Phenol content and antioxidant activity in seaweed fermented with lactic acid bacteria

Antioxidants and antimicrobials are rich in seaweed. Seaweed has been used as food for a long time and the potency to be used as a functional food. One of the most versatile foods is fermented beverages. Fermentation can increase the amount of antioxidants. This study aims to determine seaweed ( Gelidium sp . and Eucheuma cottonii ) extract fermentation with lactic acid bacteria (LAB), namely Lactobacillus plantarum and Lactobacillus acidophilus, as a starter on phenol content and antioxidant activity. The method used was an experimental laboratory. The seaweed was obtained from Yogyakarta. Seaweed extract was given a LAB starter and then fermented for 24 hrs. The samples were analyzed before and after fermentation in parameter reducing sugar, TPC, LAB viability, pH, phenol content, and antioxidant activity IC 50 tests. The result showed that both in Gelidium sp. and Eucheuma cottonii, reducing sugar does not change before

There are numerous seaweed species and have not been used optimally.Seaweed growth does not compete with agricultural land (Hou et al., 2017).Parada et al. (2019) reported that polyphenol could be utilized as an ingredient to make novel low glycemic response food.
Seaweed consumption is currently showing an increasingly interesting trend because humans are more concerned with healthy lifestyles.Many products from marine products are made and marketed as functional foods (Roohinejad et al., 2017).Seaweed stock is abundant, and the price is low.Microorganisms can ferment seaweed to increase the bioactive component (Suraiya et al., 2018).
The fermented product of seaweed has not been widely studied.Fermentation using lactic acid bacteria was reported in 2004 on Ulva spp.Besides, the Chlorophyta, Phaeophyta, and Rhodophyta groups have also been studied.Fermentation of either lactic acid or ethanolic makes the possibility of the making new fermented product (food and beverage) from seaweed and seagrass (Uchida et al., 2017).Gelidium sp. and Eucheuma cottonii are edible seaweed and abundant in Indonesia.
Antioxidants and antimicrobials are rich in seaweed.Seaweed has been used as food for a long time and has the potency to be used as a functional food.One of the functional foods is fermented drinks.Fermentation can increase antioxidants.This research focused on studying the development of the fermented product from seaweed using lactic acid bacteria.The purpose of this study was to determine the effect of lactic acid bacteria (Lactobacillus plantarum and Lactobacillus acidophilus) as a starter on phenol content and antioxidant activity during fermentation of Gelidium and Eucheuma cottonii extract.
The reducing sugar was determined following Martin et al. (2000).Aquadest was used to add one gram of sample until the volume reached 100 mL.The solution was mixed with Reagent Nelson and heated in a water bath for 30 minutes at 100°C.Allow the solution to cool before adding arsenomolibdat.At 540 nm, absorbance was measured.

Measurement of pH
The pH was determined directly using a pH meter.

Measurement of TPC and viability of lactic acid bacteria
Determination of TPC and viability of lactic acid bacteria were determined as described by Dissarapong et al. (2005).Total plate counts were determined using a plate count agar, and the dilution was using 0.85% NaCl sterile solution.A diluted sample (0.1 mL) was spread on the media's surface and incubated at 37°C.While lactic acid bacteria count was determined using Man Rogosa Sharpe agar (MRSA), and the dilution was using 0.85% NaCl.A diluted sample (1 mL) was poured into a petri dish and followed by 10 to 15 mL of MRSA media.Incubation was carried out at 37°C for 48 hrs.

Measurement of phenol
The total phenolic content was determined using the Folin-Ciocalteu method described by Saravanan and Parimelazhagan (2014).Gallic acid was used to prepare a standard calibration curve.

Statistical analysis
A completely randomized design was used throughout this study, and the experiments were done in triplicate.Data were subjected to analysis of variance (ANOVA), and mean comparison was carried out using the honest significance difference (HSD) test.Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for Windows; SPSS Inc.).

Reducing Sugar
Microorganism, including bacteria, need media or nutrition to grow.Bacteria use food and convert it to energy, grow to produce several metabolites.Like reducing sugar, the carbon source is the primary energy source (Singh et al., 2017).Component carbon and nitrogen sources usually play a crucial role in the fermentation medium (Khani et al., 2016).The increasing yield of the cell during fermentation is influenced by carbon sources (Wang et al., 2018).
Reducing sugar on 0 hrs and 24 hrs in Gelidium sp. and Eucheuma cottonii extract was shown in Table 1 and  Table 2.The Reducing sugar ranges from 0.14% to 0.18%.Reducing sugar value was low and did not show a difference value in 0 hrs and 24 hrs fermentation (p < 0.05).This phenomenon was because the fermentation condition was not suitable yet for the starter to grow and can not hydrolyze the complex carbon from seaweed into the simple carbon like reducing sugar.

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During fermentation, the starter's enzymes are expected to produce enzymes and hydrolyze the complex molecule into a simple molecule.For example, cellulose into glucose.Every enzyme has the optimum condition to hydrolyze the molecule.The factor that usually influences the enzyme activity is pH, temperature, ion, and surfactant.When the conditions are not suitable, the enzyme activity will be low (Palavesam, 2015).
Table 1 and Table 2 show that the amount of reducing sugar was meager.The reducing sugar was only approximately 0.14% to 0.18%.Low sugar content was challenging to support bacterial growth.Simple sugar is the most accessible source of energy to use during bacterial growth.L. plantarum and L. acidophilus are including a group of lactic acid bacteria.Lactic acid bacteria are fastidious.They need carbohydrates as well as other compounds to grow well (Singh et al., 2017).Total plate count (TPC) was lower than lactic acid bacteria count.It is showed that lactic acid bacteria growth was better than other bacteria in this fermentation.Lactic acid bacteria can suppress the development of different bacteria, although not optimal.During gain, lactic acid bacteria will produce acid and lower the pH.The lower pH will stop the growth of other bacteria that cannot tolerate low pH to reduce the TPC value.

Total plate count
TPC is the enumeration of aerobic, mesophilic organisms included pathogens and non-pathogen, and is used to determine the hygienic status of food production.Bacterial count (TPC) on 0 hrs and 24 hrs in Gelidium sp. and Eucheuma cottonii extract were shown in Table 3 and Table 4.The TPC of the sample ranges from 2.62 log CFU/mL to 4.53 log CFU/mL.This data showed that the count of microorganisms other than the starter is still high.This is because not all ingredient used to produce the seaweed extract was sterilized, so the contamination was still very high.
In Gelidium sp.extract, TPC did not change after 24 hrs of fermentation in both starters.While in Eucheuma cottonii extract, TPC was increased 1 log cycle after 24 hrs fermentation time.Eucheuma cottonii extract has higher reducing sugar (p < 0.05) that can support microorganisms' growth.Simple sugar, like reducing sugar, is easy to metabolize by microorganisms, including lactic acid bacteria as a starter and other bacteria present in the sample (Ficoseco et al., 2018).Table 1 and Table 2 show that the amount of reducing sugar was meager.The reducing sugar was only approximately 0.14% to 0.18%.Low sugar content was challenging to support bacterial growth.Simple sugar is the most accessible energy source to use during bacterial growth (Singh et al., 2017).

Viability of lactic acid bacteria starter
Lactic acid bacteria count of the starter on 0 hrs and 24 hrs in Gelidium sp. and Eucheuma cottonii extract was shown in Table 5 and Table 6.The data ranges from 6.7 log CFU/mL to 8.21 log CFU/mL.The bacterial count of starters increased after 24 hrs of fermentation time in both seaweed and both starter (p < 0.05).It is showed that the starter could grow in seaweed extract.The increasing microbial count was about 1 log cycle.
Eucheuma cottonii extract has higher reducing sugar (p < 0.05) that can support starter growth.Simple sugar is easy to metabolize by microorganisms, including lactic acid bacteria.Lactic acid bacteria are fastidious.It needs a carbon source like a simple sugar, a nitrogen source like amino acid or peptide, and some other compounds.(Ficoseco et al., 2018).Estevam (2018) reported that the early fermentation time of fermented milk added with seaweed extract will increase the microbial count and titratable acidity.

pH
The pH sample on 0 hrs and 24 hrs in Gelidium sp. and E. cottonii extract were shown in Table 7 and Table  8.The pH was decreased after fermentation (p < 0.05).These phenomena happen because the lactic acid bacteria starter was grown and converted the fermentation medium into lactic acid to lower the pH.
The addition of aqueous seaweed extract to fermented milk will increase the total acid, thus will reduce the pH during the early fermentation period (Estevam et al., 2018).It showed that the starter could metabolize the carbohydrates in seaweed to produce lactic acid.Wu et al. (2007) showed that some lactic acid bacteria could use the oligosaccharides from some seaweed as a fermentation medium.
Changes in pH value after 24 hrs fermentation were different between starter (p < 0.05).The changes in pH that caused by acid production during bacterial growth is strain-specific characteristic.In this research, L. acidophilus pH was lower than L. plantarum.This result was the opposite with Solval et al. (2019), in which the lactic acid bacteria count in L. acidophilus was lower than L. plantarum; thus, pH in L. acidophilus was higher than L. plantarum.

Phenol content
Phenol content on 0 hrs and 24 hrs in Gelidium sp. and Eucheuma cottonii extract were shown in Table 9 and Table 10.The phenol content showed various phenomena during the fermentation, and the value ranges are from 43.33 to 145.67.In Gelidium sp.extract, the phenol content did not change after 24 hrs of fermentation in both starters (p < 0.05).While in Eucheuma cottonii after 24 hrs of fermentation, the sample with L. plantarum showed a lower phenol content but in L. acidophilus showed a higher phenol content than before fermentation (p < 0.05).Fermentation can increase the biological functionalities include antioxidant activity (Hifney et al., 2018).
Table 9 and Table 10 show phenol content in Gelidium sp.extract and Eucheuma cottonii extract before and after fermentation.In Gelidium sp.extract, the phenol content did not change after 24 hrs of fermentation in both starters (p < 0.05).While in Eucheuma cottonii after 24 hrs of fermentation, the sample with L. plantarum showed a lower phenol content but in L. acidophilus showed a higher phenol content than before fermentation (p < 0.05).Fermentation can increase the biological functionalities include phenol content and antioxidant activity (Hifney et al., 2018;Norakma et al., 2019).Microorganisms start to modify plant constituents during fermentation.Many biochemical changes occur during fermentation, leading to an altered ratio of healthy and anti-nutritive plants' components, which affect product properties such as bioactivity and digestibility (Katina et al., 2007).The microorganism growth during fermentation may hydrolyze the seaweed tissue, contributing to phenol   (Hifney et al., 2018).But it is also reported that not all substances are increased by fermentation.
Several substances are also reduced after fermentation.The increment and reduction of phenolic content can be related to organic acids' metabolism, derivation of phenolics among themselves, and other metabolic pathways (Li et al., 2020).

Antioxidant IC 50
Antioxidant activity on 0 hrs and 24 hrs in Gelidium sp. and Eucheuma cottonii extract were shown in Table 11 and Table 12.The data showed that after fermentation, the sample has lower IC 50 when compared to before fermentation.The antioxidant activity showed slightly higher after fermentation.These phenomena happen because the lactic acid bacteria starter was grown and converted the fermentation medium into lactic acid and other products that probably function as antioxidants.During fermentation, several microbial enzymes could hydrolyze the raw material and increase the phenol content (Cheng et al., 2015;Huang et al., 2017) and flavonoid content in seaweed fermentation (Hur et al., 2014).It will increase the antioxidant activity.
Antioxidant activity of fermented seaweed extract on 0 hrs and 24 hrs in Gelidium sp. and Eucheuma cottonii extract were shown in Table 11 and  Table 12.Data IC 50 is the concentration of a sample with the ability to scavenge 50% of DPPH radicals.The antioxidant activity of seaweed extract ranges from 44667% to 38766%.This activity is still deficient.The data showed that after fermentation, it showed that the sample has a lower IC 50 .The antioxidant activity showed slightly higher after fermentation.These phenomena happen because the lactic acid bacteria starter was grown and converted the fermentation medium into lactic acid and other products that probably function as antioxidants.This data is also in line with the phenol content result (Table 9 and Table 10).After fermentation, the phenol content was slightly increased.During fermentation, several microbial enzymes could hydrolyze the raw material and increase the phenol content (Cheng et al., 2015;Huang et al., 2017) and flavonoid content in seaweed fermentation (Hur et al., 2014) and increase the antioxidant activity.

Conclusion
Fermentation on a seaweed extract with L. acidophilus and L. plantarum increased lactic acid bacteria's viability, phenol content, antioxidant activity, and reduce pH.However, the antioxidant activity was still low.Therefore, it needs to optimize the fermentation condition to obtain higher antioxidant activities.
Values are presented as mean±SD.Values with different superscripts within the row are significantly different (p < 0.05).eISSN: 2550-2166 © 2021 The Authors.Published by Rynnye Lyan Resources

Table 4 .
Total plate count in Eucheuma cottonii extract during fermentation.

Table 5 .
Viability of lactic acid bacteria in Gelidium sp.extract during fermentation.
eISSN: 2550-2166 © 2021 The Authors.Published by Rynnye Lyan Resources FULL PAPER Lack of carbon sources makes the microorganism's growth not optimal

Table 6 .
Viability of lactic acid bacteria in Eucheuma cottonii extract during fermentation.

Table 9 .
Phenol content in Gelidium sp.extract during fermentation.

Table 11 .
Antioxidant IC 50 in Gelidium sp.extract during fermentation.Values are presented as mean±SD.Values with different superscripts within the row are significantly different (p < 0.05).