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Cold Atmospheric Plasma (CAP) sterilization of powdered products: optimization and validation at alimentary and pharmaceutical levels

Periodic Reporting for period 1 - CAP-SAliPharm (Cold Atmospheric Plasma (CAP) sterilization of powdered products: optimization and validation at alimentary and pharmaceutical levels)

Reporting period: 2018-01-01 to 2019-12-31

Today, sterilization of powder products remains a challenge for the food industry due to its heat and moisture sensitivity. Therefore, the focus of the scientific community lies on novel processes under the urgent need to develop even more safe food powders (e.g. infant formulas and cereals, protein isolates, spices, nutritional supplements, among others). Bacteria are widely capable to survive in powders during long periods and might proliferate quickly after the rehydration with fatal consequences mainly in infants and elderlies. Recent cases of Salmonella spp. and Bacillus spp. contamination of infant milk formulas have been reported.
The present project aimed to improve Cold Atmospheric Plasma (CAP) treatments for the reduction of microorganisms in powdered food and pharmaceutical products. The plasma was generated in air or nitrogen by a partial ionization of the gas in a high voltage electric field. Improvements are associated with reductions in the intensity of the plasma treatment (time and plasma power) at relevant rates of inactivation. It was studied how the decrease of the treatment intensity reduces modifications of the powder matrix and thus contributes to a minimization of the formation of side products and a reduction toxicological risk.
Based on experiences with sub-atmospheric capacitively coupled and atmospheric dielectric barrier discharge plasma, a surface micro-discharge (SMD) plasma chamber was designed. This SMD device is supposed to be advantageous and relevant for an industrial application for the ambient pressure at plasma ignition, the possibility to control the process gas composition, the free choice of the distance of the sample to the source and thus reducing the plasma intensity with the distance at modified composition, the ease of scalability via a simple plasma surface extension and the ease of transition from a batch to a continuous process. Conclusions on a design with relevance for industrial application are first linked to a description of the kinetics of inactivation of microorganisms and the alteration of the powder matrix.
Gently dried cells of Salmonella spp., Franconibacter pulveris (earlier classified as Enterobacter pulveris, a surrogate of Cronobacter sakazakii) and spores of Bacillus subtilis (surrogate of B. cereus) have been used to contaminate powders of corn-starch, hydroxy-methylcellulose, whey proteins, Spirulina algae, and cricket. These binomials are relevant for the food industry and enable conclusions on inactivation principles of microorganisms.
Spores of B. subtilis are much more resistant to heat compared to cells of Franconibacter pulveris and can be inactivated by 4 log_10 cycles with a very low plasma power of 7-10 mW/cm^2 in just 5 min when exposed on a flat glass carrier. The exposition on flat glass was used as a reference inactivation measurement and for comparisons with inactivation kinetics when the microorganisms are exposed to the plasma in a powder matrix. The inactivation effectiveness is a function of the product of plasma power and treatment time, or the applied surface energy respectively. This is a key finding proved as applicable for the decontamination of powders too and enables process scaling. Contaminated powders protect bacteria to a certain extent from inactivation, but the effect can be adjusted via the applied surface energy. The relations were established for starch powder, hydroxy-methylcellulose films, and algae powder specifically. Inactivation kinetics have been modeled.
For plasma diagnostics, optical emission spectrums have been registered from the UV light to the visible range of the spectrum. In both, air and nitrogen plasma, nitrogen species of the second positive system (SPS, 320 – 400 nm) are dominant. A linear positive relationship was obtained between plasma power intensity applied (mW/cm^2) and the intensity of nitrogen bands. Nevertheless, air plasma alters a Spirulina algae powder faster and stronger than nitrogen plasma. The same trends were observed concerning the inactivation of micro-organisms in the algae powder. It was concluded, that edging (abrasion) is the dominant principle of inactivation of bacterial spores, but oxidation of outer layers of spores accelerates the inactivation. An inactivation of micro-organisms or alteration of food components by UV radiation, cannot be concluded from plasma diagnostics. The distance of the sample to the plasma source and absorption effects by the dense gas layer at ambient pressure are supposed to be the reasons.
The influence of the powder layer thickness and chemical character of the powder on the rate of inactivation of bacterial spores was studied. The distance of plasma penetration into a powder layer is limited and is a factor influencing the dimensioning of a plasma source as well as predictions on the throughput of installations using a plasma for sterilization of powdered food.
The impact on the nutritional value of CAP treated powders was evaluated through the quantification and qualification of (i) antioxidant capacity, (ii) polyphenols, (iii) chromophores, (iv) proteins and (v) lipids for algae, insects and whey protein powders. While whey proteins resist a low plasma power short time CAP treatment, the insect powder was slightly oxidized and the algae powder indicates clear modification, specifically chromophores are sensitive markers.
Long term toxicological tests are still running when writing this report. Lifespan, egg-laying, and mobility of Caernohabditis elegans is studied when feeding the worm with a CAP treated Spirulina powder.
Three papers have been submitted for publication; two are in preparation.
Many recent publications confirm the efficiency and limitations of non-thermal plasma treatments for inactivation of living cells and spores of microorganisms. The maximal inactivation level is comparable, but inactivation kinetics depend on the plasma technology and other factors. The present project contributes to the discussion by confirming that the surface energy is the dominating variable of inactivation kinetics and the key factor in process control. Most authors consider the process time as dominating variable and do not consider the real plasma power.
This project delivers a proof that a surface micro-discharge plasma (SMD) is suited for treating powders in large quantities. The present study confirms advantageous over plasma jets, dielectric barrier discharge or sub-atmospheric plasma devices. The SMD plasma can be operated at ambient pressure, with air as process gas, is suited to treat large surfaces, the inactivation of microorganisms requires rather short process time, and the applied plasma power is the lowest so far reported to the knowledge of the authors. Drawbacks are limited power layer thickness for an efficient inactivation of microorganisms and color changes of chromophores that are highly sensitive to radicals specifically from oxygen.
Plasma treatments of food powders require case-specific permissions. Advanced studies on toxicological risks linked to side products have to be delivered. Nevertheless, the decontamination of some food powders with a non-thermal plasma that does not resist heat, or moisture (such as spices or infant food formula) has a clear potential to prevent food born outbreaks of microbial infections and food waste.
Surface micro-discharge atmospheric pressure plasma (schematic)
Tuning of the inactivation effect on microorganisms in a powder matrix