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The effects of OLL1073R-1 yogurt intake on

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Cite this: Food Funct., 2019, 10, 8129

influenza incidence and immunological markers
among women healthcare workers: a randomized
controlled trial†
Tetsu Kinoshita, *a,b Koutatsu Maruyama, c Keiko Suyama,a Mariko Nishijima,a
Kimiko Akamatsu,d Akiko Jogamoto,d Kikumi Katakamid and Isao Saito e

Received 12th September 2019,
Accepted 22nd October 2019
DOI: 10.1039/c9fo02128k
rsc.li/food-function
Probiotics have been expected to enhance human immune function. The aim of this study was to evalu-
ate the effects of dietary intake of yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus
OLL1073R-1 (OLL1073R-1) on the prevention of influenza during winter and on the activation of immuno-
logical markers among women healthcare workers. 961 women aged 20–71 years were randomly
assigned to either the yogurt group (n = 479) or the control group (n = 482). Participants in the yogurt
group consumed a 112 mL yogurt drink fermented with OLL1073R-1 every day for 16 weeks, whereas
those in the control group consumed no yogurt during this period. All participants were instructed not to
consume any other kinds of yogurt or fermented dairy products throughout this trial. The cumulative inci-
dence rate of influenza was measured, and immunological markers were examined at the baseline and
after 16 weeks. No significant difference in the incidence rate of influenza was found between the two
groups (cumulative incidence rates of flu: yogurt 7.5% and control 7.7%). Natural killer (NK) cell activity did
not show a significant intervention effect ( p = 0.11), whereas the intervention effect on serum interferon
gamma (IFN-γ) production was significant ( p = 0.03). Other immunological markers did not show signifi-
cant intervention effects. Consumption of OLL1073R-1 yogurt did not show a significant preventive effect
against influenza or a significant enhancement in NK cell activity. However, intake of this yogurt showed
an increase in IFN-γ production.

1. Introduction via lactic acid-producing bacteria or substances produced from

Yogurt, which is a fermented dairy product, is generally known
to have promising health benefits for certain gastrointestinal
conditions, including lactose intolerance, constipation, inflam-
matory bowel diseases, and Helicobacter pylori infection.1,2 In
recent studies, yogurt has been expected to enhance human
immune function through changing the balance of the intesti-
nal microbiota and stimulating the intestinal immune system
a
Department of Community Health System Nursing, Ehime University Graduate
School of Medicine, Toon, Ehime, Japan. E-mail: tetsu.prospective@gmail.com;
Fax: +81-89-960-5284; Tel: +81-89-960-5283
b A previous study on yogurt fermented with Lactobacillus del-
Institute of Community Life Sciences Co., Ltd., Matsuyama, Ehime, Japan
c
Special Course of Food and Health Science, Department of Bioscience Graduate
School of Agriculture, Ehime University, Matsuyama, Ehime, Japan
d
Department of Fundamental and Clinical Nursing, Ehime University Graduate
School of Medicine, Toon, Ehime, Japan
e
Department of Public Health and Epidemiology, Faculty of Medicine, Oita
University, Yufu, Japan
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
C9FO02128K
the bacteria.3,4 However, robust evidence that dietary intake of
a fermented dairy product contributes to a reduced risk of
infectious diseases or to enhanced immune cell activity is
lacking. Previous literature is limited due to differences in the
strains of lactic acid-producing bacteria studied or due to
study design (i.e., small-scale sample size, the nature of the
study population, and investigating only subjective symptoms).
Most notably, no functional food has been shown in random-
ized controlled trials to enhance the activity of natural killer
(NK) cells in humans, which are considered major innate
immune cells combating against pathogenic viruses and
tumor cells.5,6
brueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1) showed that
this strain reduced the risk of respiratory infections in the
elderly.7 In animal experiments, the exopolysaccharides (EPSs)
produced from OLL1073R-1 had anti-influenza virus effects
and enhanced NK cell activity.8–10
Therefore, the preventive effect of OLL1073R-1 yogurt intake
against influenza infection during winter could be elucidated

This journal is © The Royal Society of Chemistry 2019 Food Funct., 2019, 10, 8129–8136 | 8129

Paper
via the enhancement of immune cell activity using an appro-
priate study design. Given that women healthcare employees
are exposed to infectious diseases during shift work and thus
their immune function might be deteriorated by their irregular
lifestyles,11 they were chosen as subjects for this trial.
2. Methods
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2.1. Power analysis and sample size
In this randomized controlled study, sample size was calculated
based on the incidence rates of influenza infection in Japanese
observational studies. Studies conducted in the 2013–2014 and
2014–2015 seasons reported influenza infection rates of 5.1%
and 7.0%, respectively, among pregnant Japanese women.12,13
In 2013–2014, the infection rate of vaccinated women was lower
than that of non-vaccinated women.12 However, in 2014–2015,
the overall infection rate did not differ significantly between
those with and without vaccination (7.3% vs. 6.7%, respect-
ively).13 The contract rate of the control group was hypothesized
to be 6.5% during this winter trial and 2.6% for the yogurt
group, based on a previous clinical trial.7 Additionally, assum-
ing an alpha error of 5%, beta error of 20%, and dropout rate
during the trial of approximately 20%, the number of partici-
pants was calculated to be 600 in each group.
2.2. Participants
Participants were recruited from 23 medical institutions in
Ehime Prefecture, Japan, from August to September, which are
not the months of flu season, 2016. Women aged 20 or older
who were currently medical or welfare-related professionals at
medical institutions in Ehime Prefecture and were capable of
understanding the study purpose were selected, with written
informed consent provided by all participants. The exclusion
criteria were as follows: (1) pregnant women, (2) women who
contracted influenza during the period of July 2016 to the date
of giving written informed consent, (3) women who show aller-
gic responses to dairy products, (4) lactose intolerant women,
(5) women who are instructed to restrict calories by a phys-
ician, (6) women with a history of diseases involving the
immune system (e.g., rheumatism, cancer, thyroid disorder,
systemic lupus erythematosus, myasthenia gravis, Graves’
disease, and scleroderma), (7) women who had participated in
other clinical trials within the past three months, and (8)
women who were judged inadequate by the principal doctor
for other reasons. Of the 1026 women who agreed to partici-
pate in this study, 20 infringed the exclusion criteria and 24
declined to participate after the agreement. Therefore, 982
women were examined at the screening session. During the
session, 20 women infringed the exclusion criteria and one
declined participation. Consequently, 961 women aged 20–71
years were enrolled in the present study.
2.3. Study design
This trial was designed as a randomized, controlled, open-
labeled study. Participants were first instructed not to
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consume any yogurt or fermented dairy foods from the day of
agreement to that of random assignment. The 961 women
were assigned to either the yogurt group (n = 479) or the
control group (n = 482) by block randomization within the
strata of the institutions they worked for, age, and having a
plan of influenza vaccination or not. The yogurt group con-
sumed the test yogurt daily for 16 weeks (from November 14,
2016, to March 5, 2017), whereas the control group consumed
no yogurt during this period. Furthermore, all participants
were instructed not to consume any other yogurt or fermented
dairy products throughout this trial. In addition, they were
directed not to change their lifestyles during this trial except
for yogurt intake. The participants underwent blood tests at
the baseline and after 16 weeks. In addition, participants were
given a “health notebook” to record any incidence of influenza
and the common cold, changes in their health condition,
changes in their lifestyles, intake of the test yogurt or not (for
the yogurt group), and intake of any other yogurt or fermented
dairy foods or not (for both groups) during the trial period.
Whether the participants received influenza vaccination or not
was left to their individual discretion. If they planned to vacci-
nate, they were prompted to receive vaccination between the
baseline blood test and the first day (Nov. 14, 2016) of test
food consumption. During the trial, four participants in the
yogurt group and 12 in the control group declined partici-
pation. In the control group, two were absent from the examin-
ation session and one did not answer the self-administered
questionnaires 16 weeks later. In addition, two participants in
the yogurt group and four in the control group revealed that
they were pregnant during the trial, thereby infringing the
exclusion criteria. Fig. 1 shows the sampling scheme through-
out this study.
2.4. Test yogurt
The test food was “Meiji Probio Yogurt R-1” drink type (Meiji
Co., Ltd, Tokyo), which is currently on the market. This yogurt
is manufactured using two lactic acid bacterial species,
L. bulgaricus OLL1073R-1 and a strain of Streptococcus thermo-
philus, originally isolated from traditional Bulgarian yogurt.
One bottle contains 112 mL drinkable yogurt and provides
76 kcal, 13.9 g carbohydrate, 0.67 g fat, 3.6 g protein, and 1.12
× 109 CFU (colony forming unit) or more of L. bulgaricus and
S. thermophilus. The total count of lactic acid bacteria is con-
firmed by colony counting using a culture medium in which
both L bulgaricus and S. thermophilus can grow. Lactic acid bac-
teria in the test drink were counted after culture in bromocre-
sol purple broth at 35–37 °C for 72 ± 3 h. Although these
culture conditions are not the most appropriate for the growth
of both bacterial species, both can be detected and that count-
ing method is universally used.
2.5. Outcome measurement
2.5.1. Cumulative incidence rates of influenza and the
common cold. The primary objective of the present study was
to examine the cumulative incidence rate of influenza within
the two groups. The participants were instructed to record
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Food & Function
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Fig. 1 Sampling scheme throughout this study.
influenza incidence in their health record only if diagnosed by
a physician. The common cold was defined as having a fever of
38 °C or more, accompanied by one or more of the following
symptoms: cough, sputum, throat pain, snivel, joint pain,
headache, and diarrhea with abdominal pain. Cumulative inci-
dence rates of influenza and the common cold were deter-
mined using the health records during the trial period (from
November 14, 2016, to March 5, 2017). However, the first week
of the trial was not subject to evaluation.
2.5.2. Immunological markers. Blood tests examining
immunological markers such as cytokines were conducted at
the baseline and after 16 weeks. Blood was sampled in each
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medical institution and examined within the sampling day.
Blood sampling was conducted in the morning (before starting
their work) after 12 hours of fast considering the diurnal vari-
ation of blood markers. The indices were NK cell activity,
serum high-sensitivity C-reactive protein (hs-CRP), serum
interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte-
macrophage colony stimulating factor (GM-CSF), interferon
gamma (IFN-γ), and tumor necrosis factor-α (TNF-α). NK cell
activities were assessed by the 51Cr release method targeting
K562 cells at Shikoku-Chuken Co. Ltd (Kagawa, Japan), hs-CRP
was gauged by latex nephelometry (N-latex CRP II, Siemens
Healthcare) at Shikoku-Chuken, and other serum immunologi-
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cal biomarkers were examined by using a BioPlex14 at Meiji Table 1 The baseline characteristics of participants
Corporation. These markers were chosen based on the pre-
vious animal study,9 which highlighted the effects of Yogurt group Control group
(n = 479) (n = 482) P value
OLL1073R-1 strain on enhancing natural killer cell activation
and on induction of producing IFN-γ. IFN-γ is an important Age (years) 39.3 (11.5) 39.4 (11.4) 0.91*
BMI (kg m−2) 22.0 (3.5) 21.8 (3.5) 0.42*
factor in eliminating infected cells, and thus we evaluated the Carrier
levels of IFN-γ and other cytokines. When the measurement Nurse 310 (64.7%) 312 (64.7%) 0.62**
Care worker 43 (9.0%) 33 (6.9%)
values were below the detection limit, half of the lowest values Physical therapist/ 36 (7.5%) 40 (8.3%)
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detected in this trial were applied. In this trial, we did not occupational therapist
examine nutritional indices, such as total protein or albumin, Others 90 (18.8%) 97 (20.1%)
Influenza vaccination
because this study focused on the changes in immunological Received 452 (94.4%) 457 (94.8%) 0.76**
functions. Did not receive 27 (5.6%) 25 (5.2%)
Irregular shift worker 220 (46.0%) 228 (47.3%) 0.67**
2.5.3. Safety. Adverse events occurring during this trial Living by herself 118 (24.6%) 120 (24.9%) 0.93**
period were evaluated for the two groups. Based on the health Current smoker 47 (9.8%) 43 (8.9%) 0.64**
record and the blood test after 16 weeks, the number of NK cell activitya (%) 26.8 (15.9–45.2) 26.7 (15.6–45.6) 0.90***
hs-CRPa (mg/dl) 0.031 (0.0086–0.11) 0.028 (0.0084–0.092) 0.21***
adverse events was counted and classified into 10 categories: IL-2a,b (pg ml−1) 0.82 (0.56–1.20) 0.80 (0.71–0.92) 0.33***
indefinite complaint (e.g. headache, fatigue, irritability, etc.), IL-4a,b (pg ml−1) 0.063 (0.016–0.25) 0.063 (0.015–0.26) 0.94***
IL-5a,b (pg ml−1) 1.16 (0.14–9.60) 1.20 (0.14–10.25) 0.83***
menstrual pain, digestive symptoms, body aches, skin symp- IL-10a,b (pg ml−1) 4.62 (1.66–12.82) 4.20 (1.49–11.88) 0.15***
toms, sleep and mental condition, allergy, respiratory symp- IL-12a,b (pg ml−1) 0.14 (0.017–1.14) 0.14 (0.017–1.21) 0.80***
toms, injury, and others. For the yogurt group, the principal IL-13a,b (pg ml−1) 2.12 (0.46–9.90) 2.20 (0.50–9.70) 0.71***
GM-CSFa,b (pg ml−1) 0.46 (0.33–0.65) 0.47 (0.31–0.70) 0.57***
doctor judged whether the adverse event had a causal relation- IFN-γa,b (pg ml−1) 2.69 (0.61–11.84) 2.65 (0.58–12.16) 0.85***
ship with the intake of the test yogurt. TNF-αa,b (pg ml−1) 0.75 (0.038–14.79) 0.74 (0.036–15.17) 0.94***

Values are expressed as means. Standard deviations or percentages are

2.6. Statistical analysis
All statistical analyses were performed with SAS version 9.4
(SAS Institute Inc., Cary, NC, USA) based on intention-to-treat
analysis. The difference in cumulative incidence rates of influ-
enza and the common cold between the two groups was ana-
lyzed by a chi-square test using the Cochran–Mantel–Haenszel
method. However, Fisher’s exact test was used when the
numbers of incidence were less than five. The variable strata
were as follows: the institutes, the yogurt group or control
group, and the outcome. If a participant dropped out during
the trial period, influenza incidence was evaluated during the
participation period since it was the primary objective, and the
common cold was excluded from evaluation.
For the adverse events, incidence rates in both groups were
analyzed in 10 categories by a chi-square test.
In all analyses, p < 0.05 (two-sided) was considered statisti-
cally significant.
2.7. Ethics statement
The present study was approved by the Institutional Review
Board (IRB) of Ehime University Hospital and the IRB of Meiji
Corporation. Written informed consent was obtained from all
participants. This trial was registered at the clinical trial regis-
try of Japan as UMIN000023932.
3. Results
Table 1 shows the baseline characteristics of the participants.
There were no significant differences identified between the
two groups for any of the indices at the baseline.
indicated in parentheses. a Geometric mean values. Lower 1SD and higher
1SD are indicated in parentheses. b Two participants were excluded from
the analysis as they were unmeasured due to the lack of blood samples at
the baseline (Yogurt: n = 478 and Control: n = 481). *Wilcoxon’s rank sum
test. **Chi-square test. ***Unpaired t-test.
3.1. Cumulative incidence rates of influenza and the
common cold
Fig. 2 indicates the Kaplan–Meier curves of the cumulative
incidence rates of influenza (A) and the common cold (B) for
each group during the 16 week trial period. No statistically sig-
nificant difference was found between the curves of the yogurt
and control groups.
The total cumulative incidence rate of influenza was 7.6%
(73/961), and all incidences were attributed to the influenza A
virus. The rate was 7.5% (36/479) for the yogurt group and
7.7% (37/482) for the control group (Table 2). Between the two
groups, there was no significant difference in the cumulative
incidence rate of influenza ( p = 0.91). With respect to the
common cold, the cumulative incidence rate was 18.4% (87/
473) for the yogurt group and 20.2% (94/466) for the control
group. Between the groups, a significant difference was not
found ( p = 0.49).
Table 2 shows the stratified analysis related to the cumulat-
ive incidence rates of influenza. Twenty-seven (5.6%) partici-
pants in the yogurt group did not receive influenza vaccination
and five (18.5%) of them developed influenza, while 25 (5.2%)
participants in the control group did not receive influenza vac-
cination and three (12.0%) of them developed influenza.
During the 16 weeks, 87 (18.2%) participants from the yogurt
group took a preventive agent for influenza (e.g., Tamiflu®,
Relenza®, and Inavir®) in accordance with the rules of the

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cell activity increased in both groups (yogurt: 26.8% to 32.1%;

control: 26.7% to 33.4%), but the intervention effect was not
statistically significant ( p = 0.11). IFN-γ production increased
in both groups (yogurt: 2.69 pg ml−1 to 6.21 pg ml−1; control:
2.65 pg ml−1 to 4.70 pg ml−1), and a significant intervention
effect was found ( p = 0.03). The corresponding significant
increase in IFN-γ production was confirmed, in case that we
regarded the IFN-γ below the detection limit as zero. For the
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other biomarkers, significant intervention effects were not

found.

Fig. 2 Kaplan–Meier curves of the cumulative incidence rates of 3.3. Safety

influenza (A) and the common cold (B) for each group during the 16
week. The straight line denotes the yogurt group, while the dotted line
denotes the control group.
Table 2 Stratified analysis of the cumulative incidence rate of influenza
ESI table† shows the number of adverse events throughout this
trial in each group. In the yogurt group, digestive symptoms
were observed significantly compared to the control group. No
serious side effects were considered to be caused by the
yogurt.

Yogurt group Control group P value

Total cumulative 36/479 (7.5%) 37/482 (7.7%) 0.91

4. Discussion
incidence of Influenza
This trial examined the effects of OLL1073R-1 yogurt intake on
Incidence in January–March, 33/479 (6.9%) 31/482 (6.5%) 0.82 the prevention of influenza and the common cold during
2017 (excluded the incidence winter and on the enhancement of immune function among
within 2016)
women who work in healthcare. Of the immunological para-
Influenza vaccination meters analyzed, IFN-γ production demonstrated a significant
Vaccinated 31/452 (6.9%) 34/457 (7.4%) 0.72 intervention effect.
Unvaccinated 5/27 (18.5%) 3/25 (12.0%) 0.59*
IFN-γ is secreted by NK cells with other cytokines and
Preventive agent for influenza enhances NK cell activity.10,15,16 Thus, IFN-γ is considered the
Took once or more 3/87 (3.5%) 6/91 (6.6%) 0.30* most critical cytokine for NK cell activity. Although IFN-γ
Did not take at all 33/392 (8.4%) 31/391 (7.9%) 0.77
might have enhanced NK cell activation to some extent,
Working shift various environmental factors, such as a change in tempera-
Irregular 13/220 (5.9%) 23/228 (10.1%) 0.10 ture with seasons,17,18 could have greatly influenced this
Day shift 23/259 (8.9%) 14/254 (5.5%) 0.08
increase. While IFN-γ levels were elevated in the OLL1073R-1
Living yogurt group, other cytokines such as GM-CSF and TNF-α were
By herself 10/118 (8.5%) 6/120 (5.0%) 0.35 not increased. Thus, it is unlikely that the elevated levels of
With family 26/361 (7.2%) 31/362 (8.6%) 0.47
IFN-γ were attributed to inflammation or infection by some
Smoking pathogens. The mechanisms of increased IFN-γ production
Current smoker 4/47 (8.5%) 4/43 (9.3%) 0.85* should be further investigated. It is known that T cells are a
Non smoker 32/432 (7.4%) 33/439 (7.5%) 0.94
major source of IFN-γ other than NK cells.19 Furthermore, pre-
Chi-square test using the Cochran–Mantel–Haenszel method. *Fisher’s vious studies have reported increased T cell activity stimulated
exact test. by OLL1073R-1 yogurt or extracellular polysaccharides pro-
duced from OLL1073R-1.7–10 In line with the association, the
consumption of the OLL1073R-1 yogurt might possibly stimu-
late IFN-γ production via T cell activation. It has been shown
institutions where they worked, and three (3.5%) of them that cell-mediated immunity evaluated by the production of
developed influenza. In the control group, 91 (18.9%) partici- IFN-γ, probably by influenza virus-specific T cells, contributes
pants took the preventive medicine and six (6.6%) developed to protection against clinical influenza infection in young chil-
influenza. In each stratum, no significant difference in the dren.20 Although we did not investigate whether it was
cumulative incidence rate of influenza was found between the antigen-specific, the increased production of IFN-γ might at
two groups. least partly contribute to protection against infection with
pathogens.
Although the preventive effect of OLL1073R-1 yogurt
3.2. Immunological markers against influenza was the primary objective, no significant
Table 3 shows the measurement values of immunological effect was observed in this trial. The cumulative incidence rate
markers at the baseline and after 16 weeks in each group. NK of influenza in the control group was 7.7%, which was 1.2%

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Table 3 Changes in the immunological indicators during the trial period

Yogurt group (n = 479) Control group (n = 482)

Baseline 16 weeks Baseline 16 weeks P value*

NK cell activity (%) 26.8 (15.9–45.2) 32.1 (18.9–54.7) 26.7 (15.6–45.6) 33.4 (20.3–54.9) 0.11
hs-CRP (mg dl−1) 0.031 (0.0086–0.11) 0.031 (0.0086–0.11) 0.028 (0.0084–0.092) 0.028 (0.0078–0.097) 0.88
IL-2a (pg ml−1) 0.82 (0.56–1.20) 0.82 (0.54–1.24) 0.80 (0.71–0.92) 0.81 (0.68–0.95) 0.77
IL-4a (pg ml−1) 0.063 (0.016–0.25) 0.11 (0.021–0.60) 0.063 (0.015–0.26) 0.11 (0.021–0.54) 0.60
IL-5a (pg ml−1)
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1.16 (0.14–9.60) 2.21 (0.32–15.15) 1.20 (0.14–10.25) 2.04 (0.30–14.02) 0.47

IL-10a (pg ml−1) 4.62 (1.66–12.82) 6.05 (2.27–16.11) 4.20 (1.49–11.88) 5.68 (2.26–14.28) 0.64
IL-12a (pg ml−1) 0.14 (0.017–1.14) 0.31 (0.029–3.19) 0.14 (0.017–1.21) 0.28 (0.029–2.75) 0.43
IL-13a (pg ml−1) 2.12 (0.46–9.90) 2.36 (0.57–9.71) 2.20 (0.50–9.70) 2.27 (0.51–10.15) 0.53
GM-CSFa (pg ml−1) 0.46 (0.33–0.65) 0.46 (0.33–0.65) 0.47 (0.31–0.70) 0.46 (0.34–0.64) 0.27
IFN-γa (pg ml−1) 2.69 (0.61–11.84) 6.21 (1.06–36.45) 2.65 (0.58–12.16) 4.70 (0.84–26.19) 0.03
TNF-αa (pg ml−1) 0.75 (0.038–14.79) 0.82 (0.034–19.68) 0.74 (0.036–15.17) 0.61 (0.025–15.04) 0.24

Values are expressed as geometric means. Standard deviations (±1SD) are indicated in parentheses. a Two participants were excluded from the
analysis as they were unmeasured due to the lack of blood samples at the baseline (Yogurt: n = 478 and Control: n = 481). *Two-way analysis of
variance with repeated measures.

higher than the hypothesized 6.5% from the power calcu-
lation. On the other hand, the rate in the yogurt group of 7.5%
was quite higher than the hypothesized 2.6%. The Kaplan–
Meier curves seemed to slightly show the preventive effects of
OLL1073R-1 yogurt; thus, it is possible that if the present
study had gone on for longer (e.g. 24–32 weeks) then a signifi-
cant effect may have been observed; however, a larger scale
study would be required in order to detect a significant effect
during 16 weeks.
The use of antiviral drugs for influenza (e.g., Tamiflu®,
Relenza®, and Inavir®) should affect the cumulative incidence
rate of influenza. In the yogurt group, 87 (18.2%) participants
took an influenza preventive agent, whereas 91 (18.9%) partici-
pants took an influenza preventive agent in the control group. In
both groups, the participants who took the preventive medicine
showed low cumulative incidence rates (3.5% and 6.5%, respect-
ively). Therefore, taking the preventive medicine seems to have
contributed to influenza prevention. However, in the analysis
among the participants who did not take the preventive medi-
cine, no significant difference in the cumulative incidence rate
was found. Thus, the preventive agents would not have affected
the difference in the cumulative incidence rate of both groups.
In the yogurt group, digestive symptoms were observed sig-
nificantly compared to the control group. The symptoms were
mainly loose stools and concentrated within 2 weeks from the
start of yogurt intake. We consider that since they had not
eaten yogurt for more than one month after they agreed to par-
ticipate in this study, a habituation period to the dairy product
was needed for some participants. Those would mean yogurt
intolerance, not lactose tolerance, because lactose tolerant
women were excluded by participants’ criteria.
The strength of this study lies in the use of a randomized
controlled design and the sample size calculation based on
reported influenza infection rates12,13 in Japanese women.
Previous studies evaluating the effects of probiotics on the pre-
vention of influenza or enhancing immune response21–26 had
limitations in study design, including small-scale, inappropri-
ate intake season, lack of basis in sample size calculation, or
the randomization was conducted among institutions but not
among individuals. The present trial was conducted using stra-
tified randomization in a large-scale population. Furthermore,
the effect of OLL1073R-1 yogurt on increased IFN-γ production
in women healthcare workers provided new evidence in the
field of nutritional immunology. Another strength is that this
large-scale study was executed under the stringent condition
that the participants do not consume any other fermented
dairy products during the 16 week trial. Despite this restric-
tion, the dropout rate was less than 3%, and over 900 partici-
pants completed the trial.
Nonetheless, this study has several limitations. First, the
participants were not blinded. Ideally, a placebo yogurt should
be used as the control food. However, since it was impossible
to prepare a placebo similar to the yogurt in appearance, taste,
and flavor, an open-label design was used. As a result, if all
participants were eager to be in the yogurt group, someone
assigned to that group might be motivated to complete the
trial period, whereas a person assigned to the control group
might feel some stress due to complete yogurt restriction.
Consequently, there is a possibility that the differences in the
mood or stress levels between the groups might affect the
results. Second, since all participants were instructed not to
consume any fermented dairy products except for the test food
for the yogurt group, there is a possibility that this rule intro-
duced selection bias. In the informed consent session, some
participants refused to participate due to this restriction. To
eliminate this influence, it might be preferable to choose par-
ticipants who do not have a habit of probiotic intake in order
to evaluate the effects of yogurt.
5. Conclusion
This large-scale clinical trial among women healthcare
workers did not show any effects of OLL1073R-1 yogurt intake
on influenza prevention and NK cell activity enhancement.
However, a significant increase in IFN-γ production was found

8134 | Food Funct., 2019, 10, 8129–8136 This journal is © The Royal Society of Chemistry 2019

Food & Function
with daily intake of OLL1073R-1 yogurt. The current findings
present novel evidence in the field of nutritional immunology
and contribute to a better understanding of the benefits of
probiotics. Further clinical trials, using an appropriate sample
size and a randomized controlled study design, are needed to
verify the effects of fermented dairy products.
Published on 24 October 2019. Downloaded by Vanderbilt University Library on 1/2/2020 9:02:19 PM.
Author contributions
The authors’ responsibilities were as follows: TK, KM, and IS
conceived and designed the study; TK, KS, MN, KA, AJ, and KK
led and conducted research; KM and IS analyzed the data; TK,
KM, an IS wrote the paper; and all authors read and approved
the final manuscript.
Conflicts of interest
The test yogurt was obtained from Meiji. Tetsu Kinoshita (first
author) is the head of the Institute of Community Life
Sciences Co., Ltd; this study was conducted based on the con-
tract agreement between Meiji and the Institute of Community
Life Sciences. Meiji had no input in the data analysis and
interpretation. The other authors had no personal or financial
conflict of interest.
Acknowledgements
The authors thank all participants, doctors and chief nurses of
23 medical institutions for cooperation in experiments. This
study was supported by funding from the Meiji Corporation
(Tokyo, Japan).
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