Growth Modeling of Bacillus cereus in Cooked Rice

  Growth Modeling of Bacillus cereus in Cooked Rice PRACTICAL TRAINING REPORT

  This practical training report is submitted for the partial requirement for Bachelor Degree

  By : Maria Jessica Arta 12.70.0037

DEPARTMENT OF FOOD TECHNOLOGY FACULTY OF AGRICULTURAL TECHNOLOGY SOEGIJAPRANATA CATHOLIC UNIVERSITY SEMARANG 2015

  

Growth Modeling of Bacillus cereus in Cooked

Rice

“Practical Training at Assumption University, Bangkok, Thailand

  

By :

Maria Jessica Arta

Student ID :12.70.0037

Faculty : Agricultural Technology

  

Department : Food Technology

This practical training report has been approved and supported by examiner in

nd

  

Practical Training Exam on 12 June, 2015

th

  Semarang, 4 July 2015 Department of Food Technology Faculty of Agricultural Technology Soegijapranata Catholic University

  Technical Advisor Practical Training Advisor

  Asst.Prof.Dr. Patchanee Yasurin Dr. Laksmi Hartayanie,M.P

  

Dean

  Dr. Victoria Kristina Ananingsih, ST., M.Sc

  

PREFACE

  Praise in the name of Lord Jesus Christ, because only His grace and bless, the author would have the opportunity to implement the practical training and internship program, so can finish the report smoothly. During the practical training, the author did the research entitled “Growth Modeling of Bacillus cereus in Cooked Rice”. This report is the complete accountability from the practical training which was done in Assumption

  th th University, Bangkok, Thailand that take place from 16 January to 26 of March, 2015.

  The author would not be able to finish all of these tasks alone. The key of success in this practical training and the report making were caused of the big support and guidance given the amazed people around the author. There are my special thanks for : 1.

  My Lord Jesus Christ who is always bless and guide me.

  2. Dr. Victoria Kristina Ananingsih, ST., MSc. for giving me this great opportunity to participate in this internship program.

  3. Dr. Patchanee Yasurin as my advisor who was teaching, advising me did this practical training and for making this report.

  4. Dr. Laksmi Hartayanie, M.P for giving me advices for making this practical work report better.

  5. All of Assumption University Biotechnology Department lecturer who are welcoming and always helping me when I did this research.

  6. P’Porn and P’Tuk as a laboratory assistant who always provide stuff and also help me in the laboratory.

  7. Natpisit Chaitachawong as a bachelor senior student who always support, accompanied, teach me during this research, and also being my partner.

  8. My family who is always support and pray for me everyday.

  9. Lorentia Santoso, Rr Panulu, Hana Melinda, Buddy Kristianto and William Wibowo as intership partners who support and accompanied me during this research until making paper.

  10. Last but not least, I would thanks to all my beloved friends who I can’t say it one by one that always support and give me spirit.

  The author realized that this report is still far from perfect and there are still many shortage due to the limitations of the author. However, the author hoped that this report can still be an inspiration and provide useful information for all the reader. The author was very grateful to be able to get a valuable and unforgettable opportunity.

  th

  Semarang, 4 July 2015 Maria Jessica Arta Author

  TABLE OF CONTENT

  4. RESEARCH METHODOLOGY………………………...……………………... 9 4.1.

  4.4.5. Statistical analysis ………………………………………………….. 12

  4.4.4. Bacteria Growth Determination……………………………………... 11

  4.4.3. Inoculum Preparation………………………………………………... 11

  4.4.2. Rice sample preparation…………………………………………….. 10

  4.4.1. Media Making…..…………………………………………………… 10

  4.4. Method…………………………………………………………................... 10

  4.3. Equipments…………………………………………………………............. 9

  4.2. Materials…………………………………………………………................. 9

  Time and Place of Practical Training………………………...………..…… 9

  Bacillus cereus………….…………………..…………………….... 8

  Page ENDORSEMENT

  Research Overview…………………………………………………………... 6 3.2. Background of Research……………………………………………………... 6 3.2.1.

  RESEARCH PROJECT…………………………………………………………. 6 3.1.

  Vision……………………………………………………………….. 4 2.3.2. Mission……………………………………………………………… 4 2.3.3. Strategy……………………………………………………………… 5 3.

  Assumption University………………………………………………………. 3 2.2. Faculty of Biotechnology……………………………………………………. 3 2.3. Vision, Mission, and Strategy of the Faculty……………………………….. 4 2.3.1.

  INSTITUTION PROFILE………………………………………………………. 3 2.1.

  Background………………………………………………………………….. 1 1.2. Purpose of Practical Training……………………………………………….. 2 2.

  INTRODUCTION……………………………………………………………….. 1 1.1.

  INDEX OF APPENDICES………………………………………………………… viii 1.

  LIST OF TABLES…………………………………………………………………. vi LIST OF FIGURES………………………………………………………………... vii

  SHEET………………………………………………………… i PREFACE……………………………………………….……….………………… ii TABLE OF CONTENTS…………………………………………..………………. iv

  5. RESULT AND DISCUSSION………………………………………………….. 13

  7. RECOMMENDATION…………………………………………………………. 22

  8. REFERENCES…………………………………………………………………... 23 9.

  APPENDICES…………………………………………………………………... 25

  LIST OF FIGURE

  Figure 1. Equipments used : Shaker Incubator (a) and Stomacher (b)........................... 9 Figure 2. Working culture overnight (Bacillus cereus) with 0,1 OD 600 ........................ 11 Figure 3. Inoculated Jasmine Rice................................................................................ 12 Figure 4. Single colony (marked with red circle) of B.cereus...................................... 12 Figure 5. Serial Dilution Technique

  ………………………………………………….. 14 Figure 6. Growth of B. cereus in Jasmine Rice.................................................

  14 Figure 7. Growth of of B. cereus 15 in Organic Rice Berry ……..............................

  LIST OF TABLE

  Table 1. Characteristics of Bacillus cereus ………………………………………… 8 Table 2. Composition of MYP Media …………………………………………………..

  10 Table 3. Mean and SD of B.cereus in Jasmine rice and Organic Rice Berry up to six hour………………………………………………………………........16 Table 4. Comparison of specific growth rate of B.cereus in Jasmine rice and Organic

  Rice Berry…………………………………………………………………17

INDEX OF APPENDICES

  Appendix 1. Map of Assumption University, Hua Mak, Bangkok………………... 25 Appendix 2.

  Research Project Schedule…………………………………………… 25

  2

1. INTRODUCTION 1.1. Background

  Food is very important for human body. This is because all body functions such as metabolic, mental, hormonal, physical or chemical cannot be performed by the body without food. Food also maintaining proper health and prevention or cure of diseases. Food will supplies the energy needed to perform all body function also growth and repair in our body cell. We get energy from essential nutrition in food such as carbohydrates, proteins, fats, minerals, and vitamins. On the other hand, human has many problem related with food, such as food crisis problem that caused hunger, food disease, food safety, agricultural problem and also food waste problem. Besides the primary demand, as the development of science and technology, the demand of food becoming more complex and diverse like simplicity of serving, good appearance and more importantly, the ability to promote health. Therefore, food technology has the very important role for answering the changing demand of food products which always increasing day by day.

  The practical training for students of Food Technology Department from Soegijapranata Catholic University in Faculty of Biotechnology, Assumption University, Thailand has become one of the ways to participate in overcoming the food problem. During this practical training, students from Soegijapranata Catholic University have a duty of learning new knowledge in their own research. And this practical training will give the student more capability in research, so the student can do something to overcoming the food problem. This program has a purpose to fit up the students with the food-related research or the experience in real world practice of food industry, either in food industries or food technology related institutions for developing skills and experience in order to face the opportunities and challenges in food technology development. Students are also encouraged to get a new experience either in food industries or food technology related institutions. Faculty of Biotechnology, Assumption University, Thailand has become a great partner of Food Technology Department, Soegijapranata Catholic University since 2007. Advance technology and facility in Biotechnology and

  3 Catholic University to take part in internship program with Assumption University. Furthermore, this practical training in Thailand will give a new experience living abroad, chance to meet people from other countries, get new friend and connection, and learn to respect culture diversity in this globalization era. Therefore, this internship program will give lot of benefit to the student, faculty and the society in the future.

1.2. Purpose of Practical Training a.

  To give the student an experience to conduct about food research in abroad, so that the student can implement the knowledge that they learned in the real industrial or scientist world.

  b.

  The student gets a better insight in the practical aspects of the laboratory.

  c.

  To give the student an opportunity to adapt with new circumstances and society.

  d.

  To strengthen relationship between Soegijapranata Catholic University and Assumption University

2. INSTITUTION PROFILE 2.1. Assumption University

  Assumption University is a private Catholic university with two campuses in the HuaMak and Suvarnabhumi areas of Bangkok, Thailand. The university is led by the Brothers of St. Gabriel, who have been active in education in Thailand since 1901.

  Assumption University is noted for attracting large numbers of foreign students from countries including Russia, China, Myanmar, India, Bangladesh, Pakistan, Malaysia, and other Asian countries. Students from China make up the largest number of foreign students, with up to 6,000 enrolled. Approximately 1,001 Indian students also attend the university. There are exchange students from the United States (Loyola) and Europe. Assumption University is also the first international university in Thailand. Assumption University became an autonomous educational institution in 1969 when Assumption Commercial College (ACC) originated Assumption School of Business. In 1972 it became Assumption Business Administration College (ABAC). In 1990 the college was granted a university status by the Ministry of University Affairs and took the name of Assumption University (AU). Recently, AU has been expanding the Suvarnabhumi campus with Gothic-style buildings and creating a nature-centered atmosphere it calls the "University in the Park". Assumption University offers many majors, such as Engineering, Information Technology, Science, Nursing, Law, Business Administration, Communication Art, Architecture, Biotechnology and Music. It is also the leading provider of E-learning in Thailand since it has established the College of Internet Distance Education on December 21, 2002.

2.2. Faculty of Biotechnology

  The Faculty of Biotechnology was founded 1993 as the ninth faculty in Assumption University to produce graduates working in biotechnology field and its related fields. The faculty has been offering two 4- year bachelor’s programs in Agro Biotechnology and Food Biotechnology. Both programs were approved of their academic standard by

  5 the Ministry of University Affairs in 1997. Since then the Faculty has produced 4 classes of graduate with the degree of BS in Agro-Industry and in Food Technology, to the country. The majorities of the Thai people are involved in agriculture and related industries. Knowledge in biotechnology can be directly implied to the utilization of agricultural products to increase the value. Therefore, it has a big impact on the living of most Thais. Biotechnology can provide agriculture with a variety of useful agents, from soil inoculants to veterinary products and possibly aqua-cultural and marine cultural products. Biotechnology implementation begins with the supplement of traditional genetic methods for the development of new or improved plant and animal strains for conventional agriculture. It also provides the food industry with bio-process and food ingredients like starter culture and enzyme, which directly involved in food processing. Main administrators of the faculty member and all the 19 full-time instructors, three teaching assistants, one administrative staff and two laboratory technicians.

2.3. Vision and Mission, and Strategies of Faculty

  In line with the university’s philosophy of education, the Faculty of Biotechnology has adopted the following vision, missions and strategies.

  2.3.1. Vision

  The application of life sciences to industrial processes yielding products and services that ultimately benefit the consumers through bio-resources and technology.

  2.3.2. Mission 1.

  To provide the nation with highly trained professionals who are able to apply basic and applied scientific principles to continuous improvement of the safety, quality and value of biotechnological services and products.

2. To produce graduates characterized by moral and ethical principles 3.

  To produce graduates who are competent to pursue advanced studies in fields related to biotechnology

  6

2.3.3. Strategy

  Thailand is a major producer of agricultural commodities used in bioprocessing and a significant food manufacturing sector. However, the food manufacturing lags behind the international average in the amount of value added in processing. Experience and state- of-the-art faculty members available in bioprocessing and technology development uniquely qualify the faculty of Biotechnology to play a major role in addressing these opportunities. The purpose of the faculty is to facilitate expansion of programs in bioprocessing, foster development of multidisciplinary efforts and encourage excellence in scientific research and academic program with the assistance of information technology.

  3.1. Research Overview

  The main topic of the research was “The Growth Modeling of Bacillus cereus in Cooked Rice”. The objective of this research was to see the growth model of Bacillus cereus in Organic Rice Berry and Jasmine Rice by using Total Plate Count method. This experiment was done from zero hour and every 1 hour after inoculation up to 6 hour at incubated temperature of Bacillus cereus.

  3.2. Background of Research Foodborne diseases caused by pathogenic bacteria are still a major threat to public health. B.

cereus is responsible for the majority of foodborne illness attributed to Bacillus. During 1993-

  1997, B. cereus was linked to 14 outbreaks and caused 691 reported cases of foodborne illness in the United States (Keith et al., 2004). B. cereus is a Gram-positive, mobile, facultative and aerobic spore former. In fact, 19 of 22 strains of B. cereus is produce enterotoxin which is known as diarrheagenic toxin, diarrheal agent, fluid accumulation factor, vascular permeability factor, dermonecrotic toxin and intestinonecrotic toxin (Spira and Goepfert, 1972).

  

Bacillus cereus is often present as an intrinsic contaminating microorganism in Refrigerated

Processed Foods of Extended Durability (REPFED), pasteurized milk, rice dishes and pastas.

  During shelf life it may become a major part of the microflora. Because of its resistant spores, significant numbers of B. cereus have also been found in herbs and spices, vegetables and dehydrated foods. The presence of both vegetative cells and spores in food commodities has been reported and their role in food safety and food spoilage elaborated (Priest, 1993).

  The main goal of any microbiological treatment is to improve food quality and extend shelf life. The safety and shelf life of many food products is dictated by the time and/or temperature required for pathogenic or spoilage microorganisms to reach a critical level

  (O’Brien, 1996). Thus, models are essential tools for helping assess and manage risk to human illness from food. Microbial modeling can predict pathogen behavior under conditions for which no data exist. Although rice is an extremely important food in many countries, very few studies to date have

  8 fewer studies directed at rice microbiological risk assessment have focused primarily on prediction of B. cereus growth, especially that of spores during storage. Because the spores can survive extreme temperatures, when conditions allow they will germinate in contaminated food and multiply relatively slowly. B. cereus vegetative cells also can grow and produce enterotoxins over a wide range of temperatures from 25 to 42°C. Most studies investigating the growth of B. cereus on rice have employed spores under nonstress conditions (Heo, 2009). Rice is a daily meal for people in the Asia

• –Pacific region. It is believed to provide more health benefits than other carbohydrate based foods, since it contains several nutrients and anti- oxidative compounds. Thai rice, especially, has gained popularity world wide for its nutrients and fragrance. Rice bran is a by-product from rice milling. This variety has a crunchy texture and is used in desserts and salads. The bran is naturally black (purple) and on cooking releases a purple color. Riceberry, a Thai black rice, has been recently developed with the aim of providing optimum nutritional benefit to general consumers, as well as supplementation to anaemic and diabetes mellitus patients, since it contains high iron and low glucose. The bran part of Riceberry is believed to be high in antioxidants and other significant constituents, such as anthocyanins, etc., that possess chemopreventive properties (Leardkamolkarn et al., 2010). Jasmine rice, called Hom Mali or KDML 105 (Khao Dawk Mali), originates from the Isaan region in northeastern Thailand. Released in 1959, Hom Mali was developed during the 1980s through a governmental initiative for export purposes. Jasmine rice cropping increased by 74% from 1990 to 1998, reaching 28.3% of rice overall acreage in Thailand, despite low yields varying from 1.9 to 2.3 tons per hectare (T/ha) (Rahman et al. 2009). Khao Dawk Mali (KDML) 105 is one of the most popular rice varieties in Thailand. In Thailand, Jasmine rice is considered as a vital crop for domestic consumers and a primary export commodity for economic growth (Sarkarung, Somrith, & Chitrakorn, 2000; Chitrakorn, 2003). Jasmine rice can be consumed in many ways, for example whole grain consumption. Some Asian peoples cook their rice in an ample amount of water to obtain desirable texture whereas, in many western cultures, rice is boiled in excess water until the centre of the grain is fully cooked (Juliano, 1982).

  9

  Bacillus cereus is a gram-positive, facultative aerobic rod shaped endospore-forming bacterium.

  Other characteristics are listed in Table 1. B. cereus has a wide distribution in nature, frequently isolated from soil and growing plants, but it is also well adapted for growth in the intestinal tract of insect and mammals. In addition to causing foodborne illness, B. cereus is also capable of causing mastitis, systemic infection, gangrene, meningitis in immunocompromised children, respiratory tract infections and other clinical problems (Pages, 2008). This is because of the enterotoxin produced by B.cereus. The enterotoxin is synthesized and released during the late logarithmic growth phase of the organism at an optimum temperature of 32-37 C (Spira&Goepfert, 1972). B. cereus growth is optimal in the presence of oxygen, but can occur under anaerobic conditions. B. cereus cells grown under aerobic conditions are less resistant to heat and acid than B. cereus cells grown anaerobically or microaerobically (Mols et al. 2009). The growth medium employed markedly affected the ability of a given strain of B.cereus to provoke a response. Food-borne illness outbreaks have been reported in European countries,

  5 American, Japan and other regions. It is generally recognized that a population of > 10

  B. cereus

• 1 cells g in the food sample taken is required for poisoning to occur (Gilbert & Kramer, 1984).

  Table 1.Characteristics of Bacillus cereus

  Property Value

  Oxygen requirement Facultative aerob Growth-pH limits

  4.9

• – 9.3 Growth-minimum a w

  0.90

• – 0.95 Growth-temperature limits

  10 C

• – 48 Growth-optimum temperature

  28 C

• – 37 Spore germination-minimum temperature 8-30 C Generation time-laboratory media at 30 C 18 - 27 min Generation time-boiled rice at 30 C 26 - 31 min Source: Gilbert et al., 1981

  4.1. Time and Place of Practical Training

  The practical training is conducted in the Microbiology Laboratory of Biotechnology Faculty,

  th

  Assumption University HuaMak Campus, Bangkok, Thailand, and take place from January, 16

  th to March, 26 2015.

  4.2. Materials

  Materials used in this research are Organic Rice Berry, Jasmine Rice, bacteria culture of Bacillus

  

cereus, MYP (Mannitol Egg Yolk Polymixin), TSB (Tryptic Soy Broth), and 0,1% Peptone

water.

  4.3. Equipments

  Equipments used in this research are, micropipette, tip, spectrophotometer, shaker incubator (Figure 1a), incubator, rice cooker, spatula, spoon, bowl, stomacher (Figure 1b) laminar air flow, autoclave, tubes, beaker glass, loop, alcohol lamp, petri dish, and sterile cotton bud.

   (a) (b)

  Figure 1. Equipments used : Shaker Incubator (a) and Stomacher (b) 4.4.

   Methods 4.4.1. Media Making

  11 bacteria. Table 2. Composition of MYP Media

  Material g/L Beef Extract

  1 Peptone

  10 D-Mannitol

  10 Sodium Chloride

  10 Phenol Red 0,025

  Agar

  15 Egg Yolk Emulsion 0,03 Polymyxin B

  0,0019 Final pH: 7.2 ± 0.2 at 25 C (Mossel, 1967) For making the media MYP, all the materials w e r e p u t a n d m i x e d into a D u r a n bottle. with tap water until all of them dissolve. Then the media was autoclaved at 121 C for 15 minutes. Before pour MYP into plates, MYP was mixed with egg yolk, NaCl 0,85%, and polymixin. After that, the sterilized media were poured into petri dish and wait until they became solid and dry. To make peptone water, 1 g/L peptone media (powder) was poured and mixed into a erlenmeyer with distilled water until all of them dissolve. Put in the tube with cap. Then autoclave it at 121 C for 15 minutes.

  4.4.2. Rice Sample Preparation

  Organic Rice Berry and Jasmine Rice were obtained from local fresh market in Bangkok, Thailand. Both of rice were wash with tap water ratio 1 : 2. Cook organic rice berry first, because it will take a longer time than jasmine rice. Three hundred grams of organic rice berry was cooked with 1,5 L tap water ratio 1 : 5. Then, 400 grams of jasmine rice was cooked with 800 ml tap water ratio 1 : 2. It would take 30 minutes for jasmine rice and 45 minutes for organic rice berry. After being cooked, both of rice was put into sterile bowl and measured 1050 kg each under aseptic condition. The bowl was covered with aluminium foil and kept in room temperature during process.

  4.4.3. Inoculum Preparation

  12 tion University’s Stock culture of B. cereus were prepared by inoculating one loopful of culture into 10 mL fresh Tryptic Soy Broth (TSB) and shake overnight by shaker incubator. Shaker incubator used to make aeration condition as Lawley (2008) said that B. cereus grow on a medium containing oxygen (aerobic) that is also known by the term aerobic sporeformers although they can grow under facultative aerobic condition. Most members of the genus Bacillus can form endospores formed intracellularly in response to unfavorable environmental conditions. Therefore, members of the genus Bacillus have a high tolerance to environmental conditions change. Then 1% v/v of overnight culture was inoculated into 40 mL of fresh TSB and shake for 100 rpm, until optical density at 600 nm reach 0.1 (SPECTRONIC, model GENESYS 5) which is in their early log phase. After this, 11 ml of culture was taken by micropipette (1% v/v of overnight culture from 1050 kilograms samples) and put on rice samples in the sterile bowl covered with aluminium foil, samples were mixed well while the culture was added.

  Figure 2. Working culture overnight (Bacillus cereus) with OD 600 is reached 0,1 4.4.4.

   Bacteria Growth Determination

  Rice samples were taken and measured 25 grams each hour, then 225 ml 0,1% peptone water was poured into plastic bag sample under aseptic condition. Stomacher was used to smashed rice and then dissolved by peptone. The bacteria growth on the samples were carried out by spread plate method. In this method, TSB were used as culture media. Before do spread plate, serial

• 1 -2 -3 -1

  dilution technique there are 10 , 10 , and 10 has done, where 10 dilution is rice sample and peptone water. 100µl of culture was swabbed on the MYP agar by loop. All plates were put on

  13 counter (Stuart Scientific). This experiment was done from zero hour and every 1 hour after inoculation up to 6 hours and were repeated 3 times with duplicate plates.

  Figure 3. Jasmine Rice was inoculated B. cereus Figure 4. Single colony with clear zone (marked with red circle) of Bacillus cereus 4.4.5.

   Statistical analysis

  The experiment was done in three replications independently. The t-test multiple comparison Duncan has been done by using R-statistic to study the growth model of B. cereus in cooked rice.

  In this research, rice samples were prepared first. Organic Rice Berry and Jasmine Rice were were washed with tap water ratio 1 : 2. Three hundred grams of organic rice berry was cooked with 1,5 L tap water ratio 1 : 5. Then, 400 grams of jasmine rice was cooked with 800 ml tap water ratio 1 : 2. It would take 30 minutes for jasmine rice and 45 minutes for organic rice berry. After being cooked, both of rice was put into sterile bowl and measured 1050 kg each under aseptic condition. The bowl was covered with aluminium foil and kept in room temperature during process. Organic rice berry should be cooked first, because it will take a longer time than jasmine rice . Rice containing high amylose produce dry rice product, otherwise the rice containing low amylose produce sticky rice and soft. Amylose content related to the amount of water absorption and development of the volume of rice during cooking. The higher the amylose content, the less sticky rice and the harder (Juliano, 1982). Furthermore, proposed by Haryadi (2008), rice containing higher protein requires more water and a longer cooking time. This relates to the structure of the seed, which is enclosed in a starch granule protein that blocks the absorption of water by the protein starch granules, and resulted in more length of time required for cooking so that gelatinization can take place perfectly. In addition, high-protein rice produce flavorful less tasty. The Standard Total Plate Count is the most common method used to quantify bacteria in foods. To perform a standard plate count, the food to be tested is suspended in liquid and a sample is then spread over the surface of a solid medium in a petri dish. Bacterial cells present will form colonies that can be counted to determine the number of cells in the original sample. When the objective is to estimate the total number of bacteria, a complex medium called Plate Count Agar is commonly used since it will support growth of many different types of bacteria. We call the results the number of Colony Forming Units (CFU), not total bacteria. This is because no single culture medium will support all different types of bacteria, we can only count those that do grow to form a visible colony (Hocking, 2003).

  Serial dilution technique used in this study. The serial dilution technique done by find the one with 30-300 colonies (Figure 4). After serial dilution technique done, continued by spread plate

  15 the growth medium. The spread plate can be used for quantitative work (colony counts) if the inoculum is a measured volume (Kiiyukia, 2003). In the spread plate method, 0,1 ml of diluted sample is pipetted onto the surface of a solidified agar medium and spread with a sterilized glass rod. Sterile spreaders are used to distribute inoculum over the surface of agar medium in plates.

  Figure 5. Serial Dilution Technique

  B. cereus growth curve in jasmine rice

  7

  6

  5 /ml

  4 CFU

  3 Series2 g Lo

  2 Linear (Series2)

  1

  1

  2

  3

  4

  

5

  6 hour (s)

  Figure 6. Growth of B. cereus in Jasmine Rice

  16

  B. cereus growth curve in organic rice berry

  8

  7

  6

  5 /ml

  4 CFU Series2 g

  3 Lo Linear (Series2)

  2

  1

  1

  2

  3

  4

  5

  6 hour (s)

  Figure 7. Growth of B. cereus in Organic Rice Berry The growth curve of B. cereus in jasmine rice and organic rice berry can be seen in Graphic 1. and Graphic 2. Based on Graphic 2, it appears that B. cereus in organic rice berry still through a phase of adaptation (lag) is at 0 hour, but in graphic 1 different occurs in B. cereus growth in jasmine rice. In this lag phase, B. cereus are in a phase of adjustment to the new environment. On microbial adaptation phase experiencing a period in which the cells become larger but the numbers remain the same or very little development occurred populations despite ongoing cell metabolism. The next phase is the growth phase logarithmic (log) achieved by B. cereus which begins after the 1 hour to achieve growth in both jasmine rice and organic rice berry. The maximum on the 5th hours of incubation. This phase is the final phase lag marked to continue splitting the microbial cells. During log phase, cells divide continuously with a constant current high growth rate numbers and log of the number of cell groups against time on a straight line (Shantharam, 1997 in Nurhajati et al., 2009). Bacillus cereus stationary phase on jasmine rice has longer time than organic rice berry which look began after the 2 hours up to 5 hours of incubation, whereas in organic rice berry look began 2 and 3 hours.

  17 hour.

  Hour Jasmine Rice Organic Rice berry

  a a

  3.51 ± 0 3.55 ± 0.0849

  a a

  1 3.63 ± 0.0306 3.86 ± 0.0755

  a b

  2 4.95 ± 0.0300 4.87 ± 0.0208

  a b

  3 5.12 ± 0.1217 4.97 ± 0.0379

  a a

  4 5.22 ± 0.0265 5.33 ± 0.0721

  b a

  5 5.33 ± 0.0306 6.31 ± 0.0503

  a a

  6 6.46 ± 0 6.46 ± 0.0058

• Remark : Different superscript within a row show significant different (P<0.05) Table 3. showed that the log CFU/ml of B.cereus in jasmine rice was significantly higher than in

  nd rd

  organic rice berry at only 2 and 3 hour. On the other hour log CFU/ml jasmine rice is lower than organic rice berry. This indicate that growth of B.cereus in jasmine rice is lower than

  7

  organic rice berry. Content B. Cereus in rice poisoning causes ranges with an average of 5 x 10

  6 CFU / g (Supardi and Sukamto, 1999). Consumption of food containing more than 10 B.cereus /

  g (USFDA, 2001) has been able to cause food poisoning, especially in food when the preparation is left without put in the refrigerator before served. B.cereus grow rapidly if the substrates contain carbohydrates. Meanwhile, when the substrates do not contain carbohydrates, growth will very slow and can not form the toxin. B. cereus toxin can reached a maximum after 4.5 hours (Supardi and Sukamto, 1999).

  18 Spesific growth rate

• 1

  (hr ) Replication 1 Replication 2 Replication3 Average SD

  b

  Jasmine Rice

  0.36

  0.34

  0.33 0.34 0.0153

  a

  Organic Rice berry

  0.87 0.95 0.645 0.82 0.1581 In table 4, the specific growth rate of B.cereus in Jasmine rice was lower than in organic rice berry. This indicate that the Jasmine rice have inhibitory effect on the growth of B.cereus. In organic rice used is sourced from organic matter and manure that comes from waste plant and animal byproducts such as compost or straw or other plant residue, whereas for the prevention and eradication of the pest is used biopesticide active ingredients derived from plant extracts (Balasubramanian and Bell, 2003). On the other hand, non-organic rice in this case jasmine rice, does not use organic materials in any eradication of pests, they use chemical compound on pesticide. That is why B. cereus growth in jasmine rice take longer time than organic rice berry. Pesticide in jasmine rice still stricken and inhibit B. cereus to grow, even though the rice is cooked.

  

Bacillus cereus is a food-poisoning bacterium that may cause two types of gastrointestinal

  disorders: the emetic syndrome, caused by ingestion of a preformed toxin in the food, and the diarrhoeal syndrome, caused by a different toxin that can be formed in the food but also in the small intestine (Granum and Lund, 1997; Granum, 2001). Due to its ubiquitous distribution in nature, B. cereus occurs frequently in a wide range of food raw materials. Rice-based products and farinaceous foods such as pasta and noodles are frequently contaminated and involved in B.

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  cereus poisoning (Kramer and Gilbert, 1989). Levels of B. cereus greater than 10 cfu/g have been found in both cooked and uncooked rice and in cereal products all over the world. Natural antimicrobial substances, such as bacteriocins, are being investigated for food preservation and to replace chemical preservatives (Cleveland et al., 2001), although nisin is currently the only bacteriocin widely used (Thomas et al., 2000). Some bacteriocins, such as nisin or lacticin 3147, prevent spore outgrowth and enterotoxin production by B. cereus (Jaquette and Beuchat, 1998).

  19 There are two types of B. cereus food poisoning. The first type, caused by an emetic toxin, results in vomiting, while the second type, caused by enterotoxins, gives diarrhoea (Kramer, 1989). In a small number of cases both types of symptoms are recorded , probably due to production of both types of toxins. Although the enterotoxins can be preformed, the number of B.

  

cereus cells in the food would be at least two orders of magnitude higher than that necessary for

  causing food poisoning, and such products would no longer be acceptable to the consumer (Granum, 2001).

  The emetic toxin causes emesis (vomiting) only and its structure has for a long time been a mystery, as the only detection system involved living primates (Kramer, 1989). The emetic toxin is resistant to heat, pH and proteolysis but is not antigenic. It is not clear if the toxin is a modified gene product or if it is enzymatically produced through modification of components in the growth medium. However, with such a structure it is most likely that cereulide is an enzymatically synthesised peptide and not a genetic product.

  

Bacillus cereus is ubiquitously present in soil, vegetation water and dust. It has been isolated