Ozon ve UV ışınlarının karabiberin mikrobiyal dekontaminasyonunda kullanım potansiyelinin araştırılması
Investigation of potential use of ozone and UV radiation in microbial decontamination of black pepper
Yazar: NAGİHAN AYVAZ
Danışman: PROF. DR. GÜRBÜZ GÜNEŞ
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2014
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Karabiber = Black pepper ; Ozon = Ozone ; Ultraviyole ışınlar = Ultraviolet rays

Özet:
İnsanlık tarihinin bilinen en eski baharatından biri olan, karakteristik tadı ve kokusu nedeniyle "baharatın kralı" olarak adlandırılan karabiberin  mikrobiyal yükü; üretim aşamalarından olan yetiştirilme, hasat, kurutma, ayıklama, öğütme, paketleme ve depolama işlemleri sırasında toprak, toz, kir, böcek, kuş ve kemirgenlerin fekal bulaşıları, işleme ve üretim sırasında kullanılan su gibi unsurlarla temas sonucu 6-8 log kob/g düzeyine kadar çıkabilmektedir. Baharatın genellikle gıdalara pişirme aşamasından sonra ilave edilmesi veya gıdalara ilave edildikten sonra kısa süreli ısıtma işlemine tabi tutulması, gıdanın bozulmasına ve ciddi sağlık sorunlarına neden olabilmektedir. Bu nedenle baharata, üretimin son basamağı olarak uygun mikrobiyal dekontaminasyon işlemi uygulamak gerekebilmektedir. Baharatın mikrobiyal dekontaminasyonunda kullanılan başlıca yöntemler; termal inaktivasyon, fumigasyon ve gamma ışınlamadır. Bu yöntemlerin sahip olduğu bazı  dezavantajlar nedeniyle alternatif olarak kullanılabilecek yöntemler üzerinde çalışılmaktadır. 
Bu çalışmada, baharatın mikrobiyal dekontaminasyonu amacıyla uygulanan ticari yöntemlere alternatif olarak üzerinde çalışılmakta olan yöntemlerden ozon ve ultraviyole (UV) uygulamalarının tane karabiberin toplam mezofilik aerobik bakteri ve E.coli yükü üzerindeki mikrobiyal inaktivasyon etkinliği, tasarlanan sabit ve akışkan yataklı sistemler kullanılarak araştırılmıştır. Ozon ve UV uygulamalarının etkinliği, öncelikle sabit yataklı sistemde belirlenmiş, yapılan denemeler sonucu çalışılması uygun görülen parametrelerle hem sabit hem akışkan yataklı sistemde, uygulamalar ayrı ayrı ve kombine şekilde çalışılmıştır. Kombine uygulamalar, ozon uygulamasının ardından UV uygulaması ve iki uygulamanın eş zamanlı uygulanması şeklinde gerçekleştirilmiştir.  
Ulaşılan bulgular incelendiğinde, sabit yataklı ozon uygulamasının tane karabiberin toplam mezofilik aerobik bakteri yükünde önemli bir azalma sağlayabilmesi için örneğin su aktivitesinin artırılması ya da yüksek konsantrasyonda uzun süre (25 ppm 3 saat) uygulama yapılması gerektiği görülmüştür. Sabit yataklı UV uygulaması söz konusu olduğunda ise önemli düzeyde inaktivasyon sağlamak için 2 saat muamele gerektiği görülmüştür. Daha düşük ozon konsantrasyonu (15 ppm) ve daha kısa sürede (1 saatte) daha fazla inaktivasyon sağlayabilmek için ozon ve UV uygulamaları sabit ve akışkan yataklı sistemde kombine edilmiştir. Sabit yataklı sistemde yapılan kombine uygulamalar, toplam mezofilik aerobik bakteri açısından inaktivasyon etkinliğini artırmamıştır. Fakat, E. coli  inaktivasyonu açısından etkili olmuştur. Sabit yataklı sistemde kombine uygulamalarla sağlanabilen en fazla inaktivasyon, toplam mezofilik aerobik bakteri için yaklaşık 0,5 log kob/g; E. coli için ise 1,5 log kob/g olarak belirlenmiştir.
Akışkan yataklı sistem, sabit yataklı sisteme göre sağlanan inaktivasyon düzeylerini artırsa da, bu artışların istatistiksel açıdan önemli olmadığı belirlenmiştir. Akışkan yataklı sistemde toplam bakteri yükünde sağlanan en fazla inaktivasyon 0,62 log kob/g olarak belirlenmiş ve yapılan kombine uygulamalar bu inaktivasyon düzeyini artırıcı etki göstermemiştir. E. coli yükünde ise kombine uygulamalar inaktivasyon etkinliğini artırmış, sağlanan en fazla inaktivasyon ise 1,72 log kob/g olarak belirlenmiştir. Yapılan tüm uygulamaların tane karabiberin renk parametreleri üzerinde önemli bir değişikliğe neden olmadığı belirlenmiştir.

Abstract:
Black pepper, one of the oldest known spices of the history of mankind,  is referred as "King of Spices", due to its characteristic flavor and aroma. It is cultivated mostly in India and also other countries with tropical climates such as Indonesia, Malaysia, Brazil, Sri Lanka, Vietnam and China. Black pepper is produced from green unripe berries of the pepper plant (Piper nigrum L.), a member of the family Piperaceae. The color of the harvested berries becomes black when they are dried. In addition to the widespread use of black pepper in food items, it is also used as drug, preservative, insecticidal and larvicidal control agent, etc. because of its active components such as piperine. Nevertheless, microbial contamination of black pepper may be as high as 6-8 log cfu/g because of its contact of soil, dust, excrement and insects during growing, collecting, processing, storage and transport. Also the high temperature and humidity in tropical countries where peppers are usually grown may favor the development of microorganisms. Spices are non-perishable commodities due to their low moisture contents, so microorganisms may not grown and multiply. However, if they get contact with water-rich food products, microorganisms might find a suitable environment to germinate and multiply. The contaminated spices that do not undergo further cooking after added to foods can cause food spoilage and foodborne diseases. In black pepper, microorganisms such as Salmonella,  Escherichia coli, Bacillus cereus and toxigenic molds and yeasts might present and they potentially creates public health risk. Between April and September 1993, a nationwide outbreak of salmonellosis occurred in Germany which was traced back to contaminated paprika and paprika powdered potato chips. Besides the estimated 1000 cases of disease, the company concerned suffered from economic and image losses. In 2009, a multi-state outbreak of salmonellosis occured in USA which was traced back to salami products that contain imported contaminated ground black pepper and red pepper. It is reported that the disease effected 272 persons in 44 states of USA. As it is seen, it is necessary to control microbial contamination of spices by applying microbial decontamination methods.   
Fumigation, irradiation and steam treatment are the commercial microbial decontamination methods applied for reducing the microbial load of spices. But these methods have some disadvantages. For example, ethylene oxide is regarded as a carcinogen and banned for usage in European Union; irradiation generally has not found acceptable by the consumer and at high doses it could cause oxidation and degradation in aromatic components of spices; steam treatment has deteriorative effect on quality of spices and moisture condensed on the surface of the spice must be removed to prevent mold growth. Hence, there is a need for development of new methods that will decontaminate the spices while maintaining the quality. High hydrostatic pressure, high pressure carbon dioxide, radio frequency, microwave, pulsed light, cold atmospheric plasma, ozone and ultraviolet radiation are alternative methods that researchers are working on for microbial decontamination of spices.
Ozone has strong microbicidal properties, can be generated cheaply, rapidly dissociates to oxygen and does not leave any residue in the treated products. Ozone was approved as generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) in 1997. With the approval of ozone contact with foods by the same authority in 2001, ozone has been started to be used intensively in food treatments. Ozone treatment is a nonthermal method. Ultraviolet (UV) treatment, is also a nonthermal method and it is approved for use as a disinfectant for surface treatment of food products (FDA 2010). Application of UV does not leave any residue in the treated products, too.
The objectives of this study were to evaluate the efficiacy of ozone and ultraviolet radiation to inactivate total mesophilic aerobic bacteria and E. coli on whole black pepper and determine the effect of these methods on the quality of black pepper by color measurements. For this purpose, a system was designed that could be used both in the fixed and fluidized bed form. At first, ozone and UV methods were applied in fixed sytem to determine the appropriate parameters, then with these parameters microbial decontamination methods were applied individually and in combination both in the fixed and fluidized bed system. Combined treatmens applied to the whole black pepper were ozone treatment (1 hour 15 ppm) followed by UV treatment (1 hour 19.8 J/cm2 in fixed bed system or 1 hour 28.8 J/cm2 in fluidized bed system) and simultaneous treatment of ozone treatment and UV treatment (1 hour in total).
According to the results, to provide a significant reduction on total mesophilic aerobic bacteria load of whole black pepper by using fixed bed ozonation system, it was necessary to increase water activity or apply high concentration and long treatment time (25 ppm 3 hours). It was found that 2 hours treatment time was required for significant total mesophilic aerobic bacteria inactivation with fixed bed UV system. Hence, ozone and UV treatments were combined to provide  more microbial inactivation with less ozone concentration (15 ppm) and treatment time (1 hour). Combined treatments applied with fixed bed system did not give contribution to the total mesophilic aerobic bacteria inactivation efficiency of individual treatments, statistically. But they increased E. coli inactivation. For ozone and UV treatments, inactivation rates of total mesophilic aerobic bacteria were 0.11 and 0.37 log cfu/g, respectively. Total mesophilic aerobic bacteria inactivation rates obtained by applying combined fixed bed systems were 0.49 and 0.41 log cfu/g  for consecutive and simultaneous treatmens. Inactivaton rates of E. coli obtained by ozone and UV treatments were 0.10 and 0.64 log cfu/g, respectively. E. coli inactivation rates obtained by applying combined fixed bed system were 1.45 and 1.32 log cfu/g for consecutive and simultaneous treatments. 
With fluidized bed system, more microbial inactivation were obtained, but it was seen that this increase was not statistically significant. Rates of microbial inactivation obtained by ozone and UV treatments for total mesophilic aerobic bacteria were 0.26 and 0.62 log cfu/g, respectively. Combined treatments did not offer additional reduction of total mesophilic aerobic bacteria, statistically. Total mesophilic aerobic bacteria inactivation rates obtained by applying combined fluidized bed systems were 0.59 and 0.51 for consecutive and simultaneous treatments. For E. coli inactivation, combined treatments had additional effects. Inactivaton rates of E. coli obtained by ozone and UV treatments were 0.87 and 0.88 log cfu/g, respectively. E. coli inactivation rates obtained by applying combined fluidized bed systems were 1.72 and 1.43 log cfu/g for consecutive and simultaneous treatments. It was observed that, all the treatments applied in both fixed and fluidized systems did not alter color parameters of black pepper, significantly.  
As fluidization increased the exposure of  whole black pepper surfaces, resulting in uniform and quick contact of the spice with ozone and UV, it was tought that better microbial decontamination might be possible by applying ozone and UV treatments to whole black pepper in a fluidized state. But the results indicated that the rates of microbial decontamination was not sufficient for commercial applications especially for total mesophilic aerobic bacteria load of whole black pepper even though the inactivation rates were found to be statistically important.  
It was found that the treatments applied to whole black pepper resulted better for E. coli inactivation than total mesophilic aerobic bacteria inactivation. In literature, it is reported that gram negative bacteria such as E. coli is more susceptible to ozone and UV treatments than gram positive bacteria such as Bacillus and Clostridium species which present in natural microbial flora of black pepper. Also naturally occuring bacteria present on black pepper might be in biofilm form and show more resistance to ozone and UV treatments. This explanations might be the reasons for the results obtained in this study. By considering especially the importance of surface properties of whole black pepper in terms of UV treatment, it would be better to treat the spices which are in form of flaked or powder such as flaked red pepper, peppermint or thyme. Furthermore, in terms of microbial decontamination, pulsed UV lamps might be more efficient than continuous UV lamps. 
In spice processing industry, implementation of integrated food safety programs that include Good Agricultural Practices (GAP), Good Manufacturing Practices (GMP) and Hazard Analysis and Critical Control Points (HACCP) is important for the production of high quality spices with low microbial load. With the implementation of these sytems, it is possible to reduce the rate of microbial inactivation of the spices and increase the applicability of alternative microbial decontamination methods.