RYŢIŲ IŠ INDIJOS, TARŠA MIKOTOKSINAIS IR TARŠOS MAŢINIMO GALIMYBĖS MYCOTOXINS

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARYACADEMY

THE FACULTY OF VETERINARY MEDICINE

ANKITA KAPOORCHAND GUPTA

RYŢIŲ IŠ INDIJOS, TARŠA MIKOTOKSINAIS IR TARŠOS

MAŢINIMO GALIMYBĖS

MYCOTOXINS

CONTAMINATION IN RICE FROM INDIA AND

PREVENTIVE MEASURES

MASTER THESIS

Of Full-time Studies of Food Sciences

The supervisor:

Assoc. prof., dr. Violeta Baliukonienė

Department of Food Safety and Quality

2

THE WORK WAS COMPLETED IN THE DEPARTMENT OF FOOD SAFETY AND QUALITY CONFIRMATION OF THE INDEPENDENCE OF THE WORK COMPLETED

I confirm that the Master Thesis presented is ―Mycotoxins Contamination in Rice from India and Preventive Measures‖.

1. Has been done by me (myself).

2. Was not used in any other Lithuanian of foreign University.

3. I have not used any resources that are not indicated in the work and I present the complete list of used literature.

11 April 2021 Ankita Gupta

(date) (author’s first and last name) (signature)

CONFIRMATION OF THE CORRECTNESS OF ENGLISH LANGUAGE IN THE WORK

I confirm the work was checked by English language editing service. 11 April 2021 Raeesabegam Usmani R. J. Usmani

(date) (editor’s first and last name) (signature)

SUPERVISOR’S CONCLUSION REGARDING THE DEFENSE OF THE MASTER THESIS

(date) (supervisor’s first and last name) (signature)

THE MASTER THESIS HAS BEEN APPROVED IN DEPARTMENT/INSTITUTE (date of approval) (first and last name of the Head of

Department/institute)

(signature)

The Reviewers of the Master Thesis

1) 2)

(first and last name) (signatures) The evaluation of defense commission of Master Thesis:

(date) first and last name of the secretary of the defense commission)

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CONTENT

SUMMARY... 4 SANTRAUKA... 5 ABBREVIATIONS ... 6 INTRODUCTION ... 7 1. REVIEW OF LITERATURE ... .9

1.1 Rice from India ... ..9

1.2 Different varieties of rice ... 10

1.3 Rice from Uttar Pradesh……….12

1.4 Rice contamination from different fungi………13

1.5 Preventive measures by improving the quality of rice ... 15

1.5.1 Chemical control of mycotoxigenic fungi………16

1.5.2 Bio-control of mycotoxigenic fungi………...16

1.5.3 Post-harvest method……….17

1.6 Methods for Prevention from Mycotoxins in rice ... 18

1.6.1 Biological detoxification……….18

1.6.2 Mycotoxin Binders………..18

1.6.3 Innovative approaches ………19

2. RESEARCH MATERIAL AND METHODS ... 21

2.1 Research material ... 22

2.2 Research methods ... 22

2.2.1 Determination of fungi colony-forming units per sample ... 22

2.2.2 Mold fungi concentration level of sample by direct platting method ... 23

2.2.3 Internal sample infection with mold fungi ... 23

2.2.4 Determination of mycotoxin concentration ... 23

2.2.5 Determination of ozonation effect………...25

2.2.6 Statistical analysis………25

3. THE RESULTS OF THE RESEARCH ... 26

3.1 Rice contamination by fungi (with /without ozonation ) ... 26

3.3.1 Total fungal count in rice ... 26

3.3.2 Rice contamination with fungal genera ... 27

3.3.3 Rice contamination with aflatoxin B1, ZEA and DON ... 32

4. DISCUSSION OF RESULTS ... 33

CONCLUSIONS ... 36

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SUMMARY

Mycotoxins Contamination in Rice from India and Preventive Measures Ankita Gupta

The Master Thesis

The supervisor: assoc. prof., dr. Violeta Baliukonienė

Place of research: The research was carried out in 2019 – 2021, in Lithuanian University of Health

Sciences, Veterinary Academy, Department of Food Safety and Quality, Mycotoxicology Laboratory.

Main topic: The Rice collected from India. The rice was divided into groups: raw (paddy) and clean. Extent: 41 pages. The paper contains 3 tables and 14 figures.

The objective of research

1. To evaluate the contamination of collected rice samples from India with mold fungi and mycotoxins and to determine the potential for reduction of mycotoxins contamination.

Research task

1. To evaluate and compare different rice (raw (paddy), clean) contamination by mold fungi; 2. To evaluate and compare rice contamination with aflatoxin B1, zearalenone, deoxynivalenol; 3. To evaluate the use of preventive measure by improving the hygienic parameters of rice from

India.

Results and conclusion: After evaluating raw (paddy) and clean rice samples from India

contaminated mold fungi, it was concluded that total fungal count in raw (paddy) rice was detected 4.22±0.263 log CFU/g on an average, compare to clean rice 6.87% higher p>0.05. On the surface of the raw (paddy) rice dominated 48.662±17.243% Fusarium spp. and 75.33±14.818% mold fungi assigned to ―other‖ group, on clean rice dominated Fusarium spp. 31.998% less and other mold fungi - 6.668% less compare to raw (paddy) rice; in the inner layers of the raw (paddy) rice dominated 62.662±14.848% Fusarium spp. and 53.33±2.789% other mold fungi, when in the inner layers of the clean rice dominated Fusarium spp. 45.998% less and 22% more mold fungi assigned to ―other‖ group. In raw (paddy) rice aflatoxin B1 concentration was detected 1.6±3.049 µg/kg, zearalenone - 314±277.366 µg/kg and deoxynivalenol - 10 ± 22.361 µg/kg on an average, in clean rice aflatoxin B1 and zearalenone concentrations were 1.03-fold lower and deoxynivalenol – 10-fold higher compare to raw (paddy) rice.

Comparing results before ozonation with after ozonation of raw (paddy) and clean rice samples was found in rice after ozonation total fungal count in raw (paddy) rice was 5.21% less (p>0.05) and in clean rice – 15.27% less (p>0.05). On the surface of the raw (paddy) rice dominated Fusarium spp. 48.21% less (p<0.05) and mold fungi assigned to ―other‖ group – 18.64% less (p>0.05), on the surface of the clean rice dominated Fusarium spp. 24.01% less (p>0.05) and mold fungi of ―other‖ group – 18.64% less (p>0.05); in the inner layers of the raw (paddy) rice dominated Alternaria spp. 46.18% more (p<0.05) and mold fungi of ―other‖ group – 18.64% less (p>0.05) and in the inner layers of the clean rice dominated Fusarium spp. 67.03% and mold fungi ―other‖ group – 51.25% less (p>0.05).

AFB1 concentration in raw (paddy) and clean rice was limits of detection (p<0.05), zearalenone – in raw (paddy) and clean rice decreased 52.3 time (p<0.05) and 4.64 time (p<0.05) respectively, deoxynivalenol concentration was limits of detection (p<0.05) in raw (paddy) rice and in clean rice decreased 1.2 time (p>0.05).

5 SANTRAUKA

Ryţių iš Indijos tarša mikotoksinais ir prevencinės priemonės Ankita Gupta

Magistro baigiamasis darbas

Darbo vadovas: doc., dr. Violeta Baliukonienė

Tyrimo atlikimo vieta: Tyrimas buvo atliktas 2019 – 2021 m. Lietuvos sveikatos mokslų universitete,

Veterinarijos akademijoje, Maisto saugos ir kokybės katedros Mikotoksikologijos laboratorijoje.

Objektas: nevalyti ir valyti ryžiai iš Indijos.

Apimtis: 41 puslapiai. Darbą sudaro 3 lentelės ir 14 paveikslai.

Darbo tikslas: įvertinti ryžių iš Indijos taršą pelėsiniais grybais ir mikotoksinais, bei mikotoksinų taršos

sumažinimą.

Darbo uţdaviniai:

1. Nustatyti ir palyginti skirtingų ryžių (nevalytų ir valytų) taršą pelėsiniais grybais; 2. Nustatyti ir įvertinti ryžių taršą aflatoksinu B1, zearalenonu, deoksinivalenoliu;

3. Įvertinti prevencinės priemonės panaudojimą, pagerinti ryžių iš Indijos higieninius rodiklius.

Rezultatai ir išvados: Po nustatymo pelėsinių grybų taršos nevalytuose ir valytuose ryžių mėginiuose,

buvo nustatyta, kad bendras pelėsinių grybų skaičius nevalytuose ryžiuose buvo 4,22±0,263 log KSV/g, t.y. 6,87 proc. didesnis, palyginus su valytais ryžiais p>0,05. Ant nevalytų ryžių dominavo Fusarium spp. 48,662±17,243 proc. ir pelėsniai grybai, priskirti ―kitiems‖ – 75,33±14,818 proc., ant valytų ryžių dominavo Fusarium spp. ir pelėsniai grybai, priskirtų ―kitiems‖ atitinkamai 31,998 proc. ir 6,668 proc. mažiau, lyginat su nevalytais ryžiais. Nevalytų ryžių vidiniuose sluoksniuose nustatyta daugiausiai

Fusarium spp. 62,662±14,848 proc. ir pelėsnių grybų, priskirtų ―kitiems – 53,33±2,789 proc., kai valytų

ryžių vidiniuose sluosniuose Fusarium spp. vyravo 45,998 proc. mažiau ir pelėsniai grybai, priskirti ―kitiems - 22 proc. daugiau. Vidutinė aflatoksino B1 koncentracija nevalytuose ryžiuose nustatyta 1,6±3,049 µg/kg, zearalenono - 314±277,366 µg/kg, deoksinivalenolio - 10±22,361 µg/kg, valytuose ryžiuose aflatoksino B1 ir zearalenono koncentracijos buvo nustatytos 1,03 karto mažesnės, deoksinivalenolio 10 kartų didesnė, lyginant su šių mikotokinų koncentracijomis nevalytuose ryžiuose. Palyginus gautus rezultatus, vertinant ryžių taršą pelėsiniais grybais ir mikotoksinais prieš ozonavimą ir po ozonavimo, buvo nustatyta, kad po ozonavimo bendras pelėsinių grybų skaičius nevalytuose ir valytuose ryžiuose sumažėjo atitinkamai 5,21 proc. (p>0,05) ir 15,27 proc. (p>0.05). Ant nevalytų ryžių nustatyta 24,01 proc. mažiau Fusarium spp. (p>0,05) ir pelėsnių grybų, priskirtų ―kitiems‖ 18,64 proc. mažiau (p>0,05). Nevalytų ryžių vidiniuose sluoksniuose vyravo Alternaria spp. 46,18 proc. daugiau (p<0,05) ir 18.64 proc. mažiau nustatyta ―kitų‖ pelėsinių grybų (p>0,05), kai valytų ryžių vidiniuose sluoksniuose dominavo atitinkamai Fusarium spp. 67,03 proc. (p>0.05) ir „kiti―pelėsiniai gyrbai 51.25 proc. (p>0,05) mažiau.

Po ozonavimo aflatoksino B1 koncentracija nevalytuose ir valytuose ryžiuose sumažėjo iki aptikimo ribos (p<0,05) zearalenono – 52,3 kartų (p<0,05) nevalytuose ryžiuose ir valytuose 4,64 kartų (p<0,05), deoksinivalenolio koncentracija nevalytuose ryžiuose nustatyta mažiau negu aptikimo riba ir valytuose ryžiuose sumažėjo 1,2 karto (p>0,05).

Raktaţodţiai: nevalyti ir valyti ryžiai, pelėsiniai grybai, aflatoksinas B1, zearalenonas,

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ABBREVIATIONS

AFB1 – aflatoxin B1

bw – body weight

CFU – colony forming units ºC – degrees Celsius

CV – Coefficient Variation

FAO – Food and Agriculture Organization DON – deoxynivalenol

ELISA – Enzyme Linked Immune Sorbent Assay HPLC – High Performance Liquid Chromatography LAB – Lactic Acid Bacteria

LOD – below the detection limit ND – not detected

mm – millimeters OTA-ochratoxin A

SDA – Sorbent Dextrose Agar SD – Standard Deviation SE – Standard Error S&Q – Safety and quality spp. – species

SPSS – Statistical Package for Social Sciences TLC – Thin-layer chromatography

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INTRODUCTION

Rice is a valuable seed which belongs to grass family where 90% of the world's population relied on rice to fulfill their daily nutritional requirements. Scientifically, rice is called Oryza sativa and is widely cultivated in Southeast Asian countries which occupy third place in rice production next to maize and sugarcane [1]. The protein present in rice called oryzenin is the second most major nutrient next to starch along with some minor nutrients [2].

Traditional rice production is practiced worldwide. Especially, black rice is widely cultivated in the region of Sri Lanka, some parts of India especially in South India, minor production practices of traditional rice can also be seen in the Philippines and Thailand. China occupies first place in the cultivation of black rice [3]. In India cultivation of red rice varieties are noticed in Kerala, Karnataka, Tamil Nadu, Bihar, Orissa, Bengal, Madhya Pradesh, and other North-eastern states with the area having an unfavorable condition such as deep water, drought, sandy soils, salinity, and cold conditions [4].

There are other rice varieties called traditional rice varieties or speciality or colored rice. Color of the rice is mainly due to the presence of anthocyanin pigment on the outermost layer of rice which imparts a red, black/purple, and brown color. Earlier, the traditional rice varieties were cultivated mainly for its nutritional and remedial purpose. Traditional rice varieties are classified based on the intensity of color present in the bran region as red, black, purple, and brown rice. The speciality of this type of rice is, even after a high degree of milling, a tinge of red color remains in the outermost region [5]. Among traditional rice varieties, red rice has got more nutritive value with the germ intact and nutty flavor.

Cultivation and consumption of traditional rice varieties are widely distributed all over the world. Some other regions in India like Matte of Kerala, Patni in Maharashtra, Jatu and Matali of Kulu valley in Himachal Pradesh grows a wide array of red rice varieties. The red rice varieties are having medicinal and nutritive value. The rice grains of traditional varieties are cooked and consumed as a whole. Apart from this, red rice varieties are converted into processed products like vinegar, tart, cosmetics, red koji, and preparation of yeast in China due to its medicinal values.

In relation to nutritional information, traditional rice varieties are rich in antioxidants, phytochemicals, phytonutrients, vitamin E, proteins, and other nutrients required for the proper functioning of the immune system and to boost memory power in children. It also helps to fight against critical diseases like cancer. The black rice, brown, and red rice are the largest reservoirs of antioxidants. It was found that traditional rice got more health benefits than white or polished rice, where most of the nutrients lost during the polishing process.

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Food contamination can pose risks to consumer health. Grains, such as rice are affected greatly by the presence of fungi and mycotoxins. Rice crop is largely cultivated in subtropical environments, which are considered favorable for fungal growth due to high temperature and humidity. During storage in silos, it is prone to contamination due to being an excellent food source for fungal growth. The presence of toxigenic fungi in rice grain from India and in other parts of the world are reported in the current literature [6]. The most important fungal genera found in rice grain are Aspergillus, Penicillium and

Fusarium. Some of these toxigenic species produce mycotoxins in the food, during storage and also the

processing stage. They are considered serious health hazards. Due to the fact that fungi contamination in stored rice grains to be a constant concern in the food industry, several preventive strategies are used such as good agricultural practices in pre-harvest, as well as appropriate storage condition in post-harvest. Fungicides and other harmful chemicals are often used however; there is an increasing public concern due to the potential risk of residues that remain in food. The development of decontamination methods safe for use in food is an attractive target for industry. Ozonation is the most promising chemical method able to inactivate microorganisms and degrade mycotoxins [7].

The objective of research

To evaluate the contamination of collected rice samples from India mold fungi and mycotoxins and to determine the potential for reduction of mycotoxins contamination.

Research tasks:

1. To evaluate and compare different rice (raw (paddy), clean) contamination by mold fungi; 2. To evaluate and compare rice contamination with aflatoxin B1, zearalenone, deoxynivalenol; 3. To evaluate the use of preventive measure by improving the hygienic parameters of rice from India.

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1. REVIEW OF LITERATURE

1.1 Rice of india

India, in South Asia, has a land area of 3,287,590 km². It measures 3,214 km from north to south between the extreme latitudes and 2,933 km from east to west between the extreme longitudes. It has a land frontier of about 15,200 km. The mainland comprises four regions: the great mountain zone, plains of the Ganges and the Indus rivers, the desert region, and the southern peninsula. On its northern frontiers, India is bounded by the Great Himalayas, which are three almost parallel ranges interspersed with large plateaus and valleys, such as the Kashmir and Kullu valleys, that are very fertile [8]. The climate of India can be described as tropical monsoon. There are four seasons: winter (December-February), summer (March-May), rainy south western monsoon (June-September), and post monsoon, also known as north eastern monsoon, in the southern peninsula (October-November).

The time of onset of winter and summer periods differs in different regions. Four broad climatic regions are identified based on rainfall. The whole of Assam and the west coast of India lying at the foot of the Western Ghats and extending from the north of Mumbai (earlier Bombay) to Thiruvanthapuram (earlier Trivandrum) are areas of high rainfall. The Rajasthan desert extending westward to Gilgit is a region of low precipitation. In between are two areas of moderately high and low rainfall, respectively. The area of high rainfall is a broad belt in the part of the peninsula merging northward with the Indian plains and southward with the coastal plains.

The low-rainfall area is a belt extending from the Punjab plains across the Vindhya Mountains into the western part of the Deccan region, widening considerably in the Mysore plateau. India is the world‘s second-largest rice producer and second most populous nation, with a population in 2010 of more than 1.2 billion, which grew at 1.4% per year from 2005 to 2010. The rural population in 2010 was above 0.8 billion, a 70% share of the total population. India‘s economy is developing into an open market economy [9]. The service sector accounts for more than half of the country‘s output. The country has become a major exporter of information technology services and software workers. The other two key sectors of the economy are industry and agriculture. Traditional village farming, modern agriculture, handicrafts, extensive modern industries, and an assortment of services make up India‘s diverse economy. Although agriculture contributes only 18% of the country‘s GDP, it provided employment to more than half (51%) of the active labour force in the country in 2010. The service sector engaged 27%, while industry had a 22% share of the workforce during the same period [10].

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1.2

Different varieties of rice

Oryza sativa

Asian rice was first domesticated from the wild rice Oryza rufipogon in China between 8,200 and 13,500 years ago, and then spread to South and Southeast Asia. Today it is cultivated on every continent save Antarctica. Worldwide there are more than 40,000 different varieties of Oryza sativa, classified into four major categories: indica, japonica, aromatic and glutinous.

Oryza sativa contains two major subspecies: the sticky, short grained japonica or sinica, and the

non-sticky, long-grained indica. Japonica varieties are usually cultivated in dry fields, in temperate East Asia, upland areas of Southeast Asia and high elevations in South Asia, while indica varieties are mainly lowland rices, grown mostly submerged, throughout tropical Asia. Recent genetic evidence show that all forms of Asian rice, both indica and japonica, come from a single domestication event that occurred 8,200–13,500 years ago in the Pearl River valley region of China.

1. Japonica

Japonica is a group of rice varieties from northern and eastern China grown extensively in some

areas of the world. It is found in the cooler zones of the subtropics and in the temperate zones. It is a relatively short plant with narrow, dark green leaves and medium-height tillers. Japonica grains are short and round, do not shatter easily and have low amylose content, making them moist and sticky when cooked [11].

 Javanica or tropical Japonica

Once thought to be a third subspecies, javanica is now known as tropical japonica.

Examples of this variety include the medium grain 'Tinawon' and 'Unoy' cultivars, which are grown in the high-elevation rice terraces of the Cordillera Mountains of northern Luzon, Philippines.

Javanica plants are tall with broad, stiff, light green leaves. The grains are long, broad, and thick,

do not shatter easily, and have low amylose content.

2. Indica

Indica rice is the major type of rice grown in the tropics and subtropics, including the Philippines,

India, Pakistan, Java, Sri Lanka, Indonesia, central and southern China, and in some African countries.

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Indica plants are tall with broad to narrow, light green leaves. The grains are long to short, slender,

somewhat flat, tend to shatter more easily and have high amylose content, making them drier and flakier when cooked than japonica varieties.

Oryza glaberrima

Oryza glaberrima, commonly known as African rice, is a domesticated rice species. African rice is

believed to have been domesticated 2,000–3,000 years ago in the inland delta of the Upper Niger river, in what is now Mali. Its ancestor, which still grows wild in Africa, is Oryza barthii.

This species is grown in West Africa. O. glaberrima shows several characteristics that make it less suitable for cultivation compared to O. sativa, such as brittle grains and poor milling quality. African rice also has lower yields than O. sativa, but it often shows more tolerance to fluctuations in water depth, iron toxicity, infertile soils, severe climatic conditions and human neglect. It also exhibits better resistance to various pests and diseases, such as nematodes, midges, viruses and the parasitic plants Striga [12]. Scientists from the Africa Rice Center managed to cross-breed African rice with Asian rice varieties to produce a rice variety called NERICA, which is an acronym for New Rice for Africa.

Fig. No. 1. Oryza sativa Fig. No. 2. Oryza glaberrima

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Fig. No. 3. Raw rice material Fig. No. 4. Rice

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Fig. No. 5. Stages of Rice

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1.3 Rice from uttar pradesh (india)

The potential of agricultural production in U.P. is far greater than what is being realized. While a major cause of rice yields appears to be the uncertainty about the availability of water in the rain fed fields, even the yields of irrigated rice are low. On the other hand, yields in large block areas ranging up to 20 hectares, grown under the close guidance of the staff of agricultural universities or Department of Agriculture, have averaged more than 4 tons/ha. Many farmers with their own irrigation facilities are getting much higher yields — even up to 6-7 tons/ha. Thus, for the irrigated rice, appropriate varieties and crop protection technology is available for obtaining high yields. Therefore, better coordination and timely action on various production practices are the most urgent needs for improving production of irrigated rice [14]. Unfortunately, however, very little progress seems to have been made on technology development for rain fed rice which constitutes nearly 70% or rice hectare age in uttarpradesh.

Uttar Pradesh is the second largest rice producing state with almost 5.86 million hectare land under rice cultivation producing about 12.5 million tonnes of rice. It is grown in the mostly area of the district Ghaziabad (Ghaziabad and Hapur) UP, India. It is the staple food of the district. ―The type of rice that should be planted in the district depends on the altitude of our district and whether. We are living in a low land area‖. ―Wet or Low land cultivation is practiced in areas which have an assured and adequate supply of water either through rainfall or irrigation. The main difference between is the fact that seeds are not directly sown put Transplanted from nurseries as small plants or sprouted [15]. Rice seeds in to puddle field. The land is ploughed thoroughly and puddle with around 3 to 5 cm of standing water to obtain a soft seed bed for the seedlings to grow quickly and reduce leaching of nutrients and weeds.‖

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17.25% of the cropped area under rice and the average production of rice in the district is about 41996 ton per annum [16].

Rice is the second staple food crop of the people of the district. It has great potential supply raw materials. There are 74 total rice mills, in which 6 are large scale. It has a great potential supply of raw materials. These mills consume paddy as their raw material and produce rice. A great amount of rice is produced in village by Dhan machine and rarely by hand in okhalies prepared by stone and wood.

The villages are located on the Indo Gangetic Plain (IGP) of the Indian subcontinent, which is known as one of the most productive agricultural areas of the world. Rice–wheat cropping systems predominate on the plain. The climate of the study area is sub-humid and characterized by three seasons: winter (October to February); summer (March to June); and rainy (end of June to the end of September). The average rainfall is approximately 1020 mm, and the western part of the region receives more rainfall than the eastern. The maximum temperature is approximately 45°C and the minimum approximately 9°C. June is the hottest month, and January is the coolest. The maximum annual rainfall (85%) occurs during the rainy season [17].

1.4 Rice contamination from mycotoxins

Fungi that produce mycotoxins are referred to as toxigenic or mycotoxigenic fungi. The main

mycotoxigenic fungi involved in the human food chain belong to three genera: Aspergillus, Fusarium

and Penicillium (Table 1). However, toxins have been detected from many other fungi under certain

growth conditions. The kind and amounts of toxin produced depend on the fungal strain, the growing conditions, as well as the presence or absence of other organisms.

Table No. 1. Principal mycotoxins produced by Aspergillus, Fusarium and Penicillium species

(https://www.google.com/search) [18]

Fungi Mycotoxins

Aspergillus α-cyclopiazonic acid; aflatoxins; echinulin; flavoglaucin;

gliotoxin; ochratoxins; patulin; sterigmatocystin; xanthoascin; xanthocillin X

Fusarium 4 -acetamido-2-buten-4-olide; apicidin; beauvericin;

chlamydospol; deoxynivalenol; enniatins; equisetin; fumonisins; fusaproliferin; fusaric acid; fusarin C; fusariocins,

fusarochromanone; moniliformin; sambutoxin; trichothecenes; wortmannin; zearalenone

Penicillium citrinin; citreoviridin; luteoskyrin; ochratoxins; patulin; penicillic acid; penitrem; PR toxin; roquefortine C; rubratoxin B; secalonic acid D

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OTA was originally described as a metabolite of A. ochraceus (Aspergillus section Circumdati) from laboratory experiments [19]. Later, the production of this toxin was repeatedly reported, mainly by

Aspergillus carbonarius and a low percentage of isolates of the closely related species A. niger Even

recently, new ochratoxigenic species in this section are emerging. Isolates from other subgenera usually produce only small amounts of OTA, or they ability to produce the toxin was not confirmed by other authors.

Natural occurrence and practical importance of OTA, however, was first linked with Penicillium species. Some of these, like P. viridicatum and P. cyclopium, have been found afterwards to do not produce OTA. Later, [20] biochemically characterized several OTA-producing strains of the genus

Penicillium, and separated them in two large groups: P. verrucosum and P. nordicum. The last deserve a

special attention as they produced more OTA than P. verrucosum under laboratory conditions. Moreover, they suggested that P. nordicum could be the source of OTA of Penicillium contaminated meat-derived products as all the isolates they examined from this food belonged to this group, whereas

P. verrucosum was only found in plant-derived material.

Mycotoxin contamination often occurs in the field prior to harvest. Post-harvest contamination can occur if the drying is delayed and during storage of the crop if moisture is allowed to exceed critical values for mold growth. Delayed harvest in rainy weather frequently leads to grain‘s sprouting on the panicle, particularly for non-dormant japonica rice. The fungi, A. flavus, A. parasiticus, A. niger, and A.

ochraceus, of which A. flavus have been identified as the primary quality deterrent, producing

aflatoxin-contaminated seeds when in storage. They explored the incidence of Aspergillus sp. in 1,200 rice samples consisting of paddy and milled rice co llected from 43 locations in 20 rice-growing states across India. The seeds collected were either from areas exposed to different weather conditions or stored at various storage conditions, namely seeds from the crop exposed to heavy rains and floods, seeds from submerged or damp conditions, seeds stored in the warehouse for 1 to 4 years, or seeds from the grain market. A. flavus and A. niger dominated in almost all the seed samples. The incidence of heavy rains during the harvesting season in India favours aflatoxin contamination of the rice crop. Fungal infection was more frequent in parboiled dried paddy and milled parboiled rice. Of the various stages, rice at the drying stage and the stage preceding milling were shown to contain aflatoxins [21]

. Aspergillus species are common contaminants in stored rice and their incidence increases with the infestation of rice weevil (Sitophilus oryzae). In addition to rice, it was reported that A. flavus incidence increases with the infestation of insects in horse gram, sorghum, and pearl millet, respectively.

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In India, the level of mycotoxins in rice differs from one location to another. This is due to various factors like temperature, relative humidity, and agricultural practices. In general, hot and humid conditions are very favourable for the growth of toxigenic fungi and mycotoxin production in agricultural produce. Many countries regulate specific mycotoxins and most countries try to limit exposure to the toxins. There are several systematic surveys on mycotoxin levels in different agricultural commodities including rice.

Fig. No. 6. Rice contamination with fungi (a, b)

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Fig. No. 7. Electron microscopic view

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1.5

Prevention methods by improving the quality of rice

1.5.1 Chemical control of mycotoxigenic fungi

Only a few studies have been conducted with regard to control strategies for mycotoxigenic fungi in rice fields. In general, chemicals have been shown to be efficient in crop protection, but they may have many negative effects. While acidifying the soil and, consequently, decreasing the occurrence of beneficial organism populations, they may potentially interfere with the plant‘s growth. Moreover, appearance of fungicide-resistant pathogenic strains suggests the need to find alternative methods to chemicals for controlling plant diseases in the field. In agricultural systems with high levels of sustainability, there is currently increasing pressure to reduce the use of insecticides, fungicides, and herbicides [23], particularly because these can even have adverse effects on human health and the environment World Health Organization [WHO].

For rice, some commercial fungicides, such as for seedling treatments, have been evaluated for their efficacy against Bakanae disease caused by Fusarium species under natural field infection and after artificial inoculation. Of the five fungicide formulations that have been assessed under natural field infection, those based on carbendazim, benzimidazole, and propiconazole were able to significantly reduce Bakanae incidence and improve grain yield. Efficacy of carbendazim and benzimidazole on Bakanae control has also been found in previous studies.

Treatment with propiconazole was the most effective in reducing the occurrence of the disease, but it caused reductions in plant height, tillering, and in grain yield. The other two fungicides applied (one containing mercury and the other containing thio-phanatemethyl) were poorly effective on reducing the spread of the disease. Considering the artificial inoculation of seeds, all the five fungicides significantly reduced foot rot, and improved plant height and grain yields. FHB caused by F. graminearum is generally managed with triazole applications that are able to reduce both disease and DON occurrence. The application of fungicide is most effective if sprayed prior to infection; however, under environmental conditions that are particularly favourable for disease development.

In an in-field study [24], studied the application of the fungicide tebuconazole (0.75 L/ha) in irrigated rice. These authors found a positive trend between mycotoxin content (AF, OTA, DON, and ZEA) and fields with fungicide application, suggesting that the fungicide acted as a stress factor, inducing mycotoxin productions and, consequently, their accumulations. This observation led to the conclusion that the choice of the fungicide should not rely just on plant productivity and plant disease, but also on the mitigation of mycotoxin production.

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1.5.2 Bio-control of mycotoxigenic fungi

With regard to possible alternative methods to limit fungi development and mycotoxin production, different microorganisms have been tested. These microorganisms may control plant dis-eases through one or more mechanisms, including competition with pathogens for space and nutrients, the production of antimicrobial compounds, the induction of host resistance to the disease, or direct antagonism to the pathogens. In this context, even microbial interactions in the rhizosphere have been shown to contribute to plant crop bio-control.

Among the microbial populations tested, lactic acid bacteria (LAB) seem to have a great potential as an agent to control fungal diseases. In particular, spraying diluted solutions of LAB onto the plant and soil have been hypothesized to support healthy plant growth. Bio-control agents have been tested as control agents for cereal diseases caused by Fusarium species [25] and the results showed efficacy in reducing Fusarium contamination.

1.5.3 Post-harvest strategies

Storage conditions play an important role in mycotoxin control, since they will influence overall fungal development. In general, high humidity and temperature can favour fungal growth and promote mycotoxin production. Storage under controlled conditions, such as packaging practices, temperature control, ventilation efficiency, and proper air humidity, will reduce fungal development and mycotoxin accumulations.

Just by controlling temperature and moisture of grains, a safer storage can easily be promoted. The assessment of moisture con-tent of grains and storage temperature of long-grain rough rice has indicated that safe storage could be achieved for 6 week sat temperatures and moistures below 27°C and 17%, respectively. For A. flavus in rice, population growth has been demonstrated to remain constant for120 days of storage at 21°C and 85% RH. At 21°C and 97% RH and at 30°C and 85% RH, fungal growth and AFs‘ production were mostly influenced by high humidity, and most significantly in brown rice, compared with rough or white rice. The same experiment for F. graminearum showed that at 85% RH (both at 21°C and 30°C) neither fungal development nor DON production were detected. In contrast, with 97% RH, even at 21°C, fungal populations and mycotoxin production were detected in the three types of rice tested.

Application of a range of techniques to inhibit A. flavus growth in stored rice grains showed that after 15 months, approximately 67%, 63%, 37%, and 6% of inhibition were achieved through the application of

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potassium sorbate, turmeric (Curcuma longa), clove (Syzygium aromaticum), and microwave heat, respectively [26].

1.6 Prevention measures for mycotoxic fungi

It has been accepted that the prevention of different mycotoxins contamination is the primary measure and alternative over the other control methods. Still numerous physical and chemical detoxification control strategies have been established to prevent the growth of toxigenic fungus and mycotoxin contamination; few strategies fulfill the standards due to their heavy cost, bio-safety risk, losses in the nutritional quality and the palatability of the products or limited binding effect. Therefore, it is necessary to develop appropriate detoxification methods to ensure food safety for human consumption. We will discuss below the biological detoxification methods and innovations for control and mitigation of mycotoxins problem [27].

1.6.1 Biological detoxification

Control of mycotoxicoses with the application of microorganisms and their enzymes is called mycotoxin modifiers which biotransform mycotoxins into less toxic metabolites. They are classified as –

 Use of microorganisms

Biological approaches for mycotoxins decontamination by using microorganisms and specific kinds of isolated yeasts have been used effectively for the management of mycotoxins in food and feeds. Mechanisms in the removal of toxins by microorganisms are still investigated and successful results have been obtained related to this method in recent years [28]. A wide range of microorganisms including bacteria, fungi, and yeasts has proved biodegradation capacity.

 Bacteria: Mycotoxin degrading bacteria have been isolated from different sources like rumen and intestinal flora, soil, and even water. Lactic acid bacteria (LAB) namely Lactobacillus, Bifidobacterium, Propionibacterium, and Lactococcus.

 Fungi and yeast: Fungal species, Aspergillus, Alternaria, Absidia, Armillariella, Candida,

Dactylium, Mucor, Penicillium, Peniophora, Pleurotus, Trichosporon, Rhizopus have been shown

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 Use of enzymes: Specific enzymes such as oxidase, peroxidase, laccase, reductase, esterase, carboxyl esterase, aminotransferase, lactono-hydrolase having the capacity of degrading mycotoxins have been purified from microbial systems.

1.6.2 Mycotoxin binders

Mycotoxin binders also known as adsorbents or sequestering agents have been used to decontaminate animal feed by binding the mycotoxin and inhibit their absorption in the gastrointestinal tract, where the bounded toxins can be eliminated via feces or urine of animal. Both inorganic (hydrated sodium calcium aluminosilicates, zeolites, bentonites, fuller's earth, diatomaceous earth, activated charcoal, kaolin, sepiolitic clay, cholestyramine) and organic binders (alfalfa fibre, oat fibers, extracted cell wall fraction of Saccharomyces cerevisiae, beta-D-glucan fraction of yeast cell wall) have been using for the control of toxin in diet [29].

1.6.3 Innovative approches

Nanobiotechnology: This technique apparently a novel promising, effective and low-cost strategy that can offer eco-friendly for the control mycotoxigenic fungi and mycotoxins in the agriculture and food industry. The nanomaterials such as nanosilver (AgNPs), Zinc Oxide nanoparticles (ZnO-NPs), Selenium nanoparticles (SNP), Copper nanoparticles (CuNPs), magnetic nanoparticles like surface active maghemite nanoparticles (SAMNs), nano clay, nanogel, nano binders, and nanodiamonds can bind and remove mycotoxins or pathogens in food and feed [30].

 Antibody-mediated technology: Development of monoclonal and recombinant fungal-specific antibodies expressed in plants can limit the distribution of the fungal pathogens in the field and ultimately minimize the mycotoxin-production load. Monoclonal antibodies with high binding specificity to Fusarium mycotoxins.

 Genetic improvement of crops: Mycotoxin contamination may both pre- and post-harvest stage which can be reduced greatly by developing disease-resistant traits through more sophisticated biotechnological approaches during the pre-harvest stage. Recently modern transgenic techniques such as Host-induce gene silencing (HIGS), RNA interference (RNAi), microRNA (miRNA)- or artificial microRNA (amiRNA)- mediated gene silencing, designer transcription activator-like effector (dTALE)-mediated up or down-regulation of gene expression, Zn-Finger nucleases, mega-nucleases, transcription activator-like effector nucleases (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, and oligonucleotide-directed mutagenesis (ODN)-based gene-editing techniques can be applied for development of mycotoxin resistant plant[31].

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 Ozone

:

Practical methods to degrade mycotoxins using ozone gas (O₃) have been limited due to low O₃ production capabilities of conventional systems and their associated costs. It is possible that the rapid delivery of high concentrations of O₃ will result in mycotoxin degradation in contaminated grainswith minimal destruction of nutrients. The major objectives of such studies were to investigate the degradation and detoxification of common mycotoxins in the presence of high concentrations of O₃. In this regard, aqueous equimolar (32 μM) solutions of aflatoxins B1, AFB2, AFG1, AFG2, cyclopiazonic acid (CPA), fumonisin B1 (FB1), ochratoxin A (OTA), patulin, secalonic acid D (SAD) and zearalenone (ZEA) were treated with 2, 10 and/or 20 weight % O₃ over a period of 5.0 min and analysed by HPLC. Results indicated that AFB1 and AFG1 were rapidly degraded using 2% O₃, while AFB2 and AFG2 were more resistant to oxidation and required higher levels of O₃ (20%) for rapid degradation. In other studies, patulin, CPA, OA, SAD and ZEA were degraded at 15 secs, with no by-products 21 detectable by HPLC. Degradation of FB1 did not correlate with detoxification, since FB1 solutions treated with O₃ were still positive in two bioassay systems [32].

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2. RESEARCH MATERIAL AND METHODS

2.1. Research materials

The research work was conducted in 2019 – 2021, in Lithuanian University of Health Sciences, Veterinary Academy, Department of Food Safety and Quality, Mycotoxicology Laboratory. The study was performed according to the scheme below Fig. 8.

Fig. No. 8. Design of Study

Sample Collection

Step:1

Fungi (CFU/g)

Fungal genera

Step:2

Mycotoxins

Concentration

Step:3

Results analysis

Step:5

Determination of

ozonation effect

Step:4

22 Rice samples were collected from Indian market.

Table No. 2. Rice samples

Rice sample code No. of samples Description of samples

Sample 1(Moti) 2 S1R1: Raw (paddy)

S1R2: Rice

Sample 2 (Moti manjuri) 2 S2R1: Raw (paddy) S2R2: Rice

Sample 3 (Chintu) 2 S3R1: Raw (paddy)

S3R2: Rice

Sample 4 (Sampura) 2 S4R1: Raw (paddy)

S4R2: Rice

Sample 5 (Damini) 2 S5R1: Raw (paddy)

S5R2: Rice

The samples were frozen immediately at –20ºC and kept until the beginning of the laboratory analyses.

2.2. Research methods

The following studies were performed:

 Mycological studies (fungi colony-forming units per sample, fungal genera).  Determination of mycotoxins concentrations by TLC (Romer Labs Inc. ®Method).  Determination of ozonation effect.

2.2.1. Determination of fungi colony forming unites per samples

Dilution method according to standard Microbiology of food and animal feedings stuffs —

Horizontal method for the enumeration of yeasts and moulds: Part 2: Colony count technique in products with water activity less than or equal to 0.95 (ISO 21527–2:2008) was used to determine total

fungal counts (CFU/g) in spice samples, in triplicates.

Ten grams of each sample were added to 90 ml portion of sterile distilled water in 250 ml Erlenmeyer flask and homogenized thoroughly on an electric shaker RS-OS 20 (Germany), at constant speed for 20 min. Ten fold serial dilutions (10-1 to 10-3) were then prepared. One milliliter from each dilution series was uniformly dispensed under the surface 15 ml Sabouraud Dextrose Agar (SDA)

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(Oxoid) fortified by 0.5 mg chloramphenicol/ml (Sigma) in Petri dishes and incubated at 26±2 ºC for 5– 10 days and examined for the growth of fungi.

Fungi colony-forming units per sample (CFU/g) was calculated:

CFU/g = (numbers of colonies x dilution factor) / volume of culture plate. CFU/g were converted into log10 (CFU/g).

2.2.2. Mold fungi contamination level of samples by direct platting method

An agar plate method was used for the estimation of rice surface infection. 100 rice were plated in Petri dishes (10 rice in one Petri dish) with a Sabouraud Dextrose Agar with Chloramphenicol and incubated for 5-7 days at 262 ºC in the dark. The genera of mold fungi were calculated in percent. Mold fungi were identified on the basis of their morphological and microscopic features using the Samson et al. (2000) [33] and Lugauskas et al. (2002) [34] key.

2.2.3. Internal sample concentration with molds

An agar plate method was used for the estimation of internal rice infection. Surface-sterilized (for 3 minutes in 1 % NaOCl solution) rice (100 per sample) were plated in Petri dishes (10 rice in one Petri dish) with a Sabouraud Dextrose Agar with Chloramphenicol and incubated for 5-7 days at 262ºC in the dark. The infection level of rice was evaluated in percent.

2.2.4. Determination of mycotoxin concentration

Concentration of aflatoxin B1, zearalenone was analyzed by Thin-layer chromatography (TLC) and described by Romer Labs Inc. ®Method (Code: a/z-tl-01-00.2) and Deoxynivalenol - Romer Labs Inc. ®Method (Code: CAM-000031-2).

Chemicals: Acetic acid, glacial (100%, p.a.), Sulfuric acid (p.a., 95-97%), Aluminum chloride (Merck, Germany), Acetonitrile (CHROMASOLV® HPLC, >99.9%), Methanol (CHROMASOLV® HPLC, >99.9%), Acetone (CHROMASOLV®, HPLC, ≥99.8%), Toluene (CHROMASOLV® HPLC, >99.9%) (Sigma Aldrich, Germany), Deionized water.

Equipment: grinder Bosch TSM6A013B (Germany), shaker RS-OS 20 (Germany), Romer®Evap- system (Romer Labs, Inc., USA), Auto SpotterTM, Model 10 (Romer Labs, Inc., USA).

Consumables: clean-up columns MycoSep®226 AflaZon+ (Romer Labs Diagnostic GmbH, Austria), MycoSep®225 Trich column (Romer Labs Diagnostic GmbH, Austria), MultiSep® 216 column (Romer Labs Diagnostic GmbH, Austria), Biopure standards: Aflatoxin B1 (2 µg/mL

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acetonitrile), zeralenone (100 µg/mL in acetonitrile), deoxynivalenol (100 µg/mL in acetonitrile) (Romer Labs Diagnostic GmbH, Austria), Silica Gel 60 F254 TLC Plates (Romer Labs Diagnostic GmbH, Austria), 100 μl syringes, Whatman No 4 filter paper (Whatman, Inc., Clifton, New Jersey, USA).

All samples of rice were dried at 60°C for 24 h in an oven and were ground.

Extract 25 grams of each sample with 100 mL acetonitrile/DI water (84/16, v/v) shake for 2 h. The extract was filtered through Whatman No 4 filter paper.

Determination of Aflatoxin B1 (AFB1) and zeralenone (ZEA) concentration: 45 μL of glacial

acetic acid was added to 4.5 mL of the filtrate and mixed well. 4.5 mL of the filtrate was pushed through clean-up column MycoSep® 226AflaZon+ (following the methodology supplied by the manufacturer). After purified 2 mL extract was evaporated to dryness under vacuum in a 60°C. Residue was dissolved with 320 µL toluene/acetonitrile (97/3, v/v). 80 μL of sample, AFB1 spotting standard: 0.05 μg/mL (10, 20, 40, 80 μL) and ZEA spotting standard 5 μg/mL (10, 20, 40, 80 μL) were spotted onto the TLC Silica gel 60 F254 plate with the AutoSpotterTM. The matrix was prepared by adding 48 μL of AFB1 spiking

standard (0.625 μg/mL each of AFB1) to 9 mL of 84/16 v/v acetonitrile/water and 90 μL of 25 μg/mL ZEA spiking standard to 9 mL of 84/16 acetonitrile/water. Plate was developed in 1/1 (v/v) toluene/acetone until solvent front was 1 cm from the top of the plate. The plate was dried in the air. The plate was viewed under a long wave of UV light. AFB1 appeared blue and has a Rf (retention factor) of approximately 0.45. ZEA appeared light green with an Rf (retention factor) value of approximately 0.9.

The plate was spread with 15% aluminum chloride in methanol. The plate was viewed again under UV light. ZEA appeared blue. The plate was spread with 10% sulfuric acid in methanol. The plate was viewed again under UV light. AFB1 appeared yellow.

Determination of of DON concentration: 10 ml of sample filtrate was transferred into a tube, pushed slightly over 4 mL through a MycoSep®225 Trich. Four mL of purified extract was transferred to the preconditioned MultiSep®216 and collected. The extract has drained through MultiSep® 216 Trich column rinse with 12 mL of 84/16 acetonitrile/DI water and was collected. Evaporated with the Romer EvapTM System. The residue was dissolved in 400 μL of 2/1 acetone/methanol. 80 µL of sample and matrix spike along with standards (100, 200, 300, 400 ng) were spotted on a silica gel TLC. The plate was developed with 1/2 toulene/acetone. The plate was sprayed with 15% aluminum chloride in methanol. The plate was heated at 150°C until standard spots were fully visible under a long wave of UV light. DON appears blue with Rf valuesof approximately 0.5. Estimate toxin in samples and spikes

compared to the standards. AFB1, ZEA, DON concentration in rice sample was calculated by Romer

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2.2.5. Determination of ozoznation effect

Ozonation of rice samples was performed at the VDU Agriculture Academy Institute of Energy and Biotechnology engineering.

The O₃ chamber was primarily cleaned with sodium hypochloride, rinsed with distilled water and dried. 100 g rice samples were loaded for ozone application. The generated O₃ gas was pumped (through the sample by a compressor (equipped with a filter to prevent the entry of moisture). The gas concentration applied was 2.5 ppm 30 min.

2.2.5. Statistical analysis

The data were analysed using the SPSS version 20.0 (IBM Corp., NY, Armonk) and ―Microsoft Office Exel 2010‖ calculating the mean of values (X), standard error (Se) standard deviation (SD), coefficient of variation (CV). The P- value of 0.05 was set as a limit for statistically significant difference in the studies.

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3. THE RESULTS OF THE RESEARCH

3.1 Rice contamination by fungi (with / without ozonation)

3.3.1 Total fungal count in rice

The samples of rice were analyzed to determine the contamination level of fungi: total fungal count and fungal genera. The determination of total fungal count in rice were determined which rice samples were most infected and which were least infected, and the average level of total fungi colony-forming units per sample in all tested samples was calculated. The samples of rice were divided in two groups: 1) Raw (paddy), 2) Clean. Also each sample was compared based on the ozonation treatment. Data shows the total fungal count in raw (paddy) and clean rice samples from India in Fig. 9.

Fig. No. 9. Total fungal count in rice samples

In rice samples from India before ozonation the total fungal count was found between 3.77-4.80 logCFU/g, on an average 4.22±0.263 log CFU/g (SD= 0.590; CV= 13.969). In clean rice samples the total fungal count was found between 3.636-4.230 log CFU/g, on an average 3.936±0.116 log CFU/g (SD= 0.261; CV= 6.639). Raw (paddy) rice samples group was contaminated 6.87% more fungal count compared to clean rice samples group p>0.05.

Ozone has effectively shown its anti-microbial and fumigation characteristics that are invaluable in food industry, where effective applications to ensure safer food products are highly prioritized. Ozone treatment on each rice sample was performed. The total fungal count in rice samples after ozonation is presented in Fig. 10. 0 1 2 3 4 5 6 Raw Clean log CF U/g Rice samples

Fungal contamination without Ozonation

S1R1 S2R1 S3R1 S4R1 S5R1

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Fig. No. 10. Fungal contamination w/o Ozonation

In raw (paddy) rice samples after ozonation the total fungal count was found between 3-4 log CFU/g, on an average 4±0.279 log CFU/g (SD= 0.624; CV=17.140). In clean rice samples after ozonation the total fungal count was found between 3.124-3.522 log CFU/g, on an average 3.334 ±0.088 log CFU/g (SD= 0.197; CV=5.913). Raw (paddy) rice samples group after ozonation was contaminated 16.75% more fungal count compared to clean rice group after ozonation p>0.05.

3.3.2 Rice contamination with fungal genera

In general, it is recommended that rice for food purposes be stored in paddy form rather provides some protection against insects and helps prevent quality deterioration. Fungal activity, especially during storage, can lead to a rapid deterioration in the nutritional quality of rice and contamination with mycotoxins. Rice is very hygroscopic. Thus, after drying they need to be effectively packaged to prevent any increase in aw, which would allow mycotoxigenic fungi of Fusarium spp., Aspergillus spp.,

Penicillium spp. and other to become active and produce mycotoxins. Raw and clean rice are very

sensitive to contamination with mycotoxigenic fungi, so there is a need for detection of fungi genera, especially those that produce aflatoxins, zearalenone, deoxynivalenol and other.

On rice various mold fungi of the genera Fusarium, Alternaria, Cladosporium were recorded, but not abundantly, except Aspergillus spp. and other fungi group. We assigned all the identified other fungi to the other fungal genera group: Rhizopus spp., Mucor spp., Acremonium spp., Scorulopsis spp. and other.

0 1 2 3 4 5 6 Raw Clean lo g CF U/g Rice samples S1R1 S2R1 S3R1 S4R1 S5R1

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Fig. No. 11. Percentage contamination of rice (before ozonation: direct plating) by fungal genera The fungal contamination count by other genera was found 68.662±9.597% on surface of raw rice samples and 75.33±14.818% on clean rice samples. Aspergillus spp. contamination on raw samples was observed 2.664±1.245% and on clean samples was 8.67% higher. Fusarium spp. contamination on raw samples was observed 48.662±17.243% and on clean samples was 31.99% less. Alternaria spp. contamination on raw samples was observed to be 8.666±5.637% and on clean samples was 0.68% less.

Cladosporium spp. contamination on raw samples was observed to be 12.664±5.906% and on clean

samples was 10.66% less.

Fig. No. 12. Percentage contamination of rice (after ozonation: direct plating) by fungal genera

-20 0 20 40 60 80 100

RAW CLEAN

PERCENTAGE (%) CONTAMINATION OF RICE Before ozonation: Direct Plating

Others Cladosporium sp. Alternaria sp. Fusarium sp. Aspergillus sp 0 20 40 60 80 RAW CLEAN

PERCENTAGE (%) CONTAMINATION OF RICE After ozonation: Direct Plating

Others

Cladosporium sp. Alternaria sp. Fusarium sp. Aspergillus sp

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After ozonation the fungal contamination count by other genera was found 55.864±22.919% on surface of raw rice samples and 36.662±8.231% on clean rice samples. Apergillus spp. contamination on raw samples was observed 5.998±3.230% and in clean samples was 0.66% less. Fusarium spp. contamination in raw samples was observed 26.664±4.367% and in clean samples was 14% less.

Alternaria spp. contamination in raw samples was observed to be 4.664±1.999% and in clean samples

was 5.332±3.265% - 0.67% less. Cladosporium spp. contamination in raw samples was observed to be 3.998±1.943% and in clean samples was 2% less.

Comparison of fungal contamination of rice surface before and after ozonation:

Raw (paddy) rice group after ozonation was contaminated 55.58% more with Aspergillus genera compared to raw rice group before ozonation p<0.05. Raw rice group after ozonation was contaminated 48.21% less with Fusarium genera compared to raw rice group before ozonation p<0.05. Raw rice group after ozonation was contaminated 46.18% less with Alternaria genera and 68.43% less with

Cladosporium genera compared to raw rice group before ozonation p<0.05. Raw rice group after

ozonation was contaminated 18.64% less with other genera compared to raw rice group before ozonation p>0.05.

Clean rice group after ozonation was contaminated 52.94% less with Aspergillus genera compared to clean rice group before ozonation p<0.05. Clean rice group after ozonation was contaminated 24.01% less with Fusarium genera compared to clean rice group before ozonation p>0.05. Clean rice group after ozonation was contaminated 33.33% less with Alternaria genera and 0.1% less with Cladosporium genera compared to clean rice group before ozonation p>0.05. Clean rice group after ozonation was contaminated 18.64% less with other genera compared to clean rice group before ozonation p>0.05. Literature sources indicate that the content of mycotoxins produced by fungi in most cases directly depends on the content of fungi in rice. To determine the rice internal contamination with mold fungi the agar plate method was applied.The data presented show that ecological alterations of mold fungi in rice can seriously affect their quality, because many mold fungi growing in a favourable substrate can intensively produce toxic substances.

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Fig. No. 13. Percentage internal contamination of rice (before ozonation) by fungal genera In Fig. 13, before ozonation the fungal internal contamination count by other genera was found 53.33±2.789% in raw rice samples and 22% higher in clean rice samples. Aspergillus spp. contamination in raw sample was observed 4.666±3.887% and in clean samples was 6.66% higher. Fusarium spp. contamination in raw sample was observed 46% higher compare to clean samples. Alternaria spp. contamination in raw sample was observed to be 19.332±5.416% and in clean samples was 11.33% less.

Cladosporium spp. contamination in raw sample was observed to be 5.332±4.545% and in clean sample

was 3.33% less.

Fig. No. 14. Percentage internal contamination of rice (after ozonation) by fungal genera

-20 0 20 40 60 80 100

RAW CLEAN

PERCENTAGE (%) CONTAMINATION OF RICE Before ozonation: internal contamination

Others Cladosporium sp. Alternaria sp. Fusarium sp. Aspergillus sp -10 0 10 20 30 40 50 RAW CLEAN

PERCENTAGE (%) CONTAMINATION OF RICE After ozonation: internal contamination

Others

Cladosporium sp. Alternaria sp. Fusarium sp. Aspergillus sp

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In Fig. 14, after ozonation, the fungal internal contamination count by other genera was found 25.998±5.517% in raw rice samples and 10.662% higher in clean rice samples. Aspergillus spp. contamination in raw sample was observed 0.666±0.666% and in clean samples was found 4.67% higher. Fusarium spp. contamination in raw sample was observed 20.662±5.812% and in clean samples was 10% less. Alternaria spp. contamination in raw samples was observed to be 21.996±14.743% and in clean samples was 16.66% less. Cladosporium spp. contamination in raw samples was observed to be 5.998±5.205% and in clean sample was 1.998±1.332%.

Comparison fungal internal contamination of rice before and after ozonation:

Raw (paddy) rice group before ozonation was contaminated 85.72% less with Aspergillus genera compared to raw rice group before ozonation p<0.05. Raw rice group before ozonation was contaminated 67.03% less with Fusarium genera compared to raw rice group before ozonation p<0.05. Raw rice group before ozonation was contaminated 13.78% more with Alternaria genera compared to raw rice group before ozonation p>0.05. Raw rice group before ozonation was contaminated 11.10% more with Cladosporium genera compared to raw rice group before ozonation p>0.05. Raw rice group before ozonation was contaminated 51.25% less with other genera compared to raw rice group before ozonation p<0.05. Clean rice group after ozonation was contaminated 52.94% less with Aspergillus genera compared to clean rice group before ozonation p<0.05. Clean rice group after ozonation was contaminated 24.02% less with Fusarium genera compared to clean rice group before ozonation p>0.05. Clean rice group after ozonation was contaminated 33.33% less with Alternaria genera and 0.1% less with Cladosporium genera compared to clean rice group before ozonation p>0.05.

3.3.3 Rice contamination with AFB1, ZEA & DON

The contamination of mycotoxins can cause a wide variety of negative impacts on health, depending on various factors on their nature and concentrations. Especially chronic exposure to mycotoxins leading to unspecific symptoms often entails serious economic losses in animal production. Aflatoxin B1 (AFB1) is a mycotoxin produced by Aspergillus flavus and A. parasiticus. AFB1 exerts toxic and carcinogenic effects and immuno-suppression. Zearalenone (ZEA), a mycotoxin produced by Fusarium culmorum and F. graminearum, has an estrogenic activity, and exerts adverse effects on reproduction system and fertilization. Deoxynivalenol (DON), a trichothecene mycotoxin produced by various species of

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Table 3. Aflatoxin B1, Zearalenone and Deoxynivalenol concentration in rice Condition Rice samples n Mycotoxins concentration, µg/kg±SD

Aflatoxins B1 Zearalenone Deoxynivalenol Before ozonation Raw (paddy) 5 1.6 ± 3.04959 314 ± 277.3658 10 ± 22.36068 Clean 5 1.54 ± 1.95141 306 ± 391.2544 100 ± 93.54143 After ozonation

Raw (paddy) 5 LOD≥1.0* 6 ± 13.41641 LOD≥10* Clean 5 LOD≥1.0* 66 ± 147.5805 80 ± 115.1086 *LOD – limit of detection

Before ozonation raw rice group was contaminated 1.6 time more with aflatoxin B1 compared to raw rice group after ozonation P<0.05. Before ozonation raw rice group was contaminated 52.3 time more with zearalenone compared to raw rice group after ozonation p<0.05. Before ozonation raw rice group was contaminated 10 time more with deoxynivalenol compared to raw rice group after ozonation p<0.05. Before ozonation clean rice group was contaminated 1.54 time more with aflatoxin B1 compared to clean rice group after ozonation p<0.05. Before ozonation clean rice group was contaminated 4.64 time more with zearalenone compared to clean rice group after ozonation p<0.05. Before ozonation clean rice group was contaminated 1.25 time more with deoxynivalenol compared to clean rice group after ozonation p>0.05.

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4. DISCUSSION OF RESULTS

Mycotoxin contamination in certain agricultural commodities has been a serious concern for human and animal health. Mycotoxins are substances produced mostly as secondary metabolites by filamentous fungi that grow on seeds, grains, and feed in the field, or in storage. Mycotoxin production is unavoidable and at times unpredictable, which makes it a unique challenge to food safety. Decontamination of mycotoxin contaminated food is not fully successful, and control of mycotoxins is the need of the hour. The development of integrated management strategies is therefore essential to ensure food safety. Mycotoxin contamination often occurs in the field prior to harvest. Post-harvest contamination can occur if the drying is delayed and during storage of the crop if moisture is allowed to exceed critical values for mold growth. Delayed harvest in rainy weather frequently leads to grain‘s sprouting on the panicle, particularly for non-dormant japonica rice. In this study it was investigated that mycotoxins are important contaminants for rice in India. There are various types of mycotoxins which are Aflatoxin B1 (AFB1), Zearalenone (ZEA) and Deoxynivalenol (DON). There were various groups of rice samples which were compared in accordance to the total fugal count and fungal genera. The rice samples were divided into two groups: (i) raw rice samples and (ii) clean rice samples. Both the groups were treated with and without ozonation and also disinfectant. In raw rice samples w/o ozonation the total fungal count for sample no. 1, 2, 3, 4, & 5 were 4.56, 3.42, 3.77, 4.54 and 4.80 log CFU/g respectively. Whereas, in clean rice samples w/o ozonation the total fungal count for sample no. 1, 2, 3, 4, & 5 were 4.23, 3.72, 4.16, 3.63 and 3.92 log CFU/g respectively. When these rice samples were treated under ozonation condition, in raw samples the total fungal count for the samples 1, 2, 3, 4, & 5 were 4.43, 3.02, 3.06, 4.04, and 3.36 log CFU/g respectively. In clean rice sample the total fungal count for sample no. 1, 2, 3, 4, & 5 were 3.13, 3.12, 3.52, 3.36 and 3.52 log CFU/g respectively. In direct platting method without ozonation, the raw rice sample average for Aspergillus spp. was 2.66%,

Fusarium spp. was 48.66%, Alternaria spp. was 8.66%, Cladosporium spp. was 12.66% and other

genera was 68.66%. Where as in clean rice samples the average of Aspergillus spp. was 11.33%,

Fusarium spp. was 16.66%, Alternaria spp. was 7.99%, Cladosporium spp. 2% and other genera was

75.33%. In the same method with ozonation, the raw rice samples had average Aspergillus spp. of 5.99%, Fusarium spp. was 26.66%, Alternaria spp. was 4.66%, Cladosporium spp. was 3.99% and other genera with 55.86%. In clean rice sample, average of Aspergillus spp. was 5.33%, Fusarium spp. was 12.66%, Alternaria spp. was 5.33%, Cladosporium spp. was 1.99% and other genera with 36.66%. When disinfected rice samples without ozonation was observed, in raw rice samples the average of