composition of Aloe vera leaf and gel, Aloe vera gel extraction, purification and ... information is available on the extraction of gel from Aloe vera leaves. However ...
CONTENTS Chapter
Title
Page
No.
No.
Contents
1
Acknowledgement
2
List of Abbreviations
3
List of Nomenclature
5
I
Introduction
7
II
Review of Literature
11
III
Materials and Methods
37
IV
Results and Discussion
50
V
Summary and Conclusion
95
References
101
Appendices
105
1
ACKNOWLEDGEMENT The author wishes to express profound gratitude and esteemed reverence to major guide Dr. A. K. Varshney, Professor & Head, Department of Agricultural Process and Food Engineering, College of Agricultural Engineering and Technology, Junagadh Agricultural University, Junagadh, minor guide Dr. A. H. Raval and advisory committee members Prof. D. M. Vyas, Dr. V. K. Poshia and Prof. V. M. Bhatt
for providing
guidance
and timely suggestions during the course of
investigation. The author also grateful to Dr. V. P. Chovatiya, Professor and Head and Mr. K.H. Dabhi Department of Botany, JAU, Junagadh for procurement of Aloe vera raw material and utilization of Cooling type Centrifuge.
The author is sincerely grateful to Prof. J. B. Savani, Principal and Dean, College of Agricultural Engineering and Technology, Junagadh Agricultural University, Junagadh for providing necessary facilities during course of studies
I record my deep sense of gratitude to Dr. N. C. Patel, Hon’ble Vice Chancellor, Junagadh Agricultural University, Junagadh for providing me an opportunity to conduct research project. I am also grateful to Dr. C. J. Dangaria, Director of Research & Dean, PG Studies, Junagadh Agricultural University, Junagadh and Dr. R. L. Shiyani, Registrar, Junagadh Agricultural University, Junagadh for their keen interest in the academic values.
The author is extremely grateful to his beloved parents and brothers for their everlasting love, during the study. Last but not the least; the author extends deep compliments to his wife Usha, and son Prashant and Harshit for relieving from home responsibilities and allowing him to complete the study successfully. Date : August 15, 2012
(V. K. Chandegara)
Juangadh 2
ABBREVIATIONS Anon.
Anonymous
Cal.
Calculated
CEG
Cold- extracted gel
Co.
Company
Com.
Commercial
FAC
Fat adsorption capacity
HEG
Hot extracted gel
HTST
High temperature short time
http
Hyper text transfer protocol
IASC
International Aloe Science Council
Inc.
Incorporation
ISI
Indian Standard Institution
J.
Journal
M/s
Messer’s
MPS
Methanol Precipitable Solids
POD
Peroxidase
PPO
Polyphenol oxidase
RCF
Relative centrifugal force
SW
Swelling
Syn.
Synonymous
TTS
Time, temperature and sanitation
TSS
Total soluble solid
Vol.
Volume
UK
United Kingdom
US
United States
WRC
Water retention capacity
WHO
World Health Organization
www
World wide web 3
NOMENCLATURE ANOVA
Analysis of Variance
Av.
Average
Abs
absolute
CaCl2
Calcium Chloride
cc
cubic centimetre
cm
Centimeter
CO2
Carbon Dioxide
C.D.
Critical Difference
C.R.D.
Completely Randomised Design
C.V.
Coefficient of Variance
ed.
Edition
d.f.
Degree of freedom
dl
Deci litre
et al.
And others
etc.
Etcetera
F
Centrifugal force
f
Function
Fig.
Figure
g
Gram
gm
Gram mole
gr
Gram
h
Hour
HCL
Hydrochloric Acid
ha
Hectare
hp
Horse power
H2SO4
Sulphuric acid
i.e.
That is
kcal
Kilo calorie 4
kg
Kilogram
kw
Kilowatt
Kwh
Kilowatt per hour
Kcal
Kilocalories
kg
Kilogram
L
Length
m
Meter
meq
Mili equivalent
mOsm
Mili osmo
min
Minute
mm
Millimetre
M.S.S.
Mean sum of squares
mm3
Cubic milimeter
mg
Milligram
min
Minute
mg/L
Mili gram per litre
ng
Nena gram
nm
Nano meter
No.
Number
Para
Paragraph
pg
Pico gram
pH
Negative logarithm (base 10) of the hydrogen ions (H+)
ppm
Parts per million
pp
Pages
r
Radius of rotation
rpm
Revolution per minute
Rs.
Rupees
Sd
Standard deviation
S.Em.
Standard error of mean
Sig.
Significant 5
S.S.
Sum of squares
Sr.
Serial
T
Thickness
t
Time
V
Volume of the leaf
vs.
Versis
viz.,
Videlicet (that is to say)
wb
Wet basis
W
Width
µg
Micro gram
µm
Micro meter
µmole/g
Micro mole per gram
LIST OF SYMBOLS
%
Per cent
@
At the rate of
0
A
Degree Angstrum
0
C
Degree Celsius
µ
Kinematic Viscosity
ω
Angular velocity
π
Pie
β
Beta
6
CHAPTER I INTRODUCTION Aloe vera is a succulent, belongs to the Liliaceae family. It is one of the 250 known species of aloes, referred to by the scientific terms of Aloe vera and Aloe barbadensis. The semi-tropical plant, Aloe vera barbadensis Miller from the Lily (Liliaceae) family has a long and illustrious history dating from biblical times. It has been mentioned throughout recorded history and given a high ranking as an allpurpose herbal plant. Aloe's thick, tapered, spiny leaves grow from a short stalk near ground level.
Aloe Juice is colourless, transparent water like juice obtained from fresh aloe leaves. It is tasteless and odourless. Aloe vera Latex commonly known as aloin having a bitter taste and purgative quality. Because of its bitter taste, it is also known as bitter aloes. Aloin is destructive to healthy tissue and cells. It is obtained from specialized cells known as pericyclictubules that occur just beneath the epidermis or rind of the same leaves from which the juice is derived. Aloe can be used in raw form or in processed forms; it can be used both externally and internally.
Dried Juice is commercially known as aloe or musavvar. This is the solidified juice coming spontaneously when the leaf is cut out of the cells in pericycle and adjacent leaf parenchyma. This juice is then dried with or without heat to give a strongly bitter substance having a characteristic disagreeable odour. This is known as aloe in the market. Three types of aloe are available in market depending upon the source plant viz. (i) Curacao aloe: a dark brown coloured substance sourced from Aloe vera (ii) Cape aloe: greenish brown coloured aloe sourced from Aloe ferox and (iii) Socotrine aloe: reddish black coloured aloe sourced from Aloe peyrii baker. The original commercial use of the Aloe plant was in the production of a latex substance called Aloin, a yellow sap used for many years as a laxative ingredient. 7
Aloe vera gel is the commercial name given to the fiber free mucilaginous exudate extracted from the hydroparenchyma of the succulent leaves of Aloe vera (Aloe barbadensis Miller). Aloe vera Gel a clear colorless, jelly-like material is derived from tissue that comprises the inner portion of the leaves. It is slightly bitter and odourless. The gel loses its transparency if extracted after 3 hours of plucking the leaves. This Aloe vera Gel, beginning in the 50's, has gained respect as a commodity used as a base for nutritional drinks, as a moisturizer, and a healing agent in cosmetics. The exudate of Aloe vera is used for numerous medical and cosmetic applications since ancient times (Morton, 1961). Commercially available aloe gel is stabilized for maintenance of its potency.
The Aloe gel is used as a moisturizer for skin care, hair care products and a healing agent in cosmetics. Medicinally it is used as antiseptic agents, natural antibiotic agent, calming agent, detoxifier and dilator to increases circulation and blood flow to the skin. It is also used as insect repellent, and a transparent pigment used in miniature painting. The cords and nets are made from the leaf fiber.
Gels, concentrates and powders are suitable for cosmetic, hair care, personal care, pharmaceutical, beverage, food, functional food, nutraceutical and dietary supplement formulations. Products are available in regular and decolorized, cosmetic and food grade preservative systems. The International Aloe Science Council has solidified its dedication to providing the world with the highest quality Aloe. The IASC has a dedicated group of professionals committed to the further growth, research and marketing of quality Aloe vera Gel and Aloe products made from this Gel. Hand filleting and whole leaf processing, the two types of Aloe vera gel extraction methods are prevalent. Gel is extracted either cold process or hot process. Combination of hand filleting and the entire whole leaf processing are used to avoid the undesirable elements, while maximizing the desired constituents. Among the 8
desirable constituents are the polysaccharides (glucomannans), glycoproteins and associated growth factors.
However, over 95 % of the Aloes on the market today still use only the inner gel and stabilize the Aloe in a high-heat process that degrades some of the enzymes, polysaccharides and mucopolysaccharides. High heat (pasteurization and/or autoclave methods) breaks down the constituents in Aloe that are the most valuable for healing. Heat also kills the live enzymes necessary for digestion. Most Aloes are heat processed (Grindlay et al., 1986).
Commercial exploitation of Aloe vera gel has been carried out for at least 50 years. Various companies in the US act as primary growers and processors of the plant and manufacture bulk supplies of the gel for domestic and export market. Many other companies are secondary processors of Aloe vera products, and cosmetics firms and chain store often buy the gel for incorporation into their own brand name products (Grindlay and Reynolds, 1986).
The cultivation of Aloe vera has acquired great commercial importance for medicinal products and cosmetics processing but information is scarce about processing of this crop. As such there is no scientific information is available in India, regarding processing of Aloe vera, its importance in nutrition, cosmetics and pharmaceutics.
Looking to the importance of the Aloe products such as creams, ointments, juices, and shampoo containing the Aloe gel, it is very much essential to study the post harvest technology of the Aloe vera plant.
The expanding Aloe industry
urgently needs to develop test procedures and a reliable database so that a product claiming to have Aloe could be tested and certified. According to Reynolds and Dweck, (1999), there is no scientific literature available for the processing of leaf gel. What so ever processes either gel extraction or gel stabilization are patented, so it is 9
essential to develop the gel extraction process, which may useful for industrial community. Despite the ideal climatic conditions for the cultivation of Aloe vera, we have not been able to exploit the excellent potential of the miraculous medicinal plant. The reasons are simple: lack of cultivation and processing know-how. Keeping above importance in view a research project was undertaken with the following specific objectives: Objectives: 1. To study the physical and chemical properties of Aloe vera leaves. 2. To develop gel extraction process. 3. To evaluate the gel extraction process
10
CHAPTER II REVIEW OF LITERATURE
This chapter deals with the review of literature on the origin and description of Aloe vera plant, production practices of Aloe vera, physical and chemical composition of Aloe vera leaf and gel, Aloe vera gel extraction, purification and stabilization technology and quality parameters used for Aloe vera gel. Very little information is available on the extraction of gel from Aloe vera leaves. However, a brief review on the available information has been presented here as under.
2.1
Aloe vera
Several species of the genus aloe has been in use under the common name of aloe viz. Aloe vera, Aloe barbadensis, Aloe ferox, Aloe chinensis, Aloe indica, Aloe peyrii, etc. Amongst these Aloe vera Linn syn. Aloe barbadensis Miller is accepted unanimously as the correct botanical source of aloe. In most reference books Aloe barbadensis Miller is regarded as the correct name but as per the WHO monograph Aloe vera (L) Burm f. is accepted as the legitimate name for this species. The genus aloe is placed taxonomically in Liliaceae family. It has also been known as Aloe vulgaris ("common aloe") and Aloe barbadensis.
2.1.1 Origin of Aloe vera
Aloe vera barbadensis a member of the lily (Liliaceae) family is a spiky, succulent, perennial plant and a native to warm dry regions, especially southern Europe, Asia, and Africa. It is indigenous to eastern and southern Africa and has been spread throughout many of the warmer regions of the world. It grows best in full sunshine and does not require much water (Denk, 2000 http://www.bio.gasou.edu/). It is popularly grown as indoors plant and cultivated almost everywhere in the world, 11
both as a houseplant and for its medicinal qualities. It is commercially cultivated in Aruba, Bonaire, Haiti, India, South Africa, the United States of America, and Venezuela. There are about 300 identified species.
2.1.2
Physical description of Aloe vera plant
Aloe vera barbadensis can grow up to 1000 mm height, although most specimens are 300 to 600 mm in height (Gilman, 1999 http://www.hort.ifas.ufl.edu). It
has
thick
leaves
that
grow
in
a
rosette
shape
(Denk,
2000
http://www.bio.gasou.edu/). The parenchyma cells of the leaves contain large quantities of pulp. It is an evergreen perennial succulent plant that looks like a cactus grown throughout the world. The fleshy leaves with serrated edges that arise from a central base and grow to nearly 300 – 500 mm long and 100 mm width at the base. Usually each plant is having 12-16 leaves, when mature and weigh about 1500 g. The Aloe leaf consists of three layers i.e. (a) the outer thick rind, (b) a viscous, jelly like mucilage layer into which the vascular bundles are attached to the inner surface of the rind, protrude and (c) the fillet proper, which has structural integrity consisting of hexagonal, structures containing the fillet fluid. This is the water storage area for the plant. (www.bonasana.com) The fibrous outer part of the leaf serves a protective function.
The plant has yellow flowers having 250 – 350 mm in length arranged in a slender loose spike; stamens frequently project beyond the perianth tube. A clear gel is colorless mucilaginous gel obtained from the parenchymatous cells in the fresh leaves of Aloe vera (Bruneton, 1995 and Grindlay et al., 1986). Gel is a part of the plant used for topical application. Anthraquinones, which exert a marked laxative effect, are contained in the bitter yellow sap of the middle leaf layer. Agarwala, (1997) studied the pharmaceutical properties of aloes and suggested that a clear distinction between substances in the colorless, tasteless parenchyma cells, is called 12
aloe gel and substances in the bitter exudate from cells associated with vascular bundles in the outer green rind of the leaf is known as the aloin.
2.1.3 Production practices of Aloe vera
Aloe vera requires tropical, sub-tropical climatic conditions with a very sunny position and a well-drained loamy soil. Plants are tolerant of poor soils (Farooqi and Sreeramu, 2001). Though Aloe vera can be cultivated on any soil for 'dry land management', sandy loamy soil is the best suited for it. It does not grow well at temperatures below 32 degree Fahrenheit or zero degree Celsius ( 0 0C).
Aloe vera is generally propagated by root suckers and planting them to the main field or it can also be propagated through rhizome cuttings. Aloe is a perennial and takes 4-5 years to mature. Plants can live and reproduce for up to 25 years (Denk, 2000). The plants can be harvested every 6 to 8 weeks by removing 3 to 4 leaves per plant. Aloe vera leaves are normally sensitive to subfreezing temperatures The weather conditions are highly affect the Aloe vera processing schedule. Pulling back on the green leaf and cutting at the white base carry out the harvesting of the Aloe vera plant. (http://www. aloecorp.com). Sachedina and Bodeker, (1999) reported the harvesting of Aloe vera leaves by hand pulling.
The potential for commercial cultivation of Aloe vera has also been considered in Tanzania by Sachedina, (1998), who recommends the encouragement of village medicinal plant gardens; followed by the establishment of Aloe growing co-operatives supplying a central processing plant for local production; and eventually a plantation, nursery and processing plant for export.
13
2.2
Handling of Aloe vera leaves
Biological activity loss is due to the microbial decay of the gel. The first exposure of the inner gel to microbes is when the leaves are harvested from the plant. Leaves in which the base is not intact and sealed will greatly increase the microbial counts in the finished product. Higher level of microbial counts significantly reduces the biological activity in the product. The other major source of microbial contamination comes from the rind of the leaf. To prevent contamination of the gel, the leaves are handled very carefully and soaked in a food grade sanitizer, which effectively reduces the microbial counts in the leaf exterior to acceptable levels. (http://www. aloecorp.com)
2.3
Physico-chemical properties of Aloe vera leaves
Wang and Strong, (1993) have studied the physical properties of Aloe vera leaves and reported that, the average weight of the individual leaves was ranging from 387 to 704 g, length 48 to 60 cm, width 8.9 to 11.5 cm and optical density 1.020 to 1.437 (abs) (Table 2.1).
Table 2.1 Characteristics of fresh Aloe vera leaves from producers averaged over a 6-month period (Wang and Strong, 1993) Grower
Weight
Leaf length
Width
Optical density
(g)
(cm)
(cm)
(abs)
1
478
53
9.7
1.095
2
611
56
11.4
1.356
3
704
60
11.5
1.020
4
387
48
8.9
1.437
14
Shih-Jen Chiou, (2003, http://ethesys.lib.pu.edu.tw/ETD-db/ETD-search/) studied the physicochemical properties of Aloe vera including vitamins, viscosity, molecular weight distribution, and monosaccharide composition of Aloe vera. The contents of vitamin C and B1 were significantly decreased after blanching treatment but those of niacin and vitamin B2 differed significantly. Heating treatment, resulting in the decomposition of Aloe vera polysaccharide, decreased the average molecule weight of Aloe vera. The viscosity of Aloe vera also declined with the increase of heating time. Changing the pH value and adding thio-compounds could efficiently inhibit the degree of nonenzymatic browning of blanched Aloe vera during storage.
2.4
Chemical composition of Aloe vera leaves
Joshi, (1998) had given the chemical constituents of Aloe vera barbadensis. The aloe plant contains 99 and 99.5 per cent water, with an average pH of 4.5. The remaining solid material contains over 75 different ingredients including vitamins, minerals, enzymes, sugars, anthraquinones or phenolic compounds, lignin, saponins, sterols, amino acids and salicylic acid. Table 2.2 present the analytical profile of Aloe vera leaves.
The main constituents of the Aloe vera leaves are: (i) Aloin: It is an irritant laxative contained in the yellow sap of Aloe, which is a constituent of the Anthraquinone complex, (ii) Methanol-Precipitable Solids (MPS): When alcohols are added to Aloe solutions about 20-25 % of the total solids come out of solution or 'precipitate'. It consists mainly Polysaccharides, Glycoprotein and salts of Organic Acids. The Polysaccharides represent about one-half to two-thirds of the MPS or about 10-15% of the total solids. (iii) Polysaccharides: There are over 200 constituents in Aloe vera, the single most important constituent being the polysaccharides. Polysaccharides consist of simple sugar molecules and are called hexoses. 15
Table 2.2 Analytical profiles of Aloe vera leaves Tests
Units
Minimum
Maximum
Aeverage
Solids
%
0.75
1.50
0.92
Water
%
98.5
99.25
99.1
Glucose
mg/dl
28.0
103.0
77.8
Purine
mg/dl
0.1
5.6
0.8
Ueea-Nitrogen
mg/dl
1.0
1.0
1.0
Creatinine
mg/dl
0.1
1.5
0.4
Sodium
meq/l
4.0
13.0
8.7
Potassium
meq/l
10.0
22.5
13.4
Chloride
meq/l
1.0
11.0
3.0
CO2
meq/l
1.0
7.0
1.7
Calcium
mg/dl
19.4
48.5
30.0
Cal. Calcium
mg/dl
23.3
52.3
33.8
Magnesium
mg/dl
3.2
4.7
3.9
Zinc
mg/dl
14.0
77.0
31.0
Phosphorus
mg/dl
0.6
1.3
1.0
Total Protein
gm/dl
0.1
0.4
0.2
Albumin
gm/dl
0.1
0.5
0.14
Globulin
gm/dl
0.0
2.0
0.06
Phosphatase
mg/dl
1.0
50.0
18.0
Sgotransaminase
mg/dl
6.0
49.0
21.0
Sgotransaminase
mg/dl
8.0
85.0
24.0
Dehydrogenase
mg/dl
0.0
9.0
3.0
Amylase
mg/dl
0.0
2.0
1.0
units/dl
0.0
1.6
0.5
Cholestrol
mg/dl
4.0
12.0
8.0
Triglycerides
mg/dl
1.0
12.0
2.4
Iron
mg/dl
3.0
30.0
15.0
B12
pg/ml
141.0
403.0
265.0
Folic Acid
ng/ml
2.7
20.0
13.2
Osmolarity
m0sm/kg
43.0
67.0
60.0
0.51
1.1
0.67
Alkaline
Lactic
Lipase
HPLC Ratio Source: http://www.drwolfe.com
16
2.5
Physico-chemical properties of Aloe vera Gel
Following companies had reported the physico-chemical properties of Aloe vera gel and shown in Table 2.3. This meets or exceeds the standards established by the International Aloe Science Council's (IASC) certification program for the determination of purity.
Table 2.3
Physico-chemical properties of Aloe vera gel
Test
http://www.aloecorp.com
Appearance
M/s Delta International (http://www.shantidatta.com)
Clear Yellow / Green Liquid
*Absorbance @ 400nm
NMT 0.500
Refractive Index
1.3340-1.3355
1.33789 - 1.34390
Specific Gravity
1.0030-1.0070
1.0221 - 1.0339
3.5-4.7
3.5 - 4.7
pH Value Total Solids
NLT 0.46% by weight
*Reported as reconstituted
Table 2.4 Chemical properties of Aloe vera gel (Wang and Strong, 1993) Grow
pH
Aloin
Soluble solids
Sugars
*Fibre
(mg/L)
(%)
(mg/L)
(%)
1
4.47
31
0.708
2555
0.077
2
4.49
39
0.610
1441
0.088
3
4.51
29
0.675
2530
0.088
4
4.54
41
0.586
1361
0.074
* Percentage of fresh weight
17
2.6
Chemical composition of Aloe vera gel
The Chemical composition of Aloe vera gel as reported by Wang and Strong, (1993) is presented in Table 2.4. Waller et al., (1978) had worked on sugar analysis of Aloe vera gel and given the sugar composition (Table 2.5). The other scientists namely Pierce (1983), Rowe and Park, (1941), Bruneton, (1995) and Devis et al., (1994) had worked time to time and reported following chemical composition of Aloe vera gel which is shown in Table 2.6. Table 2.5 Sugar content of Aloe vera gel (Waller et al., 1978) Sugar
Sugar in whole gel (µmole/g)
Total Sugar in lyophilized residue (%)
Arabinose
4.23a
4.7a
Galactose
3.60
4.3
Glucose
31.3
37.7
Mannose
39.4
47.5
Rhamnose
1.27
1.5
Xylose
4.44
4.4
a
Arabinose could not be distinguished from fructose
18
Table 2.6
Aloe gel composition Mono and polysaccharides (50-60% of solids) (Specific concentrations have not yet been determined)
Ployhexanoses Hexans
Xylose
Arabinose
Galactose
Glucose
Amino acids (ppm) Lysine
5-6
Histadine
2.8-3.3
Arginine
4.5-5.5
Threonine
5-6
Aspartic
13-15
Serine
6-7
Acid Glutamic Acid 13.5-15.5
Proline
8-9
Analine
1.0-1.3
Glycine
7-8
Valine
6.5-7.0
Methionine
1.5-2.0
Isoleucine
3.5-4.0
Leucine
8.5-9.0
Tyrosine
2.8-3.3
Pheylalanine
4.3-4.7 Vitamins (mg per 100ml)
B-1
6-7
B-2
6-7
C
47-61
Niacinamide
30-37
B-6
3.0-3.7
Choline
9.5-11.2
Enzymes (per 100 ml) Amylase
1100-1600 units
Lipase
600-800 units
Protein
0.11g / 100 gr
Fat
0.09g / 100 gr
Ash
0.25%
Crude fiber
0.10%
Calories
3.3/100 gr
Source: http://www.garudaint.com
19
2.7
Quality parameters of Aloe vera gel The quality parameters such as fiber content, viscosity, refractive index,
optical density and total soluble solid plays an important role in judging the quality and purity of extracted gel from Aloe vera leaf.
2.7.1 Fiber content The fiber content is directly related to the purity of gel and become the criteria of efficiency of gel filtration unit. More fiber content, suggests poor filtration operation. For getting pure gel fiber should be minimum. The difference between crude gel recovery and pure gel recovery gives the amount of fiber in crude gel. Wang and Strong, (1993) had found the fibre content 0.074 to 0.088 % of fresh weight of pulp.
2.7.2 Viscosity Viscosity of gel is very important factor for deciding quality in terms of activities of biological compounds. The viscosity decreases as the time passes. After some duration viscosity of gel becomes equal to the viscosity of water, the gel becomes water. Gowda et al., (1979) reported that after harvest of Aloe vera leaf, the viscous pseudoplastic nature of Aloe vera gel, mainly due to the presence of polysaccharides composed of a mixture of acetylated glucomannans was lost shortly after extraction, apparently due to enzymatic degradation. This shows there are some biological activities, which related to the viscoelastic behavior of gel. 2.7.3 Refractive index Refractive index is the physical property of gel determines the purity of gel as compared to double distilled water. Gel with lowest refractive index, is the best treatment for extraction process. More refractive index indicates the impurities in the extracted gel. 20
2.7.4 Optical density Optical density is the physical property of gel determines the purity of gel as compared to double distilled water. Gel with lowest optical density, is the best treatment for extraction process. More optical density indicates the impurities in the extracted gel. Wang and Strong, (1993) have reported optical density of 1.020 to 1.437 (abs) for Aloe vera leaves.
2.8
Method of processing Aloe leaves
2.8.1 Traditional hand filleted Aloe vera
In order to avoid contaminating the internal fillet with the yellow sap, the traditional hand- filleting method of processing Aloe leaves was developed. In this method, the lower portion i.e. 25 mm of the leaf base (the white part attached to the large rosette stem of the plant), the tapering point (50 –100 mm) of the leaf top, and the short, sharp spines located along the leaf margins are removed by a sharp knife. The mucilage layer below the green rind avoiding the vascular bundles, and the top rind is also removed with the help of knife. The bottom rind is similarly removed.
The materials of the mucilage layer, subsequent to their synthesis, are distributed to the storage cells (cellulose-reinforced hexagons) of the fillet, a process that is accompanied by dilution owing to the water (the major fillet constituent), which is stored in the fillet cells. The fillet consists of more than 99% water. The hand filleting method is very labour intensive. Owing to this fact, machines have been designed and employed which attempt to simulate the hand filleted techniques, but generally the product contains higher amounts of the anthraquinones laxatives than the traditional hand filleted approach (www.bonasana.com).
21
2.8.2
Whole leaf Aloe process
In this process, the base and tip are removed as previously delineated, and then the leaf is cut into sections and ground into particulate slurry. The material is then treated with chemicals which break down the hexagonal structure of the fillet releasing the constituents, by means of a series of coarse and screening filters, or passage through a juice press, the rind particles are removed, the expressed juice is then passed through various filtering columns which remove the undesirable laxative agents. This process, performed properly, can produce a constituent-rich juice (generally containing 3 times or more constituents than hand filleted juice), which should be virtually free of the laxative anthraquinones; this process was developed in the 1980’s (www.bonasana.com).
The present method of processing of Aloe vera is using the whole leaf, from which the undesirable elements can be selectively removed, while maximizing the desired constituents. The desirable constituents are polysaccharides (glucomannans), glycoproteins that are associated with growth factors. Table 2.7 shows the data of hand and whole leaf filleting which reveals that the quantity of desirable polysaccharides is 2.5 to 3 times higher as compare to hand filleting methods. Table 2.7 Yields and Aloe leaf processing Process Fraction
Total solids (Without preservatives or
Hand Filleting
Whole Leaf
(%)
(%)
0.45 – 0.65
1.30 – 3.50
0.12
0.16
additives) Polysaccharides Source: http://www.wholeleaf.com
22
Table 2.8 data compares various processing methods and the effect on yield (total solids), aloin concentration, and the distribution of sizes of constituents. The whole leaf method can produce an Aloe juice which is high in total solids, high in retained high dalton (molecular weight) polysaccharides with their scientifically demonstrated benefits, while the aloin concentration is at a very acceptable low level.
Table 2.8 Methods of leaf preparation and constituents Method of preparation
pH
Aloin (ppm)
H2 O (%)
Total solids (%)
(1)
(2)
(3)
(4)
(5)
Hand -filleting
4.27
6
99.25
0.48
Roller
4.30
32
99.61
0.39
Leaf Splitter
4.24
18
99.61
0.42
Whole Leaf
4.09
1
99.62
1.38
Source: http://www.wholeleaf.com
2.8.3 Total process Aloe The total process of Aloe vera is combination of hand filleting and whole leaf processing. In this process, aloe leaves are hand filleted by the traditional method. Then the green rinds and the mucilage pulp are processed separately. A combination of the products obtained by these two procedures, produces a product called Total Process Aloe. Total Process Aloe contains considerably higher concentrations of total solids, calcium, magnesium, and malic acid, which are virtually free from undesirable laxative anthraquinones. The International Aloe Science Council for certification recommends using the total process Aloe, which retains major portion of desirable constituents (www.bonasana.com).
23
2.8.4 ALOECORP process The M/s ALOECORP, an American company has suggested their own method for the processing of Aloe vera leaf. In this method, three of the outermost mature leaves are cut from each plant. Leaves are gathered in boxes, which are transported immediately to the production facility. The harvested leaves are primarily washed with hand and then conveyed
through a stainless steel conveyer to the mechanical
leaf washer. The leaves are cut and finally pass through the gel expulsion machines. The production room is kept in an ultra-sanitary state, even when not in use. Employees are required to go through a process of sanitation every time they enter the room. The entire area is thoroughly cleaned after each production run. Once the gel is expelled from the leaves, it is pumped through a de-pulping machine. The pure Aloe gel is then moved through a chilling system designed to bring the temperature of the gel below to 37 degrees Fahrenheit or 2.5 - 3 degrees Celsius. The chilled gel is stored in an insulated tank, ready to be pumped into a transport tanker for delivery to the Harlingen processing facility (Aloecorp De Mexico), (http://www. aloecorp.com). 2.8.5 American Quality Aloe, Aloe vera processing The M/s American Quality Aloe Company suggested their own methods for processing of Aloe vera leaves. The method consists of harvesting of matured Aloe vera leaves and processing within an hour to avoid biological contamination and degradation of the leaves. The mature leaves are scrubbed and rinsed several times in a special solution. The leaves are ground up and filtered to remove the large pieces and carbared filtered to remove the chlorophyll. Aloe Liquid is pumped through state of the art "high temperature/short term" heat exchange equipment for a rapid pasteurization and quick cooling to prevent degradation from a slow heat and cool down (batch method) stabilization procedure (http://www.aqaloe.com). 2.8.6
Steam distillation Waller et al., (1978) had reported the steam distillation of Aloe vera leaves. In
this process, the tough outer portion consisting of the cuticle, epidermis, and mesophyll was removed from the leaves of mature plants. The green outer portion 24
and the colorless inner part as well as the "stalk" i.e. portion of the plant above the ground remaining after the leaves had been removed and the roots were steam distilled, each separately. The distillation was allowed to proceed as long as the condensate had a definite odor; this process takes approximately 5 h. The condensate from the steam distillation was saturated with sodium chloride, extracted with diethyl ether, dried with anhydrous sodium sulfate and the ether was reduced in volume using a stream of nitrogen. 2.9 Commercial Processing One
of
the
earliest
gel
producers
was
Carrington
Laboratories,
(http://www.carringtonlabs.com) has a range of products and patented the preparation in the name of Acemannan or CarrisynTM. The well established (1973) firm is Terry Laboratories (http://www. terrylabs. com.) and is the major supplier of gel to many multinational companies. They are also the supporters of aloe research and quality control.
Aloe
vera
(http://www.aloevera.co.uk/)
Company many
U.K.
(Forever
companies
like
Living CRH
Products) International
(http;//www.aloealoe.com.) and Valley Aloe vera Inc. (http://www.quikpage.com), Bonnsana Co. (www.bonasana.com) and Aloecorp USA (http://www. aloecorp.com) have developed process for gel extraction and stabilization of gel and concentrated gel either by air-drying or freeze-drying. Basically, the ‘aloe dessert’s processing process is simple which involved sorting, grading, washing, peeling, cutting, cooking in syrup, adding flavor, packing, and pasteurizing. The most difficult part of processing is the removal of aloe and retains its original taste and its marketability. Aloe is more popular as a material for cosmetics produce purposes not as an edible produce. The process flow diagram of Aloe vera dessert as suggested by Herlina, (2001) and later developed and modified by PT. Niramas Utama Indonesia is shown in Figure 2.1. commercialized processing of Aloe vera is shown in Figure 2.2.
25
The flow chart of
Aloe vera leaves Grading, trimming and washing
Peeling and separating Aloe vera meat flesh Cutting and grading
Cooking Water Sugar Malic acid
Mixing
Citric acid Flavour agent
Packaging
Pasteurization
‘Aloe Vera’ Dessert
Figure 2.1 Processing process of ‘Aloe vera’ Dessert developed and modified by PT. Niramas Utama Indonesia
26
Fresh Aloe vera leaves
Washing Trimming
Tips and butts
Soaking & Rinsing
Whole leaf processing
Bulk Aloe pulp
Aloe oil
Cellulous pulp
Hand filleting
Filleting leaf
Aloe pulp Processing
Gel extraction
Separation
Decolorization with charcoal
Liquefaction
Concentration of Aloe juice to 32% Solids Reconstitution of 32 % concentrate into lower %
Figure 2.2
Commercial Aloe vera processing flow chart (Aloe Vera -CRH International, Inc http://www.aloealoe.com)
27
2.10
Processing of Aloe vera gel During the past several decades several basic methods of processing Aloe vera
gel have been developed.
2.10.1 Gel extraction from Aloe vera pulp
Shafi et al., (2000) developed a commercially viable process for preparing a stable and pharmacological active crystalline substance from the fresh whole leaf meal and tested the product on experimental animals and volunteers for wound healing remedy for all kinds of all damaged skin conditions. Anon., (1967) had carried out the gel extraction from Aloe vera leaves, remaining after the removal of its exudates were cut upon and its mucilage was scraped out with blunt edged knife. This mucilage was stirred vigorously in a blender to make it uniform. This solution was strained through a muslin cloth and filtered. This uniform solution was extracted for cold- extracted gel (CEG) and hot extracted gel (HEG).
Cold extracted gel (CEG) This solution was acidified with Hydro chloric acid (HCL) having pH 3.50 and the crude gel were precipitated out from the extract by adding slowly 95 % alcohol while stirring. The gel was obtained by centrifugation.
Hot extracted gel (HEG) Material left after passing the blended solution through muslin cloth, was repeatedly treated with hot water until the complete extractions of gel was affected. The crude gel (HEG) was prepared as described as above.
28
Waller et al., (1978) had reported the gel extraction process. As Aloe leaves rapidly loose their medicinal properties, the material used was either fresh or lyophilized and stored at –15
0
C. A. barbadensis leaves (35 g equivalent of dry
material) were macerated and extracted with water-acetone (1:1) and then acetone at room temperature. The combined extracts (2:1) were concentrated in a rotary evaporator (35
0
C) and the acetone free residue was extracted three times with
diethyl ether (250 m/each time).
Yaron, (1993) have extracted gel from full sized mature leaves and half size young leaves picked from the same shrub. After removal of the ‘peel’ the colourless hydroparenchyma was ground in a blender and centrifuged at 10,000 x g for 30 min at 4 0C to remove the fibers. Leaves weighing 800 g produced about 300 ml of gel. Microbial growth was inhibited by addition of a preservative, 0.05 % sodium azide or 0.1 % Girgard (Givaudan).
2.10.2 Purification of Aloe vera gel
Anon., (1967) had reported the purification of crude gel. The crude gel obtained by above method, was washed several times with ethanol till free from chlorideions. It is also stirred with absolute acetone, ether and dried over anhydrous CaCl2 in vacuum desiccators. The dried gel was also greenish tinge of chlorophyll. The gel was also de-proteinated by shaking the aqueous solution with chloroform. Dry aloe gel was white amorphous powder when dried under reduced pressure in vacuum desiccators.
2.10.3 Stabilization of Aloe vera gel
Many of the greatest benefits of Aloe vera can be lost in the processing unless great care is taken to stabilize the gel. M/s Forever Living Products has developed a stabilization method (http://www.aloevera.co.uk). 29
Yaron, (1992) had studied the fresh gel stabilization. Aloe vera gel, like most natural juices, both fruit and vegetable, is an unstable product when extracted and is subject to discoloration and spoilage from contamination by microorganisms. Sulphated polysaccharides of the red microalga Porphyridium aerugineum were obtained from the algal laboratories. An aqueous solution containing 2 % algal polysaccharide solution and 50 % Aloe vera gel was prepared and its shear stress vs. shear rate curve was generated. The solution was then stored at room temperature for 6 months for observations of the structure and homogeneity of the polysaccharides. Addition of the algal sulphated polysaccharide resulted in a homogeneous stable product: the algal polysaccharide may inhibit degradation and also browning of the aloe polysaccharide.
2.11
Quality parameters for processing of Aloe vera
Time, temperature and sanitation (TTS) are necessary to preserve these biological activities. The TTS Aloe Process not only preserves the natural biological activities of Aloe vera but also enhances the physical stability of the finished products. The TTS Aloe Process was developed with the idea of preserving the natural state of the Aloe plant throughout each stage of production by maintaining beneficial properties inherent in the plant. Data generated from these studies showed that minor compositional changes, while not significant in the chemical makeup of the product, resulted in great degrees of biological activity losses. These losses in activity were not limited to one area, but different activities were affected at different stages of production (http://www. aloecorp.com).
2.11.1 Timing of leaf process Leaves show losses of biological activity beginning at six hours following harvest when the leaves are stored at ambient temperature. Most biological activities are completely lost after 24 hours at ambient temperatures. A decrease in activity is 30
also evident when the leaves are stored refrigerated even though the rate of activity loss is greatly reduced. The losses of activity appear to be the result of enzymatic activity after the leaf is removed from the plant. In fact, it has been shown that the gel, once extracted from the leaf, has greater stability than gel, which is left in the leaf. This means that shipping of leaves, even at refrigerated temperatures, will result in loss of biological activity. The overall timing of TTS production phases is extremely critical. The processing must be completed within 36 hours of harvesting the leaves. (http://www. aloecorp.com)
2.11.2 Process Temperature
The Aloe gel processing temperature plays very important role for gel quality for cosmetic and medicinal use.
A.
Flash Cooling As a crucial step to preserve biological activity, the gel is cooled to below 5 0C
in ten to fifteen seconds following the gel extraction. Rapid cooling not only slows enzymatic and microbial deterioration of the gel, but also aids in reducing the microbial counts in the product.
B.
Pasteurization Biological activity remains essentially intact when the gel is heated at 65 0C
for periods of less than fifteen minutes. Extended periods or higher temperatures will result in greatly reduced activity levels. The best method of pasteurization is HTST (high temperature short time), which exposes the gel to elevated temperatures for periods of one to three minutes. Once heated, the gel is flashing cooled to 5 0C or below. Ashleye, (1983) had revealed that heat during pasteurization was one of the stresses imposed on the gel and there were advantages in using high temperatures for short times preferably with addition of an antioxidant such as ascorbic acid. 31
C.
Concentration The gels obtained using the pasteurization and flash-cooling methods can be
concentrated under vacuum without the loss of biological activity. The concentration operation must be conducted under 125 mm mercury vacuum at temperatures below 50 0C and must not exceed two minutes. Higher vacuums and temperatures will cause activity loss as will extend concentration times.
D.
Freeze or Spray Drying The concentrated product can then be freeze dried at temperatures between -
45 0C and 30 0C or can be spray dried with product temperatures below 60 0C without losses in biological activity.
E.
Dehydration Simal et al., (2000) had studied the effect of air-drying temperature (from 30
to 80 0C) on dehydration curves and functional properties (water retention capacity, WRC; swelling, SW; fat adsorption capacity, FAC) of Aloe vera cubes. The effective diffusivities estimated with the proposed model varied with the air-drying temperature according to the Arrhenius law except for the experiment carried out at 80 0C, where casehardening took place. The three studied functional properties exhibited a maximum when drying temperature was 40 0C decreasing either at higher or lower temperatures.
F.
Blanching Shih-Jen Chiou, (2003) in his investigation reported the nutrient contents of
Aloe vera and effect of additives on nonenzymatic browning of blanched Aloe vera and studied the nutritive compositions of the Aloe vera and the effects of pH and additives, on nonenzymatic browning of blanched Aloe vera during storage. In order to establish an optimal blanching processing of Aloe vera, the activity changes of peroxidase (POD) and polyphenol oxidase (PPO) of Aloe vera were also assayed with different heating treatment. 32
2.12
Aloe vera Product Preparations Choosing effective Aloe vera products can be challenging. Once a leaf is cut,
enzymes start to break down some of the long chain sugars which make Aloe vera gel an effective healing product, so it is important for the plant to have been properly handled and stabilized. Commercial, stabilized gel products may not work as well as the fresh gel, but cold processing is thought to best retain the beneficial properties.
Aloe vera juice is most often the form of the gel that is used internally. The nutritional composition of Aloe vera drink is shown in Table 2.9. A product that is made from the whole leaf does not necessarily contain anthraquinones from the latex layer, as those are water-soluble and can be separated out during processing. Capsules and tinctures of the gel are also available. Oral forms of the latex extract are generally capsules, as it is extremely bitter.
Salve: Remove the thin outer skin and process the leaves in a blender, add 500 units of vitamin C powder to each cup and store in refrigerator.
Dried Juice: Aloe vera juice containing the equivalent of 360 - 900 mg of dried sap is recommended by most herbalists per day.
Aloe tea and fibre tablets: Dry aloe leaves are harvested and crushed to form tea leaves. A delicious herbal tea can be brewed. Tea leaves are pressed to form fibre tablets.
33
Table 2.9 Aloe vera Drink's Nutrition fact Nutrients Unit
Thai RDI* 2 (11)
Composition of Aloe vera 4 (15)
N (c)
.
Ash
Gram
0.5
0.2
Moisture (Water)
Gram
84.4
88.3
Energy (Enerc)
Kilo Calories
61
49
Protein (Procnt)
Gram
50**
0
Fat
Gram
65**
0.6
Total available CHO (Chocdf) Gram
300**
10.9
include FIBTG Dietary Fiber (Fibtg)
Gram
25
0.2
Calcium (Ca)
Milligram
800
31
Phosphorus (P)
Milligram
800
3
Iron (Fe)
Milligram
15
-
Sodium (Na)
Milligram
2400
22
Potassium (K)
Milligram
3500
12
Copper (Cu)
Milligram
2
-
Zinc (Zn)
Milligram
15
0.1
Vitamin A (Retinol)
µgram
.
0
β-Carotene (Cartb)
µgram
.
-
800
-
Total
vitamin
A
(Retinol- µgram
Equivalent, RE)
*
Vitamin B1 (ThiA)
Milligram
1.5
-
Vitamin B2 (Ribf)
Milligram
1.7
-
Niacin (NIA)
Milligram
20**
-
Vitamin C (VitC)
Milligram
60
-
Percentage
of
Thai
Recommended
Daily
Intake
is
based
on
a
2,000
kcal
diet.
** %Energy distribution from protein, total fat and carbohydrate = 10:30:60, Total Saturated fat = 10% of total energy. Source: Thai Food Composition Tables (1999), Institute of Nutrition, Mahidol University (INMU).
34
2.13
Use of Aloe vera gel
Grindlay and Reynolds, (1986) in his review cited that the mucilaginous gel from the parenchymatous cells in the leaf pulp of Aloe vera has been used since early times for a host of curative purposes. This gel should be distinguished clearly from the bitter yellow exudates originating from the bundle sheath cells, which is used for its purgative effects. Aloe vera gel has come to play a prominent role as a contemporary folk remedy. Modern clinical use of the gel began in the 1930s, with reports of successful treatment of X-ray and radium burns, which led to further experimental studies using laboratory animals in the following decades
However, over 95 % of the Aloes on the market today still use only the inner gel and stabilize the Aloe in a high-heat process that degrades some of the enzymes, polysaccharides and mucopolysaccharides. High heat (pasteurization and/or autoclave methods) breaks down the constituents in Aloe vera that are the most valuable for healing. Heat also kills the live enzymes necessary for digestion. Most Aloes are heat processed.
Davis et al., (1989) Aloe vera preparations were evaluated for topical antiinflammatory activity using the croton oil-induced edema assay. Throughout recorded history, it has been used to keep skin beautiful and restore it to health. A frequent moisturizing ingredient in cosmetics and hair care products, it also promotes the healing of burns and superficial wounds, but should not be used on deep or surgical wounds of punctures. Topical application has been successful in treatment of sunburn, frostbite, radiation injuries, some types of dermatitis, psoriasis, cuts, insect stings, poison ivy, ulcerations, abrasions, and other dermatologic problems.
It also exerts antifungal and antibacterial effects, and thus helps to prevent wound infections. One study showed it to have a little more activity than the antiseptic silver sulfadiazine against a number of common bacteria that can infect the skin. It 35
has moisturizing and pain relieving properties for the skin lesions, in addition to healing effects.
Aloe vera gel products may also be used internally. They should not contain the laxative chemicals found in the latex layer. There is some evidence that Aloe vera juice has a beneficial effect on peptic ulcers, perhaps inhibiting the causative bacteria, Helicobacter pylori. It appears to have a soothing effect on the ulcer, and interferes with the release of hydrochloric acid by the stomach. Colitis and other conditions of the intestinal tract may also respond favorably to the internal use of gel products. Aloe vera has been shown to exert a stabilizing effect on blood sugar in studies done on mice, indicating a possible place for it in the treatment of diabetes. One study suggested that giving Aloe vera extract orally to patients with asthma, oral Aloe vera gel include prevention of kidney stones and relief of arthritis pain. 2.14 Organoleptic properties of Aloe vera gel
Aloe vera gel is viscous, colourless, odorless, taste slightly bitter. Gorloff, (1983) had reported that in gel preparation processes, organoleptic properties are important when the gel is intended for internal use. It is evident from the review of various aspects of processing of aloe vera that many stages subjective criteria during processing are followed. Therefore, there is a scope to carry out the scientific investigations on processing of aloe vera and to develop process and machineries for efficient aloe vera processing operations.
36
CHAPTER
III
MATERIALS AND METHODS
This chapter deals with the selection of raw material, procedures followed for determination of physical and chemical properties, experimental set-up, experimental and analytical technique and quality evaluation used for optimisation of process parameters for the extraction of gel from Aloe vera leaves.
3.1
Selection and procurement of Aloe vera leaves
The two years old and matured Aloe vera (Aloe barbadensis Miller) leaves were selected for the experiment. The leaves were obtained from Department of Botany, College of Agriculture, Junagadh Agricultural University, Junagadh. The leaves of Aloe barbadensis variety were cut in the early morning every day for experimentation. To avoid bio-degradation of Aloe vera leaves, each leaf is harvested by hand with knife and pulled carefully from the mother plant so as not to break the rind. The leaves were transported to the working place in a covered polyethylene bag to avoid oxidation or contamination.
3.2
Physical properties and characteristics of Aloe vera leaf
The physical properties of Aloe vera leaf namely size, shape, test weight and pulp weight were studied. The moisture content of the Aloe vera pulps and gel was measured by hot oven drying method. The 10 g of Aloe vera pulp and gel sample was kept in glass petri dish, dried at 105 0C ± 2 0C temperatures for 24 hours and its weight determined for calculating of total solid (Wang and Strong, 1993). The difference between initial weight and final oven dried weight of sample gives the moisture content of the pulp and gel. 37
3.2.1
Size and shape The maximum tri-axial dimension as shown in Figure 3.1 which were obtained
as length, width and thickness of Aloe vera leaves, were measured from 23 randomly selected leaves using metallic tape having a least count of 1 mm. The average of readings was taken as its length, width and thickness. The shape was determined by comparing the longitudinal and lateral cross-section of the Aloe vera leaves.
Figure 3.1 Diagram of the approximate geometry of Aloe vera leaves and definition of parameters for volume calculation. (L = length; W = width; T = Thickness)
3.2.2 Apparent leaf volume
The apparent volume was calculated by considering the geometry of the object similar to the geometrical shape. Knowing the values of length (L), width (W) and thickness (T), the volume of the Aloe vera leaf was calculated. The leaf volume was also calculated by considering the geometry as to be a cone with elliptical rather than circular cross section as shown in Figure 3.1.
38
The volume was calculated by the formula given by Hernandez-Cruz et al., (2002) as below:
V = (L/12) π W T.
-------------------------------(3.1)
Where; V = Volume of the leaf, mm3 L = Length of the leaf, mm W = Width of the leaf, mm T = Thickness of the leaf, mm
3.2.3
Leaf Weight
For determination of leaf weight, 23 nos. of randomly selected leaves were weighed in a precision balance (Sartorious make, 0.01 g least count) and their weights were recorded. The measurements of weights were replicated three times and the average of the readings were recorded in g.
3.2.4
Fibre content
Crude gel is defined as the gel obtained after the centrifuge operation of Aloe vera pulp, while pure gel is the gel obtained after purification of the crude gel. The fibre content is defined as the difference between the dry weight of the crude gel and that of the filtered gel (pure gel). It was measured by filtering the homogenate through a 2.0 µm muslin cloth followed by Whatman No. 4 filter paper under vacuum. Ten grams of the filtrate was placed in a dry glass petridish and dried at 105 0
C ± 2 0C for 24 hours and its dry weight determined and the difference gives the
fibre content (Wang et al. 1993). 39
3.3
Laboratory scale gel extraction set up
The laboratory scale gel extraction experimental set up consists of Blender used for making the fillets into pulp homogenate, test tubes with caps for keeping extracted gel and, Buchner flask and Buchner funnel was attached to the vacuum pump for filtration. The refrigerate box was used for handling the samples at low temperature i.e. 4 to 5 0 C.
3.3.1
Centrifuge
Centrifuge is equipment, used to separate solid particles from liquid media, based on size or shape with the action of centrifugal force. Basically, it consists of a container fixed on the central axis, is rotated with the help of 1 hp electric motor. The cooling type centrifuge (Remi Make C-24 Model) , having 31mm top radius and 37 mm bottom radius was used with maximum centrifugal force of 33500 g, at 20000 rpm, maximum rotor speed. The tube size was 100 ml and the maximum capacity of the centrifuge was 400 ml.
Centrifugation is based on the fact that when an object is moving in a circle at a steady angular velocity, is subject to an outward directed force F. The magnitude of force, depends upon the angular velocity ω, radius of rotation r. Therefore the centrifugal force is given by the following formula: F = ω2 r
......................(3.2)
Where; F = Centrifugal force in ω = Angular velocity, radians r = Radius of rotation centimetres
40
The centrifugal force F is also expressed in terms of earth’s gravitational force and it is then referred as the relative centrifugal force i.e. RCF, and commonly expressed as the number “g”. The RCF is also expressed in form following formula:(Thimmaiah, 1999). RCF = ω2r/980
.....................(3.3)
ω = π (rpm)/30 g = RCF = (1.119 X 10-5 ) ( rpm)2 ( r ) Where; RCF = Relative centrifugal force r = average radius of rotor, cm
3.3.2
Filtration unit
The filtration unit was consisted of vacuum pump, Buchner flask and Buchner funnel. The crude gel, which was obtained after centrifuge operation, was mixed with Charcoal for gel purification. The gel then was filtered in the filtration unit with the help of Whatman No. 4 filter paper for further analysis.
3.3.3 Photospectrometer
Photospectrometer was used for measuring the optical density of extracted gel. The Photospectrometer was calibrated before it was used for the experiment. The procedure consists of keeping filter slot on number 6 and then zero transmittance was set by lower knob. The Photospectrometer lower knob was kept remain in the same position till all experiments were over. Initially the Photospectrometer was calibrated with the help of distilled water by taking the optical density of distilled water as one. The filter slot was then fixed according to wavelength of material i.e. for Aloe gel 400 nm and was set at 100% transmittance for blank sample or distilled water by adjusting the side knobs. 41
3.4
Design of experiments The experiments were planned using Four Factor Completely Randomised
Design. The treatments consisted of 2 levels of Acetone, 3 levels of centrifuge temperature, 3 levels of centrifuge speed and 3 levels of centrifuge duration. The independent and dependent variables considered in this study are given below. Independent variables a. Acetone
: 2 i.e. Without addition of Acetone, and 10 % addition of Acetone 0
b. Centrifuge temperature
: 3 i.e. 5, 10, and 32 (Ambient)
C
c. Centrifuge speed
: 3 i.e. 2000, 5000 and 10,000 rpm
d. Centrifuge duration
: 3 i.e. 10, 20 and 30 min
Dependent variables a. Gel recovery, % b. Viscosity of gel, Stokes c. Refractive index of gel d. Optical density of gel, abs e. TSS content of gel, Brix Experimental details 1. Treatments 2. Replications
: Fifty four : Three
3. Total number of tests: Fifty four 4. Experimental design : Factorial Completely Randomised Design In all 54 experiments were conducted and results obtained were analysed statistically. The analysis of variance along with the level of significance was also determined. Subsequently, the principal components analysis was carried out to get the single optimum values of independent variables for the extraction gel from Aloe vera leaves. The results are presented in Chapter IV. 42
3.5
Experimental procedure
After standardizing process parameter of the extraction of gel, following experimental procedure was adopted to develop an appropriate process technology for production of gel from Aloe vera barbadensis.
The freshly harvested leaves of Aloe barbadensis variety were cut manually in the early morning for experimentation. To avoid bio-degradation the Aloe vera leaf is harvested and pulled carefully from the mother plant so as not to break the rind and immediately after cutting the leaves were kept in the icebox at 4 -5
0
C and
transported to the laboratory. The leaves were thoroughly washed with fresh water. The outer skin and the exudates of the leaves were removed manually with the help of knife to form fillet (Plate 3.6). The domestic blander (Boss Make) was used to ground the fillets for one min at low speed (700 rpm) to obtain the crushed pulp (Plate 3.7). The crushed pulp was again grounded at higher speed (1000 rpm) to obtained homogenised pulp. The 60 ml pulp on volume basis was centrifuged in cooling type centrifuge for separation of crude gel and fibre. The charcoal was mixed with crude gel for purification. The vacuum filtration method was used to obtain pure gel from crude gel. The pure gel was collected in the test tubes for further analysis. 3.6
Chemical properties of Aloe vera leaf The pure gel was used to study the chemical properties of gel namely: pH,
TSS content, sugar content etc. The chemical composition of gel ultimately affects the quality of gel. The quality of gel also affected by cultivation practices like number of irrigation applied. The method of determination of the chemical properties is given in the subsequent paragraphs.
43
3.6.1 Sugar content
For estimation of sugar content the sample was prepared by taking 0.1 g Aloe vera pure gel in a 100 ml beaker, in which 5 ml of hot, 80 % ethanol was added. The mixture was filtered (What-man No. 1 filter paper) into a beaker. The sample was kept in hot water bath at 100
o
C so that sample became dry and to evaporate the
excess ethanol. Further 10 ml of distil water was added to each sample for determination of sugar content. (Sadasivam and Manickam, 1996). The methods of their estimation are given below.
3.6.1.1 Reducing sugar Nelson-Somogiy method was used for estimation of reducing sugar. From the collected aliquot, 0.2 ml sample was taken in a test tube by pipeting and final volume was made up to 1 ml by adding distilled water. The test tubes were placed in hot water bath for 10 minutes in boiling water after adding 1 ml Alkaline Copper Tartrate reagent.
The sample was kept for cooling at room temperature.
1 ml of
Arsenomolybdate reagent was added to the solution. The volume of the solution was made up to 10 ml by adding 6 ml distilled water. The intensity of blue colour was read at 620 nm on spectrophotometer. Standard graph was prepared using Glucose (0-500 µg). The reducing sugar estimation was replicated twice and the average is taken as the reducing sugar content of Aloe vera gel and it was calculated by equation 3.4.
Sample x Glucose x Volume reading equivalent made up Reducing sugar (%) = -------------------------------------------- x 100 x 10-6 Weight of Aloe vera gel x aliquote taken ................(3.4)
44
3.6.1.2 Total sugar The phenol sulphuric acid method given by Sadasivan et al., (1996) was used to estimate total sugar. The filtrate obtained in the estimation of reducing sugar was used in total sugar estimation. 0.2 ml aliquot was taken into test tube by pipeting and final volume was made up to 2 ml by adding distilled water. 1 ml of 5 % phenol solution and 5 ml of 96 % H2SO4 was added quickly and allowed to stand for 10 minutes after mixing.
The colour of solution was read at 490 nm on the
spectrophotometer. The estimation of total sugar was carried out using standard graph prepared with glucose (0-150 µg) using following expression. The total sugar estimation was replicated twice and the average is taken as the total sugar content of Aloe vera gel and it was calculated by equation 3.5 given below. Sample x Glucose x Volume reading equivalent made up Total sugar (%) = ------------------------------------------------ x 100 x 10-6 Weight of Aloe vera gel x aliquote taken ................(3.5) 3.6.2 Total soluble solid test The total soluble solids content is the summation of all the solids dissolved in the water, beginning with sugar, salts, protein, acids, etc. The total solid contents in the extracted gel affect its quality. More will be the total solids in the gel; poor will be the quality of gel. Hand Refractometer was used for the measurement of total soluble solid content. Hand Refractometer is a simple devise used for measuring concentrations of aqueous solutions and it gives direct reading of total soluble solid content in degree Brix. The % Brix scale expresses the concentration percentage of the soluble solids content of a sample with water solution taken as reference materials. The total soluble solid content was measured by placing two drops of the Aloe vera gel on the scale of hand refractometer (0-32 0C). The averages of three readings were taken as TSS content of extracted gel and expressed in degree Brix in percent. 45
3.6.3
Determination of pH of gel The pH of a solution is the negative logarithm (base 10) of the activity or the
product of the molar concentration and the activity coefficient of the hydrogen ions (H+) in the solution. pH of Aloe vera gel was measured by a recording type pH meter. 30 ml of each sample was taken a test tube then probe was inserted in the test tube and reading was taken as the pH of the each sample. pH = - log [H+] 3.7
----------- (3.6)
Evaluation of gel quality parameters Aloe gel quality was judged by its purity. Purity of gel was determined by the
viscosity, optical density, and refractive index. 3.7.1 Viscosity The viscosity of a fluid is a very important property in the analysis of liquid behavior and fluid motion near a solid boundary. Often it is defined as the resistance to flow of a fluid. The resistance is caused by intermolecular friction exerted when layers of fluids attempts to slide by another layer. It is measured in units of poises (dyne-seconds per square centimeter), stokes or a subdivision of poises. The Oswald viscometer and Redwood viscometer were used for the measurement of viscosity of the Aloe vera gel. 3.7.1.1 Oswald type glass viscometer Viscosity of the liquid materials is measured in a calibrated glass vessel known as a viscometer. After calibration the Oswald viscometer was filled to the lower calibration mark by applying suction with a rubber bulb and drawing the liquid analyte into the apparatus. The time required for the volume of liquid between the two marks to drain from the bulb is measured. The tube at the lower end of the upper bulb has a fixed length and radius, which is used along with the pressure differential column between the upper and lower ends of the apparatus to measure the viscosity. 46
3.7.1.2 Redwood viscometer
Redwood viscometer is based on the principle of laminar flow through capillary tube of standard dimension under falling head. The Redwood No. 1 viscometer having capillary diameter.1.62 mm and length. 10.0 mm. The kinematic viscosity (µ) of liquid and the time (t) required to pass 50cc of liquid are correlated by the expression
µ = 0.0026 t – 1.175 / t
--------( 3.7)
Where, µ = Kinematic Viscosity in Stokes t = time in seconds to collect 50 cc of oil.
The standard method was used in the measurement of viscosity of Aloe vera gel as given by equation 3.7.
3.7.2 Optical density
Optical density is the measure of transparency of liquid and also a measure of quality for Aloe vera gel. Photospectrometer was used for the measurement optical density, which gives the direct reading of Absorbance. The filter slot No.6 of the Photospectrometer was set on zero transmittance by lower knob and left as such with out disturbing lower knob. The filter slot was also set according to wavelength (for Aloe gel 400 nm) and set 100% transmittance for blank sample or distilled water by side knobs.
47
3.7.3 Refractive index
Refractive Index is the ratio of the Sine of the angle of incidence to the Sine of the angle of refraction, when a ray of light of monochromatic sodium light of wavelength 589.3
0
A passes from (defined wavelength passes from) air into the
material keeping temperature as constant i.e. 27 0 C. The wavelength is 589.3 + nm corresponding with D1 and D2 lines of sodium spectrum. The Refractometer used for measurement of refractive index having range of Refractive Indices between 1.3000 and 1.7000 with an accuracy of + 0.0002. It was Calibrated with known refractive indices i.e. doubled distilled water (1.3323) at 27 0 C ± .2 0 C.
Two drops of Aloe vera gel were placed on the Refractometer prism surface and closed carefully. The mirror was adjusted until the reading was sharp. The instrument was allowed to stand for a few minutes before the reading was taken so that the sample and instrument came to equilibrium. The reading was taken when the blue and yellow shade crossed the cross mark. The results were expressed in four decimal places (Sangani, 1997).
Refractive index is the physical property of gel determines the purity of gel as compared to double distilled water. Gel with lowest refractive index, is the best treatment for extraction process. More refractive index indicates the impurities in the extracted gel. 3.8
Development of Process
In development of process each step of Aloe vera gel extraction was considered and standardised. Proportion of chemicals for addition determined. The process time for each method is fixed. After standardising the process parameters, the final gel extraction process was developed. The final process was evaluated for quality parameters of extracted gel. 48
3.9
Statistical Analysis
The statistical analysis of experiment was carried out at Statistics Department, College of Agriculture, Junagadh Agricultural University, Junagadh, with 4 Factors Completely Randomized Design.
The data of results were interpreted. The main effect, interaction and combine effect were studied. The F-test was carried out to determine whether the effect is significant or not. Critical difference and Coefficient variation were considered for the interpretation of data.
The test data of the studies conducted on different levels of acetone, centrifuge temperature, centrifuge speed and centrifuge duration for different parameters such as crude and pure gel recovery, viscosity, refractive index, optical density and TSS content are presented in Appendix A – F. The data of physical characteristics like length, width, thickness, apparent volume, weight, pulp recovery of aloe vera leaves and properties of Aloe vera gel such as m.c., fiber content, pH, sugar content were analysed and the results are presented and discussed in Chapter IV.
49
CHAPTER IV RESULTS AND DISCUSSION
This chapter deals with the results of the experiments conducted on physical and chemical properties of Aloe vera leaf and Aloe vera gel, standardization of process parameters, combine effect of process parameters on dependent variables like recovery of gel, viscosity, refractive index, optical density and TSS etc.
The
observations were analysed using Factorial CRD (Completely Randomised Design) technique and the variation in the parameters was depicted graphically. The results of the studies conducted are presented and discussed in the subsequent paragraphs.
4.1
Moisture content The moisture content of the Aloe vera pulp and gel was measured by hot oven
drying method as described in Chapter III para No.3.2 The measurement of moisture content was replicated 5 times and their average moisture content is presented in Table 4.1. The average moisture content for pulp and gel were found to be 98.88 and 99.80 % respectively. Table 4.1 Moisture content of Aloe vera leaf pulp Sr. No.
Moisture content (%)
Average
4.2
Pulp
Gel
98.88
99.80
Physical characteristics and property of Aloe vera leaf
4.2.1 Size and shape The size was determined from the measurement of length, width and thickness of Aloe vera leaf. The results of variation in size of Aloe vera leaf are presented in Table 4.2. The length, width and thickness are expressed in mm. From the Table 4.2, 50
it is clear that the length width and thickness ranges from 480 to 655, 55 to 115 and 19 to 32 mm, whereas, their averages are 55.98, 93.9 and 26.8 mm respectively. Wang and Strong, (1993), has given similar results for length, width and thickness of Aloe vera leaf. The average values and range of length, width and thickness also decides size which taken as the apparent volume of Aloe vera leaf. The average size is 371.75 cc. It can be noted from the table that the length is about 21 times to the thickness. It is said to be, that, more is the thickness better will be the pulp recovery. Therefore the thickness could be taken as one of selection criteria of Aloe vera leaf for the extraction of gel for commercial use. The shape of Aloe vera leaf may be classified as conic- tapered towards apex as per classification given by Mohsenin (1980).
4.2.2
Apparent volume Table 4.3 gives the variation in the apparent volume of Aloe vera leaf. The
average apparent volume is found to be 371.75 cc, whereas, it ranges from 127.32 to 485.73 cc. The relationship between apparent volume and fresh weight of leaves is shown in Figure 4.1. Apparent volume can be taken as a parameter for the estimation of leaf fresh weight. Taking apparent volume as a function of Aloe vera leaf weight the following linear equation was developed. Apparent volume = f (Aloe vera leaf weight) y = 0.6527x + 215.87
-------------(4.1)
The response of the predicted values is within the experimental range. 4.2.3 Leaf weight The leaf weight was measured with the help of pan balance having least count 0.01 g (Sartorious make). The leaf weight of 23 leaves were recorded and expressed in g. Table 4.3 shows variation in leaf weight of Aloe vera leaf. The maximum 51
weight of the Aloe vera leaf was recorded to be 658 g, whereas, minimum was 326 g with their standard deviation of 0.07. The average weight of the leaf was 459 g. It was also observed during the experiment that the Aloe vera leaves having higher weight, had recorded better pulp recovery. Table 4.2 Variation in length, width, thickness and size of Aloe vera leaf Average moisture content = 98.88 percent (wb) Variety = Aloe barbadensis Sr. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Average Range SD
Length (a) mm 560 520 590 620 655 530 520 620 570 590 520 530 510 480 520 590 600 550 490 500 615 565 630 55.98 480 to 655 4.97
Width (b) mm 85 100 97.5 100 55.0 65.0 60.0 100.0 95.0 110.0 105.0 115.0 110.0 105.0 115.0 97.5 95.0 102.5 100.0 97.5 85.0 70.0 95.0 93.9 55 to 115 1.67
52
Thickness (c) mm 26.7 26.0 27.0 27.0 27.8 19.0 21.0 26.2 26.5 32.0 26.6 28.8 26.7 25.8 27.3 27.7 28.0 27.2 26.4 29.0 30.0 22.0 31.0 26.8 19 to 32 0.29
Size π/12(a x b x c) cc 332.73 353.95 406.62 438.25 262.19 171.36 171.53 425.27 375.68 543.71 380.23 459.55 392.14 340.42 427.40 417.16 417.83 401.44 338.66 370.12 410.57 227.79 485.73 371.75 127.32 to 485.73 91.91
4.2.4
Pulp weight
The pulp from Aloe vera leaves was extracted as per procedure given in Chapter III section 3.5. The weight of pulp was measured after removing the skin; with the help of pan balance-having least counts 0.01 g. Pulp was extracted from single leaf each day and their pulp weight was recorded in g. Table 4.3 also presents the variation in pulp weight of Aloe vera leaf. Table 4.3
Sr. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Average Range SD
Variation in apparent volume, leaf weigh, pulp weight and recovery of Aloe vera leaf Average moisture content = 98.88 percent (wb) Variety = Aloe barbadensis Apparent volume cc 332.73 353.95 406.62 438.25 262.19 171.36 171.53 425.27 375.68 543.71 380.23 459.55 392.14 340.42 427.40 417.16 417.83 401.44 338.66 370.12 410.57 227.79 485.73 371.75 127.32 to 485.73 91.91
Leaf weight g 431 397 458 445 481 326 336 424 454 658 454 523 452 397 456 476 515 448 440 509 560 360 546 459 326 to 658 0.07 53
Pulp weight g 192 170 205 194 232 156 156 186 226 331 232 266 243 217 232 241 275 256 229 267 312 192 288 230 156 to 331 0.05
pulp
Pulp recovery % 44.55 42.82 44.76 43.60 48.23 47.85 46.43 43.87 49.78 50.30 51.10 50.86 53.76 54.66 50.88 50.63 53.40 57.14 52.05 52.46 55.71 53.33 52.75 50.04 42.82 to 57.14 4.11
700
y = 0.652x + 215.8 R² = 0.654 600
Leaf weight, g
500
400
300
200
100
0 0.00
200.00
400.00
600.00
Leaf volume, cc
Figure 4. 1 Relationship between apparent volume and fresh weight of Aloe vera leaves. The maximum pulp weight of the Aloe vera leaf was recorded to be 331 g, whereas, minimum was 156 g with their standard deviation of 0.046. The average pulp weight of the leaf was 230 g. It was observed during the experiment that the Aloe vera leaves having higher pulp weight, had recorded better gel recovery. Leaf fresh weight can be taken as a parameter for the estimation of pulp recovery. The 54
relationship between leaf weight and pulp recovery is shown in Figure 4.2. Taking leaf fresh weight as a function of pulp recovery the following linear equation was developed. Aloe vera leaf fresh weight = f (Aloe vera pulp recovery) y = 0.0172x + 42.158
-------------(4.2)
The response of the predicted values is within the experimental range.
y = 0.017x + 42.15 R² = 0.096
60
55
Pulp recovery, %
50
45
40
35
30 300
400
500 Leaf weight,g
600
700
Figure 4.2 Relationship between fresh weights of Aloe vera leaves and pulp recovery. 55
4.2.5
Pulp recovery The variation in pulp recovery of Aloe vera leaf is presented in Table 4.3. The
maximum pulp recovery of the Aloe vera leaf was recorded to be 57.14 %, whereas, minimum was 42.82 % with their standard deviation of 4.11 %. The average pulp recovery from the leaf was 50. 04 %. It was observed during the experiment that the Aloe vera leaves having higher pulp weight, had recorded better pulp recovery.
The relationship between apparent volume and pulp recovery is shown in Figure 4.3. Apparent Volume can be taken as a parameter for the estimation of leaf pulp recovery. Taking apparent volume as a function of Aloe vera leaf pulp recovery the following linear equation was developed. Apparent volume = f (Aloe vera pulp recovery) y = 0.0067x + 47.561
-------------(4.3)
The response of the predicted values is within the experimental range. 4.3
Chemical property of Aloe vera gel
The chemical properties such as pH, total soluble solid, sugar content and fiber content of extracted gel were determined. These chemical properties are required for product preparation from gel. All these parameters are discussed in the subsequent paragraphs. Table 4.4 presents the chemical properties of Aloe vera gel. From the table it is clear that the fiber content and pH of gel was found to be 0.2 % and 6.389 which is closer to the findings of Wang et al., (1993) and sugar content 1.9125 % (Total sugar) and 0.0259 % (Reducing sugar) from Aloe vera gel. M/s Delta International has found Aloe vera gel pH 3.5 - 4.7 (http://www.shantidatta.com). 56
60.00
y = 0.0067x + 47.561 R2 = 0.0223
50.00
Pulp recovery, %
40.00
30.00
20.00
10.00
0.00 0.00
200.00 400.00 Leaf volume, cc
600.00
Figure 4.3 Relationship between apparent volume and pulp recovery of Aloe vera leaf
Table 4.4 Chemical properties of Aloe vera gel Sr.
TSS
No.
(Brix)
pH
Fiber content
Sugar
(%)
(%)
Pulp
Gel
Total
Reducing
Average
1.366
6.398
1.117
0.2
1.9125
0.0259
Range
0.77
5.88
1.114
0.18
1.53
0.024
to
to
to
to
to
to
1.97
7.02
1.120
0.22
2.295
0.0278
57
4.4
Standardization of process parameters for extraction of gel from Aloe vera leaves The process parameters like acetone proportion, centrifuge temperature;
centrifuge speed and centrifuge duration for extraction process was studied. The effect of process parameters on different quality parameters such as crude and pure gel recovery, viscosity, refractive index, optical density and TSS content of gel extracted from Aloe vera leaf were recorded.
4.4.1
Effect of acetone on gel extraction process
The effect of acetone on different quality parameters such as crude and pure gel recovery, viscosity, refractive index, optical density and TSS content of gel extracted from Aloe vera leaf is presented in Table 4.5 and also shown in form of bar diagram in Figures 4.4 and 4.5. The statistical analysis is given in Appendix A – F. From the table it is clear that all the parameters increase with 10 % addition of acetone to Aloe vera pulp. The refractive index was found to be little higher in case of 10 % of acetone as compared to, without acetone in Aloe vera pulp. The distilled water was taken as reference material with the consideration that, it is one of the purest forms of liquid. The both treatment i. e, without and with 10 % acetone shows the little higher values of refractive index i.e. 1.33601 and 1.33610 respectively when compared to distilled water which is having only 1.3323 at 27 0 C ± 0.2 0C. It may be said that, the values of the refractive index closer to water is considered to be the best in terms of their purity. The refractive index of the extracted gel, without acetone had found to be closer to distilled water. It may be concluded that refractive index of the Aloe vera gel without acetone is to be considered as purest forms of gel.
It is also clear from the Figure 4.5 that, addition of 10 % acetone to pulp increases the crude gel recovery by 2.8 %, pure gel recovery by 2.3 % and viscosity by 136.5 % (Figure 4.5). It indicates favorable results because more will be the 58
viscosity of the gel active will be the biological material, but, at the same time the percent increase in refractive index by 0.0067 %, optical density 12.93 % and TSS 93.55 % (Figure 4.5) which shows un-favorable results for the extraction process because the values of the indices is little higher as compared to distilled water which has been taken as measurement of the purity of gel. Table 4.5 Effect of acetone on gel extraction process Treatments Dependent Variables Acetone
Crude gel
Pure gel
Viscosity
Refractive
Optical
TSS
Proportion
recovery
recovery
(Stokes)
index
density
(Brix)
(%)
(%)
(%)
0
57.32
40.93
0.591
1.33601
0.232
0.93
10
59.24
43.23
1.398
1.33610
0.252
1.80
S.Em.
0.171
0.131
0.004
2.15 x 10-5
2.8 x 10-4
0.01
CD @ 5 %
0.479
0.368
0.011
6.0 x 10-5
0.01
0.027
Sig.
Sig.
Sig.
Sig.
Sig.
Sig.
Test
(abs)
The statistical analysis shows the significant effect of acetone on different quality parameters such as crude and pure gel recovery, viscosity, refractive index, optical density and TSS at 5 % Cd.
Based on the results it may be concluded that the effect of 10 % acetone addition to pulp for gel extraction, results in increase in the gel recovery and viscosity, but at the same time it deteriorate the purity of gel. Hence the use of addition of acetone may be avoided.
59
70
Gel recovery (%)
60
59.24
57.32
50 43.23
40.93
40
Crude gel recovery (%) Pure gel recovery (%)
30 20 10 0 0 % acetone
10 % acetone
Proportion of Acetone (%)
Figure 4.4 Effect of Acetone on Aloe gel recovery
2 Values of quality parameters
1.8
1.8 1.6 1.33601
1.4
1.398
1.3361
1.2
Viscosity (Stokes) Optical density (abs)
0.93
1
TSS (Brix)
0.8 0.6
Refractive index
0.591
0.4 0.232
0.252
0.2 0 0 % acetone
10 % acetone
Proportion of Acetone (%)
Figure 4.5 Effect of acetone on quality parameters of Aloe gel 60
4.4.2
Effect of centrifuge temperature on gel extraction process
Temperature is considered, as one of the major factor, which ultimately affects the viscosity, optical density, refractive index and TSS content of gel extracted from Aloe vera leaves. Danhof, (2000) has reported that the sun or heat exposure and also the time of processing of Aloe vera leaves should be minimum. The effect of centrifuge temperature on different quality parameters such as crude and pure gel recovery, viscosity, refractive index, optical density and TSS content on gel extraction from Aloe vera leaf at different temperatures are presented in Table 4.6, and also given in form of bar diagram in Figure 4.6 and 4.7. The statistical analysis is given in Appendix A to F. The percentage recovery of crude and pure gel varies from 57.91 to 58.89 and 42.04 to 42.14 % respectively with the variation in temperature, which is also represented graphically in Figure 4.6. It is seen from the table that the percent recovery of gel is more or less same in both the case with the increase of temperature. This shows that there is no effect of temperature on the recovery of gel either crude or pure.
The viscosity of the extracted gel (Table 4.6) is largely affected with the changes of centrifuge temperature. The maximum viscosity (1.369 Stokes) was recorded at 5 0C and minimum (0.696 Stokes) at 32 0C (Ambient temperature). Viscosity was recorded 96.69 and 49.13 % higher at 5 0C as compared to 32 0C and 10 0C temperature. This statement is satisfied with the study conducted by Shih-Jen Chiou, (2003) that the viscosity of Aloe vera gel decreases with the increase of heating time. It is obvious that higher is the value of viscosity of Aloe vera gel, better will be the product.
61
Values of quality parameters
70
50
58.89
58.04
57.91
60
42.14
42.06
42.04
40
Crude gel recovery (%) Pure gel recovery (%)
30 20 10 0 5
10
32
Centrifuge temperature (C)
Figure 4.6 Effect of centrifuge temperature on Aloe gel recovery
Values of quality parameters
1.6 1.4
1.369 1.4
1.39
1.31
1.2 0.918
1
Viscosity(Stokes) Optical density (abs)
0.8
0.696
0.6
TSS (Brix) Refractive index
0.4 0.238
0.243
0.244
5
10
32
0.2 0 Centrifuge temperature (C)
Figure 4.7 Effect of centrifuge temperature on quality parameters of Aloe gel
62
The Refractive index and TSS increases with the increase of temperature (Table 4.6). The minimum and maximum refractive index was found to 1.33603 and 1.33610, while TSS was found 1.31 and 1.40 Brix respectively. The refractive index of the pure gel is found to be closer to distilled water at all the temperatures. It may be said that the temperature has non-significant effect on the purities of the gel. The TSS content decreases with the increase of temperature. The decrease in TSS content with temperature may be due to enzymatic degradation of Aloe vera gel at higher temperatures. Table 4.6 Effect of centrifuge temperatures on gel extraction process Treatments
Dependent Variables
Centrifuge
Crude gel Pure gel
Temperature recovery 0
recovery
Viscosity Refractive
Optical
TSS
(Stokes)
density
(Brix)
index
( C)
(%)
(%)
5
57.91
42.14
1.369
1.33610
0.238
1.40
10
58.04
42.04
0.918
1.33604
0.243
1.39
32
58.89
42.06
0.696
1.33603
0.244
1.31
S.Em.
0.213
0.164
0.005
2.68x10-5
3.51x10-4
0.012
CD @ 5 %
0.597
0.459
0.014
7.5x10-5
0.001
0.034
Sig.
NS
Sig.
NS
Sig.
Sig.
Test
(abs)
From the Table 4.6 it is seen that the optical density of the Aloe vera gel increases with the increase of temperature. The maximum value was found to be 0.244 at 32 0C and minimum at 5 0C temperatures. The increase in the value of optical density may be due to enzymatic degradation of Aloe vera gel at higher temperatures. Gel extracted at different centrifuge temperatures is shown in Plate 4.1.
The statistical analysis shows the significant effect of centrifuge temperature on different quality parameters such as crude gel recovery, viscosity, refractive index, 63
optical density and TSS, whereas for pure gel recovery it shows non-significant effect at 5 % Cd. It is concluded that 5 0C temperature yield in higher viscosity, lower refractive index and optical density with optimum recovery of gel. It may be suggested that the gel extraction may be carried out by Centrifuge at 5
0
C temperature to obtained
better quality gel.
4.4.3
Effect of centrifuge speed on gel extraction process
The centrifuge process is carried out to separate solid particles from pulp for getting crude gel. The Aloe vera gel also contains sugar molecules surrounded by gel molecules. Centrifugal force is required to break this chain of sugar molecules. Table 4.7 shows the results of effect of centrifuge speed on different quality parameters such as gel recovery, viscosity, optical density, refractive index and TSS content of gel extracted from Aloe vera leaves. Also it is represented graphically in Figure 4.8 and 4.9 and statistical analysis is given in Appendix A to F.
It is seen from the table that, the crude and pure gel recovery, viscosity, and TSS content increases with the increase of centrifuge speed, where as other parameter i.e. optical density, refractive index decreases with the increase of centrifugal speed. The maximum recovery of crude gel as well as pure gel is found to be 67.05 and 48.72 % at 10000 rpm respectively. The recovery for crude gel as well as pure gel at 2000 and 5000 centrifuge speed is 17.98 and 26.74 % and 8.33 and 14.14 % respectively lower, when compared to 10,000 rpm centrifuge speed. The higher recovery at higher speed i.e.10,000 rpm may be due the separation of solid particles from pulp or break down of the chain of sugar molecules.
The viscosity and TSS are found maximum at 10,000 rpm i.e. 1.040 strokes and 1.40 Brix respectively where as minimum values at 2000 centrifuge speed for 64
both the cases. It is said that, higher is the viscosity better will be the quality of the product. At the same time the product is considered to be biologically active. (Gowda et al., 1979). The maximum value of TSS at higher speed is found to be 1.40 Brix. This TSS value is much closer to the finding of Wang et al., (1993). Similarly the value of refractive index (1.33789 - 1.34390) at 10,000 rpm is match with the finding of the M/s. Delta International (http://www.shantidatta.com). The lower is the optical density of gel better will be the product. The 10,000 rpm centrifuge speed shows the lowest value i.e., 0.234 abs and highest at 2000 rpm speed 0.250 abs. It may be concluded that after considering all the quality parameters the 10,000 rpm centrifuge speed are found better for the gel extraction process. Extracted gel sample is shown in Plate 4.2 for different centrifuge speed.
Table 4.7 Effect of centrifuge speed on gel extraction process Treatments
Dependent Variables
Centrifuge
Crude
Pure gel
Viscosity
Refractive
Optical
TSS
speed
gel
recovery
(Stokes)
index
density
(Brix)
(rpm)
recovery
(%)
(abs)
(%) 2000
49.07
35.69
0.954
1.33689
0.250
1.33
5000
58.72
41.83
0.989
1.33599
0.242
1.37
10,000
67.05
48.72
1.040
1.33530
0.234
1.40
S.Em.
0.213
0.164
0.005
2.68x10-5
3.51x10-4
0.012
CD @ 5 %
0.597
0.459
0.014
7.5x10-5
0.001
0.034
Sig.
Sig.
Sig.
Sig.
Sig.
Sig.
Test
65
80 67.05
Gel recovery (%)
70 58.72
60 50
49.07
48.72 41.83
40
Crude gel recovery (%)
35.69
Pure gel recovery(%)
30 20 10 0 2000
5000
10000
Centrifuge speed (rpm)
Figure 4.8 Effect of Centrifuge speed on Aloe gel recovery
Values of quality parameters
1.6 1.4
1.37
1.33
1.4 1.2 1
0.954
1.04
0.989
Viscosity (Stokes) Optical density (abs)
0.8
TSS (Brix) Refractive index
0.6 0.4
0.25
0.242
0.234
0.2 0 2000
5000
10000
Centrifuge speed, (rpm)
Figure 4.9 Effect of Centrifuge speed on quality parameters of Aloe gel
66
The statistical analysis shows that all the quality parameters are found to be significant at 5 % Cd.
From the above results it is suggested that higher centrifuge speed is found suitable for all quality parameters of gel extraction except viscosity. Hence the gel extraction by centrifuge may be carried out at 10,000 rpm speed.
4.4.4 Effect of centrifuge duration on gel extraction process In gel extraction process, the duration of centrifuge plays an important role in the separation of solid particles from the crude pulp. The results of effect of centrifuge duration on different quality parameters such as gel recovery, viscosity, optical density, refractive index and TSS content of gel extracted from Aloe vera leaves are shown in Figure 4.10 and 4.11 and presented in Table 4.8. The statistical analysis is given in Appendix A to F. It is seen from the Table 4.8 that, the crude and pure gel recovery increases with the increase of centrifuge duration, where as optical density decreases with the increase of centrifuge duration. It is also seen from the table that centrifuge duration has very minor effect on the remaining parameters such as viscosity, refractive index and TSS.
The maximum recovery of crude gel as well as pure gel is found to be 61.91 and 45.13 % at 30 min respectively. The recovery for crude gel as well as pure gel at 10 and 20 min centrifuge duration is 12.21 and 5.37 % and 13.12 and 7.16 % respectively lower, when compared to 30 min centrifuge duration. Higher recovery of the gel at 30 min duration may be due the fact that all the particles are getting sufficient residual time during the extraction process. Therefore it may be concluded that sufficient time should be given to obtain optimum gel recovery from the crude pulp.
67
Viscosity as well as refractive index is higher at 10 min duration and minimum at 30 min duration. Their values are of 1.008 and 0.987 Stokes and, 1.33634 and 1.33576 at 10 and 30 min duration respectively. M/s Aloecorp, (2003) has also reported values for refractive index. Optical density of gel decreases as the duration of centrifuge increase. It was found lowest with 30 min duration i.e. 0.239 and highest with 10 min duration i.e. 0.245 abs. Lower values of optical density indicate that the product content less amount of fibrous materials. The TSS content increases with increase in centrifuge duration. It was found 1.35, 1.38 and 1.36 Brix with 10, 20 and 30 min duration respectively. Looking to the recovery of crude and pure gel extraction and other quality parameters, the 30 min centrifuge duration is found to be the best for gel extraction from pulp. Plate 4.3 shows the effect of different centrifuge duration on gel extraction process.
Table 4.8 Effect of centrifuge duration on gel extraction process Dependent Variables
Treatments Centrifuge
Crude gel
Pure gel
Viscosity
Refractive
Optical
TSS
duration
recovery
recovery
(Stokes)
index
density
(Brix)
(min)
(%)
(%)
10
54.35
39.21
1.008
1.33634
0.245
1.35
20
58.58
41.90
0.988
1.33607
0.242
1.38
30
61.91
45.13
0.987
1.33576
0.239
1.36
S.Em.
0.213
0.164
0.005
2.68x10-5 3.51x 10-4
0.012
CD @ 5 %
0.597
0.459
0.014
7.5x10-5
0.001
0.034
Sig.
Sig.
Sig.
Sig.
Sig.
NS
Test
(abs)
68
70 60
Gel recovery (%)
61.91
58.58 54.35
50 39.21
40
45.13
41.9
30
Crude gel recovery (%)
20
pure gel recovery (%)
10 0
10
20
30
Centrifuge duration (min)
Figure 4.10 Effect of Centrifuge duration of Aloe gel recovery
Values of quality parameters
1.6 1.38
1.35
1.4
1.36
1.2 1.008
0.988
1
0.987 Viscosity (Stokes) Optical density (abs)
0.8
TSS (Brix)
0.6
Refractive index
0.4 0.245
0.242
0.239
10
20
30
0.2 0
Centrifuge duration (min)
Figure 4.11 Effect of Centrifuge duration on quality parameters of Aloe gel 69
The statistical analysis shows that the effect of Centrifuge duration on different quality parameters such as crude and pure gel recovery, viscosity, refractive index and optical density are found to be significant, whereas for TSS it is nonsignificant. It is concluded that 30 min centrifuge duration may be considered optimum for gel extraction process to obtained better quality gel. The quality of gel in terms of viscosity is slightly affected by higher residual time. 4.5
Combine effect of two process parameters on gel extraction process
4.5.1
Effect of acetone and centrifuge temperature on gel extraction process Table 4.9 presents the combine effect of acetone and centrifuge temperature on
different quality parameters and their statistical analysis are given in Appendix A to F. The combine effect of acetone and centrifuge temperature on the crude and pure gel recovery, viscosity, optical density and TSS are found to be significant whereas refractive index is found to be non significant at 5 % Cd. 4.5.2
Combine effect of acetone and centrifuge speed on gel extraction process The combine effect of acetone and centrifuge speed on various dependent
variables was studied and given in Table 4.10 and also given in Appendix A to F. The statistical analysis shows significant effect of acetone and centrifuge speed on crude and pure gel recovery, viscosity, optical density and TSS and non significant on refractive index at 5 % Cd.
70
Table 4.9 Combine effect of acetone and centrifuge temperature on gel extraction process Treatments Dependent Variables Acetone CFT (0 C)
Crude Pure gel Viscosity Refractive gel recovery (Stokes) index recovery (%) (%) 56.19 40.43 0.646 1.33603
Optical density (abs)
TSS (Brix)
0.225
0.92
Without
5
Acetone
10
57.18
40.86
0.586
1.33600
0.233
0.92
32
58.58
41.50
0.542
1.33602
0.237
0.96
With
5
59.63
43.84
2.092
1.33618
0.251
1.89
10 %
10
58.90
43.23
1.250
1.33608
0.253
1.86
Acetone
32
59.20
42.63
0.850
1.33604
0.252
1.66
Sem
0.301
0.231
0.007
3.79 x 10-5
4.96 x 10-4
0.017
CD at 5 %
0.844
0.649
0.020
1.02 x 10-4
0.001
0.048
Sig.
Sig.
Sig.
NS
Sig.
Sig.
Test
CFT = Centrifuge temperature
Table 4.10 Combine effect of acetone and centrifuge speed on gel extraction process Treatments Dependent Variables Crude Pure gel Viscosity Refractive Optical TSS Acetone CFS gel recovery (Stokes) index density (Brix) (rpm) (%) recovery (abs) (%) 47.56 35.15 0.584 1.33648 0.241 0.91 Without 2000 Acetone
5000
57.99
40.40
0.590
1.33596
0.231
0.93
10000
66.41
47.25
0.600
1.33560
0.223
0.95
With
2000
50.59
36.23
1.324
1.33729
0.259
1.75
Acetone
5000
59.45
43.27
1.389
1.33601
0.252
1.81
(10 %)
10,000
67.70
50.19
1.480
1.33500
0.245
1.84
S.Em.
0.301
0.231
0.007
3.79x10-5
4.96x10-4
0.017
CD @ 5 %
0.844
0.649
0.020
1.02x10-4
0.001
0.048
Test
Sig.
Sig.
Sig.
NS
Sig.
Sig.
CFS = Centrifuge speed
71
4.5.3
Combine effect of acetone and centrifuge duration on gel extraction process Table 4.11 present the combine effect of acetone and centrifuge duration on
different quality parameters and their statistical analysis are given in Appendix A to F. The interaction of acetone and centrifuge duration on refractive index is found to be non significant and for other parameters it is found to be significant at 5 % Cd. Table 4.11 Combine effect of acetone and centrifuge duration on gel extraction process Treatments Acetone
Dependent Variables
CFD
Crude
(min)
gel recovery
Pure gel Viscosity Refractive
Optical
TSS
recovery (Stokes)
density
(Brix)
index
(%)
(abs)
(%) Without
10
52.83
37.57
0.589
1.33627
0.236
0.92
Acetone
20
58.15
41.09
0.593
1.33598
0.232
0.92
30
60.98
44.14
0.592
1.33579
0.228
0.96
With
10
55.88
40.86
1.427
1.33641
0.255
1.89
Acetone
20
59.01
42.72
1.382
1.33616
0.252
1.86
(10 %)
30
62.85
46.12
1.384
1.33573
0.249
1.66
S.Em.
0.301
0.231
0.007
3.79 x 10-5 4.96 x 10-4 0.017
CD @ 5 %
0.844
0.649
0.020
1.02 x 10-4
0.001
0.048
Sig.
Sig.
Sig.
NS
Sig.
Sig.
Test CFD = Centrifuge duration
4.5.4
Combine effect of centrifuge temperature and speed on gel extraction process For gel extraction from Aloe vera leaf, different centrifuge temperature and
speed combinations were studied and are given in Table 4.12 and Appendix A to F. 72
The interaction of centrifuge temperature and speed on all quality parameters are found to be significant except TSS, which shows non-significant effect. Table 4.12 Combine effect of centrifuge temperature and speed on gel extraction process Treatments
Dependent Variables
CFT
CFS
Crude
Pure gel Viscosity Refractive
Optical
TSS
(0 C)
(rpm)
gel
recovery
density
(Brix)
recovery
(%)
(Stokes)
index
(abs)
(%) 5
2000
49.11
35.26
1.309
1.33685
0.246
1.36
5000
57.34
41.93
1.346
1.33614
0.238
1.42
10,000
67.28
49.22
1.452
1.33531
0.231
1.43
2000
48.64
35.83
0.893
1.33698
0.250
1.36
5000
59.03
41.73
0.927
1.33591
0.243
1.37
10,000
66.45
48.56
0.934
1.33523
0.237
1.43
2000
49.46
35.98
0.660
1.33683
0.254
1.28
5000
59.79
41.84
0.694
1.33591
0.245
1.32
10,000
67.43
48.37
0.734
1.33535
0.234
1.32
S.Em.
0.368
0.283
0.009
4.6 x 10-5
6.1 x10-5
0.215
CD @ 5 %
1.034
0..795
0.025
1.3 x10-4
0.002
0.059
Sig.
Sig.
Sig.
Sig.
Sig.
Ns
10
32
Test
CFT = Centrifuge temperature CFS = Centrifuge speed
4.5.5
Combine effect of centrifuge temperature and duration on gel extraction process The statistical analysis shows that the interaction between centrifuge
temperature and duration on the crude and pure gel recovery, viscosity and refractive index and are found to be significant whereas on optical density and TSS it is non significant (Table 4.13 and Appendix A to F). 73
Table 4.13 Combine effect of centrifuge temperature and centrifuge duration on gel extraction process Treatments Dependent Variables CFT CFD Crude gel Pure gel Viscosity Refractive index (0 C) (min) recovery recovery (Stokes) (%) (%) 5 10 53.23 38.94 1.410 1.33647 20 58.15 42.41 1.350 1.33609 30 62.35 45.06 1.347 1.33574 10 10 54.37 39.38 0.927 1.33622 20 58.09 41.51 0.922 1.33609 30 61.66 45.24 0.906 1.33581 32 10 55.45 39.31 0.688 1.33633 20 59.50 41.79 0.690 1.33602 30 61.72 45.09 0.710 1.33573 0.368 0.283 0.009 4.6 x 10-5 S.Em. 1.034 0..795 0.025 1.3 x10-4 CD @ 5 % Sig. Sig. Sig. Sig. Test
Optical density (abs) 0.242 0.238 0.234 0.247 0.243 0.240 0.247 0.245 0.242 6.1 x10-5 0.002 NS
TSS (Brix) 1.37 1.41 1.43 1.38 1.40 1.38 1.31 1.34 1.28 0.215 0.059 Ns
CFT= Centrifuge temperature, CFD= Centrifuge duration
4.5.6
Combine effect of centrifuge speed and duration on gel extraction process The combine effect of centrifuge speed and duration on gel extraction process
was studied on various dependent variables and found to significant except viscosity which shows non significant interaction between two (Table 4.14 and Appendix A to F).
74
Table 4.14
Combine effect of centrifuge speed and duration on gel extraction process
Treatments CFS
CFD
(rpm) (min)
Dependent Variables Crude
Pure gel Viscosity Refractive
Optical
TSS
gel
recovery
density
(Brix)
recovery
(%)
(Stokes)
index
(abs)
(%) 2000
10
45.84
32.88
0.958
1.33736
0.254
1.37
20
48.73
35.32
0.959
1.33679
0.250
1.33
30
52.64
38.87
0.946
1.33651
0.247
1.30
10
53.06
38.45
1.007
1.33612
0.246
1.31
20
59.82
42.07
0.976
1.33603
0.242
1.40
30
63.28
44.97
0.984
1.33581
0.238
1.40
10
64.16
46.31
1.059
1.33554
0.236
1.38
20
67.19
48.31
1.027
1.33538
0.234
1.42
30
69.81
51.55
1.033
1.33497
0.231
1.39
S.Em.
0.368
0.283
0.009
4.6 x 10-5
6.1 x10-5
0.215
CD @ 5 %
1.034
0.795
0.025
1.3 x10-4
0.002
0.059
Test
Sig.
Sig.
NS
Sig.
Sig.
Sig.
5000
10000
CFS= Centrifuge speed, CFD= Centrifuge duration
4.6
Combine effect of three process parameters on gel extraction process
4.6.1
Combine effect of acetone, centrifuge temperature and speed on gel extraction process The three factors as acetone, centrifuge temperature and speed on varying
proportions were studied and the resultant effect was recorded. Table 4.15 presents the combine effect of acetone; centrifuge speed and duration on different quality 75
parameters and their statistical analysis are given in Appendix A to F. The statistical analysis shows that the combine effect of acetone; centrifuge speed and duration for all quality parameters are found to be significant. 4.6.2
Combine effect of acetone, centrifuge temperature and duration on gel extraction process The three factors as acetone, centrifuge temperature and duration on varying
proportions were studied and the resultant effect was recorded. Table 4.16 presents the combine effect of acetone; centrifuge temperature and centrifuge duration on different quality parameters and their statistical analysis are given in Appendix A to F. The statistical analysis shows that the combine effect of acetone; centrifuge temperature and duration on the pure gel recovery, viscosity and refractive index, are found to be significant, whereas on crude gel optical density and TSS the combine effect was non significant. 4.6.3
Combine effect of Acetone, centrifuge speed and duration on gel extraction process The three factors as acetone, centrifuge speed and duration on varying
proportions were studied and the resultant effect was recorded. Table 4.17 presents the combine effect of acetone; centrifuge speed and centrifuge duration on different quality parameters and their statistical analysis are given in Appendix A to F. The statistical analysis shows that the combine effect of acetone; centrifuge temperature and centrifuge duration on, crude gel recovery, refractive index and optical density are found to be significant, whereas on the pure gel recovery, viscosity and TSS the combine effect was non significant.
76
Table 4.15 Combine effect of acetone, centrifuge temperature and speed on gel extraction process. CFT= Centrifuge temperature, CFS= Centrifuge speed
Treatments
Dependent Variables
CFT
CFS
Crude gel
Pure gel
Viscosity
Refractive
Optical
TSS
(0 C)
(rpm)
recovery
recovery
(Stokes)
index
density
(Brix)
(%)
(%)
2000
46.96
33.96
0.633
1.33653
0.233
0.84
5000
54.78
39.78
0.648
1.33594
0.224
0.96
10,000
66.83
47.56
0.656
1.33560
0.219
0.96
2000
46.70
34.74
0.586
1.33640
0.240
0.96
5000
59.15
40.63
0.581
1.33603
0.232
0.88
10,000
65.69
47.20
0.591
1.33556
0.228
0.93
2000
49.00
36.74
0.533
1.33651
0.249
0.94
5000
60.04
40.78
0.540
1.33590
0.237
0.97
10,000
66.70
46.98
0.552
1.33564
0.223
0.96
2000
51.26
36.56
1.985
1.33717
0.259
1.87
10 %
5000
59.91
44.07
2.045
1.33634
0.251
1.88
Acetone
10000
67.72
50.89
2.247
1.33502
0.243
1.91
2000
50.57
36.93
1.200
1.33756
0.260
1.77
5000
58.91
42.83
1.274
1.33578
0.254
1.87
10,000
67.22
49.93
1.277
1.33491
0.245
1.93
2000
49.93
35.22
0.788
1.33714
0.259
1.61
5000
59.54
42.91
0.847
1.33592
0.252
1.68
10,000
68.15
49.76
0.916
1.33506
0.245
1.69
S.Em.
0.521
0.401
0.013
6.6x10-5
8.6x10-5
0.030
CD @ 5 %
1.462
1.124
0.035
1.8x10-4
0.002
0.084
Sig.
Sig.
Sig.
Sig.
Acetone
Without
5
Acetone
10
32
With
5
10
32
Test
CFT= Centrifuge temperature, CFS= Centrifuge speed
77
(abs)
Sig.
Sig.
Table 4.16 Combine effect of acetone, centrifuge temperature and duration on gel extraction process Treatments
Dependent Variables
CFT
CFD
Crude gel
Pure gel
Viscosity
Refractive
Optical
TSS
(0 C)
(min)
recovery
recovery
(Stokes)
index
density
(Brix)
(%)
(%)
10
50.70
36.22
0.640
1.33639
0.231
0.89
20
56.72
41.33
0.650
1.33591
0.225
0.92
30
61.15
43.74
0.647
1.33578
0.220
0.94
10
53.13
38.02
0.590
1.33617
0.238
0.92
20
57.94
40.78
0.586
1.33600
0.233
0.94
30
60.46
43.78
0.582
1.33582
0.229
0.90
10
54.65
38.46
0.536
1.33624
0.239
0.97
20
59.78
41.15
0.543
1.33603
0.237
0.99
30
61.31
44.89
0.546
1.33578
0.234
0.91
10
55.76
41.67
2.179
1.33656
0.254
1.84
10 %
20
59.57
43.48
2.051
1.33628
0.251
1.89
acetone
30
63.56
46.37
2.047
1.33570
0.248
1.92
10
55.61
40.74
1.263
1.33627
0.256
1.84
20
58.24
42.24
1.257
1.33618
0.252
1.86
30
62.85
46.70
1.230
1.33580
0.251
1.87
10
56.26
40.17
0.839
1.33642
0.255
1.64
20
59.22
42.43
0.837
1.33601
0.252
1.69
30
62.13
45.30
0.874
1.33569
0.249
1.64
S.Em.
0.521
0.401
0.013
6.6x10-5
8.6x10-5
0.030
CD @ 5 %
1.462
1.124
0.035
1.8 x10-4
0.002
0.084
NS
Sig.
Sig.
Sig.
Acetone
Without
5
acetone
10
32
With
5
10
32
Test
CFT= Centrifuge temperature, CFD= Centrifuge duration
78
(abs)
NS
NS
Table 4.17 Combine effect of acetone, centrifuge speed and duration on gel extraction process Treatments
Dependent Variables
CFS
CFD
Crude gel
Pure gel
Viscosity
Refractive
Optical
TSS
(rpm)
(min)
recovery
recovery
(Stokes)
index
density
(Brix)
(%)
(%)
10
44.02
31.89
0.580
1.33702
0.246
0.94
20
47.89
35.20
0.594
1.33630
0.241
0.92
30
50.76
38.35
0.578
1.33612
0.237
0.88
10
50.44
36.39
0.595
1.33604
0.237
0.91
20
60.61
40.81
0.588
1.33600
0.231
0.96
30
62.91
43.98
0.586
1.33583
0.225
0.93
10
64.02
44.43
0.591
1.33573
0.225
0.92
20
65.94
47.24
0.597
1.33564
0.224
0.98
30
69.26
50.07
0.611
1.33542
0.221
0.94
10
47.67
33.87
1.336
1.33769
0.262
1.79
10 %
20
49.57
35.44
1.323
1.33728
0.258
1.73
acetone
30
54.52
39.39
1.313
1.33690
0.257
1.72
10
55.67
40.52
1.420
1.33620
0.255
1.71
20
59.04
43.33
1.364
1.33607
0.252
1.84
30
63.65
45.96
1.382
1.33578
0.250
1.87
10
64.30
48.19
1.526
1.33536
0.248
1.83
20
68.43
49.37
1.458
1.33512
0.245
1.86
30
70.37
53.02
1.455
1.33451
0.242
1.84
S.Em.
0.521
0.401
0.013
6.6x10-5
8.6x10-5
0.030
CD @ 5 %
1.462
1.124
0.035
1.8 x10-4
0.002
0.084
Test
Sig.
NS
NS
Sig.
Acetone
Without
2000
acetone
5000
10000
2000
With
5000
10000
(abs)
Sig.
NS
CFS= Centrifuge speed, CFD= Centrifuge duration
4.6.4
Combine effect of centrifuge temperature, speed and duration on gel extraction process The three factors as centrifuge temperature; speed and duration on varying
proportions were studied and the resultant effect was recorded. The three factors interaction of centrifuge temperature; speed and duration was tested for F value. 79
4.6.4.1
Crude gel recovery
Table 4.18 presents the combine effect of centrifuge temperature; speed and duration on crude gel recovery and their statistical analysis are given in Appendix A. The statistical analysis shows that the combine effect of centrifuge temperature; speed and duration on, crude gel recovery is found to be significant. Table 4.18 Combine effect of centrifuge temperature, speed and duration on crude gel recovery (%) Centrifuge duration (min)
Centrifuge
Centrifuge
temperature
speed
(0 C)
(rpm)
5
2000
45.33
47.81
54.19
5000
50.69
58.39
62.94
10000
63.67
68.25
69.92
2000
45.78
48.53
51.61
5000
53.36
59.42
64.31
10000
63.97
66.33
69.06
2000
46.42
49.86
52.11
5000
55.11
61.67
62.58
10000
64.83
66.97
70.47
10
32
10
20 Crude gel recovery (%)
S.Em.
0.638
CD @ 5 %
1.790 Sig.
Test
4.6.4.2
30
Pure gel recovery
Table 4.19 presents the combine effect of centrifuge temperature; speed and duration on pure gel recovery and their statistical analysis are given in Appendix B. The statistical analysis shows that the combine effect of centrifuge temperature; speed and duration on, pure gel recovery, is found to be non significant. 80
Table 4.19 Combine effect of centrifuge temperature, speed and duration on pure gel recovery (%) Centrifuge duration (min)
Centrifuge
Centrifuge
temperature
speed
(0 C)
(rpm)
5
2000
32.11
35.61
38.06
5000
37.81
42.81
45.17
10000
46.92
48.81
51.94
2000
33.53
34.86
39.11
5000
38.56
41.61
45.03
10000
46.06
48.06
51.58
2000
33.00
35.50
39.44
5000
39.00
41.81
44.72
10000
45.94
48.06
51.11
10
32
10
20 Pure gel recovery (%)
S.Em.
0.491
CD @ 5 %
1.375 NS
Test
4.6.4.3
30
Viscosity of gel
Table 4.20 presents the combine effect of centrifuge temperature; speed and duration on viscosity of gel and their statistical analysis are given in Appendix C. The statistical analysis shows that the combine effect of centrifuge temperature; speed and duration on, viscosity of gel is found to be significant.
81
4.6.4.4
Refractive index of gel
Table 4.21 presents the combine effect of centrifuge temperature; speed and duration on refractive index of gel and their statistical analysis are given in Appendix D. The statistical analysis shows that the combine effect of centrifuge temperature; speed and duration on, refractive index of gel, is found to be significant.
Table 4.20
Combine effect of centrifuge temperature, speed and duration on viscosity of gel (Stokes) Centrifuge duration (min)
Centrifuge
Centrifuge
temperature
speed
(0 C)
(rpm)
5
2000
1.367
1.327
1.233
5000
1.367
1.297
1.375
10000
1.495
1.427
1.434
2000
0.877
0.896
0.905
5000
0.954
0.939
0.889
10000
0.949
0.930
0.924
2000
0.629
0.653
0.699
5000
0.701
0.692
0.688
10000
0.733
0.726
0.743
10
32
10
20
30
Viscosity of gel (Stokes)
S.Em.
0.150
CD @ 5 %
0.043 Sig.
Test
82
4.6.4.5
Optical density of gel
Table 4.22 presents the combine effect of centrifuge temperature; speed and duration on optical density of gel and their statistical analysis are given in Appendix E. The statistical analysis shows that the combine effect of centrifuge temperature; speed and duration on, optical density of gel, is found to be non-significant. Table 4.21 Combine effect of centrifuge temperature, speed and duration on refractive index of gel Centrifuge
Centrifuge
temperature
speed
(0 C)
(rpm)
5
2000
1.33750
1.33667
1.33638
5000
1.33630
1.33618
1.33595
10000
1.33562
1.33543
1.33488
2000
1.33718
1.33692
1.33683
5000
1.33605
1.33598
1.33568
10000
1.33542
1.33537
1.33492
2000
1.33738
1.33678
1.33632
5000
1.33602
1.33593
1.33578
10000
1.33560
1.33535
1.33510
10
32
Centrifuge duration (min) 10
20
30
Refractive index of gel
S.Em.
8.04 x 10-5
CD @ 5 %
2.26 x 10-4 Sig.
Test
83
Table 4.22 Combine effect of centrifuge temperature, speed and duration on Optical density of gel (abs) Centrifuge duration (min)
Centrifuge
Centrifuge
temperature
speed
(0 C)
(rpm)
5
2000
0.251
0.246
0.241
5000
0.243
0.238
0.233
10000
0.233
0.231
0.229
2000
0.254
0.249
0.248
5000
0.248
0.242
0.238
10000
0.239
0.237
0.234
2000
0.257
0.254
0.252
5000
0.248
0.245
0.242
10000
0.237
0.235
0.231
10
32
10
30
Optical density of gel (abs)
1.05 x 10-5
S.Em.
0.003
CD @ 5 %
NS
Test
4.6.4.6
20
TSS content of gel
Table 4.23 presents the combine effect of centrifuge temperature; speed and duration on TSS of gel and their statistical analysis are given in Appendix F. The statistical analysis shows that the combine effect of centrifuge temperature; speed and duration on, TSS content of gel is found to be non-significant.
84
Table 4.23 Combine effect of centrifuge temperature, speed and duration on TSS of gel (Brix) Centrifuge duration (min)
Centrifuge
Centrifuge
temperature
speed
(0 C)
(rpm)
5
2000
1.37
1.35
1.35
5000
1.30
1.45
1.50
10000
1.43
1.42
1.45
2000
1.38
1.38
1.32
5000
1.33
1.38
1.40
10000
1.43
1.43
1.43
2000
1.35
1.25
1.23
5000
1.30
1.37
1.30
10000
1.27
1.40
1.30
10
32
10
20 TSS of gel (Brix)
S.Em.
0.037
CD @ 5 %
0.103 NS
Test
4.7
30
Overall effect of acetone, centrifuge temperature, speed and duration on gel extraction process The overall effect of all four factors i.e. level of acetone, centrifuge
temperature; speed and duration on different quality parameters namely crude and pure gel recovery, viscosity, refractive index, optical density and TSS were studied and the resultant effect is reported in Table 4.24 to 4.29 and their statistical analysis are given in Appendix A - F.
85
4.7.1
Crude gel recovery Table 4.24 presents the overall effect of acetone, centrifuge temperature;
speed and duration on crude gel recovery and their statistical analysis are given in Appendix - A. The maximum crude gel recovery i.e. 71.33 % is found at 32 0C temperature, 10000 rpm speed and 30 min duration with 10 % of acetone treatment and minimum recovery of 42.50 % is found at 5 0 C temperature, 2000 rpm speed and 10 min duration for without acetone treatment. 10 % addition of acetone to pulp increases the crude gel recovery. It is also observed that increase in temperature; centrifuge speed and duration increases the crude gel recovery (Table 4.24). Table 4.24
Overall effect of acetone, centrifuge temperature, speed and duration on crude gel recovery (%)
Centrifuge
Centrifuge
temperature
speed
0
C
(rpm)
Centrifuge duration (min) 10
20
30
10
Without acetone
20
30
With acetone (10%)
Crude gel recovery (%) 5
10
32
2000
42.50
46.50
51.89
48.17
49.11
56.50
5000
45.72
57.33
61.28
55.67
59.44
64.61
10,000
63.89
66.33
70.28
63.44
70.17
69.56
2000
44.33
47.06
48.72
47.22
50.00
54.50
5000
51.11
61.56
64.78
55.61
57.28
63.83
10,000
63.94
65.22
67.89
64.00
67.44
70.22
2000
45.22
50.11
51.67
47.61
49.61
52.56
5000
54.50
62.94
62.67
55.72
60.39
62.50
10,000
64.22
66.28
69.61
65.44
67.67
71.33
S.Em.
0.902
CD @ 5 %
2.532 NS
Test
86
The statistical analysis shows that the overall effect of centrifuge temperature; speed and duration on, crude gel recovery is found to be non-significant at 5 % Cd. 4.7.2
Pure gel recovery The overall effect of acetone, centrifuge temperature; speed and duration on
pure gel recovery is given in Table 4.25 and statistical analysis in Appendix - B. The highest pure gel recovery (53.72 %) is recorded at 5 0C temperature, 10000 rpm speed and 30 min duration for 10 % acetone treatment and lowest recovery of 30.83 % is achieved at 5 0C temperature, 2000 rpm speed and 10 min duration for without acetone treatment. Addition of acetone to pulp increases the pure gel recovery. It is observed that increase in centrifuge temperature, speed and duration increases the pure gel recovery (Table 4.25). Table 4.25 Overall effect of acetone, centrifuge temperature, speed and duration on pure gel recovery (%) Centrifuge Centrifuge temperature speed 0 C (rpm) 5
10
32
Centrifuge duration (min) 10 20 30 10 20 30 Without acetone With acetone (10 %) Pure gel recovery (%)
2000
30.83
34.39
36.67
33.39
36.83
39.44
5000
33.67
41.28
44.39
41.94
44.33
45.94
10,000
44.17
48.33
50.17
49.67
49.28
53.72
2000
32.06
34.50
37.67
35.00
35.22
40.56
5000
37.33
40.89
43.67
39.78
42.33
46.39
10,000
44.67
46.94
50.00
47.44
49.17
53.17
2000
32.78
36.72
40.72
33.22
34.28
38.17
5000
38.17
40.28
43.89
39.83
43.33
45.56
10,000
44.44
46.44
50.06
47.44
49.67
52.17
S.Em.
0.694
CD @ 5 %
1.947 Sig.
Test 87
The statistical analysis shows that the overall effect of acetone, centrifuge temperature; speed and duration on, pure gel recovery, is found to be significant at 5 % Cd. 4.7.3 Viscosity of gel The overall effect of acetone, centrifuge temperature; speed and duration on viscosity of gel is presented in Table 4.26 and their statistical analysis is given in Appendix - C. The statistical analysis shows that the overall effect of centrifuge temperature; speed and duration on, viscosity of gel is found to be significant. The maximum viscosity was recorded 2.355 Stokes at 5 0C temperature, 10000 rpm centrifuge speed and 10 min duration for 10 % addition of acetone treatment and minimum viscosity was found 0.521 Stokes at 32 0C temperature, 2000 rpm centrifuge speed and 10 min duration for without addition of acetone treatment. It was observed that increase in centrifuge temperature decreased the viscosity of gel and addition of acetone resulted increase in viscosity of gel. There was no uniform trend in viscosity for varying centrifuge speed and duration (Table 4.26).
4.7.4
Refractive index of gel Table 4.27 presents the overall effect of acetone; centrifuge temperature; speed
and duration on refractive index of gel and their statistical analysis are given in Appendix- D. The statistical analysis shows that the overall effect of centrifuge temperature; speed and duration on, refractive index of gel is found to be significant. The minimum refractive index was recorded 1.33427 at 5 0C temperature, 10000 rpm centrifuge speed and 30 min duration for 10 % addition of acetone treatment and maximum refractive index was found 1.33760 at 10 0C centrifuge temperature and 2000 rpm centrifuge speed and 20 min duration for 10 % addition of acetone treatment. It was observed that increase in centrifuge speed, and duration decreased 88
Table 4.26 Overall effect of acetone, centrifuge temperature, speed and duration on viscosity of gel (Stokes) Centrifuge temperature (0C)
Centrifuge speed (rpm)
5
2000
Centrifuge duration (min) 10 20 30 10 20 30 Without acetone With acetone (10 %) Viscosity of gel (Stokes) 0.635 0.647 0.616 2.098 2.007 1.851
5000
0.650
0.642
0.651
2.085
1.951
2.098
10,000
0.635
0.659
0.675
2.355
2.194
2.192
2000
0.584
0.591
0.582
1.170
1.201
1.227
5000
0.592
0.584
0.567
1.315
1.295
1.211
10,000
0.595
0.584
0.596
1.304
1.276
1.252
2000
0.521
0.543
0.535
0.738
0.762
0.862
5000
0.543
0.538
0.540
0.859
0.846
0.837
10,000
0.545
0.548
0.563
0.921
0.904
0.923
10
32
S.Em.
0.022
CD @ 5 %
0.061 Sig.
Test
the refractive index of gel, and addition of acetone also results in increase in refractive index of gel. There was no uniform trend in refractive index for varying centrifuge temperatures (Table 4.27).
4.7.5
Optical density of gel Table 4.28 presents the overall effect of acetone, centrifuge temperature; speed
and duration on optical density of gel and their statistical analysis are given in Appendix - E. The statistical analysis shows that the overall effect of centrifuge temperature; speed and duration on, optical density of gel is found to be nonsignificant. The minimum optical density was recorded 0.218 at 5 0C temperature, 10000 rpm speed for 20 and 30 min duration, 5000 rpm speed for 30 min duration in without addition of acetone treatment and maximum optical density was found 0.264 89
at 10 0C centrifuge temperature and 2000 rpm speed and 10 min duration for 10 % of acetone treatment. It was observed that increase in centrifuge speed; duration decreased the optical density of gel whereas addition of Acetone results in increase in optical density of gel. Non-uniform trend in optical density was observed for varying centrifuge temperatures (Table 4.28).
Table 4.27 Overall effect of acetone, centrifuge temperature, speed and duration on refractive index of gel CFT
CFS
(0C)
(rpm)
Centrifuge duration (min) 10
20
30
10
Without acetone
20
30
With acetone (10 %)
Refractive index of gel 5
10
32
2000
1.33740
1.33613
1.33607
1.33760
1.33720
1.33670
5000
1.33607
1.33600
1.33577
1.33653
1.33637
1.33613
10,000
1.33570
1.33560
1.33550
1.33553
1.33527
1.33427
2000
1.33673
1.33623
1.33623
1.33763
1.33760
1.33743
5000
1.33613
1.33610
1.33587
1.33597
1.33587
1.33550
10,000
1.33563
1.33567
1.33537
1.33520
1.33507
1.33447
2000
1.33693
1.33653
1.33607
1.33783
1.33703
1.33657
5000
1.33593
1.33590
1.33587
1.33610
1.33597
1.33570
10,000
1.33587
1.33567
1.33540
1.33533
1.33503
1.33480
S.Em.
1.137 x 10-4
CD @ 5 %
3.19 x 10-4 Sig.
Test CFT = Centrifuge temperature CFS = Centrifuge speed
90
Table 4.28 Overall effect of acetone, centrifuge temperature, speed and duration on Optical density of gel (abs) Centrifuge
Centrifuge Centrifuge duration (min)
temperature
speed
(0C)
(rpm)
10
20
30
10
Without acetone
20
30
With acetone (10 %)
Optical density of gel (abs) 5
10
32
2000
0.241
0.233
0.225
0.261
0.259
0.257
5000
0.231
0.224
0.218
0.254
0.251
0.247
10,000
0.220
0.218
0.218
0.247
0.243
0.240
2000
0.244
0.239
0.238
0.264
0.259
0.258
5000
0.240
0.231
0.225
0.256
0.253
0.252
10,000
0.230
0.229
0.225
0.247
0.245
0.244
2000
0.251
0.250
0.247
0.262
0.258
0.257
5000
0.241
0.238
0.233
0.254
0.252
0.250
10,000
0.225
0.224
0.221
0.249
0.246
0.241
0.0014879
S.Em.
0.004
CD @ 5 %
NS
Test 4.7.6
TSS content of gel Table 4.29 presents the overall effect of all the parameters under study on TSS
content of gel and their statistical analysis are given in Appendix - F. The statistical analysis shows that the overall effect of all the parameters on TSS content of gel is found to be significant. The minimum TSS content was recorded 0.77 at 5 0C centrifuge temperatures, 5000 rpm centrifuge speed, and 10 min duration for without addition of acetone treatment and maximum TSS content was found 1.97 at 10 0C temperature and 5000 rpm centrifuge speed and 30 min duration, 10 0C temperature and 10000 rpm centrifuge speed and, 20 and 30 min duration for 10 % addition of 91
acetone treatment. Addition of acetone increased the TSS content of gel. It was observed that there was no uniform trend in TSS content for varying centrifuge temperature, speed and duration (Table 4.29). Table 4.29
Overall effect of acetone, centrifuge temperature, speed and centrifuge duration on TSS of gel (brix)
Centrifuge
Centrifuge
temperature
speed
(0C)
(rpm)
Centrifuge duration (min) 10
20
30
10
Without acetone
20
30
With acetone (10 %)
TSS of gel (Brix) 5
10
32
2000
0.87
0.87
0.80
1.87
1.83
1.90
5000
0.77
1.00
1.10
1.83
1.90
1.90
10,000
1.03
0.90
0.93
1.83
1.93
1.97
2000
0.93
1.00
0.93
1.83
1.77
1.70
5000
0.93
0.87
0.83
1.73
1.90
1.97
10,000
0.90
0.97
0.93
1.97
1.90
1.93
2000
1.03
0.90
0.90
1.67
1.60
1.57
5000
1.03
1.00
0.87
1.57
1.73
1.73
10,000
0.83
1.07
0.97
1.70
1.73
1.63
S.Em.
0.052
CD @ 5 %
0.145 Sig.
Test
92
4.8
Improved process for extraction of gel from Aloe vera leave
As a result of studies conducted on various Aloe vera leave processing parameters such as level of acetone, centrifuge temperature, centrifuge speed and centrifuge duration of gel extraction an improved technique of aloe vera leave gel extraction is suggested through a flow diagram given in Figure 4.12.
Harvest each leaf by hand with knife at 98.88 % moisture content (wb) and pulled carefully from the mother plant so as not to break the rind and also to avoid bio-degradation of Aloe vera leaves. The harvested leaves are kept in the icebox at 4 5 0C and transport to the working place in a covered polyethylene bag to avoid oxidation or contamination. The aloe vera leaves are thoroughly washed by fresh water to remove dust and foreign matter and later graded by hand. The outer skin and the exudates (20 to 30 mm part at the top and bottom of Aloe vera leaf) of the leaves are removed manually with the help of knife to form fillet. The fillets are ground in the domestic blander (Boss Make) at 1000 rpm to obtained crushed and homogenized pulp. The 60 ml pulps on volume basis are centrifuged in cooling type centrifuge at 5 0
C and 10000 rpm speed for 30 min for separation of crude gel and fiber. The 0.1 g
charcoal is mixed with 100 ml crude gel for purification. The vacuum filtration method is used to obtain pure gel from crude gel. The pure gel is collected in the bottle and shown in Plate 4.4. The packed bottles need to be stored in cool and dry place at 4 0C.
93
Aloe vera leaf
Fresh water
Washing
Dirt
Trimming
Tips and butts
Filleting
5 0C temperature 10000 rpm speed 30 min duration
Rind and exudate
Gel extraction by Centrifuge
Pulp
Crude gel
Charcoal (@ 0.1 g /100ml crude gel)
Whatman Filter No. 4
Purification
Vacuum Filtration
Viscosities:0.675 (Stokes) Refractive index:1.33550 Optical density:0.218 (abs) TSS content:0.93 (Brix)
Pure gel
Storage at 4 0C in air tight bottle Figure. 4.12 Flow chart developed for Aloe vera Gel Extraction Process
94
CHAPTER V SUMMARY AND CONCLUSION
Aloe vera is a succulent that belongs to the liliaceae family. It is one of the 250 known species of aloes, referred to by the scientific terms of Aloe vera and Aloe barbadensis. The semi-tropical plant, Aloe vera barbadensis Miller from the Lily (Liliaceae) family has a long and illustrious history dating from biblical times. It has been mentioned throughout recorded history and given a high ranking as an allpurpose herbal plant. Aloe's thick, tapered, spiny leaves grow from a short stalk near ground level.
Aloe vera gel is the commercial name given to the fiber free mucilaginous exudate extracted from the hydroparenchyma of the succulent leaves of Aloe vera (Aloe barbadensis Miller). Aloe vera Gel (a clear, jelly-like material) is derived from tissue that comprises the inner portion of the leaves. The clourless mucilaginous gel obtained from the parenchymatous cells in the fresh leaves of Aloe vera. It is slightly bitter and odourless. The gel loses its transparency if extracted after 3 hours of plucking the leaves. Commercially available aloe gel is stabilized for maintenance of its potency.
The original commercial use of the Aloe plant was in the production of a latex substance called Aloin, a yellow sap used for many years as a laxative ingredient. This terminology created much confusion later when Aloe's other main ingredient, Aloe Gel, a clear colorless, semi-solid gel, was stabilized and marketed. This Aloe vera Gel, beginning in the 50's, has gained respect as a commodity used as a base for nutritional drinks, as a moisturizer, and a healing agent in cosmetics. The exudate of Aloe vera L., Liliaceae, is used for numerous medical and cosmetic applications since ancient times. 95
Aloe vera is used as an antiseptic, bactericidal, calming agent, detoxifiers, a natural cleanser and dilates capillaries in medical science, a s moisturizer for skin care product and hair care product in cosmetics and over seventy-five nutritional compounds occur naturally within the plant, so it is used as aloe juice for nutrition purpose. The International Aloe Science Council has solidified its dedication to providing the world with the highest quality Aloe. The IASC has a dedicated group of professionals committed to the further growth, research and marketing of quality Aloe vera Gel and Aloe products made from this Gel.
Hand filleting and whole leaf processing, the two types of Aloe vera gel extraction methods are prevalent. Gel is extracted either cold process or hot process. Only recently have processing methods using the entire whole leaf been perfected so the undesirable elements can be selectively removed, while maximizing the desired constituents.
Among
the
desirable
constituents
are
the
polysaccharides
(glucomannans), glycoproteins and associated growth factors. However, over 95% of the Aloes on the market today still use only the inner gel and stabilize the Aloe in a high-heat process that degrades some of the enzymes, polysaccharides and mucopolysaccharides. High heat (pasteurisation and/or autoclave methods) breaks down the constituents in Aloe that are the most valuable for healing. Heat also kills the live enzymes necessary for digestion. Most Aloes are heat processed.
Looking to the importance of the Aloe products such as creams, ointments, juices, and shampoo containing the Aloe gel, it is very much essential to study the post harvest technology of the Aloe vera plant.
The expanding Aloe industry
urgently needs to develop test procedures and a reliable database so that a product claiming to have Aloe could be tested and certified. According to Reynolds et al., (1999), there is no scientific literature available in the processing of leaf gel. What so ever processes either gel extraction or gel stabilization is patented, so it is essential to develop the gel extraction process, which may useful for industrial community. 96
Keeping this in view, the present investigation was undertaken with the following specific objectives. • To study the physical and chemical properties of Aloe vera leaves. • To develop gel extraction process. • To evaluate the gel extraction process Aloe vera barbadensis was used as a raw material for gel extraction studies. The selection of material was based on the availability and good quality gel obtained for the end use. Different physical properties of Aloe vera leaf namely, size in terms of length, width and thickness, pulp weight, gel recovery, apparent volume were determined. Aloe vera gel physical properties like, moisture content, fibre content, refractive index, optical density was measured. Chemical properties of gel like total solids; sugar content, pH and were determined.
For the extraction of gel from Aloe vera leaves, principle of centrifugation was employed. Process parameters like centrifuge temperature, centrifuge speed and centrifuge duration on varying levels tested and optimum process parameters decided on the basis of quality parameters of extracted gel like recovery, viscosity, optical density, refractive index and TSS content.
The physico-chemical properties of extracted gel, namely; moisture content, fibre content, refractive index, optical density, viscosity, and TSS content were determined to evaluate the quality of gel. The experiment was planned using a 4 factor Completely Randomised Design. The treatments consisted of two levels of Acetone i.e. without addition of Acetone and 10 % addition of acetone, three level of centrifuge temperature i. e. 5, 10, and 32
0
C (Ambient), three level of centrifuge
speed i.e. 2000, 5000 and 10,000 rpm and three level of centrifuge duration i. e. 10, 20 and 30 min with 3 replications. The optimum proportion of Acetone, centrifuge 97
temperature, centrifuge speed and centrifuge duration was decided on the basis of quality parameters of gel. The results obtained are analysed statistically.
The following conclusions could be drawn from the present investigation. 1. The average value of length, width and thickness of Aloe vera leaf was found to be 55.98, 9.39 and 2.68 cm respectively with apparent volume of leaf 371.75 cc.
2. The average leaf weight, pulp weight and pulp recovery was found to be 0.459 kg, 0.23 kg and 50.04 % respectively.
3. The moisture content in pulp and gel was found to be 98.88% and 99.8 %, while fibre content was found to be 1.117% and 0.2% respectively.
4. The pH, TSS and Sugar content 6.389, 1.399 Brix and 1.9125 (Total sugar) and 0.0259 (Reducing sugar) from Aloe vera gel respectively.
5. The 10 % addition to pulp increased the crude gel recovery (2.8 %), pure gel recovery (2.3 %) and viscosity (136.5 %) which shown favourable results but the increase in refractive index (0.0067 %), optical density (12.93 %)and TSS (93.55 %) shown unfavourable results for the extraction process as these are indices for purity of gel.
6. Temperature is the main factor for processing of Aloe vera and particularly gel extraction process. Main effect of temperature was on viscosity of gel and also affected the gel recovery, optical density, refractive index and TSS content marginally. There was no significant difference in different temperature for crude gel recovery. It was 57.91 % at 5 0C, 58.04 % at 10 0C and 58.89 % at 32 0
C temperature gel extraction process. The pure gel recovery for different
temperature was as par and recorded as 42.14 % at 5 0C, 42.04 % at 10 0C and 98
42.06 % at 32 0C temperature gel extraction process. Total soluble solid of gel was found 1.4, 1.39 and 1.31 for 5 0C, 10 0C and 32 0C (Ambient) temperature respectively.
7. Viscosity of extracted gel was largely affected by different temperature of centrifuge operation. Viscosity was recorded 96.69 % higher at 5
0
C
temperatures than 32 0C temperatures and 49.13 % higher than the gel extraction carried out at 10 0C temperature. 8. Refractive index of gel had not shown major difference on varying temperature. It was 1.33610 for 5 0C, 1.33604 for 10 0C and 1.33603 for 32 0C (Ambient) temperature. Optical density of gel increased with increase in temperature. 9. Crude gel recovery was increased with increase in centrifuge speed. The highest crude gel recovery was found 67.05 % at 10,000 rpm, which was 17.98 % more than extraction at 2000 rpm and 8.33 % more with respect to gel extraction at 5000 rpm speed of centrifuge At 10,000 rpm speed, the pure gel recovery was highest (48.72 %) and lowest at 2000 rpm (35.69%) and at 5000 rpm 41.83 % pure gel was obtained from the Aloe vera leaf pulp. 10. There was recorded, effect on viscosity with varying speed of centrifuge. Viscosity of gel was obtained 1.04 Stokes at highest speed (10,000 rpm) and 0.954 at lowest speed (2000 rpm). At 5000 rpm speed it was found 0.989 Stokes Refractive index of gel decreased as the speed of centrifuge increased. It was found lowest at 10,000 rpm speed (1.33530) and highest at 2000 rpm speed (1.33689). But during 5000 rpm centrifuge operation refractive index was found 1.33599. Optical density of gel decreased as the speed of centrifuge increased. It was found lowest at 10,000 rpm speed (0.234) and highest at 2000 rpm speed (0.250). Total soluble solid content was increased with increase in
99
centrifuge speed. It was found 1.33, 1.37 and 1.4 for 2000, 5000 and 10,000 rpm speed respectively.
11.
The highest crude gel recovery was found 61.91 % with 30 min, which was 7.56 % more than extraction with 10 min and 3.33 % more with respect to gel extraction at 20 min duration of centrifuge With 30 min duration, the pure gel recovery was highest (45.13 %) and lowest with 10 min (39.21%). and with 20 min centrifuge duration 41.90 % pure gel was obtained from the Aloe vera leaf pulp.
12.
Viscosity of gel was obtained 1.008 Stokes with short duration and 0.987 with long duration (30 min). This shown that as the centrifuge duration increased; there was a reduction in viscosity of gel. Refractive index of gel decreased as the duration of centrifuge increased. It was found lowest at 30 min duration (1.33634) and highest at 10 min duration (1.33576). Optical density of gel decreased as the duration of centrifuge increased. It was found lowest with 30 min duration (0.239) and highest with 10 min duration (0.245). Total soluble solid content was increased with increase in centrifuge speed. It was found 1.35, 1.38 and 1.36 with 10, 20 and 30 min duration respectively.
13.
It was recommended that the extraction of gel from Aloe vera by the method of centrifuge should be carried out at 5 0C centrifuge temperatures, 10,000 rpm centrifuge speed and 30 min centrifuge duration without addition of Acetone to pulp so as to get higher gel higher gel recovery (50.17 %) and good quality of gel i.e. Viscosities: 0.675 (Stokes), Refractive index: 1.33550, Optical density: 0.218 (abs) and TSS content: 0.93 (Brix).
100
REFERENCES Anonymous, (1967). The chemical assay of aloes. Analyst 92: 593-96 Anonymous, (1999). Thai Food Composition Tables (1999), Institute of Nutrition, Mahidol University (INMU). Agarwala, O.P. (1997). Whole leaf aloe gel vs. standard aloe gel. (Differences in processing techniques yield divergent properties, costs, applications), Jr. of Drugs and Cosmetics.02/01/1997. Ashleye, F.L. (1983). Applying heat during processing the commercial Aloe vera gel. Erde International 1: 40-44 Bruneton, J. (1995). Pharmacognosy, phytochemistry, medicinal plants. Paris, Lavoisier. Davis, R.H., Donato, J.J. Hartman, G.M. and Haas, R.C. (1994). Anti-inflammatory and wound healing of growth substance in Aloe vera. Journal of the American Pediatric Medical Association, 84:77–81 Davis, R.H., Rosenthal K.Y., Cesario, L.R. and Rouw, G.A. (1989). Processed Aloe vera administered topically inhibits inflammation. J. Am. Podiatr Med. Assoc. Aug; 79(8): 395-7 Farooqi, A. A. and Sreeramu, B.S. (2001). Cultivation of Medicinal and Aromatic Crops. University Press (India) Ltd Banglore, pp: 21-26 Gorloff, D.R. (1983). Study of the organoletic properties of the exuded mucilage from the Aloe barbadensis leaves. Erde International pp: 46-59 Gowda, D.C., Neelisiddaiah, B., Anjaneyalu, Y.V. (1979). Structural Studies of polysaccharides from Aloe vera. Carbohydrates Res. 72: 201-205 Grindlay, D. and Reynolds T. (1986). The Aloe vera phenomenon: a review of the properties and modern uses of the leaf parenchyma gel. Journal of ethnopharmacology, 16:117–151 101
Herlina, L. (2001). Incorporating private - public relationship into food technology development: creating added value to “Aloe vera” product SEAG Symposium, 27.-31.8.2001, Los Banos, Philippines. Hernandez-Cruz, Luis Rodolfo, Raul Rodriguez Garcia, Diana Jasso de Rodriguez, and José Luis Angulo-Sánchez (2002). Aloe vera Response to Plastic Mulch and Nitrogen Reprinted from: Trends in new crops and new uses. 2002. J. Janick and A. Whipkey (eds.). ASHS Press, Alexandria, VA Joshi, S. P. (1998). Chemical constituents and biological activity of Aloe barbadensis: a review. Jr. of Medicinal and Aromatic Plant Sciences 20:768-773 Mohsenin, N. N. (1980). Physical properties of plant and animal materials. Gordon and Breach science publishers, New York Morton, J. F. (1961). Folk uses and commercial exploitation of Aloe leaf pulp. Economic Botany 15: 311-319 Pierce, R. F. (1983). Comparison between the nutritional contents of the aloe gel from conventional and hydroponically grown plants. Erde international, 1:37–38 Reynolds, T. and Dweck, A. C. (1999). Aloe vera leaf gel: a review update. Journal of Ethnopharmacology 68: 3-37 Rowe, T.D. and Park, L. M. (1941). Phytochemical study of Aloe vera leaf. Journal of the American Pharmaceutical Association, 30:262–266 Sachedina, H. A. (1998). An investigation of the bio-enterprise potential of endemic Tanzanian Aloe: conservation through cultivation of East African medicinal plants for integrated healthcare and sustainable development. M Sc Thesis, University of Oxford. Sachedina, H. and Bodeker, G. (1999). Wild Aloe harvesting in South Africa. J Altern Complement Med. Apr; 5(2): 121-3 102
Sadasivam, S. and Manickam, A. (1996). Biochemical Methods. 2nd Ed. New Age International (P) Ltd. New Delhi. Sangani, V. P. (1997). Studies on extraction of essential oil from cumin. Unpublished M. Tech (A.P.E.), thesis, Junagadh: 1- 83 Shafi, N., Khan, L. and Khan, G. A. (2000). Commercial extraction of gel from Aloe vera (L) leaves. J. Chem. Soc. 22(1): 47-49 Simal, S., Femenia, A., Llull, P., and Rossello, C. (2000). February. Dehydration of Aloe vera: simulation of drying curves and evaluation of functional properties. Journal of food engineering. 43(2): 109-114 Thimaiah, S. K. (1999). Standard Methods of Biochemical Analysis, Kalyani Publications, New Delhi 1999 Edition. Pp: 45-46 Waller, G.R., Mangiafico, S. and Ritchey, C. R. (1978). A Chemical investigation of Aloe barbadensis Miller. Proceedings of the Oklahoma Academy of science 58: 69-76 Wang, Y.T., and Strong, K J. (1993). Monitoring physical and chemical properties of freshly harvested field-grown Aloe vera leaves. A preliminary report. Phytotherapy Research 7: S1-S4 Yaron, A. (1993). Characterisation of Aloe vera gel composition and autodegradation and stabilization of the natural fresh gel. Phytotherapy Research 7: S11-S13 Yaron, A., Cohen E. and Arad (Malis), S. (1992). Stabilization of Aloe vera gel by interaction with sulfated polysaccharides from red microalgae with xanthan gum. J. Agrc. Food Chem. 40: 1316-1320 Web pages visited Aloe Vera - CRH International, Inc http://www.aloealoe.com/ Aloe vera Company, UK. Properties of Aloe vera constituents. http://www.aloevera.co.uk/. Accessed April, 2003 103
Aloe ferox plant. http://www.aloeferoxafrica.com Accessed July, 2001 Danhof, Ivan E. (2000). Aloe vera, The Whole Leaf Advantage Excerpts http://www.wholeleaf.com Accessed January, 2000 Denk, M. (2000). Plants with human uses. http://www.bio.gasou.edu/ Accessed March, 2003 Gilman, E. F. (1999). Aloe barbadensis. http://www.hort.ifas.ufl.edu/ Accessed 30. 3. 2003 Moore, Toni. (2001). Aloe barbadensis. http://www.ag.arizona.edu/ Accessed March, 2003 Shih-Jen, Chiou (2003). Investigation of nutrient contents of Aloe vera and effect of additives on nonenzymatic browning of blanched Aloe vera. Unpublished thesis, Food and Nutrition Department, Providence University,China http://ethesys.lib.pu.edu.tw/ETD-db/ETD-search/ The Total Process Aloe vera Story http://www. bonasana.com, Accessed December, 2001 Typical composition of Aloe vera gel, Garuda International, Inc. (1998). http://www.garudaint.com Aloe vera Processing and Product Manufacturing American Quality Aloe, Made in the U.S.A. http://www.aqaloe.com Accessed 2004 Delta International, http://www.shantidatta.com/ Accessed 2004 http://www.drwolfe.com/ Accessed 2003 http://www.carringtonlabs.com/ http://www.terrylabs.com http://www.quikpage.com http://www.aloecorp.com 104
APPENDIX- A FACTORIAL CRD HAVING 4 FACTORS ANALYSIS OF VARIANCE Effect of acetone, centrifuge temperature, speed and duration on Crude gel recovery (%) for extraction process Source variation
of
d.f.
S.S.
M.S.S.
Cal. F
A
1
150.543
150.543
61.629
5.39
0.171 0.479 Sig.
T
2
30.704
15.352
6.285
3.12
0.213 0.597 Sig.
V
2
8745.568 4372.784 1790.122
3.12
0.213 0.597 Sig.
D
2
1549.853 774.926
317.238
3.12
0.213 0.597 Sig.
AxT
2
54.339
27.169
11.123
3.12
0.301 0.844 Sig.
AxV
2
24.833
12.416
5.083
3.12
0.301 0.844 Sig.
AxD
2
32.299
16.149
6.611
3.12
0.301 0.844 Sig.
TxV
4
41.679
10.420
4.266
2.43
0.368 1.034 Sig.
TxD
4
41.916
10.479
4.290
2.43
0.368 1.034 Sig.
VxD
4
130.906
32.727
13.398
2.43
0.368 1.034 Sig.
AxTxV
4
67.984
16.996
6.958
2.43
0.521 1.462 Sig.
AxTxD
4
10.355
2.589
1.060
2.43
0.521 1.462 NS
TxVxD
8
49.757
6.220
2.546
2.01
0.638 1.790 Sig.
AxVxD
4
98.543
24.636
10.085
2.43
0.521 1.462 Sig.
A x T x V x D
8
28.322
3.540
1.449
2.01
0.902 2.532 NS
Error
108 263.815
2.443
Total
80
C.V., % = 2.68
105
S. Em. +
C.D. @ Test 5%
Tab. F 1%
APPENDIX- B FACTORIAL CRD HAVING 4 FACTORS ANALYSIS OF VARIANCE Effect of acetone, centrifuge temperature, speed and duration on Pure gel recovery (%) for gel extraction process C.D. Source of Tab. F S. Em. d.f. S.S. M.S.S. Cal. F @ Test variation 1% + 5% A
1
214.705
214.705
148.713
5.39
0.131
0.368 Sig.
T
2
0.253
0.127
0.088
3.12
0.164
0.459
V
2
4587.500 2293.750 1588.735
3.12
0.164
0.459 Sig.
D
2
947.813
473.907
328.245
3.12
0.164
0.459 Sig.
AxT
2
35.114
17.557
12.161
3.12
0.231
0.649 Sig.
AxV
2
29.976
14.988
10.381
3.12
0.231
0.649 Sig.
AxD
2
20.618
10.309
7.140
3.12
0.231
0.649 Sig.
TxV
4
12.502
3.125
2.165
2.43
0.283
0.795
NS
TxD
4
9.689
2.422
1.678
2.43
0.283
0.795
NS
VxD
4
14.621
3.655
2.532
2.43
0.283
0.795 Sig.
AxTxV
4
25.709
6.427
4.452
2.43
0.401
1.124 Sig.
AxTxD
4
17.493
4.373
3.029
2.43
0.401
1.124 Sig.
TxVxD
8
9.648
1.206
0.835
2.01
0.491
1.376
NS
AxVxD
4
3.439
0.860
0.596
2.43
0.401
1.124
NS
AxTxVx D
8
30.061
3.758
2.603
2.01
0.694
1.947 Sig.
155.926
1.444
Error
108
Total
80
C.V.,% = 2.85
106
NS
APPENDIX- C FACTORIAL CRD HAVING 4 FACTORS ANALYSIS OF VARIANCE Effect of acetone, centrifuge temperature, speed and duration on Viscosity (Stokes) of gel for gel extraction process C.D. Source of S. Em. Tes d.f. S.S. M.S.S. Cal. F Tab. F 1% @ variation + t 5% A
1
26.345
26.345
18703.853
5.39
0.004 0.011 Sig.
T
2
12.702
6.351
4508.783
3.12
0.005 0.014 Sig.
V
2
0.202
0.101
71.590
3.12
0.005 0.014 Sig.
D
2
0.015
0.008
5.367
3.12
0.005 0.014 Sig.
AxT
2
9.157
4.578
3250.468
3.12
0.007 0.020 Sig.
AxV
2
0.133
0.066
47.189
3.12
0.007 0.020 Sig.
AxD
2
0.021
0.010
7.410
3.12
0.007 0.020 Sig.
TxV
4
0.062
0.016
11.013
2.43
0.009 0.025 Sig.
TxD
4
0.039
0.010
6.927
2.43
0.009 0.025 Sig.
VxD
4
0.006
0.002
1.131
2.43
0.009 0.025 NS
AxTxV
4
0.055
0.014
9.793
2.43
0.013 0.035 Sig.
AxTxD
4
0.042
0.010
7.375
2.43
0.013 0.035 Sig.
TxVxD
8
0.069
0.009
6.167
2.01
0.015 0.043 Sig.
AxVxD
4
0.007
0.002
1.296
2.43
0.013 0.035 NS
A x T x V x D
8
0.048
0.006
4.237
2.01
0.022 0.061 Sig.
0.152
0.001
Error
108
Total
80
C.V.,% = 3.77
107
APPENDIX- D FACTORIAL CRD HAVING 4 FACTORS ANALYSIS OF VARIANCE
Effect of acetone, centrifuge temperature, speed and duration on Refractive index of gel for gel extraction process Source of C.D. @ Tab. F Test d.f. S.S. M.S.S. Cal. F S. Em. + variation 1% 5% A
1
3.0x10-7
3.0x10-7
7.803
5.39
2.1x10-5
6.0x10-5
Sig.
T
2
1.7x10-7
8.0x10-8
2.151
3.12
2.7x10-5
7.5x10-5
NS
V
2
6.8x10-5
3.4x10-5 882.212
3.12
2.7x10-5
7.5x10-5
Sig.
D
2
9.1x10-5
4.5x10-6 117.145
3.12
2.7x10-5
7.5x10-5
Sig.
AxT
2
1.1x10-7
6.0x10-8
3.12
3.8x10-5
1.1x10-4
NS
AxV
2
1.3x10-5
6.7x10-6 173.610
3.12
3.8x10-5
1.1x10-4
Sig.
AxD
2
4.6x10-7
2.3x10-7
5.887
3.12
3.8x10-5
1.1x10-4
Sig.
TxV
4
8.7x10-7
2.2x10-7
5.581
2.43
4.6x10-5
1.3x10-4
Sig.
TxD
4
5.5x10-7
1.4x10-7
3.537
2.43
4.6x10-5
1.3x10-4
Sig.
VxD
4
1.7x10-6
4.4x10-7
11.264
2.43
4.6x10-5
1.3x10-4
Sig.
AxTxV
4
1.7x10-6
4.2x10-7
10.903
2.43
6.5x10-5
1.8x10-4
Sig.
AxTxD
4
2.5x10-7
6.0x10-8
1.640
2.43
6.5x10-5
1.8x10-4
NS
TxVxD
8
9.1x10-7
1.1x10-7
2.943
2.01
6.5x10-5
2.3x10-4
Sig.
AxVxD
4
5.5x10-7
1.4x10-7
3.568
2.43
6.5x10-5
1.8x10-4
Sig.
AxTxVx D
8
9.2x10-7
1.1x10-7
2.965
2.01
1.1x10-4
3.2x10-4
Sig.
Error
108 4.2x10-6
4.0x10-8
Total
80
1.463
C.V.,% = 0.015
108
APPENDIX- E FACTORIAL CRD HAVING 4 FACTORS ANALYSIS OF VARIANCE Effect of acetone, centrifuge temperature, speed and duration on optical density (abs) of gel for gel extraction process
d.f.
S.S.
M.S.S.
Cal. F
Tab. F 1%
A
1
6.6x10-3
0.016588
2497.51
5.39
2.8 x 10-4
0.001 Sig.
T
2
1.2x10-3
0.000584
87.944
3.12
3.5 x 10-4
0.001 Sig.
V
2
7.1x10-3
0.003539
532.845
3.12
3.5 x 10-4
0.001 Sig.
D
2
1.3x10-3
0.000636
95.792
3.12
3.5 x 10-4
0.001 Sig.
AxT
2
7.2x10-4
0.000358
53.932
3.12
5.0 x 10-4 0.001 Sig.
AxV
2
7.4x10-5
0.000037
5.554
3.12
5.0 x 10-4
0.001 Sig.
AxD
2
4.3x10-5
0.000022
3.256
3.12
5.0 x 10-4
0.001 Sig.
TxV
4
2.2x10-4
0.000054
8.159
2.43
6.1 x 10-4
0.002 Sig.
TxD
4
3.7x10-5
0.000009
1.402
2.43
6.1 x 10-4
0.002
VxD
4
6.9x10-5
0.000017
2.604
2.43
6.1 x 10-4
0.002 Sig.
AxTxV
4
3.3x10-5
0.000082
12.377
2.43
8.6 x 10-4
0.002 Sig.
AxTxD
4
3.9x10-5
0.000010
1.457
2.43
8.6 x 10-4
0.002
NS
TxVxD
8
5.7x10-5
0.000007
1.064
2.01
8.6 x 10-4
0.003
NS
AxVxD
4
9.3x10-5
0.000023
3.494
2.43
8.6 x 10-4
0.002 Sig.
A x T x V x D
8
7.8x10-5
0.000010
1.474
2.01
1.5 x 10-4
0.004
Error
108
7.2x10-4
0.000007
Total
80
Source variation
of
C.V.,% = 1.07
109
S. Em. +
C.D. @ 5%
Test
NS
NS
APPENDIX- F FACTORIAL CRD HAVING 4 FACTORS ANALYSIS OF VARIANCE
Effect of acetone, centrifuge temperature, speed and duration on TSS content of gel for gel extraction process Source of Tab. F S. Em. C.D. d.f. S.S. M.S.S. Cal. F Test variation 1% + @ 5% A
1
30.507
30.507
3801.608 5.39
0.01
0.027
Sig.
T
2
0.283
0.142
17.63846 3.12
0.012
0.034
Sig.
V
2
0.115
0.057
7.161538 3.12
0.012
0.034
Sig.
D
2
0.024
0.012
1.484615 3.12
0.012
0.034
NS
AxT
2
0.553
0.277
34.48462 3.12
0.017
0.048
Sig.
AxV
2
0.028
0.014
1.715385 3.12
0.017
0.048
NS
AxD
2
0.013
0.006
0.792308 3.12
0.017
0.048
NS
TxV
4
0.024
0.006
0.734615 2.43
0.021
0.059
NS
TxD
4
0.054
0.013
1.669231 2.43
0.021
0.059
NS
VxD
4
0.125
0.031
3.896154 2.43
0.021
0.059
Sig.
AxTxV
4
0.107
0.027
3.342308 2.43
0.030
0.084
Sig.
AxTxD
4
0.003
0.001
0.10000
2.43
0.030
0.084
NS
TxVxD
8
0.088
0.011
1.363462 2.01
0.037
0.103
NS
AxVxD
4
0.035
0.009
1.080769 2.43
0.030
0.084
NS
AxTxVx 8 D
0.319
0.040
4.975
0.052
0.145
Sig.
Error
108 0.867
0.008
Total
80
C.V.,% = 6.56
110
2.01
