Identification of Bioactive Peptides with Î±-Amylase Inhibitory Potential

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Bioactive Constituents, Metabolites, and Functions

Identification of novel bioactive peptides with #-amylase inhibitory potential from enzymatic protein hydrolysates of red seaweed (Porphyra spp) Habtamu Admassu, Mohammed A. A. Gasmalla, Ruijin Yang, and Wei Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00960 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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Journal of Agricultural and Food Chemistry

Identification of novel bioactive peptides with α-amylase inhibitory potential from enzymatic protein hydrolysates of red seaweed (Porphyra spp)

Habtamu Admassu †, §, Mohammed A. A. Gasmalla4 ¶, Ruijin Yang †, ‡, Wei Zhao†, ‡ * †

State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu

Avenue, Wuxi 214122, Jiangsu, China §

Department of Food Process Engineering, Addis Ababa Science and Technology University,

P. O. Box 16417, 1000 Addis Ababa, Ethiopia. ‡

School of Food Science and Technology, Jiangnan University, 1800 Lihu Ave Wuxi, 214122

Jiangsu, China; ¶

Department of Nutrition and Food Technology, Faculty of Science and Technology, Omdurman Islamic University, P.O. Box 382, 14415, Khartoum, Sudan

*Corresponding to: Wei Zhao E-mail: [email protected], Tel: +86-13-952466350, Fax: 0510-85919150,

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ACS Paragon Plus Environment


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Abstract

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Inhibition of α-amylase enzyme is one therapeutic approach in lowering glucose level in

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the blood to manage diabetes mellitus. In this study α-amylase inhibitory peptides were

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identified from proteolytic enzymes hydrolysates of red seaweed laver (Porphyra species)

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using consecutive chromatographic techniques. In the resultant fractions from RP-HPLC

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(D1-10), D2 inhibited α-amylase activity (88.67 ± 1.05 %) significantly (p ≤ 0.5) at 1mg/mL

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protein concentration. A mass spectrometry (ESI-Q-TOF- MS) analysis was used to identify

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peptides from this fraction. Two novel peptides were identified as Gly-Gly-Ser-Lys and

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Glu-Leu-Ser. To validate their α-amylase inhibitory activity, these peptides were synthesized

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chemically. The peptides were demonstrated inhibitory activity at IC50 value: 2.58 ± 0.08 mM

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(Gly-Gly-Ser-Lys) and 2.62 ± 0.05 mM (Glu-Leu-Ser). The inhibitory kinetics revealed that

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these peptides exhibited non-competitive binding mode. Thus, laver can be a potential source

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of novel ingredients in food and pharmaceuticals in diabetes mellitus management.

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Key words: Macro algae, Laver, Bioactive peptides, α-amylase inhibition, Diabetes mellitus

15 16 17 18 19 20 21 22 23 24 25 2


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Introduction Recently, food-derived bioactive peptides with therapeutic abilities gained an increasing

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interest.1

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maintaining the onset of diet-related diseases such as diabetes mellitus (DM) has given

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particular attention.2 DM is a complex metabolic syndrome initiated by diminished production,

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insufficient bioavailability and poor sensitivity of insulin against increased plasma glucose

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level. Alpha-amylase plays a fundamental role in initiating the chemical breakdown of

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complex carbohydrates prior to their further conversion into simpler forms (glucose) and

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absorbed into blood system. Studies suggested that inhibition of α-amylase enzyme can

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considerably lessening the postprandial rise of glucose level in the blood after a mixed

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carbohydrate diet 3, thus providing an important strategy in the controlling , especially type-II

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diabetes. Cognizance of this fact, functional foods and nutraceuticals with α-amylase

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inhibitory potential has gained significant acknowledgement.

Peptides with specific amino acid sequences that are potent in decreasing and

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Previously, it has been described that food origin short chain bioactive peptides ranging

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from 2–20 amino acid residues 4 offer hormone-like physiological benefits in function beyond

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the basic nutrition.5 These peptides may possess a vast number of pharmacological or

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physiological effects including antioxidant, antidiabetics, and lowering blood pressure

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depending on composition of amino acids and their sequence.6 These amino acids are initially

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found encrypted in the original protein molecule. They can be detached and exert their

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bioactivities through the in vitro activities of proteolytic exogenous enzymes, chemical

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hydrolysis, or chemical processing and microbial fermentation of food. 4, 5

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It was reported that α-amylase inhibitory proteins have been extracted and identified from 3



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plant and animal sources.7-9 The diverse knowledge about bioactive peptides has opened up

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potential opportunities to use marine macro algae for the development of pharmaceuticals

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in recent years. Seaweeds having medicinal applications, and exhibiting bioactive properties

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have been screened.11 Among the notable species of seaweeds is laver (Porphyra spp) that

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belongs to red macro algae known as Rhodophyta. This seaweed is traditional healthy food in

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Asian countries, especially popular in Korea and Japan, which is used to make soup and wrap

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sushi with delicious taste.12

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regarded as beneficial for reducing blood sugar.13

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Moreover, the laver (Porphyra spp) has long been locally

In earlier studies, peptides of antihypertensive and antioxidant activities are isolated from 14 15

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macro algae, however, very few studies

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were reported in the literature for α-amylase

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inhibitory potential of seaweed proteins and their hydrolysed peptides. Therefore, this study

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was designed to generate, enhance peptide concentration, separate and identify peptides from

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dried laver, which are exhibiting α-amylase inhibitory properties by using consecutive

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chromatographic techniques, such as ultrafiltration (UF) membrane with various molecular

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weight cut-offs (MWCO), Sephadex gel chromatography, RP-HPLC. Identification the amino

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acid sequence of potential peptides and molecular weight were determined by using mass

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spectrometry (ESI-Q-TOF MS). The isolated and identified peptides were synthesized

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chemically and further confirmed their inhibitory activity in α-amylase bioassay.

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Materials and Methods

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Materials. The freeze dried commercial laver (Porphyra spp) was purchased from

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Rudong Laver farming and processing Industry (Nantong, Jiangsu, China). Laver was

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powdered by means of a laboratory mill IKA (A11BS25, IKA Laboratory Technology, 4


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Staufen, Germany) and sieved to obtain powder of particles size less than150 µm. α-amylase

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(EC 3.2.1.1) in porcine pancreas, with an activity of 10 U/mg solid protein, Pepsin (source:

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gastrointestinal mucosa, activity, ≥ 400 U/mg protein) and Viscozyme®L (a carbohydrase mix,

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multi-enzyme complex containing a wide range of carbohydrases, including arabinase,

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cellulase, β-glucanase, hemicellulase, and xylanase), were obtained from Sigma-Aldrich

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(Shanghai, Jiangsu, China). All the other reagents and chemicals used in this study were

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HPLC and analytical grade.

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Preparation of Laver Protein isolate (LPI). The extraction of protein was performed by

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using carbohydrase mix (Viscozyme® L) enzyme following the method reported previously 16,

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17

with slight modification: a known amount of freeze dried laver powder sample with

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particles size (< 150 µm) was blended with acetate buffer in a ratio 1:25 (w:v) in a metal

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jacketed reactor connected to a thermostatically controlled water bath. The mixture was

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agitated to produce uniform slurries. The slurries were adjusted to a working pH (4.45-4.50)

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of Viscozyme® L, and the cell wall degrading enzyme was added to all slurries and incubated

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at 50 ℃ with a continuous stirring at 300 rpm for 24h. After completion of incubation time,

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the homogenate was allowed to cool, and the solid precipitate and the supernatant was

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separated using centrifugation (Avanti J-25 Centrifuge, Beckman Coulter, USA) (10,000 ×g,

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4 °C , 45 min). The solid precipitate was re-dissolved and washed repeatedly with acidified

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water and kept in ice bath (0-4 ℃) for 1hr to isoelectric precipitation (pH 3.85-4.0) of protein.

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Then after, the centrifugation of the slurry was made at (10,000 ×g, 4 °C, 30 min), and the

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solid precipitate was retained as phase-I. The supernatants at each step were mixed together

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and adjusted to pH 9.50 using sodium hydroxide (1M) and further incubated for 30 min to 5



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solubilize the remaining proteins from the Viscozyme digestion and centrifuged as before. To

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obtain remnant protein isolates, the solid precipitate was removed and the liquid part

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(supernatant) was corrected to pH 3.85-4.0 for isoelectric precipitation as mentioned above

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and solid precipitate was separated by centrifugation (10,000 ×g, 4 °C, 30 min) as phase-II.

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Finally, the solid precipitates obtained in both phase-I and Phase-II were combined,

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thoroughly mixed in deionized H2O until all the solid precipitates are dissolved , then, the pH

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was raised to 7.0, and lyophilized. The freeze dried powder is named as protein Isolate (LPI)

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and used for hydrolysis as substrate. The total nitrogen content (%N) of the isolate was 18

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determined by using Kjeldahl apparatus

, and total protein was calculated as (%N×6.25).

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Figure 1 demonstrates the schematic representation for the extraction of protein from dried

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laver using Viscozyme® L and the consecutive processes of chromatographic techniques used

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in the isolation of α-amylase inhibitory peptides.

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Enzyme screening. The following enzymes: Alcalase, Neutrase, Pepsin and Trypsin were

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involved under their optimal conditions in preliminary experiments to screen effective

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enzyme in producing α-amylase inhibitory hydrolysates. The hydrolysis was underwent with

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Enzyme/Substrate ratio: 1:100 (w/w) and 4h hydrolysis time at recommended optimal

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temperature and pH by the manufacturers (Table 1). The hydrolysates produced were

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evaluated to measure inhibition of α-amylase activity (α-AI) as indicator variable to select

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efficient enzyme, and determine hydrolysis time.

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Hydrolysis of laver protein Isolate to produce hydrolysates (LPH). In order to

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produce α-amylase inhibitory peptides for further study, hydrolysis was conducted using a

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previously selected enzyme in the preliminary experiments under its optimal conditions 6


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described in Table 1. Each freeze-dried LPI was mixed with buffer (1:20 ratio, 8% substrate,

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pH 2, and 37 ℃ ) in a 500 ml jacketed-polyethylene glass reactor connected to a thermostat

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water bath to maintain the temperature, and stirred by magnetic bar on a magnetic stirrer. The

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pH of the dispersion has been adjusted to the working value of selected enzyme using 1.0 M

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HCL or NaOH and reacted with the enzyme at 37 ℃ for 4h. Up on completion of enzymatic

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digestion, the hydrolysate was heated to 90℃ for 15 minutes to deactivate the enzyme, then

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cooled immediately to room temperature using tape water. The hydrolysate was clarified by

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centrifugation at 8000 x g (4 ℃ , 20 min) from insoluble residues and denatured proteins, and

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the supernatants were collected. Fractionation of the supernatants was made via ultrafiltration

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(UF) membrane with molecular weight (MW) cut-offs of 10 KDa and 3 KDa (Millipore Corp.,

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Barnant co., Barrington, IL 60010, USA). The fractions obtained were categorized as:

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MW >10 kDa (LPH-I), MW = 3-10 kDa (LPH-II) and MW< 3KDa (LPH-III). All recovered

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fractions were lyophilized and analysed for their α-amylase inhibition activity. The yield of

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protein has been calculated as percentage ratio of total protein content of the hydrolysate in

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the corresponding hydrolysis time per the protein content of LPI without enzymatic

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hydrolysis.

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Degree of hydrolysis (DH %). The DH was determined using trinitrobenzenesulfonic acid (TNBS) method as described by Adler-Nissen (1979)19, and calculated as follows: DH%) =

x 100 --------------------------------------------------------- (1)

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Where, htot is the total number of peptide bonds per protein equivalent (8 meq.g-1 protein), and

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h is the number of hydrolyzed bonds.

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Measurement of α-amylase enzyme inhibitory activity. The α-amylase inhibitory effect 7



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assay experiment was carried out using the previous methods 20-22, with slight modification: In

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brief, proper amount of dilutions (0-500 µL) of the hydrolysate solution and 250 µL porcine

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pancreatic α-amylase (EC 3.2.1.1, 1U.mL-1) solution of 20 mM sodium phosphate buffer (pH

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6.9 with 6.7mM NaCl) were pre incubated at 37 °C for 20 min. Then, 250 µL soluble starch

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solution prepared as 1g/100 mL in sodium phosphate buffer (20 mM, pH 6.9 with 6.7mM

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NaCl) was added to each tube, and maintained at 37 °C for further 10 min. Then, 500µL

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DNSA coloring reagent was added to each test tube and heated at 100 ℃ for 5 min to

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terminate the reaction. The reaction mixture was cooled to room temperature and the volume

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was made to 10 mL with deionized H2O. The absorbance was read at 540 nm using a

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UV-1100 spectrophotometer. A control, prepared in using sodium phosphate buffer (pH 6.9)

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without the test sample and blank was prepared using substrate and buffer without enzyme.

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The results were reported in % inhibition of alpha-amylase as follows:

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! " #$%!& ! " %'

% Inhibition = 1 −

! " ()%& ! " %'

* X100 --------------- (2)

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Purification of α-amylase inhibitory peptides. The UF membrane fraction hydrolysate

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showing better α-amylase inhibitory potential was separated using a column packed with

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Sephadex G-15 gel filtration chromatography (10 mm × 1000 mm, id), the system was pre-

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equilibrated with deionized ultra-sonicated water and the same deionized water was used for

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eluting of the fractions. The fractions eluted at a flow rate of 0.5 ml/min and monitored using

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spectrophotometer detector (STI UV 50199, Science Technology Co., Hangzhou, China) at

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220 nm and each fraction was collected separately, lyophilized and investigated for the

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inhibition of their α-amylase activity. The lyophilized active fraction from Sephadex G-15 gel

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filtration chromatography was dissolved in demineralized water. Prior to the analysis, the 8


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samples were diluted up to a protein concentration of 10 mg/mL and filtered through 0.22 µm

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Millipore syringe. The samples were automatically injected into a J sphere ODS-H80 reverse

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phase-high performance liquid chromatography (RP-HPLC) column (C18, 10 x 250 mm,

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Waters Corporation, Milford, USA) for further purification. The conditions were: mobile

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phases (Eluent A: 0.1% TFA (trifluoroacetic acid in distilled water (v/v)), and Eluent B: 0.1%

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TFA (trifluoroacetic acid) in acetonitrile). The separation was performed with a linear gradient

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of 5% to 95% eluent B at a flow rate of 1.0 mL/min. The elution peaks of fractions were

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detected at 220 nm, and the desired peaks with the strongest α-amylase inhibition activity

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were collected, lyophilized and subjected to amino acid sequence identification and molecular

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weight determination.

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Identification of peptide sequence and molecular weight determination. The most

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active fraction of freeze dried powder of the purified peptide was loaded to a quadrupole

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time-of-flight mass spectrometer (Q-TOFMS; Waters Corporation, Milford, USA) coupled

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with an electrospray (ESI) source for the identification of amino acid sequence and molecular

172

weight determination. The freeze-dried purified peptide was dissolved in HPLC grade water

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to 1mg/mL concentration and mixed with methanol solution (1:1v/v) containing 0.1% FA

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(formic acid). The sample was filtered through 0.22 µm syringe filter, and analysed on a mass

175

spectrometer (Q-TOF), connected to an HPLC system (Waters Corp., Milford, MA, USA).

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The chromatographic separation was carried out at a flow rate of 0.2 mL/min with an

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injection volume of 10 µL on a C18 column – 100mm × 2.1 mm, 3µm particle size (Waters

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Corp., Milford, MA, USA). Peptides were separated using mobile phases comprised of

179

solvent A (0.1% FA) in water, and solvent B, (0.1% FA) in acetonitrile. The chromatographic 9



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gradient conditions was as follows: 95% of solvent A and 5% of solvent B isocratically for 5

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min, followed by a linear gradient from 95 to 50% of solvent A for 20 min. In the

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data-dependent acquisition (DDA) mode, a 1 s TOF MS scan from m/z 100 to m/z 1500 was

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performed. The tandem mass spectrometry (MS/MS) spectral data were processed using the

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MaxEnt3 algorithm to translate the spectra to molecular mass and the amino acid sequence of

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peptides were determined by using BioLynx available, MassLynxV4.1 software package.

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To further validate the α-amylase inhibitory activity of the purified peptides, the

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identified peptides, with the same sequence, were synthesized and confirmed to their

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α-amylase inhibitory activity. The purity of these peptides was measured by reverse-phase

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HPLC (Column: C18, 4.6mm X 250 mm, 5 micron). The conditions of analysis were:

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Wavelength: 220 nm, Flow Rate: 1ml/min, Injection Volume: 10 µL, buffer A: 0.1% TFA in

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water, buffer B: 0.1%TFA in acetonitrile, gradient (linear): 1%-90 % buffer B in 8min. Their

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molecular weight was measured by mass spectrometry (MS). The MS analysis of the

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synthesized peptides was performed in the following conditions: Ion source: ESI, Flow rate:

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0.2 mL/min, buffer concentration: 80 % ACN/20 % water, Run time: 1 min.

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Mode of inhibition of α-amylase enzyme. The inhibitory kinetics of peptides on

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α-amylase enzyme was conducted following the method described.23, 24

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purified peptides were incubated with α-amylase (1U/mL) solution. In another set of tubes

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α-amylase was incubated with sodium phosphate buffer (pH 6.9). Starch solution at various

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concentrations (0, 0.5,1.0, 1.5 and 2.5 mg/mL) were added to both sets of reaction mixtures,

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and then the reaction mixtures were assayed using the procedure for bioassay to measure

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α-amylase inhibitory activity. The Michaelis-Menton constant (Km), maximum enzyme 10


In this study, the

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reaction rate (Vmax) and the inhibition mode of peptides on the α-amylase-catalyzed

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hydrolysis of starch was estimated by using double reciprocal of Lineweaver–Burk plots,

204

(1/Vi versus 1/[S]) of enzyme reaction velocity and substrate concentration as follows:

205 206

V- = V./0 7

89

=

45

[2]

45 6[2]

7

85:; [2]

+

7

(Michaelis-Menton equation) --------------------- (3)

85:;

(Lineweaver-Burk equation)-------------------- (4)

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Statistical Analysis. All results are expressed as mean ± standard deviation with three

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determinations. The mean differences between each group were analysed by using SPSS

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statistical software 22.0 (SPSS Inc, Chicago, IL, USA) in one-way analysis of variance

210

(ANOVA). A P-value < 0.05 was considered statistically significant.

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Results and Discussion

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Extraction of Laver Protein isolate (LPI) from laver powder. As out lined Figure 1,

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protein was extracted using enzyme-assisted cell wall disruption technique 16, and isolated in

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the isoelectric precipitation method. The protein content was found 73.47 ± 1.65% dry mass.

215

Compared to 42.99 ± 0.50% for the dried laver powder, the protein isolate had significantly (p

216

< 0.05) higher protein content. The enzymatic extraction increased the protein content 1.74

217

times that of laver powder. Therefore, Viscozyme®L was ably digested cellular

218

polysaccharides to release protein. This isolated protein was used as substrate to produce

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α-amylase inhibitory peptides. Seaweeds contain significant amount of proteins, especially,

220

red seaweeds contain 21–47g protein/100 g dry weight.12,25,

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seaweed protein is often hindered by high degree of structural complexity, rigidity , and

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assemblies of macromolecular algal cell-wall polysaccharides crosslinked through disulphide

223

bonds.27 To enhance the recovery, the protein extraction protocols employed remains the key 11


26

However, extraction of


224

factor.

225

Production of protein hydrolysate (LPH), DH and α-amylase inhibition rate. As

226

shown in Figure 2A, among the four proteolytic enzymes involved in hydrolysis of laver

227

protein isolate to produce α-amylase inhibitory hydrolysates under their optimal conditions,

228

the hydrolysate generated by pepsin enzyme showed efficient α-amylase inhibiting activity

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(50.34%) at 1.86 mg/mL concentration, followed by hydrolysates of alcalase (31.73%),

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trypsin (26.42%), and neutrase (18.27%) at similar concentration of protein and at different

231

hydrolysis time. As observed in the results α-amylase inhibitory efficiency of the LPH was

232

affected by the type of enzyme and hydrolysis time. Thus, the hydrolysate produced by pepsin

233

enzyme was revealed that significantly (p < 0.05) potent than the other aforementioned

234

enzymes (Figure 2B). Therefore, in this experiment, pepsin enzyme was chosen as an efficient

235

enzyme in generating effective bioactive peptides from the laver protein to inhibit α-amylase

236

activity.

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Laver protein isolate was hydrolysed by pepsin enzyme for further study (1.0%

238

enzyme-substrate ratio, 8% substrate, pH 2, and 37℃). The DH, the percentage of peptide

239

bonds cleaved (h) during hydrolysis when compared with the total number of peptide bonds in

240

the original studied substrate (htot) 19, 28, was used to evaluate the extent of protein degradation.

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As demonstrated in Figure 3A, the DH% of peptic hydrolysate were 8.23% , 12.35% , 15.54%

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and 12.80% at 1, 2,3 and 4h hydrolysis time, respectively. The results showed that at 3h

243

hydrolysis, DH was highest and significantly different (p10 kDa (LPH-I), MW = 3-10 kDa (LPH-II0 and MW < 3KDa (LPH-III) were obtained.

264

The peptide fraction with MW < 3kDa (LPH-III) demonstrated the highest α-amylase

265

inhibitory effect with an IC50 value of 0.976 mg/mL (Figure 3B). Similar findings have been

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reported by Ko et.al 30 in which marine Chlorella ellipsoidea hydrolysate were fractionated by

267

using 10 and 5 kDa UF membrane and found out that the fraction with MW

Identification of Bioactive Peptides with Î±-Amylase Inhibitory Potential

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