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1 Microcapsule Design Considerations CSIRO FOOD AND NUTRITION and Characterisation Short Course on Micro- and Nano-encapsulation of Functional Ingredients in Food Products World Congress on Oils & Fats and 31 st Lectureship Series 31 st Oct 4 th November 2015, Rosario, Argentina Luz Sanguansri & MaryAnn Augustin
2 Outline Do you need microencapsulation? Why is microencapsulation required? What are important factors to consider? How is it done? Testing and characterisation of microcapsules Summary 2 Sanguansri & Augustin CSIRO
3 Do you need microencapsulation? Bioactives identified for health Is the ingredient stable in current form? NO YES Microencapsulation required Microencapsulation not required Formulate directly into Food Microencapsulated ingredient Application and Formulation into food 3 Sanguansri & Augustin CSIRO
4 Why is microencapsulation required? For stabilisation of bioactive Many are unstable once they are isolated Protection required throughout shelf-life Under storage conditions that ingredient is exposed to For enabling delivery into food Masking taste allowing addition without compromising sensory appeal Preventing undesirable interactions with other food components Stability during food processing Stability in final food product For enhancing bioavailability of bioactive Target delivery to site to exert desired physiological function Depends on intended health benefit 4 Sanguansri & Augustin CSIRO
5 Examples of potential benefits of encapsulation Nutraceutical Omega-3 fatty acids Probiotics Phenolic compounds and polyphenols Lipophilic phytochemicals (e.g. carotenoids, tocopherols) Bioactive peptides Minerals Vitamins Potential benefits of encapsulation Protection from oxidation Powder format (convenience) Taste masking Controlled release Enables incorporation in aqueous based food and beverages Improve viability during storage Protection in food product Protection from stomach acids and bile Taste masking Improved solubility Improved bioavailability Facilitates incorporation into food products Protection from oxidation Powder format (convenience) Controlled release Enables incorporation in aqueous based food and beverages Masking bitterness and astringency Controlled delivery Protection from acid environments Taste masking Avoidance of undesirable interactions (e.g. Fe-catalysed fat oxidation, Ca-induced protein precipitation) Protection against degradation Taste masking Controlled release Ease of incorporation of fat soluble vitamins in aqueous based foods and beverages 5 Sanguansri & Augustin CSIRO
6 What are the important factors to consider? Active ingredient (core) - properties Target application - format Processing conditions & capabilities Release mechanism Particle size Volume Load required Storage stability Other (legal, natural, etc) COST 6 Sanguansri & Augustin CSIRO
7 Sample questionnaire (1 of 2) Product: Core/bioactive - provided MSDS, specification and certificate of analysis o Stability to temperature, ph, shear, oxygen, moisture, light, UV o Format: liquid, powder, crystal, etc, o Purity or amount of active if it is in a carrier (specify carrier) o Shelf-life : Storage temperature, months Microencapsulated product description: o General description o Product format: (e.g. powder or liquid) o Payload (active / oil content) o Particle size o Special requirements: Stability of microencapsulated product: Acceptable % loss during shelf life (commercial ingredients) Packaging and storage conditions (temperature, months) Expected shelf-life Other requirements Application of microcapsules in final application: Food product / other (specify) Process requirements in final application Bioactive fortification level in final application per serving or per 100g Packaging and storage condition of final product Stability and shelf life of final product 7 Sanguansri & Augustin CSIRO
8 Sample questionnaire (2 of 2) Reason for encapsulation convert liquid to powder / solid protect from degradation mask taste, odour stable under high shear stable under compression provide moisture barrier controlled release increase solubility increase bioavailability Other information (if available) Do you manufacture the bioactive ingredient to be encapsulated or supplied by a third party? Does your company plan to manufacture the microencapsulated product, or by a third party? Is there an existing commercial product to match or improve? (appearance, flow properties, composition, stability, etc) What is the characteristic of the current product to match or improve? Do you need a sample for your own evaluation? (how much sample required) What would success look like during initial evaluation phase? (e.g. 5-10% loss at accelerated oxygen atmosphere, room temperature for 20 days) What is your timeline to commercialization (if successful)? 8 Sanguansri & Augustin CSIRO
9 MARKET SCIENCE ACTIVITY CAPABILITY Elements of Microcapsule Design Chemistry, Microbiology Core Material Formulation Process ME product Select/ identify Chemistry, Material Science & Engineering Existing/ New Formulation science & Chemistry Design formulations Engineering and Processing Conventional/ emerging Chemistry, Biochemistry, Food technology, Nutrition Develop specifications & applications Characterise physical & chemical Modification / Synthesis Study of interfacial behaviour Maintain/ develop processing capability Characterise physical & chemical Stability & solubility Characterise physical & chemical Study of ingredient interactions Assess effects of processing Stability of ME product e.g. heat, ph, moisture Stability of materials Triggers for release & stability of formulation Assess efficiency /consistency of process Suitability for incorporation into foods/ target delivery Work with supplier / user Establish cost / availability Compare with competitors / existing products Align with industry capability Align with end-user, Work with nutritionists CSIRO 9 Sanguansri & Augustin CSIRO
10 reformulate Generic steps to designing microcapsules 1 Design Delivery System Formulation & Manufacture Identify the core Choose encapsulant material Define microcapsule requirements Design formulation Prepare microcapsule 2 Characterise Release Properties Analyse to quantify the core Test microcapsule properties Test stability and sensory properties Test in-vitro release properties 3 Food Application & Delivery to the Gastrointestinal Tract Test stability in food application - stability during processing - stability during storage Test sensory properties in final food Test in-vitro release Test in-vivo & bioavailability Functional Food Product 10 Sanguansri & Augustin CSIRO
11 Testing & Characterisation of Microcapsules o Considerations in deciding the methodology o Physical characterisation and analysis of emulsions o Characterisation of powder microcapsules o Characterisation of microcapsules to monitor stability of the core from degradation o Release properties of microcapsules
12 Introduction Encapsulant Selection (formulation) Method of Processing (production) Method of Testing (characterisation) Choice of formulation, processing and format of the microcapsule are dependent on the active and final application... there are generic analysis that are common the task of selecting the most appropriate methodology is often dependent on final application Diversity of applications make it difficult to cover all aspects in detail Will consider the task conceptually and provide examples 12 Sanguansri & Augustin CSIRO
13 Main consideration before and during analysis - possible effect of matrix / encapsulant Must be sure that active is released so it can be measured Some matrices are hard to release the active encapsulated Incomplete release can under estimate amount of active Choice of solvent for release is important Make sure wall/matrix materials do not interfere with analysis of the active For probiotics: complete release and dispersion of cells is important to count individual cells vs. group of cells 13 Sanguansri & Augustin CSIRO
14 Other consideration during analysis For lipid extraction: Choice of solvent matrix dependent Time duration Separation method centrifuge, filter, stand For active quantification: Active extraction, separation and sample preparation Analytical method and instrumentation For probiotics viability: Rehydration and growth media Enumeration methodology 14 Sanguansri & Augustin CSIRO
15 Encapsulation Efficiency (ME) For lipid/oil encapsulation by oil extraction ME = [(total oil surface oil) / total oil ] x 100% For active encapsulation by GC or HPLC ME = [amount of active after encapsulation / amount of active added in formulation] x 100% For probiotic encapsulation total plate count ME = [viability (cfu/g) after encapsulation / viability (cfu/g) added in formulation ] x 100% 15 Sanguansri & Augustin CSIRO
16 Physical properties Emulsion Particle size Viscosity Oil-water interface Powders Moisture content Water activity Particle size Bulk density Flowability Dispersibility & solubility Morphology and structure 16 Sanguansri & Augustin CSIRO
17 Storage Stability Physical stability Emulsions - separation Powders - caking Chemical stability Lipid oxidation Active degradation Stability are influenced by storage conditions Packaging Environment temperature, humidity, oxygen, light 17 Sanguansri & Augustin CSIRO
18 Stability to stresses during processing & storage Simulate processing stresses Temperature stresses UHT, retort High shear stresses homogenisation High shear-temperature combination extrusion Simulate the environment in final food application Consider the moisture and water activity environment in final product application dry, moist or liquid food high-, low-, neutral- ph Simulate the storage and market environment Consider the temperature and relative humidity of where the product will be sold 18 Sanguansri & Augustin CSIRO
19 In-vitro release testing Simulate the release event Time Process Environment Water Simulated gastric fluid Simulated intestinal fluid Sequential exposure to simulated fluids 19 Sanguansri & Augustin CSIRO
20 Characterisation of Emulsions Examples
21 Free fat: used to optimise formulation for encapsulation of lipids Effect of protein type WPI WPC-80 a-lac NaCas SPI Effect of carbohydrate type free fat WPC:suc(40%fat) 30 Ncas:suc(40%fat) Effect of total solids 30% TS 40% TS WPC:suc(60%fat) Effect of protein: carbohydrate ratio Ncas:suc(60%fat) free fat glucose sucrose lactose DGS(DE24) Ncas:lac(40%fat) WPI:lac-suc(60%fat) 1:1 1:2 21 Sanguansri & Augustin CSIRO
22 Particle size: Used to measure quality of emulsion from reconstituted microcapsules Particle size distributions of reconstituted microcapsules at 0 (A), 1 (B), 2 (C) and 3 (D) months storage under ambient conditions 22 Sanguansri & Augustin CSIRO Polavarapu et al., 2010, Food Chem
23 Turbiscan: used to monitor emulsion stability Change in backscattering at various position along the sample measurement path indicates instability -Coalescence -Flocculation -Creaming -Sedimentation Stable emulsion Unstable emulsion Back Scattering 0:00 Back Scattering 0:00 Back Scattering 0:00 100% 100% 100% 90% 80% 0:01 90% 80% 0:01 90% 80% 0:01 70% 0:03 70% 70% 60% 60% 0:03 60% 50% 0:04 50% 50% 0:02 40% 40% 0:04 40% 30% 0:06 30% 30% 20% 10% 0:08 20% 10% 0:07 20% 10% 0:05 0% 0% 0% 0:10 0mm 20mm 40mm 60mm 0:09 0mm 20mm 40mm 60mm 0mm 20mm 40mm 60mm d(0.5) = 1.1µm d(0.5) = 3.9µm d(0.5) = 6.4µm 0:10 23 Sanguansri & Augustin CSIRO
24 Light microscopy and free fat - used to characterise emulsion properties 0.25% casein 0.1% casein 100µm 1.0% casein 100µm 100µm 24 Sanguansri & Augustin CSIRO Day et al, 2007, Food Chem
25 CLSM used to visualise emulsion structure and quality (encapsulation efficiency) Very fine structure (Protein and sugars) 50% oil loading powders Aggregated structure (Protein, sugar and pre-processed starch) Stable emulsion (Protein and pre-processed starch) Free oil visible (Protein and un-processed starch) Unencapsulated oil visible (OSA starch) Fat stained Nile Red (RED); Protein stained FITC (GREEN) 25 Sanguansri & Augustin CSIRO
26 CLSM used to visualise change in emulsion structure after addition of polysaccharide Primary emulsion (Protein sugars) Stained FITC for protein, Nile Red for fat Primary emulsion + pectin (ph7) Stained FITC(GREEN) for protein, Nile Red for fat Primary emulsion + pectin (ph7) Stained Congo Red for polysaccharide 26 Sanguansri & Augustin CSIRO
27 Characterisation of Powder Microcapsules Examples
28 DVS to measure moisture sorption properties - relates to powder flow characteristics (hygroscopicity) Relative Humidity in % Mass Change in % Change In Mass (%) - Dry Target RH (%) Influence of matrix on water uptake DVS Change In Mass (dry) Plot at 25C WPI/Hylon(1:1)/50%oil Cas/Hylon(1:1)/50%oil Target RH DVS - The Sorption Solution Time/mins Surface Measurement Systems Ltd UK Time/mins Casein and whey protein have similar MW, but different structures and inherent water binding properties 28 Sanguansri & Augustin CSIRO
29 SEM used to visualise morphology & structure of powder microcapsules Dried coacervates Spray dried formulation with starch coating Spray dried emulsion internal & external structure 29 Sanguansri & Augustin CSIRO internal & external structure internal & external structure Sanguansri & Augustin, 2010, Wiley Blackwell
30 Force (kn) Force (kn) Force (kn) Force (kn) Force (kn) Force (kn) Compression testing for mechanical strength of microcapsules 6 Formulation 1 (F1) 6 Formulation 2 (F2) 6 Formulation 3 (F3) Distance (mm) Distance (mm) Distance (mm) 6 F1 + polysaccharide 6 F2 + polysaccharide 6 F3 + polysaccharide Distance (mm) Distance (mm) Distance (mm) 30 Sanguansri & Augustin CSIRO Addition of polysaccharide improves mechanical strength of particles
31 CLSM used to visualise powder structure - location of fat and protein in the matrix Protein stained with Acridine Orange Protein stained with Acridine Orange Fat stained with Nile Red Good Powder structure (spherical particles oil inside particles) Fat stained with Nile Red Poor Powder structure (irregular particles - with free oil) 31 Sanguansri & Augustin CSIRO
32 CLSM used to visualise the location of cells within the microcapsules Protein-carbohydrate-lipid Emulsion matrix Protein matrix Resistant Starch matrix 32 Sanguansri & Augustin CSIRO
33 CLSM used to visualise probiotic cells - intact vs compromised cell membrane Live cells Live and dead cells Mostly dead cells SYTO9 stain (LIVE, GREEN) ; Propidium Iodide stain (DEAD, RED) 33 Sanguansri & Augustin CSIRO
34 Stability of the Core During Storage MUST reproduce commercial storage conditions otemperature o Relative humidity o Packaging
35 Oxipress to test resistance of sample to oxidation under accelerated storage condition IP Encapsulated omega-3 oil powders Fish oils induction period (IP) - indicates resistance of sample to oxidation Slope indicates of how fast is the reaction during IP 35 Sanguansri & Augustin CSIRO
36 Peroxide value for fat oxidation Stability of n-3 microcapsules stored at 30 C (in NaCas transglutaminase matrix) 36 Sanguansri & Augustin CSIRO Bao et al 2011, JFS
37 GC Analysis: - for quantification of omega-3 EPA and DHA DHA IS IS EPA DHA EPA 0 5 Acid methylation: triglyceride + free fatty acids Base methylation: glyceride bound fatty acids 37 Sanguansri & Augustin CSIRO
38 ELSD response (mv) mv HPLC (ELSD) or Iatroscan for Lipid class analysis e.g. mono-, di-, tri- glycerides, and phospolipids) Monoglycerides 500x Triglycerides Free fatty acids Diglycerides Elution Time (min) minutes Lipid class analysis of digested triglyceride oil (HPLC) Lipid class analysis of phospholipid rich marine oil (Iatroscan) 38 Sanguansri & Augustin CSIRO
39 Storage Stability of omega-3 powder microcapsules - sensory, headspace and fatty acid analysis Rancid Flavour Intensity Overall Quality Propanal Content (ug/g) % omega-3 and triglyceride concentration Sensory Analysis taste panel Head space analysis - GC month 3 months 6 months 9 months 12 months Storage Time 15 months 18 months 24 months month Threshold for detectible rancidity 3 months 6 months 9 months 12 months Storage Time 15 months 18 months 24 months MicroMAX (50% oil) 25 C MicroMAX (50% oil) 35 C Sensory Analysis taste panel MicroMAX (50% oil) 25 C MicroMAX (50% oil) 35 C Fatty acid analysis GC Lipid class (triglyceride) - Iatroscan month 3 months 6 months 9 months 12 months Storage Time 15 months 18 months 24 months month 3 months 6 months 9 months 12 months 15 months Storage time (months) 18 months 24 months MicroMAX (50% oil) 25 C MicroMAX (50% oil) 35 C % DHA % EPA % Triglyceride 39 Sanguansri & Augustin CSIRO
40 HPLC analysis to monitor stability of microencapsulated tocopherols Less than 10% loss over 12 months storage at 25 C (vacuum packed in aluminium foil sachets) 40 Sanguansri & Augustin CSIRO
41 mg/ g powder HPLC: analysis of resveratrol in microcapsules GC: analysis of tributyrin in microcapsules % Remaining mg/ g powder % Remaining 18 months storage stability of Resveratrol in microencapsulated powder containing a cocktail of bioactives stored at 25 C 18 months storage stability of Tributyrin in microencapsulated powder containing a cocktail of bioactives stored at 25 C Storage time (M, Axis for %R) Storage time (M, Axis for %R) M 3M 6M 9M 18M MicroMAX-I (20%TO:4.75%TB:0.25%Res) MicroMAX-I (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res) M 3M 9M 18M MicroMAX-I (20%TO:4.75%TB:0.25%Res) MicroMAX-I (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res) MIcroMAX-II (20%TO:4.75%TB:0.25%Res) 0 Shelf stability of resveratrol in a mixture of bioactive Greater than 95% remaining after 18 months storage at 25 C Shelf stability of tributyrin in a mixture of bioactive 85-90% remaining after 18 months storage at 25 C Bioactive cocktail combination: Omega-3 DHA & EPA, resveratrol, Tributyrin 41 Sanguansri & Augustin CSIRO
42 Stability of microencapsulated probiotics during storage at high Aw P ercent survival (Log 10 sca 100 Storage Stability (25 C, 50% RH) Encapsulated probiotics μm Tim e (w e e ks) Non-encapsulated probiotics 10 μm 42 Sanguansri & Augustin CSIRO Crittenden et al. 2006, AEM, 72(3),
43 Release properties of Microcapsules Test conditions MUST be under representative conditions!
44 Survival of microencapsulated probiotics during in-vitro digestion Percent survival (Log scale) 100 In-vitro survival: Encapsulated vs. non-encapsulated In-vitro Release A B 10 In gastric fluid In intestinal fluid In vitro model: US Pharmacopeia 0.01 SGF 1h ph 1.2, trypsin Non-encapsulated probiotic Encapsulated probiotic SIF 3h ph 6.8, pancreatin USP method: incubation at 37 C in SGF (ph 1.2, 2 hrs) then in SIF plus bile salts and calcium (ph 6.8, 3 hrs) 44 Sanguansri & Augustin CSIRO Crittenden et al. 2006, AEM, 72(3),
45 Acid Released (umol) ph stat used to characterise digestibility of powder microcapsules in-vitro Cas-oligo DGS MRP SPI Pectin MRP WPI Hylon MRP First Cas Hylon MRP First Cas Hylon MRP Last Intralipid SIF Time (min) In-vitro release during incubation at 37 C in simulated intestinal fluid plus bile salts and calcium (ph 6.8) for 3 hrs USP method 45 Sanguansri & Augustin CSIRO CSIRO, unpublished data
46 In-vitro release of Omega-3 from microcapsules Encapsulant: Cross Linked NaCas and Transglutaminase In-vitro release during incubation at 37 C in pepsin solution (2mg/mL citric acid, ph 2), filtered, dried, and weight of filter paper recorded 46 Sanguansri & Augustin CSIRO Bao et al 2011, JFS
47 13 C NMR used to characterise digestibility of microcapsules in-vitro (mobile components) SGF+SIF (no bile/ca) oil Encapsulant oil oil Encapsulant oil SGF In water powder Heated protein-carbohydrate with RS formulation MicroMAX Protein-carbohydrate blend formulation Non-MicroMAX 47 Sanguansri & Augustin CSIRO Burgar et al. 2009, Food Biophysics, 4, 32-41
48 CLSM used to visualise structure of microcapsules during in-vitro digestion Non-MicroMAX MicroMAX Before digestion After SGF 2h, ph 1.2 After SIF 5h, ph 6.8 Protein stained FITC (GREEN); Fat stained Nile Red (RED) 48 Sanguansri & Augustin CSIRO Chung et al. 2011, Food Chem, 124,
49 Light Microscopy: used to visualise change in structure of food matrix during in-vitro digestion Neat Powder Powder in Orange Juice Powder in Yogurt Powder in Cereal Bar SGF 2h, ph 1.2 SIF 5h, ph Sanguansri & Augustin CSIRO Shen et al. 2011, JAFC, 59,
50 % Omega-3 fatty acids Effect of food matrix release of omega-3 fatty acids B control orange juice yogurt cereal bar Digested Samples EPA DHA Recovered EPA and DHA (micro gram) EPA 2400 DHA Fish oil capsule Orange juice Yogurt Cereal bar In-vitro digestion in SGF (2hrs, ph 1.2) and SIF (5hrs, ph 6.8) In-vivo trials in ileostomy patients (<2% of dose recovered from ileal digesta) The food matrix can influence the release of long chain omega-3 fatty acids during in-vitro digestion and in-vivo trial 50 Sanguansri & Augustin CSIRO Shen et al, CSIRO, Unpublished data
51 Points to remember during testing Choice of method for testing and characterisation of microcapsule is dependent on core, encapsulant material, format and application Must be sure that active is released so it can be measured Must ensure that the encapsulant materials do not interfere with analysis Storage Stability MUST reproduce storage conditions e.g. temperature, relative humidity, packaging Release test conditions MUST be under representative conditions 51 Sanguansri & Augustin CSIRO
52 Overall Summary Microcapsule design Important to ask the right questions at the start Define the specification/requirements of the microcapsule Choose the correct method to test/characterise the microcapsule Ingredient / Product requirement Stability important for quality, shelf life Taste should not be compromised Bioavailability important for health and wellness products Commercial / Regulatory Cost still remains a major consideration for successful commercialisation Regulatory standards market jurisdiction 52 Sanguansri & Augustin CSIRO
53 Thank you Luz Sanguansri Research Team Leader t e luz.sanguansri@csiro.au w Mary Ann Augustin Research Group Leader t e maryann.augustin@csiro.au w CSIRO FOOD AND NUTRITION
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