NANOEMULSIONS/MICROEMULSIONS TECHNIQUES FOR DELIVERY OF

NUTRACEUTICALS THROUGH LIVESTOCK PRODUCTS

Dr Anita KatekhayePhD Scholar, CVSc, Hyderabad

NUTRACEUTICALS

BACKGROUND AND HISTORY

LIQUID-LIQUID SYSTEMS

MICROEMULSIONS/NANOEMULSIONS

CHARACTERIZATION OF NANOEMULSIONS

TECHNOLOGY ADVANTAGES

FABRICATION OF NANOEMULSIONS

NUTRACEUTICALS VIA DAIRY PRODUCTS

NUTRACEUTICALS VIA MEAT PRODUCTS

REFERENCES

TRENDS IN NUTRACEUTICALS & FUNCTIONAL FOODS

TRENDS IN FUNCTIONAL FOOD & NUTRACEUTICALS

• Global food encapsulation market is projected to reach about $39 billion by the year 2015 (GIA) Global Industry Analysis, Inc

• Currently, functional food market of India is estimated at $70 billion

Or • 4% of processed food market and is growing at 3 times rate

• Japan is the single largest market followed by US and Europe

• In India, around 20 companies have record of producing

nutraceuticals & marketing them globally

Ref: A. A. Patel & A. K. Singh (2012)

• In India, Nutraceuticals are marketed as Indian System of Medicine Drugs (ISMD) under the Over-

the-Counter (OTC) category

• No clinical validation of their safety or efficacy is required if therapeutically usefulness is mentioned in the text

• Nutraceutical foods are not subject to the same testing and regulations as pharmaceutical drugs

Ref: A. A. Patel & A. K. Singh (2012)

WHY THERE IS NEED OF FORTIFICATION ?

Fortification of widely consumed staple foods offer one of the simplest and most practical methods to alleviate “Hidden Hunger”

1. In 1930s Vitamin D was added to milk in US to help prevent rickets in children

2. In several countries liquid milk fortification with vit A and vit D is mandatory 3. Dried milk often fortified with Vit A and D, Calcium and Iron 4. Milk based infant formula and weaning foods are fortified with a

range of Vitamins, Minerals and other nutrients such as PUFA

Investigation is carried out at NDRI suggest possibilities of fortification of liquid milk with Ca and Iron

NUTRACEUTICALS

• Nutraceuticals is a hybrid or contraction of Nutrition and Pharmaceutical• Reportedly, it was coined in 1989 by Dr Stephen

L De Felice, founder and chairman of the “Foundation for Innovation in Medicine” in Medicine, New Jersey, USA

• Term commonly used synonymously for Designer foods, Health foods, Fortified foods, Medifoods, Vita foods, Pharma foods, Functional foods and Dietary supplements

WHAT ARE NUTRACEUTICALS ?A nutraceuticals is any substance considered as a food

or its part which in addition to its normal nutritional value provides health benefits including prevention of disease or promotion of health

OrAny non-toxic food component that has scientifically

proven health benefits, including disease treatment or prevention

Functional component of the food must be standardized in nutraceuticals product and produced under good manufacturing conditions (GMPs)

CLASSIFICATIONISOPRENOIDS (TERPENOIDS)

PHENOLIC COMPOUNDS

PROTEIN/AMINO ACID -BASE

CARBOHYDRATE & DERIVATIVES

FATTY ACIDS & STRUCTURAL LIPIDS

MINERALS

MICROBIAL

Carotenoids, Saponins, Tocopherol, Terpenes

Tannin, Lignin, Flavones, Isoflavons, Courmarins

Amino acid, Indoles, Isothiocyanates, Choline

Ascorbic acid, Oligosaccharides, Non-starch PS

n-3PUFA, CLA, MUFA, lecithin, Sphingolipids

Ca, Se, K, Cu, Zn

Probiotics, Yeast

Summary of major Lipophilic Nutraceuticals Components that need to be delivered into foods

NAME TYPES NUTRITIONAL BENEFITSFatty Acids

n-3 fatty acids, CLA, Butyric acids

Coronary Heart Disease, Bone health, Immune response disorders, Weight gain, Stroke prevention, Mental health, Cancer, Visual acuity

Carotenoids

β- Carotene, Lycopene, Lutein and Zeaxanthin

Cancer, Coronary Heart Disease, Macular degeneration and Cataract

Antioxidants

Tocopherols, Flavonoids, Polyphenols

Coronary Heart Disease, Cancer and Urinary Tract Disease

Phytosterols

Stigmasterol, β- sitosterol, Campesterol

Coronary Heart Disease

Vitamins Vit D and Oil Soluble Vit

Prevent Rickets and Osteomalacia

HISTORY• Since beginning of last century Australian housewives used

Water/Eucalyptus oil/Soap flake/White spirit mixtures to wash wool

• Schulman et al.,(1959) : First used microemulsion to describe multiphase

system (water, oil, surfactant and alcohol which form transparent solution)

• Rodawald, 1928: Discovered liquid waxes the first commercial

microemulsions

• In late 1970’s and early 1980’s interest in microemulsions really step up when it was recognized that such systems could improve oil recovery and when oil prices reached levels

• Last 20 years great deal of progress in understanding the properties of Microemulsions

EMULSION

E.g. Mayonaise (W/O), Butter

E.g. Milk, Meat Emulsion,Cream

BACKGROUND • Wide variety of delivery systems have been developed

to encapsulate lipophilic functional component including e.g. Simple solution, Association colloids, Emulsions,

Biopolymer matrices, Powders etc• Emulsion based delivery systems are getting more

attention now days- 1. Due to their targeted delivery 2. More absorption in human gut 3. Solid matrix material can be added in liquid

food system

Emulsion offers a viable option for drug delivery:- order of selective uptake lymphatic regions Oil in Water> Water in Oil> Aqueous solution

(Nishioka & Yoshino 2001)

STRUCTURAL DESIGN

PRINCIPLES

Structural components selection depends upon:-

TARGET APPLICATIONS

FINAL CONTINUOUS PHASE

CHARACTERISTCS OF BIO-ACTIVE INGREDIENTS

LIPIDS• Lipids are predominantly non-polar substances

that are highly hydrophobic• In food Industry major source of lipids are

Tryglycerols which may come from animal, fish or plant origins

• Used to solubilize non-polar lipophilic components in foods

• In Microemulsions Diglycerides are used• Nanoemulsions requires long chain triglycerides

(LCT) with some degree of unsaturation (Prevent Oswald Ripening and Health effect also)

SURFACTANTS

• Are surface-active molecules that consists of a hydrophilic head group and a lipophilic tail group

• Functional performance depends on the molecular characteristics of head and tail groups

Head group characterize – Anionic, Cationic, Zwitterionic, Non-ionic

Tail group characterize- number of tails (One or Two)

Synonyms - Emulsifier or Amphiphilic Compounds

SUITABLE NON IONIC SURFACTANTS INCLUDE

1. Polysorbates – Polyethoxyethylene sorbitan monoesters, Tween 20, Tween 40, Tween 60, Tween 65, Tween 802. Sugar Surfactant – Sucrose nanopalmitate, sucrose monolaurate,

Sucrose monostearate Crodesta F-160, Sucrose monopalmitate

3. Polyoxamers – Polyoxyethylene-polyoxypropylene block co-polymers e.g. Monolan, Lutrol etc

Preferably surfactant that acts synergistically with the hydrophilic non-ionic surfactant to alter the interfacial curvature

This lowers interfacial tension, permitting easier emulsion formation

Suitable food grade co-surfactant include: Sorbitan fatty acid esters such as sorbitan monolaurate

(Span 20), Span 40, Span 65, Span 60, Span 80, Span 85

Phospholipids egg/soy lecithin

Co-Surfactant

Purified or Ultrapure Water, Saline or Buffered Saline

More preferably balance of water 30 to 99.90 wt%

Co-solvent lowers the interfacial tension of the aqueous phase which enables the formation of smaller emulsion droplet size

• Suitable Co-solvent include C1-C10 alcohols such as methanol, ethanol ….decanol

• More preferably ethanol (C1-C4)

Aqueous Phase

Co-solvent

Additives are added to make the nanoemulsions last for

longer periods

Additives

DIFFERENT DELIVERY SYSTEMS

Fig: Different types of delivery systems that can be created based on Emulsion Technology

Conventional oil-in-water (O/W) emulsions consist of emulsifier-coated lipid droplets dispersed in an aqueous continuous phase

Formed by homogenization of oil and water phase together in the presence of a hydrophilic emulsifier

Ref: Gaonkar et al., (2014)

CONVENTIONAL EMULSIONS

MULTIPLE EMULSIONS

Multiple water in oil in water (W/O/W) emulsions consist of small water droplets contained within larger oil droplets that are dispersed in aqueous continuous phase

Two steps of formation: Preparation of W/O emulsion Homogenization of W/O emulsion and aqueous phase and water soluble emulsifier

MULTILAYER EMULSIONS

Fig. Schematic representation of formation of conventional and multilayer emulsions

Multilayer Emulsions are formed by adding polyelectrolytes to an emulsion containing oppositely charged droplets so that they adsorb and form a nano-laminated coating

TYPICAL EMULSION-BASED LIQUID-LIQUID SYSTEMS

ATTRIBUTES OF EDIBLE DELIVERY SYSTEMS

EFFICIENTLY ENCAPSULATE AND KEEPING IT ENTRAPPED

PROTECTION FROM CHEMICAL DEGRADATION

CONTROL THE RELEASE OF THE FUNCTIONAL AGENT

COMPATIBLE WITH THE SURROUNDING FOODS

Appropriate amount of functional agent

Functional Agent should be remain in its active state

e,.g. Release rate or the specific environmental stimuli that triggers release

Beverages matrix, without causing adverse effect affects on product Appearance, Rheology, Mouth feel, Flavor and Shelf life

RESIST THE ENVIRONMENTAL STRESSES

COMPLETELY PREPARED FROM GRAS INGREDIENTS

NOT ADVERSELY IMPACT THE BIOAVAILABILITY OF THE ENCAPSULATED MATERIAL

FOODS OR BEVERAGES EXPERIENCE DURING THEIR PRODUCTION, STORAGE, TRANSPORT AND UTILIZATION e.g HEATING, FREEZING ,CHILLING, DEHYDRATION OR SHEARING

USING SIMPLE COST-EFFECTIVE PROCESSING OPERATIONS

Ref: McClement, 2009

MICROEMULSIONS

Synonyms Swollen micelles, Micellar emulsion

Thermodynamically stable mixtures of water, oil and one or more amphiphils which assemble spontaneously into nanometer-scale

droplets (Flanagan & Singh, 2006)

SPONTANEOUS FORMATION OF A MICROEMULSION ARISES FROM -

SYNERGISTIC INTERACTION OF SURFACTANT, CO-SURFACTANT & CO-SOLVENT TO EFFECTIVELY SOLUBILIZE OIL MOLECULES

Water is continuous phase

Shape of droplets or particles: Spherical size or other

structures possible Thermodynamically or 20 kinetically stable liquid: Dispersion of an oil phase

and a water phase, in combination with a surfactant

Transparent or Translucent in appearance: Due to small particles or droplets dispersed

with size 5-100 nm

Microemulsions form spontaneously: Readily form without high energy input Due to ultralow interfacial tension and

favorable energy of structure formation Microbiologically stable: Due to water droplets are extremely

small Long shelf life: Particle sizes do not change with time

(brownian motion effect dominates gravity) Low calorific value: Inclusion of water reduces calorific value

of emulsions and enables water-soluble ingredients such as vitamins, flavors, salt, sugars, colorants

Regular ~ emulsions of larger water droplets can present a severe microbiological problem

Theory of Formation

Under appropriate environmental conditions, certain types of food components spontaneously assemble into well-define structures since this minimizes the free energy of the system

e.g. Micelles, vesicles, fibers, tubes, liquid crystals

SPONTANEOUS SELF ASSEMBLY SYSTEM (Bottom-up Approach)

Mcclements , 2009

Hydrophobic Effect - Primary driving force of microemulsions formation, It minimizes contact between the non-polar tails of

surfactant and water

WINSOR CLASSIFICATION SYSTEM

Ref: Winsor, P. A. (1948)

Type I: O/W Microemulsions system which is in equilibrium with an upper excess oil phase Surfactant is preferentially soluble in water (Winsor I) )

Type II: W/O Microemulsions system in equilibrium with a lower excess aqueous phase

Surfactant is mainly in the oil phase (Winsor II)

Flanagan & Singh (2006)

Type III: Equal volumes of oil and water are present Bicontinuous structure is formed

Three-phase system where a surfactant-rich middle phase coexists with both excess water and oil surfactant-poor phases (Winsor III /Middle Phase

Microemulsion)

Type IV: Macroscopically single phase

Single-phase (isotropic) micellar solution, that form upon addition of a sufficient quantity of amphiphile (Surfactant + Alcohol)

Thermodynamically stable systems are primary interest to food technologist

SINGLE PHASE MICROEMULSIONS

• Form at specific concentrations of oil, water and surfactant (co-surfactant)

• Oil concentration (<30%): oil-in-water • Aqueous concentration low : water-in-oil

• May comprise of: 1. Oil: 40-97wt~ 2. Surfactant: 1-40wt~ 3.Water phase: 0.1-25wt~ 4. Co-surfactant: 0.1-10wt~ Preferred embodiment mole ratio of Water: Surfactant is 5-70

Flanagan & Singh (2006)

STEPS IN MICROEMULSION PREPARATION

(Dissolving water soluble components) (Solids dissolve possibly by heating) (Undissolved material separated by

centrifugation)

(Dissolving oil soluble component)

(Mixed in suitable vessel and given time to equilibrate)

(Using emulsification techniques - stirring, use of membranes, applying shear, ultrasound etc)

Preparing Water Phase

Emulsifying Water & Oil Phases

Preparing Oil Phase

SHORTCOMINGS OF MICROEMULSIONS

Become physically unstable if its composition is changed e.g. upon dilution, acidification or heating in food processing

1. Contains high amount of surfactants relative to amount of oil

2. Application of microemulsions in foods is limited by the types of surfactants which are used to facilitate its formation

3. Many surfactants are not permissible in foods (bitter taste)

4. WHO and the FDA have placed restrictions on the daily intakes of many of these surfactants

MAINTAINING OPTIMUM PHYSICAL STABILITY

HIGH AMOUNT OF SURFACTANTS

NANOEMULSIONSSynonyms - (Sub-micron emulsions (SME), Mini-emulsions, Ultrafine emulsions)

Def: A nanoemulsion is an emulsion which does not

form spontaneously, but instead formed by the application of shear to a mixture of oil, water and surfactant

Oil droplet sizes >100nm (most preferably

>60nm)

Droplet size is measured by Photon Correlation Spectroscopy (Dynamic light scattering)

• It can be three types oil-in-water, water-in-oil, bicontinuous

• Kinetically stable • Spontaneously do not form need high energy input to

obtain• Steric stabilization prevents any flocculation or

coalescence• Particle size may increase over time via coalescence,

flocculation and /or ostwald ripening• Transparent / translucent appearance like

microemulsions• Metastable

SMALL SIZE AND HIGH KINETIC STABILITY MAKE NANOEMULSIONS SUITABLE FOR DELIVERY OF ACTIVE COMPOUNDS IN BEVERAGES

FORMULATION TABLE OF NANOEMULSIONS

Oil phase 0.01 to

40 Hydrophilic Non-ionic Surfactant 3 to 7 Co-surfactant 0.1 to

15 Aqueous phase 30 to

99.90 Co-solvent 15 to

45 Active Component 0.01 to

10 Additives 0 to 10Active component may be 0.01 to 50 preferably 0.01 to 10 wt %

Components Most Preferably wt %

FABRICATION OF

NANOEMULSIONS

Also known as Bottom up Approach Emulsion spontaneously form Submicron emulsion can be obtained by employing the

physico-chemical properties of the systems generally referred to as low-energy emulsification methods

LOW ENERGY APPROACHES

Phase Inversion Point

Membrane Emulsification Spontaneous

Emulsification Solvent Displacement

Emulsion Inversion Point

(Leong et al., 2009)

Disrupt oil & aqueous phases into tiny droplets using mechanical devices

Formation governed by the selected composition (Surfactant + Functional compounds) & by the quantity of energy applied

HIGH ENERGY APPROACHES

• Smaller the droplet size more surfactant/energy is required

• Some chemical get easily degraded

• Homogenizers/ High-speed devices• High Pressure Homogenizer/Microfluidizer

• Ultrasound

Devices used

Commonly used Adaptable to different processing

conditions Variety of food ingredients can be

used to prepare Nanoemulsions Effective in reducing size More control of the size distribution

and composition of resultant Nanoemulsions

HIGH ENERGY APPROACH

Less Expensive Energy Efficient alternative that

takes the advantage of the chemical energy stored in the system

Suitable for some unstable molecules such as Proteins and Peptides

LOW ENERGY APPROACH

Ref: Herrera M (2012)

PHASE TRANSITION METHODS Principle:-

Phase inversion occur when the structure of the emulsion is inverts Phase transition cause release of chemical energy that dispersed the fine droplets through emulsification method

Ref: P. Fernandez et al. (2004)

Cross Ref: P. Fernandez et al. (2004)

Uson et al.,2004 – Phase transitions produced

during the emulsification process which is carried out- At constant temperature and changing the composition

OR Izquierdo et al., 2001; Morales et al.,2003 – At constant composition and changing

the temperature These methods make use of changing spontaneous curvature of the surfactant

EMULSION PROCESSING: HOMOGENIZER

Homogenization is a unit operation using a class of processing equipment referred to as Homogenizers that are geared towards reducing the size of

droplets in liquid-liquid dispersions

HIGH PRESSURE HOMOGENIZERS

Principle - The premixed liquid passed under a high pressure (100-1500bar) through a minute gap of the homogenizing valve

Such condition generates very high shear stresses, acceleration, impact and intense turbulent flows in the liquid resulting subdivision of the dispersion phase particles or droplets and their homogeneous distribution through the mixture

Poor Productivity, Component deterioration due to generation of heat, Oil-in-water liquid nanoemulsions of less than 20% oil phase can be prepared

MICROFLUIDIZER PROCESSORS : FROM LAB TO PRODUCTION

MICROFLUIDIZER

1. Intensifying Pump - Generate high pressure (500-20000 psi)

Forces the stream through micro-channels at high velocities

Into an impingement zone

Creating an intense turbulence mixing action

2. Interaction Chamber - (Heart of microfluidizer technique) Reduces sizes of a pre-emulsion (coarse droplets)

Multiple Passes through the System is typically required to obtain the desired fine emulsion with narrow droplet size distribution Uniform emulsion compare to High Pressure Homogenization

Ref: Panagiotou T. and Fisher R. (2012). Review Article. Cahllenges 3(2), 84-113; doi:10.3390/challe3020084

ULTRASONIC HOMOGENIZERS

Principle- Utilizes ultrasonic waves for homogeneous

distribution of the dispersed phase by the actions: 1. Reducing sizes of the dispersed

particles/droplets (breaking) 2. Disintegrating the dispersed particles agglomerates 3. Blending the dispersed phase in the liquid

Use for small scale production

TECHNOLOGYADVANTAGES

Creation of transparent bioavailable beverages containing ω-3 rich Oils, Phospholipids and Minerals

Encapsulation of ω-3 fatty acids enhances the stability and bioavailability of bioactive food ingredients

OXIDATIVELY STABLE BEVERAGES (Pinto et al., 2005)

PROTECTION OF NUTRACEUTICALS

Protect against flavor, taste and nutritional degradation

• Used as vehicles for targeted nutraceuticals • Vehicles are expanded micelles in the size of<30 nm and used in clear beverages without phase separation• Potential application include Lycopene, beta-carotene, Omega-3 fatty acids, Phytosterols and Isoflavones

NANOSIZED SELF-ASSEMBLED LIQUID STRUCTURES

(Garti and Aserin, 2007)

ENHANCE BIOAVAILABILITY

1. Greater ability to dissolve/disperse poorly soluble active components at the interface

2. Faster rate of digestion by lipolysis & greater uptake (more rapid release of emulsified active ingredients)

3. Below 100 nm have greater ability to penetrate epithelial layers (Skin and Oral mucosa)

(OPTIMUM NUTRACEUTICALS DELIVERY )

TRANSPARENT TO TRANSLUCENT

1. Optically clear and applicable in beverages ( not alter visual appeal)

ENHANCE SOLUBILITY

1. Enhance solubility of bioactives at an interface

REDUCE VISCOSITY

REDUCE AMOUNT OF SURFACTANT (NANOEMULSIONS)

HYDROPHOBIC BIOACTIVE COMPONENT DELIVERY

1. Vehicle for Hydrophobic bioactive delivery

1. They do not show problem of creaming, flocculation, coalescence and sedimentation

1. Mainly use for encapsulate a diverse range of Lipophilic Compounds (Chen et al., 2006)

CONTROLLED DELIVERY OF NUTRACEUTICALS

To some extent amount of nutraceuticals release at target organ can be controlled

CHARACTERIZATION OF

NANOEMULSIONS

Determined by Photon Correlation Spectroscopy (PCS) Light scattering was monitored at 25˚C at a scattering angle of 90˚

Determined by using Brookfield DV ultra V6.0 cone and Plate Rheometer at

25±0.3˚C

Determined by using an Abbes Type Refractometer

Morphology and structure of the nanoemulsions were studied using

Transmission Electron Microscopy (TEM)

Droplet Size Analysis

Viscosity Determination

Refractive Index

Transmission Electron Microscopy

Keeping the sample at refrigerator temperature (4˚C) and room temp (25˚C) These studies performed for the specific period of time

Electrical characteristics of emulsion droplets can be measured by the electrical charge (Z-potential) of oil droplets in emulsions

Measure in Shear-Plane Z-potential predicts the stability of emulsion during storage

Color and Turbidity measurement

Optical Property

Stability Studies

Z-Potential

Scanning Electron Microscopy

Samples were fixed on stub using conductive double sided tape and then made electrically conductive by coating in a vacuum with thin layer of gold

Fig. SEM Encapsulated flavor oil in alginate solution and added in ice cream

(Ashraf et al., 2015)

NUTRACEUTICALS VIA

DAIRY PRODUCTS

FORTIFICATION IMPLICATIONS IN DAIRY

1. It is widely consumed food cover large demographic2. Various steps and process have measurable impact

on some specific nutrients3. Fulfill deficiency of milk e.g. Fe, Cu, Fiber, LCF, Vit C, E4. Easier quality control measure implementation5. Cost affordable by target population6. Higher stability and bioavailability of added

micronutriens

NUTRACEUTICALS FORMS AND NATURE OF EMULSION

Ref: Zuidam & Shimoni 2010

HYDROPHILIC NUTRACEUTICALS

LIPOPHILIC NUTRACEUTICALS

POLAR PARADOX

Water soluble Vit e.g. Ascorbic acid, Polyphenols Water-in-Oil (W/O) type emulsions

Lycopene, β-carotene, Lutein, Phytosterols, Docosahexaenoic acid (DHA), Carotenoids, Essential Fatty Acids can be encapsulated & delivered by Oil-in-

Water (O/W) type emulsions

Polar antioxidants are more active in bulk lipids than their nonpolar

counterparts Nonpolar antioxidants are more effective in oil-in-water emulsion

UHT milk with DHAHuimin et al (2014)

Studied formation and stability of Algae oil emulsion for application in UHT milk and produce UHT milk enriched with DHA - Starch hydrophobically modified with Octenyl Succinic Anhydride (OSA starch) & Corn Syrup was used as emulsifier to stabilize algae oil in UHT milk - Oxidative stability measured after heat treatment and found Algae oil-enriched milk was stable during 11 weeks of storage - Even high temp storage 40˚C does not significantly increase the oxidation process

Stable algae oil emulsion can be formed by OSA starches with Corn syrup & can be applicable for enrichment of UHT milk

PROBIOTIC ENRICHMENTLatha Sabhiki (2012)

Probiotics can be encapsulated in emulsion based delivery system containing solution of alginate (discontinuous phase) and dropped into vegetable oil (continuous phase) containing suitable emulsifier (tween 80) and surfactant (Sodium lauryl sulfate) to form beads

Later can be harvested by filtration.

Technique is Easy to scale up for large scale production and provides both encapsulated and entrapped core materials

LIMITATIONS FOR TECHNOLOGY

1. Residual oil, Emulsifier and Surfactant in the encapsulating material can affect the growth of live probiotics

2. Interact with food components3. Addition of oil may damage the organoleptic

properties and texture of the food products4. Not suitable for low fat food products

Latha Sabhiki (2012)

Work conducted at NDRI, Karnal reports the effect of microencapsulation using sodium alginate and starch on the tolerance of probiotic by emulsion

Babu el al., (2009) Probiotic Lactobacillus paracasei S233

& Sabikhi et al., (2010) Probiotic L. acidophilus

LA1

Organisms survived well in the protected form at high temperatures, high salt concentrations

& At stimulated conditions of gastric pH and at high bile salt

concentrations

VITAMIN D FORTIFICATION

• Daily recommended intake of Vit D is

Adults- 5µg/Day Children and Pregnant Woman- 10µg/Day

• Vit D is Fat soluble is hardly found in skim milk and low fat dairy products consumed largely in modern societies being an important sources of Calcium and Phosphate

• Vit D2 is largely used in pharmaceuticals industry

Ref: Patent: US 20090311329A1

• Novel approach for the nanoencapsulation and stabilization of hydrophobic biologically active compounds, particularly in non-fat or low fat edible products

• Hydrophobic domains of casein micelles stabilize the nutraceuticals in aqueous systems like milk

• Facilitate the enrichment of low fat and fat free dairy and other food products with these bioactive molecules ( vit D2, Vit K, Vit E, Vit A)

Patent: US 20090311329 A1 (2009)

Vit D2 Hydrophobic Nutraceuticals Adsorbed into Hydrophobic domains of Caseins which tend to found in the core of the micelle

VIT D IN CHEESETippetts et al., (2012)

Observed increased in retention of vit D 3 in cheddar cheese by incorporating it as a part of an oil-in-water emulsion using milk protein as emulsifier to obtain a fortification level of 280 IU/serving

Significantly retention was greater in cheese curd Vit D3 emulsified oil with NDM than control

Full fat cheese curd has little less retention compare to whole milk cheese curd

Gaysinsky et al (2007)

Conducted research on the antimicrobial activity of Eugenol microemulsions (Eugenol Encapsulated in Surfactant Micelles) in Ultra high temp pasteurized milk containing different percentages of milk fat (0, 2, 4%)He studied antimicrobial effect on L monocytogens and E coli O157:H7 strains

Results suggest that food composition, especially fat level, may affect the efficiency of targeting food borne pathogens with

surfactant-encapsulated antimicrobials

Cross Ref : Silva et al .,2012

Some of the giants of food industry such as Unilever and Nestle are also applying this technology

MEAT PRODUCTS FORTIFICATION

Salminen et al (2013)

Study provided systematic approach to evaluate the oxidative and physical stability of both the encapsulated active ingredient and subsequent inclusion into a meat product (Pork sausages)Protein stabilized oil – (25wt% oil, 0.25 wt% WPI, 50mM Sodium citrate buffer, pH 3.0) used, formed pre emulsion and incorporated in pork batter, stuffed in casing and then steam cooked to internal temp 72˚C

Emulsion provide physical and oxidative stability to base emulsionBut addition in complex meat matrices may lead to break down of base

emulsion due to differing conditions present in the meat product

CLSM images of low fat pork sausages with added with oil-in-water emulsion (25 wt% oil, 0.25wt% WPI, 50mM sod citrate buffer, pH3) a) 0 dayb) 31 days of storage

CLSM fish-in-water type emulsions (25 wt% oil, 0.25 wt% WPI, 50 mM sodium citrate buffer, pH 3) during storage

Salminen et al (2013)

SHORTCOMINGS IN MEAT PRODUCTS

Consists of complex protein matrices, salts, minerals (ingredients used such as ascorbic acid, phosphates, curing agents, spices)

1. Most food matrices differ in respect to their interactions to incorporated emulsion delivery systems

2. Ingredient interactions affects emulsion stability

MEAT IS COMPLEX FOOD

Ref: McClements & Decker, 2000

Mixing, Heating, Freezing and Freeze-thawing of meat products may likely influence the stability of emulsions based delivery systemsUpon heating proteins stabilizing the emulsion may denature

cause release of droplets interfaces to become hydrophobic and thus prone to aggregation

PROCESSING CONDITIONS

Ref: Salminen et al., (2013)

EFFECT OF SALT AND MINERALS

Overall electrostatic repulsion in emulsion droplets dependent on the ionic strength

Presence of salt (150 mM NaCl) cause flocculation of emulsion

In protein stabilized emulsions pH and ionic strength are major factors in dictating their stability

Screening of protein charges in the emulsions due to high salt and mineral concentration in the meat products may cause flocculation or coalescence of oil droplets

Ref: Djordjevic et al., 2004

FUTURE PROSPECTS1. Efficient Delivery of Herb Extract-

Technology can be used for Herb extract delivery in beverages and meat products

2. Antimicrobial Nanoemulsions in Food Industry – For decontamination of food, equipment, packaging of food (Gruere et al., 2011)

3. Potential Delivery System for PUFA- It is potential delivery system for PUFA in meat products

4. Improved Stability and Bioavailability of Nutraceuticals- Are required for the delivery of sensitive ingredients where direct contact with food matrix adversely affects the performance of bioactive compounds (Cournarie et al.,2004)

5. Novel Products-If these compounds encapsulated in multiple

nanoemulsions offer more scope for the control of encapsulation

ingredient, protection and release mechanism as compared to

conventional Emulsions (Sharma & Singh, 2012)

6. More Scope in Meat Products – As it less studied topic in meat products in preparation of functional meat products

7. Nanotherapy- Targeted delivery of nutrients for people with deficiencies

8. Totally new Taste or Mouthfeel – By encapsulating flavor

REFERENCES1. Gosh et al.,2005. Book-Clinical Aspects of Functional Foods and Nutraceuticals , CRC Press, Pp. 632. Latha Sabhiki (2014), Dairy Technology Division, Special Training Course, NDRI, Karnal3. Patent: US 20090311329A1, Yoav et al., (2007)-Casein micelles for nanoencapsulation of hydrophobic

compounds4. Tippetts et al., (2012) J. Dairy Sci.Sep;95 (9):4768-745. Salminen et al., (2013). Meat Sci. Mar93 (3)659-676. P. Fernandez et al. (2004) / Colloids and Surfaces A: Physicochem. Eng. Aspects 251 :53–587. Flanagan & Singh (2006). Critical review in food science and nutrition,46:221-2378. Cross ref: Winsor, P. A. Trans. Faraday Soc. 1948, 44, 3769. R. Adjonu et al., (2014). Journal of food Engineering 122:15-2710. Gaonkar et al., (2014). Book-Microencapsulation in the Food Industry: A Practical Implementation Guide.

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