EFFECT OF SELECTED ENZYMES ON PERFORMANCE

OF LIQUID LAUNDRY DETERGENTS

Anita Bocho-Janiszewska
University of Technology and Humanities in Radom, Faculty of Materials Science and Design,
Department of Chemistry,
Corresponding address: Chrobrego Str. 27, 26-600 Radom, Poland, a.janiszewska@uthrad.pl

Abstract: The article examines the effect of type of selected enzymes on

the performance of liquid laundry detergents. Enzymes are the catalysts of biological
processes. Like any other catalyst, an enzyme brings the reaction catalyzed to its
equilibrium position more quickly than it would occur otherwise. The most widely
used detergent enzymes are hydrolases, which remove soils consisting of proteins,
lipids, and poly-saccharides. Soil and stain components with good water solubility are
easily removed during the cleaning process. Most other stains are partially removed
by the surfactant system of a detergent, although the result is often unsatisfactory.
In most cases a suitable detergent enzyme aids the removal of soils and stains.
Whereas the detergent components have a purely physicochemical action, enzymes
act by degrading the dirt into smaller and more soluble fragments.
In the research samples of liquid laundry detergent containing selected hydrolases
(lipase, amylase and protease) were prepared. Tests of the performance of liquid
laundry detergents: viscosity, foaming properties and washing properties were
conducted. Studies were carried out at three different temperatures: 20, 30 and 40°C.
For the sake of comparison, the same tests were also performed for a commercially
available product.
The addition of the enzyme does not affect the viscosity and foaming ability
of the liquid laundry detergent. The ability to remove stains by the liquids containing
enzymes was high even at a lower temperature. Nevertheless, the complete removal
of the stain requires the joint action of the enzyme and the surfactant system.

Keywords: liquid laundry detergents, enzymes, hydrolases, washing ability, foaming

properties.

7
1. INTRODUCTION

Liquid detergents are convenient products. Compared with powdered

detergent they dissolve more rapidly, particularly in cold water, they generate
less dust and they are easier to dose. A typical liquid laundry detergent consists
all or some of the following components: surfactants, builders, optical
brighteners, polymers and enzymes [Lai 1997, Smulders 2002, Aehle 2004].
Both anionic and nonionic surfactants are used in the formulations
of liquid detergents. Surfactants are primarily responsible for wetting
the surfaces of fabrics as well as a soil, helping to lift the stains off the fabric
surface and suspend dirt particulates in solution [Broze 1999, Smulders 2002,
Tadros 2005].
Builders are introduced into detergents mainly to sequester the hardness
of the water. Common builders used are phosphorus compounds (in regions
where they are still permitted in detergent products – these compounds have
been identified as possible cause of eutro-phication of lakes and rivers and they
are severely controlled and even banned in several countries), carbonates,
zeolites, salts of polyacetic acid (EDTA) and citrates [Smulders 2002,
Mahrholz, Klein and Klein 2004, Yangxin, Jin and Bayly 2008].
Polymers are used in liquid laundry detergents as soil antiredeposition
agents, soil release agents and dye transfer inhibitors. Frequently used
polymers are carboxymethyl cellulose (CMC) derivatives, polyethylene
terephthalate and polyoxyethyleneterephthalate (PET-POET polymer)
and polyvinylpyrrolidone (PVP) [Bertleffet et al. 1998, Smulders 2002,
Lai 1997].
Enzymes improve cleaning performance by degrading large complex
molecules such as proteins, starches and fats. The reaction products are more
soluble in the washing liquor and can be removed by the surfactants more

8
efficiently. Enzymes also help to maintain whiteness and brightness and clarify
colors by removing fuzz. They even can improve fabric softness [Maase
and van Tilburg 1983, Olsen and Falholt 1998, Aehle 2004, Zhanget et al.
2014].
Enzymes are proteins. These large organic molecules are produced
by all living cells. They catalyze most chemical reactions in biological systems,
usually at low temperature and at the neutral pH, with an extreme efficacy.
They usually exhibit a very high specificity, reacting on one particular
chemical compound or even on one given bond within the molecule [Broze
1999, Aehle 2004].
Detergent enzymes are not so specific. The most widely used detergent
enzymes are hydrolases which catalyze the hydrolysis of a chemical bond.
There are four types of hydrolases currently being used in liquid laundry
detergents: protease, lipase, amylase and cellulase [Broze 1999, Aehle 2004].
Proteases support the removal of many soils commonly encountered
by the consumer such as food stains, blood and grass. These enzymes catalyze
the hydrolysis of the peptide bond found in proteins resulting in the formation
of smaller and more soluble polypeptides and amino acids [Wolff et al. 1996,
Subba et al. 2009, Singhet al. 2012, Zhang et al. 2014]. Amylase enzymes
work on food stains of the starchy variety, like rice, sauce or gravy. These
enzymes catalyze the hydrolysis of 1-4 glucosidic bonds in starch [Kravetz and
Guin 1985, Hoshino and Tanaka 2003, Hoshino, Tanaka and Kanda 2006].
Lipase enzymes target the oily, greasy stains that are some of the most difficult
stains to remove. Lipases catalyze the hydrolysis of mostly the C1 and C3
bonds in the triglyceride molecule, yielding free fatty acids and diglyceride
[Varanasi et al. 2001, Bora 2014]. Cellulases are capable of degrading
the structure of damaged cellulose fibrils which exist mostly at the surface
of cotton fibers after multicycle washing and using. Cellulases cleave β-1,4-

9
glucosidic bonds incellulose and operate directly on the natural cotton fibers.
Cellulases bring diverse benefits: fabric softening, color brightening,
antipilling, soil-release properties and antiredeposition [Calvimontes, Stamm
and Dutschk 2009, Calvimontes, Lant and Dutschk 2011].
The aim of this study was to determine the effect of various enzymes
on the performance of liquid laundry detergents. In the paper samples of liquid
laundry detergent containing selected hydrolases (lipase, amylase
and protease) were prepared. Tests of the performance of liquid laundry
detergents: viscosity, foaming properties and washing properties
were conducted. The studies were carried out at three different temperatures:
20, 30 and 40°C. For the sake of comparison, the same tests were also
performed for a commercially available product.

1. MATERIAL AND METHODS

The study examined liquid laundry detergents containing three enzymes:

protease, amylase and lipase. The formulation of the liquid laundry detergent
tested is specified in Table 1. For the purpose of comparison, an enzyme-free
laundry detergent (Base) and a commercial product (Com) were also tested.
The composition of the commercial product, based on the manufacturer’s data,
includes 5-15% anionic surfactants, <5% nonionic surfactants, phosphonates,
soap, enzymes, fragrance.

Viscosity measurement

Dynamic viscosity measurements were performed with Brookfield

DV-III rotational viscometer. Viscosity is measured with a rotating measuring
tip called the spindle, which is immersed in the test fluid. Testing was
performed at a temperature of 20°C, with the spindle rotating at 10 rpm.

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Table 1. Formulation of liquid laundry detergent tested

Composition [wt. % ]
INCI Name
Base P A L MixS Mix0

Laureth-7 10 -

Laureth-3 3 -

Sodium Laureth Sulfate 7 -

Propylene Glycol 2

Sodium Citrate 1

Protease - 0.5 - - 0.5 0.5

Amylase - - 0.5 - 0.5 0.5

Lipase - - - 0.5 0.5 0.5

Aqua to 100
Source: Authors own study

Foaming properties measurement

The foaming properties were determined using a method set out

in the Polish standard [Polish Standard – foaming properties]. The method
involved a measurement of the volume of foam produced by a free flow
of the gel laundry detergent solution from a distributor onto the surface
of the same solution inside a graduated cylinder. Measurements of volume
were performed after 30 s, 1 min and 10 min. The concentration of the test
solutions was 1 wt%.
Based on measurements, two parameters were determined:
- foaming ability, FA [cm3] – volume of foam produced after 30 s.
- foam stability, FS [%]:

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 V10
FS = ×100 [%]
V1

where: V10 – volume of foam measured after 10 min [cm3],

V1 – volume of foam measured after 1 min [cm3].

Washing properties measurement

The washing properties were determined on the basis of methodology set

out in the Polish standard [Polish Standard – washing properties]. The method
involved washing of pieces of soiled test fabric in the test washing agent
in strictly defined conditions. Pieces of cotton fabric were stained with,
previously mixed, three types of soils: tannic, fat and protein. The fabric pieces
were dried overnight in the open air. Fabrics prepared in this way were washed
separately in the liquid laundry detergent tested. The concentration of the test
liquid laundry detergent was 1 wt %. After washing, the pieces of fabric were
rinsed and ironed, and their degree of whiteness was assessed.
Based on tests, the following parameters were determined:
- washing ability, WA [%]:
X -B
WA = ×100 [%]
A- B

where: X – average degree of whiteness of soiled fabric after washing,

B – average degree of whiteness of soiled fabric before washing,
A – average degree of whiteness of unsoiled (control) fabric.

12
3. RESULTS AND DISCUSSION

Viscosity of liquid laundry detergents is shown in Figure 1. The viscosity

of the detergents tested ranged between 900-995 mPas. There was a slight
increase in the viscosity of liquids containing the enzyme tested,
but the difference was within the limit of error. It can therefore be stated
that the surfactants system has stronger impact on the fluid viscosity
and consistency than the addition of enzymes. Viscosity of the commercial
product (Com) was equal to 650 mPas and was significantly lower
than viscosity of other liquid detergents.

Figure 1. Dynamic viscosity of liquid laundry detergents tested. Source: Authors

own study

Figure 2 shows foaming ability (diamonds) and foam stability (squares)

of liquid detergents tested. All the tested detergents were characterized
by a high foaming ability (320-330 cm3) beyond commercial product for which
this parameter was much lower: 210 cm3. The foaming stability
for the detergent without enzymes (Base) and detergents containing protease

13
(P) and amylase (A) was comparable and its value was within the range
90-93 %. The addition of lipase resulted in a decrease of foam stability
o 81 % for detergent containing only lipase (L) and detergent containing
all three enzymes (MixS). Foam stability for the commercial product was
the lowest – equal to 60 %.

Figure 2. Foaming ability (dark gray diamonds) and foam stability (light gray
squares) of liquid laundry detergents tested. Source: Authors own study

The washing ability of the detergent tested is shown in Figure 3.

The pieces of soiled fabric were washed separately by solutions of various
liquid laundry detergents: detergent without enzymes (Base), detergents
containing only one enzyme – protease (P), lipase (L) and amylase (A),
detergent containing mixed three enzymes (MixS), detergent containing mix
three enzymes but without any surfactant (Mix0) and commercial detergent
(Com). The washing process was carried out at three different temperatures
20°C, 30°C and 40°C for 60 min.

14
Figure 3. Washing ability of liquid laundry detergents tested in various temperature:
20°C, 30°C and 40°C. Source: Authors own study

Even the addition of a single enzyme to the surfactant system has

a significant impact on the washing ability. This is particularly evident
for addition of protease and lipase at 20°C. Amylase has smaller impact
on the washing ability. Maximum efficiency of the stain removal was observed
for Mix S, when the mix of the three enzymes was combined with the detergent
system. In this case, the synergy effect between enzymes and between enzymes
and surfactants is very important. [Kravetz and Guin 1985, Wolffet et al. 1996,
Hoshino and Tanaka 2003, Hoshino, Tanaka and Kanda 2006, Subba
et al. 2009, Singh et al. 2012, Zhang et al. 2014]. The minimal washing ability
was observed for the liquid containing enzymes but without any surfactants
(Mix0). This result may indicate that without a surfactant system the enzymes
were destabilized and deactivated [Maaseand van Tilburg 1983, Kravetz
and Guin 1985, Hoshino and Tanaka 2003, Hoshino, Tanaka and Kanda 2006,
Zhang et al. 2014].

15
 The temperature of washing has a significant effect on the washing
ability of the liquid detergent without enzymes (Base). For liquid with enzymes
(P, A, L and MixS) the maximal washing ability was observed at 30°C
and increasing temperature to 40°C did not affect further growth
of this parameter.

4. CONCLUSIONS

The study investigated the effect of three enzymes (protease, amylase

and lipase) on performance of liquid laundry detergents. Various detergents
were tested: without enzymes (Base), containing only one enzyme – protease
(P), lipase (L) and amylase (A), containing mixed three enzymes (MixS),
containing mixed three enzymes but without any surfactant (Mix0)
and commercial detergent (Com). Tests of dynamic viscosity, foaming
properties and washing properties were conducted. The washing process was
carried out at three different temperatures 20°C, 30°C and 40°C.
Based on the tests, the following findings were made:
• the addition of the enzyme did not affect the change in viscosity
of the liquid laundry detergent tested, in this case the consistency
of the fluid detergents was primarily dependent on the surfactant
system;
• foaming ability did not depend on the addition of the enzyme;
• foam stability was very high for detergents without lipase, the addition
of this enzyme caused reduction of foam stability;
• temperature of washing had a significant effect on the washing ability
of the liquid detergent without enzymes while for liquid with enzymes
maximum washing ability was observed at 30°C. Increasing
temperature to 40°C did not affect further growth of this parameter.

16
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in Detergents, Journal of Surfactants and Detergents, vol. 1, issue 3, pp. 419-424.
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Bacillus licheniformis MTCC 2465: and Study of Its Detergent Compatibility
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Broze G. 1999. Handbook of Detergents, Marcel Dekker, Inc., New York.
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graphical Standpoint, Journal of Surfactants and Detergents, vol. 14, pp. 307–316.
Calvimontes A., Stamm M., Dutschk V. 2009. Effect of Cellulase Enzyme on
Cellulose Nano-topography, Tenside Surfactants Detergents, vol. 46, pp. 368–372.
Hoshino E., Tanaka E. 2003. Enhancement of Enzymatic Catalysis of Bacillus
amyloliquefaciens α-amylase by Nonionic Surfactant Micelles, Journal
of Surfactants and Detergents, vol. 6, issue 4, pp. 299-303.
Hoshino E., Tanaka E., Kanda T. 2006. Effects of a Nonionic Surfactant
on the Behavior of Bacillus amyloliquefaciens a-amylase in the Hydrolysis
of Malto-oligosaccharide, Journal of Surfactants and Detergents, vol. 9 issue 1,
pp. 63-68.
Kravetz, L., Guin K.F. 1985. Effect of Surfactant Structure on Stability of Enzymes
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Maase F., van Tilburg R. 1983. The Benefit of Detergent Enzymes under Changing
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on Saccharides (II) Cobuilder Performance of Ionic Allyl Glycosidepolymers

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Singh S.K., Singh S.K, Tripathi V.R., Garg S.K. 2012. Purification, Characterization
and Secondary Structure Elucidation of a Detergent Stable, Halotolerant,
Thermoalkaline Protease from Bacillus cereus SIU1, Process Biochemistry,
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STRESZCZENIE

W artykule badano wpływ stężenia oraz rodzaju wybranych enzymów na właściwości

fizykochemiczne i użytkowe płynnych środków piorących. Enzymy są katalizatorami
procesów biologicznych i biochemicznych. Podobnie jak inne katalizatory enzymy
przyspieszają reakcje obniżając ich energię aktywacji. Najczęściej stosowane
w środkach piorących enzymy to hydrolazy, które pomagają w usunięciu
zanieczyszczeń proteinowych, tłuszczowych oraz węglowodanowych. Szczególnie
w przypadkach, gdy zanieczyszczenia te są słabo rozpuszczalne w wodzie.
Rozpuszczalne w wodzie zanieczyszczenia są dosyć dobrze usuwane przez roztwór
samego związku powierzchniowo czynnego, który zwilża powierzchnię plamy,
odrywa ją od tkaniny i wprowadza do roztworu. Mechanizm takiego działania
jest czysto fizyczny. W przypadku enzymów mechanizm usuwania zanieczyszczenia
jest typowo chemiczny. Duże, nierozpuszczalne w wodzie cząsteczki są dzielone
na mniejsze, rozpuszczalne w wodzie i łatwiejsze do usunięcia przez związki
powierzchniowo czynne.
Celem badań było określenie wpływu rodzaju enzymu na właściwości użytkowe
i fizykochemiczne płynnych środków piorących. Badano trzy rodzaje enzymów:
proteazę, lipazę oraz amylazę w stężeniu 0,5 % wag. Na podstawie opracowanej
receptury sporządzono próbki płynów do prania. Dla porównania badano również
płyn handlowy oraz środek zawierający enzymy, ale bez surfaktantów. Badano wpływ
enzymu na lepkość, właściwości pianotwórcze oraz na zdolność wyprania. Badania
zdolności wyprania wykonywano w trzech temperaturach: 20°C, 30°C and 40°C.
Przeprowadzone badania wykazały, że dodatek badanych enzymów nie ma większego
wpływu na lepkość oraz zdolność pianotwórczą płynnych środków piorących.
Największy wpływ enzymów odnotowano na właściwości piorące. Zastosowanie
enzymów pozwoliło na usunięcie większości zabrudzeń nawet w temp. 20°C. Podczas
prowadzonych badań zaobserwowano efekt synergii między enzymami
oraz związkami powierzchniowo czynnymi.

Słowa kluczowe: płynne środki piorące, enzymy, hydrolazy, zdolność wyprania,

właściwości pianotwórcze

19