BIOACTIVES AND FUNCTIONAL PEPTIDES FROM SELECTED BIODIVERSIFIED RESOURCES

Abdul Salam Babji*, Masomeh Ghassem, See Siau Fern and Nur Aliah Daud

NOVEL PRODUCTS BASED ON MALAYSIAN BIODIVERSIFIED MATERIALS

BIOACTIVE PEPTIDES

COLLAGEN-GELATIN

AVIAN COLLAGEN (EDIBLE BIRD NEST)

SEAWEED ENZYME EXTRACT

PHYTOCHEMICALS EXTRACT

Bioactive peptides of Sarcoplasmic and Myofibrillar Protein From Freshwater Fish

Angiotensin Converting Enzyme (ACE) Inhibitory & Antioxidant Activities

Hypertension treatment
Medicine Vegetables Fruits Healthy foods

Hypertension medicine
Captopril Enalapril and Lasinopril

Side effects of synthetic hypertensive drugs

Dry cough, Taste disturbances Skin rashes, as well as Alterations in serum lipid metabolism

Extraction of sarcoplasmic, myofibrillar and stroma protein from fish meat

20g of sample+0.02M phosphate buffer

Hydrolysis of protein by enzymes

Degree of Hydrolysis (DH) Assay of hydrolysates

DH(Protein assay kit, SDS-PAGE)

Ultrafiltration
Removing high molecular weight protein over 10kDa in hydrolysates

Gel filtration
Separation of ACE inhibitory fraction, antimicrobial and antioxidative fraction

Collecting highest peaks

RP-HPLC separation
Select highest peak

Peptide sequencing
Confirming the structure of the highest ACEinhibitory peptide, antimicrobial and antioxidative peptides

Figure 1. Typical procedure of isolation of antihypertensive, antimicrobial and antioxidative peptides from freshwater fish meat proteins

Physiological effects of food derived bioactive peptides on major body systems

Cardiovascular system Anti-hypertensive Anti-oxidative Anti-thrombotic Nervous system Opioid agonist

Opioid antagonist

Anti-hyperlipidemic

Gastrointestinal system Mineral binding Anti-appetizing Anti-microbial

Immune system

Anti-microbial
Immunomodulatory

Cytomodulatory

ACE inhibitory peptides from Haruan (freshwater fish) Gel filtration

0.2 0.15 0.1

Sarcoplasmic Fractionation

0.05
0 0 10 20 30 40

Table 3. IC50 value of sarcoplasmic protein fractions

Samples IC 50 (g/mL)

Sarcoplasmic Protein fractions

PS1 PS2

3.3 0.51

Gel filtration

0.6 0.5 0.4

Myofibrillar Fractionation P1

0.3
0.2 0.1 0 -0.1 0 10 20 30 40

P2

P3

Table 3. IC50 value of myofibrillar protein fractions Samples IC 50 (g/mL)

Myofibrillar protein fractions

PM1
PM2 PM3

1.002 1.87889 5.224125

 According to study that has done by Ghassem et. al. (2011), two peptide sequences for the most abundant fragments were identified as VPAAPPK and NGTWFEPP, found in Haruan meat. The presence of two proline residues at the C-terminal sequence is responsible for the high ACE inhibitory activity of these peptides. The peptides that can inhibit ACE in vitro and further exert antihypertensive effect in vivo have shown great promise in the development of a novel therapeutics and functional food for preventing hypertension.

Antioxidant peptides from Patin (freshwater fish)

Alcalase-SPH-III and papain-MPH-III with MWCO 3kDa has the highest DPPH radical scavenging activity with IC50 1.26 and 1.76 times respectively higher than SPH and MPH. Alcalase hydrolysed sarcoplasmic (SI) and papain hydrolysed myofibrillar (MI) were tested for amino acid composition. Both fractions had higher amount hydrophobic and aromatic amino acids than hydrolysates. The potent fractions obtained from sarcoplasmic and myofibrillar hydrolysates showed DPPH radical scavenging activity by 1.83 and 2.97 times higher than SPH and MPH.

Amino Acid Sequence Of Antioxidant Peptides From Sarcoplasmic And Myofibrillar Protein Hydrolysates, Characterized By Swissprot Database

MPH and SPH of patin can served as source for novel peptides for natural antioxidants to be used in food products.

Collagen ?
An insoluble fibrous protein that occurs in vertebrates as chief constituent of connective tissue fibrils and in bones. Constitute >25% of protein mass in the human body.

Hydrolyzed Collagen is obtained from animal skins and bones by thermal and enzymatic hydrolysis

Source
Tendons Skin Bone Vascular system of animals The connection tissue sheaths surrounding muscle

Fish Skin Gelatin Powder Processing

Steam In Pressure Gauge

Agitator Speed Control

Reaction Medium In

Temperature Indicator

Reaction Medium Out Steam distributor

Cold Water In

Water Out

Processing of gelatin
Step 1: Raw Material Preparation Step 2: Cleaning Step 3: Alkaline Pretreatment Step 4: Acidic Pretreatment Step 4: Gelatin Extraction Step 5: Purification
The concentrated gelatin was freeze dried.

Collagen-Hydrolysate

Odorless, flavorless white powder

Cold water soluble, non gelling

Obtained by the enzymatic degradation of collagen

Mean M.W. 3.5 kD (peptides ranging from 0.5-13 kD)

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 In Malaysia, Catfish is common farm-raised, warm-water fish supplying large amounts of fish skins year-round.

Recent study reported that gelatin hydrolysates from catfish skin and bone showed high antioxidant activity including DPPH, ABTS, hydroxyl radical scavenging activities, metal chelating capacity and lipid peroxidation inhibitory activity. Functional bioactive peptides with in vitro ACE inhibitory activity have been isolated from skin and bone gelatin hydrolysate of catfish P. sutchi (Mahmoodani et al. 2012).

Edible Birdnest
The 21st centurys new health supplement

The Swiftlet Golden Triangle

What is edible bird nest?

The nests built by a few species of swiftlets The swiftlets mainly are living in deep caves or under the roofs of coves along the seashores in southeastern Asian countries. The nests are entirely out of gelatinous strands of its saliva Its known by its high nutrition and health benefits as a remarkable delicacy

Rich in nutrients and health benefits:

aiding digestion; improving voice; overall benefits to the immune system; boosting energy; raise libido; rejuvenate skin; nourishing Ying; useful in the treatment of AIDS.

Two kinds of bird nest

White-nest Swiftlet (Aerodramus fuciphogus)

red-blood nest: best and purest ones with high nutrition

Black-nest Swiftlet (Aerodramus maximus)

Birdnest products -

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Nutritional content
Parameters Protein Carbohydrate Content (% weight)

Raw EBN
58.65 0.84a 20.05 0.19b

EBN Hydrolysate
57.20 0.50b 24.95 0.01a

Moisture
Fat

17.33 0.32a
0.38 0.24a

14.18 0.32b
0.29 0.22b

Ash

3.50 0.20a

3.38 0.28a

Glycoprotein in EBN
Content: 9% sialic acid, 4.19 -7.2% galactosamine, 5.3% glucosamine, 5.03 -16.9% galactose, 0.7% fucose. Sialic acid residues conjugates are involved in biologically important ligand-receptor interactions.
Sialic acid: Specific cell-to-cell, pathogen-tocell, drug-to-cell interactions.

 Studies have been conducted for antioxidant activity on cave and house, raw and cleaned EBN. Hydrolysate of raw cave EBN form showed the highest ABTS free radical scavenging activity (34.17 0.094 mol/g) and the highest reducing power (0.683 0.01). When tested on metal chelating activity, hydrolysate of raw house EBN showed the highest activity (0.0020 0.0003 mg EDTA/g sampel) compared to others.

Since the protein content in EBN is high (56.47-60.63%), this makes birds nest a potential raw material for enzymemediated production of ACE inhibitory peptides.

Summary: Potential of EBN as supplement

High protein content per weight Contains broad range of amino acids (essential & non essential)
Water soluble amino acids Edible Birdnest

High peptide content per weight

Contains EGH important in cell division

Showed in vitro antiviral reaction Natural source for the prevention of influenza.

Marine Resources Seaweeds

March 5, 2004.

Southern Bio-products Conference

Carrageenan
Carrageenan
obtained by extraction with water or alkaline water of certain Rhodophyceae (red seaweed).

A hydrocolloid
consisting mainly of the potassium, sodium, magnesium, and calcium sulfate esters of galactose and 3.6-anhydro-galactose copolymers.

Commercial Species of Seaweed

Carrageenan Bearing Red Seaweed
Eucheuma Cottonii Eucheuma Spinosum

Agar-Agar Bearing Red Seaweed

Gracilaria Gelidium

Seaweed for carrageenan manufacture

Types of Carrageenan Extracted

Eucheuma Cottonii Kappa
Applications
Dairy Products Meat and Poultry Water gels Processed Human foods & Pet Foods Pharmaceutical Personal Care

Eucheuma Spinosum Iota

Applications
Toothpaste Other Dairy Products Pharmaceutical

Carrageenan Production

 In cooked meats carrageenan is used to improve moisture retention, cooking yields, consistency, sliceability, cohesiveness mouthfeel and juiciness, while simultaneously decreasing purge (Imeson 2000).
Kappa-carrageenan increase the hardness of meat batters when replacing fat with watergum solution, whereas iotacarrageenan improves the WHC. Chicken balls in terms of texture, hardness, elasticity, chewiness and gumminess attributes were improved with the addition of 0.5% carrageenan-kappa (Zalifah et al. 2011).

 Burger and meatball are popular foods BUT health concern by consumers due to high animal fat content.
Design & formulation of healthier meat products with less fat and salt contents and the use of edible seaweeds is a promising area of research.

Enhancement of Processed Meats Using Phytochemicals

Herbs Spices

Fruits Flower
Vegetable

Universiti Kebangsaan Malaysia

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So...

SYNTHETIC or NATURAL?

 Herbs and spices possessed anti-oxidative property and have been used to prolong shelf-life and improve taste of meat products (Mielnik et al. 2003). Herbs are commonly eaten fresh as vegetable (salad & ulam) especially Malay communities. Herbs are believed to be associated with antioxidant activities and have many beneficial effects.

Kesum (Polygonum minus)

Tenggek Burung Lemon grass (Melicope lunu-ankenda) (Cymbopogon citrates)

Indian pennywort (Centella asiatica L.)

Bitter gourd (Momordica charantia L.)

 Chan et. al. (2007) studied the antioxidant activities of 15 tested spice extracts. Spice extracts- cinnamon bark, clove buds, curry leaves and star anise exhibited high antioxidant activities (P<0.05) through DPPH scavenging activity test and ferric reducing power test.

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Antioxidant Activities Screening

Coriandrum sativum

Cuminum cyminum

Capsicum annuum

Curcuma longa

Foeniculum vulgare

Piper nigrum

Illicium verum

Zingiber officinale

Eugenia caryophylata

Brassica negra

Elettaria cardamomum Trigonella-foenum-graecum

Cinnamomum verum

Papayer somniferum

Murraya koenigii

 These four extracts also exhibited similar (P>0.05) antioxidant activities compared to synthetic lipophilic antioxidant. Through the antioxidant activities screening, cinnamon bark and clove buds extracts were chosen for further investigation.

 The recent revival of consumer interest in the therapeutic properties of herbs has led to an increase in consumer demand for herbalbased food products. Preliminary studies of screening potential herbs and spices was conducted by HudaFaujan et al. (2007), Noriham et al. (2005) and Noriham et al. (2004).

 Antioxidative properties of prepared clove buds deodorized aqueous extract (CDAE) in chicken meatballs were studied. At 200 ppm concentration, it is superior than synthetic antioxidants (BHA and BHT) in retarding the lipid oxidation and decolorization of chicken meatball, without contributing any significant impairment to sensory properties. Hence, from the present study, the herbs deodorized aqueous extract might be able to replace synthetic antioxidants in preservation of meat products from lipid oxidation.

 It is envisaged that the use of phytochemicals in meat based products will be an interesting area for the meat industry to explore as these functional extracts with antioxidative and antimicrobial properties are able to replace the current usage of synthetic food additives in the development and improvement of healthy food products.

Conclusions
R&D has resulted in exploring local resources resulting in basic scientific and technological information contributed to obtaining functional components useful to man. Application of microparticulates, hydrolysates and freeze-dried bioactives will bring forward the supplement/nutraceutical industry to global competitiveness for selected biodiversified resources. Enzymatic/physical operation process of purification, hydrolysis & idenfication of biologically active ingredient. Laboratory methods of extraction be extended to optimize microparticulates bioactives functional properties at a pilot plant and SME level.

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