83
Enzyme-Based Detergent Formulas for Fatty Soils
and Hard Surfaces in a Continuous-Flow Device
E. Jurado*, V. Bravo, J. Nez-Olea, R. Bailn, D. Altmajer-Vaz,
M. Garca-Romn, and A. Fernndez-Arteaga
Department of Chemical Engineering, Science Faculty, University of Granada, Granada, Spain, 18071
ABSTRACT: The present work analyzes the effect of incorpo-
rating a lipolytic enzyme (Lipolase 100L) into detergent formu-
las for washing fatty soils on hard surfaces. The experimental
device, which is called a bath-substrate-flow device, uses a
continuous flow on a substrate (glass spheres) soiled with tri-
olein. Washing tests were done using only the enzyme and
changing both its concentration and the temperature of the
process. The results showed that, in the presence of lipase, soil
removal was achieved through three consecutive mechanisms:
(i) fundamental removal of the soil by the bath flow through the
experimental device; (ii) emulsion of the soil in the washing
medium; and (iii) enzymatic hydrolysis of the dispersed soil. Dif-
ferent commercial surfactants were used, and detergency was
evaluated in the absence and presence of lipase. The use of sur-
factant formulas with the lipolytic enzyme showed a positive ef-
fect of the enzyme on the detergency values registered with the
fatty alcohol ethoxylate surfactants Findet 10/15 and Findet
1214N/23, and with the anionic surfactant linear alkylbenzene
sulfonate. The commercial surfactants Glucopon 600, Gluco-
pon 650, Findet 10/18, and Findet Q/21.5NF alone each pre-
sented high detergency values for fatty soils, and the effect of
the incorporation of the lipase was not significant.
Paper no. S1493 in JSD 9, 8390 (Qtr. 1, 2006).
KEY WORDS: Alkylpolyglucosides, detergents, fatty alcohol
ethoxylate, hard-surface cleaning, lipases, nonylphenol ethoxy-
late, washing process.
Detergents continue to be crucially important to the quality
of life worldwide. Their applications are broad, ranging from
domestic cleaning products and personal hygiene to the
manufacture of polymers by polymerization in emulsions (1).
On the other hand, a detergent must meet a number of
requirements, such as short-term functioning, action at low
temperatures, low environmental impact, and a reasonable
price. Therefore, frequent reviews of detergent components
are necessary. Developing specific formulas for different soil-
ing agents is a challenge in the near future for manufactur-
ers of industrial cleaning formulas, and finding effective for-
*To whom correspondence should be addressed.
E-mail: ejurado@ugr.es
Abbreviations: HLB, hydrophilic-lipophilic balance; LAS, linear alkyl-
benzene sulfonate; LU, lipase units; NF, nonylphenol ethoxylate.
COPYRIGHT 2006 BY AOCS PRESS
mulas for different soils with the most environmentally safe
components is an incentive for research in this field.
For enzymatic detergent formulas, prior work shows the
beneficial effects of amylase and protease enzymes on the
elimination of soils adhering to textiles and hard surfaces.
Notable among these is the increase in detergency at low tem-
peratures (2,3). Lipases (E.C. 3.1.1.3) are used in various
parts of the globe in diverse applications, including deter-
gents, with Lipolase (Novozymes A/S, formerly Novo-
Nordisk, Bagsvaerd, Denmark) being the first commercial en-
zyme developed for this purpose. However, they are not yet
as widespread in detergents as are proteases. Currently, re-
search into the use of lipases in detergent formulas focuses
primarily on the development of new enzymes with improved
resistance to surfactants and better performance under prac-
tical washing conditions. Studies by Hemachander and
Puvanakrishnan (4) and Fujii et al. (5) showed a 20% increase
in the effectiveness of washing in systems with lipases. Oben-
dorf et al. (6), testing commercial lipase SP1013, observed
that the hydrolysis of the triglycerides accelerated the wash-
ing process. Andree et al. (7) found that the use of lipases to-
gether with a nonionic surfactant led to detergency values
similar to those reached at concentrations greater than those
for the surfactant, revealing a synergetic effect with these sur-
factants. Washing experiments with textiles conducted by
Fujii et al. (5) have suggested that regardless of the type and
concentration of surfactant, the addition of lipase boosts the
effectiveness of the wash. In addition, assays made with a non-
ionic surfactant and lipase presented higher detergency val-
ues than those made with anionic surfactants and lipase. He-
list and Korpela (8), Xia et al. (9), and Andree et al. (7)
found that anionic surfactants, including linear alkylbenzene
sulfonate (LAS), inhibited lipases. The use of nonionic sur-
factantsespecially alcohol ethoxylates and alcohol ethoxy-
sulfatespositively influenced the activity and stability of
these enzymes.
In this work, the detersive effectiveness of the commercial
lipase Lipolase 100L was studied alone and in the presence
of different surfactants, using triolein as the soiling agent. A
continuous bath flow over a substrate soiled with fat was used
for the washing assay, simulating the behavior of different
commercial devices, as in the case of dishwashers and clean-
ing-in-place systems in industrial plants.
JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006
84
E. JURADO ET AL.

EXPERIMENTAL PROCEDURES
Materials. As components of the detergent formula, the
commercial nonionic surfactants used were Glucopon 215,
Glucopon 600, Glucopon 650 EC (Henkel KgaA, Dssel-
dorf, Germany), Findet 10/15, Findet 10/18, Findet
1214N/23, and Findet Q/21.5NF (Kao Chemicals Europe,
Barcelona, Spain). The ionic surfactant assayed was LAS
from the Kao Chemicals Europe. Table 1 lists the main char-
acteristics of these components. The hydrophilic-lipophilic
balance (HLB) values were calculated according to Griffin
(12).
The commercial lipase Lipolase 100L, from a selected
strain of Thermomyces lanuginosus, was supplied by Novozymes.
This enzyme, presented in liquid form, specifically hydrolyzes
ester bonds at positions sn-1 and -3 of the triglyceride mole-
cule, according to the process:
TG DG + FFA MG + FFA
where TG, DG, MG, FFA stand for triglycerides, diglycerides,
monoglycerides, and free fatty acids, respectively.
Triolein (Sigma, St. Louis, MO) of approximately 65% pu-
rity (technical grade) was used as the soiling agent in the
washing tests. Tributyrin (Sigma; 99% purity) was used as the
reference substrate to measure the enzymatic activity. The
other reagents used in this study (supplied by Panreac,
Barcelona, Spain) were of 99% purity or higher.
Enzyme activity. The activity of the lipase used was deter-
mined on tributyrin by the pHstat method (13) at pH 7.0 and
at several temperatures (30, 40, 50, and 60C). The value of
the lipase activity was expressed in lipase units (LU), with 1
LU being the quantity of enzyme capable of releasing one mi-
cromole of butyric acid per minute.
Bath-substrate-flow (BSF) device and washing tests. The wash-
ing tests were performed in the BSF device proposed by Ju-
rado-Alameda et al. (14) at pH 8.0. Figure 1 shows a simpli-
fied scheme of this experimental system. This device was
composed of: (a) a glass tank containing the washing bath;
(b) a packed column where the substrate was deposited after
soiling it with triolein; (c) a thermostat-controlled bath; (d) a
peristaltic pump; and (e) a paddle stirrer. The tank and col-
umn were joined in a series and connected by silicone tub-
ing, with the liquid flowing upward from the tank (a) toward
the column (b).
The temperature of the washing bath was controlled using
a thermostatic jacket on the tank (a) and column (b), where
the water circulated from the thermostatic bath (c). In addi-
tion, a mercury thermometer coupled to the tank (a) enabled
a direct reading of the temperature of the washing bath.
[1] The operating procedure for the washing assay consisted
of the following steps: (i) preparing the washing solution; (ii)
adjusting the experimental conditions (pH, temperature, re-
circulation flow, etc.); (iii) soiling the substrate (borosilicate
glass spheres 3 mm in diameter) with the soiling agent; (iv)
placing the substrate in the column; (v) beginning the deter-
sive process, i.e., sampling the washing bath (control) and
switching on the peristaltic pump to start the washing
process; and (vi) sampling at fixed intervals until the end of
the test (10 min). At that point, three samples of the washing
bath were taken. The experiments were followed by analysis
of the total fat content in the washing bath at the end of the
assay, and of the concentration of oleic acid generated ac-
cording to time during enzymatic hydrolysis.

TABLE 1
Description and Properties of the Surfactants Used in the Washing Tests
CMC
Commercial name Family (g/L, 37C)a Structureb HLB

Glucopon 215 Alkylpolyglucoside 0.241 R: 9.3
DP: 1.4 13.0
Glucopon 600 Alkylpolyglucoside 0.028 R: 11.7
DP: 1.2 11.2
Glucopon 650 EC Alkylpolyglucoside 0.073 R: 11.0
DP: 1.3 11.9
Findet 10/15 Fatty alcohol ethoxylate 0.152 R: 10
OE: 2.6 9.6
Findet 10/18 Fatty alcohol ethoxylate 0.196 R: 10
OE: 5.2 12.7
Findet 1214N/23 Fatty alcohol ethoxylate 0.021 R: 12.6
OE: 9.9 14.4
Findet Q/21.5NF Nonylphenol ethoxylate 0.034 R: 9
OE: 9.5 13
LAS Dodecyl benzenesulfonic acid (sodium salt) 1.018 R: 12
a
CMC, critical micelle concentration; from Reference 10. LAS, linear alkylbenzene sulfonate.
b
From Reference 11. R, average alkyl chain length (11); DP, degree of polymerization (11); OE, average number
of ethylene oxide units per molecule (11); HLB, hydrophilic-lipophilic balance, calculated according to Griffin (12)
and Bravo Rodriguez et al. (11).

JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006


FIG. 1. Schematic diagram of the bath-substrate-flow (BSF) device:
(a) glass tank containing the washing bath; (b) packed column con-
taining the triolein-soiled substrate; (c) thermostat-controlled bath;
(d) peristaltic pump; and (e) paddle stirrer.
The substrate was soiled by direct contact with the soiling
agent. For this step, previously established amounts of both
elements were mixed in a glass container. The soiled sub-
strate was transferred to the BSF column, where the exact
quantity of soil adhering to the substrate was determined by
the difference in weight.
Analytical procedures. Three samples from the bath (20 mL
each) were analyzed by the saponification index method to
estimate the percentage of total soil (oleic acid, monoglyc-
erides, diglycerides, and triglycerides) present in the washing
bath at the end of the detersive process (15). The titrations
required for this procedure were made with an automatic
titrator. The detersive performance, or detergency (De), was
defined as the percentage of soil in the washing bath at the
end of the process (Mbath,f) related to the total amount added
to the substrate at the beginning (Msubstrate,i):
M over time. For this, 5-mL samples were taken from the wash-
bath,f
De(%) = 100 [2] ing bath at different times (0, 1, 2, 3, 5, 7, 8, and 10 min), and
M substrate,i
The error in the detergency measurements was estimated
on the basis of three replicates from the washing assays, these
being lower than 10%.
The concentration of oleic acid in the washing bath was
determined by an adaptation of the methodology of Kwon
TABLE 2
Description of the Experiments Performed and the Experimental Conditions
Experimental conditions First stage
Washing bath Enzyme solutions
Variable studied during the process Oleic acid concentration (010 min)
Enzyme concentration (g/L) 0.0, 0.1, 0.2
Temperature (C) 40, 45, 50
Substrate Spheres of borosilicate glass, 3 mm in diameter
Triolein concentration (g/L) 15
pH 8 8
Flow rate (L/h) 30
85
ENZYME-BASED DETERGENT FORMULAS FOR FATTY SOILS
and Rhee (16). i-Octane was added to the samples taken from
the washing bath together with a small amount of 6 N HCl to
improve the extraction of oleic acid. The mixture was
brought to the boiling point to deactivate the enzyme, and
afterward the oleic acid was extracted by i-octane with cold
stirring. The oleic acid was then dissolved in the i-octane and
reacted with a solution of cupric acetate. The cupric salt
formed was determined spectrophotometrically at 715 nm.
Formation of emulsions. Triolein emulsions (5% vol/vol)
were prepared in water with Glucopon 650 and Findet
1214N/23 as emulsifiers (0.2 g/L), following the normalized
procedures described elsewhere, to determine the emulsifi-
cation capacity of the surfactants used in the washing assays
(17). The emulsification was carried out by mechanically stir-
ring the samples with a dispersion device at a rate of 13,000
rpm for 1 min.
The distribution of droplet diameters of the emulsions
prepared was determined by laser-light diffraction, with the
mean Sauter diameter (D3,2) evaluated by the following ex-
pression:
N
n d i
3
i
D 3,2 = i =1 [3]
N
ni di2
i =1
where ni is the number of particles of a certain size, corre-
sponding to a diameter di .
Experimental conditions. Table 2 presents the washing exper-
iments performed and their experimental conditions.
In the first stage, washing tests were conducted in the pres-
ence of commercial lipase, varying the enzyme concentration
in the washing solution and the temperature of the process.
As the experiment proceeded, the quantity of oleic acid gen-
erated by enzymatic hydrolysis of the triolein was determined
the content of oleic acid was analyzed in the samples.
Afterward, in the second stage, washing assays were com-
pleted at 45C using surfactant solutions of 1 g/L in the pres-
ence and absence of lipase, with the detergency determined
at 10 min into the washing process. The surfactants tested are
listed in Table 1.
Second stage
Surfactant solutions with or without enzyme
Detergency (10 min)
0.0, 0.1
45
Spheres of borosilicate glass, 3 mm in diameter
15
30
JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006
86
E. JURADO ET AL.

TABLE 3
Activity and Stability of the Enzyme Lipolase 100L
at Different Temperatures
Temperature Residual activity
(C) Activity (LU/g)a after 30 min (%)
30 110700 4500 99.7 1.8
40 120100 1000 96.5 1.8
50 121600 4600 93.0 0.5
60 97800 3900 87.0 0.3
a
LU, lipase units.

RESULTS AND DISCUSSION

Activity and stability of the enzyme Lipolase 100L. Table 3 lists the
values of the enzymatic activity on tributyrin at pH 7.0 and at
different temperatures (30 to 60C). Aqueous solutions of
the enzyme (0.01 g/L) were maintained for 30 min at tem-
peratures between 30 and 60C to assess the stability of the
enzyme. The residual activity is shown as a percentage of the
initial activity in Table 3.
The activity attained its maximum between 40 and 50C,
an interval of temperatures chosen for the washing assays, al-
though the temperature did not decisively influence the en-
zymatic activity. A decline in activity was significant only at
60C, as confirmed by the greater importance of enzymatic
deactivation at this temperature (18).
Emulsifying capacity of the surfactants. The destabilization
rate of the emulsions (the increase in Sauter diameter as a
function of time elapsed from the preparation) was constant,
on the order of 105 m/d. The D3,2 values of the emulsions
prepared were 2.29 and 2.73 m for Glucopon 650 and
Findet 1214N/23, respectively. Figure 2 presents pho-
tographs of the emulsions at 30 d from preparation, showing
the smaller size of emulsion droplets resulting from the sur-
factant Glucopon 650.
Hydrolysis kinetics of triolein in the BSF device. Experiments
were conducted using triolein as the soiling agent, with both
the enzyme concentration and temperature modified and
the oleic acid concentration in the washing bath determined
as a function of time, to develop an understanding of the
washing mechanism in the presence of the enzyme Lipolase
100L in the BSF system.
FIG. 3. Variations of the oleic acid concentration (Co) in the washing
bath with time. Washing tests were performed with distilled water as
the washing solution and at different process temperatures. The lines
are only visual guides.
Figure 3 shows the results of a control assay using distilled
water as the washing solution at different temperatures. The
oleic acid detected was an impurity in the triolein. We found
that even in the absence of enzyme, the increase in tempera-
ture raised the oleic acid concentration in the bath because
the extraction of soil increased as the temperature increased.
Another aspect reflected in this figure is the redeposition of
the soiling agent over the system surfaces. This became more
accentuated after 5 min of washing. This was evident by the
decline in oleic acid concentration in the washing bath over
time. Similar results were reported by Altmajer-Vaz (19) in
the same experimental device using a mixture of oleic,
stearic, and palmitic acids as the soil.
Figure 4 shows comparisons of the experiments con-
ducted with the two enzyme solutions (0.1 and 0.2 g/L) and
distilled water as washing solutions under the same condi-
tions of temperature (50C), recirculation flow (30 L/h), and
triolein concentration (15 g/L). This graph verifies the exis-
tence of different steps in the process of washing with en-

FIG. 4. Variations of the oleic acid concentration (Co) in the washing

FIG. 2. Photographs (60 magnification) of triolein emulsions ob- bath with time. Enzyme solutions of different concentrations (00.2
tained with the surfactants Findet 1214N/23 (left) and Glucopon 650 g/L) were used as washing solutions. Experiments were performed at
(right). 50C. The lines are only visual guides.

JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006


FIG. 5. Evolution of the oleic acid concentration (Co) in the BSF sys-
tem for different washing temperatures (4050C) using solutions of
0.2 g/L of the enzyme Lipolase 100L as washing solutions. The lines
are only visual guides. For other abbreviation see Figure 1.
zymes: (i) In the first one (03 min), the oleic acid concen-
tration increased with time independent of the enzyme con-
centration; (ii) in the period from 3 to 5 min, the oleic acid
concentration stabilized; and (c) from 5 min of washing on,
the oleic acid concentration increased considerably with time
when the enzyme was used as the washing solution, or dimin-
ished in the case of water.
Figure 5 shows the evolution of the oleic acid concentra-
tion in the BSF system for the different washing temperatures
tested using solutions of 0.2 g/L of the enzyme Lipolase
100L. A positive effect was found in the formation of oleic
acid by the enzymatic reaction with an increase in tempera-
ture. This effect may be related to greater efficiency in re-
moving the triolein from the substrate surface and dispers-
ing it in the washing bath at higher temperatures, thus
producing a larger interfacial area. The three stages of the
washing process can again be seen in this figure.
In the study of the dispersion of fatty soil during the wash-
ing process, the droplet size distribution of triolein was deter-
mined for different washing times. Figure 6 shows the results
FIG. 6. Size distribution of triolein droplets in the BSF device at differ-
ent times during washing. For abbreviation see Figure 1.
87
ENZYME-BASED DETERGENT FORMULAS FOR FATTY SOILS
FIG. 7. Evolution of the Sauter diameter (D3,2) of triolein droplets dur-
ing the washing process at 40C in the BSF device. An enzyme solu-
tion (0.1 g/L) was used as the washing solution. For other abbrevia-
tion see Figure 1.
for a washing experiment conducted with 0.1 g/L of enzyme,
at 40C, and with a recirculation flow of 30 L/h.
This figure indicates that at 3 min of washing, droplets of
between 10 and 120 m in diameter predominated, whereas
after 5 min, a large proportion of much smaller droplets
(around 1 m) appeared, and the distribution of droplets of
soil became bimodal. From 5 min onward, the droplet size
distribution was nearly constant, with the emulsion stabiliz-
ing during this stage of the washing process. This fact can be
discerned in Figure 7, in which the Sauter diameter of tri-
olein droplets dispersed in the washing bath is represented
as a function of washing time.
From these results, it can be deduced that in the rapid
washing process of 3 min, the commercial Lipolase 100L did
not contribute significantly to the detersive process. Only
after 5 min was there a considerable increase in the oleic acid
concentration in the BSF system, and this was related to
greater detergency. These results confirm the importance of
time as a limiting factor in the effectiveness of the action of
lipases in the detergent formulas.
The results indicated that the washing assays made in the
BSF device with solutions of the enzyme Lipolase 100L pre-
sented three phases: (i) In the first phase (0 to 3 min), the
elimination of soil was attributed primarily to the effect of
drawing out the soiling agent by the action of the washing
bath flow, with large fat droplets predominating at first, to-
gether with a low interfacial area and reduced enzymatic ac-
tivity. (ii) In the second phase (approx. 3 to 5 min)in which
a series of changes took place in the droplet size of the soil-
ing agent, giving rise to the formation of a large fraction of
small dropletsthe fatty soil emulsified, and the interface
area of contact between the triolein and the enzyme in-
creased. (iii) In the third phase, the soil was practically emul-
sified in the washing bath, the interfacial area of enzyme/soil
contact reached its maximum value (the droplet size distrib-
ution remained practically constant after this time), and the
enzyme acquired a greater hydrolysis capacity, releasing
greater quantities of oleic acid. This effect became more pro-
JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006
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E. JURADO ET AL.
TABLE 4
Detergency Values After 10 min of Washing at 45C with Washing Solutions Consisting of 1 g/L Solutions
of Different Surfactants, With or Without Enzyme (0.1 g/L)
Detergency (%)
Washing solution Tests without enzyme
Distilled water 32.8
Glucopon 215 57.7
Glucopon 600 98.8
Glucopon 650 EC 94.8
Findet 10/18 82.2
Findet 10/15 85.6
Findet 1214N/23 87.5
Findet Q/21.1NF 97.4
LAS 89.4
a
As calculated by Equation 4. For abbreviation see Table 1.
nounced as the enzyme concentration and temperature in-
creased.
Washing assays with surfactants and Lipolase 100L. Table 4
presents the detergency values at 10 min of washing with so-
lutions of 1 g/L of surfactant in the presence and absence of
Lipolase 100L.
In the washing assays using only distilled water, the deter-
gency value was low, indicating the difficulty of eliminating
triolein from the surface of the substrate and underscoring
the importance of redeposition. Conversely, when 0.1 g/L
Lipolase 100L was added, the detersive effectiveness rose to
50.5%, reflecting the beneficial effect of the enzyme on
cleaning the substrate as well as its antiredeposition effect.
In experiments without enzyme, we found that the fatty al-
cohol ethoxylates studied (Findet 10/15, Findet 10/18, and
Findet 1214N/23) gave similar detergency values (82 to
87%). For the alkylpolyglucosides, there was a considerable
difference between the detersive effectiveness of Glucopon
215 and those of the others (Glucopon 600 and 650). In ad-
dition, we found greater effectiveness washing with nonylphe-
nol ethoxylate (NF), although this surfactant is currently con-
troversial because of the toxicity of its degradation products
(20). In agreement with these experiments, the detergency
power of the surfactants declined in the following order: Glu-
copon 600 > NF > Glucopon 650 > LAS > Findet 1214N/23 >
Findet 10/15 > Findet 10/18 > Glucopon 215. That is, the
surfactant with the greatest detergency power for use in for-
mulas for fatty soils was Glucopon 600 (98.8%), followed by
NF (97.4%) and Glucopon 650 (94.8%).
Taking into account the results of the different emulsions
formed with the surfactants Glucopon 650 and Findet
1214N/23, we identified a relationship between the greater
detergency of the surfactant Glucopon 650 and its greater fa-
cility in emulsifying triolein in the washing bath.
Studies by Weerawardena et al. (21) with commercial sur-
factants based on mixtures of alkylpolyglucosides (Ecoteric
AS10 and Plantaren 1200, composed fundamentally of decyl
- and -D-glucoside and dodecyl - and -glucoside) re-
ported higher detergency values with the product having a
greater carbon-chain size. The results found in the present
JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006
Tests with enzyme Increase in detergency (%)a
50.5 54.0
62.1 7.5
100.0 1.2
100.0 5.5
85.6 4.1
98.2 14.7
94.6 8.2
99.5 2.2
100.0 11.8
study confirm this fact, given that as the mean size of the car-
bon chain of the alkylpolyglucoside surfactant was aug-
mented (G600 > G650 > G215), the detersive effectiveness in-
creased. This effect may be related to the fact that once the
CMC was reached, the micelles presented greater solubiliza-
tion power with an increase in the hydrophobic chain.
With respect to fatty alcohol ethoxylates, two products
were used, the surfactants Findet 10/15 and Findet 10/18,
with the same alkyl chain length (10 carbon atoms) and a dif-
ferent number of moles of ethylene oxide (3 and 6, respec-
tively). The detersive effectiveness proved lower for the sur-
factant having the highest number of ethylene oxide units, in
this case Findet F10/18, which presented lower detergency
than Findet F10/15. This result agrees with information avail-
able in the literature (22), according to which the rise in the
number of ethylene oxide units in nonionic surfactants im-
plies a more hydrophilic character for the surfactant and a
lower detergency power, owing to a lesser capacity to absorb
the surfactant on the substrate.
For a better view of the influence of the lipase on washing
in the presence of surfactant, the effect of the enzyme was
evaluated by the following expression:
De De
De = 0.1 0 *100
[4]
De0
where De0.0 and De0.1 are the detergency values found in the
absence and presence of the enzyme (0.1 g/L), and De is
the increase in detergency upon introducing the enzyme in
the washing bath. The results are shown in Table 4.
For all the surfactants assayed, the addition of 0.1 g/L of
Lipolase 100L to the washing bath positively influenced the
cleaning of the substrate, although this effect was more or
less pronounced depending on the surfactant used. Thus, for
Findet 10/15, Findet 1214N/23, and LAS, the addition of the
enzyme substantially improved the detergency value, result-
ing in increases of 14.7, 8.2, and 11.8%, respectively. In other
cases (Glucopon 600, Glucopon 650, and NF), because of the
high detergency values presented by these surfactants, the
addition of enzyme did not appreciably improve the washing
(<6% increase).

Results similar to those found in the present work were re-
ported by Fujii et al. (5), in which, regardless of the type and
concentration of surfactant, the addition of lipase increased
the effectiveness of the washing of cotton fabrics. In addition,
Andree et al. (7), Helist and Korpela (8), and Xia et al. (9)
observed that the use of nonionic surfactants, especially alco-
hol ethoxylates, positively influenced the activity and stability
of the lipases.
A notable aspect is that the results of the present work co-
incide with those of other authors (5,6,9) who used different
textile substrates. In these cases, the inner structure of the
fabric decisively influenced the effect of the enzyme, deter-
mining the transport of the enzyme to the soil located in the
interior of the fibers. According to Obendorf et al. (23), the
incorporation of lipase implies a significant increase in the
quantity of soil eliminated, regardless of the location of the
soiling agent in the fabric structure. These authors reported
that the quantity of the soil eliminated by the enzyme was sig-
nificantly greater even in structures difficult to access, such
as pores within the fiber structure. It is therefore fitting to
conclude that the incorporation of lipase implies a beneficial
effect on washing, whether of a fabric or a hard surface. In
addition, a packed column was used to place the soiled sub-
strate, so the porosity of the packed bed may also have influ-
enced the lipase action.
Therefore, an effective detergent formulation for fatty
soils on hard surfaces in short time periods could be formu-
lated with the alkylpolyglucosides Glucopon 600 or 650, given
their high level of effectiveness and facility for emulsifying
the fat in the washing bath. Similarly, incorporation of com-
mercial Lipolase 100L into the surfactant LAS or Findet
1214N/23 would give rise to equally satisfactory results, but
longer wash cycles would be advisable in these cases.
ACKNOWLEDGMENTS
Ministerio de Ciencia y Tecnologa supported the projects 1FD97-
0931 and PB1998-1293; the Ministerio de Educacin, Cultura y De-
porte, awarded study grants to Miguel G. Romn and Alejandro F.
Arteaga; and the Comissao de Aperfeioamento de Pessoal de
Nvel Superior, CAPES/Brasil, awarded study grants to Deisi A.
Vaz.
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[Received April 14, 2005; accepted October 13, 2005]
Encarnacin Jurado Alameda (Ph.D. in Chemistry, University of
Granada, Spain) earned a full professorship at the University of
JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006
90
E. JURADO ET AL.
Granada in the Department of Chemical Engineering in 1996.
Since 1999, she has been head of the Chemical Engineering Depart-
ment, where she also directs the Surfactants, Enzymes, and Emul-
sions research group. The research lines followed at present are: en-
zyme kinetics (proteases, lipases, amylases, lactases, etc.) and en-
zyme deactivation; the formulation of enzymatic detergents;
emulsion stability; biodegradation; and the toxicity of surfactants.
Vicente Bravo Rodrguez (M.S. in Chemical Sciences, University of
Granada, 1974; Ph.D. in Chemical Sciences, 1978) holds a full pro-
fessorship at the University of Granada in the Department of Chemical
Engineering. He directs the Engineering of Interfaces and Biochemical
Technology research group. The research lines followed at present in-
clude: the hydrolysis of cellulose waste and obtaining bioproducts by fer-
mentation; the desulfuring of combustion gases; enzyme kinetics and
enzyme deactivation; and the formulation of enzymatic detergents. He
is the author of three textbooks: Introduction to Chemical Engi-
neering, Fundamentals of Chemical Engineering, and Basic
Operations of Chemical Engineering.
Josefa Nez-Olea studied chemistry at the University of
Granada, where she obtained her Ph.D. in 1994. Currently, she is
working for a water supply and treatment company (EMASAGRA)
and is also a part-time associate professor at the University of
Granada. Her research interests include environmental surfactants
and investigation of their properties.
JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006
Rafael Bailn-Moreno (Ph.D. in Chemistry, University of
Granada) has been employed in the perfume industry at Sensient Fra-
grances, S.A., and is currently technical director of BMI, a manufac-
turer of industrial detergents. In addition, since 1999, he has been
an associate professor in the Department of Chemical Engineering at
the University of Granada, forming part of the Surfactants, Enzymes,
and Emulsions research group. He follows two lines of research, one
in scientometrics and the other in the physicochemistry of surfactants.
Deisi Altmajer Vaz obtained a degree in Chemical Engineering
at the University of Rio Grande (FURG), Brazil, in 1998. She ob-
tained her Ph.D. in 2004 from the University of Granada. Her field
of research is in evaluating the performance of detergent formulas
and their optimization.
Miguel Garca Romn graduated with a degree in Chemical En-
gineering from the University of Granada in 1999. He obtained his
Ph.D. in 2005 from the same university. He is currently working
there as an assistant lecturer in the Chemical Engineering Depart-
ment and conducting research into lipase kinetics and their use in
detergent formulas.
Alejandro Fernndez Arteaga graduated with a degree in Chem-
ical Engineering at the University of Granada in 2002. He is cur-
rently completing his Ph.D. thesis at the same university on the
phase behavior and properties of alkylpolyglucoside surfactants and
their use in detergents.