Preparation, characterization, and surface and biological properties of N-stearoyl amino acids

Article abstract of DOI:10.1007/s11746-001-0361-5

Several lipoamino acids were synthesized, in which n-octadecanoic acid (stearic acid) was coupled with the α-amino group of an amino acid. The products were characterized and their identities confirmed by advanced analytical techniques like Fourier transform infrared, ¹H nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. Their surface properties, such as critical micelle concentration (CMC) and foaming properties, biodegradability, and antimicrobial activity were also evaluated. The N-stearoyl amino acids (NSA) had low CMC values, and some of them showed good foaming properties. They were screened for antimicrobial activity against the gram-positive bacteria Staphylococcus aureus, Micrococcus luteus, and Bacillus cerceus, the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, and the yeast Candida albicans. All the compounds inhibited at least one of these organisms. N-Stearoyl proline was the most effective, the order of antimicrobial activity being aromatic NSA > acidic NSA > basic NSA. However, the effective inhibition by all the compounds indicates the desirability of more thorough investigation and suggests that some of these compounds may have potential utility as biostatic additives in commercial products. All NSA are highly biodegradable and can readily be removed under conditions of normal secondary sewage treatment.

Full text of DOI:10.1007/s11746-001-0361-5

Preparation, Characterization, and Surface and
Biological Properties of N-Stearoyl Amino Acids
A. Sivasamy*, M. Krishnaveni, and P.G. Rao
Chemical Engineering Division, Central Leather Research Institute, Adyar, Chennai-600 020, India
ABSTRACT: Several lipoamino acids were synthesized, in ity, and volume of the surfactant solutions. Thus, there is a
which n-octadecanoic acid (stearic acid) was coupled with the
α-amino group of an amino acid. The products were character-
ized and their identities confirmed by advanced analytical tech-
niques like Fourier transform infrared, 1H nuclear magnetic res-
onance spectroscopy, and differential scanning calorimetry.
Their surface properties, such as critical micelle concentration
(CMC) and foaming properties, biodegradability, and antimicro-
bial activity were also evaluated. The N-stearoyl amino acids
(NSA) had low CMC values, and some of them showed good
foaming properties. They were screened for antimicrobial activ-
ity against the gram-positive bacteria Staphylococcus aureus,
need to develop a suitable method to measure the foaming
properties of any surfactant molecules of any types, such as
lipoamino acids, to keep pace with advances in the surfactant
field. There is a correlation between X-ray diffraction (XRD)
and differential scanning calorimetry (DSC) data for the poly-
morphic states of surfactants (3). Hence, an attempt has been
made to measure the foaming properties of lipoamino acids
by microcalorimetric techniques.
Studies of biological properties of surfactants include
screening of antimicrobial efficacy and biodegradability. The
Micrococcus luteus, and Bacillus cerceus, the gram-negative quaternary ammonium compounds that are widely used as
bacteria Escherichia coli and Pseudomonas aeruginosa, and the
yeast Candida albicans. All the compounds inhibited at least
one of these organisms. N-Stearoyl proline was the most effec-
tive, the order of antimicrobial activity being aromatic NSA >
acidic NSA > basic NSA. However, the effective inhibition by
all the compounds indicates the desirability of more thorough
investigation and suggests that some of these compounds may
have potential utility as biostatic additives in commercial prod-
ucts. All NSA are highly biodegradable and can readily be re-
moved under conditions of normal secondary sewage treat-
ment.
germicides (4) are strongly bacteriostatic at higher dilutions
and have germicidal action in more concentrated solutions.
Earlier reports of N-palmitoylated amino acids tested for
their inhibitory action against Sendai virus fusion to lipo-
somes composed of egg phosphatidylethanolamine (5). Fatty
acids of varying chain lengths are known for their antimicro-
bial action (6,7), primarily against gram-positive bacteria and
yeast at low pH. Sheba et al. (8) studied the fungistatic action
of oleic acid, which was found to be active against a wide
spectrum of saprophytic molds and yeast. It has also been re-
ported (9) in the literature that long-chain fatty acid deriva-
tives of glycolic acid showed antimicrobial activity.
Paper no. J9653 in JAOCS 78, 897–902 (September 2001).
KEY WORDS: Amino acids, antimicrobial activity, biodegrad-
ability, critical micelle concentration, foaming property,
lipoamino acids, surface activity.
The biodegradability of surfactants has been the subject of
numerous investigations in the past few years. The hydropho-
bic groups in all nonionic surfactants like dodecyltrimethyl-
ammonium glycol monoether can be biodegradable, except
those with highly branched carbon chains. Matsumura et al.
(10) studied the biodegradability of n-alkyl glucosides, man-
nosides, and galactosides and found that these surfactants were
up to 50% biodegradable. In recent years, there has been
worldwide interest in developing surface-active molecules that
have both antimicrobial activity and environmental compati-
bility, or, in other words, that are easily biodegradable. Differ-
ent kinds of surface-active molecules (11) have been synthe-
sized, and some of them are being used in practical applica-
tions, mainly in cosmetic and food applications. Only a few
studies of their properties have been reported in the literature.
N-Acyl amino acids based on a C18 chain have not yet
been thoroughly studied with respect to their physicochemi-
cal and biological properties, in particular, their antimicrobial
activity and biodegradability. In the present work, we synthe-
sized N-octadecanoic acid-based lipoamino acids and investi-
gated their interfacial, antimicrobial, and biodegradability
properties.
Studies on lipoamino acids have been of great interest in the last
few years because of their high degree of biocompatibility. Sur-
factants have a variety of applications in detergency, solubi-
lization, emulsification, and preservation of biomaterials.
Studies on surface-active properties of different types of sur-
factants derived from lauroyl and palmitoyl derivatives of
glycine and alanine have been reported (1). Critical micelle
concentration (CMC) and foaming ability can serve to char-
acterize the physicochemical nature of any surfactant mole-
cule. The evaluation of foaming properties of surfactants by
the Ross–Miles method was reported in the literature in 1940
(2). This method involves the measurement of foam height
for comparison of relative foam stability. The measured value
depends on capillary penetration, time, temperature, solubil-
*To whom correspondence should be addressed.
Email: siva_42@hotmail.com
Copyright © 2001 by AOCS Press
897
JAOCS, Vol. 78, no. 9 (2001)

898
A. SIVASAMY ET AL.
analysis, 5–10 mg of the NSA was weighed into an aluminum
EXPERIMENTAL PROCEDURES
DSC pan. The samples were analyzed under a nitrogen atmos-
phere at a program rate of 5°C/min up to 350°C. All samples
were analyzed in duplicate. The peak integrations were per-
formed automatically by the computer program.
Materials. All the amino acids used in this study (L-glutamic
acid,
L
-aspartic acid,
L
-lysine, -histidine,
L
-arginine,
L
L-
proline,
L
-phenylalanine,
L
-tryptophan, and L-tyrosine) were
purchased from Hi Media Laboratories Pvt. Limited (Bom-
bay, India). n-Octadecanoic acid (stearic acid) was purchased
from Loba Chemie (Bombay, India), acetone from S.D. Fine
Chemicals Ltd. (Bombay, India), ethyl alcohol from Bengal
Chemicals (Calcutta, India), petroleum benzine from Ran-
baxy Fine Chemicals Limited (New Delhi, India), and the
deuterated solvent CDCl3 from Aldrich Chemicals (Milwau-
kee, WI). Sodium hydroxide and sulfuric acid were obtained
from Fischer Inorganic and Aromatics Ltd. (Madras, India).
Double distilled water was used wherever necessary. All other
chemicals used were analytical-grade reagents.
Synthesis of N-stearoyl amino acids (NSA). Synthesis of
NSA involves a two-step process, i.e., preparation of the acid
chlorides as reported elsewhere (12) followed by preparation
of the corresponding NSA. A typical method is as follows.
To an acetone and water mixture (pH 12 with NaOH), 0.24
Methodology for screening for antimicrobial activity. The
NSA prepared in this study were screened for their antimicro-
bial activity against pathogenic organisms including gram-
positive and gram-negative bacteria and a fungal strain. Six
organisms (Escherichia coli, Staphylococcus aureus, Pseudo-
monas aeruginosa, Micrococcus luteus, Bacillus cereus, and
Candida albicans) were used to study the ability of the test
compounds (at concentrations of 10 and 20 mg/mL) to inhibit
microbial growth. All the bacterial species were grown on nu-
trient agar (Hi Media Laboratory, Bombay, India); C. albi-
cans was cultivated on Sabouraud dextrose media (Hi Media
Laboratory). The cultures were incubated at 30°C. The fol-
lowing controls were employed: for E. coli, norfloxacin (5
µg); S. aureus, tetracycline (20 mg); P. aeruginosa, gen-
tamycin (20 µg,); M. luteus, erythromycin (20 µg), B. cereus,
doxycycline (10 µg); and C. albicans, ketoconazole (500 µg).
Twenty-four-hour slant cultures of the microorganism were
used to prepare suspensions for plate inoculations. The sus-
pensions served as the inoculum for the determination of an-
timicrobial activity. Agar plates were inoculated with the ap-
propriate inoculum by placing 3 drops on the agar surface and
spreading them uniformly with a sterile, bent glass rod. Filter
paper discs 6.5 mm in diameter, made from Whatman No.1
filter paper, were used to evaluate the samples. The paper
discs were wetted until they were completely saturated with
the test compound (at concentrations of 10 and 20 mg/mL)
and then placed on the surface of the agar plates inoculated
with the test organisms. N,N-Dimethylformamide was used
as carrier solvent. A minimum of two experiments was per-
formed at different times, employing duplicate plates for each
compound under test. All plates were incubated at the opti-
mal growing temperature for each organism, and readings
were taken after 24 h. The zones of inhibition were compared
to those of the controls.
mol of L-amino acids and 0.2 mol of stearoyl chloride were
added with stirring over 25 min at 0°C. After further stirring
for 30 min, the mixture was acidified with sulfuric acid to ob-
tain crystalline and semisolid N-stearoyl amino acid. Follow-
ing washing with petroleum benzine, the crystals obtained
were recrystallized from an ethanol/petroleum benzine mix-
ture.
All the prepared NSA were characterized by Fourier trans-
form infrared (FTIR) spectroscopy and proton nuclear mag-
netic resonance (NMR) spectroscopy. FTIR spectra were
recorded using a Shimadzu FTIR 8000 series Spectrometer
(Kyoto, Japan); KBr pellets were used for solid samples, and
a transmission cell with NaCl windows was used for liquid
samples. NMR spectra were recorded using a Bruker 300-
MHz FT-NMR spectrometer (Karlsruhe, Germany). Samples
were dissolved in CDCl3, and tetramethylsilane was used as
an internal standard.
Determination of CMC. The CMC of all NSA were esti-
mated by a conductivity method (Direct Conductivity Mea-
surement Meter 303; cell constant = 1.0003; Systronic Elec-
tronics, Amendabad, India). NSA solutions (10 mM) were
prepared in 0.1 N sodium hydroxide and added to a beaker
containing 40 mL of double distilled water. The specific con-
ductance was measured during gradual addition of the NSA
solution. When differences in specific electrical conductivity
were plotted against the concentration of NSA in the beakers,
two straight lines were obtained and were extrapolated to ob-
tain the concentration at their intersection, which was defined
as the CMC. All the measurements were carried out in dupli-
cate.
Evaluation of biodegradability. The 5-d biochemical oxy-
gen demand (BOD) of NSA was determined by the standard
oxygen consumption test (13) using activated sludge obtained
from a sewage treatment plant at the Central Leather Re-
search Institute (Adyar, India). All experiments were con-
ducted in triplicate.
RESULTS AND DISCUSSION
Synthesis of N-stearoyl amino acids. The NSA used in this
study were prepared by conversion of fatty acids to their acid
chlorides and further reaction with the L-amino acid under
Determination of foaming properties by differential scan-
ning calorimetry (DSC). All DSC measurements were per-
formed on a DuPont 2000 Thermal Analyzer, equipped with
a DSC cell (DuPont, Boston, MA). The peak areas were esti-
mated by the DuPont Advanced DSC (V. 4.1.C) program. For
suitable conditions. The lipoamino acids were characterized
1
by H NMR, FTIR spectroscopy and C, H, N analysis. The
characteristics of the NSA are given in Table 1.
FTIR spectroscopy. FTIR spectra of all the NSA were
recorded. The band at 1460 cm–1, assigned to the CH2 scis-
JAOCS, Vol. 78, no. 9 (2001)

STUDIES ON N-STEAROYL AMINO ACIDS
899
TABLE 1
Chemical Characteristics of the Compounds Synthesized
R in
R–CH–COOH
Analysis [%; calc. (found)]
H N
|
Amino acid
Yield
(%)
M.P.
(°C)
NHCO(CH2)16CH3
Compound
moiety
MW
C
1
2
3
4
5
6
7
8
Glutamic acid
Aspartic acid
Arginine
Lysine
Histidine
Proline
Phenylalanine
Tryptophan
Carboxymethylene
Carboxymethylene
Guanido-n-valeric acid
4-Aminobutyl
Imdazole propionic acid
Cyclic
92
90
87
50
93
50
63
92
413.594
399.567
440.668
412.654
421.621
381.596
431.656
470.693
154.75
53.56
37.26
57.40
43.77
37.94
49.34
41.28
60.11 (61.13)
59.51 (59.32)
58.87 (58.46)
34.92 (35.90)
61.53 (61.32)
36.196 (36.25)
47.33 (46.32)
68.08 (65.45)
9.4 (9.27)
9.3 (9.2)
9.8 (9.75)
5.86 (5.95)
9.25 (9.55)
5.6 (5.5)
3.05 (3.15)
3.15 (3.20)
11.44 (11.24)
3.33 (3.31)
8.9 (8.5)
1.8 (1.59)
2.04 (2.15)
5.47 (5.35)
Benzyl
3-Methyleneindole
6.61 (6.59)
9.06 (10.25)
soring motion, indicates the presence of methylene groups in N-stearoyl phenylalanine, N-stearoyl arginine, N-stearoyl pro-
the NSA. The absorption bands at around 2960 and 2930 line, and N-stearoyl tryptophan, the alkyl side chain of the
cm–1 arise from C–H stretching in methyl and methylene amino acid is bulky, resulting in restricted molecular mobility
groups, respectively. Absorptions of C=O and amide group in the aqueous environment. Therefore, NSA have the ability
were observed at 1657 and 3447 cm–1, respectively.
to form aggregates at lower concentrations, and thus their
Proton NMR spectroscopy. The 1H NMR spectra of NSA conductance becomes low. On the other hand, the overall hy-
were recorded, and the chemical shifts of the constituent pro- drophobicity of the molecules become larger and the CMC
tons (14) were as follows. A triplet was observed at a δ value lower.
of 0.8, which is characteristic of methyl protons adjacent to
DSC. The polymorphic states of surfactants are usually
methylene protons in long-chain alkyl molecules. The meth- studied by XRD, but with the advancement of microcalorime-
ylene [(–CH2)16] proton resonance was observed at δ 1–2 as try DSC techniques, it is possible to identify various polymor-
a multiplet. The resonance of the protons in the amide link- phic states of fats and fatty acids by DSC, as reported else-
age of the NSA was seen at δ of 4–6 as a doublet. For exam- where (16–18). The surfactants have four polymorphic states:
ple, in the case of N-stearoyl tryptophan, the amide NH pro- α, ∆, β, and Ω. In phase identification, XRD and DSC meth-
ton resonance appeared as a doublet (JNHCH = 5.3 Hz) with ods can be used as a tool for the assessment of various physi-
an integrated intensity of 0.75. The NMR signals of the tryp- cal properties related to attributes of the surfactants (17). The
tophan protons were spread over δ 6.8–7.8 with a total inte- following assumptions are made in order to develop this cor-
grated intensity of 4.92, which is accounted for by the five relation: The α form does not exist under the ordinary condi-
protons. Similar observations were made for all the samples. tions of preparation of the NSA (3). In the thermal absorption
The resonances of methylene protons in the amino acid moi- range below 100°C, the NSA would have the ∆ form, and
ety were observed at a δ value of 3.1–3.2. The resonances of they are assumed to be in a solid-solution or gelatinous state.
all the aromatic and heterocyclic molecules present in the At 101–180°C, the NSA would have the β form, which indi-
lipoamino acid side chains were observed at a δ value of 7–8. cates that they are in the liquid-crystalline state. The thermal
Interfacial properties of NSA. Surface-active properties of absorption range of NSA between 181 and 300°C is attrib-
NSA were evaluated in terms of CMC and foaming property uted to the Ω form, and the physical nature would be crys-
by DSC.
CMC. Specific conductance of the NSA was measured in
talline.
DSC thermal absorption data for the polymorphic states of
alkaline solution. The CMC was determined from a plot of the NSA are shown in Table 3. All thermal absorptions are re-
specific conductance vs. concentration of the NSA by extrap-
olating the two straight lines, the intersection of the lines giv-
ing the CMC value of the NSA. The values obtained are tab-
ulated in Table 2. It is inferred that below the CMC, the NSA
solutions may behave as ideal solutions. This indicates that
TABLE 2
Critical Micelle Concentration (CMC) of Sodium Salts of N-Stearoyl
Amino Acids (NSA)
Compound
Amino acid moiety
CMC (mM)
surface-active molecules are adsorbed on the surface of the
aqueous phase and form a close-packed monomolecular layer.
Subsequent addition of surfactant to the system favors
molecular aggregation or, in other words, micelle formation.
Adsorption and interfacial activity generally increase with
increasing concentration up to the CMC. Above the CMC, the
surface-active molecules behave nonideally. The shorter
the alkyl side chain in the amino acid molecules (10,15), the
higher is the CMC value. In the case of NSA (C18) like
1
2
3
4
5
6
7
8
9
Glutamic acid
Aspartic acid
Arginine
Lysine
Histidine
6.00
2.40
1.75
2.40
2.10
1.75
2.00
2.00
2.25
Proline
Phenylalanine
Tryptophan
Tyrosine
JAOCS, Vol. 78, no. 9 (2001)

900
A. SIVASAMY ET AL.
TABLE 3
Differential Scanning Calorimetry (DSC) and Lather Value (LV) Data of NSAa
∆ Phase
β Phase
Ω Phase
J/g °J/g
Amino acid
moiety
Total
°J/C
Total
°J/C
Total
°J/C
°C
J/g
°J/g
—
°C
J/g
°J/g
°C
LVb
Glutamic acid
Aspartic acid
Arginine
—
—
—
154.75
—
246.6
—
381.6
—
381.6
—
214.75
—
482.3 1518.03 1518.03 1.2513
53.56
37.26
83.93 44.950
8.458 3.151
44.950
—
—
—
—
3.151 134.23
176.0
236.24 236.24 191.67
265.27
60.82 116.57 386.61 1.1611
101.8
270.04
Lysine
57.46
92.86
62.23 35.72
40.88 37.96
73.780
—
—
—
—
212.35
258.63
117.1
72.08 186.42
148.66 435.08 1.000
Histidine
Proline
43.77
53.89 23.58
23.580 156.56
163.91
33.54
12.36
52.51
20.25
72.76 203.05
204.9
416.04 416.04 1.1748
37.94
77.10
72.05 27.33
17.80 13.72
231.770
—
—
—
—
195.04
260.05
191.9
247.6
374.28 1018.16 1.000
643.80
99.39 191.9 190.72
Phenylalanine
49.34
58.70
32.52 16.04
63.22 37.11
53.150 106.16
23.670 118.81
18.09
19.20
19.20
—
—
—
—
—
Tryptophan
Tyrosine
41.28
57.36 23.67
197.9
—
235.1
—
235.12
—
—
—
—
—
—
60.36 115.3
69.59
69.590
—
212.06
260.86
160.9
48.41 126.28
341.20 467.48 1.000
aSee Table 2 for abbreviation.
bSee Equation 2.
ported in degrees Celsius, and the corresponding integrated gram-positive bacteria S. aureus, M. luteus, and B. cereus, the
peak areas are in units of joules per gram (J/g). For the pur- gram-negative bacteria E. coli and P. aeruginosa, and the fun-
pose of normalization of these data, the °C value is multiplied gus C. albicans. N-Stearoyl proline, N-stearoyl phenylala-
by the J/g value and then divided by 100 to reduce the num- nine, and N-stearoyl tryptophan displayed inhibitory activity
ber to manageable values in units of degree joules per gram (+++) against all organisms except Pseudomonas sp. N-
(°J/g):
Stearoyl arginine showed inhibition against three microorgan-
isms, N-stearoyl lysine and N-stearoyl aspartic acid displayed
[1] inhibition against two microorganisms, and N-stearoyl tyro-
sine showed inhibition against a single microorganism. Com-
(°C) × (J/g)/100 = (°J/g)
The lather values (LV) of the lipoamino acids were pre- pounds marked + (zone of inhibition less than 0.5 cm) in
dicted in the following manner. LV is a dimensionless unit, Table 4 are not necessarily inferior in inhibitory activity to
which represents the potential of foaming. Properties of a sur-
factant are obtained (3,18) by relating the β phase (responsi-
TABLE 4
Antimicrobial Activity of NSAa
ble for lathering or foaming) with the Ω phase (responsible
for insolubility) as described in Equation 2
Antimicrobial activityb,c
A
B
C
D
E
F
LV = °J/g (Ω) + °J/g (β)/°J/g (Ω)
[2]
Stearic acid
–
++
–
++
+
+
++
+
+
–
–
–
–
–
–
–
++
+
–
–
++
++
–
++
+
–
–
–
–
+
NS-Glutamic acid
NS-Aspartic acid
NS-Arginine
NS-Lysine
On the thermograms of the NSA, the melting points of the
samples are observed below 100°C. With further heating, de-
composition is observed. For comparison purposes, the higher
the LV of a surfactant is, the greater are its surface-active
properties. The LV of NSA are given in Table 3. Among all
the NSA investigated, N-stearoyl arginine has the highest LV
and N-stearoyl histidine has the lowest.
Evaluation of antimicrobial activity of NSA. The NSA pre-
pared in this study were screened for their antimicrobial ac-
tivity against several of pathogenic organisms (7–9) includ-
ing both gram-positive and gram-negative bacteria and fungi.
Several of these compounds exhibited a broad spectrum and
high level of activity against all or most of the test organisms.
Table 4 shows the antimicrobial activity of NSA against the
NS-Histidine
NS-Proline
NS-Phenylalanine
NS-Tryptophan
NS-Tyrosine
Control
–
–
–
–
–
–
–
+++
–
–
–
–
+
+
–
+++
+++
++
++
–
+++
+++
+++
–
++
++
++
–
+++
+++
+++
+
+++
+++
+++
+++
aNS, N-stearoyl; see Table 2 for other abbreviations.
bMicroorganisms: A, Escherichia coli; B, Staphylococcus aureus; C, Pseudo-
monas aeruginosa; D, Micrococcus luteus; E, Bacillus cereus; F, Candida al-
bicans.
c+++, Zone of inhibition was at least 1 cm beyond disc area at 24 h; ++, zone
of inhibition was at least 0.5 cm beyond disc area at 24 h; +, zone of inhibition
was below 0.5 cm beyond disc area at 24 h; –, no inhibition detectable.
JAOCS, Vol. 78, no. 9 (2001)

STUDIES ON N-STEAROYL AMINO ACIDS
901
those rated ++, because the zones of inhibition may be a func- organic carbon and nitrogen are converted to carbon dioxide
tion of the ability of the compounds to diffuse through the (CO2) and ammonia (NH3), respectively. In the second and
medium beyond the point of contact with the agar surface third stages, the ammonia is oxidized sequentially to nitrite
rather than an indication of their intrinsic ability to retard mi- and nitrate. The ThOD is the total amount of the oxygen re-
crobial growth.
quired for all three steps.
The apparently large differences in the inhibitory effects
of the compounds could be related to the mode of attachment
of the amino acid moiety. The fatty acid chloride reacts with
amino acids to form an amide bond. The amide linkages may
be responsible for the inhibitory action, such that a stronger
hydrogen bond between oxygen of the fatty moiety and the
–NH group of the amino acids would result in a higher rate of
antimicrobial activity. This effect has been called the hydro-
gen belt effect in inhibition of antimicrobial action (19). The
NSA containing an aromatic amino acid were the most effec-
tive, exhibiting a strongly inhibitory action against five of the
six organisms.
A simple screening technique was used to obtain general
information on whether the compounds prepared had antimi-
crobial properties that could be useful in commercial prod-
ucts. Further testing of these compounds as biostatic agents
should be considered.
Balanced reaction for the carbonaceous oxygen demand:
CH3(CH2)16CONHCH(COOH)CH2COOH
+ 58/2 O2
→
NH3 + 22 CO2 + 19 H2O
[3]
Balanced reaction for the nitrogenous oxygen demand:
NH3 + 3/2 O2
HNO2 + 1/2 O2
NH3 + 2O2
→ HNO2 + H2O
→
HNO3
→
HNO3 + H2O
[4]
[5]
Determination of ThOD for N-stearoyl aspartic acid:
ThOD = (58/2 + 2) mol O2/mol N-stearoyl aspartic acid
= 31 mol of O2/mol N-stearoyl aspartic acid × 32 g/mol O2
= 839.09 g O2/mol N-stearoyl aspartic acid
As illustrated in Table 5 all the NSA tested in this study
were >75% in 5 d, suggesting that these compounds are read-
ily biodegradable in the environment.
Biodegradability of the lipoamino acids. The biodegrad-
ability test is mainly based on a bioassay procedure, involv-
ing measurement of O2 consumed by bacteria while stabiliz-
ing organic matter under aerobic conditions. It is necessary to
provide standard conditions of nutrient supply, pH, absence ACKNOWLEDGMENT
of microbial growth-inhibiting substances, and temperature
The authors would like to thank Dr. T. Ramasami, Director, Central
(13). A mixed group of organisms should be present in the
sample. If not, the sample has to be seeded artificially. Tem-
perature is controlled at 20°C. The test is conducted for 5 d,
as 70–80% of the samples are oxidized during this period.
Table 5 shows the 5-d biochemical oxygen demand (BOD5)
and biodegradability [BOD5/theoretical oxygen demand
(ThOD)] of NSA. The BOD was calculated per the formula re-
ported elsewhere (13). The ThOD values of the NSA were
computed from the chemical formula as illustrated for the ex-
ample of N-stearoyl aspartic acid.
Leather Research Institute, Adyar, India for his interest in this work.
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TABLE 5
Biodegradability of NSAa
c
Amino acid
Compound moiety
ThODb
BOD5
BOD5/ThOD
(%)
(g O2/mol) (g O2/mol)
1
2
3
4
5
6
7
8
9
Glutamic acid
Aspartic acid
Arginine
Lysine
Histidine
1040
992
868.50
839.09
925.26
866.75
885.36
801.15
906.47
988.26
939.96
84
84.5
73.2
73.1
74.7
74.7
74.5
74.4
78.33
1264
1184
1184
1072
1216
1328
1200
Proline
Phenylalanine
Tryptophan
Tyrosine
aSee Table 2 for abbreviations.
bTheoretical oxygen demand.
cMeasured 5-day biochemical oxygen demand.
JAOCS, Vol. 78, no. 9 (2001)

902
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[Received June 7, 2000; accepted May 31, 2001]
JAOCS, Vol. 78, no. 9 (2001)