Sensoy et al., 1997; Wouters et al., 1999;

Alvarez et al., 2000; Alvarez et al., 2003)

Specific energy

The higher inactivation, observed at constant treatment time and field strength when the conductivity of foods increases can be attributed to the increase of the specific energy input

EFFECT OF PULSE ELECTRICAL CONDUCTIVITY

Microbial resistance to PEF treatments increases as water activity

decreases (Aronsson et al., 2001; Alvarez et al., 2002)

Barbosa-Canovas et al., 2005

EFFECT OF WATER ACTIVITY

The influence of the pH on the microbial inactivation by PEF treatment is still unclear. In general, microorganisms are more resistant to any kind of stress at the optimum pH of growth (6.6-7.5). If the pH is higher or lower than the optimum value an increase of the microbial sensitivity to lethal stresses is observed

Barbosa-Canovas et al., 2005

EFFECT OF PH

MICROBIAL STRAIN CHARACTERISTICS

1

10

100

Cri

tic

al

Fie

ld S

tren

gth

[kV

/cm

]

1.0 10.0

Characteristic Cell Dimension [µm]

A

D

C

S. cerevisiae

S. senftenbergE. coli

L. monocytogenes

L. brevis

L. plantarum

B. subtilisY. enterocolitica

B

E

(Heinz et al., 2000)

Ec depends on the: type of microorganisms

size and shape

cell orientation with respect the electric field direction

characteristics of the medium

Typical values of Ec=4-14 kV/cm (Castro et al, 1993)

EFFECT OF SIZE AND SHAPE

UNEVENESS OF THE PEF TREATMENT

S. cerevisiae Treatment conditions: Trizma HCl buffer (pH 7, k=2

mS/cm); f=1Hz; t=3.11 ms;

At any E investigated the survival curves of S. cerevisiae cells show a

“tailing behavior”

-8

-7

-6

-5

-4

-3

-2

-1

0

0 300 600 900 1200 1500 1800

Treatment time, [s]

Log(

S)

E=3.3 kV/cm E=6.5 kV/cm E=8.4 kV/cmE=10.6 kV/cm E=16.0kV/cm E=22.8 kV/cmE=26.3 kV/cm E=30.9 kV/cm Control

INFLUENCE OF PULSED ELECTRIC FIELD STRENGTH AND TREATMENT TIME

CONTROL

PEF

E= 22.5 kV/cm

t=100 s

Pictures of PDA gel discs inoculated with S. cerevisiae

Treatment conditions: E=22.6 kV/cm; t=3.11 s; f=1 Hz.

Initial cells concentration: 1.2*107 CFU/ml

The distribution of the electric field strength in the treatment chamber is

not uniform (field fringing effects)

TREATED

surviving colonies

yeast colonies in solidified PDA

HETEROGENEITY OF PULSED ELECTRIC FIELD

n’ of pulses

(n’=2, 8 or 32)

Simulation of sample agitation

INFLUENCE OF SAMPLES AGITATION

S. cerevisiae Treatment conditions:

Trizma HCl buffer (pH 7, k=2 mS/cm); E=22.5 kV/cn f=1Hz; t=3.11 s; W= J/ml*pulse

INFLUENCE OF SAMPLE AGITATION

-8-7-6-5-4-3-2-10

0 400 800 1200 1600

WT, J/ml

Log

(S)

n-mix=0n-mix=32n-mix=8n-mix=2Control

Non-uniform distribution of electric field inside the

treatment chamber

L/D =0.4 (L =0.20; D =0.50 cm) L/D =2.5 (L =0.50; D =0.20 cm)

D

L

D

L

Co-field chamber: Electric field distribution

Co-field chambers have good field uniformity if the ratio L/D is sufficiently high

TREATMENT CHAMBER DESIGN CRITERIA

High voltage Pulse Generator

0-20 kV

Oscilloscope

Controller

+

HV

TIN

TOUT

MULTISTEP PROCESS


-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5 6

Nr

Lo

g(S

)

WT,R =22 J/ml

WT,R =42 J/ml

WT,R =61 J/ml

WT,R =82 J/ml

Control

-8

-7

-6

-5

-4

-3

-2

-1

0

0 1 2 3 4 5 6 7

Nr

Lo

g(S

)

WT,R=22 J/ml

WT,R=42 J/ml

WT,R=61 J/ml

WT,R=82 J/ml

Control

Treatment conditions E= 18 kV/cm f= 0-100 Hz WT= 22-42-61-82 J/ml Fow rate= 2 l/h Tin =25 °C

MICROBIAL INACTIVATION IN MULTISTEP PROCESSES

It is important to take precautions in the design of the treatment chamber in order to avoid dielectric breakdown of foods All substances, solid, liquid, or gas, are able to resist a maximum electric field strength, called dielectric intensity or strength of the product Dielectric breakdown occurs when the applied electric field strength exceeds the dielectric strength The intent of food pasteurization with PEF is to induce the dielectric breakdown of the cell membrane, not the dielectric breakdown of the fluid food. The latter, termed spark-over, is not desired in PEF pasteurization

DIELECTRIC BREAKDOWN

Dielectric breakdown of foods inside the treatment chamber is observed as a spark The passage of a spark through a food is characterized by: a large electric current flow in a narrow channel a bright luminous spark the evolution of gas bubbles solid particles deriving from food decomposition the formation of pits on the electrode surfaces an impulsive pressure through the liquid with an accompanying explosive sound (Krasuchi, 1968)


To reduce the probability of dielectric breakdown in foods, it is suggested (Zhang et al., 1995b): - using smooth electrodes surface to minimize electron emission - using round electrode edges to prevent field enhancement - optimizing the design of the treatment chamber to provide uniform electric field strength - using lower energy per pulse, short duration pulses, and/or low frequencies - degassing the product before treatments to eliminate gas bubble formation - pressuring the liquid food flowing in the treatment chamber to prevent gas bubble formation


Advantages:

- No significant heating of the food

- Minimum energy utilization

and high energy efficiency - High retention of nutrients

and vitamins - High quality - Continuous process is possible - Low processing costs

- Application is limited to liquid products with sufficient dielectric strength and proper electrical conductivity

- Low temperature storage and cold distribution chain

- Spores and many enzymes are not sensitive to PEF and require combined methods for inactivation

- High investment costs

Disadvantages:

PEF TECHNOLOGY

SAFETY ISSUES OF THE PEF TECHNOLOGY

ELECTRODES CORROSION In PEF pasteurization the electrodes are in contact with the food product The current flowing in the electrodes consists of free electrons and in the liquid of charged particles. At the interface between electrodes and liquid electrochemical reactions occur, causing electrodes corrosion

FOOD

+ HV ANODE

CATHODE

PEF TECHNOLOGY SAFETY ISSUES

Food quality Electrochemical reactions can : - cause changes of the chemical structure of the liquids near the electrodes surface - produce toxic chemicals (mostly H2O2) - cause the release of small particles of the electrodes material in the liquid Electrodes fouling Electrode-fouling, i.e. the formation of a film of food particles on the electrodes surface, can cause: - local electric field distortion and arching - electrical breakdown in the treatment chamber - fouling or contamination of the system - reduction or stopping of the product flow


Electrodes corrosion Electrodes corrosion can cause: - serious damages of the electrodes in few hours - drastic reduction of the electrodes life time

What are the variables influencing these phenomena? •Electric field strength, E

• Total specific energy input, WT

• Pulse width, t

• Pulse repetition frequency, f

• Pulse shape (exponential decay or square; mono or bipolar)

• Food characteristics (type, pH , electrical conductivity k)

• Treatment chamber configuration

• Electrodes material


Migration of Fe, Cr and Ni from electrode surface into the food were taken as measures of electrode corrosion

ICP-MS (Inductively Coupled Plasma Mass Spectrometry)


McIlvaine buffer (pH 7, k 2 mS/cm)- Stainless steel electrodes

E F F E C T

O F E

A N D

WT

0

5

10

15

20

25

30

0 50 100 150

ppb

energia specifica [J/ml]

McIlvaine : concentrazione ferro

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0 50 100 150

ppb


McIlvaine : concentrazione cromo

0

1

2

3

4

5

0 50 100 150

ppb


McIlvaine : concentrazione nichel

12.50 Kv/cm

22.03 Kv/cm

31.46 Kv/cm

rif

Fe Cr

Ni

WT, J/ml WT, J/ml

WT, J/ml

0

500

1000

1500

2000

2500

0 50 100 150

ppb


Trizma: concentrazione cromo

Trizma HCl buffer (pH 7, k 2 mS/cm) - Stainless steel electrodes

E F F E C T

O F E

A N D

WT

0

200 400 600 800

1000 1200 1400 1600 1800 2000

0 50 100 150

ppb


Trizma: concentrazione nichel

12.76 Kv/cm

21.72 Kv/cm

31.46 Kv/cm

rif

Legenda

0

2000

4000

6000

8000

10000

12000

14000

0 50 100 150

ppb

energia specifica[J]/ml

Trizma: concentrazione ferro Fe Cr

Ni

WT, J/ml WT, J/ml

WT, J/ml

McIlvaine buffer (pH 3.4 and 7)- Stainless steel electrodes

E F F E C T

O F

pH

0

2

4

6

8

10

12

0 50 100 150

ppb


confronto a ph diversi:Ni

12.71 Kv/cm K=2mS/cm ph=7

12.40 Kv/cm K=2mS/cm ph=3.4





0

10

20

30

40

50

60

70

0 20 40 60 80 100 120

ppb


confronto a diversi ph:Fe

0

5

10

15

20

25

0 20 40 60 80 100 120

ppb


confronto a ph diversi: Cr Fe Cr

Ni WT, J/ml

WT, J/ml

WT, J/ml

ANODE

CATHODE

ANODE

CATHODE

TRIZMA

McIlvaine

ELECTRODES FOULING

E F F E C T

O F E L E C T R O D E S

M A E R I A L S

McIlvaine buffer (pH 7, k 2 mS/cm) Stainless steel electrodes - Chromate electrodes surface

0

5

10

15

20

25

30

35

40

0 20 40 60 80 100 120

ppb


Confronto elettrodi sul rilascio di ferro: McILvaine ph 7 K=2 mS cm-1

0

1000

2000

3000

4000

5000

6000

7000

0 50 100 150

ppb


Confronto elettrodi sul rilascio di cromo: McILvaine ph 7 K=2 mS cm-1

0

0,5

1

1,5

2

2,5

3

3,5

0 20 40 60 80 100 120

ppb


Confronto elettrodi sul rilascio di nichel: McILvaine ph 7 K=2 mS cm-1

12.71 Kv/cm non cromati 12.50 Kv/cm cromati



Fe

Cr

Ni

WT, J/ml WT, J/ml

WT, J/ml

Trizma HCl buffer (pH 7, k 2 mS/cm) Stainless steel electrodes - Chromate electrodes surface E

F F E C T

O F E L E C T R O D E

M A E R I A L S

0

2000

4000

6000

8000

10000

12000

0 20 40 60 80 100 120

ppb


Confronto elettrodi sul rilascio di ferro:Trizma ph 7 K=2 mS cm-1

0

1000

2000

3000

4000

5000

6000

7000

0 50 100 150

ppb


Confronto elettrodi sul rilascio di cromo:Trizma ph 7 K=2 mS cm-1

0

500

1000

1500

2000

0 50 100 150

ppb


Confronto elettrodi sul rilascio di nichel:Trizma ph 7 K=2 mS /cm 12.40 Kv/cm non

cromati 12.60 Kv/cm cromati



Fe

Cr

Ni

WT, J/ml WT, J/ml

WT, J/ml

Stainless steel electrodes - Chromate electrode surface Trizma HCl buffer (pH 7, k 2 mS/cm)

Electrodes fouling (anode)

E F F E C T


M A E R I A L S

Stainless steel Chromate

-8

-7

-6

-5

-4

-3

-2

-1

0

0 30 60 90 120

Lo

g (

N/N

o)

WT, J/ml

E= 17.6 kV/cm- no Cr

E= 30.7 kV/cm - no Cr

E= 17.4 kV/cm Cr

E= 30.7 kV/cm Cr

The use of a polished chromate electrodes surface allow to: - reduce or avoid electrodes fouling

- reduce local electric field distortion

- improve PEF treatment efficiency

Microbial inactivation of S. cerevisiae E F F E C T


M A E R I A L S

COST ESTIMATION: PEF VS. THERMAL TREATMENTS

Q=200 l/h Treatment conditions E=40 kV/cm, TIN=20-35-45-55 °C WT=100-80-60-40 kJ/kg Equipment design Type of chamber co-field Chamber volume 0,034 Number of Chambers 4 Residence time 0.0025 s Repetition >=1 kHz Load voltage 25 kV Average power 20 kW Investment ca. 200 k€ Operative costs 0.0033-0.0061 €/kg

Q=200 l/h Treatment conditions TIN=20 °C Tp=85°C; t=15 s Equipment design Plate Heat exchanger Reservoir tanks 50 l Aseptic tank 50 l Investment ca. 50 k€ Operative costs 0.0089 €/kg

ESTIMATION OF TOTAL TREATMENT COSTS

PEF Pasteurization HTST Pasteurization

TRANSFER OF TECHNOLOGICAL INNOVATION

Increase the robustness of switches and other components of the PEF system

Reduce the risk of dielectric breakdown in foods Prevent/reduce electrodes corrosion and electrodes fouling Set up design criteria of the treatment chambers to ensure high

intensity electric field and uniform distribution of the electric field strength

Set up mathematical models able to predict the extent of

microbial inactivation as a function of products characteristics and processing conditions

- High investments costs

- Consumer’s concerns toward novel technologies

- Regulatory approval

FACTORS LIMITING THE USE OF PEF IN THE FOOD INDUSTRY

MASS TRANSFER ENHANCEMENT BY MEANS OF ELECTROPORATION

PEF ASSISTED EXTRACTION OF NUTRACEUTICAL COMPOUNDS FROM VEGETABLE MATRICES

PEF ASSISTED VINIFICATION: STATE OF THE ART

Fundamentals of PEF treatments applied to vinification processes

Increase of juice yield and polyphenolic content and reduction of turbidity from white grapes @ low electric field (E<1 kV/cm)

Vorobiev and coworkers (UTC – France)

Increase of polyphenolic extraction during red wine vinification @ high electric field (E>1 kV/cm)

Determination of anthocyanin profile

Influence of grape variety

Evolution of polyphenolic compounds during ageing in bottles and barrels

Raso and coworkers (UniZar – Spain)

Observation of vacuole permeabilization upon PEF application

On-line impedance measurement

Müller and coworkers (KIT – Germany)

Increase of polyphenolic extraction and antioxidant activity during red wine vinification @ low electric field (E<1.5 kV/cm)

Ferrari and coworkers (UniSA – Italy)

PEF ASSISTED VINIFICATION

Repeatability of the results over two

different grape harvests

Extending its applicability to different

Italian grape varieties

Validation of the PEF-assisted vinification

Monitoring during fermentation/maceration of the must:

Color intensity

Total polyphenols and free anthocyanins

Characterization of the fresh wine:

Physicochemical properties

Antioxidant activity

Aromatic profile

Investigation of the feasibility of the PEF treatment in the red wine

vinification

Treatments

Analysis Control Enzyme 1kV 10000 p 1.5kV 1000 p 1.5kV 2500 p

Total Polyphenols (g/l) 1.6 1.9 1.8 2.1 2.2

Free Anthocians (mg/l) 477 705 522 734 839

Color Intensity 9.85 10.8 10.4 11 11.65

Glucose and fructose (g/l) 0.61 0.69 0.65 0.6 0.62

Reducing sugars (g/l) 2.1 2.1 2.3 2.2 2.2

Antioxidant power (mg/ml of ascorbic acid)

65.18 70.3 69.87 73.35 78.52

Total Acidity (g/l of tartaric acid)

11.2 11.2 11.3 11.12 11

Alcool content (% ) 11.8 11.7 11.9 12 11.8

pH 3.21 3.19 3.17 3.2 3.2

IMPEDANCE MEASUREMENT

Signal: sinusoidal Frequency: 10 Hz e 32 MHz) Max output Voltage (rms): 1-3 V Max output Current (rms): 20-60 mA

IMPEDANCE ANALYSER (1260 Solartron, UK) SAMPLE HOLDER

Measurement of the impedance (Z)-frequency spectra as the ratio of the

voltage drop across the sample and the current crossing it during the

test.

Cell disintegration index Zuntr: absolute value of complex impedance of intact tissue, Ohm

Ztr: absolute value of complex impedance of heated tissue, Ohm

Zp=0 for intact tissue; Zp=1 for totally disintegrated material

)1()1(

)1()1(

MHztrkHzuntr

kHztrkHzuntr

pZZ

ZZZ

2.5 cm

diameter

1 cm gap

VINIFICATION PROCESS

Roller-crushing

Destemming

Addition of potassium

metabisulfite (50 ppm)

Separation of skins and must

PEF treatment of grape skins

Recombination and addition of

yeast cells

(200 ppm)

Fermentation/maceration at 25 3°C

Pressing

Enzyme addition

(20 ppm)

Fresh wine

Measurable effect of PEF

pretreatment on the release

kinetics of:

Total polyphenols (in

figure)

Free anthocyanins

Color intensity

Effect of pectolytic enzyme

is measurable but lower

than PEF

EVOLUTION OF TOTAL POLYPHENOLS

Vinification of Aglianico grapes

t (days)

0 2 4 6 8 10 12

To

tal po

lyp

hen

ols

(g

/L)

0

2

4

6

8

Untreated

E=1.5 kV/cm Wt=10 kJ/kg

E=3 kV/cm Wt=10 kJ/kg


Enzyme

t (days)

0 1 2 3 4 5 6 7 8 9

Tota

l poly

phenols

(g/L

)

0.0

0.5

1.0

1.5

2.0

2.5

Untreated





Enzyme

Vinification of Piedirosso grapes

Neither PEF pretreatment

nor enzyme addition

significantly impacted on

polyphenolic release from

Piedirosso grapes

EVOLUTION OF TOTAL POLYPHENOLS

EFFECT OF GRAPE VARIETY

Wt (kJ/kg)

0 5 10 15 20 25

ym

ax

1

2

3

4

5

6

7

8

Wt (kJ/kg)

0 5 10 15 20 25

Zp

0.0

0.2

0.4

0.6

0.8

1.0W

t (kJ/kg)

0 5 10 15 20 25

kd

(d

-1)

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Aglianico

Piedirosso

tky

yd exp

max

1

Electrical permeabilization does

not give an accurate indication

of mass transfer through cell

and vacuole membrane

)1()1(

)1()1(

MHztrkHzuntr

kHztrkHzuntr

pZZ

ZZZ

Enzyme PEF1 PEF2 PEF3

D a

nti

oxid

an

t acti

vit

y (

%)

-20

0

20

40

60


D c

olo

r in

ten

sit

y (

%)

-20

0

20

40

60

80

Aglianico

Piedirosso

Nebbiolo

Casavecchia


D t

ota

l p

oly

ph

en

ols

(%

)

-40

-20

0

20

40

60

80

100

120


D f

ree a

nth

ocyan

ins (

%)

-20

0

20

40

60

PEF

treatment

Treatment

conditions

Zp

Aglianico

Zp

Piedirosso

Zp

Nebbiolo

Zp Casavecchia

PEF1 1.5 kV/cm,

10 kJ/kg

48.6±1.5% 68.6±4.6% 67.7±5.9% 82.2±6.3%

PEF2 3.0 kV/cm,

10 kJ/kg

61.1±0.6% 91.2%±1.2% 82.4±1.3% 86.3±1.4%

PEF3 3.0 kV/cm,

20 kJ/kg

75.9±3% 94±1.2% 86.8±0.3% 91.3±0.1%

DATA CONSISTENCY OVER TWO DIFFERENT YEARS

Control

2008

PEF1

2008

Control

2009

PEF1

2009

PEF2

2009

PEF3

2009

Alcohol content

(v/v %) 11.8±0.1 12.0±0.1 9.1±0.1 10.1±0.1 11.3±0.1 10.5±0.1

pH 3.2±0.02 3.2±0.03 3.4±0.02 3.4±0.02 3.4±0.02 3.4±0.02

Total acidity

(g/l tartaric acid) 11.2±0.2 11.1±0.2 7.1±0.2 7.8±0.3 7.6±0.2 7.9±0.1

Colour intensity

(a.u) 9.8±0.05 11.0±0.06 6.8±0.06 8.3±0.04 7.5±0.04 7.4±0.07

Total polyphenols

(g/l) 1.6±0.02 2.1±0.03 1.1±0.01 2.1±0.02 2.2±0.02 2.4±0.03

Free anthocyanins

(mg/l) 477±7 734±12 300.7±10 352.9±15 387.2±11 395.4±15

Antioxidant activity

(mg/ml ascorbic

acid)

0.16±0.01 0.18±0.01 0.12±0.01 0.17±0.01 0.17±0.01 0.17±0.01

PEF treatment Treatment conditions

PEF1 1.5 kV/cm, 10 kJ/kg



PEF-wines from Aglianico grapes

Compound Composition (%)

Control PEF4 PEF5

Isoamyl alcohol (cheesy) 43.72 37.24 29.19

2-Methyl-1-butanol 9.34 12.50 10.20

Ethyl butyrate 0.11 0.10 0.08

2-Hexanol 0.30 0.27 0.28

Isohexyl alcohol 0,06 0.11 0.06

Nonyl alcohol 0,05 0.06 0.06

3-ethyl-1-butanol 0.26 0.37 0.25

1-Hexanol 3.48 4.02 3.46

Isoamyl acetate (fruity) 0.87 1.54 0.87

2-methylbutyl acetate (floral) 0.11 0.21 0.13

Heptyl alcohol 0.07 0.06 0.05

Hexanoic acid (sweaty) 0.34 0.72 0.46

Ethyl hexanoate 0.44 0.67 0.49

Ethyl isovalerate 0.40 0.47 0.28

2-Phenylethanol (rose flavor) 37.27 34.95 45.45

Ethyl succinate 0.36 0.26 0.10

Octanoic acid (cheesy) 0.48 0.76 0.67

Ethyl octanoate 0.33 0.51 0.30

Phenethyl acetate 0.32 0.32 0.27

Amyl methacrylate 0.19 0.22 0.17

Decanoic acid 0.21 0.14 0.11

Ethyl decanoate 0.08 0.09 0.04

Ethyl 2-isocyanato-2-phenylpropanoate 0.28 0.62 0.21

Tryptophol 2.82 3.29 2.09

3,4,5-Trimethoxyphenylacetic acid 0.15 0.14 0.15

Methyl 3-(indol-3-yl)propionate 0.11 0.08 0.06

Total 99.22 99.55 95.42

AROMATIC COMPOUNDS

Slight alterations

of the aromatic

profile of the

wine

PEF-wines from

Aglianico grapes

PEF

treatment

Treatment

conditions

PEF4 1.5 kV/cm,

10 kJ/kg

PEF5 1.5 kV/cm,

25 kJ/kg

IMPROVEMENT OF RED WINE VINIFICATION

4 €/ton of grapes

Unit cost: 200 €/kg Requirements: 2 g/100 kg E

nzym

e

0.7 €/ton of grapes

Energy cost: 0.12 €/kWh Requirements : 20 kJ/kg

(PEF3)

PEF

1.8 €/ton of grapes Energy cost:

0.35 €/Nm3 CH4

Requirements : 205 kJ/kg (80°C for 2 min)

Heat

High cost Addition of undesired

compounds Increase of wine

turbidity

Worsening of wine quality

High capital cost Requirement of

specialized technicians Tuning on the grape

varieties

Simple use Consolidated in the

wine industry

Good treatment for low quality wines

Improvement of the main quality parameters

Higher antioxidant activity

Cost Advantages Disadvantages

IMPROVEMENT OF RED WINE VINIFICATION

For PEF treatment ranging from E = 1.5 - 3 kV/cm and Wt = 10 - 20 kJ/kg,

Aglianico grapes were effectively permeabilized, leading to a wine with

a higher color intensity (+20%), total polyphenols (+100%) and free

anthocyanins (+30%) content as well as higher antioxidant activity

(+25%), without the addition of pectolytic enzymes.

Similar results were obtained over two harvests, confirming the

robustness and repeatability of the process.

PEF treatments need to be tuned and adapted to the biological

characteristics of the treated grapes, suggesting the need for

reliable indicators of vacuole permeabilization

PEF ASSISTED JUICE EXPRESSION FROM BLUEBERRIES

Segnale di trigger

+

HV

OSCILLOSCOPE

PG-3 Solid State Pulse Generator (ScanDinova, Sweden)

• Maximum voltage: 25 kV

• Maximum current: 500 A

• Pulse Width: 1-25 s

• Repetition frequency: 1-450 Hz

Blueberries (30g)

Electrode

Electrode

Application of Pressure

Juice collection

TRATMENT PROTOCOL Pressure

(P=0.35-1.34 bar; t=2 min)

PEF Treatment maintaining Pressure

(E=0-5 kV/cm; WT=12kJ/kg)

Pressure

(P=0.35-1.34 bar;8 min)

Juice was collected during the entire experiment

Evaluation in the resulting juice of: • total polyphenols content as mg of gallic acid equivalents (mgGA) by spectrophotometric analysis, • antioxidant activity by means of DPPH assay

PEF ASSISTED EXTRACTION OF POLYPHENOLS ARTICHOCKES BRACTS

Bracts

Head

Leaves

Stem

time (h)

0.0 0.5 1.0 12.0 24.0

concentr

ation (

mg/g

bra

cts

)

0.0

0.5

1.0

1.5

control

PEF 0.75kV/cm 0.5kJ/kg

PEF 1.5kV/cm 5kJ/kg

Water extraction

E

PEF APPLICATIONS

Electropermeabilization of cells

Microbial inactivation Improvement of mass transfer

15-50 kV/cm; 100-150 kJ/kg 2-5 kV/cm; 6-10 kJ/kg

PEF PROCESSING ENERGY CONSUMPTION

STRATEGIES TO TRANSFER INNOVATIONS

HURDLE TECHNOLOGIES

Hurdle technology provides a framework for combining a number of

factors or milder preservation techniques to achieve an enhanced level

of product safety and stability with less severe processing conditions.

35 °C

45 °C

50 °C

55 °C

60 °C

65 °C

70 °C

0

10

20

30

40

50

60

Tem

pera

ture

[°C

]

0 20 40 60 80 100

Time [s]

PEF + MODERATE HEATING

E. coli PEF Pasteurization Cycle

[Heinz, Toepfl and Knorr]

PEF + HIGH PRESSURE CARBON DIOXIDE

E<Ec

CYTOPLASM CYTOPLASM

E>Ec

CYTOPLASM

E>>Ec

PEF induces the electroporation of the cell membrane

The mass transfer of the CO2 from the outside to the inside of the electroporated cell is facilitated

The microbial inactivation rate is enhanced if compared to that of the single treatments

CO2 solubilized in the liquid matrix

CO2 solubilized in the cytoplasm

Microbial cell in contact with pressurized CO2 after PEF treatment at different conditions

PEF: 20 J/ml, 2 mS/cm

+

HPCD: 8.0 MPa for 10 minutes

6 kV/cm 9 kV/cm 12 kV/cm Effect of the electrical field strength

PEF + HIGH PRESSURE CARBON DIOXIDE

HHP Apparatus MINI FOODLAB

FPG5620

HPH Apparatus FGP7420A.275

PEF Apparatus

NOVEL TECHNOLOGIES IN COMPARISON

Reference A-PEF C-HPH A-HHP

Qua

lity s

core

s [

-]

0

2

4

6

8Antioxidant Activity

Polyphenols

PPO inactivation

Vitamin A

Vitamin C

DE

NOVEL TECHNOLOGIES IN COMPARISON

Natural compounds with high biological activity

extracted from vegetable matrices

Difficult incorporation of bioactive compounds in food matrices due to poor

solubility in aqueous phase

Polyphenols Flavonoids

Carotenoids

PUFAs

Essential oils

PEF

PEF ASSISTED EXTRACTION OF BIOACTIVE COMPOUNDS

Minimization of the impact on the organoleptic

properties of the food systems where incorporated

Increase of the biological activity through the

promotion of the mass transfer through the cell

membranes

Efficient dispersion in aqueous phase

Protection of bioactive compounds from chemical degradation

Need of efficient nanometric delivery systems

NanoTech

NanoTech

NanoTech

PEF

DESIGN OF NANODELIVERY SYSTEMS

Top-down approach

Primary emulsion

(mm size)

HSH

Secondary emulsion

(nm size)

HPH

Initial ingredients

selection

FABRICATION OF NANODELIVERY SYSTEMS

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.00.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

cream

stable O/W

stable E-O

unstable

stable W/O Em

uls

ifier (%

)

Oil (%)

Wate

r (%

)7.14236

37

0.689

1.18123

1.34120

1.25m431nm1.4

4541.72235

2.05228

FABRICATION OF NANODELIVERY SYSTEMS

PEF

INCORPORATION IN REAL FOODS

Improved infusion of nanoencapsulated bioactive

compounds into solid foods, for delivering health-

beneficial or functional properties

Improved infusion solution for more homogeneous salting

processes, therefore reducing the amount of salt needed

Reduction of allergenic power (proteins unfolding induced by PEF and

Enzymatic hydrolysis)

Combined treatments (PEF+essential oils) for enhanced

antimicrobial activity and minimized impact on quality attributes

PEF

PEF

PEF

PEF

PEF TECHNOLOGY

PULSED LIGHT TECHNOLOGY FOR MICROBIAL INACTIVATION

LP

PULSED LIGHT - HILP

- Pulsed light (PL) is considered one of the most promising non-thermal technology for rapid and effective killing of a wide variety of food pathogenic and spoilage microorganisms in foods, on food contact surface, or in thin layers of liquids (Moraru and Uesugi, 2009).

- PL treatment consists of exposing contaminated substrate to intermittent, intense short pulses of polychromatic light (100-1100 nm), emitted by a light source such as a Xenon lamp.

During PL treatment energy is stored temporarly in a capacitor and then released via Switch to the lamp filled with Xenon gas. As sequence of this, the gas ionized emitting the charachteristic light of the treatment. These are the some parameters for characterization of PL treatment. Fluence or in other words, energy dose and exposure time are the most significant ones. Also pulse width, Pulse repetition rate and peak power.

PULSED LIGHT - HILP

time

Power

PULSED LIGHT - HILP

HILP: INACTIVATION MECHANISM

The action of the PL process is attributed to the effects of the high peak power and the UV component of the broad spectrum of the flash (Rajkovic et al., 2010; Oms-Oliu et al., 2010). The lethal action of PL might be due to the coexistence of different mechanisms:

- Microbial DNA damages by thymine dimer formation (photochemical effect) (Wang et al., 2005)

Structural damages caused by the pulsing effect (photophysical effect)

Microscopic observation Krishnamurthy et al., (2008)

Protein elution (Takeshita et al., 2003)

Pulsed light

UV light

Control

Proteins

Localized overheating of microbial cells (photothermal effect) (Hiramoto 1984; Wekhof 2000; Wekhof et al., 2001)


There is no or very little information on sub-lethal damage to bacterial cells induced by PL treatment.

The relative importance of each mechanism depends on the energy dose (fluence), the type of microorganism as well as the food absorption characteristics.


- Energy dose (or fluence F, in J/cm2) experienced by the target microorganisms

- Composition of the emitted light spectrum

- Distance from the light source and position of the sample in the treatment chamber

- Substrate properties: turbidity, homogeneity, thickness, color, opacity, presence of particulate material, soluble solids content and composition

- Inoculum size

- Geometry of the treatment cell

- Number of lamps

HILP: FACTORS DETERMINING PROCESS EFFECTIVENESS

Due to the factors affecting the amount of light energy provided to the product, the

distribution of light energy may spatially vary throughout the treatment chamber as well

as within a liquid substrate leading to non-uniform treatments

(Hsu and Moraru, 2011).

Fluence decreased with increasing the distance from the lamp, in all three directions

as well as with increasing the absorption properties of the liquid substrate

Microbial inactivation and heating effects achieved at different distance and location

throughout the treatment chamber as well as within the liquid substrates are also

non-uniform

HILP: FACTORSDETERMINING PROCESS EFFECTIVENESS

Sterilization Chamber

Power Control/Module

HV Cable

Xenon Lamp

Food product

Cooling air outletCooling air inlet

Tray

Sterilization Chamber

Power Control/Module

HV Cable

Xenon Lamp

Food product

Cooling air outletCooling air inlet

Tray

COMPONENTS OF HILP EQUIPMENT

Light spectrum

SteriPulse®-XL 3000 (Xenon Corp.) Pulse width 360 s - Pulse rate 3Hz Pulse energy=1.21 J/cm2 at 1.9 cm

INTENSITA’ v.s. DISTANZA Fp vs vertical distance

EFFECT OF SPATIAL LOCATION

Tray level

n = 5 - FP = 0.70 J/cm2/pulse

n = 7 - FP = 0.51 J/cm2/pulse

n = 9 - FP = 0.37 J/cm2/pulse

n = 11 - FP = 0.271 J/cm2/pulse

Spatial locations of sample on the tray

Gram-negative ‹ Gram-positive ‹ yeast ‹ bacterial spore ‹ mold‹ virus

HILP MICROBIAL INACTIVATION

PL treatment of foods has been approved by the FDA (1996) (code 21CFR179.41).

Solid and semisolid products: Vegetables (Gómez-López et al., 2005; Izquier et al., 2011) Fruit (Marquenie et al., 2003; Bialka & Demirici, 2007, 2008b,b) Food powders seeds (Fine & Gervais, 2004; Sharma & Demirici, 2003; Jun et al., 2003) Dairy products (Dunn et al., 1991) Meat (Hierro et al., 2011) Fish (Ozer & Demirici, 2006) Honey (Hillegas & Demirici, 2003) Infant powder milk (Choi et al., 2010) Liquid foods: Fruit juices (Saurer & Moreau, 2009; Caminiti et al., 2011) Infant foods (Choi et al., 2010) Milk (Smith et al., 2002; Krishnamurthy et al., 2007)

HILP APPLICATIONS

Only very few paper deals with the processing of a liquid food in a continuous flow PL unit (Krishnamurthy et al., 2007; Caminiti et al., 2011).

FRUIT JUICES PROCESSING WITH PULSED LIGHT

Study of the lethal and sub-lethal effects of PL treatments carried out in a continuous flow system

Investigated effects: - sample heating - energy dose (F) - absorption properties (a) of two different fruit juices (apple and orange juices - type of microorganism (gram-negative, gram-positive)

PULSED LIGHT PROCESSING OF FRUIT JUICES

Light spectrum

SteriPulse®-XL 3000 (Xenon Corp.) Pulse width 360 s - Pulse rate 3Hz Pulse energy=1.21 J/cm2 at 1.9 cm

Water-Ice bath

TIN

TOUT

TCH

Sterilizzation Chamber

DL

Untreated Sample

Treated Sample

Quartz Tubes

Xenon Lamp

PC

Metal box

Quartz Tubes: 1 mm i.d., 0.5 mm wall thickness

Power/Control Module

Water-Ice bath

Water-Ethylene glycol Cooling system


Microorganisms: E. coli (DH5-) and L. innocua (11288) Initial microbial load: Co=5*106 cfu/mL

Plates Count Method - Non-Selective agar medium (TSA) - Selective agar medium (EMB for E. coli, LSA for L. innocua) - 37°C for 24h (E.coli) or 48 h (L. innocua)

Treatment medium

pH (-)

°Brix

(-) Absorption coefficient

(cm-1)

Apple juice 3.49±0.08 10.9±0.2 13.9±0.9

Orange Juice 3.78±0.6 11.1±0.5 52.4±1.2

Flow rate (ml/min)

Re

(-)

tr (s)

n (-)

F (J/cm2)

38.4 510 0.49 1.5 1.8

27.0 358 0.70 2.1 2.5

20.8 276 0.91 2.7 3.3

17.0 226 1.11 3.3 4.0

13.4 178 1.41 4.2 5.1

12.5 166 1.51 4.5 5.5

500mL

Processing conditions

1.9 cm

PL treatment

1.21 J/cm2/pulse

Tin=10°C

Tout


PL treatment

Tin

Tout

HILP treatment High Fluences

Long operative times (continuous system)

Product heating Impairing of food quality

Thermal inactivation

Light absorption and/or

Lamp heating

Blower

Water-Etylene glycol cooling system


Time (s)

0 200 400 600 800 1000 1200

DT

(°C

)

0

10

20

30

40

tr=0.91s - F=3.3 J/cm2

No cooling

Cooling

Temperature rise of the juice increased with increasing running time

The presence of a cooling system limited the heating rate of the samples.

F (J/cm2)

0 1 2 3 4 5 6

DT

(°C

)

0

10

20

30

40

50

Fruit juice

Air

The steady state temperatures increased gradually as the energy dose increased.

Tout,max=34°C

With cooling

Stedy-state Temperature


Effect of F and

Energy dose (J/cm2)

0 1 2 3 4 5 6

Lo

g(N

/No

)

-7

-6

-5

-4

-3

-2

-1

0

Energy dose (J/cm2)

0 1 2 3 4 5 6

Lo

g(N

/No

)

-7

-6

-5

-4

-3

-2

-1

0E. coli L. innocua

Orange juice

Apple juice

Orange juice

Apple juice

The lethal effect of PL intensified upon increasing energy delivered to the juice

The PL resistance of both bacteria was greater in orange juice (=52.4 cm-1) than in apple juice (=13.9 cm-1)

The occurrence of the tailing phase could be due to shading effects due to insufficient light penetration in the depth of lower transmittance juices.


Orange juice

Apple juice

Energy dose (J/cm2)

0 1 2 3 4 5 6

Lo

g(N

/No

)

-7

-6

-5

-4

-3

-2

-1

0

Energy dose (J/cm2)

0 1 2 3 4 5 6

Lo

g(N

/No

)

-7

-6

-5

-4

-3

-2

-1

0

E. coli

L. innocua

L. innocua

E. coli

Orange juice Apple juice

E. coli cells (Gram-negative) show a greater susceptibility to light pulses than L. innocua (Gram-positive) due to:

Differences in structure and composition of the cell wall

Effect of bacterial strain


Sublethal injuries

E. coli

Energy dose (J/cm2)

0 1,8 2,5 3,3 4 5,1

Lo

g1

0 c

ycl

es o

f in

act

iva

tio

n

0

1

2

3

4

5

6

7

Apple juice

Energy dose (J/cm2)

0 1,8 2,5 3,3 4 5,1

Lo

g1

0 c

ycl

es o

f in

act

iva

tio

n

0

1

2

3

4

5

6

7

Orange juice

c)

a)

L. Innocua

Energy dose (J/cm2)

0 1,8 2,5 3,3 4 5,5

Lo

g1

0 c

ycl

es o

f in

act

iva

tio

n

0

1

2

3

4

5

6

7

Energy dose (J/cm2)

0 1,8 2,5 3,3 4 5,5

Lo

g1

0 c

ycl

es o

f in

act

iva

tio

n

0

1

2

3

4

5

6

7

Orange juice

Apple juice

d)

b)

Non selective

Selective

PULSED LIGHT TECHNOLOGY TO ENHANCE THE CONTENT OF BIOCTIVE COMPOUNDS IN

VEGETABLES

Effect of UV-C and HILP on color

Brightness (L) Color intensity (C )

-12

-10

-8

-6

-4

-2

0

7 14 21

∆L

Time (days)

Control

PL 8

UV 3

-80

-70

-60

-50

-40

-30

-20

-10

0

7 14 21

∆h

Time (days)

Control

PL 8

UV-3

-5

0

5

10

15

20

25

30

35

40

45

7 14 21

∆c*

Time (days)

Control

PL 8

UV3

Tendency towards red color (H, Hue)

Color changes during 21 days , of storage

Color variation during post harvest storage: h, L, c

No significant DIFFERENCE in color of treated and untreated tomatoes during storage No color changes shortly after treatments (data not shown).

PULSED LIGHT VS. UV LIGHT EFFECTS ON VEGETABLES

0

1

2

3

4

5

6

7

Control UV1 UV2 UV3

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

Control UV1 UV2 UV3

initial pH=3.93

initial °Brix=5

initial pH=3.93

PL Treatments

0

1

2

3

4

5

6

7

Control PL1 PL 2 PL 4 PL8 PL8(9) PL 4 (9)

Brix 7 days

14 days

21 days

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

Control PL1 PL 2 PL 4 PL8 PL8(9) PL 4 (9)

pH 7 days

14 days

21 days

UV-C Treatments

Effect of UV-C and HILP on pH and °Brix

There is no significant effect of both UV-C and PL treatment on the total soluble solids

content (°Brix) and pH

0

20

40

60

80

100

120

140

160

Control PL1 PL 2 PL 4 PL8 PL8(9) PL 4 (9) UV1 UV2 UV3

Varia

tio

n o

f ly

cop

en

e c

on

ten

t

Treatments

7 Days

14 days

21 days

Lycopene content of tomatoes on the harvest day was 0.63 mg/kg FW(Fresh Weight)

Lycopene extraction

Effect of UV-C and HILP on Lycopene content


0

10

20

30

40

50

60


Varia

tio

n o

f to

tal carote

no

ids

co

nte

nt

Treatments

7 days

14 days

21 days

Effect of UV-C and HILP on Total carotenoids content


Total carotenoids content in tomatoes on the harvesting day was 4.77 μg/g fresh weight


-10

0

10

20

30

40

50

60

70


Po

lyp

hen

ols

co

nte

nt

(%

)

Treatments

7 days

14 days

21 days

Total polyphenols content of tomatoes on the harvesting day (t0) was 250 mg of gallic acid equivalents (GAE)/kg fresh weight.

Effect of UV-C and HILP on Total polyphenol content


-20

0

20

40

60

80

100

120

140

160

180


An

tio

xid

an

t acti

vit

y (

%)

Treatments

7 days

14 days

21 days

Antioxidant activity was analyzed with the inactivation of DPPH method

Effect of UV-C and HILP on Antioxidant activity

Tomatoes on the harvesting day had 25% of inactivation of DPPH

UV-C Pulsed Light

Emission of radiation belonging to a

single band

Broader spectrum of light

The lamps contain mercury vapor The lamps do not contain mercury

Very long duration of treatment (h) Very short duration of treatment (s)

Sample heating negligible Thermal effects relatively important

Poor penetration depth High penetration depth

Low emission power High emission power

Pasteurization Process Sterilization process

UV-C vs HILP

PULSED LIGHT VS. UV LIGHT PROCESSING

NOVEL TECHNOLOGIES CAN BE USED FOR:

THE PREVENTION OF DISEASES

THE DESIGN OF PERSONALIZED NUTRITION STRATEGIES

3.000 YEARS AGO: EAT A ROOT 1000 YEARS AGO: PRAY 500 YEARS AGO: DRINK A POTION 50 YEARS AGO: TAKE A PILL

TODAY: EAT A ELECTRO-PERMEABILIZED ROOT

Doc, I’ve got

a sore throat

GRAZIE Abu, Anna, Ermelinda, Francesco, Gianpiero, Mafalda, Mariangela, Mariarenata, Maria Rosaria, Mariateresa, Mauro Maria, Mertcam, Paola, Roberta, Serena

Prodal scarl c/o Università di

Salerno

Via Ponte don Melillo

84084 Fisciano (SA)

Tel: 089-964028 Fax: 089-964168

Email: [email protected]

www.prodalricerche.it

mailto:[email protected]

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