2021 Volume 27 Issue 1 Pages 57-61
In the present study, we examined the use of lactic acid bacteria as a starter for the production of pickled luffa. A starter culture of lactic acid bacteria was added to a brine containing 1% of glucose and NaCl that was used to make pickled luffa. Fermentation at 10 °C for 7 days resulted in an increase in the γ-aminobutyric acid content of pickled luffa in all the strains. In Lactococcus lactis NH-61, pickled luffa had good taste and its browning was suppressed. Lactobacillus sakei MMF-LS151 was excellent in regarding the acidity of brine, but led to the browning of pickled luffa. To the best our knowledge, this is the first report on the production of good tasting low-salt pickled luffa.
Luffa (Luffa cylindrica Roem.), called “Na-be-ra” in the Okinawa region, is a member of the Cucurbitaceae family. Luffa, together with the bitter gourd, is one of the traditional vegetables used in Okinawa as a main vegetable in summer season (Sakamoto, 2009). Various functions have been attributed to the luffa extract, including antioxidant activity (Du et al., 2006), and anti-inflammatory (Kao et al., 2012), and moisturizing effects (Umehara et al., 2018). Luffa also contains folic acid (Hirose et al., 2018) and is one of the vegetables with the best nutritional properties. However, luffa is rarely used as a vegetable outside of Okinawa. Instead, it is mainly used for skin lotion as “Hechimasui”, which is the water extracted from the plant's vascular bundle. Even in Okinawa, the use of luffa is limited to consumption as boiled and stir-fried dishes (Tahara, 2008). In addition, luffa cultivation in Okinawa is limited to the summer season (Sakamoto, 2009), and it is difficult to maintain the freshness of the fruit (Hirose et al. 2018), which is a major hindrance to expand for the vegetable. Therefore, it is necessary to develop new processing and cooking methods to increase the production of luffa. The present study focused on pickling, which have long been popular as a processing and preservation method for vegetables. Pickled vegetables can be used for a longer time than fresh vegetables. In contrast, recently, pickles with low salt content are being preferred more (Miyao, 2002). Ishikawa et al. (2003) and Sakai et al. (2014) reported the production of fermented pickles with low salt concentration using a lactic acid bacteria starter. Use of lactic acid bacteria starters can quickly decrease the pH of pickles and prevent contamination even at low salt concentrations.
Some strains of lactic acid bacteria produce γ-aminobutyric acid (GABA) using glutamic acid as a substrate (Higuchi et al., 1997). GABA is known to improve blood pressure (Inoue et al., 2003) and has been reported to reduce psychological stress (Yoto et al., 2012). Therefore, increasing GABA content during lactic acid fermentation is expected to contribute to the added nutritional value of pickled luffa.
In the present study, we examined the suitability of lactic acid bacteria for the production of low-salt pickled luffa and GABA.
Materials A commercial cultivar of luffa “Southern hechima” (Southanplant, Okinawa, Japan) was harvested when the diameter of the fruit was larger than 5 cm, during April-June 2017 at an experimental farm of the Okinawa prefectural agricultural research center. The following lactic acid bacteria strains were used in this study: Lactobacillus brevis NBRC 3345, NBRC 12005, NBRC 12520, NBRC 13110 (NITE Biological Resource Center, Chiba, Japan); Lactobacillus sakei MMF-LS151 (San-ei Sucrochemical, Aichi, Japan); Lactobacillus casei L-14, Lactococcus lactis 527 (Japan Daily Technical Association, Tokyo, Japan); Lactococcus lactis NH-61 (Okinawa Prefectural Agricultural Research Center, Okinawa, Japan) (Hirose et al., 2008). L. sakei and L. casei are now reclassified as Latilactobacillus sakei and Lacticaseibacillus casei, respectively (Zheng et al., 2020).
Production of pickled luffa with lactic acid bacteria starters Strains of L. brevis and L. sakei were cultured at 30 °C for 24 h using the MRS medium (Kanto Chemical, Tokyo, Japan). The L. casei and L. lactis strains were cultured in the same conditions using the M17 medium (Sigma-Aldrich, St. Louis, MO, USA). After incubation, cells were collected by centrifugation (1 700 × g, 10 min), washed, suspended in sterilized saline solution, and used as starters. Production of pickled luffa was performed according to the method of Ishikawa et al. (2003) with some modifications. Luffa fruits were washed in tap water for at least 10 min and then immersed in an acetic acid solution (2%) with sodium hypochlorite (100 ppm of the effective chlorine concentration) for 30 min. After washing with water thoroughly, luffa fruits were peeled and the edible parts were cut into 1 cm squares. Cut luffa (50 g) and 100 mL of sterilized brine (containing 1% of glucose and NaCl) were placed in a sterilized 200-mL glass beaker. The lactic acid bacteria starter was inoculated at 1 × 107 cfu/mL and fermented at 10 °C for 7 days. The experiments were performed in triplicate.
Preparation of an ethanol extract The shredded sample (15 g) was homogenized with 15 mL of ethanol for 1 min under ice cooling. After homogenization, the extract was separated by centrifugation (1 700 × g, 10 min). The ethanol extraction was repeated twice and the extracts were pooled and used for analyzing amino acids and absorption spectra.
Analysis of organic acids and amino acids The concentration of organic acids such as lactic acid and acetic acid in the brine was analyzed through HPLC (Hirose et al., 2017).
The concentration of amino acids such as GABA and glutamic acid in pickled luffa was analyzed by using the ultra-fast amino acid analysis system (Nexera X2, Shimadzu, Kyoto, Japan) (Kobayashi et al., 2018) equipped with a C18 HPLC column (YMC-Triart C18, 1.9 µm, 75 mm × 3.0 mm, YMC, Kyoto, Japan).
Analysis of the color Browning degree in pickled luffa was subjectively evaluated on a scale of 5 to 1 (5 = no browning, 1 = extreme browning) by trained nine adults. Absorption spectra of ethanol extract were measured at 280–400 nm (µQuant, Microplate Reader, Bio-Tek, Winooski, VT, USA).
Taste evaluation The taste of pickled luffa was evaluated by trained nine adults for flavor and overall palatability (5 = excellent, 1 = bad).
Analysis The pH of brine was analyzed by a pH meter (B-212, Horiba, Kyoto, Japan). BCP plate count agar (Merck Millipore, Darmstadt, Germany) was used for enumeration of lactic acid bacteria in brine (Hirose et al., 2017).
Statistical analysis The Steel-Dwass test (Excel statistics 2012, Social survey research information, Tokyo, Japan) was used for statistical analysis of color and taste evaluation (p < 0.05).
Production of pickled luffa Pickled luffa was produced through fermentation at 10 °C for 7 days using each lactic acid bacteria starter. In all strains, the pH stopped decreasing after 5–7 days of fermentation (data not shown). After fermentation, the viable cell count of the brine reached approximately 108 cfu/mL for all the four L. brevis and L. sakei MMF-LS151 strains (Table 1).
| Cells (cfu/mL) | pH | Organic acids | (mg/100mL) | ||
|---|---|---|---|---|---|
| Lactic acid | Acetic acid | ||||
| Lactobacillus brevis | NBRC 3345 | 3.7×108 | 4.0 | 211.5 | 86.2 |
| Lactobacillus brevis | NBRC 12005 | 3.6×108 | 4.3 | 102.2 | 32.8 |
| Lactobacillus brevis | NBRC 12520 | 4.9×108 | 3.9 | 207.1 | 75.8 |
| Lactobacillus brevis | NBRC 13110 | 4.4×108 | 3.8 | 325.7 | 80.2 |
| Lactobacillus sakei | MMF-LS151 | 9.3×107 | 3.7 | 431.2 | 24.0 |
| Lactobacillus casei | L-14 | 1.6×107 | 4.2 | 120.3 | 0.0 |
| Lactococcus lactis | 527 | 1.7×107 | 4.3 | 166.4 | 0.0 |
| Lactococcus lactis | NH-61 | 9.7×106 | 4.5 | 135.2 | 0.0 |
The starter culture was inoculated with lactic acid bacteria at 1 × 107 cfu/mL.
In the three L. brevis (NBRC 12520, NBRC 3345, and NBRC 13110) and L. sakei MMF-LS151 strains, the pH of the brines was 4.0 or lower, and the production of lactic acid was 200 mg/100mL or higher. The optimum concentration of lactic acid in pickles is 135 mg/100mL, and over 250 mg/100mL is considered too acidic (Ishikawa et al., 2003). Therefore, these four strains were expected to produce very sour pickles. In contrast, in L. casei L-14, and two strains of L. lactis (527, NH-61) strains, the viable cell count of the brine was only 106 to 107 cfu/mL. L. brevis NBRC 12005 and these three strains showed brine pH of 4.2–4.5, and the production of lactic acid was 102.2–166.4 mg/100mL. The pH of pickles is usually adjusted to 4.5–5.0 to control microbes and taste (Miyao, 2002). Furthermore, the lactic acid concentration of these pickled luffa was within the acceptable range. Therefore, these four strains were expected to result in the proper pickle sourness.
Ishikawa et al. (2003) reported that the salt concentration of hyposalt pickles is less than 3%. In the present study, pickled luffa could be produced with a salt concentration of 1%, without developing an undesired flavor. Therefore, further research would be needed to yield knowledge about the optimum salt concentration, and starter conditions and changes in bacterial flora during fermentation.
GABA production The GABA content of pickled luffa increased in all strains due to lactic acid fermentation (Table 2). L. brevis NBRC 12005, which is reported as a high GABA-producing strain (Yokoyama et al., 2002), showed a GABA content of 23.8 mg/100g in pickled luffa. Glutamic acid content increased only in L. sakei MMF-LS151. It has been reported that some strains of L. sakei do not require glutamic acid (Yamaji et al., 2005). This result suggests that this strain did not or hardly consumed glutamic acid in luffa during fermentation.
| GABA | Glutamic acid | ||
|---|---|---|---|
| Lactobacillus brevis | NBRC 3345 | 17.0 | 1.8 |
| Lactobacillus brevis | NBRC 12005 | 23.8 | 2.0 |
| Lactobacillus brevis | NBRC 12520 | 19.2 | 2.0 |
| Lactobacillus brevis | NBRC 13110 | 17.6 | 1.9 |
| Lactobacillus sakei | MMF-LS151 | 17.4 | 7.3 |
| Lactobacillus casei | L-14 | 20.9 | 1.8 |
| Lactococcus lactis | 527 | 17.8 | 1.2 |
| Lactococcus lactis | NH-61 | 18.3 | 1.7 |
| Control (before fermentation) | 5.3 | 5.8 |
Values were shown as mg/100 g.
Color of pickled luffa Lactic acid bacteria strains other than L. sakei MMF-LS151 significantly suppressed browning in pickled luffa (Table 3). Comparing the absorption spectra of ethanol extracts of pickled luffa, the absorption around 320 nm observed in the luffa before fermentation was significantly reduced in the strains such as L. sakei MMF-LS151 that showed browning (Fig. 1). Luffa is empirically known to cause browning by cutting or heating in cooking. Luffa contains phenylpropanoids (Umehara et al., 2018), which are known as substrates of enzymes responsible for the browning reaction (Nakabayashi, 1977). Particularly chlorogenic acid, one of the phenylpropanoids, shows reduced absorption at 290–370 nm due to enzymatic browning (Fujita and Tono, 1982). Furthermore, Zhu et al. (2017) observed that browning-related genes are differentially regulated in fresh-cut luffa fruits. In addition, suppression of browning by lactic acid fermentation has been reported (Imai, 1990) (Yamamoto et al., 1991). Therefore, it was hypothesized that the browning of pickled luffa was due to enzymatic browning and that lactic acid bacteria fermentation might suppress browning.
| Lactic acid bacteria | Browning degree | |
|---|---|---|
| Lactobacillus brevis | NBRC 3345 | 3.4 ± 0.7a |
| Lactobacillus brevis | NBRC 12005 | 3.0 ± 0.7a |
| Lactobacillus brevis | NBRC 12520 | 3.8 ± 0.8a |
| Lactobacillus brevis | NBRC 13110 | 3.2 ± 0.9a |
| Lactobacillus sakei | MMF-LS151 | 1.6 ± 0.5b |
| Lactobacillus casei | L-14 | 3.0 ± 0.8a |
| Lactococcus lactis | 527 | 3.8 ± 0.8a |
| Lactococcus lactis | NH-61 | 4.2 ± 0.8a |
Mean ± SD (n = 9). Browning degree in pickled luffa was subjectively evaluated on a scale of 5 to 1 (5 = no browning, 1 = extreme browning). Different letters indicate significant differences at P < 0.05 by Steel-Dwass test.
Absorption spectra of pickled luffa extracts
Luffas were fermented at 10 °C for 7 days. After fermentation, pickled luffa was extracted with ethanol. Each letter indicate: Control, before fermentation; a, Lactobacillus brevis NBRC 3345; b, L. brevis NBRC 12005; c, L. brevis NBRC 12520; d, L. brevis NBRC 13110; e, L. sakei MMF-LS151; f, L. casei L-14; g, Lactococcus lactis 527; h, L. lactis NH-61.
Taste evaluation The taste evaluation of pickled luffa showed no significant difference. However, a flavor of old pickles was perceived in L. brevis NBRC 12005, NBRC 3345, NBRC 13110, L. sakei MMF-LS151, and L. casei L-14. Therefore, their flavor scores were low (Table 4). Regarding the overall palatability, L. brevis NBRC 12520, NBRC 3345, and L. lactis NH-61 showed good scores, while L. brevis NBRC 12520 and NBRC 3345 were also perceived as strongly sour due to their high organic acid content.
| Lactic acid bacteria | Flavor | Overall palatability | |
|---|---|---|---|
| Lactobacillus brevis | NBRC 3345 | 2.9 ± 1.0 | 3.1 ± 0.7 |
| Lactobacillus brevis | NBRC 12005 | 2.9 ± 0.7 | 2.9 ± 0.9 |
| Lactobacillus brevis | NBRC 12520 | 3.7 ± 0.8 | 3.2 ± 0.4 |
| Lactobacillus brevis | NBRC 13110 | 3.1 ± 0.9 | 2.6 ± 1.0 |
| Lactobacillus sakei | MMF-LS151 | 2.9 ± 0.9 | 2.3 ± 0.9 |
| Lactobacillus casei | L-14 | 2.1 ± 1.1 | 2.6 ± 0.7 |
| Lactococcus lactis | 527 | 3.2 ± 0.8 | 2.8 ± 0.4 |
| Lactococcus lactis | NH-61 | 3.4 ± 0.8 | 3.4 ± 0.7 |
Mean ± SD (n = 9). The flavor and overall palatability of pickled luffa were subjectively rated on a score of 5 to 1 (5 = excellent, 1 = bad).
In conclusion, pickled luffa was produced using lactic acid bacteria starters. The GABA content of pickled luffa increased compared to the content before fermentation.
L.lactis NH-61 showed GABA productivity and flavorscore similar to that of other lactic acid bacteria strains. However, this strain was excellent at browning suppression and overall palatability. These finding suggested that L. lactis NH-61 was suitable for producing pickled luffa. Ishikawa et al. (2003) reported that lactic acid bacteria used in the dairy industry, and those isolated from vegetables are suitable for hyposalt pickles. Herein, L. lactis NH61 was a lactic acid bacterium isolated from a yogurt starter culture, and showed results consistent with those reported in the above-mentioned study. To the best of our knowledge, this is the first report on pickled luffa production. Using GABA content and taste as indexes, we plan to study the manufacturing conditions of pickled luffa in detail.
Acknowledgements This work was supported by a grant from the commissioned project study, Ministry of Agriculture, Fishery and Forestry, Japan (JPJ005336).
