Preservation Potential of Galangal Water Extract on Tofu

Galangal ( Alpinia galanga (L.) Willd.) has been long used for medicinal purposes. It showed promising effects on the food-spoilage microorganisms and is commonly used as spices in various cuisines. This study aimed to evaluate the microbial growth inhibitory activity and the preservation potential of galangal water extracts on tofu. The water extracts were prepared by infundation method in the concentrations of 5, 10, and 20%w/v. The phytochemical compounds in the extracts were screened as per standard methods. The tofu was preserved by immersion method for eight days under room temperature with an evaluation time point of two days. The microbial growth inhibitory activity was evaluated by indirect optical density (OD)-based enumeration, and the OD reduction by the extracts was calculated accordingly. The preservation potential was calculated from the changes of the physical characters on tofu evaluated by sensorial analysis. Galangal water extracts contained tannins and terpenoids. It concentration-dependently inhibited microbial growth on tofu, and the extract showed the best OD reduction at a concentration of 20%. It changed the color of tofu but maintained its texture. It also masked the odor of fresh tofu with the aromatic galangal scent. All three tested concentrations showed an equal preservation potential of 2 days. Our data suggested galangal water extract in the optimum concentration of 20% inhibited the microbial growth on tofu and potentially preserved it for two days. This moderate preservation effect might be attributable to its tannins and terpenoids content.


Introduction
The use of galangal (Alpinia galanga (L.) Willd., lengkuas in Indonesian, family Zingiberaceae) for medicinal purposes in the traditional systems has been widely described (Chouni and Paul, 2018;Hookheaw and Neamsuvan, 2019;Silalahi et al., 2018). The antimicrobial activities of rhizomes of this plant, among others, have been described elsewhere. The essential oil was mainly well studied for the said activity (Hamad et al., 2016;Prakatthagomol et al., 2011;Zhou et al., 2021). However, the antimicrobial activity of galangal water extract was also reported (Pillai et al., 2019;Rini et al., 2018). These microbial activities enabled it to be developed further into a natural food preservative.

PHARMACY: Jurnal Farmasi Indonesia
Galangal is also commonly used as a spice in Southeast and South Asian cuisines. It is prevalent for protein-based savory meals (Das et al., 2020). Hence, the familiarity with the aroma and taste of galangal might support it as a food preservative with a better acceptance. As the water extract was proven to be more suitable to be applied as a natural preservative for food, this study evaluated the potential use of galangal water extract as the natural food preservative on tofu (Hamad et al., 2019).
Tofu is processed from soya beans and is popular in East and Southeast Asia. It has a relatively short shelf life due to microbial spoilage as it contains water and proteins, around 85 and 8%, respectively (USDA, 2019). In addition, the reports on the contamination of tofu by pathogenic microorganisms were mentioned elsewhere (Ananchaipattana et al., 2012;Lee et al., 2017). Hence, the preservation efforts of tofu for preventing microbial decomposition and pathogenic contaminations were critically needed.
This study specifically evaluated the microbial growth inhibitory activity of galangal water extract in tofu and the effects on its physical characters during room temperature storage for eight days.

Research Method Chemical and Reagents
The galangal rhizomes (collected from Bawang, Indonesia), white firm tofu (bought from a market in East Purwokerto, Indonesia), sterilized water, nutrient broth (Oxoid, Thermo Fisher Scientific, USA), and nutrient agar (Oxoid).

Instrument
The optical density of the samples was read with the UV-Vis spectrophotometer (UV-1240, Shimadzu, Japan).

Methods
The plant materials were authenticated in the Laboratory of Plant Taxonomy, Jenderal Soedirman University, Purwokerto, Indonesia (Ref. 139/FB.Unsoed/TaksTumbVI/2015). The rhizomes were dried by direct sun-drying method and pulverized to the fine degree accordingly. According to the previous study, the water extract was prepared by infundation method in sterilized water at the concentrations of 5, 10, and 20%w/v (Hamad et al., 2017).
Phytochemical screening was subjected to the obtained water extracts. The presence of alkaloids, flavonoids, saponins, sterols, tannins, and terpenoids in the extract was detected as per standard reagents for the respective compounds (Table 1) (Patel et al., 2016;Zohra et al., 2012).
The tofu was cubed in 2x2x2 cm and immediately immersed in the boiling water to reduce the microorganism load on the surface. The cubes were aseptically placed in 100 ml of water extracts or sterilized water (as the negative control) and tightly closed afterward. They were stored at room temperature and protected from direct light and heat for eight days. On days 2, 4, 6, and 8, tofu from each sample were evaluated for microbial growth and physical characteristics as detailed in our previous works (Hamad et al., 2019;D Hartanti et al., 2019).

Data Analysis
The experiment was conducted in triplicate, and data were presented in mean value ± standard deviation (SD). The effect of extract concentration and storage time on cultured tofu's optical density (OD) was evaluated by two-way ANOVA. The OD reduction of each extract was calculated according to Formula 1. (1) ODc was the OD of the negative control, while ODe was the OD of the tested extract. The comparison between OD reductions among the extracts was calculated by one-way ANOVA. Duncan's test was used as the post-hoc for both analyses. The analysis was conducted on the SPSS ver. 19 (IBM -SPSS, USA), and the difference was considered significant at p-value < 0.05.

Results and Discussion
The galangal water extract contained tannins and terpenoids (Table  1). Tannins are polyphenol compounds with good solubility in water, particularly in the extraction with the higher temperature as in this study (Smeriglio et al., 2017). The presence of tannins in galangal was previously reported in rhizomes collected in Vijayawada (India), Sidoarjo (Indonesia), El-Fayoum (Egypt) (Johnley et al., 2020;Rashad et al., 2016;Rini et al., 2018). On the other hand, terpenoids are slightly soluble in water. However, those h lower molecular weight can be better extracted with water under the higher extraction temperature (Martins et al., 2017). The compounds were detected in the galangal extracts originated from Vijayawada as well as from Qingdao (China) and Telangana (India) (Babu et al., 2017;Johnley et al., 2020;Zhou et al., 2021). Other studies reported detecting flavonoids, alkaloids, steroids, and saponins in the galangal rhizomes extracts, which were not detected in this study (Babu et al., 2017;Rashad et al., 2016). The variation of phytochemical compounds in plant extracts is commonly reported, with the extraction Both concentration of the extract (p-value = 0.000) and storage time (p-value = 0.000) significantly affected the optical density of the media of the cultured tofu. The negative control showed the highest OD, which was comparable to the galangal water extract at a concentration of 5%. The concentration-dependent relationship was observed with the lowest OD was shown by the extract at a concentration of 20%. On the other hand, the overall OD of the media was statistically constant from day 2 to 6 and started to rise on day 8 (Figure 1) significantly. OD represented the indirect microorganism enumeration on the tofu. The higher OD indicated the presence of more microorganisms and vice versa. Hence, the galangal water extracts did not exert microbial growth inhibitory activity, while two higher concentrations dependently inhibited the growth of the microorganisms.  The hindered microbial growth on tofu until day six might indicate that the extract's actions were mediated by prolonging the lag phase of the microbial growth (Rialita, 2014). The direct tofu culture on the nutrient agar supported the OD-based microbial growth inhibitory activity of galangal water extracts. The microbial growth was clearly shown in tofu treated with negative control and galangal water extracts at two lower tested concentrations. In contrast, a much sparser profile was observed in those in the concentration of 20% group ( Figure  2).
Our result was similar to previous studies on the antimicrobial activity of galangal rhizome extracts. Galangal n-hexane, ethanol, water extracts and essential oils showed various degrees of activity against vast arrays of microorganisms (Hamad et al., 2016;Oonmetta-aree et al., 2006;Prakatthagomol et al., 2011;Rini et al., 2018). Tannins and terpenoids might contribute to the antimicrobial activity of galangal water extract. Tannins of rain tree leaves and terpenoids of lemongrass were previously mentioned to exert the said activity (Halabi and Sheikh, 2014;Sari et al., 2015;Zarin et al., 2016). The OD reduction represented the efficacy of the inhibitory activity of the galangal water extract.
The highest tested concentration of galangal water extract consistently exerted the best OD reduction in each evaluation time. It gradually increased from day 2 to day six and started to decrease on day 8. On the other hand, the extracts at 5 and 10% concentrations showed an equal OD reduction on days 2 and 4. From day six onward, the higher concentration of the extracts generated, the better OD reduction (Figure 3). The activity with concentration-dependent manner, as seen on days 6 and 8, was previously reported in the water extracts of galangal (Rini et al., 2018). The galangal water extracts changed the color of tofu from white to light brown (Figure 4). Tannins might cause the changed tofu color as they were interacted with the proteins on the surface (Wang and Heinonen, 2017).  The microbial metabolism caused the deterioration of tofu. The decomposition of the proteins caused off-odor, while the off-texture was originated from the breakdown of macromolecules (Wang et al., 2019). The extracts masked the fresh tofu odor with the hint of aromatic galangal one that delayed the start of noticeable off-odor in tofu. In the negative control, tofu started to be off-odor on day 6, while those treated with the extract in all tested concentrations were off-odor on day.
Similarly, the extracts also delayed the start of the off-texture in tofu. Those in the negative control group started to be friable in the 6, while those in galangal water extract groups remained firm until the final day of storage. A further preference testing is needed to assess if the consumer likes the changes on tofu caused by the applications of galangal water extract (Lawless and Heymann, 2010).
The preservation potential of galangal water extracts was calculated by comparing the start of any signs of tofu deterioration in the extract groups to that of the negative control. The galangal water extracts delayed the deterioration of tofu for two days. All three tested concentrations showed the same preservation potential on tofu for two days. Hence, the concentrations of extract did not affect the preservation potential. The positive effects of galangal on the preservation of milkfish have been reported elsewhere. The water extracts at the concentrations of 10% diminished the bacterial count on milkfish and generated a preferred smell, color, and texture profile. The juice also prolonged the shelf life of milkfish up to 46 hours (Florensia et al., 2012;Inayah and Bestari, 2018).
The overall results of all evaluated parameters in this study suggested that the optimum concentration of galangal water extract for the preservation of tofu was 20%. It generated the best antimicrobial inhibitory activity while maintaining the physical characters of tofu as in other tested concentrations. The best activity shown by the highest tested concentrations might relate to the higher tannins and steroids content. In the infundation process, the degree of compounds extractions was increased with the number of plant materials available up to the saturation condition is achieved (Sahne et al., 2016).

Conclusion
Galangal water extracts containing tannins and terpenoids showed a significant microbial growth inhibitory activity and a moderate preservation potential on tofu during 8day storage at room temperature. The inhibitory activity was in concentrationdependent manner with the optimum concentration of 20%.