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Keywords:

  • aquaporin8;
  • Bifidobacteria;
  • Escherichia coli ;
  • intestinal microbiota;
  • galacto-oligosaccharide;
  • prebiotics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Although studies have reported numerous effects of coffee on human health, few studies have examined its specific effects on gut microbiota. This study aimed to clarify the influence of coffee and galacto-oligosaccharide (GOS) consumption on gut microbiota and host responses. After mice consumed coffee and GOS, their intestines were sampled, and the bacterial counts were measured with quantitative RT-PCR. Results showed that GOS consumption significantly increased total bacteria counts in the proximal colon. Although Escherichia coli and Clostridium spp. counts significantly decreased in the proximal colon, Bifidobacterium spp. counts increased remarkably in the same area. A bacterial growth inhibition assay was also conducted, and the results showed that E. coli growth was inhibited only by a coffee agar. Host responses were also investigated, revealing that coffee and GOS consumption remarkably increased aquaporin8 expression in the proximal colon. In conclusion, coffee has antibiotic effects, and GOS significantly decreased E. coli and Clostridium spp. counts, but increased Bifidobacterium spp. counts remarkably. Aquaporin8 expression was also increased with a mixture of coffee and GOS consumption. This is the first study to demonstrate that coffee consumption can regulate gut microbiota and increase aquaporin8, both of which are necessary for maintaining intestinal balance.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Coffee is one of the most popular beverages worldwide (Imatoh et al., 2011) and is prepared by roasting the seeds of the coffee plant. Coffee consumption has several effects on human health (Butt & Sultan, 2011). For example, coffee consumption appears to decrease the risk of liver cancer (Larsson & Wolk, 2007). Moreover, people who consumed five cups of coffee per day decreased their emerging risk of liver cancer by 25% compared with people who did not consume coffee (Inoue et al., 2005). Coffee consumption is also associated with a significant reduction in the risk of fibrosis among steatohepatitis patients (Vitaglione et al., 2010; Molloy et al., 2012). In a large prospective study of participants with advanced hepatitis C-related liver disease, regular coffee consumption was associated with lower rates of disease progression (Freedman et al., 2009). The effects of coffee on diabetes were also studied, and coffee consumption was associated with a substantially lower risk of clinical type 2 diabetes (Van-Dam & Freskens, 2002). In addition, coffee drinkers have a lower risk of Parkinson's disease (Herman et al., 2002) and constipation (Murakami et al., 2006). Such findings have clarified the relationship between human health and coffee.

Nevertheless, only a few reports on the relationship between coffee and bacteria exist (Chaves et al., 2012). Jaquet et al. (2009) reported that coffee promoted the growth of some species of bacteria in faeces, suggesting that the probiotic bacteria that prefer oligosaccharides will propagate in the presence of the coffee oligosaccharide. In contrast, Gniechwitz et al. (2007) reported that faecal bacteria had not changed after coffee consumption compared with the faecal bacteria in the control group. Thus, studies have been unable to reach a consensus on the influence of coffee consumption on faecal microbiota. Moreover, there exist no reports on its influence on gut microbiota. Therefore, the present study investigates these issues.

The present study used galacto-oligosaccharide (GOS), a commercial product (Yakult, Tokyo, Japan), for its prebiotic testing (Kobayashi et al., 2009). Consuming GOS may relieve the symptoms of constipation (Nittynen et al., 2007). In a mouse model, GOS reduced intestinal colitis by modulating the function and trafficking of natural killer (NK) cells (Gopalakrishnan et al., 2012). Thus, GOS should help to improve intestinal balance.

Intestinal responses play an important role in host defences. The pro-inflammatory cytokines of tumour necrosis factor alpha (TNF-α) (Francés et al., 2007) and interleukin-1beta (IL-1β) (Al-Sadi & Ma, 2007) and the antimicrobial peptides of cramp (Gallo et al., 1997) are important indicators of inflamed gut epithelial cells. Increasing nitric oxidase, which is related to the inducible nitric oxide synthase (iNOS) gene, helps to induce bacterial translocation (Petors et al., 1994). Aquaporin8 is a water channel gene, and decreasing its expression seems to alleviate inflammatory bowel disease (Zahn et al., 2007) and diarrhoea (Yamamoto et al., 2007). It is still unknown, however, how coffee consumption influences these host responses. If researchers establish that coffee functions as prebiotics do, then doctors could prescribe coffee in lieu of harsher drugs, because coffee consumption could improve intestinal balance.

The aim of this study is to clarify the effects of coffee consumption on gut microbiota as well as to investigate host responses to pro-inflammatory cytokines, cramp, iNOS and aquaporin8.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains

Escherichia coli RIMD 0509763 and Enterococcus faecalis RIMD 3116001 were obtained from the Research Institute of Microbial Disease at Osaka University. The Luria–Bertani (LB) agar (Becton Dickinson, Franklin Lakes, NJ) was used for bacteria growth. These bacteria used for growth inhibition assay.

Coffee and GOS preparation

Coffea arabica (Mandelin super grade) beans were purchased from Indonesia, and were adjusted to roast degree by a roasting specialist (Tridea CO., Ltd., Ibaraki, Japan). In brief, the beans were roasted at 245 °C for 25 min. The roasted bean was powdered, and, with Adobe photoshop, the L value like was determined based on the luminosity of the coffee powder (0 black < white 255). The roasting degree was 17.3–18.5. The roasted coffee powder was kept at −30 °C before use. Over each gram of coffee powder 14 mL of boiling water was poured, and the extracted coffee was incubated at room temperature for 15 min. After incubation, the coffee was centrifuged at 3000 g for 10 min, and the supernatant was put through a 0.45-μm filter. The filtrated coffee was kept at –30 °C until use. The GOS was a commercial product of Oligomate (Yakult, Tokyo, Japan) and was prepared with Kobayashi et al.'s (2009) method.

Measurement of oligosaccharides

To establish oligosaccharide concentration, the saccharides of both coffee and GOS were measured by PAL-J (Atago, Tokyo, Japan). This study applied the unit of Brix, which is a relative measure of the amount of sucrose in a solution. For example, 1% of Brix is equivalent to a water-based solution containing 1% sucrose by weight. A total 300-μL of coffee, GOS or water was added to the dish and measured. Student's t-test was conducted as a statistical test.

Bacterial growth inhibition assay

The prepared coffee was mixed with LB agar at three different concentrations (10%, 30% and 50% coffee) and poured into a coffee-agar plate for a total of 20 mL. After subculturing, the bacteria were suspended in phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO) and adjusted for an optical density of 600 nm (OD600 nm) at 0.1, 0.5 and 1.0.

The coffee's antibacterial activity against gram-negative E. coli and gram-positive E. faecalis was investigated. After bacteria subculturing, a total of 10 μL of bacterial broth was spread on the coffee agar, and the agar was kept at 37 °C for 24 h. After 24 h of incubation, the bacterial colony was determined by three types of morphology: ‘++’ means a completely grown colony, ‘+’ means that bacteria did not grow into a full colony (partial growth) and ‘−’ means that bacteria did not grow.

Animal experiment

Seven specific pathogen-free A/J mice at 8 weeks of age (SLC, Shizuoka, Japan) were acclimatized for 1 week to the standard laboratory conditions of free access to rodent diet CE-2 (Japan clea, Tokyo, Japan) and sterile water. To avoid fighting, the experimenters selected only female mice. Room temperature was kept at 23 °C. After 1 week, each of the seven mice consumed either coffee (500 μL day−1), GOS (2000 mg kg−1 day−1) or sterile water via a feeding needle (Fuchigami, Kyoto, Japan) at the same time of day for 3 consecutive days. Then, to ensure coffee was carried to every corner of the intestine, the mice were kept at 23 °C for 24 h, after which 1 cm (0.05 g) of intestine (proximal, medium and distal of the small intestine and proximal and distal of the colon) was sampled from each mouse. The sampled tissue was washed three times in PBS. The separated tissues were kept at –80 °C. The use of animals in this study was in accordance with the guidelines of the Animal Care Committee of Osaka University.

RNA extraction and real-time RT-PCR

RNA extraction and reverse transcriptase were basically performed as described previously (Nakayama et al., 2011). In brief, the sampled intestinal tissues were washed three times in PBS, and 500 μL of PBS was added to the tissues. The tissues were then cut with scissors and homogenized with a cell strainer (Becton Dickinson Bioscience, Franklin Lakes, NJ). For RNA extraction, the homogenized tissues were mixed with trizol reagent (Invitrogen, Carlsbad, CA), glass beads and chloroform. After centrifugation, the upper phase was treated with isopropanol and 70% ethanol in turn. The extracted RNA was put through reverse transcription, and the researchers performed cDNA using a Roche cDNA Synthesis kit (Roche, Basel, Switzerland). The real-time PCR was carried out (40 cycles of 94 °C for 20 s, 53 °C for 20 s, 72 °C for 30 s) using the 7900HT fast real-time PCR system (Applied Biosystems, Paisley, UK). Real-time PCR was conducted to demonstrate the difference in the expression of beta-actin, host responses and gut-specific bacteria (Table 1). Relative count was calculated as the ratio of the target gene expressed in the distal small intestine or proximal colon (target gene/beta-actin) in the mice that consumed coffee or GOS (target gene/beta-actin) to the target gene expressed in the mice that consumed neither (target gene/beta-actin). All data were determined to be significantly different according to Student's t-test.

Table 1. Primers in this study
NameTarget gene SequenceProduct size (bp)Reference
Bacteria universal16S rRNAForward5′-ATGGCTGTCGTCAGCT-3′337Ferris et al. (1996)
Reverse5′-ACGGGCGGTGTGTAC-3′
E. coli 16S–23S rRNAForward5′-CAATTTTCGTGTCCCCTTCG-3′421Khan et al. (2007)
Reverse5′-GTTAATGATAGTGTGTCGAAAC-3′
Enterococcus spp.16S rRNAForward5′-ATCAGAGGGGGATAACACT T-3′336In this study
Reverse5′-ACTCTCATCCTTGTTCTTCTC-3′
Bifidobacterium spp.16S rRNAForward5′-AGGGTTCGATTCTGGCTCAG-3′172In this study
Reverse5′-CATCCGGCATTACCACCC-3′
Lactobacillus spp.16S rRNAForward5′-TGGAAACAGATGCTAATACCG-3′233Kang et al. (2009)
Reverse5′-GTCCATTGTGGAAGATTCCC-3′
Bacteroides spp.16S rRNAForward5′-AGTAACACGTATCCAACCTG-3′243In this study
Reverse5′-GACCAATATTCCTCACTGCT-3′
Clostridium spp.16S rRNAForward5′-AAAGGAAGATTAATACCGCATAA-3′179In this study
Reverse5′-TGGACCGTGTCTCAGTTCC-3′
Mouse TNF-alphaTNF-alphaForward5′-CATCTTCTCAAAATTCGAGTGACAA-3′235Matsumoria et al. (2004)
Reverse5′-TGGGAGTAGACAAGGTACAACCC-3′
Mouse IL-1 betaIL-1betaForward5′-CAACCAACAAGTGATATTCTCCATG-3′152Li et al. (2009)
Reverse5′-GATCCACACTCTCCAGCTGCA-3′
Mouse iNOSiNOSForward5′-AATGGCAACATCAGGTCGGCCATCACT-3′454Greenberg et al. (1997)
Reverse5′-GCTGTGTGTCACAGAAGTCTCGAACTC-3′
Mouse aquaporin8Aquaporin8Forward5′-CAGCCTTTGCCATCGTCCAGG-3′311Butler et al. (2006)
Reverse5′-CCTAATGAGCAGTCCTAGAAAG-3′
Mouse beta-actinBeta-actinForward5′-GTCCCTCACCCTCCCAAAAG-3′266He et al. (2008)
Reverse5′-GCTGCCTCAACACCTCAACCC-3′

Preparation of standards for real-time PCR

The standard for real-time PCR was achieved by making a plasmid DNA containing the target sequences. The cDNA from the mice's intestinal tissues was amplified using the specific primer. The product of correct size and sequence was purified and ligated into a vector using the TOPO TA cloning kit (Invitrogen). Competent E. coli DH5alpha were transformed with each ligated vector and, after overnight incubation, positive colonies were chosen. From each selected colony, the plasmid was purified using Miniprep (Qiagen, Hilden, Germany). Samples were diluted to 108 molecules mL−1, aliquoted and stored at −80 °C.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Influence over total bacteria

Eubacteria primer was used to quantify total bacteria. Total bacteria counts in the proximal colon significantly increased after GOS consumption compared with the counts in the mice that consumed coffee or water (Fig. 1). Total bacteria counts in the proximal and distal small intestine significantly decreased after coffee consumption compared with the counts in the mice that consumed GOS or water.

image

Figure 1. Influence of coffee, GOS or water consumption on total bacteria count in mice intestinal tracts. t-Test was conducted as a statistical analysis.

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Influence of gut microbiota

In both the distal small intestine and proximal colon, E. coli counts significantly decreased after coffee and GOS consumption compared with the counts in the control group (Fig. 2a). In the proximal colon, coffee and GOS consumption also decreased Enterococcus spp. counts compared with those in the control group (Fig. 2b). Finally, Bacteroides spp. and Clostridium spp. counts decreased in the proximal colon after coffee and GOS consumption compared with those in the control group (Fig. 2c and d). In contrast, Bifidobacterium spp. counts significantly increased after coffee and GOS consumption compared with those in the control group (Fig. 2e). Lactobacillus spp. counts also increased in the proximal colon after coffee and GOS consumption compared with those in the control group (Fig. 2f).

image

Figure 2. The presence of (a) Escherichia coli, (b) Enterococcus spp., (c) Bacteroidetes spp., (d) Clostridium spp., (e) Bifidobacterium spp. and (f) Lactobacillus spp. due to the presence of coffee or GOS in the proximal colon and distal small intestine. Mice consumed 500 μL of coffee, GOS or water per day for 3 days. The small intestinal tract of every mouse was sampled, and the RNA was extracted. The RNA was converted into cDNA, and then real-time PCR was conducted. t-Test was conducted as a statistical analysis: *P < 0.05.

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Bacterial growth inhibition

A bacterial growth inhibition assay was conducted on the coffee agar. Escherichia coli counts were inhibited by the agars with 30% and 50% coffee. Enterococcus faecalis counts were partially inhibited by the agar with 50% coffee. Neither E. coli nor E. faecalis counts were inhibited by the agar with GOS (Table 2).

Table 2. Effect of LB agar with coffee or GOS on bacterial growth inhibition
  E. faecalis E. coli
Optical density at 600 nmOptical density at 600 nm
0.10.510.10.51
  1. ++, Growth; +, partial inhibition; −, inhibition.

Percentage of coffee (%)
10++++++++++++
30++++++++++
50+++
Percentage of GOS (%)
10++++++++++++
30++++++++++++
50++++++++++++

Measurement of saccharide concentrations

Saccharide concentrations were measured with PAL-J. Results showed that GOS and coffee contained 8.83% and 2.0% Brix, respectively. The control mice consumed distilled water, which was found to contain 0.15% Brix (Fig. 3).

image

Figure 3. Concentration of oligosaccharides in coffee, GOS and water. t-Test was conducted as a statistical analysis.

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Host responses to cytokine, cramp, iNOS and aquaporin8

The cytokine expression of TNF-α and IL-1β did not differ from those of the control group after coffee or GOS consumption (Fig. 4a and b). The cramp expression in the proximal colon of the mice that consumed coffee was higher than that of the control mice and the mice that consumed GOS (Fig. 4c). The aquaporin8 expression of the mice that consumed coffee or GOS was significantly higher than that of the mice that consumed neither (Fig. 4d).

image

Figure 4. The presence of (a) IL-1β, (b) TNF-α, (c) cramp, (d) aquaporin8 and (e) iNOS due to the presence of coffee or GOS in the proximal colon and distal small intestine. Mice consumed 500 μL of coffee, GOS or water per day for 3 days. The small intestinal tract of every mouse was sampled, and the RNA was extracted. The RNA was converted into cDNA, and then real-time PCR was conducted. t-Test was conducted as a statistical analysis: *< 0.05, **< 0.01.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In response to the lack of reports on the effects of coffee consumption on gut microbiota, this study has begun to clarify these effects by comparing them with the effects of consuming GOS or water.

The total bacteria counts of mice that consumed coffee, GOS or water were investigated. The total bacteria in the intestinal tract exhibited the highest counts in the proximal colon. GOS consumption resulted in the highest total bacterial counts compared with coffee and water consumption; therefore, GOS seems to enhance bacterial growth, especially the growth of beneficial bacteria.

Specifically, the study measured the presence of E. coli, Enterococcus spp., Bifidobacterium spp., Lactobacillus spp. and the anaerobes of Bacteroides spp. and Clostridium spp. in the aforementioned mice. Coffee consumption induced E. coli and Clostridium spp. counts to decrease significantly; the Enterococcus spp. counts showed only a slight decrease. The results of the antibacterial activity assay showed that coffee inhibited the growth of E. coli and E. faecalis. Therefore, coffee may possess an antibacterial quality. Although previous studies have reported the antimicrobial activity of coffee (Daglia et al., 2007; Rufian-Henares & Cueva, 2009), the present study was the first use an in vivo model to demonstrate that coffee consumption decreases E. coli and Clostridium spp. In contrast, the presence of coffee or GOS increased Bifidobacterium spp. and Lactobacillus spp. in mouse gut. Oligosaccharide supports beneficial bacteria (Knol et al., 2005), so the oligosaccharides in coffee and GOS stimulated Bifidobacterium spp. and Lactobacillus spp., which, in turn, converted the oligosaccharides into lactic acid for acquisition of ATP. The oligosaccharides in GOS increased the counts of Bifidobacterium spp. and Lactobacillus spp. four times more than did the oligosaccharides in coffee.

Although GOS consumption caused E. coli, Enterococcus spp. and Clostridium spp. counts to decrease in the mice's colons, GOS did not have any antibacterial effect on E. coli or E. faecalis during the in vitro assay, which suggests that the other components of the gut environment helped to increase the beneficial bacteria that consumed GOS by altering the acidity of the environment. These beneficial bacteria then reduced E. coli, Enterococcus spp. and Clostridium spp. The counts for Bifidobacterium spp. and Lactobacillus spp. also increased in the mice that consumed coffee, suggesting that coffee acidifies the gut, unlike GOS, in addition to being cytotoxic against E. coli. Moreover, the mice that consumed coffee had the same number of total bacteria in their gut as the mice that consumed water although these two groups differed greatly in the composition of gut microbiota.

Host responses were also investigated in this study. Coffee contains several putative stimuli, e.g. caffeine, polyphenol and melanoidin (Daglia et al., 2007). These complex stimuli could potentially stimulate gut epithelial cells, so the researchers measured the pre-inflammatory cytokine of IL-1β and TNF-α to detect possible damage to the host. Results showed that the cytokines among the mice consuming coffee, GOS or water did not differ significantly. Thus, this study demonstrated that coffee and GOS may not be harmful to the host. Antimicrobial peptides of cramp were also investigated. There was a significant difference in cramp expression only between the coffee and GOS groups. Although these results showed no difference between coffee and water, there is a possibility that coffee could induce the anti-peptides of cramp.

Aquaporin8 is a water channel protein and an important facilitator of water transport. Some studies have suggested that diarrhoea and ulcerative colitis in humans might be closely related to a down-regulated expression of aquaporin8 (Yamamoto et al., 2007; Zahn et al., 2007). In this study, the mice that consumed coffee or GOS exhibited a much higher expression of the aquaporin8 than did the mice in the control group. This finding does not clarify the mechanism of coffee or GOS in inducing aquaporin8, but it is an interesting result that suggests that oligosaccharide might be closely related to aquaporin8 expression. Nitric oxide (NO) is produced at high levels in the immune system. It is derived from iNOS and has host-protective effects (Bogdam, 2001). This study revealed no significant difference in iNOS expression between GOS and coffee, suggesting that the intestine did not generate NO while processing either GOS or coffee.

In 2011, two of the worst E. coli poisoning outbreaks recorded in Japan and Germany occurred. Furthermore, the number of reports of persons infected with Streptococcus suis in Southeast Asia has been increasing recently. Some researchers suspect that one of the main causes of these S. suis infections could be a local traditional custom of consuming raw pork meat and fermented raw pork (Nakayama et al., 2011; Takeuchi et al., 2012).

We hope that the prebiotic function and antibacterial activity of coffee can reduce these outbreaks if used properly. The next step is to investigate whether coffee consumption eliminates such pathogenic bacteria.

In conclusion, the mice that consumed coffee had far less E. coli and Clostridium spp. and far more Bifidobacterium spp. in their colons than did the mice in the control group. Moreover, aquaporin8 and cramp expression was higher after coffee consumption. The mice that consumed GOS experienced a similar increase in beneficial bacteria. This is the first study to demonstrate that coffee consumption can regulate gut microbiota and maintain intestinal balance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by research grants from Japan Coffee Association. The authors would also like to thank Mr Nakano from Tridea Co., Ltd for his valuable comments and support.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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