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Chapter 4
Value Addition to
Coconut Skim Milk
Chapter 4A
Dehydration of
Coconut Skim Milk
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4A.1. Introduction
Oilseed proteins can be utilized as a good source of protein due to increasing
costs for hitherto known food proteins as well as increasing world population
(Cater et al., 1977). Oil seeds such as coconut are considered to be potential
sources of dietary proteins. The high production of coconut throughout the
world (around 62 million tons/year) (FAO, 2013, http://faostat.fao.org) makes it
possibly an important source of protein, despite the fresh coconut meat
containing only 4% (w/w) protein. Coconut could be a valuable source of food
grade protein if a suitable method of extraction could be employed for the
separation of oil, the major component. In the traditional process, coconut oil
is produced by subjecting copra, the dried coconut, to expelling. It is possible
to obtain oil and protein from fresh coconuts without subjecting it to long
periods of drying or high temperature. The oil obtained by this process is
known as Virgin coconut oil (VCO) and it has been gaining popularity in recent
times (Marina et al., 2009a). A process for the production of VCO from fresh
coconut employing wet processing without shear or heat was developed at
CSIR-CFTRI (Raghavendra and Raghavarao, 2011). During wet processing,
coconut residue (left after expelling of coconut milk), coconut skim milk
(CSM), essentially the aqueous phase obtained on centrifugation of the
coconut milk and insoluble protein are the major byproducts. Spent coconut
residue finds application as dietary fiber due to its high water-holding and
swelling capacities compared to any other dietary fibers (Raghavendra et al.,
2006). Many methods are reported for separation and concentration of
coconut proteins from coconut skim milk (CSM), such as heat coagulation,
isoelectric precipitation, salt precipitation, centrifugation, ultrafiltration (Kwon
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et al., 1996b) and drying (Hagenmaier et al., 1974). It would be of significant
economical as well as environmental benefit if coconut skim milk is converted
into a possible value added food ingredient.
It is required to examine its functional properties (namely, solubility,
emulsifying and foaming properties) in order to incorporate protein into
different foods. These properties are significantly affected by the method as
well as conditions employed for drying. Changes in the method or ingredients
which affects the protein solubility, may in turn alter the emulsification and
foaming capacities. Thermal processes involving heating or cooling and
mechanical processes involving shear can have an impact on the the
functional properties of proteins in corporate in different foods (Nielsen, 2010).
In the present work, the focus was to obtain coconut skim milk powder by
employing various dehydration processes namely, drum, spray and freeze
drying and to characterize the product. Further, the functional and sensory
quality aspects of powders produced by these drying methods were relatively
evaluated. This study involves conversion of CSM into a value added product
in the most effective way, which at present is let out to the environment as
waste.
4A.2. Materials and methods
4A.2.1. Materials
Fresh and mature coconuts (10-12 months) were purchased from the local
market. The analytical grade chemicals such as sulphuric acid (H2SO4),
hydrochloric acid (HCl), ethanol, petroleum ether, diethyl ether, phenol and
ammonia used were procured from Merck chemicals, Mumbai, India. Sodium
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Dodecyl Sulphate (SDS) of extra pure grade was procured from HiMedia
laboratories, Mumbai, India.
4A.2.2. Preparation of coconut skim milk
Fresh, mature and pared coconuts (80 numbers) were subjected to
disintegration using rotary wedge cutter (Krauss maffei, Germany) and milk
was expelled using a screw press. The coconut milk (13.5 kg) was centrifuged
to obtain cream, aqueous phase (CSM) and protein precipitate. CSM (7 kg)
thus obtained was subjected to different dehydration methods such as spray
drying, drum drying and freeze drying. The process flow chart for the
production of coconut skim milk powder by different drying methods is
presented Figure 4A.1.
4A.2.3. Dehydration methods
4A.2.3.1. Drum drying
CSM (2 L) at ambient temperature (25 ± 2°C) was fed manually to the heated
rolling drums of double drum dryer (Type: MASC 231, P.I.V Stufenlos,
Homburg) (heated internally by steam) in small amounts. The drum surface
temperature was maintained at 110˚C by monitoring the steam pressure. The
flakes obtained after drying were collected, ground into powder and stored in
an air tight container at 4˚C.
4A.2.3.2. Spray drying
CSM (2 L) at ambient temperature (25 ± 2°C) was fed to the spray dryer
(Model: BE1216, Bowen, USA), by a peristaltic pump at a flow rate of 30
ml/min. Nozzle type atomizer (2 mm diameter) was employed at 3 bar air
pressure in a co-current mode air flow system. The inlet air temperature was
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set at 150 ± 2°C and the outlet air temperature was about 110 ± 2°C. The
powder was collected, through a cyclone, in the collection chamber. The dried
product was stored in an air tight container at 4˚C.
4A.2.3.3. Freeze drying
CSM (2 L) at ambient temperature (25 ± 2°C) was distributed evenly in the
trays (4 nos., 60 X 29 cm) of the freeze drier (model LT5S, Lyophilisation
Systems Inc., USA). During the freeze drying process, the product was first
frozen by lowering the temperature to -30˚C. The coolants used were R404A
(44% w/w Pentafluoroethane, 52 % w/w 1,1,1- Trifluoroethane and 4% w/w
1,1,1,2- Tetrafluoroethane) and R508B (54% w/w Hexafluoroethane and 46%
w/w Trifluoromethane) at 150 psi and 200 psi, respectively. The pressure was
lowered to 3.3 X 10-4 bar for primary drying and 3.3 X 10-5 bar for secondary
drying. Heat was supplied and maintained at 25˚C to help the ice sublimate
into vapour. After 16 h of drying, the powder was collected from the trays and
the powder was stored in an air tight container at 4˚C.
The CSM powder obtained by these dehydration methods was analyzed for
their composition, functional properties such as solubility, emulsification and
foaming properties, and subjected to colour and sensory analysis.
4A.2.4. Analytical methods
4A.2.4.1. Moisture
The moisture of coconut milk, CSM and CSM powder samples was
determined according to the (AOAC, 2007) method. A quantity of 5-6 g
sample was oven dried at 100-105˚C until constant weight. The difference in
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weight of sample before and after drying was measured as moisture content
and expressed in g/kg of sample as shown below:
)1.4....(1000gin sample wet of wt.
gin sampledry of wt.- gin sample wet of wt. (g/kg)content Moisture A
4A.2.4.2. Fat
Fat in coconut milk and coconut skim milk samples was determined by
Mojonnier procedure of AOAC (2007). 10 g of coconut milk or coconut skim
milk was weighed into Mojonnier fat extraction flask. 1.5 ml NH4OH was
added and shaken vigorously. 3 drops of phenolphthalein indicator was
added. 10 ml of 95% ethanol was added and mixed. 25 ml of petroleum ether
was added and shaken vigorously and allowed to stand for 30 min for phase
separation. The ether phase was decanted. The second extraction was done
with 5 ml ethanol and 15 ml each of ethyl ether and petroleum ether. Ether
phase was allowed to separate and decanted. Third extraction was carried out
using 15 ml each of ethyl ether and petroleum ether. The ether phase were
combined and evaporated. The residual fat was weighed and expressed as
g/kg of sample.
Soxhlet method was used to estimate fat content as described in AOAC
(2007) in CSM powder samples with a few modifications. Sample was
weighed (~5 g) and transferred into cellulose extraction thimble. The thimble
was placed in a soxhlet extractor and extraction was carried out using hexane
for 16 h. The solvent was evapourated and the residual oil weight was
recorded and expressed as g/kg.
....(4A.2)........................................1000......gin sample wet of wt.
gin fat of wt. (g/kg)Fat
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4A.2.4.3. Protein
Protein was estimated by Bradford method (Bradford, 1976) using Bovine
Serum Albumin (BSA) as standard. Bradford reagent was prepared by
dissolving 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. 100
ml of 85% (w/v) phosphoric acid was added and volume was made up to 1 L
with distilled water. After thorough stirring, the solution was filtered through
Whatman no. 1 paper. 2 ml of Bradford reagent was added to 1 ml test
solution/standard BSA (10 to 100 µg/ml). The samples were incubated at
room temperature (25 ± 2C) for 15 min and absorbance recorded at 595 nm
using spectrophotometer (Shimadzu UV spectrophotometer, model 160A).
For CSM powders, micro-Kjeldahl method (AOAC, 2007) was used to
determine total nitrogen content with minor variations. Known quantity (~0.5 g)
of sample was digested using concentrated sulphuric acid (15 ml) along with
digestion mixture (1 g) (consisting of potassium sulphate, selenium dioxide
and copper sulphate) in a digestion flask until clear solution was formed. The
acid hydrolysate was neutralized with NaOH and steam distilled. The distillate
was collected in 10 ml of 2% boric acid (containing 2 drops of mixed indicator,
methyl red and bromocresol green). The distillate was titrated against 0.01N
HCl until colour changed to colourless and the titre value was recorded. A
blank (all reagents and no sample) was digested and distilled to obtain the
blank titre value. These titre values were used to calculate nitrogen content
using the equation:
)3.4...()(
4007.1)()((%) A
gsampleofWeight
HClofNormalityvaluetitreblankvaluetitrecontentNitrogen
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where 1.4007 is a nitrogen correction value. Protein content was calculated by
multiplying 6.25 (nitrogen-protein conversion factor) to nitrogen content and
expressed as g/kg of sample.
4A.2.4.4. Total Sugars and Carbohydrate
Total sugars in coconut milk and coconut skim milk was determined by Dubois
method (Dubois et al., 1956) using D-glucose as standard. To 0.5 ml
sample/standard solutions (10 to 100 µg/ml), 1.8 ml concentrated sulphuric
acid and 300 µl of 5% phenol solution were added and incubated for 15 min at
room temperature (25 ± 2C). Absorbance was recorded at 490 nm using an
UV spectrophotometer (model 160A, Shimadzu, Japan).
For powder samples, total carbohydrate content was calculated by difference
(i.e. balance left after subtracting moisture, ash, fat, and protein) and
expressed as g/kg of sample.
4A.2.4.5. Ash
Ash content in coconut milk, coconut skim milk and coconut skim milk
powders were estimated by procedure described by AOAC (2007). Known
amount of samples were placed in porcelain crucibles and charred on hot
plate till fumes were no longer produced. The crucibles were then placed in
furnace at 550C overnight. The left over ash was weighed after cooling. Ash
content (expressed as g/kg) was calculated as the ratio of weight of ash and
weight of sample.
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4A.2.5. Protein solubility
Protein solubility was determined by the method described by Zidani et al.
(2012). CSM powder was dispersed in distilled water (2% w/v) for 1 h using a
stirrer and centrifuged at 2500 g for 10 min at room temperature (25 ± 2C).
Protein, insoluble under these conditions separates out as pellet while soluble
protein remains in the supernatant. Protein solubility was measured as the
ratio of the protein in the supernatant (soluble protein) to the total protein and
expressed as percentage.
4A.2.6. Functional properties
4A.2.6.1. Emulsifying properties
Emulsifying activity index (EAI) was determined according to the method of
Pearce and Kinsella (1978). The emulsion prepared by taking 40 ml of 0.1%
(w/v) protein solution in 0.1M phosphate buffer (pH 7) and 10 ml of oil and
homogenizing (high performance dispersing instrument, model T25 basic, Ika
labotechnik, USA) at 10,000 RPM for 1 min. Aliquots of emulsion of 100 µl
were pipetted out immediately after homogenization (at 0 min) and at 10 min,
and diluted with 10 ml of 0.1% sodium dodecyl sulphate (SDS). Absorbance
of the diluted emulsion was measured at 500 nm against 0.1% SDS as blank
in a spectrophotometer (model SQ 4802, Unico, USA). Emulsifying activity
was expressed as the Emulsifying Activity Index (EAI) and calculated as
shown below:
)4.4...(........................................
10,000CV-1
factorDilution A2 2.303/g)(m EAI 5002 A
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where, ‘C’ is the weight of protein per unit volume of the aqueous phase
before emulsion formation, A500 is the absorbance at 500 nm and ‘V’ is the oil
volume fraction of the emulsion.
Emulsion Stability Index (ESI) was calculated as:
)5.4(..................................................t
(min) ESI 0 A
where, A0 is the absorbance at time 0 min and ΔA is the difference in
absorbance over the time interval (10 min).
4A.2.6.2. Foaming capacity
Foaming capacity of the samples was determined according to the method of
Coffmann and Garcia (1977). 8 g of sample was added to 100 ml distilled
water and pH of the solution was adjusted to 7.0 with dilute NaOH (0.1N).
Vigorous whipping in a blender was carried out for 1 min and the sample was
poured into a 250 ml measuring cylinder. Volumes were recorded before and
after whipping, and the percentage volume increase indicate the foaming
capacity. The later was calculated according to following equation.
6)100...(4A.(ml) whippingbefore vol.
(ml) whippingbefore vol.- (ml) pingafter whip vol. (%)Capacity Foaming
4A.2.7. Colour
CIE (Commission Internationale de L’Eclairage) L*, a*, and b* values of CSM
powder (obtained by different drying methods) were measured using a
colorimeter (model: CM-5, Konica Minolta, Japan). The values were
measured using illuminant D65 and 10° observer angle. The instrument was
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calibrated using a standard white reflector plate. Hue angle [tan−1(b*/a*)] and
chroma (a*2+b*2)1/2 were also determined.
4A.2.8. Sensory analysis
Sensory analysis for dehydrated coconut skim milk obtained by different
methods was carried out as follows. A group of 12 panellists aged 25–50
years were trained for quantitative descriptive analysis (QDA). The members
of the panel were drawn from scientific staff familiar with sensory analysis
techniques and who had earlier experience in sensory evaluation of food
products. The samples were evaluated in a sensory booth room maintained at
a temperature of 22 ± 2°C under fluorescent lighting equivalent to day light.
Descriptors typical to the product were generated in the initial evaluations
using free choice profiling. Sensory attributes such as colour (whiteness),
texture, aroma (milky, coconut, nutty, oily), sweetness, caramel flavour and
overall quality were evaluated by the panellists. The samples were served in
petri-dishes with three digit coded numbers to avoid bias. QDA method of
intensity scaling was used (Stone and Sidel, 1998). The score card consisted
of 15 cm scale where 1.25 cm was anchored as “low” and 13.75 cm as “high”.
The panel was asked to mark the intensity of the attribute by drawing a
vertical line on the scale and writing the code. The mean scores of individual
attributes were calculated and profile was drawn. Significant difference among
samples was tested using Ducan’s multiple range test (Duncan, 1955).
Significance was tested at a probability level of p ≤ 0.05.
4A.2.9. Statistical Analysis
All the physico-chemical analysis and functional property measurements were
carried out in triplicate. Results are expressed as mean ± standard deviations.
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Data was analyzed using the analysis of variance (ANOVA) using statistical
package for social science (SPSS) 16.0. The differences between mean
values were compared using Tukey’s Test with level of significance of p ≤
0.05.
4A.3. Results and Discussion
4A.3.1. Composition of coconut milk and coconut skim milk
The term ‘coconut milk’ is generically applied to the white, opaque protein–oil–
water emulsion obtained from grated or comminuted solid coconut endosperm
by expelling (Seow and Gwee, 1997). The term ‘coconut skim milk’ denotes
the aqueous phase obtained on separation of virgin oil from coconut milk
(APCC, 1994). Coconut milk is a natural (oil-in-water) stable emulsion and
extra energy (in the form of thermal, centrifugal, pH, chilling and thawing
treatments) is required to destabilize this emulsion. During centrifugation
process, phase separation occurs due to the difference in densities and the
cream and aqueous phases are collected separately. The composition, in
terms of moisture, protein, carbohydrate, fat and ash content of coconut milk
and CSM is presented in Table 4A.1. The major difference between these two
lies in the moisture and fat contents, while the protein and carbohydrate
contents remain more or less the same.
4A.3.2. Dehydration of coconut skim milk
Drum drying is a common method used for drying of liquids such as milk,
breakfast cereals, baby food, instant mashed potatoes, etc. When the CSM is
fed to the drum drier, it formed a thin film on the surface of hot drums and
during the course of revolution, the material dried due to heat transfer from
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steam through metal wall of the drums. As it reaches the other end, the
material adhered to the drums was scrapped by knife. Drum dried CSM
(porous flakes) as shown in Figure 4A.2A was observed to be light brown in
colour. It was observed to have a cooked flavour and caramelization of sugars
occurred which is often the case in drum dried products due to high heat
exposure.
Spray drying is presently one of the most widely used dehydration method in
food and pharmaceutical industry. This method enables the transformation of
feed from a fluid state into dried particulate form by spraying the feed into a
hot drying medium (air). It has several advantages like continuous operability,
adaptability to full automation and can be designed to virtually any capacity
(Gharsallaoui et al., 2007). The product obtained after spray drying of CSM
was found to be a free flowing off-white powder (Figure 4A.2B). Short time of
heat exposure, high rate of evaporation and drying taking place at wet bulb
temperature are responsible for the production of a high quality product.
Freeze drying is a method, which enables liquid or slurry to be dried under
vacuum. Freeze drying is generally known to retain original structure and
colour, negligible loss of nutrients, and excellent rehydration capability due to
the porous structure of the product (Jiang et al., 2010). The product obtained
after freeze drying was flaky (Figure 4A.2C) but formed lumps due to
absorption of atmospheric moisture.
Dehydrated CSM powders obtained by different drying methods were visually
distinctly different from each other. It can be observed from Figure 4A.1 that
174 g, 148 g and 205 g of CSM powder was obtained by drum, spray and
freeze drying of 2 kg coconut skim milk, respectively. The freeze drying has
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resulted in the highest dehydrated product yield (68.46 ± 0.09%), followed by
drum drying (58.30 ± 0.07%) while least yield observed was in case of spray
drying method (49.77 ± 0.03%). Loss due to the adherence of particles to the
walls of the spray drier is the major reason for the low product yield by spray
drying method.
4A.3.3. Proximate analysis
Proximate analysis of coconut skim milk powders dehydrated by different
methods is presented in Table 4A.2. Moisture content was found to be
significantly different among the samples. The highest moisture content was
observed in the drum dried sample while the lowest was in the spray dried
CSM powder. Protein content was about 177 g/kg in spray dried as well as
freeze dried samples, but low (~159 g/kg) in drum dried CSM powder. The oil
content was observed to be higher in freeze dried samples compared to drum
dried and spray dried CSM. Practically no difference was observed in ash and
carbohydrate contents in the CSM powders produced by the different drying
methods.
4A.3.4. Functional properties
Protein functionality has been defined as the physical and chemical properties
of protein molecules that affect their behaviour in food products during
processing, storage, and consumption. The functional properties of proteins
contribute to the quality attributes, organoleptic properties, and processing
yields of food. It is often desirable to characterize the functional properties of
food proteins to optimize their use in a food product. Three of the most
important protein functional properties in foods are protein solubility,
emulsification and foaming.
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4A.3.4.1. Protein solubility
It is desirable that proteins are usually soluble under the conditions of use for
effective functionality in different foods. CSM powders, when obtained by
different drying methods, showed significant difference in protein solubilities (p
≤0.05). The CSM powder obtained by the freeze drying method showed the
highest solubility (about 80%) when compared to spray dried product (about
65%) and drum dried product (about 62%) (Figure 4A.3A). Partial
denaturation of the protein (due to heat) during drum and spray drying might
be responsible for the lower protein solubility.
4A.3.4.2. Emulsification
Emulsifying properties can be of value when incorporating proteins in mixed
systems (water and oil). The emulsion activity index (EAI) for freeze dried
product was the highest (25.1 m2/g) where as the EAI for spray dried and
drum dried product were lower and not significantly different (14.86 m2/g and
13.94 m2/g, respectively). EAI indicates the area of interface between
aqueous and oil phases stabilized per unit weight of sample. More the
denaturation, less is the solubility of protein and accordingly protein migration
to the interface reduces making the emulsions inherently unstable. The
solubility and emulsification properties of soy as well as peanut flours were
shown to be adversely affected by moist thermal treatment (McWatters and
Holmes, 1979). Emulsion stability index was the highest for freeze dried
sample (13.55 min) followed by spray dried powder (11.36 min) and least for
drum dried powder (9.83 min) (Figure 4A.3B). It indicates that freeze dried
powder can produce more stable emulsions compared to spray dried and
drum dried samples.
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4A.3.4.3. Foaming capacity
Foams are coarse dispersions of gas bubbles in a liquid or semi-solid
continuous phase. Proteins being in the continuous phase lower the surface
tension between the two phases during foam formation and impart stability to
films formed around the gas bubbles. CSM dehydrated by different methods
exhibited significant difference (p ≤0.05) in their foaming capacities. The
highest foaming capacity of 14.75% for the freeze dried powder is an
indication of high concentration of quality protein. Foaming capacities of the
spray dried and drum dried CSM powders were observed to be 9.26% and
6.6%, respectively (Figure 4A.3A). Ibanoglu and Ibanoglu (1997) reported
similar observations of a negative influence of heat treatment on foaming
capacities in cereal foods.
The results of functional properties of CSM powders obtained by different
drying methods are presented in Figure 4A.3A and 4A.3B. The freeze dried
CSM powder exhibited the best functional properties compared to spray dried
CSM powder followed by drum dried CSM powder. Similar effects of different
methods of drying on the functional properties of enzyme treated groundnut
flour was reported by Bhagya and Srinivasan (1989). This could be attributed
to the fact that the proteins in freeze dried products do not undergo thermal
denaturation and hence are highly reconstitutable. Although the best
functional properties were exhibited by freeze dried CSM powder, it was
hygroscopic in nature. Further, freeze drying process has certain drawbacks
such as relatively long processing time and high capital cost. Spray dried
sample had very good product characteristics like free flowing nature and
appealing colour with good functional properties.
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4A.3.5. Colour analysis
The colour analysis of coconut skim milk powders dehydrated by different
drying methods is presented in Table 4A.3. Freeze dried and spray dried CSM
powders had high L* values, which indicates lightness. Similarly, a* values
were very low for freeze dried and spray dried CSM powders. Positive a*
indicates redness, which was evident in the drum dried CSM powder. The
positive b* values indicate yellowness, which was found to be low in freeze
dried and spray dried samples. Similar observations were seen when mango
powder was produced using different drying methods (Caparino et al., 2012).
These results strongly substantiate the perception of colour in the sensory
evaluation. Hue angle describes the colour perception, which was significantly
different (p ≤ 0.05) for all the CSM powders, while chroma, which indicates
saturation of colour, was similar for freeze dried and spray dried CSM
powders (p ≤ 0.05).
4A.3.6. Sensory analysis
Dehydrated CSM powder prepared using spray drying method had
characteristic milky, coconut and nutty aroma. Samples prepared by drum
drying and freeze drying methods had low score for these typical and specific
aroma notes of the product. Drum dried CSM powder was less white in colour
(p ≤ 0.05) and flaky appearance while it had a strong caramel aroma and crisp
texture. No significant difference was observed for sweetness among the
samples (p ≤ 0.05). Spray dried CSM powder scored the highest (10.8 out of
15) for overall quality which was the result of more desirable attributes such
as colour texture and aroma as seen in the Figure 4A.4.
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4A.4. Conclusion
Different methods such as drum drying, spray drying and freeze drying were
employed for the dehydration of coconut skim milk (CSM), which is a
byproduct of the virgin coconut oil industry, without the addition of any
additives. When compared to the powders obtained by other drying methods,
freeze dried CSM powder was found to have the best functional properties.
Spray drying yielded CSM powder with good quality in terms of product
characteristics and moderately good functional properties. Hence spray drying
was considered to be the most feasible method for the drying of CSM and
results indicate that spray dried CSM powder can be used as a natural food
additive and emulsifier.
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Table 4A.1: Composition of coconut milk and coconut skim milk (wet basis)
Sl.no Sample Moisture
(g/kg)
Protein
(g/kg)
Carbohydrate
(g/kg)
Fat
(g/kg)
Ash
(g/kg)
1 Coconut milk 496.31 ± 2.76 37.74 ± 0.36 51.19 ± 0.05 332.88 ± 1.58 6.54 ± 0.18
2 Coconut skim milk 857.57 ± 1.70 42.61 ± 0.35 73.31 ± 0.34 8.70 ± 0.67 8.53 ± 0.23
Values are averages ± standard deviation from three replicate analysis
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Table 4A.2: Proximate analysis of coconut skim milk powders dehydrated by different methods
Parameters Coconut skim milk powder
(g/kg) Drum dried Spray dried Freeze dried
Moisture 50.2 ± 0.18a 17.96 ± 1.34b 26.57 ± 2.15c
Protein 159.38 ± 0.70a 177.70 ± 1.05b 176.98 ± 1.37b
Fat 66.54 ± 0.16a 65.22 ± 3.44a 79.04 ± 5.27b
Ash 83.044 ± 3.46a 87.11 ± 4.52a 83.52 ± 3.21a
Carbohydrate (by difference) 640.84 ± 4.57a 656.42 ± 11.78a 633.88 ± 14.18a
Values are averages ± standard deviation from three replicate analysis
a–c Values in rows followed by same superscript letters are not significantly different (p≤0.05)
112
Table 4A.3: Colour analysis of coconut skim milk powders dehydrated by different methods
Coconut Skim Milk Powder L* a* b* Hue angle () Chroma
Drum drying 63.82 ± 0.27a 10.46 ± 0.68a 27.30 ± 1.45a 69.03 ± 0.56a 29.23 ± 1.57a
Spray drying 79.53 ± 1.27b 2.40 ± 0.63b 18.28 ± 0.40b 82.55 ± 1.78b 18.44 ± 0.47b
Freeze drying 83.77 ± 1.57c -0.06 ± 0.07c 16.39 ± 1.00b -89.80 ± 0.25c 16.39 ± 1.0b
Values are averages ± standard deviation from three replicate analysis
a–c Values in column followed by same superscript letters are not significantly different (p ≤ 0.05)
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Figure 4A.1: Mass balance flow chart for preparation of coconut skim milk
powder
114
Figure 4A.2: Pictures of coconut skim milk powder obtained by different methods: A- Drum drying, B- Spray drying, C- Freeze
drying
115
Values are averages ± standard deviation from three replicate analysis
a–c,
d-f,
g-h,
i-k Values followed by same superscripted letters are not significantly different
(p≤0.05) for protein solubility, foaming capacity, emulsion activity index and emulsion stability index, respectively.
Figure 4A.3: Functional properties (protein solubility, foaming capacity,
emulsion activity index and emulsion stability index) of
dehydrated coconut skim milk obtained by different dehydration
methods
A
B
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Figure 4A.4: Sensory profile of dehydrated coconut skim milk obtained by
different drying methods
Chapter 4B
Membrane processing of
coconut skim milk
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4B.1. Introduction
Coconuts provide a potential source of proteins with good nutritional value
and have a relatively well-balanced amino acid profile (Srinivasan et al.,
1964). Copra meal serves as cheap alternative source for protein but currently
not used for human consumption as they may be contaminated with toxic
metabolites due to poor post-harvest practices. Wet or aqueous processing of
coconut overcomes this problem and the protein obtained through this route is
of good edible quality (Woodroof, 1979). Coconut milk press cake, the spent
endosperm left over after the extraction of coconut milk, can be utilized to
extract protein under alkaline conditions. These edible proteins have been
characterized by electrophoresis and mass spectrometry (Chambal et al.,
2012). Another important source of edible protein is coconut skim milk (CSM),
the aqueous by-product obtained during production of virgin coconut oil. But
available literature on recovery of edible proteins from CSM is very low.
Coconut proteins can be concentrated by different methods described in
section 4A.1. Thus it is essential to optimize recovery and concentration of
CSM protein which is currently discarded as waste.
In response to the concerns about protein recovery, membrane filtration
technology provides exciting opportunities on a large-scale. Ultrafiltration (UF)
is one of the many membrane separation technologies used in industry and
research for purifying and concentrating macromolecular (103 - 106 daltons)
solutions, especially protein solutions. UF is employed to concentrate and
recover proteins from skim milk (Al-Akoum et al., 2002), whey (Akpinar-Bayizit
et al., 2009), soy milk (Jinapong et al., 2008) and even wastewaters (Wu et
al., 2014). However, too little attention has so far been paid on coconut
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proteins and its separation from CSM. A complex protein mixture (such as
CSM) can easily reduce the efficiency of the UF process due to the tendency
of the membrane to foul. UF studies of CSM have shown that fouling
mechanisms such as complete blocking, standard blocking, intermediate
blocking and cake formation occurred using a 20 kDa polysulfone membrane
and 60C feed temperature (Ng et al., 2014). Hence, it is important to study
effect of various process parameters in order to achieve maximum yield and
minimum fouling.
Five protein fractions, namely, albumins (21% w/w), globulins (40% w/w),
prolamines (3.3% w/w), glutelins-1 (14.4% w/w) and glutelins-2 (4.8% w/w)
were obtained from defatted coconut flour and characterized (Kwon et al.,
1996a). The major coconut protein in the endosperm is the 11S globulin or
cocosin which amounts to 86% (w/w) of total globulin while 7S was only 14%
(w/w) with native molecular weights of 326 kDa and 156 kDa, respectively
(Garcia et al., 2005). The excellent emulsifying ability of cocosin in the
absence of salt has shown to be the basis for developing new processed
foods (Angelia et al., 2010). Coconut has a great possibility of being a source
of dietary protein as consumption of coconut protein was observed to have
anti-diabetic effect in experimental rats (Salil et al., 2011), immunomodulatory
effect on mice which were immunosuppressed with cyclophosphamide (Vigila
and Baskaran, 2008), hypolipidemic and antiperoxidative effect in
hypercholesterolemic rats (Salil and Rajamohan, 2001) and cardioprotective
effect on alcohol and isoproterenol treated rats (Mini and Rajamohan, 2002).
The major factor responsible for these effects is attributed to the high content
of L-arginine present in coconut protein. The aim of the work is to concentrate
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CSM protein using membrane technology (ultrafiltration) and dehydration
(spray drying) besides quality evaluation of the final product.
4B.2. Materials and Methods
4B.2.1. Materials
Fresh and mature coconuts (10-12 months) were purchased from the local
market. Folin Ciocalteau phenol reagent and sodium azide were purchased
from Sisco Research Laboratory Pvt. Ltd., Mumbai, India. Sodium Phytate
was purchased from Sigma-Aldrich, St. Louis, USA. Membrane namely,
Pelicon TFF polyethersulfone (PES) membrane (Biomax 300) cassette of
Molecular Weight Cut Off (MWCO) of 300 kDa and ultrafiltration discs
(Biomax PES, 47 mm) of MWCO of 300 kDa, 100 kDa, 50 kDa and 30 kDa
were purchased from Millipore (India) Pvt. Ltd. Chemicals such as petroleum
ether, diethyl ether, ethanol, phenol, sulfosalicylic acid, Iron(III) chloride
hexahydrate (FeCl3∙6H2O), sulphuric acid (H2SO4), hydrochloric acid (HCl)
and ammonia of analytical grade were procured from Merck chemicals,
Mumbai, India.
4B.2.2. Preparation of coconut skim milk
For studying effects of process parameters on UF of CSM, fresh, mature and
pared coconuts (20 numbers) were subjected to disintegration using rotary
wedge cutter (Krauss maffei, Germany) and milk was expelled using hydraulic
press (B Sen Barry and Co., New India). The coconut milk (3.8 kg) was
centrifuged to obtain cream (1.8 kg), aqueous phase (CSM) (2.1 kg) and
protein precipitate (22 g). Sodium azide (0.02%) was added to CSM to avoid
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microbial growth, prefiltered through Whatman filter paper no.1 and stored at
4C until use.
For lab scale TFF of CSM, 50 coconuts were processed as mentioned above
to obtain 4.8 kg CSM. This was immediately processed without addition of
sodium azide.
4B.2.3. Ultrafiltration of coconut skim milk
4B.2.3.1. Dead-end filtration
In order to study the effect of different process parameters on UF of CSM,
solvent resistant stirred cell (model: XFUF04701, Millipore, USA) was
employed. UF discs were fitted into the unit and 50 ml CSM was filtered upto
volume concentration factor, CF=5 (i.e 10 ml) using UF membranes of MWCO
of 5 kDa, 50 kDa, 100 kDa and 300 kDa at different pH (4, 6 and 8),
transmembrane pressures (TMP) (2 bar, 3 bar and 4 bar) at fixed stirring
speed of 300 rpm. The permeate flux was measured at regular time intervals
of 10 min. The retentate and permeate were collected and analysed for
protein and carbohydrate contents.
4B.2.3.2. Tangential flow filtration
To obtain concentrated CSM, Tangential Flow Filtration (TFF) system (model:
XX42LSS12, Millipore, USA) was used, in which 2.5 L (500 ml each run) of
CSM was concentrated to 500 ml (100 ml each run) using 300 kDa
membrane. The conditions of TFF were: 2 bar TMP, feed pH 4 and
temperature 25 ± 2C (RT).
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4B.2.4. Spray drying
2 kg of CSM and 400 g of concentrated CSM (CCSM) were subjected to
spray drying. The procedure for spray drying is described in section 4A.2.3.2.
The dried powder samples from each experiment was analysed for
composition, phytate content, polyphenol content, water activity and physical
characteristics such as colour and flow properties.
4B.2.5. Analytical methods
4B.2.5.1. Proximate analysis
Proximate analysis was carried out as described in section 4A.2.4.
4B.2.5.2. Protein and Sugar estimation
The protein and sugar content in UF retentate and permeate were estimated
by methods described in sections 4A.2.4.3 and 4A.2.4.4, respectively.
4B.2.5.3. Phytate estimation
Samples of 0.50 g of powder was thoroughly mixed with 10 mL of 2.4% HCl in
50 mL tubes. Sample tubes were agitated for 16 h on shaker (model: 3040,
Tarsons, India) and centrifuged at 1000 g at 10°C for 20 min. The supernatant
from each experiment was transferred to tubes containing 1 g NaCl and the
contents were shaken to dissolve the salt. These tubes were then allowed to
rest at 4°C for 60 min followed by centrifugation at 1000 g at 10°C for 20 min.
The clear supernatant samples (referred to as the NaCl treated supernatant)
were collected for colour development. One ml of the NaCl treated
supernatant was diluted 25 times with distilled water. To 3 ml of this diluted
solution, 1 ml of modified Wade reagent (0.03% FeCl3∙6H2O + 0.3%
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sulfosalicylic acid) was added, thoroughly mixed on a vortex and centrifuged
at 1000 g at 10°C for 10 min. A series of calibration standards containing 0, 2,
4, 6, 8 and 10 µg/ml of sodium phytate were prepared to obtain standard
graph. Absorbance of colour reaction products for both samples and
standards was read at 500 nm on an UV spectrophotometer (model 160A,
Shimadzu, Japan). The phytate content in CSM and CCSM powders was
determined from standard graph (Figure 4B.1).
4B.2.5.4. Polyphenol content
Total polyphenol content was determined by Folin-Ciocateau colorimetric
method as described by Kumazawa et al. (2004). 5 g of powder was stirred in
40 ml methanol at 25 ± 2C for 1 h. The suspension was centrifuged at 3000 g
for 10 min at 25 ± 2C and supernatant was collected and volume was made
to 50 ml in volumetric flask using methanol. The extract was mixed with 2 ml
of 10% of Na2CO3 and 1 ml of 1 N Folin-Ciocateau reagent and incubated for
1 h at room temperature and absorbance was measured at 765 nm. Standard
graph was plotted using Gallic acid (Figure 4B.2) and total polyphenol content
was expressed as mg/g GAE (Gallic Acid Equivalents).
4B.2.5.5. Water activity
Water activity was measured using a portable water activity measurement
system (Pawkit, version 8, Decagon devices Inc.). Sample cup of the meter
was filled with powder samples such that the bottom of the cup was entirely
covered. After inserting the sample cup in the meter, the meter was placed on
a flat surface, switched on and not disturbed until it gives “beep sound”
123
indicating the completion of measurement. The water activity of the sample at
the corresponding temperature was recorded.
4B.2.5.6. Colour analysis
The colour analysis was carried out using a colorimeter as described in
section 4A.2.7.
4B.2.5.7. Powder flowability and cohesiveness
Powder was loaded into 100 ml tared measuring cylinder and weighed. Bulk
density (bulk) was calculated as of mass/volume. Tapped density was
measured by tapping the cylinder 1250 times with a displacement amplitude
of 3 ± 0.03 mm using tapped density meter (model ETD-1020, Electrolab,
India). Tapped density (tapped) was calculated by mass/volume after tapping.
Flowability and cohesiveness of the powder were evaluated in terms of Carr
Index (CI) (Carr, 1965) and Hausner ratio (HR) (Hausner, 1967), respectively
and determined as following equations:
)1.4..(..................................................100 BCItapped
bulktapped
)2.4......(...................................................................... BHRbulk
tapped
4B.3. Results and Discussion
4B.3.1. Effect of process parameters on ultrafiltration of CSM
Membrane filtration can be a very efficient process of separating the
components that are suspended or dissolved in a liquid if the process
parameters are optimized to achieve maximum yield. The choice of
parameters is made with respect to permeation and retention of the key
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components. Different process parameters considered in this study were:
Molecular Weight Cut Off (MWCO) of membrane, feed pH and
Transmembrane Pressure (TMP). Their effect was studied on transmembrane
flux, protein retention and sugar removal.
4B.3.1.1. Effect of membrane molecular weight cut off
The effect of MWCO of UF membranes on transmembrane flux, protein
retention and sugar removal is shown in Figure 4B.3. The TMP was
maintained at 3 bar and stirring speed at 300 rpm. It can be seen from the
figure that the flux is much higher during UF for membrane of high MWCO.
The initial as well as final permeate flux was high for UF using 300 kDa
membrane as it offered the least resistance to passage of water along with
dissolved salts and low molecular weight components. The required
concentration factor (CF=5) was obtained about 3 times faster using 300 kDa
membrane compared to 5 kDa membrane. In terms of protein retention, lower
MWCO membranes were obviously found more effective in retaining protein
in the retentate. About 14% (w/w) protein was lost in permeate using 300 kDa
membrane while 2-3% loss was observed in the permeates using 100 kDa, 50
kDa and 5 kDa membrane. Kwon et al. (1996b) reported protein loss in
permeates to be ~20% and ~10% using 10 and 5 kDa membranes,
respectively, during production of coconut protein concentrate from 1%
coconut protein solution at 1 bar TMP using pilot scale hollow fiber
ultrafiltration unit. Ultrafiltration is typically used to separate proteins for
concentration, desalting or buffer exchange. It can be also used for removal of
sugars, non-aqueous solvents, low molecular weight compounds etc. (Basile
and Nunes, 2011). The sugar removal was the highest using 300 kDa
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membrane (~84%) while it ranged from 63-65% (w/w) for other MWCO
membranes. Taking into account the least processing time, the highest sugar
removal and ~83% protein retention, 300 kDa membrane was selected for
subsequent experiments.
4B.3.1.2. Effect of feed pH
In general, the pH of CSM is 6 and can be easily altered by addition of acids
or bases. In order to study the effect of feed pH (4, 6 and 8) on permeate flux,
protein retention and sugar removal, UF was carried out using 300 kDa
membrane at constant TMP of 3 bar and stirring speed of 300 rpm. From
Figure 4B.4(A), it can be observed that the initial flux at feed pH 4 to be twice
as that at feed pH 6 and 8. The high permeate flux at feed pH 4 led to faster
UF of CSM i.e. it consumed only about 2/3rd time compared to UF of CSM of
pH 6 and 8. The flux was almost similar for UF for feed pH 6 and 8 and fairly
constant throughout the UF process. Most coconut proteins have their
isoelectric point (pI) at about pH 4 (Tangsuphoom and Coupland, 2009) and
thus show minimum solubility at this pH (Naik et al., 2012). The pI is the pH of
a solution at which the net primary charge of a protein becomes zero. At pH
below and above the pI, the proteins will have predominantly positive and
negative net charge, respectively. This leads to electrostatic repulsion among
proteins. But at pI, where the net surface charge is zero, the surface of the
protein will be least solvated or hydrated leading to aggregation of protein
molecules. These aggregates precipitate out of the solution and is known
“isoelectric precipitation” (Nakai and Modler, 1996). Formation of large
aggregates at pI (pH 4 for coconut protein) during UF led to the highest
protein retention (~87%, w/w) compared to that of pH 6 (~81% w/w) and pH 8
126
(~74%, w/w) as shown in Figure 4B.4(B). The sugar removal was similar for
feed pH 4 and 6 (~83% to ~84% w/w) while for feed pH 8, only ~79% (w/w)
permeated through membrane (Figure 4B.4(C)). In view of the highest flux,
the highest protein retention and sugar removal, the next set of experiments
were performed using feed pH 4 and 300 kDa MWCO membrane.
4B.3.1.3. Effect of transmembrane pressure
The effect of TMP on transmembrane flux, protein retention and sugar
removal during UF of CSM using 300 kDa MWCO membrane and feed pH 4
is shown in Figure 4B.5. It can be observed from the figure that initial flux is
higher at higher TMP. However, after 20 min, fluxes for 2 bar and 3 bar TMP
was almost similar. High flux and low processing time can be achieved by UF
of CSM using high TMP. Protein retention was found to be similar for 2 and 3
bar TMP (~86% and ~87% (w/w), respectively) but slightly reduced at 4 bar
TMP (~83%). Sugar removal was maximum at 4 bar (~86%) and decreased
with a decrease in TMP. Therefore, it can be inferred that although high flux
and sugar removal is possible by increasing TMP, it results in some loss of
protein in the permeate during UF of CSM.
4B.3.2. Spray dried concentrated coconut skim milk
Tangential flow filtration (TFF) was employed to concentrate CSM to obtain
CCSM. CSM (2 kg) and CCSM (400 g) were spray dried to yield 103.5 g and
80.2 g of powder products, respectively. The proximate analysis of CSM,
CCSM, Spray Dried Coconut Skim Milk (SDCSM) and Spray Dried
Concentrated Coconut Skim Milk (SDCCSM) are presented in Table 4B.1. It
can be observed that protein and fat contents in CCSM were observed to
127
increase 2 fold compared to CSM (from 2.52% and 0.49% (w/w) to 6.93% and
0.75%, (w/w) respectively). On the other hand, ash and carbohydrate contents
were found to reduce slightly after UF. Comparison between SDCSM and
SDCCSM composition shows no difference in moisture content (~3%, w/w
w.b.) while protein and fat contents have were observed to increase 2 fold
(from 21% to 46% (w/w) and ~5% to ~9% (w/w)). The ash and carbohydrate
contents were found to reduce by 31% and 43% (w/w), respectively, after
concentration by UF and spray drying. Similar trends were observed when
Soy protein concentrate was produced by UF followed by freeze drying (Rao
et al., 2002) and UF followed by spray drying (Jinapong et al., 2008).
4B.3.2.1. Polyphenol content, phytate content, water activity and powder
properties
Antinutritional factors such as polyphenols and phytic acid limit the use of
oilseed proteins for human consumption (Tan et al., 2011). In many oilseed
protein sources, polyphenolic compounds are responsible for development of
adverse flavours and colors in food products. They also bind to essential
nutrients and alter their chemical and functional properties especially of
proteins (Sosulski, 1979). From Table 4B.2, it can be observed that the
amount of total polyphenols reduced significantly (from 2.56 mg/g to 1.84
mg/g) after ultrafiltration.
Phytic acid (hexaorthomonophosphate ester of myo-inositol) occurs in
legumes, cereals and oil seeds as the calcium magnesium salt, phytin. Phytic
acid is known for its metal chelating properties and thus decreases the
bioavailability of many essential minerals by interacting with multivalent
cations and/or proteins to form complexes that may be insoluble or otherwise
128
unavailable under physiologic conditions (Cheryan and Rackis, 1980). Both,
spray dried CSM and CCSM powders contain about 0.2% (w/w) phytate which
is much lower than 2 to 3% (w/w) present in commercial soy protein isolates
(Okubo et al., 1975). Ultrafiltration was unable to lower the phytic acid
content in CCSM powder. This may be due to the presence of protein-phytic
acid complex in CSM.
Most of the unit operations used in food processing involve stabilization of
food material by removal of water either by drying or concentrating and water
activity serves as an index of determining the efficiency of controlling the
behaviour of water in food systems (Rockland, 1987). The water activity
values of both CSM and CCSM powders were similar (0.26 and 0.27,
respectively) at 25.7C which indicates that the powders as microbiological
stable.
Classification of flowability and cohesiveness of dried CSM and CCSM
powder are presented in Tables 4B.3 and 4B.4, respectively. Flow properties
of both the powders were not much different indicated by Carr Index (~33
which indicates fair flowability). Similarly, the cohesiveness was almost
identical (~1.5 which indicated high cohesiveness) for CSM and CCSM
powders based on Hausner ratio. Colour analysis, as indicated in Table 4B.5,
revealed less lightness and more yellow colour (indicated by lower L* and
higher b* values in CIE colour measurement system) in dehydrated CCSM
compared to dehydrated CSM. Both the powders looked alike and off-white in
colour when seen visually.
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4B.4. Conclusion
The process parameters such as Molecular Weight Cut Off of membrane,
feed pH and Transmembrane Pressure (TMP) were found to have
considerable effect on TMF, protein retention and sugar removal during
ultrafiltration of CSM. The standardized conditions for UF were membrane
MWCO 300 kDa, feed pH 4 and transmembrane pressure 4 bar with respect
to transmembrane flux, protein retention efficiency and removal of sugars.
Coconut skim milk could be concentrated for high protein content using
ultrafiltration. Proximate analysis of the spray dried powders indicated that
protein content nearly doubled in CCSM (46%, w/w) when compared to CSM
(21%, w/w). In contrast, ash and carbohydrate contents reduced nearly to
half.
130
Table 4B.1: Proximate analysis of different byproducts of coconut
Parameters
(%)
Byproduct sample*
CSM CCSM SDCSM SDCCSM
Moisture 84.88 ± 0.39 79.18 ± 0.30 2.99 ± 0.21 3.09 ± 0.27
Protein 2.52 ± 0.16 6.93 ± 0.61 21.43 ± 0.34 45.49 ± 1.94
Fat 0.49 ± 0.09 0.75 ± 0.01 4.78 ± 0.22 9.49 ± 0.12
Ash 1.08 ± 0.06 0.99 ± 0.00 11.52 ± 0.39 7.93 ± 0.37
Carbohydrate 6.34 ± 0.44 6.11 ± 0.18 59.27 ± 1.53 34.00 ± 3.80
*Byproduct Sample:
CSM- Coconut Skim Milk
CCSM- Concentrated Coconut Skim Milk
SDCSM- Spray Dried Coconut Skim Milk
SDCCSM- Spray Dried Concentrated Coconut Skim Milk
Values are averages ± standard deviation from three replicate analysis
131
Table 4B.2: Polyphenol content, phytate content and water activity of
byproducts of coconut
Byproduct
Sample*
Polyphenol Content (mg/g)
Phytate content (mg/g)
Water activity
(at 25.7C)
SDCSM 2.56 ± 0.08 2.11 ± 0.01 0.26 ± 0.01
SDCCSM 1.84 ± 0.04 2.15 ± 0.03 0.27 ± 0.00
*Byproduct Sample:
SDCSM- Spray Dried Coconut Skim Milk
SDCCSM- Spray Dried Concentrated Coconut Skim Milk
Values are averages ± standard deviation from three replicate analysis
132
Table 4B.3: Classification of byproducts of coconut flowability based on Carr
Index
Byproduct sample* Carr Index
(CI %) Flowability
SDCSM SDCCSM
<15 Very good
15–20 Good
33.14 32.95 20–35 Fair
35–45 Bad
>45 Very bad
*Byproduct Samples:
SDCSM- Spray Dried Coconut Skim Milk
SDCCSM- Spray Dried Concentrated Coconut Skim Milk
133
Table 4B.4: Classification of byproducts of coconut cohesiveness based on
Hausner Ratio
Byproduct sample* Hausner Ratio
(HR) Cohesiveness
SDCSM SDCCSM
<1.2 Low
1.2–1.4 Intermediate
1.47 1.5 >1.4 High
*Byproduct Samples:
SDCSM- Spray Dried Coconut Skim Milk
SDCCSM- Spray Dried Concentrated Coconut Skim Milk
134
Table 4B.5: Colour analysis of byproducts of coconut
Parameter SDCSM SDCCSM
L* 83.81 ± 0.10 80.70 ± 0.05
a* −0.83 ± 0.02 0.54 ± 0.01
b* 12.37±0.08 15.21±0.02
*Byproduct Samples:
SDCSM- Spray Dried Coconut Skim Milk
SDCCSM- Spray Dried Concentrated Coconut Skim Milk
Values are averages ± standard deviation from three replicate analysis
135
Figure 4B.1: Standard graph for phytate estimation
y = -0.0489x + 0.7657 R² = 0.9994
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 2 4 6 8 10 12
Ab
so
rba
nc
e @
50
0 n
m
Phytate Concentration (µg/ml)
136
Figure 4B.2: Standard graph for total polyphenol content
y = 0.0329x - 0.1519 R² = 0.999
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 10 20 30 40 50 60
Ab
so
rban
ce
@ 7
65
nm
Gallic Acid concentration (µg/ml)
137
Figure 4B.3: Effect of ultrafiltration membrane molecular weight cut off
(MWCO) on (A) transmembrane flux, (B) protein retention and
(C) sugar removal
0
2
4
6
8
10
12
14
16
18
20
0 50 100 150 200 250 300
Flu
x (L
m-2
h-1
)
Time (min)
300 kDa
100 kDa
50 kDa
5 kDa
0
20
40
60
80
100
300 kDa 100 kDa
50 kDa 5 kDa
Pro
tein
Co
nte
nt
(%,
w/w
)
Permeate
Retentate
0
20
40
60
80
100
300 kDa 100 kDa
50 kDa 5 kDa
Suga
r C
on
ten
t (%
, w/w
)
Retentate
Permeate
A
B
C
138
Figure 4B.4: Effect of feed pH (using 300 kDa MWCO membrane) on (A)
transmembrane flux, (B) protein retention and (C) sugar
removal
0
5
10
15
20
25
30
35
0 20 40 60 80 100 120
Flu
x (L
m-2
h-1
)
Time (min)
pH 6 (control)
pH 4
pH 8
0
20
40
60
80
100
pH 4
pH 6 (control) pH 8
Pro
tein
Co
nte
nt
(%,
w/w
)
Permeate
Retentate
0
20
40
60
80
100
pH 4
pH 6 (control) pH 8
Suga
r C
on
ten
t (%
, w
/w)
Retentate
Permeate
B
A
C
139
Figure 4B.5: Effect of transmembrane pressure (using 300 kDa MWCO
membrane and feed pH 4) on (A) transmembrane flux, (B)
protein retention and (C) sugar removal
0
10
20
30
40
50
0 10 20 30 40 50 60 70
Flu
x (L
m-2
h-1
)
Time (min)
2 bar
3 bar
4 bar
0
20
40
60
80
100
2 bar 3 bar
4 bar
Pro
tein
Co
nte
nt
(%,
w/w
)
Permeate
Retentate
0
20
40
60
80
100
2 bar 3 bar
4 bar
Suga
r C
on
ten
t (%
, w
/w)
Retentate
Permeate
B
C
A
