2014 Volume 20 Issue 2 Pages 479-483
To determine whether fermented rice extract (FRe) has bifidogenic growth-stimulating effects in vitro, the effects of FRe on the growth of lactic acid bacteria and bifidobacteria were evaluated. Treatment with 0.2% FRe stimulated the growth of three strains of lactic acid bacteria (Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium lactis), significantly increasing the number of cells by 11 – 53 fold after 12 – 18-h incubation compared with the control (p < 0.05). HPLC analysis indicated that the panose content of the FRe powder was 6.7%. The growth-stimulating effects of FRe on bifidobacteria and lactic acid bacteria were likely due to panose, and additional unknown and/or known substances in the FRe. Based on these results, we propose that FRe could be used as a growth stimulator for bifidogenic and lactic acid bacteria in pharmaceutical and food applications.
Bifidobacteria are the predominant type of bacteria found in the gastrointestinal tract, and have various beneficial effects on health (Picard et al., 2005). Intensive research efforts are currently focused on modifying the composition of intestinal flora towards a more beneficial community. One way to achieve this may be the use of prebiotics and probiotics that function as bifidogenic growth stimulators. Various oligosaccharides, and bifidobacteria themselves, can be used to promote the growth of bifidobacteria (Hopkins et al., 1998; Gibson and Wang 1994). In addition, several bifidogenic growth stimulators have been reported, including 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), 1,4-dihydroxy-2-naphthoic acid (DHNA) (Isawa et al., 2002), and O-α-d-Glucopyranosyl-(1→6)-O-α-d-glucopyranosyl-(1→4)-α-d-glucose (panose) (Park et al., 1992; Mäkeläinen et al., 2009).
Fermented rice extracts (FRes) have various beneficial effects on health, including anti-osteoporotic (Cho et al., 2010), hypoglycemic (Lu et al., 2010), anti-tumor (Ho et al., 2010), neuro-protective (Lee et al., 2010), anti-atherosclerotic (Setnikar et al., 2005), anti-stress, anti-fatigue (Kim et al., 2002), and chemopreventive (Akihisa et al., 2005) activities, compared with non-fermented extracts. However, whether FRes have bifidogenic growth stimulating effects in vitro is not known. We therefore assessed the effects of FRe on the growth of lactic acid bacteria and bifidobacteria, and identified the main active compounds in FRe responsible for the growth-stimulating effects.
Materials FRe production was carried out in three fermentation steps. During the first step (saccharification), washed, non-glutinous rice (1 kg) was soaked for 6 h, drained for 30 min, and then steamed for 15 min at 121°C. After rapid cooling, 10-g malt powder (SG Food, Kimhae, Korea) and 4 L of water were added, and the mixture was fermented in a 20-L sterile glass container at 55°C for 12 h to produce the fermentate. In the second fermentation step, 20-mL Saccharomyces cerevisiae (ATCC 9804) suspension were added to the fermentate, mixed well, and incubated at 30°C for 48 h. Finally, 20-mL lactic acid bacteria, Weissella paramesenteroides (KACC 91704), were added to the second fermentate, mixed well, and incubated at 30°C for 48 h. This final fermentate was autoclaved at 150°C for 15 min, and then filtered through a 40-mesh sieve to obtain the final filtrate, which was freeze-dried for 2 days to a moisture content of ∼0.8%. The freeze-dried FRes were ground in a mill, passed through a 500-mesh sieve, and stored at −20°C until use.
Nutritional composition analysis The nutritional composition (carbohydrate, reduced sugar, protein, crude fat, saturated fatty acids, unsaturated fatty acids, cholesterol, and ash) and sodium content of the FRes were determined based on the methods of Korea Food and Drug Administration (KFDA; 2012).
High-performance anion-exchange chromatography (HPAEC) Panose was identified as an active compound following the methods of Kwon et al. with slight modifications. Briefly, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was carried out on a Dionex 500 System (Thermo Fisher Scientific Inc., Waltham, MA) with a pulsed amperometric detector, and an SP gradient pump. The samples were filtered (0.45-µm membrane) and injected (10 µL) on to a CarboPac PA-1 guard column (2 × 50 mm; Thermo Fisher Scientific Inc.) attached to a CarboPac PA-1 anion-exchange analytical column (2 × 250 mm). For the detection of carbohydrates, a Dionex ED40 Electrochemical Detector with an Au working electrode and an Ag/AgCl reference electrode was used in pulsed amperometry mode. The mobile phase consisted of 150 mM NaOH (A) and 150 mM sodium hydroxide/500 mM sodium acetate (B), using a gradient elution of 0 – 30 min (0 – 100% B). All eluents were constantly purged with helium to avoid the formation of carbonate from carbon dioxide. The eluent flow rate was held at 1.0 mL/min. Data were collected and analyzed using the Dionex Chromeleon 6.4 software. For quantitative analysis, standard D-panose (P2407, Sigma-Aldrich, Schnelldorf, Germany) was analyzed using the HPAEC system under identical conditions.
Microorganisms The lactic acid bacteria (LAB) Lactobacillus acidophilus, Streptococcus thermophilus, and Bifidobacterium lactis were isolated from fermented milk cultures (YO-MIX™, DANISCO Deutschland, GmbH, Germany). L. acidophilus and S. thermophilus were incubated in MRS broth (Difco Laboratories, Detroit, MI) with 1% maltose at 37°C for 24 h under aerobic conditions. B. lactis was cultured in RCM broth (Difco Laboratories) at 37°C for 24 h under anaerobic conditions of 5% H2, 5% CO2, and N2 90%, using the Bactron Anaerobic Chamber system (SHELLAB, USA).
Measuring growth stimulation To confirm the growth-stimulating effects of FRe, L. acidophilus and S. thermophilus were cultured in MRS broth containing 1% maltose, and MRS broth supplemented with 1% maltose and 0.2% FRe, respectively, at 37°C under aerobic conditions. In contrast, Bifidobacterium lactis was cultured in RCM broth with 0.2% FRe at 37°C under anaerobic conditions. Cultures of identical media and conditions, but without FRe, were prepared as negative controls. In addition, panose (0.02%, 0.04%, and 0.2%; Sigma-Aldrich P2407) and DHNA (0.001%, and 0.01%; Sigma-Aldrich 281255) were used as positive controls.
Inocula of each strain were cultivated aerobic or anaerobically in the appropriate medium at 37°C for 24 h, and the turbidity of suspensions was adjusted to an absorbance of 0.3 at 625 nm. One hundred microliters of culture broth were then inoculated into 10-mL test medium, and samples were incubated at 37°C. Microbial numbers were determined by the spread-plate method after 12- and 18-h incubations.
Statistical analyses All data are presented as means ± standard deviation, and are the results of at least three independent experiments. All statistical analyses were performed using Student's t-test.
Nutritional composition of FRe The nutritional composition of FRe is shown in Table 1. The major component of FRe was carbohydrate (83.4%), while the reducing sugar content was 2.07%. The content of proteins and ash, the minor components, were 8.5% and 1.1%, respectively. In contrast, crude fat, saturated and unsaturated fatty acids, and cholesterol were not detected (Table 2).
| Composition | Unit | Contents |
|---|---|---|
| Calories | Kcal | 368 |
| Carbohydrate | % | 83.4 |
| Sugars | % | 20.7 |
| Proteins | % | 8.5 |
| Crude fat | % | 0.0 |
| Saturated fatty acids | % | 0.0 |
| Unsaturated fatty acids | % | 0.0 |
| Cholesterol | mg/100 g | 0.0 |
| Ash | % | 1.1 |
| Sodium | mg/100 g | 47.1 |
| Water | % | 7.0 |
| Strain | Time (h) | Control | FRe (0.2%) | Panose (0.02%) | Panose (0.04%) | Panose (0.2%) | DHNA (0.001%) | DHNA (0.01%) |
|---|---|---|---|---|---|---|---|---|
| Lactobacillus acidophilus | 12 | 4.0 × 104 | 7.16 × 105 | 3.96 × 104 | 4.26 × 104 | 7.26 × 104 | 8.8 × 104 | 4.9 × 105 |
| ±0.3 × 104 | ±0.49 × 105a | ±0.7 × 104 | ±0.25 × 104 | ±0.4 × 104c | ±1.06 × 104b | ±1.22 × 105b | ||
| 18 | 2.1 × 106 | 6.1 × 107 | 2.43 × 106 | 3.63 × 106 | 8.56 × 106 | 3.0 × 106 | 3.1 × 107 | |
| ±0.3 × 106 | ±1.38 × 107b | ±0.3 × 106 | ±0.37 × 106b | ±0.2 × 106a | ±0.36 × 106c | ±0.26 × 107a | ||
| Streptococcus thermophilus | 12 | 2.4 × 104 | 4.73 × 105 | 2.33 × 104 | 2.26 × 104 | 4.3 × 104 | 5.1 × 104 | 3.3 × 105 |
| ±0.5 × 104 | ±0.80 × 105a | ±0.2 × 104 | ±0.55 × 104 | ±0.45 × 104 | ±0.7 × 104b | ±0.2 × 105a | ||
| 18 | 2.3 × 106 | 5.63 × 107 | 2.4 × 106 | 2.7 × 106 | 7.83 × 106 | 6.5 × 106 | 3.2 × 107 | |
| ±0.6 × 106 | ±1.88 × 107b | ±0.45 × 106 | ±0.43 × 106 | ±0.37 × 106a | ±0.7 × 106b | ±0.26 × 107a | ||
| Bifidobacterium lactis | 12 | 3.03 × 104 | 3.6 × 105 | 3.46 × 104 | 4.06 × 104 | 9.16 × 104 | 9.4 × 104 | 1.6 × 105 |
| ±0.2 × 104 | ±0.6 × 105a | ±0.5 × 104 | ±0.3 × 104b | ±0.2 × 104a | ±0.4 × 104a | ±0.2 × 105a | ||
| 18 | 8.13 × 105 | 4.36 × 107 | 8.26 × 105 | 9.03 × 105 | 4.03 × 106 | 8.1 × 106 | 1.3 × 107 | |
| ±0.35 × 105 | ±0.83 × 107a | ±0.47 × 105 | ±0.25 × 105c | ±0.2 × 106a | ±0.17 × 106a | ±0.1 × 107a |
All measurements were performed in triplicate, and data are the means of three replicates. Statistical significance: ap < 0.001, bp < 0.01, and cp < 0.05 compared with the control.
Panose content of FRe To identify the presence of LAB growth stimulators in the FRs, the levels of the branched oligosaccharides panose and DHNA were measured. Based on the HPAEC analysis, the panose content of the FRe powder was 67.0 ± 1.4 mg/g, or 6.7%. In contrast, DHNA levels were below the limit of detection of 0.1 µg/g (data not shown). Chromatograms of control panose and FRe measured by high-performance anion-exchange chromatography are shown in Figure 1A and B, respectively. The relative peak area of panose increased significantly upon its exogenous addition to FRe (Figure 1C).
Chromatogram of panose in FRe by high-performance anion-exchange chromatography. A. authentic panose (Sigma-Aldrich, P2407), B. FRe and C. FRe added authentic panose.
Growth-stimulating effects FRe (0.2%) stimulated the growth of the lactic acid bacteria, L. acidophilus, S. thermophilus, and Bifidobacterium lactis. Specifically, the number of bacteria increased significantly to 11 – 53 fold after 12 – 18 h incubation, compared with negative control significant (p < 0.05; Table 2). In the positive control panose group, the numbers of L. acidophilus, S. thermophilus, and B. lactis increased in a dose-dependent manner when exposed to 0.02 – 0.2% panose. The number of L. acidophilus in media containing 0.2% panose increased approximately two- and four fold compared with the control after 12- and 18-h incubations, respectively. The number of S. thermophilus in media containing 0.2% panose was increased approximately two- and three fold compared with control after incubation for 12 and 18 h, respectively. Finally, the number of B. lactis in media supplemented with 0.2% panose was increased approximately three- and five fold compared with control after incubations of 12 and 18 h, respectively. Panose therefore stimulated the growth of B. lactis to a greater extent than either L. acidophilus or S. thermophilus (Table 2). After treatment with 0.01% and 0.1% DHNA, the number of bacteria was increased 13.8 – 16 fold compared with the control (p < 0.05; Table 2). The same effects were seen in each bacterial strain after both 12- and 16-h incubations, except that the growth of B. lactis was increased only after incubation for 18 h.
Several bifidogenic growth stimulators have been identified, including Sake-kazu (Furuta et al., 2007), ACNQ, DHNA (Isawa et al., 2002), and panose (Park et al., 1992; Mäkeläinen et al., 2009). Specifically, panose exerted bifidogenic effects, and significantly increased the growth of Bifidobacterium sp. in vitro (Mäkeläinen et al., 2009). In this study, FRe and DHNA stimulated the growth of all tested LAB (L. acidophilus, S. thermophilus, and B. lactis) compared with the control, promoting an increase of 13.8 – 16 fold after incubation for 12 and 18 h. In addition, supplementing the media with 0.2% panose stimulated the growth of L. acidophilus and S. thermophilus only two- to threefold compared with the negative control, showing that panose has weaker growth-stimulating effects than FRe. Similarly, the addition of 0.2% panose stimulated the growth of B. lactis more than the negative control, but less than FRe. These data are consistent with the observations of Mäkeläinen et al. who reported that panose enhanced the growth of bifidobacteria, especially Bifidobacterium lactis, decreased the growth of Bacteroides sp., and did not affect the growth of Lactobacillus sp. compared with the negative control.
In our study, supplementing growth media with exogenous DHNA stimulated the growth of all tested microorganisms. This is consistent with previous reports that and DHNA stimulated exclusively the growth of bifidobacteria among intestinal bacteria, including Clostridium sp., Lactobacillus acidophilus, and staphylococcus aureus (Isawa et al., 2002).
Panose is bifidogenic growth stimulator whose effects are specific to bifidobacteria. Compared with the growth rates of LABs in 0.04% panose and 0.2% FRe groups, the growth stimulating effect of 0.2% FRe (containing 0.014% panose) was superior to 0.04% panose against LABs. In addition, FRe strongly stimulated the growth of both bifidobacteria and lactic acid bacteria, including L. acidophilus, and S. thermophilus, suggesting that known or unknown substances, in addition to panose, mediate its growth-stimulating effects. We therefore suggest that FRe can be used as a growth stimulator of both bifidobacteria and lactic acid bacteria in pharmaceutical and food applications.
Acknowledgements This study was supported by a grant (B0012284) from the Busan Tradition Liquor Industry RIS, and was also supported by a grant (R0002840) the Ministry of Trade, Industry and Energy, Republic of Korea.
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