Monascus spp. fermented brown seaweeds extracts enhance bio-functional activities

Author’s Accepted Manuscript Monascus spp. fermented brown seaweeds extracts enhance bio-functional activities Sharmin Suraiya, Jong Min Lee, Hwa Jin Cho, Won Je Jang, Dong-Gyun Kim, Young-Ok Kim, In-Soo Kong www.elsevier.com/locate/sdj

PII: DOI: Reference:

S2212-4292(17)30649-1 https://doi.org/10.1016/j.fbio.2017.12.005 FBIO247

To appear in: Food Bioscience Received date: 14 September 2017 Revised date: 20 November 2017 Accepted date: 11 December 2017 Cite this article as: Sharmin Suraiya, Jong Min Lee, Hwa Jin Cho, Won Je Jang, Dong-Gyun Kim, Young-Ok Kim and In-Soo Kong, Monascus spp. fermented brown seaweeds extracts enhance bio-functional activities, Food Bioscience, https://doi.org/10.1016/j.fbio.2017.12.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Monascus spp. fermented brown seaweeds extracts enhance bio-functional activities Sharmin Suraiya1, Jong Min Lee2, Hwa Jin Cho1, Won Je Jang1, Dong-Gyun Kim3, Young-Ok Kim3, In-Soo Kong1* 1

Department of Biotechnology, College of Fisheries Science, Pukyong National University, 45

Yongso-ro, Nam-gu, Busan, 48513, Republic of Korea 2

Industrial Bio-material Research Centre, Korea Research Institute of Bioscience and

Biotechnology, Daejeon, 34141, Republic of Korea 3

Biotechnology Research Division, National Fisheries Research and Development Institute,

Busan, 46083, Republic of Korea

*

Corresponding author,

Professor In-Soo Kong Department of Biotechnology College of Fisheries Science, Pukyong National University 45, Yongso-ro, Nam-gu, Busan, 48513, Republic of Korea Tel: +82-51-629-5865, Fax: +82-51-629-5863 E-mail: [email protected]

Abstract 1

Two species of brown seaweeds, Saccharina japonica and Undaria pinnatifida were fermented by the red molds; Monascus purpureus and Monascus kaoliang to increase their bio-functional properties. The phenolic contents of S. japonica fermented by M. purpureus (SjMp) and M. kaoliang (SjMk) were the highest 71.53 ± 2.25 and 66.50 ± 4.64 mg gallic acid equivalent/g extract, respectively, whereas the highest flavonoid content was evident in S. japonica fermented by M. purpureus (SjMp) and U. pinnatifida fermented by M. purpureus (UpMp) (27.93 ± 0.28 and 26.88 ± 1.24 mg quercetin equivalent/g extract, respectively). Reducing sugar, protein and essential fatty acids levels also increased in fermented seaweeds. The antioxidant activities of fermented seaweed extracts exhibited significantly (p < 0.05) lower IC50 values than those of unfermented extracts. S. japonica fermented by M. purpureus (SjMp) exhibited the lowest IC50 values of antidiabetic activities mediated by α-amylase and rat intestinal α-glucosidase (maltose and sucrose): 0.98 ± 0.10, 0.02 ± 0.07 and 0.08 ± 0.13 mg/mL, respectively. M. purpureus fermented S. japonica extract at 4.58 ± 0.85 μg/mL afforded 50% inhibition of lipase, which was the most effective of all samples tested in this regard. Extracts from brown seaweeds fermented by Monascus spp. exhibited increased phenolics and flavonoids contents associated with strong in vitro bio-potential, DNA protection and the absence of any toxic effect on intestinal epithelial Caco-2 cells. Thus, fermented seaweed extracts may be recommended as food ingredients or therapeutic diet for patient suffering from oxidative stress, hyperglycemia and hyperlipidemia.

Graphical Abstract

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Keywords Brown seaweed, Red molds, Fermentation, Bio-functional, Phenolic and Flavonoid

1. Introduction Seaweeds are promising renewable marine resources. Seaweeds contain many bioactive compounds and nutrients including polysaccharides, carotenoids, protein, dietary fiber, phenolics, flavonoids, vitamins, minerals, and polyunsaturated fatty acids (PUFAs), which are considered valuable in growing children and young and pregnant women (Athukorala et al., 2006). Seaweed 3

is very cheap (being abundant) and can be subjected to microbial fermentation and conversion to increase the levels of bioactive compounds (Uchida et al., 2017). Moreover, seaweeds are a very productive source of biomass; the absence of hard lignocellulosic material facilitates easy depolymerization (Guneratnam et al., 2017; Tabassum et al., 2017). Seaweeds are rich in cellulose, laminarin, glucan, mannitol, alginate and agar all of which can be readily hydrolyzed to simple sugars upon fermentation with suitable microorganisms (Tabassum et al., 2017). Brown seaweeds contain high amounts of moisture (70−90% wet weight), polysaccharides 48−61% (dry weight), protein 5−21% and lipid 1−4% of dry weight (Holdt & Kraan, 2011) and can be subjected to solid state fermentation, which has long been applied to improve the shelf life and enhance organoleptic and nutritional qualities (Cheng et al., 2011). Fermentation changes the ratio of nutritive to anti-nutritive components, affects bioactivity and digestibility, and converts water-insoluble materials to water-soluble components using various microbial enzymes (Kim et al., 2013). According to Tabassum et al. (2017), molecular biologists have proven that fungi converted all polysaccharides of brown seaweed to fermentable sugars. Fermentation of food materials by fungal species is associated with the synthesis of important phytochemicals, affording major health benefits. Of the many fungal species, Monascus spp. are known to produce health-promoting compounds during fermentation of food materials; these include pigments, an anti-hypercholesterolemic agent (lovastatin), an anti-hypotensive material (γ-aminobutyric acid), antioxidants including dimerumic acid and antimicrobial compounds (Cheng et al., 2015; Dikshit & Tallapragada, 2016). In East Asian countries, Monascus spp. have been extensively used to ferment red beans; to produce red alcoholic beverages; to color fish paste, surimi and tomato ketchup; and for medicinal purposes (Huang et al., 2017).

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Fermentation of many food materials by Monascus spp. improves antioxidant properties. In the human body, free radicals such as the hydroxyl radical (•OH), the superoxide anion (O2•−) and hydrogen peroxide (H2O2) are generated via oxygen metabolism and other biochemical reactions. Free radicals degrade proteins, the cell membrane and cellular components such as DNA (Juan & Chou, 2010) causing many diseases/conditions including neurological disorders, cancer, aging, rheumatoid arthritis, atherosclerosis, cataracts, muscular dystrophy etc. (Ruberto et al., 2001). Antioxidants prevent free radical formation and thus retard rancidity and harmful oxidation of food components, ensure nutritional quality, and prolong food life (Shahidi & Naczk, 2003). As natural antioxidants are not in sufficient amount, people are using synthetic antioxidants. However, these materials are associated with side effects and toxicities; both investigators and consumers thus focus on natural antioxidants (Dudonné et al., 2009). Vitamin E and carotenoids (natural antioxidants) are water-insoluble. Also, both vitamins C and E are heat-sensitive and thus readily destroyed. Seaweed-based bioactive materials are thus in demand. Recently, fungal fermentation has been used to increase the antioxidant and anti-cholesterolemic activities of food and medical compounds and also for food preservation (Xiao et al., 2014). Many bioactive compounds are formed during seaweed fermentation; these scavenge free radicals and reactive oxygen species (Bae & Kim, 2010; Uchida et al., 2017).

The anti-diabetic potential afforded by Monascus fermentation has been explored by several researchers (Shi & Pan, 2012). On the other hand, brown seaweeds also inhibit α-amylase and αglucosidase activities, attributable to the contained phenolics, flavonoids, fatty acids and other beneficial compounds (Sharifuddin et al., 2015). The phenolic and flavonoid levels correlate positively with the extents of α-glucosidase inhibition and antilipase activity (Ryan et al., 2016).

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Monascus fermentates are rich sources of essential fatty acids such as oleic, linoleic, palmitic and stearic acids (Wei et al., 2003), necessary for normal growth, dermal integrity and skin moisturization; these fatty acids also prevent cancer, asthma and cardiovascular disease and improve brain health (Moharram et al., 2012). As, seaweeds are rich source of fatty acids it also inhibit lipases and exert anti-hyperlipidemia activities (Sharifuddin et al., 2015). So, fermentation process could be beneficial to increase phenolic, flavonoid, protein, reducing sugar and fatty acids levels of brown seaweeds and prevention of diabetics and hyperlipidemia.

However, some Monascus spp. produce citrinin (a cytotoxic agent). Thus, any cytotoxic effect of Monascus-fermented foods designed for human consumption must be assessed. In vitro testing is very efficient and is a well-established method by which to evaluate cytotoxicity (Gutleb et al., 2002). In vitro assays are cheaper and quicker than animal studies (Gad, 2000). Cytotoxicity assays can be used to explore the acute toxicities of bioactive compounds (Rasmussen et al., 2011). The Caco-2 cell model has been extensively used to investigate the toxicities of bioactive compounds (O’Sullivan et al., 2012). Fernandes et al. (2012) used the Caco-2 cytotoxicities of compounds/ substances as measure of the intestinal permeabilities of such substances. So why, in this research work we conducted a study on the in vitro toxic effect of the fermented sample on Caco-2 cell. Although, brown seaweeds and Monascus-fermented extracts from different substrates individually have shown profound bio-potential activities, no report has yet evaluated brown seaweed extracts fermented by Monascus spp. It is necessary to measure various bio-functional activities when seeking to develop new products for the food and pharmaceutical industries. Therefore, we evaluated the effects of solid-state fermentation of the brown seaweeds,

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Saccharina japonica and Undaria pinnatifida by M. purpureus and M. kaoliang in terms of antioxidant status assessed based on scavenging of 2,2'-azino-bis (3-ethylbenzothiazoline-6sulphonic acid) (ABTS+), ferric reducing antioxidant power (FRAP), hydroxyl radical (•OH), and nitric oxide (NO); and on DNA protection, anti-diabetic and anti-lipase activities. We also measured the levels of phenolics and flavonoids, protein, reducing sugar, and fatty acids and the cytotoxicities of the fermented products.

2. Materials and Methods 2.1. Red mold cultures and inoculum preparation Monascus purpureus KCCM 60168 and Monascus kaoliang KCCM 60154 were obtained from the Korean Culture Centre of Microorganisms (KCCM). M. purpureus and M. kaoliang were cultured on potato dextrose agar (PDA) and yeast extract agar (YEA), respectively at 30 °C for 12 days. Fungal colonies were stored at 4 °C and sub-cultured monthly. Sterile distilled water (10 mL) was added to an agar plate, fully covered with fungal growth, scraped aseptically and kept at room temperature for 5 min to ensure a homogenous spore suspension, which was then used for inoculation.

2.2. Fermentation and extract preparation S. japonica and U. pinnatifida were purchased from a local market in Busan, Republic of Korea. A sterile blender (Shinil Multi mixer SMX-757CM, Seoul, Republic of Korea) was used to grind the dried seaweeds and the powders were passed through a 0.5-mm-pore-size stainless steel sieve. Fermentation proceeded using the method of Suraiya et al. (2017) with some modifications. Five grams of dry substrate was placed in a 100-mL Erlenmeyer flask containing

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0.4% (w/w) peptone as the organic nitrogen source. Distilled water was added to maintain the initial moisture content at 50% (w/w). The flask contents were thoroughly mixed, autoclaved (121°C, 20 min), cooled at room temperature and glucose (prepared aseptically on a clean bench using syringe-Minisart filters of pore size 0.2 μm; Sartorius stedim, Göttingen, Germany) was added to 0.8% (w/w). Then, M. purpureus and M. kaoliang were inoculated (into different flasks) followed by incubation at 30 °C for 20 days. Samples (0.25 g) of fermented freeze-dried material were extracted into 5 mL of distilled water by sonication (Sonifier® S-250A, duty cycle 35% and output control 35%, Branson Ultrasonics Corporation, Danbury, CT, USA) in the cold (4 °C) for 20 min. The extracts were then centrifuged at 15,000× g for 15 min and the supernatants freeze-dried at −48 °C (Eyela FDU-1200, Tokyo Rikakikai Co., Ltd, Tokyo, Japan). Extracts of S. japonica and U. pinnatifida fermented by M. purpureus and M. kaoliang were abbreviated below as SjMp, SjMk, UpMp and UpMk, respectively, whereas unfermented S. japonica and U. pinnatifida were abbreviated as SjU and UpU respectively. Freeze-dried extract powders were diluted to various concentrations in distilled water prior to biochemical analysis.

2.3. Total phenolic content (TPC) Total phenolic contents of fermented and unfermented samples were determined using the Folin– Ciocalteu reagent as described by Chew et al. (2011); and absorbance was measured at 765 nm using a microplate spectrophotometer (Infinite M200 nanoquant, Tecan, Zurich, Switzerland). Gallic acid (Sigma-Aldrich) was used as a standard. To measure the total gallic-acid equivalent phenolic content a high performance liquid chromatography (HPLC) instrument equipped with a model 484 UV/VIS detector and a C18 column (5 μm, 4.6×250 mm) was used. We employed the method of Dinh et al. (2017) with gallic acid as the standard. 8

2.4. Total flavonoid content (TFC) Total flavonoid contents of fermented and unfermented seaweeds were determined as described by Ozsoy et al. (2008) with minor modifications. Briefly, 0.375 mL of solution (40 mg sample in 1 mL distilled water) was mixed with 1.88 mL of water, and 112 μL of 5% (w/v) NaNO2 added, followed by incubation at room temperature for 6 min and addition of 225 μL of 10% (w/v) AlCl3. Next, 0.75 mL of 1 M NaOH and 0.41 mL of distilled water were added and the absorbance read at 510 nm using a microplate spectrophotometer (Infinite M200 nanoquant, Tecan, Zurich, Switzerland). Quercetin (Sigma-Aldrich) served as the standard.

2.5. Reducing sugar, protein and fatty acid contents Reducing sugars were estimated using the dinitrosalicylic acid (DNS) method of Miller (1959). Protein contents were analyzed employing the Bradford method (1976). For fatty acid analysis, 5 g of freeze-dried fermented and unfermented samples were placed into thimble and extracted with 100 mL of n-hexane (97.8% v/v) in a Soxhlet apparatus. Each flask was heated at 65 ºC using a heating mantle for 16 h. The solvent was evaporated by using rotary evaporator at 40 ºC and extracted oils were stored at −60 ºC for analysis. Fatty acids were analyzed via gas Chromatography (GC) and expressed as percentage of total fatty acids (% TFA) following the method of Haq et al. (2017). 2.6. Radical-scavenging assay The ABTS+ radical-scavenging activities of fermented and unfermented samples were determined using the method of Xiao et al. (2014) with some changes. The ABTS+ stock solution was prepared by mixing 7 mM aqueous ABTS with 2.45 mM aqueous potassium persulfate (1:1

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ratio; v/v) followed by incubation for 16 h at room temperature in the dark. ABTS+ working solution was prepared from the ABTS+ stock solution by diluting with ethanol to an absorbance of 0.85 (±0.02) at 734 nm. Then, 125-μL amounts of samples of different concentrations (0.0195−2.5 mg/mL) were mixed with 850-μL of ABTS+ working solution, incubated for 6 min and the absorbance at 734 nm measured using a spectrophotometer (Mecasys, Optizen, Republic of Korea). Modified method of Benzie & Strain (1996) was followed for measuring the FRAP assay of fermented and unfermented seaweed samples. Ten milliliters of 2,4,6-tripyridyltriazine (TPTZ) solution (10 mM, in 40 mM HCl), 10 mL of 20 mM FeCl3 and 100 mL of 0.3 M acetate buffer (pH 3.6) were mixed and constituted the FRAP reagent. Sample solutions (150-μL amounts) at different concentrations (0.078−5 mg/mL) were added to 0.85-mL amounts of the FRAP reagent, followed by incubation at 37 °C for 20 min. Then, absorbance at 593 nm was measured against a blank. Hydroxyl radical-scavenging activity was measured following the method of Li et al. (2014). Sample of different concentrations (0.3125−10 mg/mL) were used for this study. Hydroxyl radical scavenging activity absorbance was assayed by absorbance at 536 nm. The NO scavenging assay was performed using 50 μL amount of sample solution (0.039−1.25 mg/mL), which were mixed with 450-μL amounts of sodium nitroprusside (SNP) solution (10 mM), followed by incubation for 4 h. Then, 500 μL of Griess reagent were added and the tubes kept at room temperature for 10 min. A pink chromophore formed because of diazotization of the nitrite with sulfanilamide followed by coupling with naphthyl ethylene diamine. Then the absorbance was measured at 546 nm. Nitrite radical scavenging activity was determined by measuring the reduction in absorbance as antioxidants donated protons to the nitrite radical. Ascorbic acid (Sigma-aldrich) was used as a positive control for all radicalscavenging assays.

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2.7. DNA-protective activity Oxidative DNA damage was measured by Fenton’s reagent and the protective effects of fermented and unfermented seaweed samples were assayed using the method of Suraiya et al. (2017) with slight modifications. Each reaction mixture contained 12 μL of sample solution (2 mg/mL in distilled water), 4 μL λ DNA (Takara, Cat No. # 3010; 400 µg; 300 ng/µL) diluted to 200 ng/μL and 4 μL Fenton’s reagent (20 mM FeCl3, 12.5 mM ascorbic acid and 7.5 mM H2O2). Each mixture was incubated at 37 °C for 30 min and DNA examined via 1% (w/v) agarose gel electrophoresis, and visualization and photography under a UV-transilluminator (Vilber Lourmat, France). A tube with λ DNA and distilled water instead of the sample served as the positive control. A tube with λ DNA, distilled water and Fenton’s reagent was used as negative control.

2.8. Pancreatic α-amylase inhibition According to the procedure of Adisakwattana et al. (2012), pancreatic α-amylase assay was performed using different concentration (0.156−5 mg/mL) of fermented and unfermented sample. The reaction was terminated by addition of dinitrosalicyclic acid (DNS), followed by boiling for 5 min and cooling. Each sample was then diluted and absorbance at 540 nm measured spectrophotometerically (Mecasys, Optizen, Republic of Korea). Acarbose (Sigma-Aldrich) served as the positive control. We calculated: % Inhibition=

×100

Where, Abssamples was the absorbance of the sample, AbsControl was the absorbance without the sample.

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2.9. Intestinal α-glucosidase inhibition The method of Adisakwattana et al. (2012) was used to assay inhibition of intestinal αglucosidase activity by various concentrations (0.038−2.5 mg/mL) of fermented and unfermented samples. The reaction was terminated by addition of DNS, followed by boiling for 5 min and cooling. Each sample was then diluted and absorbance at 540 nm was measured spectrophotometrically. Acarbose served as the positive control. We calculated: % Inhibition=

×100

Where, Abssamples was the absorbance of the sample, AbsControl was the absorbance without the sample.

2.10. Pancreatic lipase inhibition The pancreatic lipase inhibitory activities of fermented and unfermented sample were assayed as described by Kim et al. (2010) using various sample concentrations (0.488−125 μg/mL). We spectrophotometerically measured the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at an absorbance of 405 nm (Mecasys, Optizen, Republic of Korea). Orlistat served as the positive control. We calculated: % Inhibition = Where Abs

Control

×100 was the absorbance without the sample, Abs

samples

was the absorbance of the

sample extract.

2.11. MTS cytotoxicity (Non-radioactive cell proliferation) assay To explore the cytotoxicities of fermented and unfermented brown seaweed extracts on Caco-2 cells, we purchased Caco-2 cell line (KCLB 30037) from the Korean Cell Line Bank (KCLB) 12

and grew the cells at 37 ⁰C under 95% air and 5% CO2 (both v/v) in Dulbecco’s Eagle Medium (DMEM, Gibco, Grand Island, NY) supplemented with 10% (w/v) Fetal Bovine serum (FBS) and 1% (w/v) streptomycin-penicillin (100 μg/mL and 100 IU/mL, respectively). Cell viability was measured using the cell proliferation assay kit (Celltiter 96® AQueous non-radioactive cell proliferation assay, Promega, Germany). Cells were seeded (6.2 × 103 cells/well) into a 96-well plate with a clear flat bottom, incubated for 24 h and the adherent cells then treated with extracts at various concentrations, followed by further 24 h of incubation. Then, 20 µL of MTS/PMS (35 µL PMS in 700 µL MTS, prepared just before use) solution was added to each well (containing 100 µL of medium), followed by incubation for 2 h. The MTS assay is based on the conversion of a yellow tetrazolium salt (MTS) in the presence of phenazine methosulfonate (PMS) to soluble purple formazan formed by the actions of dehydrogenases in metabolically active cells. The formazan level was measured by absorbance at 490 nm using a microplate spectrophotometer (Infinite M200 nanoquant, Tecan, Zurich, Switzerland); the absorbance is directly proportional to the number of living cells.

2.12. Data analysis We calculated IC50 values by logarithmically plotting percentage (%) of inhibition against the concentrations of sample or standards using Microsoft Excel 2013. All data are expressed as means ± standard deviations (SDs), including the phenolic and flavonoid contents. Data were analyzed using IBM SPSS software ver. 20 and the 5% level was considered significant (p < 0.05).

3. Results 3.1. Total phenolic contents

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The total phenolic contents of fermented extracts differed significantly from those of unfermented samples (Table 1). Of the fermented extracts, SjMp exhibited the highest phenolic content (71.53 ± 2.25 mg gallic acid equivalent/g extract), followed by SjMk (66.50 ± 4.64 mg gallic acid equivalent/g extract). UpMk yielded the lowest value (57.37 ± 6.40 mg gallic acid equivalent/g extract). Unfermented seaweed contained very low levels of phenolics: 13.64 ± 0.60 and 20.09 ± 0.15 mg gallic acid equivalent/g extract in SjU and UpU, respectively. The presence of gallic acid in both fermented and unfermented brown seaweeds was confirmed using HPLC (data not shown).

3.2. Total flavonoid contents The total flavonoid contents of SjMp and SjMk were 27.93 ± 0.28 and 25.74 ± 1.90 mg quercetin equivalents/g of extract, and those of UpMp and UpMk 26.88 ± 1.24 and 23.92 ± 0.06 mg querectin equivalents/g of extract, respectively. Unfermented S. japonica and U. pinnatifida had very low flavonoid contents of 15.16 ± 0.80 and 18.36 ± 0.14 mg quercetin equivalent/g of extract, respectively (Table 1).

3.3. Reducing sugar, protein and fatty acid contents Fermentation increased reducing sugar and protein contents (Table 1). Fermentation of brown seaweed by Monascus spp. increased the level of fatty acids including palmitic acid (16:1), stearic acid (18:0), oleic acid (18:1n9c), linoleic acid (18:2n6c) and arachidic acid (20:0) (Table 2). The palmitic acid levels of SjU and UpU were 15.82% and 16.47% in respectively, whereas those of SjMp, UpMp, and UpMk were 27.65%, 41.53% and 40.47%, respectively. The stearic acid level of SjMp, SjMk, UpMp and UpMk increased to 9.44%, 10.34%, 4.97% and 4.74%, respectively, from the 1.17 and 2.06% of SjU and UpU (respectively). Unfermented seaweeds 14

contained oleic acid at 20.25% (SjU) and 7.85% (UpU); these levels increased in fermented samples to 34.98% (SjMp), 43.93% (SjMk), 28.29% (UpMp) and 29.23% (UpMk). The linoleic acid contents of SjMp and SjMk increased to 12.78% and 19.11%, respectively from the 8.37% of SjU. Seaweed fermentation by Monascus spp. reduced omega-3 fatty acid levels these were used as carbon source. The GC chromatograms of fermented and unfermented S. japonica and U. pinnatifida are shown in Fig. 1, with the retention times.

3.4. Antioxidant activities The ABTS+ radical-scavenging activities of fermented and unfermented samples varied significantly (p < 0.05) (Fig. 2a). An elevated antioxidant activity correlated with increases in the phenolic and flavonoid contents, these materials scavenge free radicals. At extract concentration of 2.5 mg/mL, the ABTS+ radical scavenging activities of SjMk, SjMp and ascorbic acid, UpMk, UpMp, SjU and UpU were 86.2%, 85.2%, 81.9%, 79.3%, 72.8% and 66.3%, respectively. The IC50 values of fermented samples were significantly lower (p < 0.05) than those of unfermented samples (Table 3). FRAP radical-scavenging activities of Monascus-fermented brown seaweeds are shown in Fig. 2b. Monascus-fermented S. japonica exhibited a higher scavenging activity than Monascus-fermented U. pinnatifida. At a sample concentration of 5 mg/mL, the highest FRAP-scavenging activity was 83.9% in SjMp, followed by 81.0%, 78.2%, 77.3%, 64.7% and 56.1% in SjMk, UpMk, UpMp, SjU and UpU, respectively. Ascorbic acid had the lowest FRAP IC50 value at 1.21 ± 0.86 mg/mL, followed by levels of 1.69 ± 0.54 and 1.98±0.18 mg/mL for SjMp and SjMk, respectively (Table 3). The hydroxyl radical-scavenging activities of fermented and unfermented seaweed samples increased in a concentration-dependent manner (Fig. 2c). At concentrations of 10 mg/mL, ascorbic acid, SjMk and UpMk exhibited the highest hydroxyl radical-scavenging activities of 95.1%, 87.9% and 87.1%, respectively. In terms of OH− radical15

scavenging activity, ascorbic acid had an IC50 value similar to that of UpMp. The IC50 values for other samples were (in ascending order): SjMp
3.5. DNA-protective activity Hydroxyl radicals are generated by Fenton’s reagent and cause oxidative breakdown of DNA. Exposure of λ DNA to Fenton’s reagent caused DNA nicking or disappearance (Fig. 3a). The positive control (lane 1), DNA treated with SjMp and SjMk (lanes 3, 4) and DNA treated with UpMp and UpMk (lanes 6, 7) were significantly protected compared with DNA treated with SjU and UpU (lanes 5, 8). DNA disappeared in the negative control (lane 2). Fermention significantly prevented oxidative damage to DNA because the fermented samples had higher phenolic and flavonoid levels than the unfermented samples. SjMp, SjMk, UpMp, UpMk, SjU and UpU inhibited DNA damage by 69.5%, 67.6%, 59.7%, 45.9%, 40.7% and 27.6%, respectively (Fig. 3b).

3.6. α-amylase and rat intestinal α-glucosidase inhibition The α-amylase inhibitory activities of fermented and unfermented extracts are shown in Table 4. The α-amylase inhibitory activity of SjMp had a significantly (p < 0.05) lower IC50 value (0.98 ± 0.10 mg/mL; p < 0.05) than those of other samples. The IC50 values for α-amylase inhibition by UpMp and UpMk were similar to that of acarbose. Unfermented samples exhibited lower α-

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amylase inhibition activities; the IC50 value of SjU and UpU were 3.71 ± 0.22 and 4.46 ± 0.05 mg/mL, respectively. The IC50 values in terms of intestinal sucrase and maltase activities were <1 mg/mL, indicating that Monascus-fermented extracts effectively inhibited intestinal maltase and sucrase. Fermented samples varied significant (p < 0.05) in terms of their ability to inhibit αglucosidase, more so than unfermented samples.

3.7. Lipase inhibition assay Fermented samples exhibited significantly higher (p < 0.05) inhibition of pancreatic lipase than unfermented samples (Table 4). Orlistat and SjMp exhibited the higher pancreatic lipase inhibitory activities; the IC50 values were 3.67 ± 0.19 and 4.58 ± 0.85 μg/mL, respectively.

3.8. MTS cytotoxicity (Non-radioactive cell proliferation) assay The cytotoxicities of fermented and unfermented samples to Caco-2 cells (percentage of cell viabilities) were calculated (Fig. 4). UpU and UpMk (10 µg/mL) reduced cell viabilities; however, all other samples afforded >93% cell viability at various concentrations.

4. Discussion Fermentation is an ecofriendly process by which macromolecules are catabolized to simpler compounds by microorganisms. Fermentation of S. japonica and U. pinnatifida by Monascus spp. triggered various compositional and functional changes. Enhancement of the phenolic and flavonoid contents of fermented seaweeds is attributable to the fact that enzymes released by Monascus spp. break down seaweed cell walls, releasing phenolics and flavonoids.

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Our present results were consistent with the result of Eom et al. (2011) showed that fermentation of brown algae by the yeast, Eisenia bicycilis enhanced the phenolic content. Therefore, S. japonica fermentation by Aspergillus oryzae increased antioxidant activity and the total phenolic content (Rafiquzzaman et al., 2015). Phenolics are normally bound to sugars and thus lack bioavailability. However, during fermentation, complex phenolics are hydrolyzed to simple soluble phenols which are bio-active, readily absorbable and increase antioxidant activity (Cheng et al., 2015; Huang et al., 2017).

Various microbial enzymes including amylases, cellulases, esterases, glucosidases, invertases, lipases and tannases produced during fermentation break down of seaweed cell walls and facilitate flavonoid extraction (Hur et al., 2014). During microbial fermentation, glycosylation, deglycosylation, methylation and sulfate conjugation increase the flavonoid contents of fermented product (Huynh et al., 2014). Flavonoids are mainly found from plant and secondary metabolites of fungus. Cheng et al., 2015, Lee et al., 2013; Das & Rosazza, 2006 also found that flavonoid levels increased during fermentation by several fungal species including Aspergillus, Penicillium, Rhizopus and Monascus. Thus, Monascus fermentation is a favorable approach to increase the phenolic and flavonoid contents of fermented brown seaweed.

Increases in reducing sugar levels during fermentation are attributable to various enzymatic reactions (Afoakwa et al., 2013). Also, the protein content of Monascus-fermented seaweed increased. Bayitese et al. (2015) found that fermentation of cassava peel, pineapples and cocoyam by Saccharomyces cerevisae and A. oryzae enriched protein levels. Seaweeds are rich in PUFAs that exhibit anti-arteriosclerosis, anti-hypertension, anti-inflammation and

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immunoregulatory effects (Holdt & Kraan, 2011). We found that fermentation reduced the levels of some seaweed fatty acids because these were used by the fungi, but the levels of certain fatty acids with bio-potential increased. Moharram et al. (2012) found that the essential fatty acids of M. ruber prevented cardiovascular diseases and depression. Oleic, palmitic, stearic and linoleic acids preserve dermal integrity and renal function and are essential for the normal growth. Additionally, some fatty acids play critical roles in the anti-inflammatory response; prevent arthritis, asthama, cancer, lupus; and improve brain function (Moharram et al., 2012). Fraga et al. (2008) found that Aspergillus spp. were rich in palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2), which together represented 95% of all fatty acids.

ABTS+ radical scavenging activity is based on hydrogen atom transfer. Pyo & Lee (2007) found that soybeans fermented by M. pilosus exhibited significantly higher (p < 0.01) ABTS+ radical scavenging activity than the unfermented extract. Kim et al. (2013) reported that the elevated ABTS+ radical-scavenging activity was attributable to increases in the total phenolic and flavonoid contents. Microbial fermentation enhanced FRAP activity (Xiao et al., 2014). Yao et al. (2010) also reported that Bacillus-fermented legume extracts exhibited higher FRAP activity, perhaps attributable to release of iron chelating compounds. Phenolics in the extracts reduced the of TPTZ–Fe3+ complex to the TPTZ–Fe2+ form (Vadivel et al., 2011). Our results were similar; the Monascus fermentation process produced secondary metabolites (pigment, lovastatin) and also increased phenolic and flavonoid contents.

19

Hydroxyl radical-scavenging is very important; OH− reacts with the amino acids, sugars, nucleotides and lipids of living cells. Many studies have reported that OH− radicals damage cells and that phenolics and flavonoids exhibit hydroxyl radical-scavenging activity (Chandrasekara & Shahidi, 2012; Liyana-Pathirana et al., 2006).

In human, NO is synthesized from amino acids of neuron, vascular endothelial cells, phagocytes. The NO radical creates many physiological issues including diabetes, cancer, multiple sclerosis, ulcerative colitis and Alzheimer’s disease because of its cytotoxic effect. NO is directly scavenged by flavonoids. Xu et al. (2011) found that fermented tea effectively scavenged NO.

Fermented samples inhibited DNA oxidation by Fenton’s reagent to greater extents than unfermented samples due to presence of more phenolic and flavonoid content, similar result was reported previously by Xiao et al. (2014). Different studies showed that phenolic and flavonoid levels were increased by fermentation, bound to redox-active metal ions and prevented redox reaction (Liyana-Pathirana et al., 2006). Chandrasekara & Shahidi (2012) found that hydroxyl radical-induced DNA damage could be prevented by chelating ferrous ions and either decomposing or scavenging H2O2. DNA Disruption and damage are triggered by free radicals, eventually causing carcinogenesis, cytotoxicity and mutagenesis. Monascus-fermented seaweed was rich in polyphenols and flavonoids, protecting DNA from damage by scavenging free radicals.

Hyperglycemia and diabetes mellitus are very common; both are characterized by high blood sugar, triglyceride and cholesterol levels, associated with consumption of high-fat diets and

20

adaptation to modern lifestyle. Acarbose, voglibose and miglitol (synthetic hypoglycemic agents) exert antidiabetic effects associated with gastrointestinal problems including diarrhoea, abdominal discomfort and flatulence. Consequently, it is very important to find new plant αamylase and α-glucosidase inhibitors with few or no side-effects. (Shai et al., 2010). In this case, seaweed based fermented products may effectively treat metabolic disorders. Monascusfermented, phenolic-rich, durian seed extract inhibited α-glucosidase activity (Srianta et al., 2013).

Dietary fats are digested to free fatty acids and glycerol by duodenal pancreatic lipases. In addition, the gastric lipase of the human stomach plays a role in fat digestion. Natural bioactive compounds that inhibit pancreatic lipase are attracting attention because there are no side-effects (Birari & Bhutani, 2007). Moreno et al. (2003) and De la Garza et al. (2011) showed that lipase inhibitor effects of polyphenol-rich grape seed extract and various plant extracts reduced dietary fat absorption and fat accumulation in adipose tissue. Orlistat is approved by the Food and Drug Administration to treat obesity. The IC50 values of orlistat and an aqueous extract of Opuntia ficus-indica in terms of inhibiting pancreatic lipase were 1.57 µg/mL and 588.5 µg/mL, respectively (Padilla-Camberos et al., 2015). Flavonoids, phytosterols and essential fatty acids were found potential in reducing blood sugar and triglycerides, and elevate high density lipoprotein, effectively treating metabolic syndrome (Ryan et al., 2016; Moharram et al., 2012; Wei et al., 2003).

Cytotoxicity assays are commonly used in the food and pharmaceutical industries. The human intestinal Caco-2 cell line is employed extensively for in vitro toxicology studies (Artursson et

21

al., 2001; Sambuy et al., 2005; Videmann et al., 2008). This line was used by Fernandes et al. (2012) to screen new oral drugs in terms of permeability and toxicity; substances highly toxic to intestinal epithelial cells cannot serve drugs. Fernandes et al. (2009) found that a crude fungal extract of Alternaria alternate was moderately toxic to HeLa cells. Banjerdpongchai & Kongtawelert (2011) found that water extract of fermented and unfermented Houttuynia cordata were moderately toxic to the human HL-60, Molt-4 cell lines. In the present study, we found that Monascus-fermented and unfermented extract were not cytotoxic, may thus serve as functional foods.

5. Conclusion Today, natural and healthy foods are very attractive. Seaweed-based products are important because seaweeds contain many bioactive compound and nutrients. Here, we fermented brown seaweeds by red molds (Monascus spp.) to enrich the levels of bioactive compounds to attempt to develop seaweed-based foods. The phenolic, flavonoid, antioxidant, anti-diabetic and antilipase activity in Monascus-fermented seaweed extracts were significantly (p < 0.05) higher than those of unfermented seaweed. Fermented extracts exhibited higher DNA-protective activities and were not toxic to intestinal epithelial Caco-2 cells. Fermented seaweed extracts may be useful foods or as therapeutic diets for patients suffering from oxidative stress, hyperglycemia and/or hyperlipidemia.

Acknowledgments The research was supported by a grant from the National Fisheries Research and Development Institute (R2017022), Republic of Korea.

Conflict of Interest 22

The authors declared no conflict of interest.

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Fig. 1. Fatty acid compositional analysis of fermented and unfermented seaweeds with retention time. SjU (S. japonica unfermented); SjMp (S. japonica fermented by M. purpureus); SjMk (S. japonica fermented by M. kaoliang); UpU (U. pinnatifida unfermented).

32

Fig. 2. Antioxidant activities of fermented (M. purpureus and M. kaoliang) and unfermented brown seaweed. (a) ABTS radical scavenging activity, (b) FRAP scavenging activity, (c) OH radical scavenging activity, (d) NO scavenging activity

33

Band intensity

300000

b

250000 200000 150000 100000 50000 0

1

2

3

4

5

6

7

8

Lane

Fig. 3. (a) Agarose gel electrophoretic separation of damaged DNA and the protective effect of Monascus spp. fermented and unfermented S. japonica and U. pinnatifida extract. Lane 1: λ DNA+ DW (+ control); Lane 2: λ DNA+ DW+ Fenton’s reagent (- control); Lane 3: λ DNA + water extracted of SjMp + Fenton’s reagent; Lane 4: λ DNA + water extracted of SjMk + Fenton’s reagent; Lane 5: λ DNA+ SjU+ Fenton’s reagent; Lane 6: λ DNA+ water extracted of UpMp + Fenton’s reagent; Lane 7: λ DNA+ water extracted of UpMk + Fenton’s reagent; Lane 8: λ DNA+ water extracted of UpU + Fenton’s reagent. SjMp (S. japonica fermented by M. purpureus); SjMk (S. japonica fermented by M. kaoliang); SjU (S. japonica unfermented); UpMp (U. pinnatifida fermented by M. purpureus); UpMk (U. pinnatifida fermented by M. kaoliang); UpU (U. pinnatifida unfermented). S: supercoiled DNA strands; N: nicked DNA strands. (b) Band intensity analysis by GelQuant 1.8.2

34

Fig. 4. Cell proliferation (% of viability) of Caco-2 cells incubated for 24 hours with unfermented brown seaweed extracts and Monascus spp. fermented brown seaweed exracts and assessed by using MTS (non-radioactive cell proliferation assay) technique. SjMp (S. japonica fermented by M. purpureus); SjMk (S. japonica fermented by M. kaoliang); SjU (S. japonica unfermented); UpMp (U. pinnatifida fermented by M. purpureus); UpMk (U. pinnatifida fermented by M. kaoliang); UpU (U. pinnatifida unfermented). The data were expressed as mean ± standard deviation (SD) (n=3).

35

Table 1 Total phenolic, flavonoid, reducing sugar and protein content of Monascus spp. fermented S. japonica and U. pinnatifida and unfermented seaweed extract. Sample

Phenolic content

Flavonoid content

Reducing sugar content

Protein content

(mg gallic acid/g extract)

(mg quercetin/g extract)

(mg/g extract)

(mg/g extract)

SjU

13.64 ± 0.60a

15.16 ± 0.80a

75.95 ± 3.63b

0.39 ± 0.04a

SjMp

71.53 ± 2.25d

27.93 ± 0.28e

87.92 ± 5.97d

0.64 ± 0.02cd

SjMk

66.50 ± 4.64d

25.74 ± 1.90d

87.43 ± 4.63d

0.60 ± 0.14cd

UpU

20.09 ± 0.15b

18.36 ± 0.14b

61.81 ± 2.63a

0.52 ± 0.04bc

UpMp

60.28 ± 1.81c

26.88 ± 1.24de

83.39 ± 0.36cd

0.69 ± 0.11d

UpMk

57.37 ± 6.40c

23.92 ± 0.06c

80.04 ± 2.59bc

0.68 ± 0.05d

Values with the same letter in each column are not significantly different (p < 0.05) according to Duncan’s test Mean ± SD (n=3). SjU (S. japonica unfermented); SjMp (S. japonica fermented by M. purpureus); SjMk (S. japonica fermented by M. kaoliang); UpU (U. pinnatifida unfermented); UpMp (U. pinnatifida fermented by M. purpureus); UpMk (U. pinnatifida fermented by M. kaoliang).

Table 2 Fatty acid content (%) and composition of fermented and unfermented samples analyzed by gas chromatography (GC) Fatty acid (%)

Retention time

SjU

SjMp

SjMk

UpU UpMp UpMk

Caproic Acid (C6:0)

10.91

0.00

2.43

0.84

0.00

0.00

0.00

Caprylic Acid (C8:0)

12.11

0.00

3.57

1.26

0.00

0.00

0.00

Mystric Acid (C14:0)

20.06

8.72

5.25

2.40

3.13

5.74

5.54

Myristoleic Acid (C14:1)

21.93

1.24

0.00

0.00

0.00

0.00

0.00

Pentadaecanoic Acid (C15:0)

21.65

0.00

0.00

0.00

2.99

0.18

0.17

Cis-10-Pentadecanoic acid (C15:1)

23.17

3.60

0.00

0.00

0.00

0.00

0.00

Palmitic Acid (C16:0)

23.42

15.82

27.65

14.56

16.47 41.53 40.47

Palmitoleic Acid (C16:1)

24.62

3.47

0.00

1.63

0.00

0.50

0.53

Heptadecanoic Acid (C17:0)

25.02

0.00

0.00

0.00

0.00

0.00

0.83

Stearic Acid (C18:0)

26.59

1.17

9.44

10.34

2.06

4.97

4.74

Oleic Acid(C18:1n9c)

27.69

20.25

34.98

43.92

7.85

28.29 29.23

Linoleic Acid (C18:2n6c)

29.13

8.37

12.78

19.11

8.29

5.89

6.20

Arachidic Acid (C20:0)

29.54

0.51

0.76

0.78

0.67

1.11

1.09

r-Linoleic Acid (C18:3n6)

30.22

3.55

0.00

0.64

2.24

0.82

0.85

Linolenic Acid (C18:3n3)

30.81

4.40

0.00

0.57

9.22

0.73

0.94

Cis-11,14 Eicosadienoic Acid (C20:2)

31.99

7.35

1.65

1.79

22.23

1.94

1.43

36

Behenic Acid (C22:0)

32.42

0.00

0.00

0.00

0.00

0.99

0.67

Cis-8,11,14-Eicosatrienoic Acid (C20:3n6)

33.10

0.56

0.00

0.00

0.00

0.45

0.47

Tricosanoic Acid (C23:0)

33.99

14.38

1.50

2.18

14.37

5.06

4.77

cis-13,16-Docosadienoic Acid (C22:2)

34.93

0.49

0.00

0.00

0.00

0.00

0.00

cis-5,8,11,14,17-Eicosapentanoic Acid (C20:5n3)

36.00

6.12

0.00

0.65

10.47

1.80

2.07

SjU (S. japonica unfermented); SjMp (S. japonica fermented by M. purpureus); SjMk (S. japonica fermented by M. kaoliang); UpU (U. pinnatifida unfermented); UpMp (U. pinnatifida fermented by M. purpureus); UpMk (U. pinnatifida fermented by M. kaoliang).

Table 3 IC50 values of antioxidant, antidiabetic and lipase inhibition activity of fermented and unfermented samples Samples

IC50 (mg/mL)

IC50 (µg/mL)

ABTS

SjU

FRAP

OH

NO

α-amylase

Rat

Rat

intestinal

intestinal

α-

α-

glucosidase

glucosidase

(Maltose)

(Sucrose)

Lipase

0.02 ±

3.38 ±

2.16 ±

2.81 ±

3.71 ±

0.29 ±

0.97 ±

8.03 ±

0.17c

0.03e

0.25d

0.96c

0.22d

0.25c

0.10b

0.02d

0.01 ±

1.69 ±

1.18 ±

1.53 ±

0.98 ±

0.02 ±

0.08 ±

4.58 ±

0.14a

0.54b

0.11b

0.03ab

0.10a

0.07a

0.13a

0.85b

0.02 ±

1.98 ±

1.79 ±

1.66 ±

1.90 ±

0.03 ±

0.07 ±

5.39 ±

0.13b

0.18c

0.13c

0.41ab

0.03c

0.16a

0.23a

0.28c

0.03 ±

4.43 ±

2.89 ±

7.18 ±

4.46 ±

0.46 ±

1.30 ±

10.48 ±

0.29d

0.27f

0.30e

0.23d

0.05e

0.12d

0.65b

0.24f

0.01 ±

2.44 ±

0.95 ±

1.79 ±

1.58 ±

0.16 ±

0.25 ±

5.13 ±

0.05a

0.71d

0.10ab

0.15b

0.10b

0.86b

0.06a

0.28c

0.01 ±

2.45 ±

1.65 ±

1.30 ±

1.51 ±

0.27 ±

0.33 ±

7.35 ±

0.06a

0.90d

0.19c

0.07a

0.37b

0.19c

0.82a

0.12e

Ascorbic

0.02 ±

1.21 ±

0.76 ±

2.51 ±

-

-

-

-

acid

0.07b

0.86a

0.02a

0.02c

SjMp

SjMk

UpU

UpMp

UpMk

37

Acarbose

Orlistat

-

-

-

-

-

-

-

-

1.45 ±

1.59 ±

0.35 ±

0.03b

0.15e

0.02a

-

-

-

-

3.67 ± 0.19a

Values with the same letter in each column is not significantly different (p < 0.05) according to Duncan’s test Mean ± SD (n=3). SjU (S. japonica unfermented); SjMp (S. japonica fermented by M. purpureus); SjMk (S. japonica fermented by M. kaoliang); UpU (U. pinnatifida unfermented); UpMp (U. pinnatifida fermented by M. purpureus); UpMk (U. pinnatifida fermented by M. kaoliang).

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