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

AVIAN COLLAGEN (EDIBLE BIRD NEST)

COLLAGEN-GELATIN

SEAWEED ENZYME EXTRACT

PHYTOCHEMICALS EXTRACT

BIOACTIVE PEPTIDES

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

20g of sample+0.02M phosphate buffer

Gel filtration

DH(Protein assay kit, SDS-PAGE)

Hydrolysis of protein by enzymes

Removing high molecular weight protein over

10kDa in hydrolysates

Ultrafiltration

Extraction of sarcoplasmic, myofibrillar and stroma

protein from fish meat

Degree of Hydrolysis (DH) Assay of hydrolysates

Collecting highest peaks

RP-HPLC separation

Peptide sequencing

Separation of ACE inhibitory fraction, antimicrobial and

antioxidative fraction

Select highest peak

Confirming the structure of the highest ACE-

inhibitory 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

Nervous system

Opioid agonist

Opioid antagonist

Immune system

Anti-microbial

Immunomodulatory

Cytomodulatory

Gastrointestinal system

Mineral binding

Anti-appetizing

Anti-microbial

Cardiovascular system

Anti-hypertensive

Anti-oxidative

Anti-thrombotic

Anti-hyperlipidemic

Gel filtration

Samples IC 50 (µg/mL)

Sarcoplasmic Protein fractions

PS1 3.3 PS2 0.51

0

0.05

0.1

0.15

0.2

0 10 20 30 40

Sarcoplasmic

Fractionation

Table 3.

IC50 value of sarcoplasmic protein fractions

ACE inhibitory peptides from Haruan (freshwater fish)

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40

Myofibrillar

Fractionation P1

P2 P3

Gel filtration

Samples IC 50 (µg/mL)

Myofibrillar protein fractions

PM1 1.002 PM2 1.87889 PM3 5.224125

Table 3.

IC50 value of myofibrillar protein fractions

• 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 ?

“Hydrolyzed 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.

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

Pressure Gauge

Steam In

Water Out Cold Water In

Steam distributor

Reaction Medium In

Agitator Speed Control

Temperature

Indicator

Reaction

Medium 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.

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Collagen-Hydrolysate

Obtained by the enzymatic degradation of collagen

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

Cold water soluble, non gelling

Odorless, flavorless white powder

• 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 century’s 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 • It’s 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 Content (% weight)

Raw EBN EBN Hydrolysate

Protein 58.65 ± 0.84a 57.20 ± 0.50b

Carbohydrate 20.05 ± 0.19b 24.95 ± 0.01a

Moisture 17.33 ± 0.32a 14.18 ± 0.32b

Fat 0.38 ± 0.24a 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-to-cell, 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 µmol/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 bird’s nest a potential raw material for enzyme-mediated production of ACE inhibitory peptides.

Summary: Potential of EBN as supplement

Edible Birdnest

High protein content per

weight

Contains broad range of amino

acids (essential & non essential)

Water soluble

amino acids

Showed in vitro antiviral

reaction

Contains EGH – important in cell division

High peptide content per

weight

“Natural source for the prevention

of influenza.”

March 5, 2004. Southern Bio-products Conference

Marine Resources – Seaweeds

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 water–gum solution, whereas iota-carrageenan 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.

Universiti Kebangsaan Malaysia 37

Herbs Spices

Fruits Flower

Vegetable

Enhancement of Processed Meats Using Phytochemicals

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 (Melicope lunu-ankenda)

Indian pennywort (Centella asiatica L.)

Bitter gourd (Momordica charantia L.)

Lemon grass (Cymbopogon citrates)

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• 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.

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 herbal–based food products.

• Preliminary studies of screening potential herbs and spices was conducted by Huda-Faujan 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.

THANK

YOU