Post on 29-Nov-2015
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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.
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