Daily intake of fermented milk with Lactobacillus casei strain Shirota reduces the incidence and duration of upper respiratory tract infections in healthy middle-aged office workers

Abstract

Purpose:
Although several studies have demonstrated the efficacy of probiotics for preventing upper respiratory tract infections (URTIs) in at-risk populations, including children and the elderly, few studies have investigated the efficacy of probiotics in healthy adults living normal, everyday lives. Thus, we tried to evaluate the effects of Lactobacillus casei strain Shirota-fermented milk (LcS-FM) on the incidence of URTIs in healthy middle-aged office workers.

Methods:
In a randomized controlled trial, 96 eligible male workers aged 30-49 years consumed LcS-FM containing 1.0 × 10(11) viable LcS cells or control milk (CM) once daily for 12 weeks during the winter season. URTI episodes were evaluated by a physician via a questionnaire of URTI symptoms.

Results:
The incidence of URTIs during the intervention period was significantly lower in the LcS-FM group than in the CM group (22.4 vs. 53.2 %, P = 0.002). The time-to-event analysis showed that the LcS-FM group had a significantly higher URTI-free rate than the CM group over the test period (log-rank test: χ (2) 11.25, P = 0.0008). The cumulative number of URTI episodes and cumulative days with URTI symptoms per person was lower in the LcS-FM group, and the duration per episode was shorter. Inhibition of both reductions in NK cell activity in peripheral blood mononuclear cells and increases in salivary cortisol levels was observed in the LcS-FM group.

Conclusion:
The results suggest that the daily intake of fermented milk with LcS may reduce the risk of URTIs in healthy middle-aged office workers, probably through modulation of the immune system.

Full text

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Eur J Nutr (2017) 56:45–53
DOI 10.1007/s00394-015-1056-1
ORIGINAL CONTRIBUTION
Daily intake of fermented milk with Lactobacillus casei strain
Shirota reduces the incidence and duration of upper respiratory
tract infections in healthy middle‑aged office workers
Kan Shida1 · Tadashi Sato1 · Ryoko Iizuka1 · Ryotaro Hoshi2 · Osamu Watanabe2 ·
Tomoki Igarashi2 · Kouji Miyazaki1 · Masanobu Nanno1 · Fumiyasu Ishikawa1
Received: 10 June 2015 / Accepted: 23 September 2015 / Published online: 29 September 2015
© The Author(s) 2015. This article is published with open access at Springerlink.com
mononuclear cells and increases in salivary cortisol levels
was observed in the LcS-FM group.
Conclusion The results suggest that the daily intake of
fermented milk with LcS may reduce the risk of URTIs
in healthy middle-aged office workers, probably through
modulation of the immune system.
Keywords Probiotics · Lactobacillus casei strain
Shirota · Upper respiratory tract infection · Common cold ·
NK cell activity · Cortisol
Introduction
The human intestinal tract harbors more than 100 trillion
bacteria, and the commensal gut microbiota plays a pivotal
role in maintaining the health of the host [1]. Thus, much
attention has been given to probiotics, which can survive
the intestinal tract and recover/maintain balanced gut
microbiota when ingested orally [2, 3]. Some strains of lac-
tobacilli and bifidobacteria are popular probiotics, usually
consumed as fermented dairy products or supplements [4].
Among the health benefits of probiotics, the prevention or
control of infectious diseases is one of the most promising
targets [5, 6]. Several studies have demonstrated that some
probiotics are effective against not only infections in the
gastrointestinal tract, but also those in the respiratory tract
[7, 8].
Many clinical trials against upper respiratory tract infec-
tions (URTIs), such as the common cold and influenza,
have evaluated various probiotic strains, and many of these
have demonstrated efficacy against URTIs [7, 8]. The tar-
get populations in these clinical trials have generally been
infants, children, students, and the elderly [9–12]. Since
the immune defenses in these populations are relatively
Abstract
Purpose Although several studies have demonstrated the
efficacy of probiotics for preventing upper respiratory tract
infections (URTIs) in at-risk populations, including chil-
dren and the elderly, few studies have investigated the effi-
cacy of probiotics in healthy adults living normal, everyday
lives. Thus, we tried to evaluate the effects of Lactobacillus
casei strain Shirota-fermented milk (LcS-FM) on the inci-
dence of URTIs in healthy middle-aged office workers.
Methods In a randomized controlled trial, 96 eligible
male workers aged 30–49 years consumed LcS-FM con-
taining 1.0 × 1011 viable LcS cells or control milk (CM)
once daily for 12 weeks during the winter season. URTI
episodes were evaluated by a physician via a questionnaire
of URTI symptoms.
Results The incidence of URTIs during the intervention
period was significantly lower in the LcS-FM group than
in the CM group (22.4 vs. 53.2 %, P = 0.002). The time-
to-event analysis showed that the LcS-FM group had a sig-
nificantly higher URTI-free rate than the CM group over
the test period (log-rank test: χ2 11.25, P = 0.0008). The
cumulative number of URTI episodes and cumulative days
with URTI symptoms per person was lower in the LcS-FM
group, and the duration per episode was shorter. Inhibition
of both reductions in NK cell activity in peripheral blood
Kan Shida, Tadashi Sato, and Ryoko Iizuka have contributed
equally to this work.
* Kan Shida
kan-shida@yakult.co.jp
1 Yakult Central Institute, 5-11 Izumi, Kunitachi-shi, Tokyo
186-8650, Japan
2 Faculty of Research and Development, Yakult Honsha,
Shinbashi, Tokyo 105-8660, Japan
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46 Eur J Nutr (2017) 56:45–53
1 3
weak, the use of probiotics might be pertinent. However,
few studies have investigated the efficacy of probiotics in
healthy adults [13, 14].
Maintaining the immune defense system within a normal
healthy state lowers the risk of URTIs. NK cell activity and
salivary immunoglobulin A (IgA) are considered important
in the prevention of URTIs [15, 16]. However, several envi-
ronmental factors, including a stressful lifestyle, are likely
to weaken the immune defense system [17], which may
result in an increase in the risk of URTIs. There is some
evidence to suggest that some probiotic strains restore NK
cell activity and salivary IgA levels [18–20]. Thus, daily
ingestion of a probiotic beverage might maintain normal
immune function and control URTIs.
Lactobacillus casei strain Shirota (LcS) is a probiotic
that can survive within the intestinal tract and recover bal-
anced gut microbiota [21]. The immunomodulatory activi-
ties of the strain have been studied extensively in animal
models and human studies [18, 22]. Studies in healthy sub-
jects with low NK cell activity showed that ingestion of
LcS-fermented milk (LcS-FM) recovered the activity [18].
Clinical trials have shown that LcS effectively reduces the
risk of bladder cancer and colorectal tumor recurrences,
and it has been proposed that immunomodulatory activi-
ties, including the recovery of NK cell activity, are among
the underlying mechanisms [23]. Therefore, accumulating
evidence implies that this probiotic has potential use as an
immunostimulatory food material.
Some clinical trials with LcS have targeted URTIs. A
double-blind, randomized, placebo-controlled trial in
healthy elderly people attending day care facilities showed
that consumption of LcS-FM reduced the duration of each
URTI episode, but not the incidence rate [24]. Another
trial conducted in healthy elderly nursing home residents
showed a nonsignificant decreasing trend in the incidence
of upper respiratory symptoms via LcS-FM ingestion
[25]. On the other hand, a study conducted among healthy
young athletes of endurance-based sports showed that LcS
was effective in preventing URTIs [20]. In the latter study,
daily consumption of LcS-FM significantly reduced the
incidence of URTIs and the cumulative number of URTI
episodes. Variations in the efficacy of LcS-FM might be
due to differences among study populations.
We think that it is important to evaluate the efficacy of
LcS in normal healthy adults, because events in daily life
can lead to weakened immune defense responses. Thus,
healthy middle-aged office workers were selected for this
study. We measured the effect of the daily consumption
of LcS-FM on the incidence of URTIs as the primary out-
come. Additionally, we analyzed both NK cell activity and
salivary IgA levels as immunological markers, and the lev-
els of cortisol in saliva as a stress marker.
Materials and methods
Participants
Healthy male workers living in Tokyo or its suburbs, aged
30–49 years, and working within office buildings were
recruited for this study via Web site advertising. The exclu-
sion criteria were as follows: (1) working outside the office
building twice or more a week; (2) difficulty providing
saliva and blood samples; (3) pollinosis, chronic rhinitis,
asthma, or milk allergy; (4) periodontitis or gingivitis; (5)
history of serious liver, kidney, heart, lung, or gut disease;
(6) receiving current medical treatment; (7) regularly con-
suming probiotics or fermented milk; (8) taking drugs or
supplements that might affect the outcome of the study; (9)
history of influenza vaccination or infection within the last
6 months; and (10) being deemed ineligible for this study
by a physician, based on blood chemistry, blood pressure,
pulse rate, or other reasons.
Participants were given a detailed explanation of the
purpose and potential risks of the study, and 217 workers
provided written consent and participated in a screening
test, in which blood, urine, and saliva analysis, a physical
examination, measurements of blood pressure and pulse,
and a questionnaire regarding lifestyle and working pro-
file were performed. Seventy-eight participants met the
exclusion criteria, 10 with salivary IgA concentrations of
509 μg/ml or more were excluded, and 29 declined to par-
ticipate prior to allocation. The remaining 100 participants
were randomly allocated to two groups: the LcS-FM group
and the control milk (CM) group.
Sample size calculation
The sample size was calculated by assuming that the inci-
dence of URTIs during the test period of 12 weeks would
be 80 % and LcS-FM would reduce the incidence by 35 %
[13, 20, 26]. At a significance level of 0.05 with 80 %
power, it was estimated that 44 participants were needed
per group to detect significant differences between the
groups by the Chi-square test. Thus, 100 participants were
included in this study.
Study design
We conducted a randomized, controlled trial from December
8, 2012, through March 5, 2013, at the Chiyoda Paramedi-
cal Care Clinic (Tokyo, Japan). The study was conducted
according to the guidelines laid down in the Declaration of
Helsinki. All procedures were approved by the Institutional
Review Board of the Chiyoda Paramedical Care Clinic. Writ-
ten, informed consent was obtained from all participants.
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47Eur J Nutr (2017) 56:45–53
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Test drinks
LcS-FM consisted of skimmed milk, high-fructose corn
syrup, sugar, flavoring, and a minimum of 1.0 × 1011 live
LcS cells. Probiotic strain LcS (YIT 9029) was obtained
from the Culture Collection Research Laboratory of the
Yakult Central Institute. Milk was used as a control bever-
age [12]. LcS-FM and CM drinks were placed into plastic
bottles. The test drinks were delivered to each participant
once a week under refrigeration and stored in a refrigerator
until consumption.
Procedures
Participants were allocated randomly to either the LcS-
FM or CM group. Both groups were asked to consume
one bottle of the test drink every day for 12 weeks and
to refrain from consuming any other probiotic foods and
supplements.
Participants kept a health diary during the intervention
period each day before bedtime, recording body tempera-
ture, any symptoms of illness, and the impact of any symp-
toms on their daily activities by rating the impact as none/
light, moderate, or severe. The test drinks consumed and all
prohibited foods and supplements were also recorded in the
diary. Participants were also asked to record possible symp-
toms of URTIs on the URTI symptom questionnaire on a
daily basis.
Participants visited the clinic at weeks 0, 6, and 12 dur-
ing the intervention period, and saliva and blood samples
were collected. Participants were free to consult any doctor
of their choice when experiencing symptoms of an illness,
but were required to record the details of any diagnosis pro-
vided, hospital care given, prescribed and non-prescribed
medications taken, and the results of influenza virus tests
via influenza diagnosis kits if applicable in the health diary.
Evaluation of URTIs
URTI episodes were evaluated by a physician at the clinic
based on the information recorded in the health diary, the
URTI symptom questionnaire, and interviews at 6 and
12 weeks after the intervention period commenced [12,
20]. The URTI symptom questionnaire consisted of ques-
tions on the following 16 symptoms: (1) fever; (2) chill;
(3) headache; (4) runny nose; (5) stuffy nose; (6) sneezing;
(7) cough; (8) sore throat; (9) sputum; (10) malaise; (11)
muscular pain; (12) joint pain; (13) nausea; (14) diarrhea;
(15) stomach ache; and (16) itchy eyes. The non-numerical
ratings of none/light, moderate, and severe were scored as
1, 2, and 3, respectively, for each item. The daily symptom
severity score for URTIs was calculated by summing all
the scores for each item, except those for nausea, diarrhea,
stomach ache, and itchy eyes, which were set to distinguish
a URTI episode from gastrointestinal infections and pollin-
osis. Thus, the maximum score for daily symptom severity
was 36. Symptoms separated by more than 2 consecutive
symptom-free days were recorded as separate episodes.
Influenza was diagnosed according to the results of
influenza virus test kits conducted as required at any hos-
pital and was counted as a URTI episode. URTIs other than
influenza were recorded as the common cold.
NK cell activity
Peripheral blood mononuclear cells were freshly isolated,
and NK cell activity was measured by the chromium
release assay using K562 target cells. Peripheral blood
mononuclear cells and 51Cr-labeled K562 cells were incu-
bated at a ratio of 20:1 for 3.5 h, and radioactivity released
from lysed target cells was measured. The percentage of
specific lysis was calculated as NK cell activity using the
following formula: specific lysis (%) = (experimental
release − spontaneous release)/(maximal release − sponta-
neous release) × 100.
Saliva analysis
Saliva was collected between 09:00 and 11:00 at the clinic
by the passive drool method. Participants were asked to
allow saliva to pool in the mouth for 3 min before transfer-
ring it via a straw to a collection tube. Saliva collection was
repeated four times with 1-min intervals. The pooled saliva
was centrifuged at 1500×g for 15 min, and the supernatant
was stored below −20 °C until analysis.
The levels of secretory IgA and cortisol were deter-
mined by immunoassay kits (Salimetrics, PA) according to
the instruction manuals.
Adverse events
General biochemical and hematological testing of blood,
urinary qualitative examinations, and physiological tests
were performed at 0 and 12 weeks, and the results before
and after the intervention period were compared. Any
adverse health events were recorded by participants in their
health diaries during the intervention period and confirmed
by the clinic physician at weeks 6 and 12.
Statistical analyses
The difference in URTI incidence between the groups
was analyzed by the Chi-square test. Time-to-episode
curves for the first URTI in the groups were described
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48 Eur J Nutr (2017) 56:45–53
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by the Kaplan–Meier method, and the difference was
analyzed by the log-rank test (27). A hazard ratio was
estimated using unadjusted Cox proportional hazard
model. The risk reduction efficacy was calculated using
the following formula: efficacy (%) = (1 − hazard
ratio) × 100. Comparisons of the cumulative numbers of
URTI episodes per person and levels of symptom sever-
ity between the groups were performed by the Wilcoxon
two-sample test. Comparisons of cumulative days with
symptoms per person and the durations of URTI epi-
sodes were achieved using the unpaired Student’s t test.
Unpaired and paired Student’s t tests were, respectively,
used for intergroup and intragroup comparisons of NK
cell activity, salivary IgA secretion, and cortisol levels.
Two-tailed P values <0.05 were considered statistically
significant. All analyses were performed using IBM
SPSS software version 20.0 (IBM Japan, Tokyo, Japan)
and SAS software version 8.2 (SAS Institute Japan,
Tokyo, Japan).
Results
Participant flow and characteristics
The participant flow diagram for this study is shown in
Fig. 1. Eligibility was assessed in 217 male workers.
Among them, 100 participants were recruited to the study
and allocated to either the LcS-FM or CM group. One par-
ticipant in the CM group did not start the intervention due
to personal reasons, and 99 participants completed it. Of
the 99 participants, 3 were excluded from the analysis for
the following reasons: one started medication for hyperten-
sion during the intervention period; one consumed prohib-
ited probiotic drinks on 10 days during the test period; and
one was found to meet an exclusion criterion of the study.
Thus, 96 participants comprising 49 participants in the
LcS-FM group and 47 participants in the CM group were
used for data analysis.
The baseline characteristics of the 96 eligible partici-
pants from the two groups are summarized in Table 1. No
significant differences in age, body mass index, living with
a child, smoking habits, or salivary IgA levels were found
between the groups. Both groups showed very good com-
pliance in terms of test drink consumption.
Incidence of URTIs
The primary outcome measure of this study was URTI inci-
dence, the results of which are shown in Table 2. The incidence
rates of URTIs during the intervention period (weeks 1–12)
were significantly different at 53.2 and 22.4 % for the CM
and LcS-FM groups, respectively (P = 0.002). The incidence
rate of the common cold was significantly lower in the LcS-
FM group than in the CM group (18.4 vs. 44.7 %, P = 0.005).
The incidence of influenza was 10.6 % in the CM group and
4.1 % in the LcS-FM group, although this difference did not
Assessed for eligibility (n = 217)
Excluded (n = 117)
-Meeting exclusion criteria (n = 78)
-Having higher sIgA (n = 10)
-Declined to participate (n = 29)
Randomised (n = 100)
Allocated to CM (n = 50)
-Declined to participate (n = 1)
Allocated to LcS-FM (n = 50)
-Declined to participate (n = 0)
Completed (n = 49)
-Analyzed (n = 47)
-Excluded from analysis (n = 2)
Completed (n = 50)
-Analyzed (n = 49)
-Excluded from analysis (n = 1)
Fig. 1 Participant flow diagram for this study
Table 1 Baseline
characteristics of participants
CM control milk, LcS-FM L. casei strain Shirota-fermented milk, BMI body mass index, SIgA salivary
immunoglobulin A
a No. of participants living with one or more children attending elementary/junior high school
b P values analyzed by the unpaired Student’s t test
c P values analyzed by the Chi-square test
CM (n = 47) LcS-FM (n = 49) P value
Mean (SD) n (%) Mean (SD) n (%)
Age (years) 40.5 (5.9) 40.6 (5.3) 0.931b
BMI (kg/m2) 23.6 (2.7) 22.8 (2.8) 0.184b
No. living with a childa17 (36.2) 19 (38.8) 0.792c
No. of smokers 9 (19.1) 16 (32.7) 0.306c
SIgA secretion rate (μg/min) 57.4 (23.7) 50.8 (31.0) 0.245b
Product compliance (%) 99.7 (1.1) 99.7 (0.9) 0.930b
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49Eur J Nutr (2017) 56:45–53
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reach statistical significance. Time-to-episode curves for the
first URTI are shown in Fig. 2. The curve of the LcS-FM group
was significantly higher than that of the CM group over the test
period (log-rank test: χ2 11.25, P = 0.0008). The URTI-free
rates were 0.78 (95 % CI 0.66–0.89) and 0.47 (95 % CI 0.33–
0.61) in the LcS-FM group and the CM group, respectively.
The hazard ratio was calculated 0.32 (95 % CI 0.16–0.65).
We further analyzed the incidence rates of URTIs dur-
ing the following three periods: 1st period (weeks 1–4); 2nd
period (weeks 5–8); 3rd period (weeks 9–12). As shown in
Table 2, the incidence of URTIs was significantly reduced in
both the 1st and the 2nd periods (P = 0.017 and P = 0.049,
respectively) but not in the 3rd period in the LcS-FM group.
Characteristics of URTI episodes
The effects of the test drinks on the cumulative number
of episodes, the cumulative days with symptoms, and the
duration and severity of URTIs were analyzed. The cumu-
lative numbers of URTI episodes per person during the
intervention period were 0.7 in the CM group and 0.3 in
the LcS-FM group (P = 0.004; Table 3). The LcS-FM
group had fewer cumulative days with URTI symptoms
per person during the test period, and the duration of each
URTI episode was shorter compared with the CM group
(P = 0.001 and P = 0.002, respectively). The mean and
peak daily severity scores of URTIs did not differ between
the two groups.
Blood and saliva parameters
Since immune defense mechanisms are important for host
resistance to viral infections, we measured NK cell activ-
ity in peripheral blood mononuclear cells and IgA secre-
tion rates in saliva at weeks 0, 6, and 12 of the interven-
tion period. NK cell activity decreased at week 6 compared
with week 0 in the CM group, but not in the LcS-FM group
(Fig. 3). Thus, NK cell activity was significantly higher
at week 6 in the LcS-FM group than in the CM group
(P = 0.013). Salivary IgA secretion increased in weeks
6 and 12 in both groups, and the secretion did not differ
between the groups.
Table 2 Primary outcomes: incidence of URTIs
URTI upper respiratory tract infection, CM control milk, LcS-FM L.
casei strain Shirota-fermented milk
a No. of participants with URTIs/common cold/influenza
b P values analyzed by the Chi-square test; P < 0.05 is considered
statistically significant
CM (n = 47) LcS-FM (n = 49) P valueb
na (%) na (%)
Whole period (1–12 weeks)
URTIs 25 (53.2) 11 (22.4) 0.002
Common cold 21 (44.7) 9 (18.4) 0.005
Influenza 5 (10.6) 2 (4.1) 0.201
1st period (1–4 weeks)
URTIs 11 (23.4) 3 (6.1) 0.017
Common cold 11 (23.4) 3 (6.1) 0.017
Influenza 0 (0.0) 0 (0.0) –
2nd period (5–8 weeks)
URTIs 12 (25.5) 5 (10.2) 0.049
Common cold 10 (21.3) 4 (8.2) 0.069
Influenza 3 (6.4) 1 (2.0) 0.293
3rd period (9–12 weeks)
URTIs 7 (14.9) 6 (12.2) 0.705
Common cold 5 (10.6) 5 (10.2) 0.603
Influenza 2 (4.3) 1 (2.0) 0.484
0
0.2
0.4
0.6
0.8
1
01020304050607080
Pr
oporon of subjects
not having an URTI
Time to event (days)
Fig. 2 Kaplan–Meier time-to-event curves for the first URTI. The
URTI-free rates were 0.78 (95 % CI 0.66–0.89) and 0.47 (95 % CI
0.33–0.61) in the LcS-FM (thick line) and the CM (thin line) groups,
respectively
Table 3 Secondary outcomes: episode number per person, total days
with symptoms per person, duration and severity of URTIs
URTI upper respiratory tract infection, CM control milk, LcS-FM L.
casei strain Shirota-fermented milk
a P values analyzed by the Wilcoxon two-sample test; P < 0.05 is
considered statistically significant
b P values analyzed by the unpaired Student’s t test; P < 0.05 is con-
sidered statistically significant
c Mean values of daily severity score during the period of each URTI
episode
d Peak values of daily severity score of URTIs
CM (n = 47) LcS-FM (n = 49) P value
Mean (SD) Mean (SD)
Cumulative number of
URTI episodes
0.7 (0.7) 0.3 (0.8) 0.004a
Cumulative days with
symptoms (days)
3.4 (4.3) 1.0 (2.1) 0.001b
Duration per episode
(days)
5.0 (2.5) 2.8 (1.6) 0.002b
Mean severity scorec15.8 (2.8) 15.9 (2.6) 0.966a
Peak severity scored18.2 (4.2) 17.4 (3.6) 0.882a
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50 Eur J Nutr (2017) 56:45–53
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Salivary levels of the stress marker cortisol were ana-
lyzed. In contrast to the change in NK cell activity, cortisol
levels were increased at week 6 in the CM group but not
in the LcS-FM group, and the difference was significant
between the groups (P = 0.045).
Safety
There were no adverse effects associated with consumption
of the test drinks during the test period, as confirmed by
the physician following examinations of blood and urine,
physiological tests, analysis of health diaries, and inter-
views (data not shown).
Discussion
This randomized controlled trial conducted in healthy mid-
dle-aged male office workers clearly demonstrated that the
daily intake of probiotic fermented milk, LcS-FM, reduced
the risk of URTIs. The time to first URTI episode analy-
sis showed that the risk reduction efficacy for LcS-FM was
68 % (95 % CI 35–84 %) compared with CM. The analy-
sis of URTI incidence for divided three periods showed that
the positive health effects of LcS-FM were more evident in
earlier test periods. The incidence of the common cold in
the LcS-FM group (18.4 %) during the intervention period
of 12 weeks decreased by more than half compared with
the control group (44.7 %). The reduction in the incidence
of influenza did not reach statistical significance, which
might be due to the low incidence rates of both groups
(10.6 % for the control; 4.1 % for the LcS-FM group). In
addition to the reduced risk for URTIs, LcS-FM reduced
the cumulative number of days with URTI symptoms and
shortened the duration of each episode.
Intervention studies using similar LcS-FM drinks
against URTIs have been conducted previously. The results
obtained from two previous trials in elderly participants
aged over 80 years did not show a significant reduction in
URTI incidence rates [24, 25]. In contrast, the present study
clearly demonstrated that LcS-FM effectively reduced the
risk of URTIs. These results are consistent with the results
of a trial conducted in athletes aged 18–55 years [20]. The
ages of the participants in the latter study were similar to
those in the present study, and thus, the different age groups
among these studies might explain the conflicting results.
Another difference between the present and the previous
studies should be noted. The viable number of LcS cells
ingested daily was a minimum of 1.0 × 1011 cells in this
study, while in the aforementioned studies with elderly par-
ticipants, the numbers of viable cells were 1.3 × 1010 or
4.0 × 1010. In general, a higher number of ingested viable
probiotic cells will tend to lead to a more beneficial out-
come. The dose–response associated with improved effi-
cacy against URTIs has been reported. De Vrese et al. [28]
showed that while the intake of a multivitamin and mineral
tablet containing 5 × 107 viable lactobacillus and bifido-
bacterium cells shortened the duration of common cold
infections, it had no effect on the incidence rate; however,
another study showed that the similar tablet containing
5 × 108 viable probiotic cells reduced the incidence rate
[29]. The relationship between viable LcS cell numbers
and efficacy should be examined and discussed in future
studies.
Many probiotic intervention studies that have demon-
strated efficacy against URTIs have been conducted in
infants, children, students, and the elderly [9–12]. Physi-
cally active individuals, including rugby players and ath-
letes of endurance-based sports, might also be targets for
probiotics [20, 27, 30]. Since immune defense activities are
relatively weak in such populations, they generally experi-
ence more infections. The present study demonstrated that
0
20
40
60
0w 6w 12w
NK acvity (%)
#
*
0
0.2
0.4
0.6
Corsol (µg/dL)
#
** *
0
40
80
120
0w 6w 12w
0w 6w 12w
IgA (µg/min)
** **
** **
a
b
c
Fig. 3 Changes in NK cell activity in peripheral blood mononuclear
cells, IgA secretion rate, and cortisol levels in saliva throughout the
intervention period of 12 weeks. Data are expressed as means ± SD.
Open circles, CM group (n = 47); closed circles, LcS-FM group
(n = 49). *P < 0.05; **P < 0.01 versus week 0 in the same group;
#P < 0.05 versus CM group
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51Eur J Nutr (2017) 56:45–53
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healthy middle-aged office workers are also a promising
target population for probiotics. A clinical trial in employ-
ees at a manufacturing company in Sweden showed that
daily consumption of L. reuteri ATCC 55730 reduced the
incidence of sick leave due to respiratory or gastrointesti-
nal illnesses during the 80 days of intervention [13]. The
efficacy of probiotics was more evident in a subgroup of
shift workers [13, 14]. The present study demonstrated the
efficacy of probiotics in typical desk workers. In general,
such employees work under pressure and stress every day,
which might be a risk factor for lowered immune defenses.
Daily consumption of certain probiotics at higher doses
may prevent disturbances in immune function resulting
from stressful events in daily life. Thus, these results sug-
gested the potential use of probiotics to improve health in
the workplace.
In the present study, LcS-FM consumption led to
improvements in immunological parameters and a stress
marker. At week 6 of the intervention period, NK cell activ-
ity decreased and the salivary levels of cortisol increased in
the control group, but these parameters kept within baseline
levels in the LcS-FM group. NK cells play a very important
role in the prevention of viral infections, including URTIs
[15]. Inhibition of reduced NK cell activity might increase
resistance to URTIs. The reason for the changes in immu-
nological parameters and stress markers in the control
group is not clear. However, the participants were assigned
unusual daily tasks, including the recording of health infor-
mation of many parameters during the intervention period,
which might have affected their immune and endocrine sys-
tems, especially in the first half of the intervention period.
At a later period, the participants might no longer feel
such stress because getting used to the daily tasks and the
parameters might return to the baseline levels. If the case
is true, intake of LcS-FM may prevent decrease in immune
defense activities caused by certain events, rather than aug-
ment their baseline levels. This may be the reason why
improvements in the parameters by LcS-FM intake were
detected only at week 6. In fact, a previous study in healthy
adults with the normal levels of NK cell activity showed
that LcS intake had no effect on their NK cell activity [31].
Previous studies have revealed that the daily consump-
tion of LcS-FM helps to recover low NK cell activity [18].
In vitro studies with human peripheral blood mononuclear
cells suggest that LcS stimulates monocytes/macrophages
to produce IL-12 and augments NK cell activity [32, 33].
The direct regulation of immune function by LcS might be
realized in the present clinical trial, which in turn might
reduce the incidence of URTIs.
LcS-FM may mediate another protective mechanism
whereby the ingestion of the probiotic drink reduces
stress-related host responses. In the present study, LcS-
FM inhibited increases in the levels of the stress hormone
cortisol, which has the ability to decrease NK cell activity
[34]. Some clinical trials with probiotics or prebiotics have
already focused on psychological stress-related decreases
of host defense mechanisms. The prevention of URTIs by
probiotic or prebiotic supplementation among university
students under stress due to final exams was reported [35,
36]. However, in contrast to the present study, the levels of
cortisol were not examined in these trials.
Increasing evidence suggests that probiotics and the
intestinal microbiota affect the nervous system and brain
function [37]. Recently, Tanida et al. [38, 39] showed that
some probiotic strains including LcS suppress the neural
activity of sympathetic nerves in rats. The nervous system
is closely related to the immune system [40]. Future studies
should focus on nervous system-related actions of probiot-
ics as well as their direct immune modulatory activities to
obtain a better understanding of the precise mechanisms of
preventing viral infections via probiotics.
In conclusion, the findings obtained in this study sug-
gest that the daily intake of fermented milk with LcS may
reduce the risk of URTIs in healthy middle-aged office
workers, probably through modulation of the immune
system.
Acknowledgments We thank the staff of the Creative Pioneer for
Clinical Challenge Co Ltd (Tokyo, Japan) for their help with partici-
pant management, sample analysis, and data analysis. We acknowl-
edge Dr. Mitsuhisa Kawai, the Yakult Central Institute, for the calcu-
lation of sample size and the statistical analyses. We also thank the
participants in this study. This study received no specific grant from
any funding agency.
Compliance with ethical standards
Conflict of interest All of the authors are employed by the Yakult
Honsha, which produces fermented dairy products using the probiotic
strain LcS.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea-
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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