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UNCORRECTED PROOF
ORIGINAL ARTICLE
Efficacy of a
s1
-casein hydrolysate on stress-related
symptoms in women
JH Kim
1
, D Desor
2
, Y-T Kim
3
, W-J Yoon
3
, K-S Kim
4
, JS Jun
5
, KH Pyun
1
and I Shim
1
1
Department of Integrative Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea;
2
Laboratoire de
Neurosciences Comportementales, Unite
´de recherche sur l’Animal et les Produits Animaux, Universite
´Henri Poincare
´, Institut National
Polytechnique de Lorraine/Institut National de la Recherche Agronomique, Faculte
´des Sciences, Vandoeuvre Les Nancy Cedex, France;
3
Department of Pharmaceuticals and Health Foods, Lotte R&D Center, Seoul, Korea;
4
Department of Family Medicine, College of
Medicine, The Catholic University of Korea, Seoul, Korea and
5
Department of Psychiatry, Inha University Hospital, Incheon, Korea
Objective: To examine the effects of a
s1
-casein hydrolysate on females with stress-related symptoms.
Design: Double-blind, randomized, crossover, placebo-controlled trial.
Setting: The a
s1
-casein hydrolysate was manufactured by INGREDIA (Arras, France) and the placebo was manufactured by
DIETAROMA (Bourg, France). Study was designed and performed at PROCLAIM (Rennes, France), and the statistical analyses
were performed by D Desor (Nancy, France).
Subjects: A total of 63 female volunteers suffering from at least one disorder that may be related to stress such as anxiety, sleep
problems and general fatigue.
Interventions: A total of 63 volunteers participated in a double-blind, randomized, crossover, placebo-controlled study.
Subjects were randomly allocated to receive either tablets containing a
s1
-casein hydrolysate or placebo at the dose of 150 mg/
day for 30 days. After a 3 weeks washout period, they were crossed over for a new 30-day period of tablets intake. The outcome
measure was a questionnaire including 44 items of symptoms that may be related stress in which the severity of each sign was
evaluated using a 10-degree scale. These measures were studied repeatedly at the day of 0, 15 and 30 after the start of each
interventional period.
Results: The 30-day treatment by a
s1
-casein hydrolysate in females with stress-related symptoms reduced their symptoms,
particularly in digestion (Po0.01), cardiovascular (Po0.05), intellectual (Po0.01), emotional (Po0.05) and social problems
(Po0.05).
Conclusion: This study showed that a 30-day ingestion of a
s1
-casein hydrolysate decreased the stress-related symptoms in
females suggesting that this product may be used as an effective functional ingredient alleviating such symptoms.
Sponsorship: This study was partially supported by the INGREDIA of France and Neurobiology Research Program from the
Korea Ministry of Science and Technology (2004-01757) of Korea.
European Journal of Clinical Nutrition (2006) 0, 000–000. doi:10.1038/sj.ejcn.1602553
Keywords: casein; a
s1
-casein hydrolysate; stress; anxiety; women
Introduction
Stress refers to the response to any physical or psychological
stimulus that disrupts ongoing homeostasis (Pacak and
Palkovits, 2001). The stress response comprises adaptive
physiological processes activated in animals and humans in
challenging situations (Carlson, 1994). In a stressful condi-
tion, the sympathetic branch of the autonomic nerve system
is active, and the adrenal gland secretes epinephrine,
norepinephrine and steroid stress hormones (Carlson, 1994).
Prolonged stress can be noxious in various areas: sleep
(Buguet et al., 1994), memory (de Quervain et al., 1998) and
feeding behavior (Hotta et al., 1999). Subjects presenting
long-term high-serum concentration of glucocorticoids may
suffer from fatal lesions of the central nervous system
(McEwen, 2005). Sympathetic stimulation by stress activates
Journal: EJCN Disk used Despatch Date: 13/11/2006
Article : npg_ejcn_1602553 Pages: 1–6 OP: KGU ED: SUJA
Received 15 August 2005; revised 4 September 2006; accepted 25 September
2006
Correspondence: Dr I Shim, Department of Integrative Medicine, College of
Medicine, The Catholic University of Korea, 505 Banpo-Dong, Seocho-Ku,
Seoul 137-701, Korea.
E-mail: ishim@catholic.ac.kr
Q1
European Journal of Clinical Nutrition (2006) 0, 000– 000
&
2006 Nature Publishing Group All rights reserved 0954-3007/06 $
30.00
www.nature.com/ejcn
UNCORRECTED PROOF
atherogenesis appearance, facilitates platelet aggregation,
induces arrhythmia and can lead to myocardial ischemia
onset by increasing the needs of the myocardium for oxygen
and metabolites and by reducing its contribution (Engler and
Engler, 1995). In addition, many studies have highlighted
the role of the professional or psychosocial stress in some
types of hypertension (Vrijkotte et al., 2000). Evidence is now
accumulating that psychological and social stress may
contribute to the apparent excess mortality risk in women’s
coronary disease. Women are known to be more frequently
diagnosed with depression, and to more frequently display
depressive symptoms than men (Weissman and Klerman,
1992). Stress also modifies digestive secretion and motility,
leads to gastro-duodenal ulcer (Murison, 2000) and affects
the endocrine (Sapolsky, 1997) and immune systems (Ca-
cioppo et al., 1995).
As a consequence, stress can increase the risk of various
diseases, exacerbate many medical disorders, and change
healthy life-style behaviors. Therefore, effective manage-
ment of stress is essential to decrease vulnerability to
illnesses and to improve the quality-of-life.
Nowadays, milk proteins are among the most widely
consumed human food proteins. Milk protein consists of
soluble whey protein and insoluble colloidal casein. Caseins
account for 76–86% of the total milk proteins and can be
represented by major gene products: a
s1
-casein, a
s2
-casein, b-
casein and k-casein (Odagiri, 1985). Many peptides stem-
ming specifically from bovine a
s1
-casein have already been
identified as having biological activity (Maruyama et al.,
1987; Le
´onil et al., 2001).
A milk a
s1
-casein hydrolysate (lactium) and a bioactive
decapeptide (a
s1
-casein-(f91–100), a-casozepine), a fragment
of this hydrolysate that has been spatially modeled (Lecou-
vey et al., 1997a, b), have already shown an anxiolytic-like
profile in the conditioned defensive burying (CDB) test and
in the elevated plus maze in rats, two well-known models
used to study anxiolytic agents in rodents (Miclo et al.,
2001). Moreover, the effect of lactium (the industrial a
s1
-
casein hydrolysate containing the bioactive a-casozepine) on
hemodynamic responses was evaluated in healthy human
volunteers facing successive mental and physical stress
situations (Messaoudi et al., 2004).
The purpose of this study was to investigate whether oral
intake of a
s1
-casein hydrolysate (a
s1
-CH) in females with
stress-related symptoms would modify various symptoms in
physiological, psychological and social areas according to
their responses to self-report questionnaires.
Methods
Subjects
Subjects were recruited by way of advertisements in public
places (University Henri Poincare
´, Vandoeuvre-le
`s-Nancy,
France). A total of 63 volunteer women aged of more than 18
years old who pretend to suffer from at least one symptom
that may be stress-related: anxiety, sleep, general fatigue
were recruited after announcements in public places such as
a halls of public institutions. They participated after
informed consent by filling the questionnaire. At the
beginning of the study 69 subjects were selected, but six of
them did not complete the trial for various reasons not in
relation with the treatment. All the data concerning the
subjects were in accordance with the rules of the Commision
Nationale de l’Informatique et des Liberte
´s (CNIL). Subjects
were free of any known primary alimentary allergic problems
or medication by anti-depressant, anxiolytic, vegetal seda-
tive or hypnotic treatment in progress or in the 15 days
before the beginning of the study. They had no history of
any illness, no excessive consumption of alcohol or tobacco
or no body mass index over 25. The descriptive socio-
professional status of subjects was shown (Table 1).
Tested products
a
s1
-CH (lactium), consisting of a tryptic hydrolysate of a a
s1
-
casein enriched protein fraction, was supplied by INGREDIA
(Arras, France) and the placebo composed of only bovine
skimmed milk powder was supplied by DIETAROMA (Bourg,
France). Both groups were administered similar looking
white tablets containing either 75 mg of a
s1
-CH or 75 mg of
placebo.
Experimental schedule
The study was based on a randomized, double-blind, cross-
over schedule. Each subject ingested two tablets containing
either a
s1
-CH or placebo (150 mg/day) every evening during
the treatment periods. One group received the product a
s1
-
CH for 30 days, and then had a wash-out period of 3 weeks
after which they received the placebo product for a period of
30 days. The other group received the placebo product for 30
days, had a wash-out period of 3 weeks, and then they
received the a
s1
-CH for a period of 30 days.
Evaluation of the efficacy of a
s1
-CH
The questionnaire covered three main areas potentially
affected by stress: Physical and physiological area (digestive
tract, respiratory system, cardiovascular system, locomotor
system, other physical symptoms of stress); psychological
area (intellectual functions, emotional area); social life. The
NPG_EJCN_1602553
Table 1 Socio-professional characteristics of subjects
Socio-professional status nAge (mean7s.d.) (min,max)
Active women 22 63% 37710 (20, 59)
Inactive or jobless women 10 16% 3478 (26, 50)
Student 23 36% 21711 (18, 23)
Retired 8 13% 5774 (50, 61)
Whole sample 63 100% 33714 (18, 61)
Q2
Efficacy of a
s1
-casein hydrolysate
JH Kim et al
2
European Journal of Clinical Nutrition
UNCORRECTED PROOF
questionnaire was made up from items from the Hamilton
Anxiety Scale (Hamilton, 1967; Maier et al., 1988) and Ferreri
Anxiety Rating Diagram (FARD) (Ferreri et al., 1988) partly
for assessing stress symptoms including anxiety, depression
and internal tension. Totally, the questionnaire included 44
items of stress symptoms.
The efficacy of the products was investigated by the use of
the above-mentioned questionnaire in which the severity of
each sign was evaluated using a visual 10-degree scale
(0 ¼not at all, 9 ¼excessively). We applied the format used
in a classical perceived stress scale in French adapted from
Lafleur and Be
´liveau (1994). The scoring method was
changed, from a 4-point scale to a 10-point one (0–9). All
subjects filled in the questionnaire repeatedly at day 0, for
example, before any intake of the products, and at days 15
and 30 of each intake period after the start of this study.
Statistics
Statistical analysis of the data was performed using the SPSS
statistical software program.
The effect of the a
s1
-CH was investigated relatively to the
symptoms of each area through the detection of the
maximum variation of major symptom because someone
who suffers from stress mainly hopes a decrease of his/her
most unpleasant symptoms.
Given that the distribution of the behavioral scores and
percentages are generally not Gaussian, and when the values
are o30 or 470%, non parametric statistics were used. The
Wilcoxon test was used to analyze the difference between
treatment groups for major stress symptoms. Differences
were considered to be significant when Po0.05.
First of all, the stability of the baseline was investigated.
When the response was stable, it was possible to perform
direct comparisons using raw values of the scores. If the
values of the scores were not stable from one period to the
other, the variations of the scores (in terms of percentages
relatively to day 0 of each period) were compared.
Further relationship between predictor valuables and
major stress symptoms was analyzed using discriminant
function analysis. The discriminant analysis was conducted
to show if treatment by a
s1
-CH could be discriminated from
the placebo substance by taking into account the results in
the eight studied areas, the dimensions of digestive,
respiratory, cardiovascular, locomotion, physical, intellec-
tual, emotional and social symptoms. For each dimension,
the percentages of variation under a
s1
-CH and placebo were
used. When the initial value was equal to 0, the computation
of the percentage of variation was impossible and the value
was suppressed in this analysis.
Results
Stability of the baseline for period 1 and 2
Between the first day of the first period and the first day of
the second period of treatment, and whatever the treatment
received during period 1, the intensity of 30 symptoms out
of 44 ( ¼68.2%) significantly decreased. It indicated a strong
effect of time and/or participation to the study on the
perceived symptoms of stress.
Comparison of the effect of treatment by a
s1
-CH and placebo on
the development of various aspects of stress between day 0 and 15
The results of the statistical comparisons of the magnitude
variation of the major stress symptoms between day 0 and
day 15 of treatment in the two groups (a
s1
-CH and placebo)
were shown (Table 2). The intensity of all the stress
symptoms between day 0 and day 15 of treatment was
decreased in both groups. Concerning the whole population,
the only significant improvement was found for cardiovas-
cular symptom. Improvement in this dimension was found
in the subjects treated by a
s1
-CH (49.0%) versus the subjects
treated by placebo (24.3%).
The results of the statistical comparisons in subjects with
the highest initial intensities (intensity 44 on day 0) for the
symptoms, between a
s1
-CH and placebo groups, on days 0
and 15 of treatment, were shown (Table 3). The intensity was
decreased in both groups. Significant improvements of
symptoms were found for the product a
s1
-CH for dimensions
of digestive, cardiovascular, and physical symptoms was
significantly greater in the subjects treated by a
s1
-CH than in
the subjects treated by placebo.
Comparison of the effect of treatment by a
s1
-CH and placebo on
the development of various aspects of stress between day 0 and 30
The results of the statistical comparisons of the magnitude
variation of the symptoms between day 0 and day 30 of
treatment in the two groups (a
s1
-CH and placebo) were
shown (Table 4). The intensity of the measured symptoms
NPG_EJCN_1602553
Table 2 Percentage of improvement of the major symptoms evaluated
on the whole samples at day 15 of the treatments
Symptom items % Improvement Difference P-value
a
S1
-CH Placebo
Digestion 49.9 41.4 8.5
Respiration 50.4 31.4 19.0
Cardiovascular 49.0 24.3 24.7 o0.05
Locomotion 53.6 54.5 0.9
Physical area 44.3 38.2 6.1
Intellectual area 41.0 49.0 8.0
Emotional area 31.7 30.1 1.6
Social life 40.9 48.9 8.0
Abbreviation: a
S1
-CH: a
S1
-casein hydrolysate.
Efficacy of a
s1
-casein hydrolysate
JH Kim et al
3
European Journal of Clinical Nutrition
UNCORRECTED PROOF
between day 0 and day 30 of treatment was decreased in the
a
s1
-CH and placebo groups. The improvement was signifi-
cant for digestive symptom and intellectual problems in the
subjects treated by a
s1
-CH versus in the subjects treated by
placebo.
The results of statistical comparisons of the intensity of
symptoms in the subjects with the highest symptom
intensities (intensity 44 on day 0) in the a
s1
-CH and placebo
groups, on the day 0 and day 30 of treatment, were shown
(Table 5). The intensity of the symptoms between day 0 and
day 30 of treatment was significantly decreased in both
groups. Concerning the subjects with the highest symptom
intensities, significant improvements could be demonstrated
in the a
s1
-CH group after a 30-day long treatment for
digestion (36.6% for placebo/66.1% for a
s1
-CH), cardiovas-
cular symptom (35.5% for placebo/48.0% for a
s1
-CH),
intellectual problems (36.7% for placebo/64.8% for a
s1
-CH),
problems in emotion (23.5% placebo/43.8% for a
s1
-CH) and
social life (22.5% for placebo/36.7% for a
s1
-CH) (Figure 1).
There were no significant differences for the subjects treated
with a
s1
-CH versus the subjects treated with placebo for
respiratory symptom, locomotion or other physical symp-
toms.
Discriminant analysis
Discriminant function can be represented as a discriminant
axis, gathered in s.d. units. In the present case, a gap was
seen between the two groups on the discriminant axis
NPG_EJCN_1602553
Table 3 Percentage of improvement of the major symptoms evaluated
on the subjects with highest intensities of the symptoms at day 15 of the
treatments
Symptom items % Improvement Difference P-value
a
S1
-CH Placebo
Digestion 58.0 38.6 19.4 o0.05
Respiration 53.2 51.0 2.2
Cardiovascular 53.4 21.8 31.6 o0.001
Locomotion 61.8 59.5 2.3
Physical area 61.5 43.5 18.0 o0.05
Intellectual area 50.8 42.8 8.0
Emotional area 31.9 24.4 7.5
Social life 41.2 44.6 3.4
Abbreviation: a
S1
-CH: a
S1
-casein hydrolysate.
Table 4 Percentage of improvement of the major symptoms evaluated
on the whole samples at day 30 of the treatments
Symptom items % Improvement Difference P-value
a
S1
-CH Placebo
Digestion 65.6 44.6 21 o0.01
Respiration 51.5 60.4 8.9
Cardiovascular 48.9 39.0 9.9
Locomotion 61.1 64.7 3.6
Physical area 45.3 37.6 7.7
Intellectual area 62.5 46.2 16.3 o0.05
Emotional area 39.7 34.1 5.6
Social life 40.2 30.5 9.7
Abbreviation: a
S1
-CH: a
S1
-casein hydrolysate.
Table 5 Percentage of improvement of the major symptoms evaluated on the subjects with highest intensities of the symptoms at day 30 of the
treatments
Symptom items Number of subjects Number of subjects % Improvement Difference P-value
a
S1
-CH placebo a
S1
-CH placebo
Digestion 37 35 66.1 36.6 29.5 o0.01
Respiration 9 8 68.9 43.1 25.8
Cardiovascular 34 33 48.0 35.5 12.5 o0.05
Locomotion 34 32 65.8 63.9 1.9
Physical area 32 30 53.8 41.0 12.8
Intellectual area 34 32 64.8 36.7 28.1 o0.01
Emotional area 38 37 43.8 23.5 20.3 o0.05
Social life 51 50 36.7 22.5 14.2 o0.05
Abbreviation: a
S1
-CH: a
S1
-casein hydrolysate.
alphas1-CH
PLACEBO DIGESTION (*)
SOCIAL (*)
EMOTIONAL (*)
INTELLECTUAL (*)
PHYSICAL
LOCOMOTION
CARDIO-VASCULAR (*)
RESPIRATION
0
10
20
30
40
50
60
70
Figure 1 Graphical representation of percentage improvement of
the major symptoms evaluated on the subjects with highest
intensities of the symptoms at day 30 of the treatments.
Efficacy of a
s1
-casein hydrolysate
JH Kim et al
4
European Journal of Clinical Nutrition
UNCORRECTED PROOF
according to treatments. The ordinates in standard deviation
units of the barycenters of the groups treated by a
s1
-CH and
placebo on the discriminant axis was 0.481 and 0.435,
respectively. It indicated that the subjects treated with a
s1
-
CH had globally greater percentages of improvement of their
symptoms than the placebo subjects. The quality of the
classification can be evaluated through the final classifica-
tion matrix computed from the discriminant equation for
each subjects, the probability of being in one or the other
group. The result was compared to the real group of the
subject. In consequence, discriminant analysis correctly
classified 67.8% of the subjects. The following Fisher’s exact
test showed that the null hypothesis is P¼0.0089. It
indicated that the treatments by a
s1
-CH and placebo led to
highly significant differences, with greater improvements in
the a
s1
-CH treatment group compared to the placebo group.
Discussion
The aim of the present study was to evaluate the effects of
orally taken a
s1
-CH on women who suffered from at least one
symptom that may be stress-related according to their
responses to a questionnaire. Various tools have been used
to characterize the different components of stress reaction,
questionnaires of psychology (Cohen questionnaire; Spiel-
berger questionnaire; Thayer questionnaire), endocrine assay
(Ilardo et al., 2001) and physiological measurements (Boiten
et al., 1994; Larson et al., 2001). In this study, the tool used is
a questionnaire covering three main areas, physical and
physiological, psychological area, and social life by reason
that stress is linked to the concept of tension, which is itself
related to the muscular and postural systems other physio-
logical systems, and internal state with negatively affective
intellectual and the relational functions.
The present study demonstrated that a
s1
-CH reduced the
stress-related symptoms, in various tension aspects. In
addition, there was a considerable effect of the placebo
treatment ranging from 15% to more than 40% of improve-
ment of the major symptoms. It is interesting to point out
that the intensity of some of the assessed symptoms was
significantly decreased also in the subjects treated by
placebo. Stress, anxiety, depression are well known for
having an important psychological component and placebo
response is often very high in clinical studies concerning
them (Berk and Dodd, 2005). The ‘HURIET-SERUSCLAT’ law,
which is concerned with trials on human subjects in France,
implies that the subjects sign an ‘informed consent’ form
before the beginning of the experiment. This means that
when the study begins, they know that they will be treated
by a substance (a
S1
-CH), or by placebo, during the two
periods, and that a
S1
-CH can possibly reverse their symptoms
of stress. During the study, the subjects’ compliance was not
monitored. Participation in this study might have conferred
a placebo effect.
The effect of a
s1
-CH could be detected on the 15th day of
treatment in the cardiovascular system and on the 30th day
of treatment in the digestive system and intellectual
symptoms. The supplementation with a
s1
-CH at the dose
of 150 mg/day, was particularly efficient on the subjects who
demonstrated the highest initial intensities (44) for their
major symptoms. The cardiovascular, the digestive system
and the physical major symptoms were improved on the
highest intensity of the symptom at day 15 of the treatment
and the digestive, the cardiovascular, the intellectual, the
emotional and the social symptoms/problems were im-
proved on the highest intensity of the symptom at day 30
of the treatment.
Taken together, a
s1
-CH could have a regulating effect in
the field of stress-related symptoms, namely, the tension
related physiological system (cardiovascular and digestive
systems), internal state that negatively interacts with the
affective emotional troubles, the intellectual functions and
the relational functions (social behavior) of the subjects.
Cow’s milk has long been considered a tranquilizing
beverage with sleep-inducing capacity, but the molecular
causes of this potential action are not known. Numerous
bioactive peptides which are identified in milk protein may
be released after enzymatic digestion (Meisel, 1997; Clare
and Swaisgood, 2000). The functional peptides from milk
showed many physiological effects, such as calcium bio-
transfer activity (Lee et al., 1979), opiate activity (Meisel and
FitzGerald, 2000), immunomodulating activity (Pitt et al
1974; Totima et al., 1994), anti-hypertensive activity (Mar-
uyama et al., 1987), and anti-thrombotic activity (Chabance
et al., 1995). The a
s1
-CH has also been reported to exhibit a
benzodiazepine (BDZ)-like activity of the GABA
A
receptor
without side effects (Miclo et al., 2001). However, there are
no data on clinical human studies.
The placebo (skimmed milk powder) contains casein and
particularly a
S1
-casein (entire proteins), but the bioactive
peptide identified in the a
S1
-CH, namely a
S1
-casein (f91–100)
cannot be significantly released (from it) because in vivo
digestion can be assimilated as a multi-enzymatic process
including first a pepsic hydrolysis in the stomach. Pepsin
cleaves 91–92, 95–96, 98–99 and 99–100 bonds of a
S1
-casein
(Mercier et al., 1970) preventing a
S1
-casein (f91–100) release
from the entire protein contained in placebo. The significant
content of the bioactive peptide in the a
S1
-CH and its relative
small size may protect it from further digestion and may
explain the significant effects after in vivo intake. In this
study, the a
S1
-CH results from a mono-enzymatic digestion
of purified a
S1
-casein.
The results of discriminant analysis showed that the
percentages of variations of the magnitude of the symptoms
in the eight areas contained enough information to
discriminate the two groups according to the treatments. It
could therefore be considered that the treatments by a
s1
-CH
and placebo lead to significant differences with greater
improvements in the a
s1
-CH treatment group compared to
the placebo group.
NPG_EJCN_1602553
Q3
Efficacy of a
s1
-casein hydrolysate
JH Kim et al
5
European Journal of Clinical Nutrition
UNCORRECTED PROOF
In conclusion, this study provided evidence that a 30-day
ingestion of a
s1
-CH displayed positive effect in females with
self-reported complaints similar to many clinical stress
symptoms suggesting that a
s1
-CH may be used as an effective
functional ingredient alleviating stress. Further studies are
needed to investigate if the conclusions drawn from this
population can be generalized to males and to non-clinical
populations with similar symptomatology, and if the effects
can be observed on a long-term basis.
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NPG_EJCN_1602553
Q4
Efficacy of a
s1
-casein hydrolysate
JH Kim et al
6
European Journal of Clinical Nutrition
