Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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journal homepage: www.elsevier.com/locate/job
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Combining prebiotics and probiotics to develop novel synbiotics that suppress
13 oral pathogens
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Q1 Yukako Kojima a, Tomoko Ohshima b,n, Chaminda Jayampath Seneviratne c, Nobuko Maeda b
16
a
17 Department of Oral Microbiology, Graduate School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Kanagawa 230-8501, Japan
b
Department of Oral Microbiology, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Kanagawa 230-8501, Japan
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Oral Sciences, Faculty of Dentistry, National University of Singapore, Singapore
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art ic l e i nf o a b s t r a c t
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Article history: Objective: Synbiotics, resulting from a synergistic combination of prebiotics and probiotics, is a new
24 Received 2 April 2015 concept in clinical studies that is known to promote intestinal health. However, the beneficial effects of
25 Received in revised form synbiotics on oral health have not been investigated. The present study attempted to develop new
26 9 August 2015
synbiotics against oral pathogens (bacterial and fungal).
Accepted 17 August 2015
27 Methods: Prebiotic screening was carried out by sugar assimilation tests using 12 saccharides. About 40
28 strains of lactobacilli were used for probiotic screening. Standard in vitro assays were performed against
29 Keywords: oral pathogens, such as Candida albicans, Streptococcus mutans, and Porphyromonas gingivalis. Growth
Synbiotics inhibition and biofilm formation assays were conducted for C. albicans using lactobacilli in co-culture or
30
Probiotics
31 with the culture supernatant (-CS). Subsequently, the disc diffusion assay was used as a growth inhibi-
Prebiotics
32 tory test against P. gingivalis and the amount of insoluble glucan produced by S. mutans was determined
Oral pathogens
by phenol-sulfate staining.
33
Results: The results showed that arabinose, xylose, and xylitol are the saccharides with a strong potential
34
to be used as prebiotics and five lactobacilli strains isolated from the oral cavity have the potential to be
35 used as probiotics. These strains inhibited the growth of C. albicans and P. gingivalis, and had an inhibitory
36 effect on the production of insoluble glucan by S. mutans. Lactobacilli strains isolated from dairy foods did
37 not show a significant effect on human oral microbiota.
38 Conclusion: The present study indicates that prebiotics and probiotics can potentially be developed into
39 novel synbiotics against oral pathogens in future.
40 & 2015 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology.
41
42 67
43 68
44 1. Introduction shown that probiotics have benefits beyond promoting intestinal 69
45 health. Therefore, Salminen et al. defined a probiotic as “A viable 70
46 Major oral diseases such as dental caries, periodontal disease, and microbial food supplement which beneficially influences the 71
47 oral candidiasis are infectious in nature and these oral diseases are health of the host” [3] while, FAO/WHO define probiotics as “live 72
48 caused by oral pathogens Streptococcus mutans, Porphyromonas gin- microorganisms when administered in adequate amounts confer a 73
49 givalis, and Candida albicans, respectively. Oral diseases affect millions health benefit on the host” [4]. In addition, the health benefits of 74
50 of people worldwide and the incidence of oral diseases is expected to oral probiotics have been a focus of several clinical trials [23]. 75
51 rise in the next decade due to an increase in the number of elderly In 1995, Gibson and Roberfroid defined “Prebiotics” as “a non- 76
52 people and immunocompromised patients. digestible food ingredient that beneficially affects the host by 77
53 Prebiotics and probiotics are well known for their beneficial selectively stimulating the growth and/or activity of one or a 78
54 effects in promoting the health of the human gastrointestinal tract. limited number of bacteria in the colon, and thus improves host 79
55 health” [5]. However, there are only limited studies on oral pre- 80
The term “Probiotics” was coined by Kollath in the 1950s and
56 biotics. Gibson and Roberfroid also introduced the concept of 81
subsequently Lilly and Stillwell employed the term in 1965 [1]. In
57 “synbiotics” in the same report and defined synbiotics as “a mix- 82
1989, Fuller redefined a probiotic as “A live microbial feed sup-
58 ture of probiotics and prebiotics that beneficially affects the host 83
plement which beneficially affects the host animal by improving
59 by improving the survival and implantation of live microbial 84
its intestinal microbial balance” [2]. However, later it was later dietary supplements in the gastrointestinal tract, by selectively
60 85
61 stimulating the growth and/or by activating the metabolism of one 86
62 n
Corresponding author. Tel.: þ 81 45 580 8441; fax: þ 81 45 572 3516. or a limited number of health-promoting bacteria, and thus 87
63 E-mail address: ohshima-t@fs.tsurumi-u.ac.jp (T. Ohshima). improving host welfare” [1]. 88
64 89
http://dx.doi.org/10.1016/j.job.2015.08.004
65 1349-0079/& 2015 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology. 90
66 91

Please cite this article as: Kojima Y, et al. Combining prebiotics and probiotics to develop novel synbiotics that suppress oral pathogens.
J Oral Biosci (2015), http://dx.doi.org/10.1016/j.job.2015.08.004i
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1 The activities of probiotics and synbiotics have been studied Table 2 67

2 extensively [24] however, little is known about the mechanism of 40 Lactobacilli strains for this study. 68
3 their action on oral health. Therefore, in the present study we have 69
Reference strains
4 attempted to discover probiotic and prebiotic candidates that can Lactobacillus animalis ATCC35046 70
5 be used to develop a novel synbiotics against oral bacterial and L. salivarius LS1 71
6 fungal pathogens. L. reuteri PTA 72
7 L. reuteri DSM 73
L. brevis (Labostrain, Tsurumi Univ. Dental Med. Dept. Oral Microbiol.)
8 L. murinus ATCC35020
74
9 L. casei ATCC393 75
2. Material and methods
10 76
Isolated from dairy foods
11 11. Lactobacillus spp. Yogurt 77
2.1. Saccharides
12 13L. L. casei YIT9029-L Yogurt 78
13 13S. L. casei YIT9029-S Yogurt 79
A total of 12 saccharides were screened to identify suitable 14. Lactobacillus spp. Mozzarella cheese
14 80
prebiotic candidates (Table 1). 17L. Lactobacillus spp. Red Cheddar cheese
15 17S. Lactobacillus spp. Red Cheddar cheese
81
16 19L. Lactobacillus spp. Red Cheddar cheese 82
17 2.2. Bacterial strains and culture conditions 19S. Lactobacillus spp. Red Cheddar cheese 83
18 20. Lactobacillus spp. Gouda cheese 84
We used three oral pathogenic microbes: Candida albicans 23. Lactobacillus spp. Brie Hermitage cheese
19 85
24. Lactobacillus spp. Foume d'Ambert cheese
20 (ATCC18804), Streptococcus mutans (ATCC25175), and Porphyr- 86
21 omonas gingivalis (ATCC33277), stored at  80 °C at the Depart- Isolated from oral flora 87
101. Lactobacillus casei
22 ment of Oral Microbiology, Tsurumi University School of Dental 88
102. L. gasseri
23 Medicine. 103. L. fermentum 89
24 C. albicans was cultured on Sabouraud dextrose agar (Nissui, 104. L. crispatus 90
25 Tokyo, Japan) at 30 °C for 2 days under aerobic conditions, then one 105. L. salivalius 91
106. L. gasseri
26 inoculation loop-full of the colony was subcultured in tryptic soy 92
107. L. crispatus
27 broth (Becton Dickinson and Company, Sparks, MD, USA) supple- 108. L. plantarum 93
28 mented with 5% dextrose anhydrous (TSBD), and incubated under 109. L. salivalius 94
29 aerobic conditions at 30 °C overnight (14–16 h) with constant shaking 110. L. casei 95
30 111. L. fermentum 96
to obtain a late logarithmic growth phase culture (the turbidity was
112. L. paracasei
31 2.4 and the OD was measured at 620 nm (Ultrospec 4000 UV/vis 113. Lactobacillus spp.
97
32 spectrophotometer, Pharmacia Biotech Inc., NJ, USA). The pH was 5.0). 114. Lactobacillus spp. 98
33 S. mutans was defrosted and inoculated in brain heart infusion 115. Lactobacillus spp. 99
34 116. Lactobacillus spp. 100
broth (BHI) (Becton Dickinson and Company) and incubated under
117. L. paracasei
35 anaerobic conditions at 37 °C overnight (14–16 h), then sub- 101
118. Lactobacillus spp.
36 cultured in fresh BHI under aerobic conditions with constant 119. Lactobacillus spp. 102
37 shaking for 6 h. This subculture was incubated overnight (14–16 h) 120. Lactobacillus spp. 103
38 to obtain a late logarithmic growth phase culture (the turbidity 121. Lactobacillus spp. 104
39 122. L. plantarum 105
was 1.2 and the OD was measured at 620 nm. The pH was 5.4).
40 P. gingivalis was defrosted and inoculated in BHI supplemented 106
41 with 0.5% yeast extract, 0.05% cysteine–HCI, 5 μg/mL hemin, and 107
42 100 μg/mL Vitamin K under anaerobic conditions at 37 °C for 2 days. Reference strains and clinical strains from the human oral 108
43 It was then subcultured in fresh medium and incubated for another cavity were stored at  80 °C at the Department of Oral Micro- 109
44 2 days to obtain a late logarithmic growth phase culture (the turbidity biology, Tsurumi University School of Dental Medicine until used. 110
45 All lactobacilli strains were subcultured in MRS Broth (Becton 111
was 1.1 and OD was measured at 620 nm. The pH was 7.7).
46 Dickinson and company Sparks, MD, USA) at 37 °C under anaerobic 112
A total of 40 strains of lactobacilli were used to identify
47 conditions for 2 days. 113
potential candidates for use as probiotics (Table 2). This included
48 114
seven reference strains, 22 strains isolated from the human oral
49 2.3. Saccharide assimilation tests of lactobacilli: C. albicans or S. 115
cavity [13] and 11 strains isolated from dairy foods by selective
50 mutans 116
cultivation on BBL™ LBS Agar (Becton Dickinson and company)
51 117
supplemented with glacial acetic acid under anaerobic conditions
52 Saccharide assimilation tests were performed on lactobacilli and 118
at 37 °C for 2 days.
53 oral pathogens C. albicans and S. mutans to identify candidates that 119
54 can be used as prebiotics. Microbial suspensions of lactobacilli 120
Table 1
55
12 Saccharides used in this study. (1  104–106 CFU/10 μL), C. albicans (1  104–105 CFU/10 μL), and S. 121
56 mutans (  104 CFU/10 μL) were each added to 190 μL of Tryptic soy 122
57 Saccharides broth (TSB) without dextrose supplemented with 4% of one of the 123
58 mono- or oligo-saccharides shown in Table 1. The turbidities of cul- 124
Glucosea Maltosea
59 ture solutions were determined as OD at 620 nm at 24, 48 and 72 h 125
Galactosea Lactoseb
60 Xylosea Treharosea by using a microplate reader (Multiskans Multisoft Labsystems). 126
61 Xylitola Arabinosea 127
62 Cellobiosea Melezitosec 2.4. Co-culture test for detecting growth inhibitory effect of lacto- 128
63 Sucrosea Raffinosea bacilli on C. albicans 129
64 a
Wako Pure Chemical Industries, Ltd. Japan.
130
65 b
E. Merck, Darmstadt Germany. Lactobacilli cultured for 24 h in TSBD under anaerobic condi- 131
66 c
DIFCO LABORATORIES Detroit Michigan USA. tions were used for growth inhibitory assays. 190 μL of lactobacilli 132

Please cite this article as: Kojima Y, et al. Combining prebiotics and probiotics to develop novel synbiotics that suppress oral pathogens.
J Oral Biosci (2015), http://dx.doi.org/10.1016/j.job.2015.08.004i
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1 suspension was co-cultured under aerobic conditions for 24 h with 67

2 10 μL of C. albicans suspension. The turbidity of the C. albicans Turbidity OD620nm C.albicans 68
3 suspension was then estimated (OD measured at 620 nm) by 1.2 glucose 69
4 subtracting from the initial OD value. galactose 70
5 1 xylose 71
6 2.5. CFU assay or turbidity assay on C. albicans as were used as xylitol 72
7 culture tests with lactobacilli culture supernatant (-CS) was used 0.8 cellobiose 73
8 sucrose
74
9 A culture suspension of lactobacilli was centrifuged (Avanti™ 0.6 maltose
75
10 HP-20, Beckman Coulter, Inc., CA, USA) at 7000 rpm at 4 °C for 76
lactose
11 20 min and the resulting supernatant was collected. 100 μL of the 0.4 treharose
77
12 yeast form of C. albicans suspension was added to 750 μL of each arabinose
78
13 lactobacilli-CS and MRS 750 μL and incubated under aerobics 0.2 melezitose
79
14 condition overnight. Thereafter, 100 μL of each sample was plated raffinose
80
15 on Sabouraud dextrose agar plates and incubated for 2 days. The 81
0
16 number of live cells was determined by counting the Colony 0hr 24hr 48hr 72hr 82
17 Forming Units (CFU). 83
18 For performing hyphal inhibition and biofilm formation assays, 84
Turbidity S.mutans
19 100 μL of neutralized culture supernatant of lactobacilli was added OD 620nm glucose 85
to 100 μL of C. albicans diluted 2 fold with concentrated RPMI 1640
20 0.6 86
galactose
21 broths (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine 87
xylose
22 0.5 88
serum (Gibco™, Invitrogen Corporation, Grand Island, N.Y., USA).
xylitol
23 C. albicans were incubated under aerobic conditions in 96 well 89
24 0.4 cellobiose 90
plates in lactobacilli-CS and medium supplemented with serum at
25 37 °C for 24 h with constant shaking. The amount of biofilm sucrose 91
26 0.3 92
formed was determined by measuring the turbidity (OD measured maltose
27 at 620 nm). 93
0.2 lactose
28 94
29 treharose 95
2.6. Disc diffusion assay for detecting any growth inhibitory effect of 0.1
30 arabinose 96
lactobacilli-CS on P. gingivalis
31 melezitose
97
32 0 98
Inoculum of 100 μL P. gingivalis was inoculated on Brucella HK 0hr 24hr 48hr 72hr raffinose
33 99
agar (Kyokuto Pharmaceutical Co., Ltd., Japan) supplemented with
34 Fig. 1. Saccharide assimilation tests. (A) Saccharide assimilation test of C.albicans. X 100
5% defibrinated sheep blood (Nihon Bio-test Inc., Japan). Sterile axis shows incubation time and Y axis shows turbidity. (B) Saccharide assimilation
35 101
paper discs (ADVANTECs, Toyo Roshi Kaisya, Ltd., Japan) were test of S. mutans X axis shows incubation time and Y axis shows turbidity.
36 102
placed on the surface of the agar plates and 50 μL of NaOH-neu-
37 103
tralized lactobacilli-CS was dropped on the discs. The diameter of grow when incubated with three saccharides i.e. xylose, xylitol,
38 104
the growth inhibitory zone (halo) was measured after cultivation and arabinose (Fig. 1B).
39 105
under anaerobic conditions for 5 days.
40 106
41 3.2. Growth inhibitory effect of lactobacilli on C. albicans 107
42 2.7. Determining the amount of insoluble glucan produced by S. 108
43 mutans using phenol-sulfate staining Data from the co-culture assay of the yeast form of C. albicans 109
44 showed that more than half of all lactobacilli strains (29 out of 40 110
45 A 1  10 CFU/μL suspension of S.mutans was added to a mix-
6
strains) had an inhibitory effect on the growth of C. albicans 111
46 ture of 100 μL of Tryptic soy broth (TSB) supplemented with 4% 112
(Fig. 2A). The results from the turbidity assay when lactobacillus-
47 sucrose and 100 μL of each lactobacilli-CS in 96 well plates. The 113
CS was used were confirmed by the CFU assay (Fig. 2B). Notably,
48 plates were then incubated under aerobic conditions at 37 °C for 114
eight lactobacilli strains: 102, 103, 105, 108, 109, 112, 120, and 122
49 72 h. A total of 25 μL of each sample was stirred with 25 μL of 5% 115
had a significant inhibitory effect on the growth of C. albicans.
50 phenol for 30 s in each well of the 96 well plates. The plates were 116
Hence, these eight lactobacilli strains were considered as the first
51 placed on ice and then 125 μL of concentrated sulfuric acid was 117
candidates for use as probiotics. Further, data from the turbidity
52 added to each well and stirred for 30 s. This was followed by 118
assay showed that five strains were found to exhibit an inhibitory
53 incubation at 80 °C for 30 min and the absorbance was then 119
effect on the formation of biofilm by C. albicans (Fig. 2C).
54 determined at 620 nm (Fox et al. [14]). 120
55 121
56 3.3. The growth inhibitory effect of lactobacilli-CS on P. gingivalis by 122
57 3. Results disc diffusion assay 123
58 124
59 3.1. Saccharide assimilation tests of lactobacilli, C.albicans, and S. We determined the inhibitory effect of the C. albicans inhibitory 125
60 mutans lactobacilli on P. gingivalis. P. gingivalis is very susceptible to low 126
61 pH therefore, lactobacilli-CS was neutralized before use in the disc 127
62 The data from the saccharide assimilation tests for all lacto- diffusion assay. 128
63 bacilli strains are shown in Table 2. All lactobacilli exhibited Fig. 3 shows that the CS of seven lactobacilli strains: 102, 103, 129
64 growth with all 12 saccharides shown on Table 1. 108,109, 112, 120, and 122 produced a larger growth inhibitory 130
65 Fig. 1A shows that C. albicans did not grow when incubated area for P. gingivalis around the discs when compared to the 131
66 with eight of the 12 saccharides (Fig. 1A), and S. mutans did not negative control (uncultured medium). 132

Please cite this article as: Kojima Y, et al. Combining prebiotics and probiotics to develop novel synbiotics that suppress oral pathogens.
J Oral Biosci (2015), http://dx.doi.org/10.1016/j.job.2015.08.004i
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1 67
Increase of turbidity
2 68
0.8
3 69
4 0.7 70
5 0.6
71
6 72
7 0.5 73
8 0.4 74
9 75
0.3
10 76
11 0.2 77
12 78
0.1
13 79
14 0 80

CTL
13S

17S

19S
13L

17L

19L

101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
11

14

20
23
24
1
2
3
4
5
6
7

15 81
-0.1
16 82
17 83
18 log CFU 84
19 8.0 85
20 7.0 86
21 87
22 6.0 88
23 5.0 89
24 90
25 4.0 91
26 3.0 92
27 93
28 2.0 94
29 1.0 95
30 96
0.0
31 97

CTL
13S

17S

19S
13L

17L

19L

101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
11

14

20
23
24
1
2
3
4
5
6
7

32 98
33 99
34 Increase of turbidity 100
0.9
35 101
36 0.8 102
37 103
38 0.7 104
39 105
0.6
40 106
41 0.5 107
42 108
43 0.4 109
44 110
0.3
45 111
46 0.2 112
47 113
0.1
48 114
49 0
115
50 103 108 112 120 122 CTL 116
51 117
Fig. 2. Growth inhibitory effect of lactobacillus on C. albicans. (A) The growth inhibitory effect indicated by co-culture test. X axis shows no. of LAB strains and Y axis shows
52 increase in turbidity of MRS by measuring OD at 620 nm. Kruskal–Wallis p o 0.01. (B) The growth inhibitory effect evaluated by the CFU assay. X axis shows no. of lacto-
118
53 bacillus strains and Y axis shows log CFU. Kruskal–Wallis p o 0.001. (C) Inhibitory effect of lactobacillus-CS on C. albicans biofilm formation. X axis shows no. of LAB strains 119
54 and Y axis shows increase in turbidity of MRS by measuring OD at 620 nm. Kruskal–Wallis p o 0.001. 120
55 121
56 122
57 123
58 3.4. The inhibitory effect of lactobacilli-CS on insoluble glucan pro- 4. Discussion 124
59 duction by S. mutans using phenol-sulfate staining 125
60 The concept of prebiotics and that synbiotics were a combi- 126
61 Phenol-sulfate staining data measuring the amount of insoluble nation of pre and probiotics was first introduced by Gibson et al. in 127
62 glucan produced by S. mutans showed that (Fig. 4), five strains: 1995. Since then probiotics have been the focus of various clinical 128
63 103, 108, 112, 120, and 122 out of six lactobacilli strains possess studies [9–12]. However, most of these studies focus on the 129
64 inhibitory effects. These five lactobacilli strains were determined functions of probiotics either in maintaining overall health by 130
65 to be the candidates to be used as probiotics. restoring the normal functioning of the immune system and 131
66 132

Please cite this article as: Kojima Y, et al. Combining prebiotics and probiotics to develop novel synbiotics that suppress oral pathogens.
J Oral Biosci (2015), http://dx.doi.org/10.1016/j.job.2015.08.004i
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1 mm Diameter of inhibitory area effects. In contrast to the data shown in the API 50CHL system: D- 67
2 13 xylitol is not assimilated by L. paracasei and only 2% is assimilated 68
3 by L. plantarum and arabinose is not assimilated by L. fementum, 69
12
4 the strains we used in the present study could assimilate these 70
5 11 saccharides. 71
6 To identify probiotic candidates, we evaluated the growth 72
7 10 inhibitory effects of C. albicans, P. gingivalis, and S. mutans by either 73
8 using lactobacilli cells or their culture supernatant. When cultured 74
9
9 together, eight out of the 40 lactobacilli strains demonstrated an 75
10 8 inhibitory effect on C. albicans (Fig. 2A and B). Further, we deter- 76
11 mined the growth inhibitory effect of the lactobacilli-CS of these 77
12 7 eight candidate strains on P. gingivalis (Fig. 3). Lactobacilli-CS was 78
13 102 103 105 108 109 112 120 122 CTL neutralized prior to the test as P. gingivalis is very sensitive and 79
14 Fig. 3. Growth inhibitory effect of lactobacilli-CS on P. gingivalis using the disc difficult to grow under low pH conditions [15]. For practical 80
15 diffusion method. X axis shows no. of lactobacilli strains and Y axis shows diameter applications of lactobacilli however, they should be able exhibit 81
16 of inhibitory area. these inhibitory effects in neutral pH or there would be an 82
17 increased risk of dental caries. Thereafter, the inhibitory effects of 83
18 OD620nm lactobacilli were evaluated on biofilm formation by C. albicans and 84
2
19 S. mutans (Fig. 4 or 2C). S. mutans biofilms are encased in a matrix 85
20 1.8 of insoluble glucan, which entrap acids that contribute to the 86
21 1.6 pathogenesis of dental caries. On the other hand, C. albicans grows 87
22 1.4 in two major forms i.e. yeast or hypha. This organism usually 88
23 1.2 grows in the yeast form in culture media rich in saccharine like 89
24 1 TSBD however, in media that are in rich animal proteins it grows in 90
25 0.8 the hyphal form. Hyphal formation is a major virulence attribute of 91
26 0.6
C. albicans [16,17]. Candida biofilm is generally comprised of both 92
27 yeast and hyphal forms. The data on the inhibitory effects on 93
0.4
28 biofilm formation shows that the best candidates for probiotics are 94
0.2
29 five lactobacilli strains: 103, 108, 112, 120, and 122. 95
0
30 Data derived from the present study suggests that different 96
102 103 108 112 120 122 CTL
31 conditions are required by synbiotics for intestinal microbiota and 97
32 Fig. 4. Inhibitory effect of lactobacilli-CS on insoluble glucan production by S. oral microbiota. Oligosaccharides, especially oligofructose and 98
mutans using phenol-sulfate staining. X axis shows no. of LAB strains and Y axis
33 inulin have been studied as prebiotics for intestinal microbiota by 99
shows turbidity by OD620 nm. Kruskal–Wallis p o 0.05.
34 many researchers [6,7]. Notably, these oligosaccharides undergo 100
35 digestion in the intestines and convert to mono- or small oligo- 101
36 improving the specta of human intestinal microbiota or as adju- saccharides that can be utilized by probiotic microorganisms. 102
37 vants in the treatment for Crohn's disease, food allergies, immu- Therefore, both prebiotics and probiotics fulfill the criteria of 103
38 nological disease, and other intestinal inflammatory conditions. synbiotics in the intestine [8]. On the other hand, it is difficult to 104
39 Recently, a number of studies have focused on the growth inhi- find prebiotics with similar properties because there is very little 105
40 bitory effects probiotics have on oral pathogenic organisms par- time for digestion of food to happen in the oral cavity. The target of 106
41 ticularly, S. mutans, P. gingivalis, and C. albicans. These studies have the present study was to identify synbiotics for the oral micro- 107
42 identified preventive and therapeutic roles of probiotics in oral biota. Therefore, mono-saccharides or disaccharides were con- 108
43 diseases such as dental caries, periodontal disease, and candidiasis sidered as alternative prebiotics candidates. We expected that 109
44 [15,19,20–22,31]. However, none of the studies have reported a prebiotics would inhibit the growth of pathogenic oral micro- 110
45 single probiotic strain with a growth inhibitory effect against all organisms or would not act as a nutritional source for them. The 111
46 the three oral pathogens mentioned above. Further, comparative candidate saccharides identified in the present study promoted 112
47 and synergistic effects of prebiotics and probiotics have not been the growth of probiotic bacteria and also inhibited the growth of 113
48 explored thus far. oral pathogenic microorganisms. 114
49 In order to find candidates that can be used as prebiotics, we Five lactobacilli strains isolated from the human oral cavity 115
50 tested 12 potential saccharides that were identified from previous were identified as suitable probiotic candidates. Lactobacilli strains 116
51 studies and from literature on food science [28–30]. As shown by isolated from dairy products did not have a strong effect on oral 117
52 sugar assimilation tests (Fig. 1), the candidates with the highest microbiota although they may carry health benefits for intestinal 118
53 potential to function as prebiotics were arabinose, xylose, and microbiota. Taken together, these results identified a panel of 119
54 xylitol. These three saccharides functioned as prebiotics by sup- candidates suitable for development as oral synbiotics in the 120
55 porting the growth of lactobacilli. In addition, they did not support future. These synbiotics are expected to provide the much-needed 121
56 the growth of known pathogenic microorganisms like S. mutans oral health benefits to the community. 122
57 and C. albicans. These results support data from previous studies Live probiotic microorganisms have been used for human 123
58 showing that xylitol and arabinose do not support the growth of health benefits for a long time. However, recent reports have 124
59 these pathogenic microorganisms [25–27,32]. A number of studies suggested that live bacteria may not be as essential for health since 125
60 have investigated the relationship between xylitol and oral dead cells or cell components may be able to provide similar 126
61 pathogenic organisms including the inhibitory effects of xylitol on health-promoting benefits [18]. In the present study, we demon- 127
62 the growth of S. mutans and C. albicans despite the presence of strated that not only live probiotic bacteria, but also neutralized 128
63 glucose [26,33]. Since the other two saccharides: arabinose and lactobacilli-CS could promote the health of the oral cavity by 129
64 xylose (members of the class aldopentose or its reduced deriva- inhibiting pathogenic oral microorganisms. Further studies are 130
65 tives) are similar to xylitol in their molecular structure, therefore, warranted to determine the optimal combination of saccharides 131
66 it is possible that they may be able to exhibit similar inhibitory and lactobacilli to develop synbiotics that can be employed in 132

Please cite this article as: Kojima Y, et al. Combining prebiotics and probiotics to develop novel synbiotics that suppress oral pathogens.
J Oral Biosci (2015), http://dx.doi.org/10.1016/j.job.2015.08.004i
 6 Y. Kojima et al. / Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Please cite this article as: Kojima Y, et al. Combining prebiotics and probiotics to develop novel synbiotics that suppress oral pathogens.
J Oral Biosci (2015), http://dx.doi.org/10.1016/j.job.2015.08.004i