Microsoft Word - 3 Journal nr 4 2010 din 8 noiembrie_108-113.doc Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Year IX, No. 4 - 2010 112 INTENSE LIGHT PULSES EFFECT ON FUNGAL BURDEN OF MUSTARD AND BLACK PEPPER Elena Alexandra ONICIUC1, Liliana GÎTIN1, Sorin CIORTAN2 Nicoleta MAFTEI (ARON)1, Anca NICOLAU1 1Faculty of Food Science and Engineering, Bioengineering Dept., University Dunarea de Jos of Galati, Romania 2Faculty of Mechanical Engineering, Mechanical Design and Graphics Dept., University Dunarea de Jos of Galati, Romania 111 Domneasca Street, 800201, Galati, Romania oniciuc_elena@yahoo.com Abstract: Mustard seeds and black pepper berries were used as test materials in an experiment that aimed to put the basis of a decontamination procedure for spices and condiments using a non-thermal minimal processing method based on Intense Light Pulses. The method wants to be a better solution to actual decontamination procedures of spices, food ingredients that have to fulfill the safety requirements regarding fungal burden and mycotoxin content. The need for the development of innovative technologies for the production of high quality spices is widely recognized by different authorities involved in food safety. The tests were performed on an installation prototype consisting of a vibratory sieve (800 rpm) and IFP 800 flash lamp discharging in Xenon gas. Different energetic densities (0.170 J/cm2, 0.783 J/cm2 and 1.393 J/cm2) and different pulse regimes (10·10-3 s, 20·10-3 s and 30·10-3 s) were used in order to establish the most suitable regime for fungal decontamination. The experimental results showed that it is possible to obtain 100% fungal decontamination of mustard seeds by setting appropriate regimes that take into consideration the initial fungal burden, the seeds quantity per sieve squared centimeter, the energetic density and the pulse regime. For black pepper berries, contamination was significantly reduced (1.7 log), but total fungal decontamination was not achieved. Comparatively to mustard seeds, black pepper berries display a similar roughness but a different undulation, the last one being the potential responsible for mould spores protection against light pulses. Key words: moulds, decontamination, mustard, black pepper, intense light pulses Introduction Most spices contain a high number of microorganisms among which moulds represent a significant part. Having been dried material from plant origin, spices are commonly heavily contaminated with xerophilic storage moulds [1]. The most frequent fungal contaminants of spices are species from the genera Aspergillus and Penicillium [2]. Some species that belong to these genera are known as potential producers of different toxic substances such as aflatoxins, ochratoxins and sterigmatocystine, i.e. mycotoxins that exhibit toxic, mutagenic, teratogenic and carcinogenic effects in humans and animals [3]. Mycotoxins synthesized by moulds in spices pose an important health risk in the end-products, being so resistant (degrade at 265oC) that cannot be eliminated during Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Year IX, No. 4 - 2010 113 food processing. For example, the contamination of processed meat with aflatoxin was shown to correlate with the addition of spices to fresh meat [4]. To minimize the hygiene and health risks of moulds presence on spices, the European Spice Association (ESA) has set up quality minima (QM), establishing requirements on the minimum quality for spices entering the EU market. In some respects, the quality minima are additional to legislation, in other respects, they further define legal requirements. Thus, ESA stipulates for spices a content of 105 yeast and moulds per gram as target and 106 yeast and moulds per gram as absolute maximum [5]. Only few conventional technologies, such fumigation and irradiation, are efficient for spices sanitation. However, applications such as the fumigation with ethylene oxide are restricted and even banned by law in the European Union [6], while gamma irradiation of spices (10 kGy) is allowed by the European legislation, but frighten consumers who are afraid of formation of low-molecular-weight volatile or non- volatile radiolysis products that are potentially harmful and may migrate into the food and impair food flavor. Modern technologies as high hydrostatic pressures ranging from 100 MPa to 1000 MPa are not efficient on products with water activities below 0.66 as spices are. As a consequence, there is a need for the development of innovative technologies for the production of high quality spices. While Lilie and coworkers [7] propose as novel decontamination method of spices a modification of the well-known vacuum- steam-vacuum (VSV) procedures characterized by a rapid evacuation after a short-steam treatment, other solutions should be taken into discussions. In this respect, the present paper aims to put the basis of a new decontamination procedure for spices and condiments using a non- thermal minimal processing method based on Intense Light Pulses. Materials and Methods Materials Two spices were used in the experiments: mustard seeds and black pepper berries. Mustard seeds packed in 100 g bags by SC ORLANDO IMPORT EXPORT 2001 Ltd., a company that is certified IFS and ISO 9001/2000, were sampled from a local food store. All sampled bags belonged to the same lot (297 014 - LOT 12), but even this, the content of the bags was mixed in order to obtain a homogenous laboratory sample, which was then kept in a sterile plastic bag at room temperature. Black pepper berries were kindly provided by INTERSERVICES SA, a company that provide insurance related services, case handling and assistance in shipping and trading of goods. A part of these berries were artificially contaminated with spores of Penicillium camemberti, in order to obtain a higher fungal contamination. Equipments The tests were performed on a prototype of an installation consisting of a vibratory sieve (400 rpm and 800 rpm) driven by an electric motor with eccentric, and an IFP 800 flash lamp discharging in Xenon gas (Figure 1). The distance between the flash lamp and sample was set at 0.10 m. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Year IX, No. 4 - 2010 114 Figure 1. Experimental installation for irradiation of condiments with intense light pulses 1 - flash lamp; 2 - protective enclosure; 3 - tray for seeds; 4 - vibrating platter; 5 - support site; 6 – platter holder (rigid), 7 - engine support (elastic), 8 - electric motor, 9 - platter support (elastic), 10 – eccentric. Methods Seeds/berries treatments Amounts of 20 g of mustard seeds or black pepper berries were aseptically taken, placed on the vibrating sieve and submitted to ILP treatments. The speed of the vibrating sieve was set at 800 rpm, in order to made 1600 flights per minute and to ensure seeds and berries exposure to light pulses on all sides. Different energetic densities (0.170 J/cm2, 0.783 J/cm2 and 1.393 J/cm2) and different pulse regimes (10·10-3s, 20·10-3s and 30·10-3s) were used in order to establish the most suitable regime for fungal decontamination. Tests were performed in duplicates. Microbiological analysis Total fungal counts were performed according to SR ISO 21527-2:2009 (Microbiology of food and fodder. Horizontal method for enumeration of yeasts and molds. Part 2: Colony count technique in products with less than or equal to aw 0.95). Rose Bengal (Scharlau Chemie, Spain) was used as culture medium. After inoculation, the Petri dishes were incubated at 25°C for 5 days. The genus of the isolated moulds was determined based on the characteristics of the colonies and the microscopic aspect of the mycelium. Results and discussion Mustard seeds and black pepper berries used in these experiments had similar fungal contamination levels (103 per gram), and displayed similarities regarding the quality of their microbiota (figure 2 and figure 3), whether considering the presence of Aspergillus and Penicillium genera as toxicogenic moulds. Representatives of Penicillium genus were present in proportion of 26% on mustard seeds and 20% on black pepper berries, while representatives of Aspergillus genus were present in proportion of 26% on mustard seeds and 79% on black pepper berries. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Year IX, No. 4 - 2010 115 26% 26% 13% 35% Penicillium Aspergillius Rhizopus Others Figure 2. Initial fungal microbiota of mustard seeds 20% 79% 1% Penicillium Aspergillius Mucor Figure 3. Initial fungal microbiota of black pepper berries The initial level of the existing fungal microbiota on mustard seeds (1.1∙103 CFU∙g-1) was gradually decreased during ILP treatments. A treatment consisted in applying a certain number of pulses per 10-3 second at different tensions. The results obtained for 10·10-3 s and 30·10-3 s at 700 V, 1500 V and 2000 V are presented in figure 4a and figure 4b. It is noticed that when tension increases, the fungal burden of the seeds decreases. The decontamination effect is more powerful when a higher number of pulses are applied. At a tension of 2000 V during 10·10-3 s destroyed 90 % of the fungal population, while 30 pulses per 10-3 second destroyed 96 % of the fungal population. 0 200 400 600 800 1000 1200 1400 C o ntro l s a m ple 700 1500 2000 Source tension, V N um be r of c ol on ie s, lo g C FU xg -1 Figure 4a. Evolution of the fungi level on mustard seeds as result of ILP treatments at the regime of impulse 10∙10-3s 0 200 400 600 800 1000 1200 1400 C o ntro l s a m ple 700 1500 2000 Source t ension, V N um be r of c ol on ie s, lo g C FU xg -1 Figure 4b. Evolution of the fungi level on mustard seeds as result of ILP treatments at the regime of impulse 30∙10-3s Total fungal decontamination of mustard seeds is possible to be achieved by setting appropriate ILP regimes. The initial level of the existing fungal microbiota on black pepper berries (6.5∙103 CFU∙g-1) was not significantly decreased, despite the fact that different ILP treatments were tested and their intensity was gradually increased (700 V / 10·10-3s, 700 V / 30·10-3s, 1500 V / 10·10-3s, 1500 V / 30·10-3s). In all these cases the fungal contamination level was not decreased beside 103 CFU∙g-1, which means that less 1 log decreasing of fungal burden was obtained (figure 5). Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Year IX, No. 4 - 2010 116 1 10 100 1000 10000 Control sample 700 V 10x10-3 700 V 30x10-3 1500 V 10x10-3 1500 V 30 x10-3 Treatment variants N um be r of c ol on ie s, lo g C FU x g-1 Figure 5. Evolution of the fungi level on naturally contaminated black pepper berries as result of ILP treatments The experiments were repeated on black pepper berries that were artificially contaminated with Penicillium camemberti spores in order to obtain a fungal burden above the limit indicated by ESA (European Spicy Association) as target value (105 CFU∙g-1). The results of ILP treatments on these artificially contaminated black pepper berries (8∙106 CFU∙g-1 initial fungal burden) is shown in figure 6. It was necessary a heavy treatment (2000 V / 30·10-3 s) to reduce the fungal burden with almost 1.7 log. 1 10 100 1000 10000 100000 1000000 10000000 Control sample 700 V 10x10-3 700 V 30x10-3 2000 V 10x10-3 2000 V 30 x10-3 Treatment variants N um be r o f c ol on ie s, lo g C FU xg -1 Figure 6. Evolution of the fungi level on artificially contaminated black pepper berries as result of ILP treatments To reduce the fungal burden of black pepper berries it is necessary to use much intense ILP treatments that in the case of mustard seeds. It is possible that the surface roughness of the black pepper berries to exert a protection against light pulses for the mould spores present on their surface. Conclusions An installation consisting of a vibratory sieve (400 rpm and 800 rpm and an IFP 800 flash lamp discharging in Xenon gas was successfully tested for fungal decontamination of mustard seeds and black pepper berries. The treatments using intense light pulses succeeded to totally reduce the fungal burden of mustard seeds and to reduce the fungal burden of black pepper berries below the target value indicated by the European Spicy Association. The study demonstrated that ILP treatments could be a solution to actual decontamination procedures of spices, responding in this way to the need of developing innovative technologies for the production of high quality spices. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Year IX, No. 4 - 2010 117 References: 1. ROMAGNOLI, B., MENNA, V., GRUPPIONI, N., BERGAMINI, C. 2007. Aflatoxins in spices, aromatic herbs, herbs – teas and medicinal plants marketed in Italy. Food Control, 18, 697-701 2. ŠARIÃ, L. Ã., ŠKRINJAR, M. M. 2008. Share of aflatoxicogenic moulds from genera Aspergillus and Penicilllium in mycopopulations isolated from spices from meat processing industry, Matica Srpska Proceedings for Natural Sciences, 114, 115—122 3. HEPERKAN, D., ERMIS, O.C., 2004. Mycotoxins in spices in Meeting the mycotoxin menace (Editors: D. Barug, H. van Egmond, R. Lopez-Garcia, T. van Osenbruggen and A. Visconti), Wageningen Academic Publishers, p.206 4. Hampikyan, H., Baris Bingol E., Colak, H., AYDIN, A. 2009. The evaluation of microbiological profile of some spices used in Turkish meat industry, Journal of Food, Agriculture & Environment, 7 (3&4): 111-115. 5.***2004, European Spice Association Quality Minima Document, page 9 (http://www.esa- spices.org/content/pdfs/ESAQualityMinimaDocum ent191104.pdf) 6. SCHWEIGGERT, U., CARLE R., SCHIEBER, A. 2007. Conventional and alternative processes for spice production – a review, Trends in Food Science & Technology, 18 (5): 260-268 7. LILIE, M., HEIN, S., WILHELM, P. 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