1. Power ultrasound, high pressure and thermal processing for the inactivation of microbial spores
Evelyn and Filipa V. M. Silva
ICEF 12
Quebec City, Canada, June 14-18, 2015
Good afternoon everyone…
My name is Evelyn. First I would like to thanks ICEF conference committee that giving us opportunity today to present our research.
Today my presentation is about Power ultrasound, high pressure and thermal processing for the inactivation of microbial spores.

2. Food safety and preservation
-potential health risk
from consumption
Food preservation:
-inhibition spoilage
-inactivation spoilage & pathogenic microorganisms
Consumers’ demand:
-safe
-fresh appearance
-flavour
-texture
-shelf-life
First I would like to speak about a little bit background of our research.
Increasing consumers demands on safe, healthy, fresh-tasting and minimally processed with a longer shelf life and changes in people eating habits (fewer people eat foods produced on their lands) have placed food safety and food preservation become an increasingly important components of the food industry.
Food safety and Food preservation/processing relates with the inactivation of microorganisms including their vegetative and spore forms (bacterial and fungal).
Or refers to one of a number of techniques/processes of treating and handling food to prevent food spoilage between the time of production and the time of consumption.
Food preservation usually involve preventing (inhibit or inactivate) the growth of microorganisms (bacteria, yeast and moulds):
to prevent foodborne illness since some of these organisms can secrete poisonous substances or toxins which are dangerous to human health and can even be fatal.
to prevent quality degradation/prevent oxidation of essential biochemical components which causes food destruction since these microorganisms
can attack food constituents, breaking down/degrading the food constituents and use them to grow.
SHORTEN IT !!

3. food safety or spoilage concerns
BACTERIAL SPORES and
MOULD ASCOSPORES –
food safety or spoilage concerns
Food
Pasteurization
Low acid cold stored foods (pH>4.6)
High acid ambient stored foods (pH<4.6)
Thus, foods are commonly pasteurized.
Food pasteurization traditionally refers to a mild heat treatment to inactivate vegetative forms of pathogenic and spoilage microorganisms (thermal method).
Now food pasteurization refers to any process, treatment (including non thermal methods), or combination thereof, that is applied to food to reduce the most resistant microorganism of public health significance to a level that is not likely to present a public health risk under normal conditions of distribution and storage. This indicates only partial destruction of microbes or the existence of bacterial spores and mould ascospores after the pasteurization process.
For high-acid foods (pH < 4.6), the foods are stable at ambient conditions after a pasteurization process, because the acidic food environment is not conducive to the growth of harmful microorganisms and microbial spores in the pasteurized food. For these types of foods, a pasteurization process allows a long shelf-life (months) at room temperature, and if refrigerated storage is used, a milder pasteurization may be applied and the product quality is improved.
In these types of foods, microbial spores of concern are:
- Byssochlamys spp., Talaromyces spp., Neosartorya spp. and Eupenicillium spp. (since spores may activate to germinate after the mild treatments)
- Alicyclobacillus acidoterrestris (since spores can germinate and grow in high acidic fruit products ).
With respect to low-acid food products (pH > 4.6, e.g. milk), a shorter shelf-life (days) is obtained after pasteurization, and foods are usually cold stored/refrigerated to maintain product safety by restricting the growth of surviving pathogens.
Microbial spores of concern are:
- psychrotrophic non proteolytic Clostridium botulinum
- psychrotrophic Bacillus cereus
Psychrotrophic microorganisms are defined as microorganisms that being able to germinate and grow at temperatures of 7°C or less (during refrigerated storage).
SHORTEN IT!!
-psychrotrophic Clostridium botulinum
-psychrotrophic Bacillus cereus
-Byssochlamys spp., Talaromyces spp., Neosartorya spp., Eupenicillium spp.
-Alicyclobacillus acidoterrestris

4. Food Preservation Processes
Thermal
Non-Thermal
Pasteurization
High pressure processing
Ohmic heating
Pulsed electric field
Microwave heating
Ultrasonication
Infrared heating
Pulsed light
Blanching
Irradiation
Two methods in Food preservation/processing: Thermal and Non thermal
Few examples for thermal (heat addition): pasteurization, ohmic, microwave and infrared heating, blanching
Sterilization at high temperature often diminish food constituents.
Pasteurization is the common process, in which the final product has a storage life of some weeks (generally under refrigeration). However, vitamins, taste, color, and other sensorial characteristics are decreased with this treatment. High temperature is responsible for these effects and can be observed in the loss of nutritional components and changes in flavor, taste, and texture, often creating the need for additives to
improve the product.
Nowadays, non-thermal methods has been increasingly evaluated and adopted for replacing thermal treatments and allow most foods to retain their original sensory, nutritional, and functional properties during processing. Examples for non thermal methods are: high pressure processing (HPP), Pulsed electric field (PEF) and ultrasonication.
Ozone
Cold plasma
Electron beam
Oscillating magnetic field

5. Ultrasound technology
Power ultrasound
(≥10 W/cm2, kHz)
Food preservation: research stage
Power ultrasound
Power ultrasound is one emerging non thermal food preservation methods that relies on the application of sound waves (>10 W/cm2, 20 to 100 kHz) to the food/beverage, causing microbial cell death. This phenomenon called cavitation that creates microgas bubbles due to regions of pressure change. Microbial killing involves the thinning of the cell membranes, localized heating, and the production of free radicals, which induces adverse chemical changes in the DNA or protein denaturation.
Ultrasound is a novel technology for food processing that has been explored in the lab with successful results but it is still under development (research stage). The application involves: extraction, drying, crystallization, filtration, defoaming, homogenization, meat tenderization and food preservation. 
With respect to food preservation (inhibition or inactivation), ultrasound most likely have to be used in conjunction with heat treatment (thermosonication), pressure treatment (manosonication), or both (manothermosonication).
For thermosonication, the effectiveness of an ultrasound treatment is dependent on the type of microorganism being treated. Other factors that are known to affect the effectiveness of microbial inactivation are amplitude of the ultrasonic waves, exposure/contact time, volume of food being processed, the composition of the food and the treatment temperature.
Thermal
Thermosonication (TS)

6. High pressure processing (HPP)
Generally, MPa
Well developed commercial
food preservation process
HPP
High pressure processing or HPP ( MPa at chilled or mild temperatures, from ms to over than 20 min treatment) is a well developed commercial process for preservation shelf stable high acid foods with minimal effects on taste, texture, appearance, or nutritional value.
E.g. strawberry, apple, and kiwi jams, avocado products (guacamole), tomato salsa, applesauce, and orange juice. 
HPP cannot yet be used to make shelf-stable versions of low-acid products such as vegetables, milk, or soups because of limitation of this process to destroy spores without added heat.
Therefore, pasteurization of low acid foods is usually achieved through combination of HPP with heat (HPP-Thermal) to obtain an increased rate of inactivation of microbial spores.
The effectiveness of HPP-thermal treatment is also dependent on the type of microorganism being treated, pressure, temperature, exposure/contact time, and properties of the treated foods.
Thermal
HPP-Thermal

7. Our work Technologies: TS HPP-Thermal Thermal Spores of:
Byssochlamys nivea in strawberry puree
Psychrotrophic Bacillus cereus in milk
In this work, inactivation of 2 microbial spores of concern i.e Byssochlamys nivea and psychrotrophic Bacillus cereus were investigated and 3 technologies were compared and the kinetics was modeled.
Byssochlamys nivea and Bacillus cereus were chosen because they are a heat resistant sporeformers. Byssochlamys nivea can grow over a wide range of temperature and pH as most fungi, may grow under reduced oxygen conditions inside the food packs and is a concern to human and animal health since it can produce the mycotoxins patulin which may act in the central neural system causing sustained tremors and convulsions, whereas psychrotrophic Bacillus cereus is able to regenerate to large numbers at refrigerated temperatures and also can produce toxins in the foods (causing diarrhea or emesis).
Strawberry puree was chosen as the treatment medium for Byssochlamys nivea and milk for the psychrotrophic Bacillus cereus since the two medium are prone to the contamination by these microbes.

8. Enumeration of survivors - N in Potato dextrose agar
B. nivea: Comparing different technologies for the inactivation at 75°C
75°C
75°C
75°C
0.33 W/mL
600 MPa
Our first experiment was the inactivation of Byssochlamys nivea ascospores in strawberry puree:
TS, HPP and thermal inactivation of the ascospores at 75°C for up to 30 min was investigated. Then, the ascospore survivors were enumerated on potato dextrose agar (PDA) for 5 days.
For HPP-thermal and thermal: 3.5 ml puree containing fungal spore suspension was submitted to the 75°C-Thermal and 600 Mpa HPP-75°C.
TS: 100 mL puree containing fungal spore suspension was submitted to 200 W, 24 kHz, 0.33 W/mL ultrasonic processor
Times up to 30 min
Enumeration of survivors - N in Potato dextrose agar

9. B. nivea spores inactivation at 75°C
The results: (Do you think that I should mention about the thermal pretreatment for TS or better not??)
As can be seen from the graph, for thermal processing, significant spore activation (initial increase in the number of spores with the processing rather than reduction) in the B. nivea ascospores was registered with 75°C for up to 30 min (reaching 1.2 log at 30 min). With fungal spores, activation is a mechanism caused by the application of heat, a chemical or other factor under certain conditions, which causes breaking of the spore dormancy for germination, leading to an increase in the viable counts by several logs. Other investigators have also observed thermal activation of B. nivea ascospores in fruit juice or nectar at temperatures between 75 and 85°C.
For 10 min process, 1.4 log reductions in B. nivea spores was obtained for HPP-thermal (the spores reduced steadily with the HPP-T processing time) vs. no reductions for TS and thermal. Suggesting for short time treatments (≤ 10 min), HPP-T was the best techniques among the others.
However, for a 75°C and 15 min process, TS was more effective followed by HPP-T and lastly sole thermal.
TS process showed a sharp decline in the spore population following an activation at a maximum of +1.0 log at 5 min.
After 15 min treatment at 75°C for B. nivea, TS and HPP-T caused ≥1.8 log reductions vs no inactivation for thermal.

10. TS – Modeling B. nivea spores inactivation in strawberry puree
TS 65°C
T (°C) a b c d R2
MSE 75
-9.1± 6.1
10.1± 6.0
5.1± 0.3
15.9± 6.5
0.994
0.03 70
-3.9± 0.7
6.1± 0.6
11.6± 0.6
16.7± 2.5
0.983
0.07 65
-2.7± 1.2
5.7± 1.1
17.7± 1.5
24.0± 6.5
0.940
0.25
Since TS demonstrated the best results after 15 min, then TS inactivation of B. nivea ascospores at three temperatures (65, 70 and 75°C) was studied and the kinetics was modeled.
The figure shows the log survivors of spores contained in strawberry puree by TS.
TS at 75°C, the maximum temperature supported by the ultrasound equipment, was the best.
Short time treatments did not inactivate the spores since activation shoulders were observed for all TS temperatures tested (75, 70, and 65°C) with a maximum at 5 min for 75°C, 10 min for 70°C and 15 min for 65°C. The peaks in the log counts were followed by approximately linear spore inactivation. There was no spores reduction at TS ≤ 55°C after 60 min (not shown).
Higher spore activation for longer periods was obtained when lowering the TS temperature. Overall, while 15 min at 75°C achieved ≃ 2 log reductions, 35 min at 70°C and ˃ 60 min at 65°C were required to obtain the same spore inactivation.
Due to the activation shoulders observed in all the TS spore survival curves the first order kinetics was not appropriate and modeling was a challenge. Initially, four non-linear models (double Weibullian, Peleg, logistic and Lorentzian using TableCurve 2D software) were attempted. However, the double Weibullian and Peleg’s models used for heat activated Bacillus spores were inappropriate, presenting high standard errors for the estimated parameters (results not shown). On the contrary, four parameters logistic and Lorentzian models worked well. The Lorentzian distribution was a better model (showing 0.025−0.248 MSE and 0.940−0.994 R2), although the performance slightly decreased at 65°C. The Lorentzian b parameters increased from 5.7 to 10.1 as the temperature was increased from 65 to 75°C, whereas the Lorentzian a, c, and d parameters decreased with the TS temperature, exhibiting temperature dependence (R2>0.81).
TS 70°C

11. Enumeration of survivors - N in nutrient agar
B. cereus: Comparing different technologies for the inactivation at 70°C
70°C
70°C
70°C
0.33 W/mL
600 MPa
The second experiment was the inactivation of Bacillus cereus spores in reconstituted milk:
TS, HPP and thermal inactivation of the spores at 70°C for up to 20 min was investigated. Then, the spore survivors were enumerated on nutrient agar (NA) for 2 days.
For HPP-thermal and thermal: 3 ml milk containing B. cereus spore suspension was submitted to 70°C-Thermal and 600 Mpa HPP-70°C.
TS: 100 mL puree containing B. cereus spore suspension was submitted to 200 W, 24 kHz, 0.33 W/mL ultrasonic processor
Times up to 20 min
Enumeration of survivors - N in nutrient agar

12. B. cereus spores inactivation at 70°C
First order: 𝒍𝒐𝒈 𝑵 𝑵 𝟎 =− 𝒕 𝑫 𝑻
Thermal
Processes
D70°C ± SD
Thermal
8.64 ± 0.30
HPP-Thermal
4.75 ± 0.09 TS
2.93 ± 0.03
The results:
From the figure, it can be concluded that TS was more effective followed by HPP-T and lastly sole thermal.
This is supported by the first order kinetic values obtained for the 3 methods attempted, in which TS D70°C < HPP-T D70°C < Thermal D70°C.
For 10 min process at 70°C for B. cereus, 3.5 log reductions in B. cereus spores was obtained for TS vs. 2.4 log for HPP-T and 1 log for thermal.
TS caused ≥5 log reductions vs 3.2 log for HPP-T and 2 log for thermal after 15 min treatment at 70°C. TS
HPP-Thermal

13. TS – Modeling B. cereus spores inactivation in milk
TS 50°C
T (°C)
Skim milk: B. cereus spores
DT-value ± SD (min) R2
MSE 70
2.93 ± 0.03
0.980
0.03 60
4.00 ± 0.04
0.996
0.01 50
8.05 ± 0.11
0.988
0.02
z-value ± SE (⁰C)
45.7 ± 0.07
0.960
TS 60°C
Since TS also demonstrated the best results for the spore inactivation after 10 min, then TS inactivation of B. cereus spores at three temperatures (50, 60 and 70°C) was studied and the kinetics was modeled using the first order kinetics.
TS at room T showed negligible effect on the spore inactivation (results not shown).
From the figure, TS at 70°C was the best.
As expected, the higher the temperature, the higher the spore inactivation rate. This was confirmed by the lower D-values obtained with the increasing temperature (from 8.1 min at 50°C to 2.9 at 70°C).
The first order kinetics was supported by high R2 ( ) and low MSE values ( ).

14. Comparison of TS inactivation of B. nivea and B. cereus
In this figure, TS log survivors of B. nivea ascospores at 75°C and B. cereus spores at 70°C were represented and the inactivation was compared.
For 10 min TS treatment, 3.5 log was obtained with B. cereus and no inactivation for B. nivea at 75°C, suggesting that the B. nivea ascospores were more resistant to TS than B. cereus spores.

15. Conclusions Ultrasound or HPP alone were ineffective.
For B. cereus, TS was more effective followed by HPP-T and lastly sole thermal.
For the mould spores, HPP-T worked better since activation shoulders were observed with the TS.
The mould ascospores were more resistant than the bacterial spores.
TS inactivation: B. nivea - Lorentzian model
B. cereus spores - first order kinetics
Finally, the conclusions of this research are……………..
…for both spores
2. - For 15 min treatment at 75°C for B. nivea, TS and HPP-T caused ≥1.8 log reductions vs no inactivation for thermal.
- For 15 min treatment at 70°C for B. cereus, TS caused ≥5 log reductions vs 3.2 log for HPP-T and 2 log for thermal.
3. the 10 min thermal treatment resulted in 3.5 log in B. cereus spores at 70°C and no inactivation in B. nivea ascospores at 75°C 4.

16. Limitation and further research
Temperature ~75°C was the maximum temperature supported by the ultrasound equipment, thus further research must be made to optimise the process conditions by designing an ultrasound probe that can withstand higher temperatures.

17. Acknowledgement
PhD grant from Directorate General of Higher Education Ministry of Education and Culture of Indonesia is acknowledged.
Project “Non thermal pasteurization of foods”, Faculty Research Development Fund, Faculty of Engineering, University of Auckland is recognized.
The support from laboratory and administrative staff from Chemicals and Materials Engineering Department, University of Auckland is also appreciated.
Lastly, I would like to acknowledge ……………………………

18. THANK YOU
And thank you very much for listening to my presentation.