ABSTRACT
Rice kefiran is superior in functionality, has high concentration of mucilaginous polysaccharide, and low lipid content, compared to conventional kefiran. However, reports on its physiological functionality, especially studies on life expectancy and aging, in model organisms are rare. In this study, nematodes were used as model organisms that were fed rice kefiran, along with Escherichia coli OP50, as a result of which, the lifespan of nematodes was extended and age-related retardation of mobility was suppressed. It also increased the heat stress resistance in nematodes. Experiments using daf-16 deletion mutant revealed that rice kefiran functions via DAF-16. Thus, this study revealed the longevity, anti-aging and heat stress tolerance effects of rice kefiran in nematodes.
Rice kefiran fed C. elegans prolonged life span, increased motility and enhanced heat stress tolerance. These anti-aging effects was mediated through DAF-16.
Kefir is a kind of fermented food that is consumed commonly in the Caucasus region, which is famous for higher longevity. Kefir grain, few centimeters in size, is fermented in milk from bovine animals or goats. It is a bacterial mass that contains bacteria, such as lactobacilli, and yeasts. Recently, many effects of lactobacilli and yeasts have been clarified, including the regulation of intestinal function and induction of probiotic effects. Since kefir contains these microbes, it is also expected to exert beneficial functions as a healthy food [1–3].
The mucopolysaccharide kefiran is considered to be one such main compound that exerts the health benefits of kefir [4]. Kefiran is produced by Lactobacillus kefiranofaciens and included in diets as functional food and in cosmetics as moisturizing agent [5]. Rice kefiran is a fermented product made by culturing Lactobacillus kefiranofaciens purely isolated from kefir in a medium containing rice [6]. Rice kefiran contains more than 5 mg/g of kefiran. Since kefir is a fermented milk drink, it contains lipids and contains alcohol by fermentation. On the other hand, rice kefiran is a fermented product of rice and contains little lipid, and Lactobacillus kefiranofaciens is a homofermentative lactobacillus, therefore it does not contain alcohol.
Till date, many physiological effects of kefiran have been reported, such as the regulation of intestinal function, immune function, anti-inflammation, cardiac disease, and so on [7–11]. Despite the superior features of rice kefiran, the number of physiological reports is not as many [8,12–14]. Moreover, the physiological effects on life span and aging have not been reported yet. In this study, we aimed to analyze the physiological effects of rice kefiran on life span and aging.
We selected C. elegans as a model organism for studying aging. C. elegans is a commonly used model organism owing to its transparent body and ease of culture [15]. It is widely applied in aging-related studies, as a model organism for various biological assays, due to its short life span of approximately a month. Its genome and cell lineages are well described and its digestive system has been shown to be similar to that of higher animals. Recently, it has been used to evaluate the effect of certain foods and their related functional components, including the anti-aging effects of Bifidobacterium and nucleoprotein, extracted from salmon milt, by feeding them on those, along with E. coli [16,17]. Their genetic similarity with higher animals would enable us to apply the obtained results in higher animals in the future.
Many genes related to longevity, such as daf-2, daf-16, sir-2.1, age-1, and skn-1, are already known in nematodes [18–20]; we especially focused on daf-16 in this study. DAF-16 is a kind of transcription factor and a homolog of FOXO in higher animals [21]; DAF-16/FOXO is strongly related to longevity [21]. Activated DAF-16 localizes in the nucleus, functions as a transcription factor, and induces the transcription of target genes [22], such as hsp-12.6 (related to heat stress tolerance) and sod-3 (related to oxidative stress tolerance) [23,24]. Up-regulation of stress tolerance by activated DAF-16 induces longevity and anti-aging features.
In this study, we analyzed rice kefiran with dextrin (KD), along with dextrin (D) as control, since the industrially produced rice kefiran inherently contains dextrin. In order to understand the effects of pure rice kefiran, we also analyzed rice kefiran without dextrin (K).
Materials & methods
Nematode and rice kefiran
C. elegans strains used in this study were: N2 Bristol (wild type) and daf-16 (mgDf50, obtained from the Caenorhabditis Genetics Center [CGC], University of Minnesota, MN). The nematodes were cultured at 20°C on NGM plates, with E. coli (OP50) spread on it [15]. Rice kefiran mixed with dextrin (KD), dextrin (D), and rice kefiran (K) was provided from Daiwa Pharmaceutical Co., Ltd. (Tokyo, Japan).
Nematode synchronization
To collect eggs, adult nematodes were crushed in NaClO solution (1:10 of 10 N NaOH [Wako Pure Chemical Industries, Ltd., Osaka, Japan] and NaClO [Haiter; KAO, Tokyo, Japan]); this process synchronized the nematode growth level.
Evaluation of life span
Synchronized worms were cultured on NGM plates (with OP50 spread; OP plates) at 20°C for 96 h, and then transferred to OP, KD, D, or K plates at various concentrations (KD plates; 5.0 mg/mL, D plates; 4.0 mg/mL, K plates; 0.1, 1.0, or 10.0 mg/mL) and culture continued at 20°C. KD samples contained dextrin at approximately 80% of total mass. The day of initial transfer to OP or KD and K plates was designated as day 0. The worms were then transferred to new plates every 2 days. Live and dead worms were counted on each transfer day. Worms that displayed no movement upon gentle probing with a platinum picker were judged as dead. To prevent offspring generation, 0.5 mg/mL of FUdR (2’-deoxy-5-fluorouridine [Wako Pure Chemical Industries, Ltd.]) was added to the plates at days −1, 0, 2, and 4. Besides the life span of wild type worms, that of mgDf50 worms was also assessed. The survival rate of 40 worms was determined per group.
Evaluation of nematode motility
Synchronized worms were cultured on OP plates at 20°C for 96 h, and then transferred to OP, KD, D, or K plates at various concentrations (KD plates; 0.1, 0.5, or 5.0 mg/mL, D plates; 0.08, 0.4, or 4.0 mg/mL, K plates; 1.0 or 10.0 mg/mL) and culture continued at 20°C. The day of initial transfer to OP, KD, or K plate was designated as day 0. The worms were transferred to new plates every 3 days and their thrashing movement was counted on each transfer day. As mentioned earlier, 0.5 mg/mL of FUdR was added to the plates at days −1, 0, and 3 to prevent offspring generation. In addition to the movement of wild type worms, that of mgDf50 worms was also assessed. The movement at day 0 was set as 100% and 10 worms were evaluated per group.
Assessment of heat stress tolerance; viability
Synchronized worms were cultured on OP, KD, D, or K plates at different concentrations (KD plates; 5.0 mg/mL, D plates; 4.0 mg/mL, K plates; 0.1, 1.0, or 10.0 mg/mL) at 20°C for 96 h, and then incubated at 35°C for 10 h. The time point of heat application was designated as 0 h. The survival rate of 40 worms per group was evaluated every 2 h. Ten hours after heat application, the survival rate was evaluated.
Assessment of heat stress tolerance; motility
Generally, heat stress decreases the movement of worms. To measure the recovery rate from heat stress, synchronized worms were cultured on OP, KD, D, or K plates at several concentrations (KD plates; 5.0 mg/mL, D plates; 4.0 mg/mL, K plates; 0.1, 1.0, or 10.0 mg/mL) at 20°C for 96 h, transferred to OP plates, and then incubated at 35°C for 4 h. The time of heat application was designated as 0 h, and thrashing movement was counted every 12 h. In parallel, the movement of worms kept at 20°C (instead of 35°C) was counted. The ratio of movement count in the worms cultured at 35°C to that at 20°C was calculated, and results presented. Movement of 10 worms was counted per group. Besides the recovery in wild type worms, that in mgDf50 mutant worms was also evaluated.
Statistical processing
Data are shown as the mean ± SEM, analyzed with Tukey’s test. Survival rate was analyzed with the log-rank test. Statistical tests were performed by IBM SPSS Statistics (International Business Machines Corporation, NY). Graphs were generated using Microsoft Excel (Microsoft Corp., Redmond, WA) and Microsoft PowerPoint (Microsoft Corp.). P-values of <0.05 indicated statistically significant differences.
Results
In this study, rice kefiran with dextrin (KD) contained dextrin at 80% of total mass; therefore, in the control, we used dextrin (D) at 80% of KD mass. In order to clarify the effects of rice kefiran alone, we also examined rice kefiran without dextrin (K).
Effect of rice kefiran on life span
Although there was no significant difference (p = 0.13), 5.0 mg/mL rice kefiran with dextrin (KD) tended to extend the life span of wild type nematodes, whereas 4.0 mg/mL dextrin as control (D) did not (Figure 1(a)). However, KD (5.0 mg/mL) and D (4.0 mg/mL) did not extend the life span of daf-16 gene-deficient mutants (mgDf50) (Figure 1(b)). Rice kefiran without dextrin (K) (1.0 and 10.0 mg/mL) significantly induced longevity in wild type nematodes (Figure 1(c)); however, it could not do so for mgDf50 (Figure 1(d)).
Life span of worms fed on rice kefiran. Life span of wild type N2 (a) and MgDf50 (b) fed on dextrin (D) and rice kefiran with dextrin (KD). Life span of wild type N2 (c) and MgDf50 (d) fed on several concentrations of rice kefiran (K). To prevent offspring generation, 0.5 mg/mL of FUdR was added to the plates at days −1, 0, 2, and 4. N = 40 per group, *P < 0.05 by log-rank test.
Life span of worms fed on rice kefiran. Life span of wild type N2 (a) and MgDf50 (b) fed on dextrin (D) and rice kefiran with dextrin (KD). Life span of wild type N2 (c) and MgDf50 (d) fed on several concentrations of rice kefiran (K). To prevent offspring generation, 0.5 mg/mL of FUdR was added to the plates at days −1, 0, 2, and 4. N = 40 per group, *P < 0.05 by log-rank test.
Effect of rice kefiran on age-dependent movement
Movement of nematodes decreases with age. KD (0.5 mg/mL) was seen to suppress the age-related movement reduction in wild type nematodes at day 9 while D (4.0 mg/mL) did not (Figure 2(a)). K (1.0 and 10.0 mg/mL) significantly suppressed the decline of movement in wild type nematodes at days 3, 6, and 9 (Figure 2(b)), but did not suppress that in mgDf50 on similar days (Figure 2(c)).
Movement of worms fed on rice kefiran. Movement of wild type N2 (a) fed on several concentrations of dextrin (D) and rice kefiran with dextrin (KD). Movement of wild type N2 (b) and MgDf50 (c) fed on several concentrations of rice kefiran (K). To prevent offspring generation, 0.5 mg/mL of FUdR was added to the plates at days −1, 0, and 3. Movement count at day 0 was set as 100%. Data are presented as the mean ± SEM, N = 10 per group, *P < 0.05; ***P < 0.005 by Tukey’s t-test.
Movement of worms fed on rice kefiran. Movement of wild type N2 (a) fed on several concentrations of dextrin (D) and rice kefiran with dextrin (KD). Movement of wild type N2 (b) and MgDf50 (c) fed on several concentrations of rice kefiran (K). To prevent offspring generation, 0.5 mg/mL of FUdR was added to the plates at days −1, 0, and 3. Movement count at day 0 was set as 100%. Data are presented as the mean ± SEM, N = 10 per group, *P < 0.05; ***P < 0.005 by Tukey’s t-test.
Effect of rice kefiran on heat stress tolerance
Generally, C. elegans is cultured at 20°C. In this study, 35°C was adopted as heat stress. After 10-h incubation at 35°C, survival rate was measured every 2 h (Figure 3). Neither of D (4.0 mg/mL), KD (5.0 mg/mL), or K changed the survival rate of wild type nematodes under heat stress.
Heat stress tolerance of worms fed on rice kefiran (viability). The survival rate of wild type N2 (a), fed on dextrin (D) and rice kefiran with dextrin (KD), after heat stress. Movement of worms (b) fed on several concentrations of rice kefiran (K). N = 40 per group, not significant by log-rank test.
Heat stress tolerance of worms fed on rice kefiran (viability). The survival rate of wild type N2 (a), fed on dextrin (D) and rice kefiran with dextrin (KD), after heat stress. Movement of worms (b) fed on several concentrations of rice kefiran (K). N = 40 per group, not significant by log-rank test.
Next, the recovery of movement, following 4-h heat stress, was measured (Figure 4). Although there was no significant difference (p= 0.054), KD increased the movement of wild type nematodes 12 h after heat stress, whereas D (4.0 mg/mL) did not (Figure 4(a)). Neither of KD (5.0 mg/mL) or D (4.0 mg/mL) changed the movement of mgDf50 after heat stress (Figure 4(b)). K (1.0 and 10.0 mg/mL) significantly enhanced the movement of wild type nematodes 12 h after heat stress (Figure 4(c)), whereas it did not do so for mgDf50 at 12 or 24 h (Figure 4(d)).
Heat stress tolerance of worms fed on rice kefiran (motility). Thrashing rate, after heat stress, of worms (a) and MgDf50 (b) fed on dextrin (D) and rice kefiran with dextrin (KD). Thrashing rate, after heat stress, of worms (c) and MgDf50 (d) fed on several concentrations of rice kefiran (K). Data are presented as the mean ± SEM, N = 10 per group, *P < 0.05; ***P < 0.005 by Tukey’s t-test.
Heat stress tolerance of worms fed on rice kefiran (motility). Thrashing rate, after heat stress, of worms (a) and MgDf50 (b) fed on dextrin (D) and rice kefiran with dextrin (KD). Thrashing rate, after heat stress, of worms (c) and MgDf50 (d) fed on several concentrations of rice kefiran (K). Data are presented as the mean ± SEM, N = 10 per group, *P < 0.05; ***P < 0.005 by Tukey’s t-test.
Discussion
This study analyzed the effects of industrially produced rice kefiran on aging and heat stress tolerance. Compared to previous kefir, rice kefiran is a new product fermented in a medium using rice hydrolysate. Till date, several effects of kefiran have been reported, such as regulation of intestinal function, immune function, and anti-inflammatory function [7–11], and some studies on rice kefiran had reported on structure, fat accumulation, and blood pressure changes [8,12–14]. However, there had been no prior research on the effect of rice kefiran on longevity and aging. In this study, we fed rice kefiran to C. elegans. There was no precedence of rice kefiran feeding to nematodes till now; however, we adopted it for the first time, considering that nematodes have a short life span, and are commonly used as model organisms to evaluate the effect of certain foods and their components.
Rice kefiran without dextrin (K) prolonged the life span of nematodes and suppressed the age-dependent retardation of movement (Figures 1(c) and 2(b)), thereby demonstrating the anti-aging effect of rice kefiran. Although K did not increase the survival rate of nematodes after heat stress (Figure 3(b)), it did improve the recovery of movement from heat stress (Figure 4(c)). The results of rice kefiran with dextrin (KD) tended to be similar, although some phenotypes were not significantly so (Figures 1(a), 2(a) and 4(a)). Dextrin, adopted as control, suppressed these phenotypes (Figure 1(a), 2(a), 4(a)). Based on these results, it was supposed that these effects did not arise from dextrin, but from pure rice kefiran. In order to clarify the mechanism, daf-16-deficient mutant (mgDf50) was used in similar assays; the phenotypes in wild type nematodes were suppressed in mgDf50 (Figure 1(b,d), 2(c) and 4(b,d)). The results suggested that the transcription factor DAF-16 was necessary to prolong life span, suppress the retardation of motility, and recover from heat stress. Anti-aging and stress tolerance to heat, oxidation, and heavy metals were reported to be up-regulated by DAF-16 [23–25]. Stimulated DAF-16 localizes in nuclei and accelerates the transcription of target genes related to heat stress tolerance, such as hsp-12.6 [24]. This up-regulation of gene transcription promotes the recovery from heat stress; sod-3 is also a target gene of DAF-16 and can scavenge reactive oxygen species (ROS) [23]. ROS accumulate in mitochondria and cytoplasm, in an age-dependent manner, and cause cellular damages and diseases [26]. Rice kefiran activates DAF-16 and may induce the reduction of ROS. Consequently, rice kefiran could induce the extension of life span and improvement of motility [27].
In lifespan assay (Figure 1(a,c)), KD did not show similar phenotype significantly with K, even though 5.0 mg/mL KD contained same mass of rice kefiran with 1.0 mg/mL K. Since dextrin is indigestible, it is possible that dextrin might have affected the absorption of kefiran. However, so far, there is no knowledge as to whether dextrin affects the absorption of substances in the intestine of nematodes. Also, there is no report on the decomposition of dextrin in the nematode body. On the other hand, there is a prior study that glucose reduces the activity of DAF-16 [28]. However, in our study, we did not add as much dextrin as glucose equivalent to the previous studies. Although dextrin is indigestible and is thought to be hardly decomposed into glucose, the possibility that dextrin influenced the signal pathway of nematode cannot be completely ruled out. Therefore, although it is unlikely that the activity of DAF-16 was reduced by dextrin, it is conceivable that dextrin may have suppressed the increase of the activity of DAF-16.
This study clearly demonstrated that rice kefiran prolongs the life span, induces anti-aging effects, and up-regulates heat stress tolerance of C. elegans through DAF-16. DAF-16 acts as a transcription factor, stimulated by the signals from insulin-like signaling (IIS) pathway [22]. This suggested that digested rice kefiran activated upstream of IIS pathway and resulted in the activation of DAF-16. Consequently, activated DAF-16 induced the transcription of numerous genes that possibly resulted in the physiological effects revealed in this study.
We plan to extend our study by feeding rice kefiran to higher animals in future. Higher animals, such as mice, have a homolog of transcription factor DAF-16, FOXO [19]. We hope to see similar longevity and anti-aging effects in higher animals feeding on rice kefiran.
Author Contributions
TS and KS conceived and designed the study; TS, TF, KS, MAbe, KK, and MArai performed experiments; KS provided essential tools and reagents; TS, TF, KS, MAbe, KK, MArai,and KS analyzed data,; TS and KS wrote the paper; TS and KS made manuscript revisions; and KS supervised the study as a principal investigator. All authors read and approved the final manuscript.
Disclosure statement
No potential conflict of interest was reported by the authors.
