The Effect of Potato Polyphenols in Combination with Cellulose or Inulin in a Mixed Culture of Swine Fecal Bacteria

*Corresponding author: Michihiro Fukushima, Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 0808555, Japan. Tel: 0155 49 5557/ Fax: 0155 49 5577; E-mail: fukushim@obihiro.ac.jp 1Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan 2Upland Farming Resource Research Division, NARO Hokkaido Agricultural Research Center, Minami 9-4, Shinsei, Memuro, Kasai, 082-0071 Hokkaido, Japan


Introduction
Dietary fibers are edible plant components or carbohydrates that are not digested and absorbed in the small intestine but fermented in the large intestine by colonic microbiota [1][2][3] . Epidemiological studies have shown a positive correlation between high intake of dietary fiber and health benefits. Studies have indicated that dietary fibers are instrumental in regulating body weight, immune function and contribute to diabetes control [2,4,5] . Likewise, lowering of depressive symptoms may be attributed to higher dietary fiber intake [6] . Furthermore, there is an association between adequate intake of dietary fiber and prevention of non-communicable diseases such as colon, rectal and breast cancers including coronary heart diseases [7][8][9] .
Inulin is a soluble dietary fiber found +in various plants [10] and proven to be positively affecting the microbiota composition. Microbial response to various dietary fibers varies to some degrees and this determines the type and amount of products produced at the end of the fermentation process. With the existence of ß-(2-1)-d-fructosyl fructose bonds between the fructose units along with ß-configuration, the digestibility of inulin is impossible in the human small intestine [11] . Although indigestible in the small intestine, almost 90 % escaped and being digested in the colon [12] . Studies confirmed and widely accepted that inulin is regarded as a prebiotic compound due to its prebiotic efficacy [13] . According to studies by Pompei et al [14] and van de Wiele et al [15] , inulin selectively increases the population of bifidobacteria Copyright: © 2018 Fukushima, M. This is an Open access article distributed under the terms of Creative Commons Attribution 4.0 International License.

Statistical analysis
All data are presented as a mean and Standard Error (SE). The in vitro fermentation was conducted in a block design with replicates of five. Two-way ANOVA was performed to assess the effect of fiber, extract, and their combination. Differences of p < 0.05 and p < 0.1 were taken to be statistically significant and tended to be significant respectively. If the variance was observed in the main effect of the group, Tukey's test was used for this comparison (P < 0.05). [ Table 1] summarizes the bacteria populations in the fermenters during in vitro colonic fermentation. The populations of bifidobacteria and anaerobic bacteria in the fermenters were significantly (p < 0.05) higher for INU and IP groups compared with CEL and Short title: Potato Polyphenols in Combination with Cellulose or Inulin CP groups respectively, although CP was significantly (p < 0.05) higher than CEL. The interaction between INU and potato polyphenols were significant (p < 0.05) for bifidobacteria at 24 and 48h. While coliform bacteria was significantly higher (p < 0.05) for CEL and CP groups, IP group was significantly (p < 0.05) lower at 24 and 48h, even lower than INU group. The populations for Lactic acid bacteria and Lactobacillus were higher for IP and INU groups compared to CP and CEL groups. There were no significant differences in the population of Cl. perfringens amongst the groups although survival can be seen only for CEL group. Each value represents the mean and standard error (n = 5). Two-way ANOVA was performed to assess the effect of fiber (CEL and INU), Potato polyphenols and their combination. Differences of p < 0.05 and p < 0.1 were taken to be statistically significant and tended to be significant respectively. Mean values (a -c) with unlike letters within the same point are significantly different (p < 0.05), as determined by ANOVA with Tukey's test.

SCFA concentration in fermenters
[ Table 2] shows the cumulative concentrations of SCFA in the fermenters during in vitro colonic fermentation. The acetic acid concentration for IP group was significantly higher (p < 0.05) than the rest of the groups at any given time points. On the other hand, CEL group was significantly (p < 0.05) lower compared with IP, INU and CP groups. It can also be noted that acetate concentration for CP group was significantly higher (p < 0.05) at 6h compared to INU group, although the concentration was significantly higher for INU group at 24 and 48h time points. The interaction between INU and potato polyphenols were significant (p < 0.05) for acetate at 12 and 24h. Propionate concentrations were significantly higher (p < 0.05) for IP group than the rest of the groups. There were no significant differences amongst other groups at 12 and 24h, although propionate concentration for INU group was significantly higher at 48h compared with CP and CEL groups. Further, the interaction between INU and potato polyphenols were significant (p < 0.05) for propionate at 12, 24 and 48h. Butyrate concentration was significantly higher (p < 0.05) at 6 and 12h time points for IP group than the rest of the groups. At 24h, there were no significant differences in the butyrate concentrations for IP and INU groups respectively. Interestingly, at 48h, the butyrate concentration for IP group was significantly (p < 0.05) lower than INU group. The interaction between INU and potato polyphenols were significant (p < 0.05) for butyrate at 48h. Likewise, significant difference (p < 0.05) for the interaction between INU and potato polyphenols can be noticed for total SCFA at 12 and 24h. The production of total SCFA were significantly higher in order; IP > INU > CP > CEL.

pH in fermenters
[ Figure 1] shows the pH value at different time points for each sample treatments during the fermentation. The interaction between INU and potato polyphenols were significant (p < 0.05) for pH at 6h. The pH value for INU and IP groups were significantly (p < 0.05) lower compared with CEL and CP groups, although CP group was significantly (p < 0.05) lower than CEL group at 6 and 12h time points respectively. The pH values for INU and IP groups were maintained below 5.6. It can be noted at 6h that pH values make a sudden drop for all groups.

Ammonia concentrations in the fermenters
[ Figure. 2] shows the ammonia concentrations of different samples treatments during in vitro colonic fermentation. The interaction between INU and potato polyphenols were significant (p < 0.05) for ammonia at 12, 24 and 48h. The ammonia concentration for IP was significantly (p < 0.05) lower than the rest of the groups at 12 and 24h time points. Although no significant difference between CEL and CP groups at 12 and 24h, at 48h, the ammonia concentration for CP group increases significantly (p < 0.05). While there was no significant difference between IP and INU groups at 48h, there was a slight decrease in ammonia concentration for IP group.

Discussions
The effects of potato polyphenols in combination with cellulose or inulin on colonic fermentation in a mixed culture of swine fecal bacteria were investigated in the current study. The total polyphenol content of potato extract was 6.5%, which is polyphenol-rich. A study by Han et al [25] , reported the findings to be quite similar to the current result.
Studies have indicated that dietary fibers can influence or modulate the bacterial community structures of the gut [28] . Further, Russell et al [29] . Highlighted that diet is the principal factor shaping the gut bacterial composition and functionality. Consequently, Desai et al [30] . and Sonnenburg et al [31] . stated that beneficial health outcomes can be attributed to dietary fibers. Although dietary fibers may vary in structure and composition, selective consumption will determine which microbial groups are favored in the gut [32,33] . For example, inulin is a soluble dietary fiber and regarded as prebiotic [10] . Almost 90% escaped digestion and fermented in the large intestine by the colonic microbiome [34] . It was reported that upon fermentation, inulin stimulates the development of beneficial bacteria such as bifidobacteria and lactobacillus while inhibiting the pathogenic ones [35,36] . Likewise, polyphenols also stimulates beneficial bacteria by reshaping the gut microbial community and enhance beneficial effects such as weight loss [37] . Further, Parker et al [38] . reported that polyphenols can stimulate the proliferation of Short title: Potato Polyphenols in Combination with Cellulose or Inulin bifidobacteria and production of SCFA in vitro and Vendrame et al [39]. showed that fiber and polyphenols can increase bifidobacteria species.
Furthermore, a human intervention studies by Tuohy et al [40] . Reported that consuming dietary fiber and polyphenol-rich chocolate will simultaneously up regulate commensal bacteria and prevent degenerative diseases. The current study showed similar results with the population of bifidobacteria and anaerobic bacteria being higher for IP group than any other groups. When combining inulin and potato polyphenols, the populations of beneficial bacteria were increased. It is worth noticing that the interaction between INU and potato polyphenols were significant (p < 0.05) for bifidobacteria at 24 and 48h. This indicates that an increase in population was attributed to the interactions of INU and potato polyphenols. This was also supported by Parker et al [38] . In addition, Zenon et al. [24] stated that combining polyphenols and inulin increase the population of beneficial bacteria. Further, Vendrame et al. [39] showed that fiber and polyphenols can increase bifidobacteria species. Interestingly, the population of coliform bacteria was significantly (p < 0.05) lower for IP group. One possibility could be the antimicrobial effect of potato polyphenols, which needs to be further elucidated. The other possibility could be the antimicrobial effect of other species of bacteria against Coliforms. This could be the case in the current study because Francesco et al. [41] reported that Lactobacillus strains possess antimicrobial effect against gas-forming coliforms. In addition, Richard et al. [42] reported a synbiotic combination of resistant starch and bifidobacterium lactis lowered the total coli form counts. Healthy cecal environment can also be indicated by the increase of lactic acid bacteria and Lactobacillus populations. Our current result also confirmed this. In light of polyphenol consumption and CL.perfringens suppression, it was speculated previously that polyphenol consumption reduces CL.perfringens. CL.perfringens is a pathogen responsible for many gastrointestinal illnesses [43] . A study by Lopez-Legarrea et al. [44] reported that intake of polyphenol-rich fruits and vegetables increases the counts of beneficial bacteria and decreasing the population of CL.perfringens. Our current study also confirmed this. Although there was no significant difference amongst the groups, by 24h however, no CL.perfringens populations for INU and IP groups compared with CEL and CP groups.
Colonic microbiota can ferment either resistant starch or dietary fiber to SCFA at different rates and amounts, depending upon their origin [45] . An epidemiological study have also shown that SCFA (Acetate, Propionate and Butyrate) formation during colonic fermentation has been proven to exert beneficial health properties to the host [46] . Acetate, being one of the most common and abundant SCFA in human colon, metabolized in systematic areas like muscles and protect epithelial cells against pathogenic bacteria like E.coli [47][48][49] . According to Scheppach [50], colonic production of acetate can also enhance the ileal motility and increases colonic blood flow. Our current study showed that production of acetate was significantly (p < 0.05) higher for IP amongst the group at any given time point. The interaction between INU and potato polyphenols were significant at 12 and 24h for acetate, indicating that acetate production was influenced by the interactions of both INU and potato polyphenols. Zenon et al [24] also reported an increase in acetic acid pool when combining grapefruit flavonoids and inulin. It is worth noticing that acetic acid was the major SCFA produce followed by propionate and butyric acid. A similar result was reported by Ding et al. [51] . It was reported elsewhere that propionate also exerts some beneficial properties for the host health [52][53][54][55] . In our current study, propionate production by IP group increases significantly (p < 0.05) compared to other groups. The current result correlates well with a study by Han et al [56] . on spent turmeric powder which shows increase propionic production during colonic fermentation. The current study indicated an interaction between INU and potato polyphenols were significant at 12, 24 and 48h for propionate, highlighting the fact that propionate production was influenced by the interaction of both INU and potato polyphenols. Zenon et al [24] also reported the combination of grapefruit flavonoids and inulin increase propionate production. Health promoting effects of butyrate have also been reported elsewhere [57][58][59][60] .Our current study showed increased production of butyrate during colonic fermentation except for CEL group. It is interesting to note that while CP increases butyrate production, it was suppressed by IP group, although significantly higher but lower (p < 0.05) than INU. There is a possibility that polyphenols in IP group might suppress butyrate production during colonic fermentation, although detail mechanisms need to be further elucidated. A similar finding by Zenon et al [24] also reported that combining Inulin and polyphenols significantly (p < 0.05) lowered the butyrate acid pool compared to Inulin alone. According to Negi et al [61] . Grapefruit peel extract has strong antimicrobial activity, which could possibly suppress enzymes responsible for butyrate production. In another study, Tebib et al [62] reported that grape seed tannins decreased the activity of certain bacterial enzymes. This could be the possible reason in our current study because the interaction of INU and potato polyphenols tended to be significant (p < 0.1) and significant (p < 0.05) at 24 and 48h respectively. The polyphenols in IP group might decrease the activity of bacterial enzymes responsible for butyrate production, although yet to be elucidated. Although there was a slight suppression of butyrate production, this does not affect the total SCFA production during colonic fermentation. The total SCFA production was highest (p < 0.05) for IP group in our study. In addition, the interaction of INU and potato polyphenols were significant (p < 0.05) at 12 and 24h. This is expected because an interaction of INU and potato polyphenols were significant (p < 0.05) for each SCFA.
Our current study also highlighted the effect of CP and IP during colonic fermentation. Compared to their single supplements of CEL and INU respectively, it can be noticed that pH values and ammonia concentrations were significantly (p < 0.05) higher for CP and lower (p < 0.05) for IP respectively. This could indicate potentially that combining polyphenols with dietary fiber (INU) may possibly suppress the detrimental effects exert by the former. This study corresponded well with a study by Zenon et al [24] . They reported that flavonoid extract from grapefruit used as a single dietary supplement did not display a beneficial influence on cecal metabolism, increased pH and lowered bacterial activity and SCFA concentration. Further, they stated that intake of polyphenols and Inulin simultaneously could reduce the detrimental effects of the former on cecal fermentation. This could be the case with the current study.
The colon is an active site where the microbiota normally acts upon a complex polyphenolic compounds to an absorbable molecule. The type of colonic environment will depend very much on the type and amount of substrates available in the colon. In this study, the colonic pH seems to be correlated well with the production of SCFA. It can be noted at 6h that there was a sudden drop in pH values for all treated samples. This indicates the production of SCFA during fermentation. As the production of SCFA increases, the pH value decreases. While the pH value for the positive control (INU) and IP [ Figure.1] were consistent at around 5.5 (p < 0.05), CEL and CP were significantly (p < 0.05) higher. It is interesting to note that interaction of INU and potato polyphenols were significant (p < 0.05) at 6h time point. This indicates the reduction of pH is attributed by the interaction of potato polyphenols and INU because Tuohy et al. [40] highlighted that consuming dietary fiber and polyphenol-rich chocolate will simultaneously upregulate acidophilic bacteria which will ultimately reduce the colonic pH. These results may suggest that the production of different metabolites during colonic fermentation may possibly affect the colonic pH. Zenon et al [24] showed that dietary addition of inulin to the flavonoid-diet significantly decreased the pH of digest.
While lowering of colonic pH may reduce pathogenic bacteria and promote beneficial bacteria, an increase in ammonia concentration in the colon may be an important factor for colonic health as well. The colonic pH in this experiment shows the ammonia concentration during the course of fermentation of four samples. Any studies have indicated that high ammonia concentration in the colon may potentially harmful to the host. Davila et al. [63] highlighted that an increase in ammonia concentration can affect the energy metabolism of colonic epithelial cells. Further, Mitsuoka [64] stated that putrefactive compounds such as ammonia were regarded to be harmfully related to colon cancer and aging. This study showed that ammonia concentration for CP group was quite higher than the rest of the groups. Interestingly, the ammonia concentration for IP group was significantly lower (p < 0.05), even lower than control group (INU). This could indicate that addition of potato polyphenols with Inulin may suppress the formation of ammonia-producing bacteria while increasing the beneficial bacteria by lowering of colonic pH. A study conducted by Vanhoof and De Schrijver [65] reported that when inulin added to a non-purified diet on nitrogen metabolism in rats and ileum-fistulated pigs, cecal ammonia concentration decreases. In addition, Tebib et al. [62] reported that bacterial enzyme activities decrease when treated with grape seed tannins. Our current study also confirmed this because an interaction of INU and potato polyphenols were significant (p < 0.05) at 12, 24 and 48h. This could be the possible reason for the current study. While polyphenols suppress bacterial enzyme responsible for ammonia production, inulin can simultaneously reduce the colonic pH, thus, reducing the chances for pathogenic bacteria survival and maintaining a stable and healthy colonic environment simultaneously.
In conclusion, the effect of INU and IP induced significant changes in the colonic micro biota, SCFA concentrations and ammonia concentrations by reducing the colonic pH and increasing the populations of beneficial bacteria compared to CEL and CP. Therefore, our results suggested that combining potato polyphenols with Inulin can alleviate the detrimental effects of the former during colonic fermentation and maintaining a conducive environment for colonic health. Although our study highlighted the positive impact IP might have on colonic microflora, however, it is difficult to conclude with human settings because of the use of pig feces and not human feces. Further research is needed.

Conclusion
In our study, the effect of INU and IP induced significant changes in the colonic micro biota by reducing the colonic pH and increasing the populations of beneficial bacteria compared to CEL and CP. After 48h of fermentation, the production of total SCFA was significantly higher in order; IP > INU > CP > CEL. Compared to INU, IP can potentially suppress bacteria as indicated by the decrease in butyrate production and coliform populations. While the pH value and ammonia concentrations for CP increase, IP reduces these to a significant amount. Therefore, our results suggested that combining potato polyphenols with Inulin can alleviate the detrimental effects of the former during colonic fermentation and maintaining a conducive environment for colonic health.