Anti-glycation properties of the aqueous extract solutions of dried algae products and effect of lactic acid fermentation on the properties

Food Chemistry 192 (2016) 1109–1115

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Anti-glycation properties of the aqueous extract solutions of dried algae products and effect of lactic acid fermentation on the properties Takashi Kuda a,⇑, Mika Eda a, Manami Kataoka a, Maki Nemoto a, Miho Kawahara a, Satoshi Oshio b, Hajime Takahashi a, Bon Kimura a a b

Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-city, Tokyo 108-8477, Japan TERRADA Warehouse Company, 2-6-10 Higashi-Shinagawa, Shinagawa-city, Tokyo 140-0002, Japan

a r t i c l e

i n f o

Article history: Received 17 April 2015 Received in revised form 23 June 2015 Accepted 17 July 2015 Available online 18 July 2015 Keywords: Edible algae Anti-glycation Antioxidant Lactobacillus plantarum

a b s t r a c t The antioxidant and anti-glycation properties in aqueous extract solutions (AESs) of 11 dried algae products were investigated. AESs of brown algae Ecklonia kurome (kurome) and Ecklonia stolonifera (tsuruarame) showed a strong DPPH radical-scavenging capacity and Fe-reducing power with high total phenolic compound content. On the other hand, superoxide anion radical-scavenging capacities of Porphyra sp. (iwanori, red alga), sporophyll of Undaria pinnatifida (mekabu, brown alga), and Gelidiaceae sp. (tengusa, red alga) were also high. Anti-glycation activities in BSA-fructose and BSA-methylglyoxal glycation were also high in kurome, while iwanori showed high activity. Results of the BSA-fructose model agreed with those of superoxide anion radical-scavenging. On the other hand, those of the BSA-methylglyoxal model agreed with those of the phenolic content, DPPH radical-scavenging capacity, and Fe-reducing power. Anti-glycation activities of iwanori, U. pinnatifida (wakame), and mekabu in the BSA-fructose model were clearly increased by fermentation with Lactobacillus plantarum AN6. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Since ancient times, the inhabitants of coastal regions of Far Eastern countries, such as Korea and Japan, have discovered and collected edible algae from beach cast (Ikehara & Hayashida, 2003). Particularly in the Noto peninsula coastal region, which faces the middle of the East Sea (the Sea of Japan), 100 or more species of algae can be collected, of which approximately 40 are edible (Kuda & Ikemori, 2009). the Ministry of the Environment, Government of Japan, defines a Satoumi as a coastal area where biological productivity and biodiversity have increased as a consequence of human activity (Berque & Matsuda, 2013). The traditional eating habit of various algae is considered one of the features of the Noto Satoumi region. It is known that these algae, particularly the brown algae Ecklonia cava, Ecklonia stolonifera, and Ecklonia kurome, contain notable bioactive compounds, such as high content of phlorotannins (algal polyphenols), that have antiviral, antibacterial, and antioxidative properties (Kuda, Kunii, Goto, Suzuki, & Yano, 2007; Kwon et al., 2013). In recent years, anti-glycation properties have attracted attention as having food function (Deetae, Parichanon, Trakunleewatthana, Chanseetis, & Lertsiri, 2012; Ho, Wu, Lin, & ⇑ Corresponding author. E-mail address: [email protected] (T. Kuda). http://dx.doi.org/10.1016/j.foodchem.2015.07.073 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

Tang, 2010). Glycation is a non-enzymatic reaction of reducing sugars with amino acids and/or proteins in processed food and in vivo (Anguizola et al., 2013). Advanced glycation end products (AGEs), such as carboxylmethyl lysine and carboxylethyl lysine are generated after various intermediates such as glyoxal, methylglyoxal (MGO), and 3-deoxyglucosone (Nemet, Varga-Defterdarovic, & Turk, 2006). AGE formation is irreversible. AGEs are thought to induce diabetes and other diabetes- and ageing-related illnesses such as retinopathy, cataracts, arteriosclerosis, and renal dysfunction (Semba, Nicklett, & Ferrucci, 2010). Because there are oxidation reactions in several parts in the glycation reactions for AGE generation, antioxidants are considered inhibitory materials for medicines and treatment diets that prevent AGE formation (Nagai, Mori, Yamamoto, Kaji, & Yonei, 2010). In the case of food materials, various foodstuffs are reported to be anti-glycative materials with antioxidant properties (Deetae et al., 2012; Ho et al., 2010). As mentioned above, some edible macroalgae have strong antioxidant activities; however, their anti-glycation properties have yet to be elucidated. On the other hand, some lactic acid bacteria (LAB) including probiotics also have antioxidant and anti-inflammatory activities in vitro and in mice (Kanno, Kuda, An, Takahashi, & Kimura, 2012; Kuda, Kawahara, Nemoto, Takahashi, & Kimura, 2014). Some LABs can increase the antioxidant capacities of vegetables, milk, and soy milk during the fermentation (Kanno et al., 2012;

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Kawahara et al., 2015; Kuda, Kaneko, Yano, & Mori, 2010). The anti-glycation properties of the probiotics Lactobacillus acidophilus and Bifidobacterium infantis were recently reported (Stancu, Sanda, Rogoz, & Sima, 2012). Furthermore, one study reported that the prebiotics inulin and oligofructose ameliorated the AGE-related pathology of adult volunteers with pre-diabetes (Kellow, Coughlan, Savige, & Reid, 2014). In this study, to clarify the anti-glycation effect of traditional edible algae, we determined the inhibitory effect of aqueous extract solutions of dried algae products obtained from the Noto Satoumi region on glycation in bovine serum albumin-fructose (BSA-Fru), BSA-methylglyoxal (BSA-MGO), and lysine-glucose (Maillard reaction) models. Furthermore, to examine the additive or synergistic effect of the food materials and LAB or lactic acid fermentation, the effect of the Lactobacillus plantarum isolated from the Noto Satoumi region on the anti-glycation capacities of the selected algae samples was also investigated. 2. Materials and methods

The dried samples were milled using a blender (Oster 16 Speed Blender; Osaka Chemical Co., Osaka, Japan) and sieved through 1-mm2 mesh. The algae powder (5 g) was added to 200 mL of distilled water and heated at 105 °C for 15 min using an autoclave. After cooling with tap water, the algae suspension was centrifuged at 3000g for 10 min at 4 °C. The collected supernatant was used in the algal aqueous extract solutions (AESs) and stored at 20 °C. 2.3. Noto Satoumi lactic acid bacteria (LAB) strains Two Noto Satoumi LAB strains Lb. plantarum AN6 and Lactococcus lactis subsp. lactis Noto-SU1 were used in this study. Lb. plantarum AN6 was isolated from aji-narezushi, a fermented horse mackerel with cooked rice made in the Noto Satoumi region (Kuda, Yazaki, Ono, Takahashi, & Kimura, 2013). Lc. lactis Noto-SU1 was isolated, in present study, from algal beach cast on a coast and was selected as an acid- and bile-resistant strain. These LAB strains were pre-cultured at 30 °C for 48 h with de Man, Rogosa, and Sharpe (MRS) broth (Oxoid; Basingstoke, UK).

2.1. Chemicals 2.4. Total phenolic content and antioxidant properties of the AESs (+)-Catechin, Folin–Ciocalteu’s phenol reagent, the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical phenazine methosulphate (PMS), 3-(2-pyridyl)-5,6-di(p-sulfophenyl)1,2,4-triazine disodium salt (ferrozine), b-nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium salt (NBT), and MGO were purchased from Sigma–Aldrich (St. Louis, MO). Phloroglucinol dihydrate (PG), potassium ferricyanide, trichloroacetic acid (TCA), BSA, lysine, D-glucose (Glu), and D-fructose (Fru) were purchased from Wako Chemicals (Osaka, Japan), while 1,10-phenanthroline was purchased from Nacalai Tesque (Kyoto, Japan). The other reagents were of analytical grade. 2.2. Preparation of aqueous extract solutions from dried algae solutions A total of 11 dried products (Table 1) of five species of Phaeophyta, E. stolonifera (tsuruarame), E. kurome (kurome), Undaria pinnatifida (wakame) (the frond part), mekabu (sporophyll of wakame), and Chorda filum (tsurumo), as well as three species of Rhodophyta, Gelidiaceae sp. (tengusa), Campylaephora hypnaeoides (ego), and Porphyra sp. (iwanori), were purchased from retail shops in the Noto peninsula region.

Table 1 Dried algal samples used in this study. Scientific name Pahaeophyta Ecklonia stronifera E. kurome Undaria pinnatifida (frond part) U. pinnatifida (frond part) U. pinnatifida (sporophyll part) U. pinnatifida (sporophyll part) Chorda filum Rhodophyta Gelidiaceae sp. Gelidiaceae sp. Campylaephora hypnaeoides Porphyra

Market name (Japanese)

Harvest place (city)

Abbreviation

Kajime Kajime Wakame

Wajima Suzu Wajima

E1 E2 Up1

Wakame

Suzu

Up2

Mekabu

Wajima

Up’1

Mekabu

Suzu

Up’2

Tsurumo

Wajima

Cf

Tengusa Tengusa Ego

Wajima Suzu Wajima

G1 G2 Ch

Iwanori

Suzu

P

2.4.1. Phenolic compounds Total phenolic content as polyphenol content level was determined as described previously (Kuda & Ikemori, 2009) with slight modifications. Briefly, 0.03 mL of a diluted sample solution and 0.06 mL of 10% Folin–Ciocalteu solution were placed in a 96-well microplate. After 3 min, 0.12 mL of 10% sodium carbonate was added. The mixture was allowed to stand for 60 min at ambient temperature and the absorbance was measured at 750 nm using a grating microplate reader (SH-1000 Lab; Corona Electric, Hitachinaka; Ibaraki, Japan). The phenolic content is expressed as PG equivalents PGEq/mL. The experiments to determine phenolic content, antioxidant properties, and anti-glycation properties were conducted in triplicate. 2.4.2. DPPH radical-scavenging capacity DPPH radical-scavenging capacity was determined as described previously (Kuda & Yano, 2009) with slight modification. Briefly, sample diluted solution (0.1 mL) and ethanol (0.1 mL) were put into a 96-well microplate, and absorbance at 517 nm (Abs1) was measured using the microplate-reader (SH-1000 Lab). Next, 1 mmol/l DPPH radical in was added and incubated at 37 °C for 30 min and the absorbance (Abs2) was measured again. The DPPH radical-scavenging capacity was calculated using the following formula: Radical scavenging capacity ð%Þ ¼ ð1  ðAbs2 of sample  Abs1 of sampleÞ=ðAbs2 of control  Abs1 of controlÞ  100

2.4.3. Superoxide anion radical-scavenging activity Superoxide anion radical-scavenging activity was measured using a non-enzymatic method (Kuda & Ikemori, 2009). The sample solution (0.1 mL) was treated with 0.05 mL of 250 mmol/l phosphate buffer (pH 7.2), 2 mmol/l NADH (0.025 mL), and 0.5 mmol/l NBT (0.025 mL), while absorbance at 560 nm was measured as a blank value. After a 5-min incubation at ambient temperature with 0.025 mL of 0.03 mmol/l PMS, the absorbance was measured again. The radical-scavenging capacity was calculated using the above formula.

T. Kuda et al. / Food Chemistry 192 (2016) 1109–1115

2.4.4. Ferrous reducing power The reducing power was determined as described in our previous report (Kuda & Yano, 2009) with slight modification. Briefly, each 0.05 mL of the sample solution, 0.025 mL of 0.1 mol/l phosphate buffer (pH 7.2), and 0.025 mL of 10 g/l potassium ferricyanide were placed in a 96-well microplate. After incubation at 37 °C for 60 min, 0.025 mL of 10% TCA and 0.1 mL of distilled water were added and the absorbance was measured at 700 nm (Abs1). Next, 0.025 mL of 0.1% FeCl3 was added to the mixture and the absorbance was measured again (Abs2). Ferrous reducing power was calculated using the following formula: Reducing power ðOD 700 nmÞ ¼ ðAbs2 of sample  Abs1 of sampleÞ  ðAbs2 of control  Abs1 of controlÞ

1111

2.6. Algal fermentation properties of Noto Satoumi LABs The pre-cultured strains (0.03 mL) were inoculated in 3 mL of the AESs and incubated at 30 °C for 2 days. If turbidity could be observed by the naked eye, the pH value was determined using a pH meter (Twin pH; Horiba; Kyoto, Japan). The anti-glycation assays in the BSA-Fru and BSA-MGO models were measured as described above. 2.7. Statistical analysis Data of the antioxidant activity properties of the selected strains are presented as means and standard errors. Data pertaining to the anti-glycation capacities before versus after fermentation were subjected to Student’s t-test using statistical software (Excel Statistic Ver. 6; Esumi Co., Tokyo, Japan).

2.5. Anti-glycation properties of the AESs 2.5.1. Lys-Glu Maillard reaction The effect of the AESs on the Lys-Glu Maillard reaction was determined as described previously (Kuda & Yano, 2014) with slight modifications. Both of Glu and L-lysine (2.0 mol/l, 0.5 mL) were mixed with 0.5 mL of AES and 0.5 mL of 0.25 mol/l sodium phosphate buffer in screw-capped test tubes and kept at 50 °C in a water bath for 3 h. The absorbance was measured at 465 nm using the grating microplate reader. The percentage of Maillard reaction inhibition was calculated using the following equation:

Maillard reaction inhibition ð%Þ ¼ ð1  ðAbs 3 h of sample  Abs 0 h of sampleÞ= ðAbs 3 h of blank  Abs 0 h of blankÞÞ  100

2.5.2. BSA-Fru glycation model The anti-glycation assay in the BSA-Fru model was determined using the method of Wang, Yagiz, Buran, Nunes, and Gu (2011) with slight modification. This model evaluates all stages of protein glycation. Fru (1.5 mol/l, 0.5 mL) was mixed with 0.5 mL of AES and 0.5 mL of sodium phosphate buffer (50 mmol, pH 7.4, with 0.02% sodium azide) in screw-capped test tubes and kept at 37 °C for 2 h. BSA (30 mg/mL, 0.5 mL) was added to each test tube and the mixtures were incubated at 37 °C for 5 days. Fluorescent AGEs were monitored on a multiple microplate reader (SH-9000; Corona Electric) using 340 and 420 nm as the excitation and emission wavelengths. Percentage of the AGE inhibition was calculated by the following equation:

3. Results and discussion 3.1. Total phenolic content The total phenolic contents in the AESs of the 11 dried algae products are shown in Fig. 1A. The phenolic concentrations in the sample AES of kurome E. kurome (E1), tsuruarame E. stolonifera (E2), and mekabu sporophyll of U. pinnatifida (Up’1) were high at approximately 8, 4, and 3 PGEq/mL AES, respectively, corresponding to 320, 160, and 120 lmol PGEq/g of the dried products, respectively. Iwanori Porphyra sp. P AES showed phenolic contents of approximately 1.6 PGEq/mL. The phenolic compound contents of the other AESs were <1 lmol PGEq/mL. The high content of the total phenolic contents in the AESs of two Ecklonia (E1 and E2) and iwanori P agreed with those of our previous reports (Kuda & Ikemori, 2009; Kuda, Tsunekawa, Goto, & Araki, 2005; Kuda et al., 2007). On the other hand, the contents of the two mekabu products in the AESs differed from each other. Mekabu is a sporophyll of U. pinnatifida. The contents of various compounds, including polysaccharides and polyphenols, in mekabu were thought to change drastically during the annual stages (Hodt & Kraan, 2011). 3.2. Antioxidant properties

FI 0 d and 5 d represents fluorescent intensity after the reaction for 0 and 5 days, respectively.

3.2.1. DPPH radical-scavenging capacity DPPH has been used extensively as a free radical to evaluate the reduction of substances in various foods including edible algae (Kuda & Ikemori, 2009; Kuda et al., 2005). The percentage of DPPH-scavenging activities at the concentration of 3.125 mg AES/mL is shown in Fig. 1B. Among the 11 algal AES samples, the scavenging activity was high in AESs of kurome E2 followed by tsuruarame E1, mekabu Up’1, and tsurumo Cf. The order appeared to agree with the phenolic content results (Fig. 1A), although the capacity of iwanori P was not as high. The scavenging capacity of the other five AESs was unclear. These results agreed with those of our previous study (Kuda & Ikemori, 2009; Kuda et al., 2005).

2.5.3. BSA-MGO glycation model The anti-glycation assay in the BSA-MGO model was performed also using the method of Wang et al. (2011) with slight modification. This model evaluates the middle stage of protein glycation. MGO (60 mmol/l, 0.5 mL) was mixed with 0.5 mL of AES and 0.5 mL of the sodium phosphate buffer and kept at 37 °C for 2 h. BSA (30 mg/mL, 0.5 mL) was added to each test tube and incubated at 37 °C for 5 days. Fluorescent AGEs were monitored on the multiple microplate reader using 340 and 380 nm as the excitation and emission wavelengths. The percentage of AGE inhibition was calculated using the same equation as that in the BSA-Fru model.

3.2.2. Superoxide anion radical-scavenging capacity The capacity of the AESs to scavenge superoxide radicals was confirmed when they were generated by a chemical system composed of PMS, NADH, and oxygen (Fig. 1C). The scavenging capacities of kurome E2, tsuruarame E1, and mekabu Up’1, which showed high phenolic content and a high DPPH radical-scavenging capacity, were also high. On the other hand, the scavenging capacity of the AESs of mekabu Up’2 and tengusa Gelidiaceae sp. G1 was also high, although their phenolic content was not as high. Furthermore, the capacity of iwanori P was also high. The capacity was low in AESs of frond of wakame Up1 and

Anti-glycation capacity ð%Þ ¼ ð1  ðFI 5 d of sample  FI 0 d of sampleÞ=ðFI 5 d of blank  FI 0 d of blankÞÞ  100

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µmol phloroglucinol -1 Equivalent mL AES

400 10.0

Total phenolic compounds

300 7.5 200 5.0 2.5 100 00

Scavenging capacity (%)

B 100

DPPH radical scavenging

80 60 40 20 0 E2 E2

E1 E1

Up'1 Up’1

Cs Cf

Up2 Up2

P P

Samples*

100

Superoxide anion radicals scavenging

80 60 40 20 0

E2 E1 Up'2 G2 Up1 Up1 E2 Up’1 Up’2 Cs Cf E1 E1 G1 G1 PP Up'1 Ch G2

Cs Up2 Up2 Up'2 Cf Up’2 Up1 Up1 Ch Ch G2 G2 G1 G1

D Absorbance (OD at 700nm)

E2 E1 E1 Up’1 Up'1 P E2 P

Scavenging capacity (%)

C

A

2.5

Fe-reducing power

2.0 1.5 1.0 0.5 0.0 E2 E2

E1 E1

P Up'2 Up’1 Up’2 CsCf Up2 Up1 Up1 Up2

Samples*

Fig. 1. Total phenolic compound content (A), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radical-scavenging capacity (B and C), and Fe-reducing power (D) of aqueous extract solution of dried algae products. ⁄See Table 1. Sample contents in B, C, and D were 3, 0.8, and 3 mg, respectively, in dried sample equivalent/mL. Values are mean and standard error of the mean (n = 3).

Up2. In our previous report (Kuda & Ikemori, 2009), the correlation between superoxide anion radical-scavenging activity and the phenolic compound content was moderate (r2 = 0.611). The superoxide anion radical-scavenging activity of the AESs was caused not only by the phenolic compounds but also by other water-soluble compounds such as peptides, polysaccharides, and Maillard reaction products (Kuda, Hishi, & Maekawa, 2006; Shao, Chen, & Sun, 2013). In most organisms, superoxide anion radicals are converted to hydrogen peroxide by superoxide dismutase. In the absence of transition metal ions, hydrogen peroxide is stable. However, hydroxyl radicals can be formed by the reaction of superoxide with hydrogen peroxide in the presence of metal ions, usually ferrous or copper (Macdonald, Galley, & Webster, 2003). Hydroxyl free radicals are much more reactive (toxic) than superoxide anions. The superoxide anion-scavenging activity of AESs suggests that the edible algae shown in Fig. 1C, even Up’2, showed low phenolic content and DPPH radical-scavenging capacity, have the benefits of decreasing not only of superoxide anion, but also of hydrogen peroxide and hydroxyl radicals.

3.2.3. Ferrous reducing power Most non-enzymatic antioxidant activities such as the scavenging of free radicals and the inhibition of peroxidation are mediated by redox reactions (Zhu, Hackman, Ensuma, Holt, & Keen, 2002). Compounds with reducing power are electron donors that can reduce the oxidized intermediates of lipid peroxidation processes and thereby act as primary and secondary antioxidants (Yen & Chen, 1995). As shown in Fig. 1D, the highest amount of the reducing power was obtained in AES of kurome E2, followed by tsuruarame E1, which supported the phenolic content and DPPH radical scavenging results.

3.3. Anti-glycation properties 3.3.1. Effects on Lys-Glu Maillard reaction The Maillard reaction during the heating at 50 °C for 3 h was detected as browning observed by absorbance at 465 nm. The effects of the AESs of the edible algae are summarized in Fig. 2A. The AESs of tengusa G1 and G2 suppressed the browning by about 43% and 33%, respectively. The AESs of ego Ch and iwanori P also inhibited about 24% of the browning. These four samples are red algae Rhodophyta. On the other hand, kurome E2 strongly promoted the browning, while mekabu Up’1 also promoted the browning. These two brown algae Phaeophyta showed high phenolic content and radical-scavenging capacities (Fig. 1A–C). In the case of cocoa, the decreased reduction of sugars and amino acids by the Maillard reaction during the roasting was accelerated by the addition of polyphenol in a dose-dependent manner (Noor-Soffalina, Jinap, Nazamid, & Nazimah, 2009). In the case of the Lys-Glu model in test tubes, the browning was also accelerated by seawater bittern (nigari) solution, which mainly consists of Mg2+ and Cl (Kuda & Yano, 2014). Furthermore, the addition of phosphate clearly increased the browning. Kurome and tsuruarame are rich in both polyphenols and minerals (Kuda & Ikemori, 2009). In this experiment, the heating temperature was very high. Therefore, it is considered that this phenomenon reflected a cooking effect rather than glycation in the body. Some Maillard-reacted brown products are considered to reflect food functions, including antioxidant capacity (Kuda & Yano, 2014; Kuda et al., 2006).

3.3.2. Anti-glycation property in the BSA-Fru model We used BSA in this study to determine the anti-glycation property. Serum albumin is abundant in the serum, and it can be glycated at multiple sites (Wautier & Guillausseau, 2001). The

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C 50

100

0

80

-50 -100 -150

Maillard reaction (Lysine-Glucose)

-200

Inhibition (%)

Inhibition (%)

A

60 40 20

-250

0 E2 E2

G1 G2 Ch P Up’2 Up1 Cf Up2 E1 Up’1 E2

B

P E1 E1 Up’1 Up'1 Cf CsUp’2 Up'2Up2 Up2 Up1 Up1 G2 G2 Ch Ch G1 G1 P

D BSA-Fru

80 60 40

III

100

Inhibition (%)

100

Inhibition (%)

BSA-MGO

E2

80

Up’1 E1

IV

Up’2 60 G1

I

40 20

20

II

P

Cf

0

0 E2 PP E1 E1Up’1 Up'1Up’2 Up'2 G1 G1 Cf Cs Up1 Up1 Up2 Up2 Ch Ch G2 G2 E2

Samples*

0

5

10

15

20

25

30

Total phenolic compounds (mmol phloroglucinol Equivalent /g)

Fig. 2. Effects of aqueous solutions of dried algae products on Maillard reaction (A), glycation of bovine serum albumin (BSA) with fructose (Fru; B) and methylglyoxal (MGO; C). ⁄See Table 1. Values are mean and standard error of the mean (n = 3). Figure D shows the correlations between total phenolic compounds and inhibition activities in BSAFru (open circles and I: r2 = 0.536) and BSA-MGO (closed circles and III: r2 = 0.782) models. Lines II and IV show the correlation without Rhodophyta (r2 = 0.679 in BSA-Fru, and 0.983 in BSA-MGO).

BSA-Fru model system was used to simulate the protein glycation that occurs at an accelerated rate in vivo under non-physiological conditions, accounting for some of the complications of hyperglycaemia and diabetes (Wang et al., 2011). In the BSA-Fru model (Fig. 2B), the AESs of kurome E2, tsuruarame E1, and mekabu Up’1 had high DPPH radical-scavenging capacity (Fig. 1B) and showed high anti-glycation capacities of 98%, 72%, and 71%, respectively. However, iwanori P and mekabu Up’2 also showed clear anti-glycation capacities of 78% and 64%, respectively. This result agrees with the superoxide anion radical-scavenging capacity (Fig. 1C) rather than phenolic content, DPPH radical-scavenging capacity, and ferrous reducing power. 3.3.3. Anti-glycation property in the BSA-MGO model This model evaluates the middle stage of protein glycation (Wang et al., 2011). MGO is a well-known intermediator for AGE formation. MGO can react with serum albumin as well as other extracellular and intracellular proteins within the tissues. Furthermore, some MGO-modified proteins can be toxic to cells (Takeuchi et al., 2001). In the BSA-MGO model (Fig. 2C), as in the BSA-Fru model, AESs of kurome E2, iwanori P, tsuruarame E1, and mekabu Up’1 showed high anti-glycation capacities of 96%, 75%, 65%, and 55%, respectively. The inhibitory effects of the other AESs were not as high. This result is in agreement with the phenolic content (Fig. 1A), DPPH radical-scavenging capacity (Fig. 1B), and Fe-reducing power (Fig. 1D) rather than superoxide anion radical-scavenging capacity. Fig. 2D shows the correlation between the phenolic content and the anti-glycation capacities. That between BSA-Fru inhibition and the phenolic content (r2 = 0.536) was lower than that between

BSA-MGO and the phenolic content (r2 = 0.782). Considering Phaeophyta without Rhodophyta, the decision coefficient (r2) values in BSA-Fru and BSA-MGO were 0.679 and 0.983, respectively. In the case of brown algae Phaeophyta, the inhibitory effect on BSA-MGO glycation might be caused by their polyphenol compounds called phlorotannins, which have potential pharmacological properties such as anti-diabetic and anti-cancer activities (Thomas & Kim, 2011). 3.4. Anti-glycation properties of the AESs fermented with Noto Satoumi LABs 3.4.1. Fermentation properties We determined the growth of Lc. lactis subsp. lactis Noto-SU1 and Lb. plantarum AN6 in the 11 AES samples. Among them, six algae – wakame Up1, mekabu Up’2, tsurumo Cf, iwanori P, tengusa G1, and ego Ch – could be fermented by both of the Satoumi LABs. On the other hand, the other five AESs – from kurome E2, tsuruarame E1, wakame Up2, mekabu Up’1 and tengusa G2 – could not be fermented by the LAB strains. These algae, particularly in kurome E2, had rich phenolic content (Fig. 1A). Their bactericidal effects, particularly on Gram-positive bacteria, have been reported (Kuda et al., 2007). The pH values of the six AESs after 48 h of fermentation are summarized in Table 2. The fermentation activity, estimated by pH lowering, of Lb. plantarum AN6 was stronger than that of Lc. lactis subsp. lactis Noto-SU1. Each AES of wakame and mekabu (Up1 and Up’2) was easy fermented by Lb. plantarum AN6 from pH 6.4 to 3.5. However, the other AESs of the algae (Up2 and Up’1) were not fermented. Although mekabu Up’1 showed a high polyphenol content

T. Kuda et al. / Food Chemistry 192 (2016) 1109–1115

(Fig. 1A), the wakame Up2 content was not so high. Interestingly, although the pH value of tsurumo Cf AES was low at pH 4.5, Lb. plantarum AN6 could ferment and lowered the pH to 3.9 and 3.5.

3.4.2. Anti-glycation properties of the fermented aqueous solutions and suspensions The AES samples showed fermentation by the Satoumi LABs, and induction of the anti-glycation capacities was determined as described above. In the BSA-MGO model, no significant effect of fermentation was shown. In the BSA-Fru model, the anti-glycation capacities of the AESs of iwanori P, wakame Up1, and mekabu Up’2 were increased by the fermentation with Lb. plantarum AN6 (Fig. 3B). On the other hand, there were no significant effects of Lc. lactis subsp. lactis Noto-SU1 on the anti-glycation of any algae samples. As shown in Figs. 1 and 2, the anti-glycation capacities in BSA-Fru model was agreed with result of superoxide anion radical-scavenging capacity, rather than DPPH radical scavenging capacity, Fe-reducing power and also total phenolic compound content. We previously reported that the fermentation by several Lb. plantarum strains isolated from the Satoumi region induced the superoxide anion radical-scavenging and anti-inflammation capacities in MRS broth, Japanese white radish, milk, and soybean milk (Kawahara et al., 2015; Kuda et al., 2010). In this study, we determined the anti-glycation properties of 11 dried products of edible algae obtained from the Noto peninsula. Kurome and tsuruarame, which showed significantly strong anti-glycation and antioxidant activities and high polyphenol content, are considered useful food materials for human health, particularly in diabetes. Furthermore, the anti-glycation capacity of some of the samples, such as iwanori and mekabu, was increased by the fermentation with Lb. plantarum AN6. The anti-glycation capacity of the fermented algal AESs was the same as kurome or tsuruarame. Although we have not directly investigated the absorption of algal polyphenols and other compounds from intestinal epithelial cells, it is had been reported that small polyphenols such as apigenin and resveratrol could be absorbed rapidly by human entero-epithelial Caco-2 cells, likely via transporters (Teng et al., 2012). As mentioned above, brown algae have polyphenols called phlorotannins that show a wide molecular size range (Thomas & Kim, 2011). Furthermore, in our previous study, the polyphenols and antioxidant capacity were detected in both the high

Table 2 Values of pH in the aqueous solution of algae fermented by lactic acid bacteria (LABs) isolated from Noto. Algae

Phaeophyta Undaria pinnatifida (frond part) U. pinnatifida (sporophyll part) Chorda filum Rhodophyta Porphyra sp. Gelidiaceae sp. Campylaephora hypnaeoides

Abbreviation

Control without LABs

Lactococcus lactis N-SU1

Lactobacillus plantarum AN-6

Up1

6.37 ± 0.06

5.30 ± 0.00

3.50 ± 0.10

Up’2

6.47 ± 0.06

4.89 ± 0.29

3.50 ± 0.00

Cf

4.50 ± 0.00

4.40 ± 0.00

3.47 ± 0.12

P G1 Ch

6.57 ± 0.06 7.30 ± 0.00 7.40 ± 0.10

4.70 ± 0.00 5.99 ± 0.06 5.11 ± 0.10

4.50 ± 0.00 4.63 ± 0.06 4.57 ± 0.06

The aqueous solutions inoculated LABs were incubated at 30 °C for 48 h. Value are mean and SD (n = 3). E. stronifera (E1), E. kurome (E2), U. pinnatifida (Up2, Up’1) and Gelidiaceae sp. (G2) were could not be fermented.

A

B 100 Inhibition (%)

1114

80

BSA-Fru

**

*

*

60 40 20

0

Iwanori Wakame (P) (Up1)

Mekabu (Up’2)

Fig. 3. Image of Lactobacillus plantarum AN-6 (A) and the increasing anti-glycation capacity in BSA-Fru model of edible algae by AN6 fermentation (B). The cell shape was observed under a table top scanning electron microscope (TM3030; Hitachi, Tokyo, Japan) without any treatment or staining. The anti-glycation capacity of iwanori was measured after dilution with three volumes of water. (B) Values are mean and standard error of the mean (n = 3). ⁄ and ⁄⁄: p < 0.05 and 0.01, respectively, when the fermented aqueous extract solutions (AESs) (closed columns) were compared to the intact AESs (open columns).

(>300 kDa) and low (<3 kDa) molecular-weight fractions of brown algae (Kuda et al., 2006). The receptor for advanced glycation end products is a multiligand cell-surface molecule of the immunoglobulin superfamily. It was originally described as a receptor for protein adducts formed by AGEs that accumulate in diseases such as diabetes and renal failure (Zill, Günther, Erbersdobler, Fölschb, & Faista, 2001). Recently, the inhibitory effect of dietary prebiotic supplementation on advanced glycation was found to be correlated with intestinal microbiota (Kellow et al., 2014). It can be considered that not only low-molecular-weight compounds but also high-molecularweight compounds in the algal AESs, as well as LAB, ameliorate the AGE-related damages with some intestinal microbiota. Thus, further studies related to anti-glycation properties using human cell cultures and in vivo experiments, and for the purification of active compounds in intact and fermented AESs of edible algae are needed. 4. Conclusion In conclusion, here we determined the antioxidant and anti-glycation properties of the AESs of dried algae products made in the Noto peninsula. The AESs of brown algae kurome E2 and tsuruarame E1 showed strong DPPH radical-scavenging capacities and Fe-reducing powers with high total phenolic compound contents. On the other hand, the superoxide anion radical-scavenging capacities of iwanori P, mekabu Up’2, and tengusa G1 were also high. Although the anti-glycation activities shown by the BSA-Fru and BSA-MGO models were also high in kurome, a Rhodophyta (iwanori P) showed high activity. The results of the BSA-Fru model, which evaluates all stages of protein glycation, agreed with the superoxide anion radical-scavenging results. On the other hand, that of the BSA-MGO model, which evaluates the middle stage of protein glycation, agreed with results of total phenolic content, DPPH radical-scavenging capacity, and Fe-reducing power. The anti-glycation activities of iwanori P, wakame Up1, and mekabu Up’2 in the BSA-Fru model were clearly increased by fermentation with Lb. plantarum AN6. The present study’s results suggest that, once fermented with LAB, kurome and other edible algae have profitable functions for preventing diabetes- and ageing-related glycation. Acknowledgements The present study was supported by The Towa Foundation for Food Science & Research.

T. Kuda et al. / Food Chemistry 192 (2016) 1109–1115

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