Seaweeds are rich in minerals and dietary fiber and are thus typically used as a health food. One such seaweed,
Fermentation by living beneficial microorganisms may be useful in achieving this goal. Generally speaking, fermentation is a biochemical reaction that metabolizes high-molecular weight organic compounds into simpler molecules. Fermentation not only enhances the nutrient content of foods through the biosynthesis of vitamins, essential amino acids, and proteins, it also improves protein quality and fiber digestibility. Furthermore, fermentation improves micronutrient bioavailability and aids in the degradation of anti-nutritional factors (Achinewhu et al., 1998; Adewusi et al., 1999; Bae and Kim, 2010). Here, we report a microbial strain that effectively improved the functional qualities of
The microbial strains used in this study were isolated from traditional Korean fermented foods and maintained at the Food Microbiology Laboratory, Department of Food Science and Technology, Pukyong National University. Selection of a strain for fermentation was done by assaying the total phenolic compound (TP) content and antioxidant activity during fermentation of the
The TP content was determined according to a modified version of the Folin-Ciocalteu method using phloroglucinol as the standard (Kim et al., 2006a; Bae and Kim, 2010). The samples were diluted to match the measurable range of the spectrophotometer. A 0.1 mL aliquot of the diluted sample was mixed in an Eppendorf tube with 0.5 mL of 1 N Folin-Ciocalteu reagent. The mixture was allowed to stand for 3 min following the addition of 0.4 mL of 20% Na2CO3. The samples were incubated in the dark at room temperature for 45 min and then centrifuged at 1,600 g for 8 min. The optical density of the supernatant was measured spectrophotometrically with a GENios® microplate reader (Tecan Austria GmbH, Grodig, Austria) at 765 nm. The TP content (%) was calculated using a standard graph. The TP content in the non-fermented or fermented extract was calculated using the following linear equation based on the calibration curve:
Y=0.5337X+0.3615, r2 = 0.9874
where, Y is the absorbance and X is the total phenolic contents in milligram phloroglucinol equivalents per gram extract.
According to the manufacturer’s instructions, portions of each yeast strain were picked up with a sterile loop from the YM plates and then suspended in API basal medium ampoules (Gunde？ et al., 2001). The suspensions were used to fill the cupules of the test strip (API 20C AUX system; bioMerieux, Marcy l’Etoile, France). The API trays were inoculated and incubated for 72 h at 30°C. The growth in each well was recorded after 24, 48, and 72 h of incubation. A profile number based on the reactions was assigned and identifications were made by reference to the API Analytical Profile Index (bioMerieux). Cupules showing turbidity that was significantly greater than that in the negative control cupules were considered positive (Fenn et al., 1994).
Filtered extracts were analyzed for pH, salinity, and proximate composition. The moisture content was measured by oven-drying at 105°C to a constant weight. The contents of crude ash, crude protein, and crude lipid were determined by standard methods (Association of Official Analytical Chemicals, 1995). Reducing sugars were measured by the Somogy method with a minor modification (Somogyi, 1952). All experiments were carried out in triplicate. The data are shown as mean values.
The pH was measured with a tip probe electrode. Salinity was measured by converting the chlorine content into sodium chloride using a YSI 63 pH meter (YSI Inc., Yellow Springs, OH, USA).
The determination of FAAs was done by ion-exchange chromatography using a Hitachi model L-8900 amino acid analyzer (Hitachi Co. Ltd., Tokyo, Japan) with methods adapted from the literature. A total of 10 μL of supernatant filtered through a 0.45-μm syringe filter (Advantec, Tokyo, Japan) was applied to an ion exchange column (#2622SC; Hitachi Co. Ltd.) using lithium citrate buffer.
DPPH radical scavenging activity was measured as described by Qureshi et al. (2010). A total of 1 mL of non-fermented or fermented extract (diluted 100-fold) was added to 1 mL of 0.2 mM DPPH in ethanol. After mixing vigorously for 30 s, the solution was incubated at 37°C for 30 min. The DPPH radical scavenging activity of the solution was measured using a GENios® microplate reader (Tecan Austria GmbH) at 517 nm. The DPPH radical scavenging activity was calculated using the following equation, in which
Seaweeds possess metabolites with useful biological activi-
Change of total phenolic contents in Eisenia bicyclis water extract by microbial fermentation
ties, including antifungal, antioxidant, antiviral, and antitumor activities (Noda et al., 1989; Nagayama et al., 2002; Kim et al., 2006b). It was previously reported that a seaweed extract containing commercial enzymes showed enhanced antioxidant (Heo et al., 2005) and anticoagulant activities (Athukorala et al., 2006). However, such enzymes are expensive. Therefore, our laboratory has focused on the microbial fermentation of seaweeds as an alternative method (Bae and Kim, 2010; Eom et al., 2010; Song et al., 2011b). The biological activities of plant materials have been reported to relate to their content of phenolic compounds (Lee et al., 2009), and these activities are based on the physiological functionalities of polymers of polyphenol (McDougall et al., 2005). In this study, we used an increase in TP content to judge the effectiveness of microbial fermentation. The change in antioxidant activity during fermentation of the extract was monitored as an indicator of the biological activity of the sample.
As shown in Table 1, the TP content of the
Polyphenolic compounds are known to possess antioxidant activity (Jimenez-Escrig et al., 2001). We measured the antioxidant activity of the extract before and after microbial fermentation. As shown in Fig. 1, the DPPH radical scavenging activity rose as the TP content of the
[Fig. 1.] Effect of microbial fermentation on DPPH radical scavenging activity of Eisenia bicyclis water extract. Experiments were repeated three times. LB-1 LB-2 and LB-3 stand for Lactobacillus sp. LB-1 LB-2 and LB-3 respectively. BS-1 and BS-2 are Bacillus sp. BS-1 and BS-2 respectively. YM-1 2 and 3 are yeast strains isolated in this study.
Physiological characteristics of yeast YM-1 strain
observed in the other samples, reflecting the different physiological functions of the enzymes from the other microorganisms (De Mot and Verachtert, 1985; Bar-Shimon et al., 2004). Considering the enhanced TP content and DPPH radical scavenging activity obtained through microbial fermentation, we selected yeast strain YM-1 for our subsequent experiments.
The selected strain (YM-1) was identified based on an analysis of its physiological characteristics and the API Analytical Profile Index as described in the Materials and Methods. The physiological characteristics of the strain are given in Table 2. YM-1 exhibited 94.9% identity with
Microbial fermentation causes changes in food constituents (Jeng et al., 2007). Accordingly, it was expected that a change in the properties of
We additionally investigated the effect of fermentation by YM-1 on the FAA content of
The FAA content was also affected by
Change of proximate components pH and salinity by Candida utilis YM-1 fermentation in Eisenia bicyclis water extract
Effect of Candida utilis YM-1 fermentation on free amino acid content of Eisenia bicyclis water extract
progressed (Table 4). Similar to the reducing sugar content, the FAAs appeared to be used as a substrate for yeast growth during fermentation. For example, the phosphorylated serine content, which has proposed anti-obesity effects (Cremades et al., 2004), increased until 5 days of fermentation.
Considering the results obtained in the current study, we conclude that