Original papers
Screening of Phosphate-accumulating Probiotics for Potential Use in Chronic Kidney Disorder
2019 年 25 巻 1 号 p. 89-96
詳細
2019 年 25 巻 1 号 p. 89-96
Hyperphosphatemia is a secondary health issue that arises during chronic kidney disorder (CKD). Phosphate binders and dialysis are prescribed in later stages of CKD, although they may lead to harmful side-effects and worsen quality of life. Therefore, we examined the potential of intestinal bacteria (lactic acid bacteria and bifidobacteria) as phosphate-accumulating organisms (PAOs), whether PAO-formulated food can prevent CKD at earlier stages. Among the experimental organisms, Bifidobacterium adolescentis JCM1275 was the most effective. The effects of prebiotic and soy peptides were also evaluated where fructo-oligosaccharide was found to further enhance phosphate accumulation by B. adolescentis JCM1275, resulting in the best phosphate accumulation activity, identified in the study. Therefore, certain bifidobacteria and lactic acid bacteria strains have the potential to act as biological phosphate accumulators and contribute to the prevention of CKD pathology and improve patient outcome by care (not cure by medicines).
Chronic kidney disease (CKD) is an increasingly common disorder with a global distribution. As any decrease in kidney function is largely permanent and unrecoverable, prevention is particularly important. One of the most important health issues that can develop during CKD is hyperphosphatemia, in which serum phosphate levels are elevated above 6.5 mg/dL (Block et al., 2004). Currently, restrictions on phosphate intake, phosphate-binding agents, and dialysis under the supervision of medical practitioners are the most common treatments used to control hyperphosphatemia (Emmett, 2004). However, these approaches can have unwanted side effects. For example, the phosphate- and protein-restricted diets recommended for patients with CKD can promote protein deficiency and associated diseases, in addition to limiting food choice that can affect quality of life. Moreover, dialysis consists of restricted, frequent, and long procedures that can be painful, again affecting quality of life (Webster et al., 2017). Finally, the phosphate binders often prescribed for the advanced stages of CKD, such as calcium carbonate, lanthanum carbonate, ferric citrate, and Sevelamer, can lead to hypercalcemia, lanthanum bone deposition, and gastric dysfunction such as constipation and vomiting (Lopes et al., 2012). This restricts their long-term application for many patients.
As an alternative approach to chemical-based therapies, phosphorus removal by intestinal microbes (probiotics) has not yet been fully investigated. Many microbes that remove phosphorus have been reported, particularly in the field of wastewater treatment. Screening of these microbes has revealed several that show potential for a wide range of applications, such as the bioremediation of phosphate from waste water streams (Liu et al., 2006). In the initial stages of these studies, acetate agar-based enrichment screening revealed Acinetobacter spp. to be potent phosphate accumulating organisms (PAOs) (Fuhs and Chen, 1975). Likewise, yeast cultures have been enriched by phosphate-rich culture, revealing that these organisms also have the potential to remove phosphate from waste streams (Watanabe et al., 2008). More recently, Serratia marcescens has been examined in detail, demonstrating that the species is a potent PAO when grown in media supplemented with citrate, Mg2+, Ca2+, and phosphate (Chaudhry and Nautiyal, 2011). Unfortunately, these previously described PAOs cannot be applied to restoring serum phosphate balance in the gut as they are not safe to consume by humans.
Chemical binders, pharmacy and dialysis treatments are prescribed in the later stages of the disease, when kidney condition is almost irreversible, making serum phosphate levels difficult to maintain and worsen the quality of life with life-long side effects. However, if edible intestinal bacteria were identified that can remove phosphorus, they may be able to prevent hyperphosphatemia at preliminary stages of CKD. Probiotic-formulated foods do not require a medical doctor's prescription and are already available on the market. In addition, probiotics offer a safer approach than current treatments and may also confer additional healthy attributes while maintaining quality of life.
To this end, we screened several probiotics (including both lactic acid bacteria and bifidobacteria strains) to examine whether any had the potential to act as a PAO. This would offer a new preventive approach to reduce CKD-associated hyperphosphatemia at early stages of CKD and protect kidney function by balancing serum phosphate load. We also examined the effects of different carbon and nitrogen sources under static culture conditions. Our data indicate that several bifidobacteria and lactic acid bacteria strains can act as biological PAOs and may contribute to the prevention of some CKD symptoms.
Chemicals and equipment De Man, Rogosa, and Sharpe broth (MRS; Difco Laboratories, Tokyo, Japan), hipolypeptone (Nihon Seiyaku, Tokyo, Japan), beef extract (MP Biomedicals, Santa Ana, CA, USA), and yeast extract (BD, Franklin Lakes, NJ, USA) were used for the culture of bacteria. Glucose, Tween-80, K2HPO4, sodium ascorbate, L-cysteine-HCl, and 4′,6-diamidino-2-phenylindole (DAPI) dye were all procured from Wako Pure Chemicals Industries (Osaka, Japan). Fructooligosaccharide (FOS) and lactosucrose (LS) were purchased from Wako, whereas LP-90S was provided by Ensuiko Sugar Refining (Tokyo, Japan). Polypeptone was procured from the Nihon Pharmaceutical Company (Tokyo, Japan) and the soy peptides HK and AM were kindly supplied by the Fuji Oil Company (Osaka, Japan). For analysis, a V-550 spectrophotometer (JASCO, Tokyo, Japan), a DMRXA/RD fluorescence microscope (Leica, Wetzlar, Germany), and an HM-25G pH meter (TOA-DKK, Tokyo, Japan) were used.
Subculture and maintenance of microbes The studied microbes are either probiotics or originate from food, as follows: Lactobacilli (Lb. acidophilus JCM1028 [Asuda et al., 2005], Lb. casei JCM1134 [Ogawa et al., 2006], Lb. delbrueckii subsp. lactis JCM1010 [Miyamoto et al., 2000], Lb. gasseri JCM1025 [Asuda et al., 2005], Lb. plantarum JCM1055 [Guzman et al., 2018], Lb. reuteri JCM1112 [Morita et al., 2008] and Lb. rhamnosus JCM1136 [Panigrahi et al., 2004]), Lactococci (Lc. lactis subsp. cremoris JCM16167 [Schleifer et al., 1985] and Lc. lactis subsp. lactis JCM5805 [Xia et al., 2018]), Streptococcus salivarius subsp. thermophilus JCM20026 (Kitahara, 1938) and Bifidobacteria (B. adolescentis JCM1275 [O'Callaghan and van Sinderen, 2016], B. animalis subsp. animalis JCM1190 [Ruiz-Moyano et al., 2012], B. bifidum JCM1254 [Nishiyama et al., 2017], B. breve JCM1192 [Dinoto et al., 2006], B. breve JCM1273 [Sañudo Otero et al., 2017], B. longum subsp. infantis JCM1210 [URL cited (i)], B. longum subsp. longum JCM1217 [O'Callaghan and van Sinderen, 2016], B. pseudolongum subsp. pseudolongum JCM1205 [Liu et al., 2013] and B. thermophilum JCM1207 [Parvez et al., 2006]).
Starter cultures of Lactobacilli, Lactococci, Bifidobacteria, and Streptococci were inoculated from frozen glycerol stock cultures into 300-mL Erlenmeyer flasks containing 100 mL of sterilized MRS medium or Bifidobacterium medium (containing (w/v) 1 % hipolypeptone, 0.5 % beef extract, 0.5 % yeast extract, 1 % glucose, 0.1 % tween 80, 0.3 % K2HPO4, 1 % filter sterilized sodium ascorbate, and 0.05 % L-cysteine HCl). These were grown for either 18 h or 24 h at 37 °C. Generally, lactic acid bacteria are cultured at 37 °C, which is the optimal temperature for their growth (Hujanen et al., 2001). Lactococci species were grown for 24 h at 30 °C and sub-cultured for further studies.
Screening of potential PAOs among lactic acid bacteria and bifidobacteria strains During a preliminary experiment, the phosphate accumulation performances of B. breve JCM1273, B. longum subsp. infantis JCM1210, Lb. acidophilus JCM1028, Lc. lactis subsp. lactis JCM5805 (negative control) and Lb. casei JCM1134 were evaluated. After cultivation in growth media, the UOD680 and phosphate levels (g/L) were measured after 0, 6, 12 and 24 h of incubation. A previously published ascorbic acid method was applied to estimate phosphate concentration in the collected samples, with some modification (Coutinho, 1996). For later experiments, all 19 probiotics were cultivated, and their phosphate accumulation performance was evaluated. After 24 h of cultivation, dry cell concentration (g/L) and phosphate (mg/mg-dry cell) were assessed for all collected samples.
Phosphate content was estimated using the ascorbic acid method (Eaton et al., 2005). Briefly, collected samples were appropriately diluted and hydrolyzed using peroxodisulphuric acid potassium (0.25 g) and 5 N sulphuric acid (0.54 mL) at 121 °C for 30 min. After hydrolysis, samples were filtered through a 0.2-µm filter and assessed for phosphoric acid by adding 0.8 mL of reagent mixture (5 N Sulphuric acid: K2(SbO)2C8H4O10· 3H2O, 0.1372 g/100 mL: (NH4)6Mo7O24·4H2O, 4.0 g/100 mL: (ascorbic acid, 1.32 g/75 mL + formic acid, 0.6 g (0.4954 mL) + EDTA, 25 mg) =10: 3: 6: 1) to 4.2 mL of filtered samples. Phosphoric acid was used as a standard and after reaction for 10 min at room temperature, absorbance (A880) was taken using spectrophotometer.
Polyphosphate staining with DAPI B. adolescentis JCM1275, B. breve JCM1192, Lb. acidophilus JCM1028, and Lb. casei JCM1134 were stained with DAPI, using a previously published procedure with some modification (Streichan et al., 1990). Stained samples were observed under a fluorescence microscope, with stored polyphosphate granules highlighted in yellow and DNA highlighted in blue.
Effects of different carbon sources on phosphate accumulation Our study indicated that several bifidobacteria strains (B. adolescentis JCM1275, B. breve JCM1192 and B. breve JCM1273) were potential PAOs. We next evaluated the effects of different carbon sources on phosphate accumulation. These active cultures were inoculated into sterilized Bifidobacterium media in which glucose was replaced by either fructooligosaccharide (FOS) or LS (lactosucrose). Cultures were incubated under growth conditions, as mentioned above. After incubation, samples were collected and evaluated for phosphate accumulation (mg/mg-dry cell).
Effects of different nitrogen sources on phosphate accumulation Finally, selected bifidobacteria strains were further evaluated to assess the effects of different nitrogen sources on phosphate accumulation. These active cultures were inoculated into sterilized Bifidobacterium media in which nitrogen sources were replaced by either HK or AM soy peptides (Kitagawa et al., 2008), and were incubated under the growth conditions described above. After incubation, samples were collected and evaluated for phosphate accumulation (mg/mg-dry cell).
Effects of different carbon and nitrogen sources combination on phosphate accumulation B. adolescentis JCM1275 was evaluated to assess the effects of different carbon and nitrogen sources on phosphate accumulation. The active cultures were inoculated into sterilized Bifidobacterium media in which carbon and nitrogen sources were replaced by FOS+AM, FOS+HK, LS+AM and LS+HK; and incubated under growth conditions, as mentioned above. After incubation, samples were collected and evaluated for dry cell concentration (g/L) and phosphate accumulation (mg/mg-dry cell).
Statistical analysis All experiments were performed in triplicate and the results are expressed as means. Standard errors are expressed as error bars (p < 0.05 using ANOVA analysis in MS-Excel 2010 and Tukey Kramer post-hoc test for null hypothesis analysis). Two-way ANOVA analysis was performed for Figs. 4 and 5.
Comparison of specific phosphate reduction (mg/mg-dry cell) by various bifidobacteria strains after 24 h cultivation with different carbon (glucose, fructo-oligosaccharides [FOS], or lactosucrose [LS]) and nitrogen sources (polypeptone, or soy peptides HK and AM). Black bar: B. adolescentis JCM1275; white bar: B. breve JCM1192; gray bar: B. breve JCM1273. Specific phosphate reduction was defined as the decrease in phosphate concentration in the media (mg-PO43−/L)/dry cell concentration (mg-dry cell/L). Standard errors are expressed as error bars. The significant difference with in bacterial groups of different carbon and nitrogen sources for specific phosphate reduction (mg/mg-dry cell) are marked as * (p values are <0.05). The significant difference between different carbon and nitrogen sources for specific phosphate reduction (mg/mg-dry cell) are marked as # (p values are <0.05).
Effects of different combinations of carbon and nitrogen sources on dry cell concentration and specific phosphate reduction for B. adolescentis JCM1275 after 24 h cultivation. Standard errors are expressed as error bars and significant difference between different combinations of carbon and nitrogen sources for dry cell concentration (g/L) and specific phosphate reduction (mg/mg-dry cell) are represented as # and * respectively where p values are <0.05.
Screening of potential PAOs among various probiotic lactic acid bacteria and bifidobacteria strains Four strains, B. breve JCM1273, B. longum subsp. infantis JCM1210, Lb. acidophilus JCM1028, and Lb. casei JCM1134, were evaluated for growth characteristics and reduction in phosphate levels (Fig. 1), indicating a capacity to remove phosphate. Lc. lactis subsp. lactis JCM5805 was used as a negative control, as it did not remove significant levels of phosphate from broth (Fig. 1). The bacterial groups for cell growth were found to be significantly different, as represented by different letters (a→ Lb. casei JCM1134; b→ Lb. acidophilus JCM1208; c→ Lc. lactis subsp. lactis JCM5805, B. breve JCM1273 and B. longum subsp. infants JCM1210) with p values of <0.05 (Fig. 1A). Phosphate concentrations (g/L) between bacterial groups were significantly reduced from 0 h to 12 h, as represented by different letters (d→ B. breve JCM1273; e→ Lb. casei JCM1134), with p values of <0.05 (Fig. 1B).
(A) Cell growth and (B) phosphate concentration under static culture of bifidobacteria and lactic acid bacteria strains; black square: Bifidobacterium breve JCM1273; white square: Bifidobacterium longum subsp. infants JCM1210; Black triangle: Lactobacillus acidophilus JCM1208; white triangle: Lactobacillus casei JCM1134; Black diamond: Lactococcus lactis subsp. lactis JCM5805 was used as negative control. Standard errors are expressed as error bars. (A) The significant difference for cell growth between bacterial groups were represented by different letters (a→ Lb. casei JCM1134; b→ Lb. acidophilus JCM1208; c→ Lc. lactis subsp. lactis JCM5805, B. breve JCM1273 and B. longum subsp. infants JCM1210) where p values are <0.05. (B) The significant reduction for phosphate concentrations (g/L) between bacterial groups from 0h to 12h were represented by different letters (d→ B. breve JCM1273; e→ Lb. casei JCM1134) where p values are <0.05.
Several strains (B. breve JCM1273, B. longum subsp. infants JCM1210, L. acidophilus JCM1208, and Lb. casei JCM1134) were investigated further. These data are also the first to show that intestinal bacteria have the ability to accumulate or remove phosphorus, similarly to environmental strains (Fuhs and Chen, 1975; Liu et al., 2006; Watanabe et al., 2008). We next screened the phosphate accumulation capacity of several bifidobacteria and lactic acid bacteria strains by examining phosphate removal from culture medium (Fig. 2). It has previously been shown that bifidobacteria can act as potent PAOs by removing phosphate under growth conditions. The tested bifidobacteria strains were classified into three categories; (A) growth-associated phosphate removal (B. breve JCM1192 and B. breve JCM1273), (B) less growth but increased phosphate removal (B. adolescentis JCM1275), and (C) high growth but reduced phosphate removal (B. longum subsp. infantis JCM1210). Growth-associated phosphate removal occurs in the exponential phase of microbial growth and the phosphate is consumed by microbes for metabolism, storage and maintenance. While non-growth associated phosphate removal occurs in the stationary phase of microbial growth or adverse conditions when nutrient supply is limited, and the phosphate is consumed by microbes as energy storage and is used for metabolism (van Loosdrecht et al., 1997).
Comparison of dry cell concentration (g/L) and specific phosphate reduction (mg/mg-dry cell) in various bifidobacteria and lactic acid bacteria strains; 1: B. breve JCM1273; 2: B. longum subsp. infantis JCM1210; 3: B. adolescentis JCM1275; 4: B. animalis subsp. animalis JCM1190; 5: B. bifidum JCM1254; 6: B. breve JCM1192; 7: B. longum sub sp. longum JCM1217; 8: B. pseudolongum subsp. pseudolongum JCM1205; 9: B. thermophilum JCM1207; 10: Lb. acidophillus JCM1028; 11: Lb. casei JCM1134; 12: Lb. delbrueckii subsp. lactis JCM1010; 13: Lb. gasseri JCM1025; 14: Lb. plantarum JCM1055; 15: Lb. reuteri JCM1112; 16: Lb. rhamnosus JCM1136; 17: Lc. lactis subsp. cremoris JCM16167; 18: Lc. lactis subsp. lactis JCM5805 and 19: S. salivarius subsp. thermophilus JCM20026. Specific phosphate reduction (mg/mg-dry cell) was defined as the decrease in phosphate concentration in the media (mg-PO4−3/L)/dry cell concentration (mg-dry cell/L). Standard errors are expressed as error bars. The significant difference with in bifidobacteria and lactic acid bacteria for specific phosphate reductions (mg/mg-dry cell) are marked as a and b respectively where p values are <0.05. While significant difference between bifidobacteria (a) and lactic acid bacteria (b) is expressed with p value <0.01.
When compared to lactic acid bacteria, bifidobacteria were more potent PAOs, with p values of <0.01 (Fig. 2), and this was supported by DAPI staining (Fig. 3). Among bifidobacteria, B. thermophilum JCM1207, B. adolescentis JCM1275, B. breve JCM1192 and B. breve JCM1273 showed significantly different positive specific phosphate reduction, while B. longum subsp. infantis JCM1210 showed significantly different negative specific phosphate reduction, with p values of <0.05 (Fig. 2). Among lactic acid bacteria, Lb. plantarum JCM1055 showed significantly different positive specific phosphate reduction, while Lc. lactis subsp. cremoris JCM16167, Lc. lactis subsp. lactis JCM5805 and S. salivarius subsp. thermophilus JCM20026 showed significantly different negative specific phosphate reduction, with p values of <0.05 (Fig. 2).
Fluorescence micrographs (A) B. adolescentis JCM1275, (B) B. breve JCM1192, (C) Lb. acidophilus JCM1028 and (D) Lb. casei JCM1134 stained using DAPI. Polyphosphate granules are stained in yellow. The black bar represents 5 µm.
Specific examination of dry cell concentration (g/L) and phosphate concentration (mg/mg-dry cell) indicated that the best PAO among the tested bifidobacteria strains was B. adolescentis JCM1275, followed by B. breve JCM1192 and B. breve JCM1273 (Fig. 2). A similar trend was shown by DAPI staining, in which polyphosphate granules were stained yellow (Fig. 3). B. adolescentis JCM1275 showed significantly higher fluorescence when compared to B. breve JCM1273, B. breve JCM 1192 and Lb. acidophilus JCM1028 (Fig. 3). This is the first report indicating that Bifidobacteria spp. can accumulate phosphorus in the form of polyphosphate granules (polyphosphate). As they showed potential as PAOs, B. adolescentis JCM1275, B. breve JCM1192, and B. breve JCM1273 were studied further to evaluate the effects of different carbon and nitrogen sources on phosphate accumulation in culture.
Effects of different carbon sources on phosphate accumulation Fructooligosaccharides (FOS; also known as oligofructan or oligofructose) are naturally occurring prebiotic molecules. They are polymers of D-fructose residues linked by β (2→1) bonds with a terminal α(1→2) linked D-glucose that can also be used as artificial sweeteners (Lorenzoni et al., 2014). Lactosucrose (LS; lactosylfructoside or O-β-D-galactopyranosyl-(1→4)-O-α-Dglucopyranosyl (1→2)-β-D-fructofuranoside) is an oligosaccharide consisting of galactose, glucose, and fructose. LS is also used as an artificial sweetener and functional food ingredient [URL cited (ii)]. FOS and LS are both prebiotics that are selectively processed by certain probiotic species, affecting immunity and enhancing gut health (Pandey et al., 2015). We therefore examined the effects of prebiotics (FOS and LS) as carbon sources on the PAO properties of B. adolescentis JCM1275, B. breve JCM1192, and B. breve JCM1273 by measuring phosphate accumulation in the culture media. When compared to the control conditions that used a glucose carbon source, phosphate accumulation in the three potential PAOs was higher when cultivated with prebiotics (Fig. 4). Of the tested prebiotics, FOS treatment promoted greater accumulation than LS or glucose, particularly in B. adolescentis JCM1275 and B. breve JCM1273, although B. breve JCM1192 in LS was still higher than in controls. Despite being lower than FOS, LS treatment also increased phosphate accumulation by the potential PAOs relative to the control, but the only significant result was obtained with B. adolescentis JCM1275 (Fig. 4). Only modest levels of phosphate accumulation were observed for B. breve JCM1192 and B. breve JCM1273. Compared to B. breve JCM1273 and B. breve JCM1192, B. adolescentis JCM1275 showed significantly increased phosphate accumulation with the supplementation of carbon sources (glucose, FOS and LS), with p values of <0.05. Overall, no negative effects on phosphate accumulation were observed after cultivation of probiotics with prebiotics, except in the case of B. breve JCM1192 with FOS. Our data indicate that phosphate accumulation of the most effective PAO (B. adolescentis JCM1275) was improved by cultivation with prebiotics rather than with glucose as a carbon source. We hypothesize that this is because prebiotic exposure accelerated either phosphate polymerization or phosphate storage in the organisms.
Effects of different nitrogen sources on phosphate accumulation The hydrolysis of soy protein isolates or soy peptides, and their constituent amino acids, is very tightly regulated in bacteria. Soy peptides are of particular interest, as they are marketed as isotonic drinks and incorporated in various functional foods. Importantly, absorption and assimilation of soy peptides is easier and quicker than soy protein isolates (Kitagawa et al., 2008). In this study, we examined the effects of cultivation with soy peptides and whether these could further elevate phosphate accumulation capacities in bifidobacterial strains. We first examined the soy peptide HK and AM, demonstrating that it enhanced phosphate accumulation in B. adolescentis JCM1275 more effectively, with p values of <0.05, although only a modest change in phosphate accumulation was observed for B. breve JCM1273 and B. breve JCM1192 (Fig. 4). Although HK has been found to be a better growth stimulant than AM, and this was supported by our data (Fig. 4). The effect of FOS and LS on phosphate accumulation by B. adolescentis JCM1275 was found to be significant, with p values of <0.05 (Fig. 4).
We also examined combinations of carbon and nitrogen sources, demonstrating that FOS led to the best phosphate accumulation values (0.33 mg/mg-dry cell) for B. adolescentis JCM1275 and its growth (0.65 g/L), with p values of <0.01 (Fig. 5). Among different combinations of carbon and nitrogen sources, HK with LS showed significantly different positive dry cell concentration while FOS and LS showed significantly different negative dry cell concentration for B. adolescentis JCM1275, with p values of <0.05 (Fig. 5). Among the different combinations of carbon and nitrogen sources, AM+FOS and AM+LS showed significantly different negative specific phosphate reduction for B. adolescentis JCM1275, with p values of <0.05 (Fig. 5). Other combinations resulted in either a marginal increase or a reduction in phosphate accumulation (Fig. 5).
As per two-way ANOVA analysis, the influence of FOS and LS are significant in accelerating phosphate accumulation by B. adolescentis JCM1275, with p values of <0.05, while the influence of HK and AM is milder but significant, with p values of <0.05, as shown in Fig. 4. The effect of carbon and nitrogen source interaction on phosphate accumulation was also studied, and was found to be significantly negative, with p values of <0.05, as shown in Fig. 5. Among FOS and LS, the effect of FOS alone to significantly accelerate phosphate accumulation by B. adolescentis JCM1275 was most predominant and showed the lowest p value (p < 0.01). Therefore, we may conclude that, among the studied carbon and nitrogen sources, FOS is the most effective carbon source for increasing phosphate accumulation in B. adolescentis JCM1275.
Previously, several strains have been suggested to be good PAOs that could serve to accumulate phosphate; for example, B. longum has been reported to decrease serum phosphate levels in CKD patients receiving hemodialysis (Ogawa et al., 2012). In that study, B. longum was used in conjunction with hemodialysis to reduce serum phosphate levels and the strategy was implemented for CKD patients as curative, not as preventative. However, the present study screened safe and potential intestinal microbes as PAO for the prevention of CKD at preliminary stages and the implementation of successful strains is easy to introduce into the food market, as consumption of probiotic-formulated food is safe and works as care, not a cure.
Other reports for PAOs include Halobacterium salinarium, Halorubrum distributum, and Brevibacterium antiquum, which can store up to 9.5 %, 10 %, or 13 % (w/v) polyphosphate, respectively. In addition, these species can also accumulate between 70 % to 90 % (w/v) of 8–11 mM orthophosphate (Vaziri et al., 2013). We hypothesize that these organisms likely require more free phosphate than polyphosphate, although this would depend on environmental conditions. Brevibacterium casei, Acetobacter xylinum, and Cryptococcus humicola have also been documented as potential PAOs that can store up to 95 %, 86 %, or 55 % (w/v) of phosphate respectively from media containing a 5 mM KH2PO4, 5 mM MgSO4, 30 mM glucose, and 5 g/L Difco amino acid mixture ([URL cited (iii)]; Breus et al., 2012; Kulakovskaya et al., 2012; Ryazanova et al., 2009, 2007). An additional study reported that Saccharomyces cerevisiae, Brevibacterium casei, and Acetobacter xylinum accumulate 0.2, 0.03, or 0.05 mg phosphate per mg dcw of phosphate, respectively (Ryazanova et al., 2009). Nitrogen deficiency can also accelerate phosphate accumulation by Kuraishia capsulate and Saccharomyces cerevisiae, by up to 14 % and 70 % (w/v), respectively (Ryazanova et al., 2009). Therefore, our data and these other studies indicate that phosphate accumulation by these potential PAOs depends on environmental compositions and conditions. To maximize phosphate removal, a strategy to develop PAOs that can survive in the human gut, and also efficiently remove phosphate from partially digested food and inhibit phosphate assimilation into the blood, should be followed. These ‘elite’ PAOs could be used individually or in combination with prebiotics to enhance phosphate removal. Furthermore, these organisms would also confer probiotic attributes that would contribute to gut health in patients with CKD.
A variety of procedures and chemical binders are prescribed by medical practitioners for the treatment of advanced CKD. However, these therapies can have harmful side-effects and affect patient quality of life. In addition, during the advanced stages of CKD, kidneys are severely damaged, and it is difficult to elicit recovery using treatment. However, the probiotic approach advanced in this study has identified several potential PAOs that remove phosphate in vitro. If applied in vivo, these strains could potentially reduce kidney burden and CKD progression at preliminary stages. Probiotic use to prevent hyperphosphatemia at early stages of CKD would have several advantages over current methods, such as the generally recognized as safe (GRAS) designation, making treatment more easily accepted by patients and reducing the requirement for prescription by medical practitioners. Probiotic use may also confer other benefits to promote gut health.
In summary, B. adolescentis JCM1275 was the best PAO among the tested organisms and conditions. Cultivation with FOS (prebiotics) showed the highest enhancement of PAO phosphate accumulation capability. These data demonstrate that bifidobacteria and lactic acid bacteria strains have the potential to be used as biological phosphate accumulators that could contribute to prevent CKD-associated hyperphosphatemia. The organisms and prebiotics highlighted in our study should therefore be explored in more detail to clarify the best combinations to utilize in the most effective and efficient means to prevent CKD at preliminary stages. In addition, the potential PAOs can be taken as foods or used to design functional food formulations (not medicines) that can prevent CKD in early stages and maintain quality of life.
Acknowledgements A.A. was supported by a scholarship grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. A.A. also studied under the Global Food Security Partnership Program at the University of Tsukuba. This work was supported in part by the Sumitomo Electric Industries Group Corporate Social Responsibility Foundation (grant to H. A.).
