High Pressure Processeslecture in the course

Advanced Processes in Food Technology ERASMUS

Dr. Milan HouškaFood Research Institute Prague, p.r.o.

E-mail:m.houska@vupp.cz

Lectured and reviewed by Prof. Zdeněk BubníkInstitute of Chemical Technology, Prague

E-mail : zdenek.bubnik@vscht.cz

Lecture outline - 1

-Introduction (why new technologies, NovelQ EU project)

-High pressure pasteurisation

-Principles and known technologies

-Pressure and foods (known effects)

-Examples of currently produced foods by high pressure

-Main producers of the machinery of the industrial scale

-Arrangement of selected units

- Overview of application and publications in the world

Lecture outline - 2

- Czech Republic situation and selected research results onapple broccoli juices

- Fruit and vegetables allergens and high pressure

- Germinated seeds and legumes

- Results available for application

- Conclusions (recommended literature, thesis challenge)

High pressure pasteurisationPrinciples and technologies

- Technology known more than 100 years

- Food packed into the flexible container or pouch

- Packed food is placed into the high pressure chamber

- The chamber is filled with drinking water or with other pressure transmitting fluid

- Chamber is closed and more fluid is pumped into the chamber to get the pressure 4000 or more bars (in industrial scale feasible pressure range is 4000-6000 bars)

- Pressure holding time from 1 to 15 minutes

- Control pressure release to avoid the microorganism cell rupture

High pressure pasteurisationPrinciples and technologies

- Technology known more than 100 years

- Food packed into the flexible container or pouch

- Packed food is placed into the high pressure chamber

- The chamber is filled with drinking water or with other pressure transmitting fluid

- Chamber is closed and more fluid is pumped into the chamber to get the pressure 4000 or more bars (in industrial scale feasible pressure range is 4000-6000 bars)

- Pressure holding time from 1 to 15 minutes

- Control pressure release to avoid the microorganism cell rupture

Known effects of the high pressure on foodsAdvantages- inactivation of viable microorganisms without heating, in combination with heating there is feasible the pressure assisted sterilisation

- keeps natural character, colour, nutritional substances, aroma

- partial coagulation of proteins in milk determined for production of yoghurt or cheese – higher curd gel strength without necessity to add more dry milk or higher dose of the enzyme

- keeps some enzyme active (sometimes advantage, sometimes disadvantage, see case study of germinated legumes)

- Saving the bioactive substances in vegetables

Disadvantages- minor effect on microbial spores (only pasteurisation at cold conditions)

- chamber is relatively small, batch process, less productivity than traditional pasteurisation, higher costs

Known effects of the high pressure on foodsAdvantages- inactivation of viable microorganisms without heating, in combination with heating there is feasible the pressure assisted sterilisation

- keeps natural character, colour, nutritional substances, aroma

- partial coagulation of proteins in milk determined for production of yoghurt or cheese – higher curd gel strength without necessity to add more dry milk or higher dose of the enzyme

- keeps some enzyme active (sometimes advantage, sometimes disadvantage, see case study of germinated legumes)

- Saving the bioactive substances in vegetables

Disadvantages- minor effect on microbial spores (only pasteurisation at cold conditions)

- chamber is relatively small, batch process, less productivity than traditional pasteurisation, higher costs

High pressure assisted sterilisation of foods- inactivation of viable microorganisms and spores in combination of highpressure and heating

Advantages- Utilisation of the compression heating (water about 3°C per each 100 MPa)

- Shorter heating up and cooling down times

- Better quality due to the above mentioned effects

- Treatment in packaging

- Saving more vitamines and other nutritional components

Disadvantages- Preheating outside necessary

- Additional cooling necessary

High pressure assisted sterilisation of foods- inactivation of viable microorganisms and spores in combination of highpressure and heating

Advantages- Utilisation of the compression heating (water about 3°C per each 100 MPa)

- Shorter heating up and cooling down times

- Better quality due to the above mentioned effects

- Treatment in packaging

- Saving more vitamines and other nutritional components

Disadvantages- Preheating outside necessary

- Additional cooling necessary

Pilot or full scale equipment available:

• High pressure (chamber volume from 0.2 ml to 125 litres)

• High voltage pulses (power from 0.2 kW to 800 kW)

• Cold plasma

• Intensive light flashes

• Ohmic heating of heterogeneous foods

The denaturation of proteins and combined effect of pressure and temperature

Examples of foods produced by high pressure- Quacamole (Avomex, U.S.A.) thick sauce from butter pear

- Salsa Mexicana (Avomex, U.S.A.)

- Chicken Fajita Dinner (Avomex, U.S.A.) ready to eat chicken chilled food

- Gold Oysters (Motivatit Seafoods, U.S.A.) (easy to open, inactivation of Vibrio cholerae)

- Organic apple juice (Frubaca, Portugal)

- Orange juice (Orchard House, UK) shelf life 21 days

- Solofrutta (ATA, Italy) fruit juices and pureé

- Cooked rice (Eschigo Seika, Japan) ready to reheat in microwave oven - Stewed ham (Espuňa, Spain) sliced ham vacuum packed treated by high pressure to prolong the shelf life declared in U.S.A. as „listeria free“product

- Ham Prosciutto (Abraham Brothers, Germany)

Examples of foods produced by high pressure- Quacamole (Avomex, U.S.A.) thick sauce from butter pear

- Salsa Mexicana (Avomex, U.S.A.)

- Chicken Fajita Dinner (Avomex, U.S.A.) ready to eat chicken chilled food

- Gold Oysters (Motivatit Seafoods, U.S.A.) (easy to open, inactivation of Vibrio cholerae)

- Organic apple juice (Frubaca, Portugal)

- Orange juice (Orchard House, UK) shelf life 21 days

- Solofrutta (ATA, Italy) fruit juices and pureé

- Cooked rice (Eschigo Seika, Japan) ready to reheat in microwave oven - Stewed ham (Espuňa, Spain) sliced ham vacuum packed treated by high pressure to prolong the shelf life declared in U.S.A. as „listeria free“product

- Ham Prosciutto (Abraham Brothers, Germany)

- Nicolas Corea (NC) Hyperbaric, Spain- AWURE former FLOW (USA) - COBE Steel (Japan) - Žďas a.s. – prototype with chamber 125 litres (CR)

Main producers of the full scale equipmentMain producers of the full scale equipment

NC HYPERBARIC designs, manufactures and markets industrial High Pressure Processing equipments for food processing.

Intensifiers

Electric cabinet

Yoke

Unloading line

Plugs

Vessel

Loading Line Buttons

THE NC HYPERBARIC CONCEPT

19901991

19921993

19941995

19961997

19981999

20002001

20022003

20042005

2006Total

26

2135524

11

11

24

466

223

1

18

23332

1

1 1 1 1

17

424

42

1

6

221

1

Vegetable products

Meat products

Juices and beverages

Seafood and fish

Others products

Branches of the food industry where the high pressure technology was applied: number of installations and year of installation

Total overview of the full scale equipments (number of equipments, year of installation, continent)

91 HPP machines in 55 companies producing more than 150 different products

Total production in 2005 : 100 000 to 120 000 Tons

19901991

19921993

19941995

19961997

19981999

20002001

20022003

20042005

2006Total

9191

79

68

5242

3220

121087771 2 3 5

56

8610799

4111

19

24431211

1

14

21

14

11 1 1 2

211

TOTALAmericaEuropaAsiaOceania

Y

Situation in the Czech Republic- In the Czech Republic the technology is utilised by the join stock company Beskyd Fryčovice. Production started by the March 2005.

- Thanks to the grant of Ministry of Industry and Retail the first high pressure equipment was build and tested. Each product is carefully tested and safety programme HACCP applied. These products found already their customers.

- Products are based on fruit and vegetables.

Situation in the Czech Republic- In the Czech Republic the technology is utilised by the join stock company Beskyd Fryčovice. Production started by the March 2005.

- Thanks to the grant of Ministry of Industry and Retail the first high pressure equipment was build and tested. Each product is carefully tested and safety programme HACCP applied. These products found already their customers.

- Products are based on fruit and vegetables.

Tested foods in the CR

- cauliflower juice with antimutagenic effects

- vegetable pieces in pickle

- fruit pieces in sweet pickle (fruit salad)

- cooked chicken meat

- apple and carrot juice

- orange juice

- pureé from apple and carrot

- vegetable juice

- broccoli juice, germinated legumes etc.

Tested foods in the CR

- cauliflower juice with antimutagenic effects

- vegetable pieces in pickle

- fruit pieces in sweet pickle (fruit salad)

- cooked chicken meat

- apple and carrot juice

- orange juice

- pureé from apple and carrot

- vegetable juice

- broccoli juice, germinated legumes etc.

Why fruit - vegetable juices???Nutricionists recommendation: There is urgent need to increase the consumption of fruit and vegetables in the Czech Republic up to 600 g per capita and day (best in the fresh state).

How to achieve this goal?

- Fresh ready to use raw materials for home cooking

- Ready to eat fresh fruit and vegetable salads

- Fresh fruit and vegetable juices (much easier to drink than eat)

How to preserve these juices and save their biological value?

Solution:

High Pressure Treatment – one of the most gentle preservation method prolonging the chilled storage time.

Why fruit - vegetable juices???Nutricionists recommendation: There is urgent need to increase the consumption of fruit and vegetables in the Czech Republic up to 600 g per capita and day (best in the fresh state).

How to achieve this goal?

- Fresh ready to use raw materials for home cooking

- Ready to eat fresh fruit and vegetable salads

- Fresh fruit and vegetable juices (much easier to drink than eat)

How to preserve these juices and save their biological value?

Solution:

High Pressure Treatment – one of the most gentle preservation method prolonging the chilled storage time.

Low daily intake of glucosinolates in the Czech Republic

- Average daily intake of glucosinolates in the Czech Republic isabout 10 mg per capita and day (only due to consumption of cabbage and cauliflower)

- Japan 112 mg per capita and day

- Great Britain 46 mg per capita and day.

It can be related to much higher occurence of breast and prostate cancer in the Czech Republic in comparison to the mentioned states.

Low daily intake of glucosinolates in the Czech Republic

- Average daily intake of glucosinolates in the Czech Republic isabout 10 mg per capita and day (only due to consumption of cabbage and cauliflower)

- Japan 112 mg per capita and day

- Great Britain 46 mg per capita and day.

It can be related to much higher occurence of breast and prostate cancer in the Czech Republic in comparison to the mentioned states.

Cruciferous vegetables - the source of glucosinolates

- Juices from this type of vegetable can be the source of isothiocyanates that are bioactive in chemoprevention.

- Broccoli has fair composition of glucosinolates (progoitrin that can affect the thyroid gland is not present)

- Broccoli is the main source of glucoraphanin that can be hydrolyzed by the myrosinase to isothiocyanate - sulforaphane (it has tested very high antimutagenic antigenotoxic activity)

Cruciferous vegetables - the source of glucosinolates

- Juices from this type of vegetable can be the source of isothiocyanates that are bioactive in chemoprevention.

- Broccoli has fair composition of glucosinolates (progoitrin that can affect the thyroid gland is not present)

- Broccoli is the main source of glucoraphanin that can be hydrolyzed by the myrosinase to isothiocyanate - sulforaphane (it has tested very high antimutagenic antigenotoxic activity)

Apple broccoli juice

The main condition of the consumption – sensorial acceptability

- Therefore it was developed the recipe of apple-broccoli juice combined with orange concentrate and acidified with lemon juice

- 50 % fresh apple pulp juice

- 30 % fresh broccoli pulp juice

- 17 % frozen orange concentrate

- 3 % lemon juice

Packaging into PET bottles with volume 330 ml.

Apple broccoli juice

The main condition of the consumption – sensorial acceptability

- Therefore it was developed the recipe of apple-broccoli juice combined with orange concentrate and acidified with lemon juice

- 50 % fresh apple pulp juice

- 30 % fresh broccoli pulp juice

- 17 % frozen orange concentrate

- 3 % lemon juice

Packaging into PET bottles with volume 330 ml.

Frequency of consume of tested package size (0.33 litres)

0

5

10

15

20

25

30

35

num

ber o

f con

sum

ers

broccoli

white cabbage

carrot

broccoli 5 22 13 16 1 3

white cabbage 15 31 7 4 0 3

carrot 15 14 17 7 4 3

daily on alternate day twice a week once a week one a month never

Daily convenient packaging size for consuming

0

5

10

15

20

25

30

35

40

45

litres

num

ber o

f con

sum

ers

broccoli

white cabbage

carrot

broccoli 3 13 31 6 4 1 2

white cabbage 2 10 39 2 4 1 2

carrot 1 12 26 3 1 0 0

0,1 0,2 0,3 0,4 0,5 0,6 0

0

10

20

30

40

50

60

day of consum ( days)

Num

ber o

f con

sum

ers

improvement great 0 1 1 2 2 2 3 4 4 5

improvement small 0 0 4 10 15 15 20 18 18 18

steady state 57 56 53 45 41 40 34 33 33 32

worsening small 3 3 2 3 1 2 2 4 4 4

worsening great 0 0 0 0 1 1 1 1 1 1

1 2 3 4 5 6 7 8 9 10

Feeling of the total condition of person during 10 days consum of apple-white cabbage-lemon juice

The anti-mutagenic activity of the vegetable juices was studied under model conditions using bacterial strains of Salmonella typhimuriumTA-98 (Ames test)

Model mutagens

- aflatoxin B1

- 2-amino-3-methylimidazo [4, 5-f] quinoline (IQ)

- Nitroso-methyl-urea

The anti-mutagenic activity of the vegetable juices was studied under model conditions using bacterial strains of Salmonella typhimuriumTA-98 (Ames test)

Model mutagens

- aflatoxin B1

- 2-amino-3-methylimidazo [4, 5-f] quinoline (IQ)

- Nitroso-methyl-urea

The inhibition rate was calculated from number of revertants Rt using:

Inhibition rate [%] = 100 – [(Rt (juice + mutagen) / Rt (mutagen)) .100]

The inhibition rate is ranged by the following way:

0 – 20% negative,

20 – 40% weak positive,

40 – 60% positive,

60 – 90% strongly positive,

> 90% probably toxic

The inhibition rate was calculated from number of revertants Rt using:

Inhibition rate [%] = 100 – [(Rt (juice + mutagen) / Rt (mutagen)) .100]

The inhibition rate is ranged by the following way:

0 – 20% negative,

20 – 40% weak positive,

40 – 60% positive,

60 – 90% strongly positive,

> 90% probably toxic

Sulforaphane - inhibition of mutagenicity

0

10

20

30

40

50

60

70

1 2,5 5 10 20

dose [ug]

I [%

] MNUIQAFTB1

Antimutagenic activity of juices : mutagen Aflatoxine B1 (mould toxine)‏

Inhibition of mutagenityProcessingJuice

Weakly positiveNegativeWeakly positive up positive

FrozenPasteurisedPressurized

Red cabbage

Weakly positive up positivenegative Weakly positive up positive

FrozenPasteurisedPressurized

Cauliflower

negativenegativenegative

FrozenPasteurisedPressurized

White cabbage

positive up strongly positiveweakly positivepositive up strongly positive

FrozenPasteurisedPressurized

Broccoli

Weakly positive up positivenegativepositive up strongly positive

FrozenPasteurisedPressurized

Brussels sprouts

Food safety – HACCP methodology

- Hazard analysis and critical control points for apple-broccoli juice

- physical, microbial and chemical risk

- Prediction of anti-mutagenic activity of individual vegetable juices

- Ames test

- Juice preparation for Ames tests

- chilled vegetables, ROTOR Vitamat, Champion and Green Power juicers have been used, pH adjusted to 4 by lemon juice

- compared samples: frozen (-18°C), heat pasteurized (80°C for 10 minutes) and high pressure treated (500 MPa, 10 minutes)

Food safety – HACCP methodology

- Hazard analysis and critical control points for apple-broccoli juice

- physical, microbial and chemical risk

- Prediction of anti-mutagenic activity of individual vegetable juices

- Ames test

- Juice preparation for Ames tests

- chilled vegetables, ROTOR Vitamat, Champion and Green Power juicers have been used, pH adjusted to 4 by lemon juice

- compared samples: frozen (-18°C), heat pasteurized (80°C for 10 minutes) and high pressure treated (500 MPa, 10 minutes)

Daily intake of glucosinolates represented by one bottle of apple-broccoli juice

-The volume of 330 ml of juice contains about 100 ml of broccoli juice. This volume can be prepared by juicing of 167 g of fresh broccoli (juice extraction rate about 60%).

-The average glucosinolate content in broccoli is about 860 mg per 1 kg of broccoli, Hrncirik and Velisek (1999).

-In 167 grams of the fresh broccoli is about 144 mg glucosinolates.

- If we adopt the juice extraction rate the same as the glucosinolatesextraction rate 60 % the juice will contain about 86 mg glucosinolates. This intake is between Japan and Great Britain.

Daily intake of glucosinolates represented by one bottle of apple-broccoli juice

-The volume of 330 ml of juice contains about 100 ml of broccoli juice. This volume can be prepared by juicing of 167 g of fresh broccoli (juice extraction rate about 60%).

-The average glucosinolate content in broccoli is about 860 mg per 1 kg of broccoli, Hrncirik and Velisek (1999).

-In 167 grams of the fresh broccoli is about 144 mg glucosinolates.

- If we adopt the juice extraction rate the same as the glucosinolatesextraction rate 60 % the juice will contain about 86 mg glucosinolates. This intake is between Japan and Great Britain.

Sulforaphane content in one bottle of apple broccoli juice

-It was found that the sulforaphane content in commercially produced apple-broccoli juice varies between 0.40 – 1.34 µg/ml.

- In one bottle with volume 330 ml is between 132 - 442 µg of sulforaphane.

-There is the company in USA purchasing the tablets with broccoliextract that contains 120 µg of sulforaphane in each tablet, recommended daily intake is one tablet.

-The other components that have synergic effect are not probably present in the tablets.

Sulforaphane content in one bottle of apple broccoli juice

-It was found that the sulforaphane content in commercially produced apple-broccoli juice varies between 0.40 – 1.34 µg/ml.

- In one bottle with volume 330 ml is between 132 - 442 µg of sulforaphane.

-There is the company in USA purchasing the tablets with broccoliextract that contains 120 µg of sulforaphane in each tablet, recommended daily intake is one tablet.

-The other components that have synergic effect are not probably present in the tablets.

Risk elimination by CCP - GMP and GHP is adopted in the production plant.

- Chemical contamination risks eliminated at the acceptance of raw material step (documents, preliminary analysis).

- Pest and microbial risk is under the control of chilled storage of raw materials (e.g. moulds growth)

- Foreign body and microbial spoilage risk is eliminated at washing and inspection step of raw materials

- Germination of spores of clostridia is eliminated by chilled storage and acidification of juice below pH 4.2

- Microbial risk of viable pathogens (target microbe Esch. coli) is eliminated by high pressure treatment (5 log decades inactivation rate demonstrated by the challenge test)

- Microbial spoilage risk eliminated also by high pressure treatment step (L. plantarum target microbe)

Risk elimination by CCP - GMP and GHP is adopted in the production plant.

- Chemical contamination risks eliminated at the acceptance of raw material step (documents, preliminary analysis).

- Pest and microbial risk is under the control of chilled storage of raw materials (e.g. moulds growth)

- Foreign body and microbial spoilage risk is eliminated at washing and inspection step of raw materials

- Germination of spores of clostridia is eliminated by chilled storage and acidification of juice below pH 4.2

- Microbial risk of viable pathogens (target microbe Esch. coli) is eliminated by high pressure treatment (5 log decades inactivation rate demonstrated by the challenge test)

- Microbial spoilage risk eliminated also by high pressure treatment step (L. plantarum target microbe)

Fruit –vegetable juices treated by high pressure

New product marketed at the begin of the year 2007: white cabbage - apple juice

Fruit –vegetable juices treated by high pressure

New product marketed at the begin of the year 2007: white cabbage - apple juice

- Germinated plant seeds- radish seeds- alfa-alfa seeds- wheat seeds

-Germinated legume seeds- lentils- chickpea- Cicera- pea

Advantages:-HP inactivates microorganisms-Does not inactivate alpha-galactosidase –during storage it hydrolyses galactosides to simple saccharides:

- removal of flatulence- fresh like characted, sweet at the end of storage

Germinated legume seeds - chickpea

Germinated lentil seeds

1 day 2 days 3 days

4 days 5 days

Three kinds of vegetable salads prepared with germinated mung beans

Allergens in foods and high pressure

- Hope for high pressure effects on allergens- Selected results of the team VÚPP, Imumed, Charles University hospital Královské Vinohrady

-20

-15

-10

-5

0

5

10

15

20

25

30

35

185 195 205 215 225 235 245 255

wave length [nm]

CD

[mde

g]

0000-00-25

5010-30-21

5010-40-22

5010-50-23

Figure 1 ECDFigure 1 ECD spectra of allergen spectra of allergen rMalrMal d1 d1

00000000--0000--25 untreated sample, 25 untreated sample,

50105010--3030--21 sample treated 500 21 sample treated 500 MPaMPa for for 10 minutes at 3010 minutes at 30°°C, C,

50105010--4040--22 sample treated 500 22 sample treated 500 MPaMPa for for 10 minutes at 4010 minutes at 40°°C,C,

50105010--5050--23 sample treated 500 23 sample treated 500 MPaMPa for for 10 minutes at 5010 minutes at 50°°C)C)

Basophile activation testBasophile activation test

Activation (degranulation) of basophiles –membrane markers

CD63 - membrane of basophile granules

Flow cytometry

Positive test >15% CD203 + CD 63 +

Prick to prick testPrick to prick test

-- SafetySafety-- Quantitative test resultsQuantitative test results-- Positive test: wheal diameter Positive test: wheal diameter >> 3 mm3 mm

Blot Gel

5 4 3 2 1 5 4 3 2 1 1 2 3 4 1 2 3 4 55

1 1 –– control control rMalrMal d1d12 2 –– untreated untreated rMalrMal d1 solutiond1 solution3 3 –– 500 500 MPaMPa, 10 minutes, 30, 10 minutes, 30°°CC4 4 –– 500 500 MPaMPa, 10 minutes 40, 10 minutes 40°°CC5 5 –– 500 500 MPaMPa, 10 minutes 50, 10 minutes 50°°CC

ResultsResults

Apple juice and homogenateApple juice and homogenate-- Basophile activation testBasophile activation test-- Skin Prick to prick testSkin Prick to prick test-- Western blot testWestern blot test-- Double blind placebo control oral provocation testDouble blind placebo control oral provocation test

Mald1

6 5 4 3 2 8 9 10 Mal d1

developed with serum mixture

Blot:2 – standard Mal d13 – without skin - untreated with HP4 – without skin - HPT5 – with skin - untreated with HP6 – with skin - HPTGel:8 – without skin - untreated with HP9 – without skin - HPT10 –juice without ascorbic acid - untreated with HP

Figure 3 Influence of HPT 550 MPa for 10 minutes and presence of apple skin on apple juice allergenicity – method Western blot

GelBlot

Research results available for commercialisation

Pressure treated homogenates of hop- FRIP Patent pending and know how

- no drying necessary- no loss of compounds- fresh like hop for one year- chilling only necessary

Pressure treated homogenates of herbsand spice plants

- Basil (very strong homogenate) - fresh like- one year of chilled storage tested

Conclusions

Recommended literature

D. A. Ledward, D. E. Johnston, R. G., Earnshaw, & A. P. M. Hasting (Eds.), High pressure processing of foods, Nottingham: Nottingham University Press, 1995

Hendrickx M.E.G., Knorr D., editors, Ultra High Pressure Treatments of Foods, KluwerAcademic/Plenum Publishers, New York, Boston, Dordrecht, London, Moscow, 2002

journal papers

Challenge of diploma thesis assistence at VÚPP agreement with Prof. Bubník and VÚPP.