2019 年 25 巻 6 号 p. 785-791
Dendropanax morbifera (DM) extract was co-fermented by Bacillus subtilis HA and Lactobacillus plantarum EJ2014 for producing poly-γ-glutamic acid (γ-PGA) and γ-aminobutyric acid (GABA). The first fermentation with B. subtilis HA resulted in a viscous broth with pH 7.85, 0.01% acidity, 0.72 mg/g peptides, 17.39 U/g protease activity, 8.44 log CFU/mL B. subtilis, 4.96 Paosn consistency index, and 45.84 mg/mL mucilage. For the second fermentation with L. plantarum, the broth was enriched with skim milk. The co-fermented broth indicated 0.5% acidity and pH 5.1. The viable cell counts of B. subtilis decreased, whereas those of L. plantarum increased, as did the peptide content. The MSG precursor was effectively converted to GABA, which showed a concentration of 12.80 mg/mL. Therefore, the co-fermented DM extract was enriched with γ-PGA, GABA, peptides, and probiotics by the serial co-fermentation, and could be used as a potential ingredient for developing functional foods.
Dendropanax morbifera (DM) is a plant endemic to Jeju Island and the southern coast of Korea. The roots, leaves, seeds, and stems of DM are used in Oriental medicine for treating headaches, infectious diseases, skin diseases, and other maladies (Moon, 2011). DM stem extracts exhibit strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity as well as cytotoxic activities against human tumor cell lines, whereas methanolic leaf and stem extracts contain antioxidant and anticancer compounds (Hyun et al., 2013), suggesting that the plant is a potential source of natural drugs for treating various diseases.
Bacillus strains produce poly-γ-glutamic acid (γ-PGA) as a capsular or extracellular viscous biopolymer composed of units of D-glutamic acid, L-glutamic acid, or both. γ-PGA is biodegradable, non-toxic, and non-allergenic, and is used in the food, medical, and wastewater industries (Ogunleye et al., 2015). The selection of carbon sources for γ-PGA production is strain-dependent, although glucose and glycerol have been shown to be effective, particularly when used in combination (Ko et al., 1998). Nevertheless, some strains also require glutamate in the medium to be able to produce γ-PGA (Xu et al., 2005).
Lactic acid bacteria (LAB) include the Lactobacillus and Leuconostoc genera, which are known for their health-promoting properties (Fijan et al., 2014). Lactic acid bacteria such as Lactobacillus brevis, L. plantarum, L. delbrueckii, L. paracasei, and Lactococcus lactis have shown glutamate decarboxylase activity and ability to produce GABA (Das et al., 2015; Kwon et al., 2016). GABA acts as an inhibitory neurotransmitter in the mammalian central nervous system and has also been reported to have antihypertensive, anticancer, and tranquilizing activities (Cha et al., 2011; Cho et al., 2007; Joye et al., 2011). Thus, enrichment of foods with GABA is desirable, and several studies have reported GABA-fortified herbal extracts, seaweed, and soybean (Cha et al., 2011; Kim et al., 2014; Kwon et al., 2016; Lim et al., 2016).
Therefore, the aim of the current study was to optimize the production of both γ-PGA and GABA in DM extract, using Bacillus subtilis and Lactobacillus plantarum in a serial co-fermentation. The physicochemical properties of the fermented DM extract were determined. The production of bioactive compounds including γ-PGA, GABA, peptides, and probiotics was analyzed. It is expected that the developed fermented product could be used as an ingredient for functional food development.
Materials Dendropanax morbifera was purchased from BKbio (Jeju, Korea). Monosodium-l-glutamate (MSG) and glucose were obtained from CJ CheilJedang Corporation (Seoul, Korea) and Samyang Genex Corporation (Incheon, Korea), respectively. Skim milk was purchased from Seoul Milk ICA (Seoul, Korea). Angiotensin-converting enzyme (ACE) and hippuryl-L-histidyl-L-leucine (HHL) were purchased from Sigma (St. Louis, MO, USA). All chemicals used were of analytical grade.
Strains and starter cultures Lactobacillus plantarum EJ2014 was grown on Difco™ Lactobacilli MRS agar (BD, Sparks, MD, USA) at 30 °C for 24 h. Then, a single colony was inoculated in sterilized MRS broth and cultured at 30 °C for 24 h to prepare the starter culture.
The starter of B. subtilis HA (KCCM 10775P) was cultured in nutrient broth (NB) at 42 °C for 24 h. The cells were harvested and suspended in 10 times the volume of sterilized distilled water.
Preparation and composition of the DM extract Stems and leaves (1:1) of DM were mixed with 10 times the volume of water and heated at 95 °C for 8 h for producing the DM extract. The extract was not evaporated, and was used in its original liquid form.
The soluble solids, reducing sugar, polyphenol and flavonoid content of the DM extract were determined using a standard methodology (Nielsen, 2010). The mineral content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES; Optima 700DV, Perkin Elmer, Waltham, MA, USA). As a pretreatment, the DM extract was heated at 550–600 °C for 12 h, solubilized with 10 mL of HCl, and filtered to obtain a soluble fraction. The concentrations were measured using a standard curve.
Co-fermentation process To prepare the culture medium, the DM extract was sterilized by heating at 121 °C for 15 min, and then mixed with sterilized 30% MSG and 30% glucose solutions, adjusting the final concentrations to 5% MSG and 3% glucose. The B. subtilis HA starter was inoculated at a 5% concentration, and incubated by shaking (160 rpm) at 42 °C for 2 days. Subsequently, for the second fermentation, glucose (1.5%) and skim milk (0.0–5.0%) were added. The starter culture of L. plantarum EJ2014 was inoculated at a 1% concentration in MRS agar, followed by incubation at 30 °C for 5 days.
pH and acidity The pH of the fermented DM extract was determined by pH-meter (Model 420+, Thermo Orion, Beverly, MA, USA). Titratable acidity (%, w/v) was determined by measuring the amount of 0.1 N NaOH required to reach a pH of 8.3, and expressed as concentration of lactic acid (%, v/v).
Mucilage analysis The fermented DM extract (10 mL) was centrifuged at 24,948 ×g for 20 min. The supernatant was mixed with twice the volume of isopropanol, and the precipitate was washed with 95% ethanol and dried at 50 °C for 24 h to yield crude mucilage. The consistency properties of the fermented broth were determined using a rheometer (HAKKE RheoStress 1, Karlsruhe, Germany) fitted with a cone plate device (Plate PP35Ti, diameter 3.5 cm, 1.0 mm gap) at 20 °C. The sample (1 mL) was loaded between the plate and cone plate device, and the consistency index was expressed as Pa•sn.
The molecular weight of the mucilage was determined by gel permeation chromatography. The crude mucilage was dissolved in distilled water and centrifuged at 15,710 ×g for 10 min. The sample was analyzed on a Knauer K-501 HPLC system (Berlin, Germany) fitted with an RI-detector K2301 and Shodex OHpak SB-805 column (Showa Denko, Tokyo, Japan). The mobile phase was distilled water, pumped at a flow rate of 1 mL/min. The molecular weight of crude mucilage was calculated using the peak retention time obtained. Purified dextran (50 kDa; American Polymer Standards, Mentor, OH, USA) was used as a standard.
Viable bacterial counts To determine the viable bacterial counts of B. subtilis, the fermented DM extract was serially diluted with sterilized water. Subsequently, a 20 µL sample was plated onto an MRS agar plate and cultured at 42 °C for 24 h to confirm colony forming units (CFU). To selectively determine the viable bacterial counts of L. plantarum EJ2014 after the co-fermentation, the MRS agar plate with mixed strains was cultured at 30 °C for 48 h to delay the growth of B. subtilis, and the colonies of L. plantarum were counted.
GABA and amino acid analysis The GABA and glutamic acid contents in the co-fermented DM extract were determined by HPLC. using a modified Waters AccQ Tag Method (Armenta et al., 2010).
As a pre-treatment, the sample was hydrolyzed in 6 N HCl at 110 °C for 22 h and filtered through a 0.45-µm syringe filter. Amino acid derivatization with AccQ•Tag reagents (Waters, Milton, MA, USA) was conducted according to the manufacturer's protocol. Hydrolyzed sample (10 µL) was mixed with 70 µL of AccQ•Tag Ultra borate buffer, and 20 µL of AccQ-Fluor reagent, followed by heating for 10 min at 55 °C. The sample was analyzed by HPLC (Alliance 2695, Waters) using an AccQ•Tag column (Waters; 3.9 × 150 mm) at 25 °C. A gradient elution was performed at a flow rate of 1 mL/min for 55 min, using a mixture of Waters AccQ•Tag Eluent A: distilled water (1:10) as eluent A, acetonitrile as eluent B, and distilled water as eluent C. Absorbance was measured at λ excitation (250 nm) and λ emission (395 nm) using a fluorescence detector.
Analysis of protein hydrolysis The protein in the co-fermented DM extract was analyzed by SDS-PAGE. As a pretreatment, 12.5 µL of supernatant was centrifuged at 15,710 ×g for 10 min, mixed with an equal volume of 2× Laemmli sample buffer-β-mercaptoethanol (19: 1), and heated at 100 °C for 10 min to induce denaturation. Electrophoresis was performed at 120 V for 50 min. The resulting gel was stained with InstantBlue solution, and the molecular weights of the proteins were estimated visually by their mobilization in the gel. A Page Ruler Prestained Protein Ladder (10–180 kDa; Thermo Scientific, Waltham, MA, USA) was used as the molecular weight marker.
To determine the peptide content, a modified Anson's method was applied. Briefly, the fermented broth (4 mL) was diluted 10-fold using distilled water and centrifuged at 15,710 ×g for 15 min. The supernatant (0.7 mL) was mixed with 0.7 mL of 0.44 M trichloroacetic acid (TCA) and incubated at 37 °C for 30 min. The precipitate was removed by centrifugation at 15,710 ×g for 10 min, and the supernatant (1 mL) was mixed with 2.5 mL of 0.55 M Na2CO3 and 0.5 mL of Folin-phenol reagent. After incubating at 37 °C for 30 min, absorbance was measured using a spectrophotometer (Ultrospec®2100 pro, Amersham Biosciences, Piscataway, NJ, USA) at 660 nm. The peptide content in the supernatant was determined using tyrosine as a standard.
Angiotensin-converting enzyme inhibition To evaluate the inhibitory effect of the angiotensin-converting enzyme (ACE), the sterilized co-fermented DM extract was mixed with twice the volume of isopropanol, and the supernatant was concentrated. The substrate (HHL) was dissolved in 50 mM boric acid buffer (pH 8.3), and ACE was prepared using a previously published method (Cushman and Cheung, 1971) Briefly, 50 µL of the 7.6-M HHL substrate was mixed with 15 µL of supernatant and 50 µL of 0.1-unit ACE solution, stirred, and incubated at 37 °C for 40 min. Then, 125 µL of 1 N HCl was added to stop the reaction, followed by adding 1 mL ethyl acetate, stirring for 10 min, and centrifuging at 9296 ×g for 10 min. A supernatant aliquot (0.5 mL) was collected, dried, and dissolved in 1 mL of water. The optical density of the sample was measured at 228 nm and calculated using Eq. 1:
 |
Where Ec is the initial absorbance of distilled water, Ecb is the absorbance of distilled water after stopping the reaction, Es is the initial absorbance of the sample, and Esb is the absorbance of the sample after stopping the reaction.
The Bradford assay was performed to determine the degree of hydrolysis (DH) of the milk protein, calculated using Eq. 2:
 |
Where CH is the casein hydrolysate and TCH is the TCA-treated casein hydrolysate.
Statistical analysis Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS Version 23.0, SPSS Inc. Chicago, IL, USA). The mean and standard deviation were calculated. Measurements were performed in triplicate. Significant differences among means were determined by one way-ANOVA and Duncan's multiple range test (p < 0.05).
Physicochemical properties of the DM extract The DM extract had dark brown color and typical herbal flavor with slight sourness. The physicochemical properties of the DM extract are summarized in Table 1. The extract showed pH 5.01, 0.23% acidity, 45.5 mg/g soluble solids, and it also contained various minerals among which potassium and calcium predominated.
| Component | Value | |
|---|---|---|
| pH | 5.01±0.01 | |
| Acidity (% lactic acid) | 0.23±0.01 | |
| Soluble solids content (mg/g) | 45.5±1.60 | |
| Reducing sugars (mg/g) | 8.80±0.01 | |
| Polyphenols (mg/g) | 6.02±0.06 | |
| Flavonoids (mg/g) | 1.38±0.01 | |
| Minerals (µg/g) |
Na | 154.53 |
| Ca | 366.29 | |
| K | 1424.52 | |
| Mg | 182.22 | |
| P | 186.16 | |
| Fe | 2.11 | |
| Mn | 44.54 | |
pH, acidity, and viable cell counts after the alkaline fermentation The pH of the DM extract fermented by B. subtilis tended to increase, indicating 7.85 after 2 days of fermentation (Table 2). By contrast, the acidity of the fermented DM extract decreased from 0.12% at the start of the fermentation to 0.01% after 2 days. B. subtilis HA produces an alkaline protease that is responsible for the generation of peptides and various metabolites that result in alkaline conditions (Kembhavi et al., 1993). This increase in alkalinity agrees with our previous results (Yoon et al., 2018).
| Fermentation time (days) | pH | Acidity (% lactic acid) | Consistency index (Pa·sn) | Flow behavior (n value) | Mucilage content (mg/mL) | Viable bacterial counts (log CFU/mL) |
|---|---|---|---|---|---|---|
| 0 | 6.16±0.01c | 0.12±0.02a | 0.00±0.00c | 0.87 | 0.00±0.00b | 7.42±0.21c |
| 1 | 6.56±0.01b | 0.11±0.02a | 4.96±0.23a | 0.24 | 45.84±0.81a | 8.89±0.10a |
| 2 | 7.85±0.01a | 0.01±0.00b | 4.63±0.04b | 0.25 | 44.87±1.46a | 8.44±0.30b |
The initial viable bacterial count of B. subtilis HA was 7.42 log CFU/mL, which increased during the fermentation, indicating 8.44 log CFU/mL after 2 days (Table 2). This increase is similar to that attained using Oriental raisin extract as a growth medium (Yoon et al., 2018) and indicates that herbal extracts are suitable for fermentation with B. subtilis HA.
Mucilage and consistency analysis The mucilage production and consistency index of the first-fermented DM extract are shown in Table 2.
Addition of glutamic acid and glucose to the DM broth for fermenting by B. subtilis has been reported to boost the production of γ-PGA in a co-fermentation (Kunioka and Goto, 1994; Yu and Oh, 2011). For this reason, the DM extract used here was enriched with both glucose and MSG.
During the first fermentation, the viscosity of the DM extract increased as a result of the production of mucilage, which indicated 45.84 mg/mL and a consistency index of 4.96 Pa·sn after 1 day. Moreover, HPLC analysis revealed that the crude mucilage had a γ-PGA content of 574.6 mg/g, which was higher than that reported in a previous study (8.3 mg/g) that used B. subtilis HA and Leuconostoc mesenteroides subsp. dextranicum for fermenting carrot pomace (Jung and Lee, 2009). Nevertheless, further fermentation resulted in a slight decrease in the mucilage content and consistency, which may have been due to the enzymatic degradation of γ-PGA (Seo et al., 2008).
The molecular weight (MW) of γ-PGA is closely related to mucilage consistency, and it can vary depending on the pH, temperature, and culture method (Seo et al., 2008; Shih and Van, 2001). The MW of γ-PGA produced by fermentation with B. subtilis HA of a defined medium consisting of glutamate, glucose and salts was 1220 kDa (Seo et al., 2008), whereas the MW of the γ-PGA isolated here from crude mucilage was 1788 ± 66.67 kDa. Thus, alkaline fermentation using B. subtilis HA is able to produce γ-PGA with a high MW.
pH and acidity of the co-fermented DM extract The co-fermentation using B. subtilis and L. plantarum can provide the synergy needed to facilitate GABA production by lactic fermentation because B. subtilis synthesizes pyridoxal 5-phosphate (Belitsky, 2004), which is a cofactor of glutamate decarboxylase. After 2 days of the first fermentation with B. subtilis HA, the DM extract was enriched with skim milk and subsequently fermented by L. plantarum for 5 days. As shown in Fig. 1, the pH of the DM extract co-fermented without skim milk decreased from 7.07 to 5.47. On the other hand, the pH of the DM extract co-fermented with 2.5% and 5% skim milk drastically decreased after 1 day of co-fermentation, but then it slightly increased. Generally, lactic fermentation in the presence of fermenting sugars decreases the pH because of the lactic acid production. However, lactic fermentation with MSG as a precursor for GABA production resulted in a pH increase.
Changes in (a) pH and (b) acidity of DM extract fortified with skim milk (0%, 2.5%, 5%) and co-fermented by B. subtilis HA and L. plantarum EJ2014. Different letters in the same group indicate significant differences by Duncan's multiple range test (p < 0.05)
The acidity changes of the co-fermented DM extract are shown in Fig. 1. The acidity of the DM extract co-fermented without skim milk increased from 0.14% to 0.4%. By contrast, the acidity of the DM extract supplemented with skim milk exhibited a substantial increase after 1 day of co-fermentation, followed by a decrease. The higher acidity of the DM extract containing skim milk suggests that the lactose content in skim milk may affect the lactic acid production in the final co-fermented product. Thus, the acidity in the fermented DM extract could be regulated by adding lactose as a fermenting sugar.
Viable bacterial counts of the co-fermented DM extract The effect of the skim milk content on the viable bacterial counts in the co-fermented DM extract is displayed in Fig. 2.
Viable bacterial counts of DM extract fortified with skim milk (0%, 2.5%, 5%) and co-fermented by B. subtilis HA and L. plantarum EJ2014
In the co-fermented DM extract without skim milk, the viable cells of B. subtilis HA gradually decreased for 1 day, but then increased gradually to 7.74 log CFU/mL after 5 days. However, the viable bacterial counts of L. plantarum EJ2014 decreased from 8.40 log CFU/mL to 7.93 log CFU/mL. On the other hand, the co-fermented DM extracts containing skim milk showed a different bacterial growth pattern, with B. subtilis HA decreasing gradually and L. plantarum EJ2014 increasing for 1 day and then remaining stable or showing only a slight decrease. The co-fermented DM extract with 5% skim milk showed the highest viable cell counts of L. plantarum and the lowest viable cell counts of B. subtilis after 5 days. Thus, addition of skim milk facilitated the second (lactic) fermentation by encouraging the growth of L. plantarum and inhibited the first fermentation by decreasing the growth of B. subtilis.
GABA and amino acid content in the co-fermented DM extract Free amino acids in the co-fermented DM extract were determined using HPLC, revealing the complete conversion of glutamic acid to GABA. In the broth with 2.5% skim milk, the initial glutamic acid content was 16.15 mg/mL, which decreased to 0 mg/mL by the end of the co-fermentation. By contrast, the GABA content increased from 0 to 12.80 mg/mL. The GABA production attained here was higher than that obtained using a dropwort-based broth co-fermented by Leuconostoc mesenteroides SM and L. plantarum K154 (Kwon et al., 2016), although slightly lower than that obtained by a similar co-fermentation using an Oriental raisin-based broth (Yoon et al., 2018). The high production of both γ-PGA and GABA attained here indicates that MSG as a precursor was efficiently bioconverted by the serial co-fermentation using B. subtilis and L. plantarum.
D. morbifera, and in particular its ethanol extract, has been reported to contain antimicrobial compounds (Nakamura et al., 2018). However, the aqueous extract prepared here did not affect the growth of the lactic acid bacteria used in the fermentation. Furthermore, fermentation of herbal extracts has been reported to enhance their therapeutic properties through changes in their composition including fortification with bioactive compounds produced by the fermentative microorganisms (Hussain et al., 2016). Even though a thorough analysis of the composition of the DM extract before and after fermentation is beyond the scope of this study, it is expected that the fermented extract would provide additional health benefits because of its content of GABA, mucilage, and probiotics.
Peptide content of the co-fermented DM extract The effect of skim milk addition on the production of peptides is shown in Fig. 3. The peptide content tended to increase during the fermentation, suggesting that the protease activity increased. This is also related to the pH of the fermented DM extract, which markedly decreased when skim milk was added (Fig. 1). The DM extract with 5% skim milk indicated the highest peptide content (0.95 mg/g), whereas that without skim milk showed the lowest.
Peptide content of DM extract during the first fermentation and after fortification with skim milk (0%, 2.5%, 5%) for the co-fermentation with B. subtilis HA and L. plantarum EJ2014
The presence of peptides in the co-fermented DM extract suggests that the protease produced by B. subtilis HA hydrolyzes the milk protein. Thus, supplementation of skim milk during the co-fermentation can be a strategy to enhance the production of peptides. The SDS-PAGE analysis of the co-fermented DM extract is shown in Fig. 4. The casein fraction and β-lactoglobulin disappeared, whereas low molecular weight peptides (<10 kDa) may have been generated as suggested by the results of peptide hydrolysis (Fig. 3) although they could not be detected in the gel.
SDS polyacrylamide gel electrophoretic patterns of co-fermented DM extract. ST = molecular weight marker. The casein and β-lactoglobulin bands are highlighted
Angiotensin-converting enzyme (ACE inhibitory effect) Fermented soybean products such as natto are known to contain angiotensin-converting enzyme (ACE) inhibitors, which show anti-hypertensive effects (Okamoto et al., 1995). The ACE inhibitory effect of the co-fermented DM extract is shown in Table 3. The highest peptide concentration (166.77 ± 26.29 mg/mL) corresponded to the highest ACE inhibitory activity (90.82 ± 1.87%). This result agrees with previous research, suggesting that skim milk can be a source of bioactive peptides with ACE inhibitory activity (Ricci et al., 2010). Because milk protein has to be hydrolyzed to release those peptides, the degree of hydrolysis (DH) was calculated and yielded a value of 83.45 ± 3.28%, indicating that most of the milk protein was hydrolyzed during the co-fermentation.
| Peptide content (mg/mL) | ACE inhibition rate (%) |
|---|---|
| 166.77±26.29a | 90.82±1.87a |
| 16.67±2.63b | 53.15±0.26b |
| 8.34±1.32c | 49.82±0.08c |
| 1.66±0.26d | 43.76±1.08d |
Co-fermentation of DM extract by B. subtilis and L. plantarum was performed in the presence of MSG and skim milk, resulting in the production of bioactive components such as γ-PGA, GABA, peptides, and probiotics. The co-fermentation was optimized by adding 5% MSG and 3% glucose to the DM extract fermented by B. subtilis, and adding 2.5% skim milk for the second fermentation with L. plantarum. The MSG precursor was effectively converted to both γ-PGA and GABA by the serial co-fermentation. The high production of bioactive components attained demonstrates that serial co-fermentation using B. subtilis and L. plantarum could be a novel strategy for increasing the value of natural products.
Acknowledgements This research was financially supported by the Ministry of Trade, Industry and Energy (MOTIE) and the Korea Institute for Advancement of Technology (KIAT) through the Community Business Activation Program (P0200600004 Development of High-Value Region Specialized Food and Customized Commercialization for Activating Social Economy).
