Thạc Sĩ Quality Improvement of Bixin from Annatto Seed by Modified Extraction Methods

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

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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








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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




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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

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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.

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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 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.