quality improvement of bixin from annatto seed by modified extraction methods

Master's Quality Improvement of Bixin from Annatto Seed by Modified Extraction Methods Department of Food Science and Technology Graduate School of Chonnam National University Hoang Van Chuyen Directed by Professor Jong-Bang Eun August 2011 i CONTENTS Page ABSTRACT……………………………………………………………. 1 INTRODUCTION…………………………………………………… 3 MATERIALS AND METHODS………………………………………. 14 1. Materials……………………………………………………………………… 2. Measurement of moisture content in annatto seeds……………………………. 3. Determination of total content of bixin in annatto seed……………………… 4. Extraction of bixin…………………………………………………………… 5. Determination of bixin degradation………………………………………… 6. Analysis of bixin content in annatto extracts………………………………… 7. Gas chromatography (GC) analysis of toluene and m-xylene…………………. 8. Data analysis………………………………………………………………… 14 14 14 15 16 17 17 18 RESULTS AND DISCUSSION 1. Moisture content and total bixin content of annatto seeds…………………… 2. Extraction yield of bixin from annatto seeds by three methods……………… 3. Degradation of bixin during three extraction processes……………………… 4. Total amount of bixin extracted out from annatto seeds……………………… 5. Amounts of toluene and m-xylene produced during extraction processes…… 6. Effects of temperature and volume of sodium hydroxide solution on the extraction yield of bixin……………………………………………………… 20 20 20 21 25 27 32 ii 7. Effects of temperature, volume of soybean oil and light on the extraction yield of bixin and level of toluene and m-xylene in oil extracts……………………. 8. Effects of extraction method, volume of acetone and light on the extraction yield of bixin and content of volatile compounds in extracts…………………. 9. Extraction of bixin from annatto seed by combinations of sodium hydroxide solution and soybean oil……………………………………………………… 36 42 46 CONCLUSION………………………………………………………… 51 REFERENCES…………………………………………………………. 52 ABSTRACT IN KOREAN…………………………………………… 56 ACKNOWLEDGEMENTS……………………………………………. 58 iii LIST OF TABLES Table Page 1. Estimated world production of annatto seed……………………………………… 6 2 . Annatto products and their uses………………………………………………… 7 3. Codes and synonyms of annatto pigment………………………………………… 8 4. Moisture content and total bixin content of annatto seeds during storage……… 22 5. Concentration of toluene in annatto extracts during extraction processes by sodium hydroxide solution, soybean oil, and acetone……………………………. 30 6. Concentration of m-xylene in annatto extracts during extraction processes by sodium hydroxide solution, soybean oil, and acetone……………………………. 31 7. Concentration of toluene and m-xylene in soybean oil extracts in daylight and in darkness ………………………………………………………………………… 41 8. Concentration of toluene and m-xylene in acetone extracts in daylight and in darkness………………………………………………………………………… 45 9. Toluene and m-xylene contents in the extracts of combination 1……………… 49 10. Toluene and m-xylene contents in the extracts of combination 2 …………… 50 iv LIST OF FIGURES Figure Page 1. Annatto tree, fruits, and seeds……………………………………………………… 5 2 . The structural formula of cis- and trans-bixin…………………………………… 11 3 . Sample preparation procedure for analysis of toluene and m-xylene in annatto extracts by gas chromatography……………………………………………………. 19 4 . Bixin yield during extraction processes by soybean oil, acetone, and sodium hydroxide solution………………………………………………………………… 23 5 . Degradation of bixin during extraction processes with vegetable oil, acetone, and sodium hydroxide solution…………………………………………………………. 24 6 . Factual percentage of bixin extracted out from annatto seeds by vegetable oil, acetone, and sodium hydroxide solution…………………………………………… 26 7 . GC-FID chromatogram of toluene (1), ethylbenzene (2), m-xylene (3), and o- xylene (4) in annatto extract……………………………………………………… 29 8. Extraction yield of bixin at different temperature by sodium hydroxide solution 33 9. Extraction yield of bixin with different ratio of annatto seed weight/volume of sodium hydroxide solution…………………………………………………………. 35 10. Extraction yield of bixin by soybean oil at 80 and 100 o C……………………… 37 11. Extraction yield of bixin with different ratio of annatto seed weight/volume of soybean oil……………………………………………………………………… 39 12. Yield of bixin extracted by submerging in acetone at 50 o C with different ratio of annatto seed weight/volume of acetone and in the dark condition……………… 43 13. Percentage of bixin extracted from annatto seed at stages of the extraction combinations between sodium hydroxide solution and soybean oil……………… 47 1 Quality Improvement of Bixin from Annatto Seed by Modified Extraction Methods Hoang Van Chuyen Department of Food Science and Technology Graduate School of Chonnam National University (Directed by Professor Jong-Bang Eun) ABSTRACT The extraction yield, degradation of bixin, and the contents of toluene and m-xylene in annatto extracts during extractions by acetone, soybean oil and sodium hydroxide solution were investigated. Pigment from annatto seeds was extracted by immersing seeds in soybean oil at 120 o C, by acetone in Soxhlet extractor, and by sodium hydroxide solution at 50 o C. The bixin extraction yield by acetone was the highest (81.3%) and followed by the extraction by sodium hydroxide (45.4%). The extraction by soybean oil had the lowest bixin extraction yield (24.7%), and it decreased gradually after 40 min of extraction process. The losses of bixin in the extraction by acetone and sodium hydroxide increased slowly at quite low levels. These were only 12.3% and 14.5% after 120 min, respectively. In contrast, the extraction by soybean oil caused severe degradation of bixin, and it grew up rapidly during process to 63.4%. All three methods produced trace amounts of toluene, which was the highest in sodium hydroxide extract 2 at the level of 18.25 mg/L. Concentration of m-xylene in acetone extracts was very high and remained unchanged during extraction process in the range of 90-110 mg/L. Amount of m- xylene produced by soybean oil extraction also increased rapidly in a time-dependent manner to 80.88 mg/L at the end of extraction, whereas appearance of this compound in sodium hydroxide extracts was very low. The replacement of extraction in Soxhlet extractor by immersion of annatto seeds in acetone at 50 o C with exclusion of light effects resulted in a great reduction of m-xylene content in acetone extracts (15.74 mg/L). Moreover, it also achieved quite high extraction yield of bixin (67.2%) after only a short time of extraction (40 min) without significant effects of acetone volume to extraction yield. Besides, this method also required simple equipment for extraction process than the Soxhlet system. The combination of 40-min extraction by sodium hydroxide solution at 50 o C and 20-min extraction by soybean oil at 100 o C in darkness significantly lowered concentration of volatile compounds compared to those of single extraction methods. Total bixin yield of this combined extraction (53.7% in 60 min) was significantly improved, compared to 45.4% in 120 min of sodium hydroxide extraction and maximum bixin yield (30.2%) of soybean oil extraction. Moreover, the reduction of solvent volume in this combination produced extracts with high concentration of pigment, which provide benefit in cost saving for production of annatto powder and distribution of oil-soluble annatto. Therefore, it is suggested that this combined extraction is a potential replacement for single extractions by sodium hydroxide solution and soybean oil. 3 I. INTRODUCTION Annatto is a natural colorant, which imparts a range of color from yellow to red, due to content of color compounds in the solution. This pigment is obtained from the seed coat of the tropical shrub Bixa orellana L. This tree is native to tropical South America, where it has been a traditional part of some foods for centuries. But it is also cultivated in many countries of Central America (Mexico, Guatemala, Caribbean), Africa (Kenya, Tanzania, Côte d’Ivoire, Angola), and South Asia (India, Sri Lanka, Philippines, Vietnam), especially in coffee-growing areas. The Bixa orellana L. plant grows from two to five meters tall when mature. The fruits of the tree Bixa orellana L. consist of a pod covered with fleshy spines, varying in size and shape. The inside of the pod is usually divided into two valves containing between ten to fifty small seeds (Figure 1). Fruits become mature after pollination 5–6 months. Production of seed reaches a maximum in 4– 12-year-old plants, which can remain productive for more than 20 years. Seed yield is reported to be as high as 3-5 t/ha. Annatto seeds that are properly harvested and dried to suitable moisture content retain their quality for a long time provided they are stored in a cool, dark and dry place. Color content of the seed varies from1.5 to 4% due to variety, cultivation conditions, and postharvest techniques (Bechtold and Mussak, 2009). The principle export form of annatto is seed. However, several suppliers have carried out partial processing/refining of annatto seeds into concentrates to increase export values. The main market for annatto is the USA, Western Europe and Japan. Besides, there is also considerable inter-trade (in seeds) between the Central and South American suppliers. The quantity of annatto seed harvested in the producing countries is estimated at 14,500 metric tons. Brazil, Peru, Ecuador and Kenya contribute the major proportion of this production (Table 1). It is likely that around 7,500 tons of annatto seed are used annually as a food color worldwide and, assuming an 4 average color content of 2%, this equates to 150 tones of bixin available for extraction. The remaining 7,000 metric tons of annatto seed is consumed locally in Brazil, Peru and Ecuador mainly as a spice/condiment. Annatto seeds and extracts have been used for over 200 years to impart a range of color from yellow to red. Although commercial production occurred in South and Central America from the beginning of 19 th century, the production of commercial extracts in Europe and the United States started until the 1870s, primarily to color butter and cheese. In modern times, annatto color is also used worldwide in other foods like meat and fish products, soft drinks, sugar confectionery, margarine, ice cream, soups, etc. (Table 2). Annatto plays a significant role in the industry of food color. It is considered that annatto extracts rank as the second most economically important natural color in the world and it is the most frequently used natural color in the food industry in the United Kingdom (FAO, 1995; Lauro & Francis, 2000; Cannon, 2003). Variety of codes and synonyms is used for annatto pigment due to each region, organization, and country (Table 3). In the European Union and the United States, annatto is permitted to use in foods, drugs, and cosmetics. 5 Figure 1. Annatto tree, fruits, seeds, and powder. (http://davesgarden.com) [...]... loss of bixin in annatto seeds is corresponding to the results of a study of Balaswamy et al (2006) This showed a slight decrease of bixin content in annatto seeds stored at room temperature in jute bags from 1.69% to 1.43% for 360 days of storage 2 Extraction yield of bixin from annatto seeds by three methods The extraction yield of bixin in three extraction methods is shown in Fig 4 The amount of bixin. .. and bixin content was analyzed by a spectrophotometer (Optizen 2120UV, Mecasys Co.Ltd, Daejeon, South Korea) The content of bixin in annatto seed was calculated using the following formula: 14 = m 100 M(100 − W) C: Content of bixin in annatto seed (%) m: Total weight of extracted bixin (mg) M: Weight of annatto seed sample (mg) W: Moisture content of annatto seeds (%) 4 Extraction of bixin 4.1 Extraction. .. stability of bixin and levels of volatile compounds are produced in the extraction processes to find methods for improving extraction quality of bixin from annatto seed In the present study, we investigated the degradation, extraction yield of bixin, and formation of toluene and m-xylene in annatto extracts during three extractions by soybean oil, acetone, and sodium hydroxide solution Effects of some... Degradation of bixin during extraction processes with vegetable oil, acetone, and sodium hydroxide solution 24 4 Total amount of bixin extracted out from annatto seeds The total amounts of bixin, which was freed out from annatto seeds into the extracts by the three methods, are showed in Fig 6 After calculating the bixin yield and its loss, it was observed that the factual percentage of bixin transferred from. .. material was removed from annatto seed in the extraction by hot vegetable oil Although obtained bixin of the extraction by sodium hydroxide solution was only 23.6% after 20 min, it grew steadily during the process and reached to 45.4% at the end of the extraction 3 Degradation of bixin during three extraction processes Fig 5 shows the loss of bixin during the extraction processes by soybean oil, acetone,... soybean oil Extraction by acetone Acetone 40 NaOH Extraction by NaOH 30 20 10 0 20 40 60 80 100 120 Extraction time (min) Figure 4 Bixin yield during extraction processes by soybean oil, acetone, and sodium hydroxide solution 23 70 Bixin degradation (%) 60 50 40 deg Extraction by soybean oil degraded bixin acetone Extraction by 30 Extraction degraded by NaOH 20 10 0 20 40 60 80 100 120 Extraction time... to bixin extraction yield and content of volatile compounds were also investigated A modification of extraction by acetone and two combinations of sodium hydroxide solution and soybean oil were carried out to improve extraction yield and extraction quality of bixin 13 II MATERIALS AND METHODS 1 Materials Annatto seeds were purchased from local market in Vietnam Soybean oil (Ottogi) was purchased from. .. transferred from seed coat of annatto into acetone extracts and soybean oil extracts really high Because of the very high bixin yield obtained from the extraction by acetone, the total amount of bixin was free from seed by this method was the highest among the methods throughout extraction time; it was 44% at the period of 20 min and rose to 92.7% after 120 min This high extracted amount was the result of the... solubility of bixin in acetone and the continuously washed process of annatto seeds by pure solvent in the Soxhlet extractor The total extracted amount of bixin by soybean oil was also significantly higher than that of sodium hydroxide extraction (p . Content of bixin in annatto seed (%) m: Total weight of extracted bixin (mg) M: Weight of annatto seed sample (mg) W: Moisture content of annatto seeds (%) 4. Extraction of bixin 4.1. Extraction. Moisture content and total bixin content of annatto seeds…………………… 2. Extraction yield of bixin from annatto seeds by three methods …………… 3. Degradation of bixin during three extraction processes………………………. extracted from annatto seed at stages of the extraction combinations between sodium hydroxide solution and soybean oil……………… 47 1 Quality Improvement of Bixin from Annatto Seed by Modified