Phương pháp tạo màng calcium alginate and calcium pectinate films

The second focus of this project was to determine thin film diffusion and permeability coefficients of the created polymer films.... The ESEM film thickness measurements demonstrated a 2

Preparation of Calcium Alginate and Calcium Pectinate Films and

Determinations of Their Permeabilities

This work and its defense approved by:

of the requirements for the degree of

MASTER OF SCIENCE in the Department of

Chemistry

by

Robert B Wieland

Bachelors of Science in Chemical Technology University of Cincinnati, June 2001

Committee Chair: Dr James E Mar

Small amounts of polymers are typically used in flavor and food applications

Polymers are typically applied in thin coatings which allow for a cost-effective encapsulation with desirable barrier properties Understanding the properties of thin barrier coatings is essential to obtaining optimal encapsulation performance Many of the polymers used in the flavor and food industry are cross-linked hydrogels, which are water insoluble but water swellable Hydrogel barriers allow water soluble components to be extracted from the encapsulation system Flavor components having a large affinity for water will be extracted from the encapsulation system while more hydrophobic flavor components will remain encapsulated Preferential flavor extraction is a large problem for the flavor industry because flavors are complicated mixtures of both hydrophilic and hydrophobic components

Understanding diffusion and permeability coefficients is desirable for creating optimized encapsulation systems However, creating thin uniform films reproducibly can be challenging and expensive In the past, thick polymer films were cast onto a metal sheet and cross-linked with the appropriate chemicals The method produced wrinkled and inconsistent film thicknesses The inconsistent films produced irreproducible diffusion and permeability coefficient data New testing methods were developed to understand flavor partitioning across thin hydrogel membranes One focus of the present work was to create 10^m to 50^m polymer films reproducibly

with uniform thicknesses The second focus of this project was to determine thin film diffusion and permeability coefficients of the created polymer films.

The first portion of this thesis discusses the creation of thin polymer films

Calciumalginate and calcium pectinate films were created using a lightly scuffed metal sheet The sheet was then used in a leveling apparatus which provided a level surface for film casting The polymer films were characterized by micrometer measurement, environmental scanning electron microscopy (ESEM) and swelling ratio experiments Micrometer measurements demonstrated the successful preparation of 21 to 23 (+/- 1) calcium alginate films and 19 to 20 (+/- 1) calcium pectinate films The 4 to 6% relative standard deviation was considered acceptable for the present work The calcium

alginate and calcium pectinate films were also analyzed by ESEM Both sides of the films were analyzed at 200X and 1500X magnifications The polymer film surface

exposed to the scuffed metal sheet produced a rough and irregular surface The

polymer film surface not exposed to the scuffed metal sheet had a smooth and uniform surface Film thickness measurements were also performed using the ESEM computer software to further verify the film thickness measurements obtained from the

micrometer The ESEM film thickness measurements demonstrated a 20.9 (+/- 1.1) calcium alginate film and a 20.2 (+/- 0.7) calcium pectinate film had been produced Both films demonstrated an average relative standard deviation of 4 to 6% which was considered acceptable for the present work The ESEM measurements of film thickness demonstrate the methodology for creating thin polymer films is reproducible and within the desired thickness range However, the scuffed metal sheet creates films that are rough on one side and smooth on the other side Preliminary polymer swelling ratio experiments in distilled water showed calcium alginate films swell to 2.4 times their original dry weight and calcium pectinate films swell by a factor of 3.8 The large

swelling ratios for the films indicated that distilled water was an appropriate solvent for determining film permeability and diffusion coefficients

The second portion of this thesis focused on determining film diffusion and permeability coefficients A new thin-film diffusion cell (TFD) was built and coupled to a UV/VIS spectrophotometer fitted with a fiber optic probe which allowed for in-situ measurement of analytes which absorb ultraviolet radiation such as benzaldehyde Permeability measurements using benzaldehyde demonstrated a permeability coefficient of 3X 10-5 cm/sec (+/- 5%) for the 22 (+/- 1) calcium alginate film and 2 X 10-4 cm/sec (+/-6%) for the 20 (+/- 1) calcium pectinate film Diffusion coefficients were then calculated for the two films The diffusion coefficient for a 22 (+/-1) calcium alginate film was 6.5 X 10-8 (+/- 11%) cm2/sec while the diffusion coefficient for a 20 (+/-1) calcium pectinate film was 3.9 X 10-7 (+/- 12%) cm2/sec The relative standard

deviations for the permeability and diffusion coefficients were considered acceptable for this study The permeability and diffusion coefficients indicated

a calcium pectinate film is more permeable than a calcium alginate film of

equal thickness.ACKNOWLEDGEMENTS

I would like to express my gratitude to Dr Dave Siegel for the guidance and support shown to me while obtaining a graduate degree I acknowledge my graduate achievement would not have been possible without the patience, flexibility and understanding shown by this man I would also like to thank Dr Robert Eilerman for his flexibility allowing me to achieve an academic milestone while maintaining full time duties at the Givaudan Flavor Corporation

I would like to acknowledge the financial support of the Givaudan Flavor Corporation The financial support allowed for critical glassware to be purchased and allowed me to obtain a graduate degree

I would like to give a heartfelt thank you to Dr Jing Zhang for helping me understand diffusion and permeability In this way, Dr Zhang helped me become a better chemist with an increased knowledge of polymer

6 6

I appreciate the guidance Dr James Mark has given while writing my graduate thesis and during my academic career His insight has been valuable and informative

I would like to thank my mother Brenda Wilson for her endless encouragement Your determination, work ethic and loving support has enabled me to be successful in the workplace and in academia

I am grateful for the support and encouragement shown by my wife Jessica Wieland Without her support this achievement would not be possible Thank you for understanding the extra hours at school and the extra hours of homework which kept us apart I could not ask for a more supportive and inspiring wife

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF TABLES

7 7

Table 9: Benzaldehyde absorbance measurements across calcium alginate films 34 Table 10: Benzaldehyde absorbance measurements across

calcium alginate films.3

8 8

9 INTRODUCTION

4Both flavors and active ingredients such as vitamins impart important

characteristics to products desirable to consumers in the marketplace

However, flavors and active ingredients can be lost or degraded during food processing, with the result of losing consumer benefit However, critical

components can be encapsulated using polymeric materials to prevent such losses Polymers are typically applied in thin coatings which allows for a cost effective encapsulation with functional properties1 Understanding the

properties of thin barrier coatings is essential to obtain optimal encapsulation performance The food and flavor industry have used polymeric materials for decades as bulking agents, viscosifiers, and barrier materials for various encapsulation systems Materials such as sugar, maltodextrin, pectin and alginate can be used to create water soluble encapsulation systems2 Pectin and alginate materials are of great interest to the flavor industry due to the cross-linkable nature of these natural polymer materials3 Cross-linked pectin and alginate form hydrogel barriers which are water insoluble but water

swellable4 The swelling properties of hydrogel barriers can be manipulated by varying levels of chemical cross-linking along these carbohydrate chains5 Highly cross-linked polymer materials typically demonstrate minimized

diffusion properties which creates an effective barrier reducing flavor loss during cooking processes Lightly cross-linked polymers become

less effective barriers due to increased diffusion properties

Flavor encapsulation systems containing hydrogels have been utilized in food

to create products with increased flavor perception6,7 However, flavors encapsulated in hydrogel systems typically need to be reformulated to preserve such a desirable perception The swelling property of hydrogel barriers allows flavor components having a large affinity for water to be extracted from the encapsulation system while more hydrophobic flavor components remain encapsulated The swelling property of hydrogel barriers

is a large problem for the flavor industry because flavors are complicated 1Gutcho, M.H Microcapsules and Other Capsules Noyes Data Corporation: Park Ridge, NJ, 1979

2 Risch, S.J (1995) Encapsulation: Overview of Uses and Techniques In Risch, S.J and Reineccius, G.A (Ed.) Encapsulation

and Controlled Release of Food Ingredients (pp 2-7) Washington, DC: American Chemical Society

3 Schlemmor, U (1989) Studies of the binding of copper, zinc

and calcium to pectin, alginate, carrageenan and gum guar in

HCO"3 - CO2 buffer Food Chemistry, 32 (3), pg 223-234.

4 Bajpai, S.K.; Sharma, S (2004) Investigation of swelling/degradation behavior of alginate beads crosslinked with Ca2+ and Ba + ions

Reactive & Functional Polymers, 59 (2004), pg 129-140.

5 Flory, P.J Principles of Polymer Chemistry Cornell University Press:

Ithaca, NY, 1953

i

10 INTRODUCTION

mixtures of both hydrophilic and hydrophobic components For example, typical fruit flavors contain numerous individual ingredients which impart a delicate balance and flavor profile Individual cherry flavor ingredients have vegetable oil:water partition coefficients ranging from 4 to 1 A partition coefficient, denoted as P in this document, is the concentration ratio of a compound in two immiscible solvents at equilibrium8,9 The P coefficient in this study is a measure of differential compound solubility between vegetable oil and water The higher the P value the more hydrophobic the compound Since most food applications are exposed to water over time, maintaining a

balanced flavor profile is difficult Creatin

i

gan encapsulation system with reduced flavor diffusion properties would be beneficial for the flavor industry.

The flavor industry creates encapsulation systems to address various food processing issues Analyzing an encapsulation system typically entails "in-use” tests which only demonstrate whether or not the encapsulation system provides a benefit6 A typical "in-use” test consists of making an encapsulation containing a flavor The encapsulated flavor is then added to a food application and processed under normal cooking conditions These tests only provide a result which is negative or positive Since the encapsulation system is a complex product no data is provided on what aspect of the technology is providing the result Also, the food application is very complex and also affects how the encapsulation performs These facts leave the researcher asking

"Have we created a better encapsulation or merely used the encapsulation in a more desirable environment?”

Analytical experiments, such as encapsulation-dissolution testing, have been created

to characterize the individual encapsulation systems in model food application

environments Valuable data has thus been obtained which can predict encapsulation performance in various applications7 However, encapsulation performance is highly dependent on capsule particle size, polymer barrier thickness, polymer permeability, capsule structure and encapsulation makeup Capsule performance is measured, with account for all the variables that affect encapsulation performance.Previously at the Givaudan Flavor Corporation attempts were made to study the diffusion and

permeability properties of hydrogel films containing clay8 Films approximately 25 to

200 microns were cast and cross-linked with the appropriate reagents The method produced wrinkled films and inconsistent film thicknesses that only allowed for small pieces of the films to be analyzed The irregular films produced irreproducible diffusion

6 Mark, J.E.; Allcock, H.R.; West, R Inorganic Polymers 2nd ed Oxford University

Press: New York, NY, 2005

7 Martin, C.A., (2003) Evaluating the Utility of Fiber Optic Analysis for Dissolution

Testing of Drug Products Dissolution Technologies, pg 37-39.

8 Vale, J.M (2004) Modification of Calcium Alginate Membranes with Montmorillonite Clay to Alter the Diffusion Coefficient (Masters Thesis, University of Cincinnati, Department of Chemistry, 2004)

and permeability measurements The analytical methodology used to characterize the films was tedious and involved the use of multiple analytical techniques Each

analytical technique contributed compounded errors which affected the accuracy of the data

The primary goal of the present study is to create thin hydrogel films whose diffusion and permeability properties can be measured easily with relative accuracy Understanding flavor diffusion across thin hydrogel membranes will provide the basic knowledge for hydrogel encapsulation development The films created were approximately 20^m thick, which replicates typical coating thicknesses used in the flavor industry Creating thin films can be challenging due to the following circumstances: thin films become very brittle, brittle and cracked films lead to ineffective barriers, and thin films are hard to cast uniformly The thin films created were uniform and reproducible which ensured a robust method and reliable data

For the present work, two calcium cross-linkable polymers were chosen for study Alginate and pectin were chosen for their acceptance in the food and flavor industry The thin film hydrogels were characterized by micrometer film thickness measurements, Environmental Scanning Electron Microscope (ESEM), swelling ratio

experiments and Thin Film Diffusion (TFD) - Ultraviolet-Visible (UV/VIS) analysis

1.1 Pectin: Sourcing, Manufacture and Functionality

Pectin and cellulose are abundant in fruits such as oranges, lemons, limes and apples Pectin and cellulose associate creating a macromolecule called protopectin which binds or absorbs water Cellulose provides mechanical rigidity and pectin provides flexibility in the fruit and plant stock The mechanical properties of protopectin allow the plant to weather environmental changes during seasonal changes9

Large farming ventures and food companies process fruits such as oranges and apples, thus creating waste streams of citrus peel and apple pomace The waste streams are typically created in the regional areas where the crop is grown For instance, large waste streams of orange peel are created in Mexico, California and Florida The waste streams are collected and processed to yield valuable extracts such as pectin and cellulose

Pectin is produced worldwide by major manufacturing companies such as Cargill and CP Kelco Extraction techniques performed on the citrus peel and apple pomace can be altered to produce pectin with different functionalities Typically, the citrus peel or apple pomace is added to a hot acid solution where the protopectin undergoes hydrolysis The hydrolysis step liberates the pectin from the cellulose

9

www.cargilltexturizing.com/products/hydrocolloids/pectins/cts_prod_hydro_pec_fun.shtml

The citrus peel or apple pomace is then separated from the hot acid solution by pressing, filtration and concentration processes The concentrated solution is added

to ethanol where the pectin precipitates This precipitate is then washed, dried, and milled to a

specific particle size Processing conditions are constantly being modified and

optimized to meet specific customer needs10

Citrus peel and apple pommace processing allows for three types of pectin to be manufactured The hydrolysis processing step applied to citrus peel and apple pomace alters the degree of esterification (DE) found in the pectin Thermodynamic properties such as glass transition temperature (Tg), melting point (Tm) and setting rates can be drastically altered by the degree of esterification Pectin with a DE value greater than 50 is referred to as high-methoxyl pectin (HM)15,16 Typical HM-pectin’s have a 55-85% degree of esterification and form thermostable gels in acidic

pH solutions containing 60% sugar HM-pectin is used to stabilize milk by reducing protein flocculation and enhancing beverage viscosities High-methoxyl pectin has other valuable uses such as minimizing ice crystal formation, thus enhancing confectionary freeze-thaw properties Pectin with a DE value below 50 is referred to

as low-methoxyl pectin (LM) Typical LM-pectin’s have a 15-45% degree of

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esterification and form thermoreversible gels under acidic or basic processing conditions Thixotropic solutions for ice cream can be created using LM-pectin LM- pectin also has the ability to be crosslinked by divalent cations such as calcium and magnesium Crosslinked pectin typically becomes water insoluble and is useful for film and encapsulation purposes This pectin can also be extracted under basic conditions using ammonia producing amidated low methoxyl pectin (LMA) Typical

LMA-pectin’s have a 15-45% degree of esterification and a 5-25% degree of amidation (DA) The addition of the amide groups to the pectin molecule changes the rheological properties and reduces pectin’s ability to be crosslinked by divalent cations LMA-pectin produces thermoreversible gels which are typically used in glazes and fruit preparations Many different forms of pectin can be created to meet specific customer needs The variety of pectin’s produced allows for new and innovative consumer products to be developed

1.2 Pectin: Structure

Pectin is a natural polysaccharide containing up to1000 saccharide units in a like configuration Pectin molecules have a linear backbone composed of units of (1, 4)-linked a-D-galacturonic acid and its methyl ester11 Figure1.2-1 illustrates the basic structure of a pectin molecule

chain-11 www.cpkelco.com/food/pectin.html

Figure 1.2-1: Structure of the pectin molecule.

The galacturonic acid units may be in the salt form (galacturonate), which allows the pectin to be an anionic polymer The galacturonic acid residues can be esterified with methanol When 50% or more of the carboxyl groups contained in the polymer are methylated the pectin is considered high-methoxyl pectin This pectin is not cross-linkable with divalent cations and has limited uses for flavor encapsulation systems Less than 50% methylation produces pectin which is cross-linkable with divalent cations such as calcium Figure1.2-2 illustrates the basic structure of a calcium pectin molecule The pectin structure also contains L-rhamnose and methylpentose The addition of these sugars to the pectin polymer structure creates

a branched molecule which has much reduced linearity The average molecular weight of pectin is typically 50,000 to 150,000 g/mol

1.3 Alginate: Sourcing, Manufacture and Functionality

Seaweed has been used to obtain natural polymers such

as alginate, agar and carrageenan over the last 50 years Alginate provides rigidity for the seaweed plant by association with sodium chloride in ocean water Alginate also acts as a humectant to reduce water loss in the plant in changing tidal conditions12 Alginate is found in the Phaeophycaea brown algae family Seaweed varieties such as Macrocystis pyrifera, Ascophyllum Nodosum and Laminaria are useful for alginate production12 Comercially important seaweed is typically harvested in the coastal waters of California, Australia, Norway and Japan Seaweed from different coastal regions produces alginate with different properties due to structural differences in the extracted polymer.

The manufacturing process to obtain alginate begins with harvesting seaweed in the desired coastal region Boats are built to a special design to skim the ocean surface and retrieve the top three feet of the seaweed plant The harvested seaweed is gently dried and milled to desired specifications for optimal processing The milled seaweed is then added to hot water and sodium carbonate under mixing conditions The caustic treatment allows the seaweed to swell and form a viscous solution The highly viscous solution is diluted and the insoluble residues are removed Chlorine is added to the liquid fraction containing the alginate and treated with calcium chloride to form a water insoluble precipitate The calcium alginate is then recovered, pressed to remove excess water and treated with a hydrochloric acid solution The acid-washed alginate cake is then pressed and washed with a basic solution to produce sodium alginate which is water soluble The solubilized alginate is then spray dried and sieved to the

desired customer specifications20,21

12 Cosgrove, D.J., (2005) Growth of the plant cell wall Molecular Cell Biology, 5,

pg 850-861

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.Alginate is generally used as a cold-setting gel that is thermally stable when

crosslinked The food industry typically uses alginate as a viscosity control agent for products such as jellies, jams, pastes, beverages, soups and ice-cream22,23,24

Industries such as pharmaceutical and biomedicine companies use alginate to

encapsulate drugs and cellular materials13 Alginate has also been used to dehydrate products such as paper and textiles, as flame retardants for fabrics, and as blood

detoxifiers

1.4 Alginate: Structure

Alginate is a complex carbohydrate comprised of glucuronic and mannuronic acid monomers Based on the seaweed source and processing, alginate can be produced with varying glucuronic and mannuronic acid contents14 Figure 1.4-1 illustrates the monomers contained in alginate

Alginates containing large percentages of glucuronic acid content are called high-G

alginates15 High-G alginates typically produce gels that are strong and exhibit good heat stability However, the gels are brittle and this can create a product with little impact strength Alginates containing large percentages of mannuronic acid content are called high-M alginates These alginates produce gels that are elastic, and this increases freeze-thaw stability The block sequences of glucuronic acid and mannuronic acid monomer units also affect alginate functionality Varying block lengths such as GG, MG and MM produce gels with blended properties and regimes

13 Milanovanovic, A.; Bozic, N and Vujcic Z (2007) Cell wall invertase

immobilization within calcium alginate beads Food Chemistry, 104 (1), pg 81-86.

14

www.cargilltexturizing.com/products/hydrocolloids/alginates/cts_prod_hydro_alg_mol.shtml

15 Sime, Wilma J., (1990J Alginates Limewood, Raunds, Northhamptonshire NN9

6NG, UK p 53-60

(1,4) P — D mannuronate (1,4) a—Lguluronate

Figure 1.4-1: Alginate monomers

of localized crystallization when glucuronic acid units are crosslinked with divalent cations16 Figure 1.4-2 illustrates alginate glucuronic and mannuronic block types.

Figure 1.4-2: Alginate block types

The divalent cation fits into the D-glucuronic acid block structure like eggs in an egg box This binds the alginate polymers together by forming junction zones which results in gelation17 Figure 1.4-3 illustrates the calcium binding site of D-glucuronate and Figure 1.4-4 illustrates the egg box structure of calcium crosslinked alginate

16

www.fmcbioploymer.com/food/ingredients/Alginates/PGA/Introduction/tabid/2410/fault/aspx

17

www.fmcbioploymer.com/food/ingredients/Alginates/PGA/Introduction/tabid/2412/fault/aspx

DOST

Guluronate block-QOC

Mannuronate blockMannuronate-Guluronate-Mannuronate block

Figure 1.4-3: Calcium binding site in polyguluronate dimer

Figure 1.4-4: Calcium alginate cross- linked "Egg box” model

2 Experimental Section

2.1 Experimental Objectives

The objectives of the present work are as follows:

Prepare uniform films of calcium alginate reproducibly in a 5 to 50pmthickness range.

a) Prepare uniform films of calcium pectinate reproducibly in a 5 to 50^m

thickness range

b) Analytically measure the diffusion of benzaldehyde across the

hydrogel barriers to determine permeability and diffusion coefficients

2.2 Raw Materials

1. Sodium alginate extracted from brown seaweed (Phaeophyceae) was chosen

to create thin films The sodium alginate is cross-linkable with divalent cations such as Ca and Mg2+2+ The material is a whitish tan powder containing 1 to 3% moisture The average molecular weight is 75,000 g/mol The sodium alginate trade name is Keltone LV and was supplied by ISP (International Specialty Products) 1361 Alps Road, Wayne, New Jersey 07470

2. Pectin extracted from lemon peel was chosen to create additional thin films The pectin is also cross-linkable with divalent cations such as Ca2+ and Mg2+ The material is a white powder containing approximately 3 to 4% moisture and average molecular weight was 95,000 g/mol The pectin trade name is TIC Gum-32 and was supplied by TIC Gums, 4609 Richlynn Drive, Belcamp, MD 21017

3. Calcium chloride was used as the crosslinking material for sodium alginate

and pectin The material is a white granular powder containing approximately 2% moisture The calcium chloride was supplied by the Givaudan Flavor Corporation, 1199 Edison Drive, Cincinnati, Ohio 45126

4. Benzaldehyde was used to measure diffusion across the calcium alginate and calcium pectin films The benzaldehyde was 98% pure with a boiling point of

435.1 K Benzaldehyde has a Log P value of 1.78 which is considered to be relatively water soluble in the flavor industry The benzaldehyde was supplied

by the Givaudan Flavor Corporation, 1199 Edison Drive, Cincinnati, Ohio 45126

5. Miglyol 812 (Medium chain triglycerides) was used to prepare dilute benzaldehyde solutions It is

an oxidative-stable vegetable oil which was supplied by Givaudan Flavor Corporation 1199 Edison Drive, Cincinnati, Ohio 45126

Figure 2.2-1: Structure of benzaldehyde

2.3 Procedures - Laboratory Testing Equipment Preparation

Three Baker’s Secret medium cookie sheets were purchased from a local grocery store The measurements of the cookie sheets were 43.2cm X 27.9cm X 1.9cm with

an estimated surface area of 1205.3 cm2 The cookie sheets were lightly scuffed under water with an abrasive sponge to partially remove the Teflon™ coating from the sheets Partially removing the Teflon™ coating allowed the polymer solutions to wet the surface creating a uniform polymer film

Film Sheet Apparatus

A leveling apparatus was created to ensure a level surface for use with the film sheets The leveling apparatus was created using medium-density fiberboard and spring-loaded clamps The base measurements of the leveling apparatus were 96.5 cm X 63.5 cm X 2.0 cm The clamping system consisted of two pieces of 86.4 cm fiberboard strips attached to the base of the leveling apparatus 43.5 cm apart Three spring-loaded clamps were applied to each strip at the desired Figure 2.3.1-1: Scuffed cookie sheet