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IMPROVEMENT OF ACTIVE PACKAGING MATERIALS
BASED ON POLY(LACTIC ACID) CARRYING
ENCAPSULATED ANTIMICROBIAL VOLATILES

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MinJung Joo

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IMPROVEMENT OF ACTIVE PACKAGING MATERIALS BASED ON
POLY(LACTIC ACID) CARRYING ENCAPSULATED ANTIMICROBIAL
VOLATILES
By

MinJung Joo

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Packaging

2010

ABSTRACT
IMPROVEMENT OF ACTIVE PACKAGING MATERIALS BASED ON
POLY(LACTIC ACID) CARRYING ENCAPSULATED ANTIMICROBIAL
VOLATILES
By

MinJung Joo
Bio-based blends made of poly(lactic acid) (PLA), an aliphatic thermoplastic polyester,
and B—cyclodextrins (B-CDs), an enzymatically modified starch, are stiff and brittle due to
the incompatibility which limits their applications. The same limitations are expected for
bio-based antimicrobial materials created by blending PLA with inclusion complexes
(ICs) which serve as a carrier for natural antimicrobial volatile, trans-2-hexenal based on
B-CDs. The objective of this study was to overcome limitations by enhancing the
compatibility of the carrier with PLS by using a masterbatch. The study was divided into
two phases. In phase one, the interfacial adhesion of PLA and B-CDs at various ratios
was investigated and the effectiveness of using a masterbatch to improve the adhesion
was studied. In phase two, the materbatch was used to develop an antimicrobial material
based on a PLA/ICs blend carrying trans-Z-hexenal. The use of the masterbatch
significantly enhanced the compatibility between PLA and B—CDs, and improved the
thermal, mechanical, optical, and barrier properties of the blends. The antimicrobial
PLA/ICs-trans-Z-hexenal blend has been shown to be effective against Alternaria Solam'.
The exposure of the ICs to high heat and relative humidity during processing caused a
premature loss of the antimicrobial compound encapsulated in the B-CD molecules for

later release, and resulted in a reduced antimicrobial activity.

ACKNOWLEGEMENTS

I would like to thank my adviser, Dr. Eva Almenar. During my master‘s program, she
provided me valuable guidance on my research and taught how to think about research. I
am very happy that I had the opportunity to be a member of her group. Also, I want to
thank to Dr. Rafael Auras and Dr. Ramani Narayan for their invaluable feedback during
my experiments and their revision to my thesis. I want to acknowledge to Dr.Laurent
Matuana for permission to use his equipment and Dr. Randolph Beaudry‘s comments
regarding inclusion complexes.

Moreover, I am very thankful to my family. They sincerely supported me and encouraged
me to persevere. I hope the completion of my thesis can inspire June in his life. To my
special D, thank you.

I appreciate to Dr. Rai Deepak from India for sharing the kinetic study with me. Carol
Flegler and Abigail Tirrell from Microscopy center helped me a lot to take SEM pictures.
Andre Legowo and Atul Singla were helped to make blends with extruders. Zhiguan
Yang from Chemical Engineering gave me many comments about extruding materials
with a pilot-scale extruder. Jin Shan from Forestry helped me to process blends with an
extruder. I am thankful to DongHo Kang for helping analysis of samples. I want to
acknowledge to our research group under the direction of Dr. Almenar. Hayati Samsudin,
my fi'iend, helped me a lot during my masters and OH and Lee gave me lots of comments.
Also, thank to all Korean members (Dr.Big Shin, Dr.Shin in LA, Dr.Shim, JK, Prof Min,
Prof Jung, Wook bro, Oh bro, Keun, JiYoung, Wontai, Tae, Jonghun, Kang, KK,

Kyungmi). Master‘s program was so fun because we were together.

iii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. vi
LIST OF FIGURES .......................................................................................................... vii
ABBREVIATIONS ........................................................................................................... ix
CHAPTER 1. INTRODUCTION ........................................................................................ 1
1.1 Introduction and motivation ...................................................................................... 1
1.2 Objectives .................................................................................................................. 4
CHAPTER 2. LITERATURE REVIEW ............................................................................. 5
2.1 Active packaging ....................................................................................................... 5
2.1.1 Active packaging market .................................................................................... 5
2.1.2 Types of active packaging .................................................................................. 6

2.2 Antimicrobial packaging ........................................................................................... 6
2.2.1 Antimicrobials .................................................................................................... 7
2.2.1.1 Naturally occurring antimicrobials .............................................................. 8
2.2.1.1.1 Trans-2-hexenal .................................................................................... 8

2.2.2 Antimicrobial polymers .................................................................................... 10
2.2.2.1 Antimicrobial polymer market .................................................................. 11
2.2.2.2 Antimicrobial polymers and processing .................................................... 11
2.2.2.2.] Extrusion ............................................................................................. 12
2.2.2.2.].1 Factors affecting extrusion ............................................................ 14

2.2.2.3 Antimicrobial polymers and materials ....................................................... 14
2.2.2.3.] Petroleum-based antimicrobial polymers ........................................... 14

2.2.2.3.2 Bio-based antimicrobial polymers ...................................................... 15
2.2.2.321 Poly(lactic acid), PLA ................................................................... 15
2.2.2.3.2.1.1. PLA blends and masterbatches .............................................. l6

2.2.2.322 Cyclodrextrins ............................................................................... l8
2.2.2.3.2.2.1 Micro-encapsulation ............................................................... 19

22.2.3222 Controlled release ................................................................... 20

CHAPTER 3. PREPARATION AND CHARACTERIZATION OF BLENDS MADE OF
POLY(LACTIC ACID) AND AN ENZYMATICALLY MODIFIED STARCH WITH A
CAPACITY TO CARRY HYDROPHOBIC MOLECULES: IMPROVEMENT OF THE

BLEND PROPERTIES BY USING A MASTERBATCH ............................................... 21
INTRODUCTION ......................................................................................................... 22
EXPERIMENTAL ......................................................................................................... 25

Materials .................................................................................................................... 25
Formulation and preparation of pellets ...................................................................... 25
Preparation of sheets .................................................................................................. 27
Analyses ..................................................................................................................... 27

Mechanical characterization .................................................................................. 27

iv

Thermal characterization ....................................................................................... 28

Optical characterization ......................................................................................... 29
Morphological characterization ............................................................................. 3O
Barrier characterization ......................................................................................... 30
Statistical methods ................................................................................................. 31
RESULTS AND DISCUSSION .................................................................................... 31
Mechanical characterization ...................................................................................... 31
Thermal characterization ........................................................................................... 33
Optical characterization ............................................................................................. 41
Morphological characterization ................................................................................. 44
Barrier characterization ............................................................................................. 46
CONCLUSIONS ........................................................................................................... 49

CHAPTER 4. DEVELOPMENT AND CHARACTERIZATION OF A BIO-BASED
ANTIMICROBIAL POLYMER MADE OF POLY(LACTIC ACID) AND
ENZYMATICALLY MODIFIED STARCH CONTAINING TRAPPED TRANS-2-

HEXENAL ........................................................................................................................ 51
1. Introduction ................................................................................................................ 52

2. Experimental Section ................................................................................................. 54
2.1 Materials. ............................................................................................................. 55

2.2 Microbial strains. ................................................................................................. 55

2.3 Synthesis and characterization of inclusion complexes (ICs). ............................ 56

2.4 Preparation of the antimicrobial masterbatch ...................................................... 58

2.5 Preparation of the antimicrobial sheet ................................................................. 58

2.6 Antimicrobial effectiveness of ICs, pellets and sheets. ....................................... 59

2.7 Characterization of the antimicrobial sheet ......................................................... 61

2.8 Statistical analysis. ............................................................................................... 63

3. Results and discussion ............................................................................................... 64
3.1 Antimicrobial effectiveness of ICs made of B-CD and trans-2-hexenal .............. 64

3.2 Antimicrobial effectiveness of pellets and sheets made of PLA and B-CD-trans-
2-hexenal .................................................................................................................... 70

3.3 Physical, mechanical, and barrier characterization of the antimicrobial sheet....75

4. Conclusions ................................................................................................................ 77
CHAPTER 5. CONCLUSIONS ........................................................................................ 79
CHAPTER 6. REFERENCES ........................................................................................... 81

 

LIST OF TABLES
Table 1. Main characteristics of trans-2-hexenal ............................................................... 10
Table 2. Physicochemical characteristics of cyclodextrins ............................................... 18

Table 3. Sample codes for neat PLA and PLA/B-CDs blends (produced with or without

masterbatch (MB)) ............................................................................................................. 27
Table 4. Tensile strength, modulus of elasticity and elongation at break of the neat PLA
and the PLA/B-CDS blends ................................................................................................ 33
Table 5. Glass transition temperatures and melting temperatures of the neat PLA and the
PLA/B-CDS blends ............................................................................................................. 35
Table 6. AE of the PLA-BCDS blends with respect to PLA as reference .......................... 44
Table 7. Characterization of the neat PLA sheet and the antimicrobial sheet ................... 77

vi

LIST OF FIGURES

Figure 1. Chemical structure of trans-2-hexenal ................................................................. 9
Figure 2. Chemical structure of PLA ................................................................................. 16
Figure 3. Chemical structure of B-cyclodextrin ................................................................. 18
Figure 4. Preparation steps of the four different types of sheets: plain PLA (steps 1, 2, 3,
7, 8), PLA-B—CDs (steps 1, 2, 3, 7, 8), PLA-B-CDs-MB (steps 1, 2, 3, 4, 5, 6, 7, 8) ........ 25
Figure 5. DSC thermograms of the neat PLA and the PLA/B-CDs blends (lSt cycle) ...... 36
Figure 6. Percent of crystallinity (%) of the neat PLA and the PLA/B-CDS blends (a, and
b indicate significant differences between samples) .......................................................... 36
Figure 7. Weight change (%) as a function of the temperature (°C) of the neat PLA and
the PLA/B-CDS blends ....................................................................................................... 38
Figure 8. Derivative weight (%/°C) as a function of the change of temperature (°C) of the
neat PLA and the PLA/B-CDs blends ................................................................................ 38
Figure 9. Storage modulus changes of neat PLA and PLA/B-CDs blends ........................ 40
Figure 10. Tan delta changes of neat PLA and PLA/B-CDs blends .................................. 40

Figure 11. Lightness of neat PLA and PLA/B-CDS blends (a,b and c indicate significant
differences between samples) ............................................................................................ 43

Figure 12. a* and b“ values of neat PLA and PLA/B-CDs blends (a, b, c, d and A, B, and
C indicates significant differences between samples) ....................................................... 44

Figure 13. Scanning electron micrographics of the surface of fractured neat PLA and
PLA/B-CDS pellets. Panel (a) shows neat PLA while panels (b), (c), (d) show PLA-B-
CD15%, PLA-[3-CD15%-MB, PLA-B-CD3O (arrows pointing to the conglomerates) ..... 46

Figure 14. Oxygen permeability coefficient of the neat PLA and of the PLA/B-CDs
blends at 23°C and 0%RH (a,b,c, and d indicate significant differences between samples)
........................................................................................................................................... 47
Figure 15. Water permeability coefficient of the neat PLA and of the PLA/B-CDs blends
at 23°C and 37.8°C, and 100%RH (Not data for PLA-BCD515%, PLA-BCDs30% at
378°C) (a, b, c, d and A B indicate statistical difference among samples) ....................... 47

Figure 16. Chemical structures: a. trans-Z-hexenal, b. PLA, and c. B-CD ........................ 55

vii

Figure 17. Synthesis and characterization of the ICs ........................................................ 57
Figure 18. Processing and characterization of pellets and antimicrobial sheets ................ 59

Figure 19. Left: (-o-) Inhibition of the growth of the postharvest decay fungi Alternaria
solani, Aspergillus niger, Boh'ytis cinerea, Colletotrichum acutatum and Penicillium Sp.
during exposure to trans-2-hexenal released from ICs over a seven day storage period at
23°C. (4-) Growth rate of the same fungi without exposure to trans-2-hexenal over a
seven day storage period at 23°C (controls). (-A-) Inhibition of the growth of the same
fungi after those being transferred to trans-2-hexenal-free atmosphere bioassay systems in
new media over a seven day storage period at 23°C. (-A-) Growth rate of the controls
after those being transferred to an trans-2-hexenal-free atmosphere bioassay systems in
new media over a seven day storage period at 23°C. Right: (-A-) Concentration of trans-
2-hexenal released from ICs over a seven day storage period at 23°C. ............................. 67

Figure 20. Scanning electron micrographics of the surface of fractured B-CDs and ICs.
Panel (a) shows B-CDs, (b) shows B-CDs after simulating the IC synthesis but without
using trans-2-hexenal, (c) shows ICs after complexation with trans-2-hexenal, (d) shows
ICs after release of trans-2-hexenal (24 hrs exposed to 100% RH). ................................. 70

Figure 21. Colony diameter of Alternaria Solani cultures in the presence (-A-) and
absence (-o-) of trans-2-hexenal released from antimicrobial pellets (a) and sheets (b)
during a 7 days storage period at 23°C . ............................................................................ 71

Figure 22. Alternaria Solani in the absence (a) and presence (b) of antimicrobial pellets
afier 7 days at 23°C. ........................................................................................................... 73

Figure 23. Evolution of trans-2-hexenal released from pellets (-O-) and sheets (-D-) in the

headspace of the bioassay systems containing Alternaria Solani cultures during 7 days of
storage at 23°C. .................................................................................................................. 74

viii

1'

ASTM
AN OVA
B-CDS
BOPP
CD
CDs
DMA
DSC
EVA
GF SE
HDPE
HSD
ICs
LDPE

LLDPE

OTR
PE

PET
PLA

PLGA

ABBREVIATIONS

American Society of Testing and Materials
univariate analysis of variance
B-cyclodextrins

biaxially oriented poly(propylene)
cyclodextrin

cyclodextrins

dynamic mechanical analysis
differential scanning calorimeter
ethylene vinyl acetate

grapefruit seed extract

high density poly(ethylene)
honestly significant difference
inclusion complexes

low density poly(ethylene)

linear low density poly(ethylene)
masterbatch

oxygen transmission rate
poly(ethylene)

poly(ethylene terephthalate)
poly(lactic acid)

poly(lactic-co-glycolide)

ix

PP

PS

PVC

PVOH

SEM

TGA

TPS

WVPC

WVTR

poly(propylene)

poly(styrene)

poly(vinyl chloride)

poly(vinyl alcohol)

relative humidity

scanning electron micrOSCOpy
thermogravirnetric analysis
thermoplastic starch

water vapor permeability coefficient

water vapor transmission rate

CHAPTER 1. INTRODUCTION

1.1 Introduction and motivation

Active packaging is a technology that promotes product quality and safety by adding
specific compounds to the package. Many different types of active packaging have been
proposed and these include the control of gases (oxygen, carbon dioxide, ethylene, etc.),
moisture, and microbial contamination [1].

Antimicrobial packaging is a type of active packaging that retards/inhibits mold or
bacteria growth, generally by contacting or releasing active compounds with
antimicrobial properties [2]. Since the use of antimicrobial packaging offers some
advantages compared with the direct addition of antimicrobial agents to food products as
the preservative agents are applied to the packaging material in a way than only low
levels of preservative come into contact with the food [3], the food and packaging
industry has shown a growing interest in antimicrobial packaging in the last years [4].
The number of recently published articles and patents suggest that research on the
incorporation of antimicrobials into packaging for food applications has more than
doubled in the past 5 years [2]. There are many ways to make packages antimicrobial, but
just two basic mechanisms: (1) placing the antimicrobial compound inside the package
along with the product to be packed (e.g.: sachets and labels), and (2) making the
antimicrobial compound part of the materials that form the package itself. The latter can
be achieved by entrapping antimicrobial agents into the polymeric matrix, or by polymer

surface modification, or by coating [5].

Thermal processing methods such as extrusion can be used to incorporate antimicrobials
into polymers [2]. The choice of the antimicrobial is often limited by the incompatibility
of the component with the packaging material or by the heat liability of the component
during extrusion [6-7]. For example, thermally unstable antimicrobials such as volatiles
cannot be directly extruded. They need to be processed in a protected state.
Microencapsulation is a relatively new technology that is used for protection,
stabilization and slow release of food ingredients. The encapsulating material generally
consist of starch derivatives, proteins, gums, lipids or any combination thereof [8].
Cyclodextrins (CDs) are cyclic oligosaccharides produced by enzymatic fermentation of
starch that have successfully been used for pharmaceutical formulation and delivery [9-
10]. The most notable feature of cyclodextrins is their ability to form inclusion complexes
(host-guest complexes) (ICs) in solution with a very wide range of solid, liquid and
gaseous compounds by a phenomenon of molecular complexation. Among the different
types of CDs, B-cyclodextrins (B-CDs) are the most accessible, the lowest priced and
generally useful. [9]. B-CDs have been used for encapsulation and controlled release of
naturally occurring volatile compounds such as hexanal, and acetaldehyde in order to
reduce fungal growth [11-12]. In this study, B-CDs have been chosen to develop
antimicrobial packaging materials due to their capability to: (1) carry antimicrobial
volatiles by forming ICs, (2) release these depending on the surrounding conditions
(temperature and relative hmnidity) [13], and (3) protect the antimicrobial volatile from
the high temperatures achieved during polymer processing.

Antimicrobial compounds have been incorporated into paper, thermoplastics and

thennosets, and have been tested against a wide variety of bacteria and molds [2]. Most

 

of these studies have been done using commercial petroleum-based polymeric matrixes
such as linear low density poly(ethylene) (LLDPE), ethylene vinyl acetate (EVA), nylon,
etc. Bio-based polymers have the potential to become viable alternatives to the
petroleum-based ones for active packaging applications. Bio-based polymers contain raw
materials originating from agricultural and marine sources and they can be directly
extracted from biomass like starch, cellulose, or synthesized from bio-derived monomers,
or produced directly by natural or genetically modified microorganisms [l4]. Bio-based
polymers with antimicrobial activity have been developed by using chitosan, nisin,.starch,
etc [15-17]. Poly(lactic acid), PLA, is a biocompatible and biodegradable semi-crystalline
polyester with promising further developments because its mechanical and barrier
properties are comparable to those of conventional polymers [18-19]. In this study, ICs
based on B-CDs carrying trans-2-hexenal, a naturally occurring antimicrobial volatile
found in plant tissue and products, have been blended with PLA to develop a bio-based
antimicrobial polymeric material. The antimicrobial capacity is based on the release of
the antimicrobial volatile in function of temperature and relative humidity during storage.

In the development of polymer blends, polymer compatibility is an important factor since
this governs the morphology of the blends and therefore, affects the mechanical and
physical properties of these [20]. Blending starch with PLA reduces the cost and
increases the biodegradability of the blend, however, the mechanical strength of the blend
is reduced due to the poor interfacial adhesion between materials [18]. The resulting
blend is brittle and rigid, both major drawbacks in many fields of application. Similar
results were expected for blend of CDs and PLA. In this study, the interfacial adhesion of

PLA and B-CDs at different weight ratios was investigated and the approach of using a

masterbatch (PLA with high [fl-CD content) to improve the adhesion was studied. In
addition, it was studied whether the exposure to relative humidity and to high heat during
processing causes a premature loss of the antimicrobial compounds encapsulated in the B-
CD molecules for later release, as this would reduce the antimicrobial properties of the

active material.

1.2 Objectives

The main objective of this study was to improve active packaging materials based on
poly(lactic acid) carrying encapsulated antimicrobial volatiles by enhancing the
dispersion of the active compound and the compatibility between phases.
Specific objectives were to:
characterize the effect of the B—CDs content on the mechanical, thermal, barrier,
morphological, and optical properties of the PLA/B-CDs blends,
study the effect of the masterbatch on the mechanical, thermal, barrier,
morphological, and optical properties of blends made from PLA and B-CDs,
reduce the stiffness and brittleness of the polymer material,
improve the dispersion of the ICs,
develop an antimicrobial polymer material based on bio-based materials, and to
verify its antimicrobial capacity,
study the effects of processing on the antimicrobial activity of the films, and to
quantify this,

enhance the commercial applicability of this new polymeric material.

CHAPTER 2. LITERATURE REVIEW

2.1 Active packaging

There is a need of new food conservation technologies to ensure quality and safety of
packaged food. Modified atmosphere packaging (MAP), which is the most commonly
used packaging technology to prolong food shelf life by surrounding the food product
with and optimal atmosphere, can be adversely affected by changes in temperature and
relative humidity throughout the cold chain. These changes can negatively affect the
quality and safety of the packaged food. Active packaging transforms the concept of
packaging from being a mere container or ‘passive package’ to an ‘active package’ that
plays an active role in maintaining or even improving the quality of the packed product [1,
21]. Active packaging can be described as a new-food conservation technology based on
the interactions within a food/package/environment system to improve the quality and

safety of the packaged food and to increase its shelf life [1, 5].

2.1.1 Active packaging market

Active packaging technologies have received a great deal of attention during the last
decade. Changes in retail and distribution practices such as centralization of activities,
new trends (e. g. intemet-shopping), and globalization of markets, all resulting in
increased distribution distances and longer storage times of different products with

different temperature requirements, are putting huge demands on the packaging industry.

In addition, consumers are increasingly demanding mildly preserved convenience foods
that have better fresh—like qualities [22-23]. Thus, active packaging is gaining interest
fiom researchers and industry due to its potential to satisfy these needs. The number of
packaging products that are developed and used in commercial applications is growing.
Active packaging comprised approximately 27% of the global market in 2008 [24]. In
2010, the US market for “active, controlled and smart” packaging for food and beverages

is an estimated $38 billion and is expected to surpass $54 billion by 2015 [25].

2.1.2 Types of active packaging

A variety of active packaging technologies have been developed to provide better quality,
wholesome and safe foods, and also to limit package related environmental pollution and
disposal problems [26]. Different types of active packaging include oxygen scavengers,
carbon dioxide emitters/ absorbers, moisture regulators, ethylene absorbers, ethanol
emitters, flavor releasing/absorbing systems, as well as antioxidant release, antimicrobial

containing, and gas permeable/breathable films [21-22, 24, 26].

2.2 Antimicrobial packaging

Antimicrobial packaging is a system that can kill or inhibit the growth of micro-
organisms to extend the shelf life and to enhance the safety of packed products [3, 27]..
Research on antimicrobial packaging started with the development of packaging

materials containing chemicals with antimicrobial capacity in their macromolecular

structures using common packaging materials [27]. In recent years, a lot of research is
being devoted to the development of different types of antimicrobial delivery systems and

packaging material-product combinations to maximize the efficacy of the system [28].
2.2.1 Antimicrobials

There is a wide variety of substances with antimicrobial activity like organic acids, spice
extracts, enzymes, proteins, antibiotics and metals [29]. They can be classified into three
groups: chemical agents, natural agents, and probiotics [30]. Chemical agents are the
most common substances used in the industry and some examples of these include
organic aids, and alcohols [27]. They offer additional benefits in preventing post-process
contamination and in strengthening inhibitory effects against microorganisms. Natural
agents are mostly extracted from plant and animal sources and they have been proposed
and/or tested for antimicrobial activity in packaging [31]. Probiotics are naturally
produced antimicrobials that can inhibit the growth of other bacteria. They can
effectively control competitive undesirable microorganisms [30]. The effectiveness of

antimicrobial agents can differ as follows [30]:

Microbiocidal effect

The antimicrobial agent is expected to kill the target spoilage and pathogenic
microorganism. This type of antimicrobial agent can eliminate micro-organisms from a
food/packaging system. The specific nomenclature is bactericidal and fungicidal for those

that kill bacteria and fungi, respectively.

Microbiostatic effect

The antimicrobial agent, applied above a certain critical concentration, can inhibit the
growth of microorganisms. However, at concentrations below the critical one, or if the
agent is removed from the packaging systems through a seal defect, leakage, Opening or
any other means, the suppressed microorganisms can grow or their spores germinate. The
specific nomenclature is bacteriostatic and fungistatic for those that kill bacteria and

fungi, respectively.

2.2.1.1 Naturally occurring antimicrobials

Natural antimicrobial compounds have been used for centuries in food preservation.
Research concerning plant-origin compounds has increased since the 1990s [32]. The
mechanism of natural antimicrobials is to interrupt metabolic pathways in the cell wall
membrane/structure. For example, nisin interacts with the sulfiIr containing compounds
in the bacterial membrane, disrupting their function as a semipermeable membrane and
causing lysing of the cells. Antimicrobials also can reduce the water activity within the
cell. Since water is a key compound in the metabolism of a cell, any disruption of the

fluid flow causes growth suppression [31].

2.2. 1.1 .1 Trans-2-hexenal

Trans-Z-hexenal (Figure 1) is a naturally occurring compound of plant origin that plays
an important role in the aroma of many fruits and vegetables including pome fruit, stone

fruit, strawberries, grapes and vegetables [33-34].

\

CH3 H

Figure 1. Chemical structure of trans-Z-hexenal [35]

The antimicrobial effectiveness of trans—2-hexenal has been widely studied in vitro [36-
39]. The antifimgal activity of trans-2-hexenal vapor in vivo has been confirmed against
Pichia subpelliculosa in sliced apples [40], Penicillium expansum in pears and apples [37,
4142] Boaytis cinerea in strawberries [38], and Escherichia coli, Salmonella enteritidis
and Listeria monocytogenes in sliced apples [43].

Table 1 summarizes the main characteristics of the compound trans-2-hexenal. The vapor
pressure of trans-2-hexenal is 10mmHg at 20°C (www.sigmaaldrich.com) and the
antimicrobial activity of Trans-2-hexenal may be due to its high reactivity and volatility
[37]. The precise mechanism of the protective action of trans-2-hexenal is not clear yet,
but likely they permeate by passive diffusion across the plasma membrane, and once
inside the cell, the a, B-unsaturated aldehyde moiety reacts with biologically important
nucleophilic groups which play a key role in living cells [44]. The combination of its
antimicrobial capacity and the fact that it is approved as food additive permitted for direct
addition to food for human consumption by the US Food and Drug Administration [45]
makes trans-2-hexenal particularly interesting as an antimicrobial compound for food

9

applications. According to US Food and Drug Administration, toxic LDso of trans-2-

hexenal is 780 mg/kg [46].

Table 1. Main characteristics of trans-2-hexenal [35]

 

 

 

 

 

Linear structure C6H100
Boiling point 146-149°C
Flash point 35°C
Density 0.846g/mL at 25°C
Molecular weight 98.14 g/mol

 

2.2.2 Antimicrobial polymers

Antimicrobial polymers are one of the most promising concepts for active packaging.
Antimicrobial polymers are mainly classified into the following types: (1) those where
the antimicrobial agent is directly incorporated into the packaging material and (2)
coatings which act as a carrier for the antimicrobial agent. The antimicrobial agent is
released from the polymer into the product to inhibit microbial growth [47]. Some
antimicrobial agents can migrate from the surface of the package material to the product
and others are effective without migration of the active agents to the product [2, 26, 31].

Sachets and pads are the main trend to deliver antimicrobials due to their high
effectiveness in suppressing surface microbial growth [26]. However, they are not
broadly accepted by consumers due to fears of ingestion by children and accidental
consumption with package contents. [2]. The development and use of antimicrobial
polymers in the form of thin films is expected to increase in the next decade [26].There

are promising alternatives such as immobilization of antimicrobials by ion or covalent

10

linkages to polymers and polymers that are inherently antimicrobial polymers are more
effective than the direct addition of the antimicrobial agent since they slow the migration
of the agents away fiom the surface, thus helping to maintain high concentrations where

these are needed [48].

2.2.2.1 Antimicrobial polymer market

Research in the use of antimicrobial packaging materials as an alternative method for
controlling microbial contamination has significantly increased during the last few years
[3, 49]. The global market demand for specialty antimicrobials in plastics was $130
million at manufacturing level and $231 million at end-user level in 2001. The overall
grth of the industry was around 3.5-4% per year. The expected grth of the global
plastic antimicrobial industry was 4% per year up to 2005 [50]. In 2006, Japan was the
leader in the commercialization of antimicrobial films. The commercialization of these
films was limited outside of Asia because of their strong odor due to the

allylisothiocyanate content [51].

2.2.2.2 Antimicrobial polymers and processing

Polymer processing can be defined as a group of operations carried out on polymeric
materials to shape them into useful products [52]. Classic polymer technological
processes (such as extrusion) are generally used to process antimicrobial polymers [53].

Thermally stable antimicrobials such as silver substituted zeolites are used with thermal

ll

processing methods such as extrusion and injection molding [2]. However, other
antimicrobial compounds are thermally instable and therefore, these active agents may
partially or completely be lost, or may partially or completely lose its antimicrobial
activity if incorporated into the polymeric matrix due to the high pressure and
temperature conditions in the extruder [7, 53]. In addition, the antimicrobial activity of an
incorporated active substance in a polymeric matrix may deteriorate during converting,
storage, and/or distribution of the active packaging material. The residual antimicrobial
activity is defined as the effective activity of the antimicrobial agent after the casting
(extrusion or coating) and the converting processes [31]. Besides chemical degradation,
the loss of the antimicrobial substances by volatilization is another reason for
antimicrobial activity reduction during casting and storage [7]. For example, the volatility
of antimicrobial compounds like naturally occurring plant volatiles cases them to be lost
when exposed to high temperatures. The treatment of the polymer or the derivatization of
active agents prior to their addition to the polymer may be necessary to increase the

compatibility between the active agent and the polymer [26].

2.2.2.2.] Extrusion

Extrusion is the process of shaping a molten polymeric material by forcing it though a die
[54] using a piece of equipment called an extruder. The extruder takes the raw material
and compresses, heats, melts, and conveys it [55]. The parts of an extruder are hopper,
screw, barrel, and die. The screw performs a number of functions such as conveying,

melting and mixing. L/D ratio indicates the relative length of the screw and 24:1 is the

12

most common value. This could go up to 32:1 for better mixing or more output.

Depending on the number of screws, extruders are divided into single, and twin screw

extruders [54]. The barrel is the reservoir that hosts the extruder. The barrel of any

extruder has three sections: feed, compression, and metering. The feed section is the first

part of the barrel that conveys the pellets and where the melting process starts; the

compression section is where the material gets fully {melted and mixed, and the metering

section is the area where the molten material is conveyed. The die is the part of the

extruder where the molten material is shaped and this varies in size, shape and format

[55].

Most plastic resins are processed through the extrusion process. This process is

accountable for significant quantities of plastic products, such as films, sheets and

profiles, and it is also used to produce the plastic pellets that are later used by other

plastics manufacturing processes [56].

There are different types of extrusion. Blow and cast extrusion are the most commonly

used to process sheets and films. The major difference between blown and cast films is

the method by which the film is formed, which is dictated by the shape of the die. [56].

Blow film extrusion involves extruding a polymer through a ring-shaped die, through
either the bottom or the side. The melt is forced through the die opening and forms a
comparatively thick-walled tube [56]. Cast extrusion is widely used to produce polymer
fihns. In cast extrusion, a molten polymer is extruded through a flat die, then stretched in
air and cooled on a chill roll [57]. Cast extrusion allows a polymer to be manufactured

into a film with better tolerance control than can be achieved in blow film extrusion.

13

2.2.2.2.1.1 Factors affecting extrusion

The control of extrusion factors such as temperature, time, and pressure results in a
higher-quality, and a more efficient extrusion of the plastic material. Temperature is a
difficult factor to control because of the various heating elements throughout the extruder.
Time is also critical since this parameter determines the effect of processing on the
resulting extrudate [5 8]. Extrusion conditions can be tailored to produce extrudates with

the desired properties [59].

2.2.2.3 Antimicrobial polymers and materials

2.2.2.3.1 Petroleum-based antimicrobial polymers

Most antimicrobial films have been reported in the literature as made from synthetic
polymers such as poly(ethylene) (PE), low density poly(ethylene) (LDPE), ethylene vinyl
acetate/linear low density poly(ethylene) (EVA/LLDPE), poly(vinyl chloride) (PVC),
poly(vinyl alcohol) (PVOH), nylon, or biaxially oriented poly(propylene) (BOPP) [60].
Most research has been carried out with LDPE films with incorporated antimicrobial
components such as silver-substituted zeolite [61], imazalil [62], sorbate [7], nisin [63],
chitosan [47], grapefruit seed extract (GFSE) [64-65], poly(propylene) (PP) with essential
oils [66], poly(vinyl alcohol) (PVOH) with iysozyme [67], and poly(styrene) (PS), and

poly(ethylene terephthalate) (PET) with sodium benzoate [29].

14

2.2.2.3.2 Bio-based antimicrobial polymers

Bio-based polymers are defined as polymers that contains natural raw materials which
could be extracted directly from natural raw materials such as starch, and protein, or
produced by chemical synthesis from bioderived monomers, or produced by micro-
organisms [68]. The desire for natural ingredients and the realization that plants harbor
compounds of an antimicrobial nature has led to the production of a number of films with
extracts from plants [60]. Currently, incorporating food preservatives [69] and natural
antimicrobial agents into bio-based polymers is becoming a popular area of packaging
research [31]. Bio-based polymers are being widely studied as edible coatings or film
materials. Antimicrobial polymers have been developed by blending polylactide-co-
polycaprolactone with encapsulated allyl isothiocyanate [70], and by blending l-
polylactide and l-polylactide-polycaprolactone with nanoclays [71]. Other bio-based
materials with a potential for developing antimicrobial polymers are chitosan [15] and

starch [72]

2.2.2.3.2.1 Poly(lactic acid), PLA

Poly(lactic acid), PLA (Figure 2), is a linear aliphatic thermoplastic polyester obtained
from the fermentation of sugar feed stocks [73] and produced by controlled
depolymerization of the lactic acid monomer [74]. PLA is biodegradable and
compostable. It breaks down to biomass and C02 and water in a given time period and

environment under the action of biological enzymes [75]. The use of PLA may be a

15

promising way to reduce the solid waste disposal problem [76-77] so it is attractive as

plastic substitute.

 

 

r 0 CH3 1
o
X /
o
I. _ n
CH3 0

Figure 2. Chemical structure of PLA [76]

The stereochemistry of PLA is complex because of the chiral nature of the lactic acid
monomer. The L/D ratio of the lactic acid unit varies PLA properties such as melting
temperature and crystallinity [18, 74].

PLA has comparable mechanical and barrier performance to that of some petroleum-
based polyester. It has also a good processability with good shaping and molding
capability which are desirable for a number of applications [18, 78]. The use of PLA has
been limited because of its high cost, and low availability [78], however, the increase of
the oil price and the implementation of environmental policies such as green tax might
expand the use of this polymer [78]. Main drawbacks of PLA are rigidity, brittleness, and

a low glass transition temperature [18, 79].
2.2.2.321 . 1. PLA blends and masterbatches

Blending PLA with other polymers (biodegradable and non-biodegradable) or with
additives is probably the most extensively used methodology to improve PLA mechanical

l6

properties such as brittleness [80]. Polymers blends are produced when the physical,
chemical, or rheological properties required for processing or for application cannot be
achieved using a single polymer. Blends are also produced to reduce cost. The blending
of polymers is a complicated process due to possible incompatibilities between them.
Starch has been used as filler for environmentally friendly plastics because it is
inexpensive and renewable [81]. PLA has been blended with starch mostly because PLA
is still more expensive than conventional plastics and the degradation rate of PLA is not
completely satisfactory in some instances [82]. In previous studies, as a low-cost
alternative, native starch has been added to PLA with poly(ethylene glycol) [83] and
thermoplastic starch to PLA with glycerol [81] to create a low-cost alternative material
without sacrificing mechanical properties. Starches with different amylase levels [82],
moisture contents [83] and ratios [79] have been blended with PLA to investigate the
water sensitivity of PLA-starch blends. Numerous studies have been done on the
influence of plasticizers [84] and compatabilizers [85] on the thermal, mechanical and
morphological properties of PLA.

A masterbatch can be used to improve the blending process. A therm0plastic masterbatch
is a complex blend based on a thermoplastic resin containing a high amount of additive
which is then blended with a polymer to improve its properties [86]. The addition of a
masterbatch to the PLA has been shown to improve the mechanical properties of the
blend by reducing the brittleness, and increasing the impact strength [87]. Investigations
of the effect of a masterbatch on the PLA properties are relatively scarce, and further
studies on the influence of the masterbatch on the PLA processing conditions and others

are necessary.

17

2.2.2.322 Cyclodrextrins

Starch is a renewable, degradable, carbohydrate biopolymer derived from different
sources like rice, wheat, corn, cassava and potatoes. All these properties allow it to be
considered as a possible substitute for petroleum-based plastics [79]. Starch has been
blended with petroleum-lbio-based polymers to increase their biodegradability, and to
reduce the usage of the petroleum-based or more expensive bio-based polymer. Blends

made fiom PLA-starch [88], and LDPE-starch [89] have been developed and

characterized.

 

 

HO OH J7

Figure 3. Chemical structure of B-cyclodextrin [90]

Table 2. Physicochemical characteristics of cyclodextrins [91]

 

 

 

 

 

 

Attribute u -cyclodextn°n B-cyclodextl‘in Y'CYCIOdeXtrin
Glucopyranose 6 7 8
(units)
Molecular weight 972 1135 1297
(g/mol)
Solubility H20
(g/lOOmL) 14.5 1'85 23.2
Cavrty(21)ameter 4.7-5.3 6.0-6. 5 7.5-8.3
Cavity volume 2 2
(AP 174 6 427

 

18

Cyclodextrins, CDs (Figure 3), produced by fermentation of starch, are cyclic, non-
reducing oligosaccharides built up from six, seven, or eight glucopyranose units. [3-
cyclodextrin, B-CDs (Table 2), the most important and most readily accessible member of
this group, is comprised of seven glucopyranose units [92].

CDs are typical “host” molecules that can trap a great variety of molecules that have the
size of one or two benzene rings, or a side chain of comparable size in the case of even
larger compounds, to form crystalline inclusion complexes [92]. Water molecules filling
the CD cavity are replaced by the “guest” when this is less polar than water and its size
and shape are compatible with that of the host. As long as the complexes do not

dissociate in a highly diluted aqueous solution, the enclosed molecules are protected [92].

222.322.] Micro-encapsulation

Microencapsulation is a process by which a substance or a mixture is coated or entrapped
in another material [93]. There is a lot of research done in a wide range of cores
(encapsulatants), wall-forming materials (encapsulating agents), and technologies for
controlling the interaction of ingredients for manufacturing microcapsules and
microparticles of different size and shape [94]. The retention of flavor compounds is
governed by factors related to the chemical nature of the core, including its molecular
weight, chemical functionality, polarity and relative volatility, to the wall material
properties, and to the nature and the parameters of the encapsulation technology [95].
Inclusion encapsulation generally refers to the supra-molecular association of a ligand

(the ‘encapsulated’ ingredient) into a cavity-bearing substrate (‘shell’ material) by an

19

hydrophobic effect [96]. Different aroma compounds have been encapsulated into CDs
[93, 97]. Some encapsulated aroma compounds have been shown to prevent or inhibit

fungal growth [13].

2.223222 Controlled release

The European Directive 3AQl9a defines controlled release as a “modification of the rate
or place at which an active substance is released [94]. The aim of controlled release
systems is to transfer the antimicrobial agent to the packed food at a specific rate in order
to maintain a predetermined concentration of the active compound in the release medium
for a predetermined period of time [98]. For matrix systems encapsulating volatile
compounds, the release depends on several mutually dependent processes, such as
diffusion of the volatile compound through the matrix, type and geometry of the particle,
transfer fi'om the matrix to the environment and degradation/dissolution of the matrix
material [99]. CDs are used to provide a controlled release of volatiles over the last years.
The release can be affected by the surrounding relative humidity and temperature.

PLA has been investigated as a biodegradable polymeric device for controlled drug
release /delivery. Drug release from PLA and poly(lactic-co-glycolide) (PLGA)
microspheres has been examined at various temperatures in order to clarify the effect of
temperature on mechanisms drug release [100-101]. PLA blends such as PLA-

poly(ethylene glycol) has been also investigated as a biodegradable drug delivery system

[102].

20

 

CHAPTER 3. PREPARATION AND CHARACTERIZATION OF BLENDS
MADE OF POLY(LACTIC ACID) AND AN ENZYMATICALLY MODIFIED
STARCH WITH A CAPACITY TO CARRY HYDROPHOBIC MOLECULES:

IMPROVEMENT OF THE BLEND PROPERTIES BY USING A
MASTERBATCH*
Joo, M.J., Auras, R., Narayan, R., Almenar, E.

*to be submitted for publication to MACROMOLECULES

ABSTRACT: Cyclodextrins (CDs) are enzymatically modified starch with a hydrophilic
exterior and a hydrophobic cavity, which enables them to form inclusion complexes with
a variety of hydrophobic molecules. The incorporation of CDs in a polymer matrix
enables new applications for polymers in the food and pharmaceutical packaging area. In
this study, B-CDs, one of the most common CDs, were mixed with poly(lactic acid) (PLA)
with the goal of creating a biodegradable and bio-based polymer with the capacity to
carry hydrophobic molecules. The materials were extruded, pelletized, and cast into
sheets. The effect of different B-CD contents (0, 15, or 30%), and of the use of a
masterbatch (MB) on the morphology and the physical pr0perties of the PLA/CD3 blends
was investigated. The MB consisted of pellets containing 30% B-CD content and was
used in an attempt to improve the dispersion of the B-CDs into the PLA matrix. Testing
of mechanical, thermal, and barrier properties revealed that B-CDs and PLA are
incompatible. Thermal stability, tensile strength, modulus of elasticity, elongation at
break, and oxygen and water-barrier capacity of the PLA decreased with the B-CD

content. In all cases, the decrease was notably reduced when the B-CDs were added as a

21

masterbatch and this showed that the use of the masterbatch increased the compatibility
of the PLA and the B-CDs. Also the crystalline content and color change increased with
the B-CD content but not in the blend obtained using the masterbatch. In SEM images the
blends showed an uneven distribution of the B-CDS in the polymeric matrix.

KEYWORDS: Poly(lactic acid), B-cyclodexuins, masterbatch, preparation,

characterization

INTRODUCTION

Public concern about the environment have stimulated interests in biodegradable
polymers as alternatives to conventional polymers [79]. Poly(lactic acid) (PLA) is one of
the more widely studied biodegradable polymers, because many of its properties are
equivalent or superior to those of some petroleum-based plastics, which makes this
polymer suitable for a variety of applications [80].

PLA is a linear aliphatic thermoplastic polyester derived from lactic acid and it is mainly
synthesized from the commercial fermentation of sugar feedstock [73] which means it is
a compostable polymer at specific conditions [103]. PLA has a transparent and glossy
finish [104] and heat seal capacity [104]. PLA in its semicrystalline form is used in
applications where high mechanical strength and toughness are required [87]. The C02,
02, and N2, permeability coefficients of PLA are reasonable: they are lower than those for
poly(styrene) (PS), but higher than those for poly(ethylene terephthalate) (PET) [76].
PLA is suitable for a wide range of processing technologies including injection molding,

blow molding, thermoforrning, cast film and sheet, extrusion blow film, foaming, and

22

others [78]. The toxicity of PLA is low, which makes it an adequate material for food
packaging applications [105]. All these pr0perties make PLA more attractive than other
bio-based materials as a biodegradable plastic substitute. However, PLA is more
expensive than commonly used plastics [78]. PLA has a relatively low glass transition
temperature and any further reduction thereof limits the application of the polymer
further [106] and its impact strength and elongation at break are smaller than those of
widely used polymers such as high density poly(ethylene), poly(propylene), and
poly(styrene) [104]. Based on some of these properties, PLA offers vast potential for the
development of biodegradable packaging systems that carry active compounds for food
and pharmaceutical applications. While some aspects of the potential for active
packaging have been explored [107], further research and development is needed.
Cyclodextrins (CDs) are cyclic oligosaccharides composed of B-(1,4) linked
glucopyranose subunits. They are produced by enzymatic degradation of starch by
bacteria. CDs have a cage-like supramolecular structure that allows them to interact with
molecules, ions, and radicals [9]. CDs also enable the controlled release of these
molecules as shown by [1 1-13]. Therefore, the blending of this modified starch with
other polymers might be a new approach to increase the number of food and
pharmaceutical applications for these polymers.

Generally, CDs are used to synthesize copolymers with PLA for drug delivery or for
improving the miscibility of PLA with others [108-109], however, information on
blending this modified starch with PLA is scarce. In contrast, PLA/starch blends have
been widely studied since blending starch with PLA is a promising approach [110]:

starch is abundant and cheap, and PLA has good mechanical properties and can reduce

23

the sensitivity of starch to moisture [82, 111]. In addition, the resulting blend is
biodegradable and is derived from renewable sources. However, hydrophobic PLA and
hydrophilic starch are thermodynamically immiscible because of the hydroxyl and
carboxyl end groups in the PLA and the numerous hydroxyl groups in the starch [18].
Starch remains in a separate conglomerate form in a PLA matrix [112]. Due to this weak
interfacial adhesion between phases, the mechanical strength of PLA/starch blends is low
and the material is weak and brittle [18, 79]. Numerous studies have been undertaken to

improve the interfacial interactions of these blends through the use of compatibilizers and

other additives [1 13-1 14].

Masterbatch is another way to improve properties in a polymer blend and it has been

broadly used with polymers like polypropylene [115], poly(ethylene terephthalate) [116],

and others. Four commercial masterbatches have been blended with PLA and some

properties of PLA such as clarity and impact resistance have been improved by the

addition of the masterbatch. However, the four masterbatches had a slight negative effect

on the tensile strength of PLA [87]. PLA and poly(butylenes adipate-co-butylene

terephthalate) has been blended by using masterbatches as muticompatibilizer to enhance

compatibility [1 17].

In this study, B-CDs were mixed with PLA in order to create a bio-based polymer with a

capacity of carrying hydrophobic molecules. Different blends were created with the

objective to achieve specific prOperties that make these blends fit for a number of
packaging applications. Due to the expected weak interfacial adhesion between phases
(PLA and modified starch), a masterbatch was tested in an attempt to improve the

dispersion of the B-CDs into the PLA matrix and thus to enhance the compatibility and

24

mechanical properties of the blend. The effect of the B-CD content (0, 15, or 30%), and

the use of a masterbatch (MB) on the morphology and on the physical properties of the

blends PLA-B-CDS has been investigated.

EXPERIMENTAL

Materials

Poly(lactic acid) 40501) (PLA 405013) resin (95% L-lactide, Density=1.24g/cm3) was

kindly provided by Wilkinson Inc. (Calhoun, NE, US). B-cyclodextrin (B-CD) (purity

>99%, Density=0.5g/cm3, and Food grade) was purchased from Wacker Chemical

Corporation (Adrian, MI, US).

Formulation and preparation of pellets

Step 1/4. Premix and blend

PLA

%

BCDs

 

15% w/ MB

 

 

 

 

 

 

15%

 

 

30%

 

 

 

 

 

 

A

\

Step 2/5. Pelletizer Step 3/6. Dry
Pellets

I PET-3% % :> Step8. Dry
3

@
a

Q Sheets
I—P o 53

Step 7. Cast process

 

 

 

 

 

 

Figure 4. Preparation steps of the four different types of sheets: plain PLA (steps 1, 2, 3,

7, 8), PLA-B-CDs (steps 1, 2, 3, 7, 8), PLA-B-CDs-MB (steps 1, 2, 3, 4, 5, 6, 7, 8)

25

PLA 4050D resin and fi-CDs were dried in a vacuum oven (1430 VMR International,
Cornelius, OR, US) at 65°C for 4 and 24 hrs, respectively . Quantities of PLA resin and
B-CDs according to the calculated compositions (Table 3) were premixed and
subsequently blended (Figure 4. Step 1). The premix was extruded using a pilot scale
extruder (Century extruders CX-30, Traverse city, MI, US) with 30 mm diameter twin
screws with a L/D ratio of 42: l , and ten separate barrel zones temperature controlled with
a water system and set from feeder to die to temperatures of 27 °C, 81 °C, 129 °C, 144 °C,
150 °C, 161 °C, 161 °C, 160 °C, 161 °C, and 161 °C. The screw speed was 100 rpm and
the level of torque was controlled at 70~75%. The blend was collected from the die and
allowed to cool down prior to being granulated into pellets using a pelletizer (Scheer bay
Co. N. Euclid, Bay city, MI, US) ((Figure 4. Step 2). The pellets containing 0 (PLA), 15
(PLA-B-CD15%), and 30% (PLA-B-CD30%) CDs were stored until use in a desiccator at
23 °C (Figure 4. Step 3).

The masterbatch (MB) consisted of pellets containing 30% B-CD content. Quantities of
PLA and MB pellets according to the calculated compositions (Table 3) were premixed
and subsequently blended (Figure 4. Step 4) using the same equipment and processing as
described above. The blend was collected from the die and allowed to cool down prior to
being granulated into pellets using the same equipment as previously described (Figure 4.
Step 5). The pellets containing 15% B-CDs (PLA-B-CD15%-MB) were stored until use in

a desiccator at 23 °C (Figure 4. Step 6)-

26

 

Table 3. Sample codes for neat PLA and PLA/B-CDS blends (produced with or without

 

 

masterbatch (M3))
PLA B-CDs PLA-B-CD30% o *
Sample Code (wt%) (wt%) (wt%) B-CD content (wt /o)
PLA 100 O O 0
PLA-B-CD15% 85 15 O 15
PLA-B-CD30% 70 3O 0 30
PLA-B-CDI 5%-MB 50 O 50 1 5

 

* nominal values

Preparation of sheets

All four different pellets (Table 3) were first dried in a vacuum oven prior to use to
remove remaining moisture (Figure 4. Step 3/6). The pellets were converted into sheets
by cold cast process (Figure 4. Step 7). A single screw microextruder (Randcastle RCP-
0625 Microextruder, Randcastle extrusion system Inc., Cedar Grove, NJ, US) with a
screw diameter of 15mm and a length to diameter ratio (L/D ratio) of 24:1 was used. The
screw Speed of the extruder was 35 rpm. The temperature profile of the extruder was
controlled by a water cooling system and set from the feeder zone to the die zone at 149°
C, 160° C, 177° C, 177° C and 1770 C. Chill rolls temperature was 52° C. Ambient air was
used to cool down the sheets on top of the roll. All samples were stored in a desiccator at

23° C to prevent moisture absorption (Figure 4. Step 8).

Analyses

Mechanical characterization

27

Neat PLA and PLA/B-CDS Sheets were shaped into specimens with a width of 1 in and a
length of 8 in prior to use. The thickness of the specimens (0.17 mm) was determined by
averaging of three readings taken at different points. The tensile properties: tensile
strength, elongation at break, and modulus of elasticity were measured according to
ASTM D882-02 [”8], using a Instron Universal Testing Machine (Model 5565, Instron,
Norwood, MA, US) with a crosshead speed of 0.5 in/min, and a gage length of 5 in.
Tensile properties of neat PLA and PLA/B-CDS blends were determined from an average

of 10 samples.

Thermal characterization

Thermal transitions of neat PLA and PLA/B-CDS sheets were determined using a TA
Instruments QIOO V 9.8 Differential Scanning Calorimeter (TA instruments, New Castle,
DE, US). The temperature calibration of the equipment was performed in accordance
with ASTM E967-03 [119] and the heat flow calibration was performed in accordance
with ASTM E968-02 [120] Transition glass temperature (Tg), and melting temperature
(Tm) were measured and calculated in accordance with ASTM D3418-03 [121]. The
degree of crystallinity (%Xc) for neat PLA and PLA/B-CDS sheets was calculated as

follows [82]:

AHm+AH,]X100

 

%Xc =

 

28

where AH", , AHC, and X p1,; are the melting enthalpy, crystallization enthalpy, and PLA

content, respectively. A value of 93 J/g for the melting enthalpy for 100% crystalline
PLA was obtained from the literature [122].

An amount between 7-9 g of PLA and PLA/B-CDS sheets was used for each experience.
Samples were heated from 40 to 190 °C at a rate of 10 °C/min. The B-CD content was
taken into consideration when determining %Xc. Three replications of each type of sheet

were tested.

Mass loss or gain due to the decomposition of the neat PLA and PLA/B-CDS sheets was
measured using a therrnogravimetric analyzer (Hi Res TGA 2950, TA instruments, New
Castle, DE, US) under a nitrogen flow of 70 in/min. The samples were scanned with a

constant heating rate of 10°C/min from 40°C to 600°C.

Dynamic mechanical analysis was used to characterize and to compare the viscoelastic
nature of neat PLA and PLA/B-CDS sheets. Storage modulus (E'), loss modulus (E') and
damping coefficient (tan5) of all materials were measured as, a function of temperature in
accordance to ASTM D4065-06 [123] by using a TA Instruments Model Q800 dynamic
mechanical analyzer equipped with tension clamps and operating at a heating rate of
3°C/min (from -20°C to 120°C), and a frequency of 1.0 Hz. All specimens were 40 mm

long, 5 mm wide, and 0.17 mm thick. Three samples for each type of material were tested.

Optical characterization

29

The color of the neat PLA and of the PLA/B-CDS sheets was measured using a
colorimeter (LabScan XE, HunterLab, Reston, VA, US) with a 17-mm-diameter
measuring area. The CIELAB color system was used to characterize the color. The
different sheets were square-shaped (60 x 60 m2) and then placed on the measuring area
where CIE L*, a*, and b* values were measured. The three color coordinates ranged
from L*=0 (black) to L*=100 (white), -a* (greenness) to +a* (redness), and -b*
(blueness) to +b* (yellowness). Total color difference (AB) was calculated by using the

equation below with respect to neat PLA as reference [76].

 

AB = JAL’ +Aa2 +Ab2

Morphological characterization

A scanning electron microscope (J SM-model 6400 SEM, JEOL, Peabody, MA, US) with
an accelerating voltage of 12 kV and a working distance of 25 mm at X500 magnification
was used to assess the morphology of plain PLA and PLA/B-CDS sheets. Prior to analysis,

the samples were fractured, stored in a desiccator, and finally coated with 3 nm of gold.

Barrier characterization

Water vapor transmission rates (WVTR) of neat PLA and PLA/B-CDS sheets were
measured in accordance to ASTM F1249-05 [124] using a Permatran W Model 3/33
Water Permeability Analyzer (MOCON, Minneapolis, MN, US). Three to five sheets of
each type were tested at 23 and 37.8°C, and 100% RH (using wet sponges). Oxygen

transmission rates (OTR) of the sheets were measured in accordance to ASTM D3985-05

3O

[125] using a 8001 Oxygen Permeation Analyzer Cytocon, Minneapolis, MN, US). Three
to five sheets of each type were tested at 23 °C and 0% RH. All samples were masked
with adhesive type aluminum foil (McMaster-carr, Aurora, Ohio, US), leaving an

uncovered test area of 3.14 cmz.

Statistical methods

Statistical analysis of the results was performed using a univariate analysis of variance
(AN OVA). Means were separated using the Tukey honestly significant difference (HSD)
test (p < 0.05) in the analytical software SPSS version 15 (SPSS Inc., Chicago, IL, USA).
The data were analyzed and graphically plotted using Sigrna-plot software version 10

(Systat Software Inc., Richmond, CA).

RESULTS AND DISCUSSION

Mechanical characterization

The mechanical properties tensile strength (machine direction (MD) and cross-machine
direction (CD)), modulus of elasticity, and elongation at break of neat PLA and PLA/B-
CDS blends have been measured and they are listed in Table 4. In general, all mechanical
properties of the PLA changed significantly with the addition of the B-CDs. When the [3-
CDs were directly blended with the PLA (PLA-B-CD15% and PLA-B-CD30%), the
tensile strength, elongation at break, and modulus of elasticity of the PLA decreased by
about a factor of three, two, and two, respectively. This happened because B-CDs acted as

fillers in the PLA continuous matrix and due to the poor interfacial interaction between

31

 

the hydrophilic B-CD and hydrOphobic PLA the continuous matrix became discontinuous.
These perturbations of the otherwise continuous matrix resulted in changes of the
mechanical properties. A similar incompatibility has been reported for blends of PLA and
starch [81]. A reduction of the strength and of the elongation at break in the PLA/starch
blends has been observed regardless of starch type and composition [79, 126]. It has also
been reported that the reduction of the tensile strength and of the elongation of the
PLA/starch blends is related to the nucleating effect of the starch on the PLA matrix
[127]. This could also be applicable to B-CDs since those plays a role as a nucleating
agent for PLA [128].

With increasing starch content in the PLA/starch blends, the PLA matrix become more
discontinuous resulting in decreases of strength and elongation [79, 126]. In contrast,
strength and elongation at break in the PLA/B-CDS blends did not depend on the B-CD
content (15% vs. 30%). The tensile strength of the PLA/B-CDS blends PLA-B-CD15%
and PLA-B-CD30% was 11.55i1.26 MPa vs. 11.31:bl.34 MPa, and 6.60:l:0.78 MPa vs.
3.82:l:1.16 MPA, for MD and CD, respectively. No effect of the CDS content on the
elongation at break of the PLA/B-CD blends observed. All samples ranged around a value
of 2% and 1% for MD and CD, respectively. The tensile strength and elongation at break
of other bio-based blends, like blends of PLA and TPS have been reported to depend on
its composition. In contrast, the modulus of elasticity of the blends was slightly affected
by the amount of B-CDS. This was more evident in the CD results.

The mechanical prOperties of the -blends improved with the use of the masterbatch to
incorporate the B-CDs. This indicated better mixing between PLA and B-CDS. The tensile

strength of the blends was increased by the use of the masterbatch. PLA-B-CD15%-MB

32

showed approximately twice the tensile strength of the blends PLA-B-CD15% and PLA-
B-CD30%. The modulus of elasticity of the blends was also improved with the use of the
masterbatch. In this case, PLA-B-CD15%-MB showed 2-3 times the modulus of elasticity
of the blends PLA-B-CD15% and PLA-B-CD30%. However, no effect of the masterbatch
on the elongation at break of the blends was observed. These results indicate that the

blends were less breakable and more flexible in the presence of the masterbatch.

Table 4. Tensile strength, modulus of elasticity and elongation at break of the neat PLA

and the PLA/B-CDS blends

 

 

 

 

 

 

 

 

 

. . Modulus of
Sample code Tenszfigtgngth Efrgiitzngt 61:53:23),
PLA 37.9:t0.9 a 3.5:t0.4 a 2.4i0.0 a
MD PLA-B-CD15% 11.6:I:l.3 b 1.6:I:O.I b 0.9:t0.l b
PLA-B-CD15%-MB l9.7:l:2.3 c 1.9:t0.l b 1.4:I:0.l C
PLA-B-CD30% l 1.3:lzl.3 b 2.1:h0.5 b 0.5:I:O.2 b
PLA 29.2il.8 a 1.7:t0.3 a 2.3i0.0 a
PLA-B-CDIS‘VO 6.6:t0.8 bc l.l:l:0.l b 0.8:h0.l b
CD PLA' [1.315% 10.5¢1.5 b 1.3i0.2 b 1.3:t0.1 c
PLA-B-CD30% 3.8:t1.2 c 1.13200 b 0.7i0.2 d

 

a,b,c, and (1 indicate significant differences between samples

Thermal characterization
Table 5 summarizes some of the DSC results for the neat PLA and for the PLA/B-CDS
blends. The neat PLA exhibited a glass transition temperature (Tg) of 57 °C and a melting

temperature (Tm) of 148 °C. The addition of l3-CDs slightly decreased both of these

33

thermal transitions in the PLA. The Tg of all the PLA/B-CDS blends were about a value of
54°C and all Tm ranged about a value of 147°C. Similarly, Ke and Sun (2000) did not
observe a significant difference in T8 among polymer blends of PLA and corn or wheat
starches at various ratios [79]. Figure 5 shows in detail how crystallinity and melting
peaks were modified with the addition of B-CDS. As the amounts of B-CDS increased, the
melting peak become broader and deeper and the crystallization peak become larger than
those of the neat PLA. The broader melting peak was possibly caused by a wide range of
sizes of the crystals formed by the interaction between the PLA and the B-CDs. The latter
had a Tm of 155°C (data not shown). These broader melting peaks in the PLA/B-CDS
blends may lead to higher hot tacks under a broader range of Tm, especially at lower ones.
The larger crystallinization peak was caused by the nucleating capacity of the B-CDS in
the PLA matrix [128]. The fraction of crystallinity of the neat PLA and of the blends
PLA/B-CDS is presented in figure 6. In the calculation of the crystallinity of the blends
only PLA was considered since PLA was the main load-bearing phase. The crystallinity
of the blends was relatively low but higher than that of the neat PLA. The slight increase
was correlated to the amount of B-CDs. The higher amount of B-CDS, the higher the
crystallinity. This agrees with Almenar, Auras, Harte, Rubino (2009) who reported that
the crystallinity of PLA increased with the increase of the B-CD content. Starch has been
identified as a nucleating agent for PLA, too [88, 128-129]. However, it has been
reported that the crystallinity of blends with starch contents of <20% is the same as that
of extruded pure PLA, and that the crystallinity decreases slightly as the starch content
increases to 40% [79]. The crystalline contents of PLA-B-CD15%-MB and neat PLA did

not differ. The reduction of the crystalline content of the PLA in the PLA-B-CD15%-MB

34

blend compared with that of the PLA/B-CD15% blend was due to the use of the MB.
Lower crystalline contents have been related to higher impact resistance and ductivility
[130]. A reduction in crystallinity of the PLA with the addition of commercial
masterbatches has been reported [87].

A hysteresis peak (63°C) associated with the T8 of neat PLA and its blends is also shown
in Figure 5. This peak was an aging peak [79] and resulted from an increase in the excess
enthalpy of relaxation during processing [131]. The use of the masterbatch did not affect
the thermal properties of the PLA in the blends. The same Tg and Tm were observed for
PLA-B-CD15%-MB and for PLA-B-CD15% (Figure 5 and Table 5). Similarly, Byme,
Ward, Kennedy, Irnaz, Hughes, Dowling (2000) who evaluated four commertial
masterbatches in order to improve the impact strenght, flexibility, and clarity of PLA

reported no effect of the masterbatch on the Tg and on the Tm of the PLA [87].

Table 5. Glass transition temperatures and melting temperatures of the neat PLA and the

PLA/B-CDS blends

 

 

 

 

 

Sample code Tg (°C) Tm (°C)
PLA 57.0d:0.9 a 148.2:t0.4 a
PLA-B-CD15% 53.9i0.4 b 147.5i0.3 ab
PLA-B-CD15%-MB 54.3:t0.5 b 147.9:1:0.2 ab
PLA-B-CD30% 54.2:1205 b l47.l:l:0.6 c

 

a, b, and c indicate Significant differences between samples

35

 

 

Heat flow Exo (ng)

 

--------- PLA-BCDtS°/o
........................... PLA-BCD15°/o-MB
—- ————— PLA-BCD30%

 

 

 

I I I

20 40 60 80 100 120 140 160 180 200
Temperature(%)

Figure 5. DSC therrnograms of the neat PLA and the PLA/B-CDS blends (1St cycle)

 

10

 

 

 

a?
V b
3‘ b
2: 51 I
E {
a
t:
O
'6 g t
§ 0- a a
it
0 withoutMB
0 with MB
-5 ,— u l
0 15 30

Percent of cyclodextrin (%)
Figure 6. Percent of crystallinity (%) of the neat PLA and the PLA/B-CDS blends (a, and

b indicate significant differences between samples)

36

 

TGA thermograms of the neat PLA and of the PLA/B-CDS blends are presented in
Figures 7 and 8. The decomposition of the neat PLA started at around 366°C and ended at
411°C (Figure 7).The PLA/B-CDS blends showed a lower thermal stability than that of
the neat PLA and this was B-CDS content dependent. The higher the B-CDS content the
lower the thermal stability. No effect of the masterbatch on the thermal stability of the
blends was observed. Figure 8 Shows the rates of decomposition for the neat PLA and for
the PLA/BCDS blends. The rates of decomposition for PLA/BCDs blends were faster than
for the neat PLA. PLA-B-CD 30% showed the fastest rate of decomposition. The
derivative thermogravimetric curve shows the temperature at the maximum rate of weight
loss, and this corresponds to the decomposition temperature [81]. PLA-B-CD15% and
PLA-B-CD15%-MB showed similar rates of decomposition as observed in figure 8. The
remaining water in the B-CDS was possibly the reason for a faster decomposition rate for
the blends than for the plain PLA. It has been reported that the initial moisture content of
the starch had a significant effect on the decomposition of PLA during processing of
PLA/starch blends. The PLA gets slightly degraded in the presence of water under the
high levels of heat used during processing [82]. The derivative thermogravirnetric curves
of the PLA/B-CDS blends showed a shoulder which results from the degradation of the [3-
CDs. This has been confirmed by performing the same study on B-CDS (data not shown)
which resulted in the same bimodal peak. Further confirmation comes from a study of
the derivative weight of poly(B-CD/Z benzoxazine IC) under nitrogen which showed a

bimodal peak at about 300~350°C [132].

37

 

100

 

 

 

 

 

 

 

 

 

 

 

100~ . .
90I
80 -
so
70 . '-
25 sol 60 g \
E 300 320 340 360 380 400
O)
.5
; 40J
PLA
20 - ——- PLA-BCD15%
............ PLA-BCD15o/o-MB
- ----- PLA-0c030%
0 f ' I
100 200 300

 

Temperature (°C)
Figure 7. Weight change (%) as a function of the temperature (°C) of the neat PLA and

the PLA/B-CDS blends

 

 

 

 

 

 

 

   

 

 

 

 

 

 

3.5
3.0
3.0 -
2.54
6‘ 2.5 -
§ 2.0-
E 2.0 '
.9 ] 1.5
‘3’ 1.5 360
0
>
g ml PLA
E —— PLA-BCD15% ]
8 05- ............ PLA-BC015%-MB I
_._.- PLA-BCD30% L
0.0 L
'0.5 I f I T
100 200 300 400 500 600
Temperature (°C)

Figure 8. Derivative weight (%/°C) as a function of the change of temperature (°C) of the

neat PLA and the PLA/B-CDs blends

38

Dynamic mechanical characterization was performed on the neat PLA and on its blends.
Dynamic mechanical analysis mainly measures viscoelastic behaviors and compares the
stress and strain signals as the in-phase (storage) and out-of-phase (loss) components,
from which storage or elastic (E'), loss (E") moduli as well as the tan 5 = E"/E' are
obtained as functions of temperature [81]. E' and tan 8 of all samples are presented as a
function of temperature in figures 9 and 10.

As shown in figure 9, the PLA blends showed higher E' than that of the plain PLA over
the entire temperature range. This confirms that the PLA matrix is restrained by the
loading of B-CDS because of the incompatibility between components. Therefore, E'
increased proportional to the concentration of B-CDS. These results are in agreement with
those reported by Almenar et a1 (2009). No effect of the masterbatch on E’ was observed.
Therefore, the use of the masterbatch didn’t improve the applicability of the blends in
applications that require rigorous processing conditions. E' dropped at 60~65°C because
of the increase in the segment mobility of the polymer chain at around the glass transition

temperature.

39

 

 

     
 

1200 j T"T”"'~--..-
..... CNN-

;- I.
g m... ‘
7 800 # -M\.~-‘.N ............ I
a T ‘
a i
8
E
,7, — — PLA-BCD15%

............ PLA-BCD15%-MB

— ----- - PLA-BCDSO%

0

 

 

 

 

I I l I T l T r

-20 0 20 40 60 80 1 00 1 20

Temperature(°C)

Figure 9. Storage modulus changes of neat PLA and PLA/B-CDS blends

 

 

 

 

 

2.5
—— PLA
20] ---— PLA-[3CD 15%
' """""" PLA-[3CD 15%-MB
— """ PLA-8CD 30%
1.5 ‘
3
8
C
.2 1.0 T
0.5 r
0.0 ‘
-20 20 40

 

Temperature(°C)

Figure 10. Tan delta changes of neat PLA and PLA/B-CDS blends

40

The neat PLA showed the highest value of tan delta and this decreased in the following
order: PLA-B-CDl5%-MB>-B-CD15%>-B-CD30%. It has been reported that the neat
PLA showed a very sharp and intense peak because there was no restriction to the chain
motion [133]. The reduction of height of tan delta confirmed the hindrance of the chain
mobility by the presence of B-CDs. Less reduction in height was observed in the blend
containing the masterbatch. The masterbatch seems to improve the chain motion and
therefore, the processability of the PLA/B-CDS blends. The glass transition temperature
of the blends obtained from the DMA curve was a little higher than that obtained from
the DSC curve. DMA monitors the temperature dependence of mechanical stress/strain
and loss behavior (tan delta) as a function of the frequency of the oscillating deformation
force applied. For a perfectly elastic material, the applied stress and measured strain is
perfectly in phase and thus no losses occur. As Tg is approached, mobility of the polymer
chains increases until, at a given temperature, the relaxation time for chain mobility
coincides with the frequency of the applied stress. At this particular point, the polymer
absorbs mechanical energy and the dammng factor (loss tan delta) reaches a maximum
value. Thus the glass transition temperature can be identified as the point of maximum
damping on a plot of tan delta versus temperature at the particular frequency under

consideration [1 34] .

Optical characterization
CIELAB describes the optical prOperties using the parameter L*, a*, and b*. In the
CIELAB Space, the parameter L* is a measure of the lightness of a sample, and ranges

from 0 (black) to 100 (white) [135]. Figure 11 shows the lightness of the neat PLA and of

41

the PLA/B-CDS blends. As observed, the blends presented a higher lightness than the neat
PLA (92.53:!:0.16 vs. 93.24i0.15 and 93.34zl:0.21, for PLA, PLA-B-CD15%, and PLA-B-
CD30%). There are two reasons for this lightness increase. The first one is the natural
white color of the B-CDs which became part of the polymer matrix during processing and,
the second reason is the higher crystallinity content in the blends due to the nucleating
agent capacity of the B-CDs. In general, the higher the crystalline content in a polymer,
the higher its lightness is. However, there were no significant differences between PLA-
B-CD15%, and PLA-B-CD30% because the reduced mobility of the PLA molecules
caused a similar crystallinity of the material in Spite of the different amounts of
nucleating agent. Similarly, extrudates of PLA/starch are opaque and of white color [82].
Ke et a1 (2000) reported PLA/starch blends with 20% of starch as light gray and it turned
into off-white as the starch contents increased to 240% [79]. PLA-fi-CD15%-MB showed
less lightness compared with PLA-[3-CD15%. This is in agreement with the reduction of
the crystallinity resulting from the use of the masterbatch (figure 6). Therefore, the use of
the masterbatch improved to see through the new material. .

In the CIELAB space, variables a* and b* define the degree of greenness (- a*) or
redness (+ a*), and blueness (- b*) or yellowness (+ b*) [76]. Figure 12 shows the color
variables a* and b* of the neat PLA and of the PLA/B-CDS blends. All samples exhibited
a“ high b* value due to the natural yellow color of the PLA [76]. Both b* and a*
increased as B-CD content increased. Therefore, the blends became redder and more
yellow and with a total color more saturated. Of the blends, PLA-B-CD15%-MB showed
the lowest redness, close to that of the neat PLA, while at the same time PLA-B-CD15%-

MB showed the same yellowness as PLA-B-CD30%. This was due to the presence of [3-

42

CDs in the polymer matrix. Byrne et a1 (2009) also reported an increase of the yellowness
index of PLA with the use of the commercial masterbatches Biomax strong, and PLA
chSlS-N [87].

A slightly modified way to calculate AB was used [136]. In this study, AE denotes the
overall difference in color between the samples (blends) and a reference (neat PLA), and
the larger the value of AB, the larger the differences in color. According to Sharma
(2003), a AB of approximately 2.3 corresponds to a just noticeable difference [137]. The
values of AB were less than 2.3 for all the blends and therefore, there were no differences
in overall color among them. Although the color values L*, a“, and b* of the blends have

been shown to be dependent on the B-CDS content, this did not happen for the overall

 

 

 

 

color of the blends.
94.0
93.5 -
bc c
-I 93.0 i
ab
92.5 - £3
0 without MB
0 with MB
92.0 . . .
0 15 30
Percent of cyclodextrin (%)

Figure 11. Lightness of neat PLA and PLA/B-CDS blends (a,b and 0 indicate significant

differences between samples)

43

4.0

 

3.5 1

3.0 - C

E c
c
n 2.5 d b + d
+ b
2.0 - a
1 5 . + a 0 without MB
' O with MB

1.0 I I 1 I I I I I
-0.14 -0.12 -0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04

 

 

 

Figure 12. a* and b* values of neat PLA and PLA/B-CDS blends (a, b, c, d and A, B, and

C indicates significant differences between samples)

Table 6. AE of the PLA-BCDS blends with respect to PLA as reference

 

PLA PLA-B-CD15% PLA-B-CD15%-MB PLA-B-CD3 0%
AE - 0.91 1.25 1.27

 

 

Morphological characterization

Scanning electron micrographics of the surface of fractured neat PLA and PLA/B-CDS
blends were taken to better understand the interaction between PLA and B-CDS. All
samples were prepared in the same way, and all micrographics were taken under the same
conditions. The results are shown in Figure 13. Panel (a) shows the continuous phase of
the neat PLA, while panels (b), (c), and ((1) show the micrographics for PLA with
different B-CD contents and how this continuous phase became discontinuous with the

addition of B-CDs. The mix of PLA and B-CDS produced a two-phase system. This claim
44

was based on: (1) B-CDs were not completely covered by the PLA phase. A larger
amount of uncovered B-CDS is present in the blend with the highest B-CD content (30%)
(d) which might be due to the increased size of the B-CDs conglomerate in this material.
It seems that larger B-CDS conglomerates (approximately 15 um width) were formed by
interaction between smaller ones during processing and that higher amounts of B-CDS
lead to the formation of larger conglomerates (figure 13, arrows). This would be in
agreement with previous study done with CDs that reports the ability of CD3 to form
supramolecular complexes by linking covalently or non covalently specifically to other
CDs [9]. (2) The presence of cavities between the PLA and the B-CDS. The higher the
amount of B-CDs, the larger the cavities were. This illustrates the poor adhesion between
the phases PLA and B-CDS. These results are supported by similar ones published for
PLA/starch blends. Microscopic observations revealed non-unifome dispersed holes in
TPS/PLA blends which indicated a separation of phases between TPS and PLA [84].
Some gaps between PLA and starch in PLA/starch blends were observed which indicated
likely poor adhesion between the two phases [79]. Comparing the micrographics of
PLA/starch with those of PLA/ B-CDS for the same content of filler shows that the
miscibility between PLA and B-CDS was higher than that of PLA and starch.

The micrographic of PLA-BCD1 5%-MB (c) looks like a combination of the micrographic
of the PLA-BCD30% (d) and of the neat PLA (a) since some areas still showed
conglomerates although these were smaller than the ones in the PLA-BCD30% blend.
Also, the number of cavities was lower. Therefore, processing improved the adhesion

between the two phases due to the reduction of the conglomerate size and of the number

45

of cavities. The micrographic of PLA-BCD15%-MB (c) does not show improved

interfacial adhesion compared to PLA-BCDIS% (b).

 

Figure 13. Scanning electron micrographics of the surface of fractured neat PLA and

PLA/B-CDs pellets. Panel (a) shows neat PLA while panels (b), (c), (d) show PLA-B-

CD15%, PLA-B-CD15%-MB, PLA-B-CD30 (arrows pointing to the conglomerates)

Barrier characterization

46

 

 

 

 

& 30-14

3 d

0

E

- 2e-14

e l

n

:‘5

g 1e-14-

% no

0

E o o ‘0 o withoutMB

0- a . withMB
O 15 30

Percent of cyclodextrin (%)

Figure 14. Oxygen permeability coefficient of the neat PLA and of the PLA/B-CDS

blends at 23°C and 0%RH (a,b,c, and d indicate significant differences between samples)

 

 

 

 

3.5e-14
Q d
3.0e-14 -
it?
n.
g 2.5e-14 -
a“ §c
E 2.0e-14 -
E
g 1.5e-14 -
g 1.09.14 -
(I
o 4 I B o 23°C without MB
g 59°45 I b o 23°c with M8
a g g 0 318°C without MB
0.0 1 I 37.8°Cwith MB
0 15 30

Percent of cyclodextrin (%)

Figure 15. Water permeability coefficient of the neat PLA and of the PLA/B-CDS blends
at 23°C and 37.8°C, and 100%RH (Not data for PLA-BCDsl 5%, PLA-BCDS30% at

378°C) (a, b, c, d and A B indicate statistical difference among samples)

47

 

The oxygen permeability coefficients of neat PLA and its blends are presented in figure
14. The permeability of the neat PLA was 8.71><10'18:l:1.17><10'18 kg-rn/mz-s-Pa at 23°C
and this value was in agreement with the value of 6.0><10'18 kg-m/mz-s-Pa reported in the
literature for PLA films exposed to the same temperature [7 6]. For higher temperature
(25°C), oxygen permeability of PLA films has been reported as 3.3><10'l7 kg-m/mz-s-Pa
[138]. The values for the PLA/B-CDS blends were over several orders of magnitude larger
than the neat PLA, and this difference was more pronounced for the higher B-CD content
(6.69x10"5a4.10x10"6 and 2.83><10‘”:i:8.62x10"5 kg'm/mZ'S, for PLA-B-CD15% and
PLA-[S-CD30%, respectively). The increased permeability was caused by the
discontinuity between the B-CDs and the PLA. The voids between phases allowed the
oxygen molecules to diffuse faster through the matrix. The use of the masterbatch
improved the barrier properties of the blends due to the reduction of discontinuities in the
polymer matrix. PLA-B-CD15%-MB showed a lower permeability than PLA-B-CD15%
(2.43><10"5i1.64>< 10'16 kg-m/m2~s-Pa vs. the data above).

Figure 15 shows the water vapor permeability coefficients of neat PLA and of the PLA/B-
CDs blends. The permeability of neat PLA was 1.11><10'l°i1.34><10'1° kg-rn/mzsPa and
2.43><10"5i3.00><10'1° kg-m/mz-s-Pa, at 23°C and at 37.8°C, respectively. Higher
permeability values for water vapor have been reported in the literature (2.0><10'l4
kg-m/mz-s Pa at 20°C and 90%RH) [76]. This might be due to differences in processing
history, and other parameters. The permeability of the blends increased over that of the
neat PLA with the increase of the B-CD content. The immiscibility of phases in the
blends allowed more water molecules to penetrate. Some of the water molecules were

possibly absorbed by the polymer matrix since part of them was removed in the drying

48

 

process before the processing of the material. Similarly, it has been reported that water
can penetrate the PLA/starch blends through the voids between phases and that some
waster was absorbed by the starch (<20%) [79]. The permeability of the PLA-0-CD30%
blends at 37.8°C was so high that it couldn’t be measured. The use of the masterbatch
increased the barrier properties of the blends. The PLA-B-CD15%-MB blend has a water
vapor permeability of 3.58X10'152E8.34><10'1° kg-m/mz-s-Pa and 7.07><10"51:4.64><10'l6
kg'm/mz-s-Pa at 23°C, and at 37.8°C. Both values were lower than 2.21><10'I4d:1.31><10"5,
the value obtained for PLA-B-CD15%.

In general, oxygen and water vapor permeability coefficients were a little lower than
those reported in the literature, which might be caused by differences in thickness,

processing and parameters.

CONCLUSIONS

In this study, a bio-based polymer with the capacity to carry hydrophobic molecules has
been developed and characterized. Testing of mechanical, thermal, barrier, optical, and
morphological properties revealed that B-CDs and PLA are incompatible. The higher the
B-CD content, the higher the incompatibility due to the increase in size of the B-CDs
conglomerates, and the poor adhesion between phases. The use of a masterbatch (PLA
containing high B-CD content) is an effective way to improve the compatibility between

PLA and B-CDS, and the properties of PLA/B-CDS blends.

49

The author thanks to Dr. Laurent Matuana for allowing us to use both the extruder and
the pelletizer. Also, the authors thank Atul Singla and Jin Shan with their help with

processing of the samples.

50

CHAPTER 4. DEVELOPMENT AND CHARACTERIZATION OF A BIO-
BASED ANTIMICROBIAL POLYMER MADE OF POLY(LACTIC ACID) AND
ENZYMATICALLY MODIFIED STARCH CONTAINING TRAPPED TRANS-2-

HEXENAL*
Joo, M.J., Merkel, C., Auras, R., Almenar, E.

*to be submitted for publication to BIOMACROMOLECULES

The present study aims at developing and characterizing a bio-based antimicrobial
polymer with potential use in food and pharmaceutical packaging applications, but also
as an inherently sterile material by itself. Trans-2-hexenal, a naturally occurring plant
volatile with antimicrobial capacity, was trapped into B-cyclodextrins (B-CDS),
enzymatically modified starch, and indirectly incorporated into a poly(lactic acid) (PLA)
matrix in order to develop a bio-based antimicrobial polymer. A masterbatch was used
prior to casting of the polymer in order to improve the dispersion of the antimicrobial
compound in the PLA matrix. Physical, mechanical, barrier, and antimicrobial properties
of antimicrobial and neat PLA sheets were determined in order to study the effect of the
antimicrobial on the polymer properties. The antimicrobial capacity of the sheets was
tested against the fungus Alternaria solani. Incorporation of trans-2-hexenal trapped into
B-CDs to the PLA matrix did not affect the physical properties, but did affect the
mechanical, barrier and antimicrobial capacity, and the new polymer was effective in
inhibiting the growth of micro-organisms. Processing operations resulted in a dramatic
loss of antimicrobial capacity. The masterbatch showed 90% more antimicrobial capacity

than the polymer sheet.

51

1. Introduction

Antimicrobial polymers are a type of active polymers that can reduce, retard or inhibit the
growth of spoilage and pathogenic micro-organisms, generally by contacting or releasing
active compounds with antimicrobial properties. The antimicrobials can be incorporated
into the polymer, coated or immobilized onto the polymer, or be an inherent part of the
polymer [2, 139-140]. There are many antimicrobials that potentially can be used to
develop antimicrobial polymers. In recent years, much interest has been attracted by the
ones that can be found in nature.

Some naturally occurring plant volatiles are well-know to provide protection against
microbial proliferation in wounded areas of plants [33]. Their use as antimicrobial has
been thoroughly explored in in vitro and in vivo systems, and among these the highest
effectiveness has been reported for aldehydes [141]. The precise mechanism of the
protective action of aldehydes is not clear yet, but likely they permeate by passive
diffusion across the plasma membrane, and once inside the cell, the (1., B-unsaturated
aldehyde moiety reacts with biologically important nucleophilic groups which play a key
role in living cells [44]. Trans-2-hexenal is one of the aldehydes most successfully tested
to date. This naturally occurring six-carbon plant volatile produced through the
lipoxygenase pathway has been successfully employed against bacteria and molds in in
vitro and in vivo systems [36, 43-44, 142-143]. The combination of its antimicrobial
capacity and the fact that it is approved as food additive permitted for direct addition to
food for human consumption by the US Food and Drug Administration [45] makes trans-

2-hexenal particularly interesting as an antimicrobial compound for food applications.

52

However, its direct application on a substrate has limited benefits because of its rapid
volatilization. Cyclodextrins (CDs) are a possible alternative to trap, and to control the
release of trans-2-hexenal.

CDs are cyclic oligosaccharides composed of 0t-1,4-coupled D-glucose units produced
enzymatically from starch. They have a hydrophilic exterior and a hydrophobic cavity,
which enables them to form inclusion complexes (ICS) with a variety of hydrophobic
molecules [144]. The trapping of naturally occurring volatiles into CDs allows for great
application flexibility. For example, CDs have been used as carriers and releasers of
aldehydes like acetaldehyde and hexanal to reduce and/or inhibit spoilage micro-
organisms [1 1-12].

Antimicrobials can be directly incorporated into a polymeric matrix leading to
antimicrobial polymers as mentioned above [139]. This direct insertion is not possible for
nattually occurring plant volatiles because of their rapid volatilization. The use of CDs
will allow the incorporation of naturally occurring plant volatiles into the polymeric
matrix since the volatiles are handled as a powder. To date, most antimicrobial polymers
are petroleum-based. Polymers made from natural resources with antimicrobial properties
are practically restricted to edible films and coatings [60, 68]. Bio-based polymers with
antimicrobial activity have been developed by using starch, cellulose, alginates,
carrageenan, chitosan, agar, wey, zein, gluten, soy, gelatin, waxes, and glycerides [68].
Poly(lactic acid), PLA, is a biocompatible and biodegradable semi-crystalline polyester
with potential for further developments because its mechanical and barrier properties are
comparable to those of conventional polymers [18-19]. PLA is a plastic material made

from natural resources such as corn or sugar cane and is transparent and glossy [76], is

53

approved for food contact by the US Food and Drug Administration [145], and can be
degraded in a composting system [146]. These preperties make PLA particularly
interesting as a bio-based viable alternative matrix for antimicrobial polymers for food
and pharmaceutical packaging applications. In this study, trans-2-hexenal was trapped
into CDs and was indirectly incorporated into a poly(lactic acid) matrix, yielding a bio-
based antimicrobial polymer. Since ICs are sensitive to external stimuli, such as
temperature changes, shear forces, and the presence of competing CD-binding molecules
[91, 144], the antimicrobial capacity of the polymer may change during processing [7]
and the immiscibility between PLA and B-CDs affects the characteristics of antimicrobial
polymer. Joo et a1. (2010) reported that the use of a masterbatch made of PLA containing
high B-CD content improved the distribution of the CDs in the polymeric matrix and the
miscibility between PLA and CDs [147]. In addition, Han (2000) recommend the use of a
masterbatch to process antimicrobial polymers to minimize the deterioration of the
antimicrobial agent during film fabrication, distribution and storage [27]. Thus, a
masterbatch was used prior to casting of the antimicrobial polymer in order to improve
the dispersion of the antimicrobial compound in the PLA matrix.

The purpose of this study was: (1) to develop a bio-based antimicrobial polymer using a
masterbatch, (2) to characterize the new polymer in terms of mechanical, physical, barrier,
and antimicrobial properties, and (3) to study the effect of processing on the antimicrobial

capacity of the new polymer.

2. Experimental Section

54

2.1 Materials. The volatile compound, trarrs-Z-hexenal (purity >95%, Food grade), was
purchased fiom Sigma-Aldrich Corp. (Saint Louis, MO, US) (figure 16 1a). Poly(lactic
acid) 4042D (PLA 4042 D) (92% L-lactide, D=l.24g/cc) resin was purchased from
NatureWorks, LLC (Blair, NE, US) (figure 16b). B-cyclodextrin (B-CDs) (purity >99%,
Food grade) were purchased from Wacker Chemical Corporation (Adrian, MI, US)

(figure 16c).

O
a CH3 H
0 CH3
0
0
n
b CH3 0
I_ -1
HO
_._L
O
C. __ HO OH J7

 

 

Figure 16. Chemical structures: a. trans-2-hexenal, b. PLA, and c. B-CD

2.2 Microbial strains. Single spore isolates of Botrytis cinerea, and Colletotrichum
acutatum were originally isolated from diseased blueberry fruit, and of Penicillium sp.
from spoiled bread. Aspergillus niger and Alternaria solani spore isolates were provided

by the Department of Plant Pathology, MSU, East Lansing, MI, USA.
55

2.3 Synthesis and characterization of inclusion complexes (ICS). An amount of 5.67 g
of B-CDS was weighed using an electronic balance (AdventurerTM precision balance,
OHAUS, NJ, US) and then added to a beaker containing 56 ml of 100 °C distilled water
(figure 17a). The mix was stirred for a few seconds on a hot plate stirrer (Therrnolyne®
MirakTM hot plate stirrer; Sigma-Aldrich Corp.(Saint Louis, MO, US)) prior to the
addition of 315 pl of pure-trans-Z-hexenal. The molar ratio of B-CDS and trans-2-hexenal
was 5:3. The solution was stirred for two hours at 130 rpm, and then cooled down using a
second stirrer without heating (Therrnolyne Nuova II stir plate, Barnstead International,
Testware, Sparks, NY, US). The mix was centrifuged at 1600 rpm for 1 hr and then dried
at the 90°C in a conventional oven for 24hr (10-180 Incubator, QL, Quincy Lab, Inc,
Chicago, US). The ICS were maintained in a desiccator at 23°C until use.

To determine the amount of trapped volatile (figure 17b), 2 ml glass vials were filled with
ICS (0.01 g) and then positioned at the bottom of 40 ml glass vials containing 1 ml of
distilled water (placed at the bottom) each. The vials immediately were tightly closed
with Mininert® valves (Supelco, Bellefonte, PA, US). Approximately 24 hours later, a
65-um PDMS/DVB SPME fiber (Supelco, Bellefonte, PA) was exposed to the vial
headspace for 10 minutes, and then was inserted into the splitless injection port of a GC
Hewlett-Packard 6890 series esc (Agilent technology, Palo Alto, CA, US) equipped with
FID and an HP-5 column (30 m><0.32 mm><0.25 um, Hewlett—Packard, Agilent
Technology, Palo Alto, CA, US). The initial oven temperature was 40 °C for 5 min. It
was then increased to 230 °C at 5 °C/min and maintained for 10 min. The injector and
detector temperatures were 230 and 270 °C, respectively. The amount of trans-2-hexenal

released from the ICS was evaluated in triplicate, and quantified on the basis of prepared

56

calibration curve. A calibration curve was prepared by measuring the chromatographic
area of accurately known trans-2-hexenal solutions and by plotting the different amounts
of trans-2-hexenal (concentration) on the X-axis vs. their corresponding measured
responses (chromatographic area) on the Y-axis.

A scanning electron microsc0pe (J SM-model 6400 SEM, JEOL, Peabody, MA, US) with
an accelerating voltage of 12 kV and a working distance of 25 mm at x500 magnification
was used to assess the morphology of the B-CDS and of the ICS during their synthesis,
simulating their synthesis but without trans-2-hexenal, and after the release of the trans-2-

hexenal. Prior to analysis, the samples were coated with Osmium.

o b. Characterization

AN: - ° W I—_—)

Inclusion complexes

 

 

 

 

 

 

 

 

 

 

no at J?
L Trans-2-hexenal B-cyclodextn’n I T
J, 8. Synthesis
’ Oven Drying
\ i Centnfugation
I: Heating 1: Cooling PP? Em

 

00

 

 

ITI

00

 

 

t: :
F E

 

 

 

 

 

 

 

 

 

< TIITIF

 

 

 

 

Figure 17. Synthesis and characterization of the ICS

57

2.4 Preparation of the antimicrobial masterbatch. A masterbatch was prepared to
improve the dispersion of the antimicrobial compound and the properties of the PLA
matrix. Antimicrobial masterbatch was prepared as follows (figure 18a):

PLA 4042D dried for 4 hrs at in a conventional oven (1430 VMR International, Cornelius,
OR, US), was mixed with ICS (70:30 w/w) using a 32 mm conical counter-rotating twin-
screw extruder (C.W. Brabender Instruments, South Hackensack, NJ, US) with a MD
ratio of 13:1 and four separated temperature controlled barrel zones set from feeder to die
at 170 °C, 175 °C, 175 °C, and 170 °C. The screw speed was set at 40 rpm. The blend
was collected from the die and allowed to cool down prior to being granulated into pellets
(JC-S, Conair Wortex, Franklin, PA, US). The resulting pellets were used as
antimicrobial masterbatch pellets for the casting process. The pellets were maintained in
a desiccator at 23 °C until use.

2.5 Preparation of the antimicrobial sheet. The antimicrobial masterbach and plain
PLA 4042D pellets were mixed (50:50 w/w) and pelletized using the same processing
conditions as described in figure 18b. The pellets were transferred to the hopper of a
casting film twin extruder (RCP-0625 Microtuder control panel, RandCastle extrusion
system, INC, Cedar Grove, NJ, US) with four separated temperature controlled barrel
zones set from feeder to die at 160 °C, 165 °C, 170 °C, 170 °C and 170 °C, and a screw
speed of 35 rpm. The resultant melt was extruded through a slit die to form a sheet, and
the extrudate was cooled on chill rolls kept at a constant temperature of 52 °C. The width
and thickness of the resulting antimicrobial sheet were 19 cm and 0.019 cm, respectively.

The antimicrobial sheets were maintained in a desiccator at 23 °C until use.

58

 

Inclusion completes [- Poly(lactic acid)

MVVOKJ'. TI +12 . ..

 

 

 

 

 

0

a. Preparation of the masterbatdi '7

Antimicrobial pellets

33%

 

 

w c. Characterization

0

b. Prep aration of the antimicrobial sheet
Control

 

 

Antimicrobial sheets

$8 :7—

Figure 18. Processing and characterization of pellets and antimicrobial sheets

 

 

 

 

2.6 Antimicrobial effectiveness of ICS, pellets and sheets. Culture preparation and
bioassay (figure 18c): Five kinds of fungi: Alternaria Solani, Aspergillus niger, Botrytis
cinerea, Colletotrichum acutatum, Penicilium spp. were cultured on Potato Dextrose
Agar (PDA) (Sigma-Aldrich Corp. Saint Louis, MO, USA) in plastic petri dishes (0 9 cm)
for 14 days at 23 °C. An amount of 10mL of sterile distilled water and of 1 mL of
glycerol were added to each petri dish in order to allow the removal of mycelia by means
of a spatula. Each conidial suspension was transferred to a plastic tub and then vigorously
shaken to dislodge the spores from the mycelia. The number of spores was concentrated

to 1 x 10° spores/mL using the Neubauer improved method (Bright-Line Hemacytometer,

59

Hausser Scientific, Horshan, PA, USA). Each conidial suspension was divided into
several microtubes and then frozen in a refrigerator.

For the bioassay, conidial suspensions were defrozen and then an amount of 3.5 ,uL of
each conidial suspension was placed in the center of a small petri dish (0 5.5 cm)
containing PDA using a 100 ,uL Oxford autoclavable Benchmate pipet (N ichiryo, Japan).
The petri dishes were then placed inside 500 mL sterile glass jars, which were closed
with screw caps and stored at 23 °C. For the control bioassay systems, regular screw caps
were used. For treatment bioassay systems, modified caps were used to allow the
withdrawal of the volatile [12]. Petri dishes were placed on supports made from their own
lids but perforated to allow volatile flow. The supports were inserted into the jars prior to
the petri dishes. Treatment bioassays systems were divided into three groups. One group
was used to test the ICS, another to test the antimicrobial masterbatch, and the remaining
one to test the antimicrobial sheets. The amount of ICS (4 g) added to the bioassay was
determined based on previous microbial studies using pure trans-2-hexenal (data not
shown) and on the amount of volatile released from the ICS during the characterization
process (see above). To test the antimicrobial masterbatch pellets and sheets, same
amount as that of ICS was placed inside the jars. This reduced the amount of ICS to 1.2,
and 0.6 g for pellets and sheets, respectively. In all treatments, the volatile was released
due to the rich humidity environment generated by the petri dish in the closed system.
Controls and treatments were stored at 23 °C. Three bioassays systems were tested per
fungus and per treatment.

Measurement of fungal growth and trans-2-hexenal levels: Growth of the cultures in both

control and treatment bioassay systems was evaluated daily over a 7 day period at 23 °C

60

by measuring the diameter of the colony on the agar surface. Measurement of diameters
was made using a conventional ruler and reported in centimeters. Because of the optical
transparency of both the glass and Petri dish, these measurements could be made without
opening the jars. Petri dishes with no fungal growth after exposure to trans-2-hexenal
were moved from the bioassay systems and transferred to a new bioassay system
containing a headspace atmosphere free of volatile. Prior to their insertion into the jars,
the growth-free Petri dishes were modified by transferring 5 mm agar plugs from the
center of the plate to new Petri dishes containing fresh agar and then inserted into the new
jars. Radial growth of the cultures was evaluated over a 7 day period at 23 °C as
mentioned above.

The levels of trans-2-hexenal released from ICS, materbatch pellets, and sheets were
determined by using a SPBM fiber and gas chromatography as reported above.

2.7 Characterization of the antimicrobial sheet. Physical, mechanical, and barrier
properties of the antimicrobial sheet and the plain PLA sheet were measured as follows
(figure 18c):

Thermal transitions of the neat PLA sheet and the antimicrobial sheet were determined
using a TA Instruments Q100 V 9.8 Differential Scanning Calorimeter (TA instruments,
New Castle, DE, US). The temperature calibration of the equipment was performed in
accordance with ASTM E967-03 [119] and the heat flow calibration was performed in
accordance with ASTM E968-02 [120]. Transition glass temperature (T3), and melting
temperature (Tm) were measured and calculated in accordance with ASTM D3418-03
[148]. The degree of crystallinity (%Xc) for neat PLA and antimicrobial pellets and sheets

was calculated as follows [79]:

61

AJTm4-AJTC
93xk§u

0

Axe =

 

 

1x100

where AH", , AHC , and X1?“ are the melting enthalpy, crystallization enthalpy, and PLA

content, respectively. A value of 93 J/g for the melting enthalpy for 100% crystalline
PLA was obtained from the literature [122].

An amount between 7-9 g of PLA and PLA/B-CDS sheets was used for each experience.
Samples were heated from 40 to 190 °C at a rate of 10 °C/min. The B-CD content was
taken into consideration when determining °/oXc. Three replications of each type of sheet
were tested. In the calculation of the crystallinity of the blends only PLA was considered
since PLA was the main load-bearing phase.

Neat and antimicrobial PLA Sheets were maintained in a in a desiccator at 23 °C until use
and then shaped into specimens with a width of 1 in and a length of 8 in prior to use. The
thickness of the specimens (0.18 mm) was determined by averaging of three readings
taken at different points. The tensile preperties: tensile strength, elongation at break, and
modulus of elasticity were measured according to ASTM D882-02 [118], using a Instron
Universal Testing Machine (Model 5565, Instron, Norwood, MA, US) with a crosshead
speed of 0.5 in/min, and a gage length of 5 in (ASTM D882-02 [118]). Tensile properties
of neat and antimicrobial sheets PLA was determined from an average of 10 tests at 23°C
and 50%RH. Ten replications of each type of sheet were tested.

Permeation cells made from poly(methylmethacrylate) were used to determine

gravimetrically by using a modification of ASTM Standard Method B 96-80 [149] the

62

water vapor transmission rate (WVTR) of neat and antimicrobial PLA. Cells were filled
with desiccant and placed in an environmental chamber set at 37.8°C and 85% RH. The
weights of the cups were recorded daily. Linear regression-derived lepes of the steady
state (linear) portion of weight loss versus time curves were used to estimate WVTR. The
mean of three replications of each type of sheet were tested and the initial and final
stagnant air gap height was used in the calculations. The permeability (kg-m/mz-s-Pa)

was calculated as:

=WVIRxL
AP

P

 

where L is the mean film specimen thickness (In) and AP partial water vapor pressure

difference (Pa).

2.8 Statistical analysis.

Statistical analysis of the results was performed using a univariate analysis of variance
(ANOVA). Means were separated using the Tukey honestly significant difference (HSD)
test (p < 0.05) and a t-test was used to analyze statistical differences between the sheets in
the analytical software SPSS version 15 (SPSS Inc., Chicago, IL, USA). The data were
analyzed and graphically plotted using Sigma-plot software version 10 (Systat Software

Inc., Richmond, CA).

63

3. Results and discussion

3.1 Antimicrobial effectiveness of ICs made of fl-CD and trans-Z-hexenal

Previous studies carried out by this research group have shown the effectiveness of pure
trans-2-hexenal against the firngi Alternaria solani, Botrytis cinerea, Colletotrichum
acutatum, Penicillium sp., and Aspergillus niger [150]. Based on these results and on the
characterization of the release of trans-2-hexenal from ICs, 4 g of ICs were calculated to
release enough trans-2-hexenal into a volume of 0.5 L of air (bioassay system) to prevent
the growth of all the fimgi listed above. The high relative humidity necessary to release
the trans-2-hexenal from the ICS was provided by the culture media enclosed in the
bioassay system.

Effectively, 4 g of ICS generated a concentration of approx. 1 [IL trans-2-hexenal/L air
figure 4 (panel on the right) which was sufficient to avoid fungal development over a
seven day storage period at 23°C (figure 4, panel on the left). This occurred in all
bioassay systems but the ones containing Penicillum Sp where a lower trans-2-hexenal
concentration was observed. This was possibly due to the higher affinity of this volatile
for the cell membrane of the Penicillium sp. spores. Inouye et a1 (2001) showed that
different essential oils have a different gaseous contact with bacteria depending on their
permeability and/or interaction with the cell membrane [151]. In agreement, Almenar et
al reported that toxic volatile molecules have different affinities to different fungal cell
membranes [11]. Independently of the initial trans-2-hexenal concentration, the volatile
decreased in all bioassays due to its absorption by the culture medium as has been

reported to happen for other naturally occurring volatiles like acetaldehyde and hexanal

64

[1 1-12]. Remaining concentrations of trans-Z-hexenal in the bioassay systems were
similar for all fungi (figure 4, panel on the right).

The antimicrobial effect of ICS of trans-2-hexenal has been investigated earlier [36].
Trans-Z-hexenal at a concentration of 30 ug/mL showed a bactericidal effect against
Staphylococcus aureus IF012732. The results of this study cannot be compared with our
results directly since the volatile was encapsulated into a different type of CDs, and only
the bactericidal capacity was investigated. ICS of other naturally occurring volatiles also
have been tested against fungal growth. An amount of 1.2 g of ICs of hexanal was tested
against Colletotrichum Acutatum. Since fungal growth was observed, it was concluded
that the amount of IC necessary to prevent all growth of this fungus is higher than 1.2 g
[12]. An amount of 0.7 g of ICS of acetaldehyde was tested against Alternaria alternata.
No fungal growth was observed over a seven day storage period at 23°C [11]. In our
study, the remaining amount of trans-2-hexenal in the headspace of the bioassay systems
was lower than those reported for hexanal and for acetaldehyde. This is possibly
explained by the higher affinity of trans-2-hexenal to the fungal membrane. A higher
effectiveness has been reported for trans-2-hexenal than for hexanal or for acetaldehyde
and the volatile effectiveness has been related to the affinity of the volatile to the cell
membrane [1 1].

The fungicidal and/or fungistatic effect of trans-Z-hexenal was investigated and the
results are presented in Figure 19 (panel on the left). Trans-Z-hexenal is considered
fungistatic if growth of the fungus occurred after it was transferred from the bioassay
system containing the volatile to media in a jar free of the volatile. If no growth is

observed after transfer to an empty jar, then it is considered fungicidal. Figure 19 (panel

65

on the left) shows that trans-2-hexenal had fungicidal activity against the five fungi
tested. Therefore, trans-2-hexenal can be considered fungicidal at a concentration as low
as 1 [IL trans-2-hexenal/L air. Higher values have been reported in the literature for other
aldehydes in model systems. Hexanal, at a concentration of 3.3 pL/L air had a fungicidal
effect against C. acutatum but not against B. cinerea [12]. In real systems, trans-2-
hexenal has been shown more effective against fungal and bacterial grth than other
naturally occurring volatiles. An amount of 20 uL/L of trans-2-hexenal proofed
bactericidal for Listeria monocytogenes inoculated fresh-cut apples, while 150 uL/L of
hexyl acetate were necessary to achieve the death of this fungus. These results show that
trans-2-hexenal is more effective against major postharvest pathogens than other
naturally occurring volatiles. In Figure 4 (panel on the left) is also observed that controls
showed different fungal growth patterns after these were transferred to media in ajar free
of the volatile. The difference resulted from the fact that the initial spores were 14 day
old while the spores in the plugs transferred from one bioassay to another contained
spores only 7 days old, and this affects the germination process.

It has been reported that the antifungal activity of aroma compounds is dependent on their
vapor pressure rather than their concentration in the system which means that factors
such as temperature which are able to increase the vapor pressure of these substances can
enhance their antimicrobial activity [39]. Therefore, further research is necessary to find
key factors able to increase the vapor pressure and therefore the antimicrobial activity of

ICs of trans-2-hexenal.

66

Length of colony (cm)
O—waibOIO—th-bOIO-INOD-bmOdeAmO-le-bm

 

p
h

p
h
In

 

 
  
    
 

—+— Without ICs first week
——0— With le
+ \Mthout ICs

—A— With le

 

 

J

 

 

 

Days at 23°C

 

Altemarla Solani

- 1.0
- 0.8
. 0.6
i 0.4
- 0.2

 

Asperglllus nlger

Woe

0.0
) 1.0

r 0.8

r 0.4
. 0.2

 

second week

Botrytls cinerea

pl trans-2-hexenal IL air

0.0
. 1.0

i 0.8
. 0.6
. 0.4
r 0.2

 

Colletotn'chum acutatum

0.0
~ 1.0

l 0.8
i 0.6
- 0.4
- 0.2

 

Penicillium sp.

0.0
- 1.0

- 0.8
r 0.6
r 0.4
I 0.2

 

 

F

012345678

Days at 23°C

0.0

Figure 19. Left: (-0-) Inhibition of the growth of the postharvest decay fungi

67

Figure 19 (continued). Alternaria solani, Aspergillus niger, Botrytis cinerea,
Colletotrichum acutatum and Penicillium sp. during exposure to trans-2-hexenal released
from ICs over a seven day storage period at 23°C. (-e-) Growth rate of the same fungi
without exposure to trans-2-hexenal over a seven day storage period at 23°C (controls). (-
A-) Inhibition of the growth of the same fungi after those being transferred to trans-2-
hexenal-free atmosphere bioassay systems in new media over a seven day storage period
at 23°C. (- A-) Growth rate of the controls after those being transferred to an trans-2-
hexenal-free atmosphere bioassay systems in new media over a seven day storage period
at 23°C. Right: (-A-) Concentration of trans-2-hexena1 released from [CS over a seven day

storage period at 23°C.

Scanning electron micrographics of the surface of fractured B-CD and ICs during their
synthesis (with or without trans-2-hexenal), and after release of trans-2-hexenal were
taken to better understand the interaction between B-CDs and trans-2-hexenal. All
samples were prepared in the same way, and all micrographics were taken under the same
conditions. The results are shown in Figure 20. Panel (a) shows B-CDS, while panels (b),
(c), and (d) show the micrographics for ICs during their synthesis (with or without trans-
2-hexenal) and after the release of trans-2-hexenal. B-CDS are naturally of the size of A,
which is below the resolution of SEM with a magnification of x500. Since the molecules
shown in panel (a) are much larger than the individual B-CDS, it can be concluded that [3-
CDs occur in nature as conglomerates and not as a single molecule. This might cause due
to the surrounding relative humidity which interacts with the single molecules and helps

to form a more complex structure. Comparing panel (a) with panels (b) and (c) reveals

68

that in presence of water the B-CDs form a much larger conglomerate which seems to
have a more crystal-like structure. If panels (b) and (c) are compared, panel (b) shows
more similarity to the B-CDs conglomerates observed in panel (a) than can be found in
panel (c). Therefore, the complexation of the trans-2-hexenal into the B-CDs results in a
more pronounced change in the structure of the conglomerate, towards a more crystal-
like structure. This agrees with Szejtli (1998) who reported that formed inclusion
complexes can be isolated as stable crystalline substances [152]. Panel (d), which shows
the ICs after the release of the entrapped volatile, illustrates the reduction of the size of
the conglomerate. This reduction may be due to the breakdown of the crystalline structure
when exposed to relative humidity and two possible reasons could be given: (1) the water
interacts with the crystal and breaks it down, or (2) the release of the volatile creates a

less stable structure which breaks down. Further studies such as X-ray diffraction are

necessary to verify the crystal sizes.

69

 

Figure 20. Scanning electron micrographics of the surface of fractured B-CDS and ICs.

Panel (a) shows B-CDS, (b) shows B-CDS after simulating the IC synthesis but without
using trans-2-hexenal, (c) shows ICs after complexation with trans-2-hexenal, (d) shows

ICs after release of trans-2-hexenal (24 hrs exposed to 100% RH).

3.2 Antimicrobial effectiveness of pellets and sheets made of PLA and B-CD-trans-Z-

hexenal

70

 

 

 

 

 

 

 

 

 

 

 

5 ‘ (a)
A 4
g i
>
5
T: 31
0
3
i3. 2 i
5
'l -0- Control

1 i -¢— Treatment

.2 a; a. 4:. 3% A
o I I I I I I I
0 1 2 3 4 5 6 7 8
Days at 23°C

5 ‘ (b)
e 4-
3
>0
5
a 3 '
0
'6
S 2 -
5
.i

1 ..

0 I I I I I7 I I

o 1 2 3 4 5 6 7 8

Days at 23°C
Figure 21. Colony diameter of Alternaria Solani cultures in the presence (-A-) and
absence (-O-) of trans-2-hexenal released from antimicrobial pellets (a) and sheets (b)

during a 7 days storage period at 23°C .

71

Since Alternaria solani was the most sensitive to trans-2-hexenal of the five fungi listed
above (data not shown), the antimicrobial effectiveness of PLA/B-CD-trans-2-hexenal
pellets and sheets was determined for this fungus only. Figure 21 shows the colony
diameter of Alternaria Solani cultures in the presence and absence of trans-2-hexenal
released from antimicrobial pellets (a) and sheets (b) during a 7 days storage period at
23°C. As is evident from panel (a), PLA/B-CD-trans-Z-hexenal pellets inhibited
completely the growth of Alternaria Solani during 1 week at 23°C while panel (b) shows
that the PLA/B-CD-trans-2-hexenal sheet only delayed the Alternaria growth. The
complete inhibition of growth due to the release of trans-2-hexenal from pellets is
visualized in figure 22. The concentration achieved in the sealed jars containing
antimicrobial pellets after 24 hrs was 008le air (figure 23). Therefore, a concentration
as low as 0.08 uL/L air can suppress Alternaria grth during at least 1 week at room
temperature. This concentration is much lower than that reported above as effective for
trans-2-hexenal but this is because the above reported IC amount was chosen high
enough to inhibit all five fungi. Alternaria grth in the presence or absence of the
antimicrobial sheet showed statistical differences until day 3. The fungal growth was
reduced by 21% at day 2 and by 7% at day 3. The different antimicrobial effect between
pellets and sheets was due to the different amounts of trans-2-hexenal released from the
polymeric matrixes (figure 23). This difference was expected since the pellets contained
twice the amount of ICs than the sheets (the pellets were used as a masterbatch to form
the antimicrobial sheet). However, the concentration of trans-2-hexenal achieved in the
bioassay system containing the antimicrobial sheet was four orders of magnitude lower

than the expected level of 50% of the value achieved using the antimicrobial pellets. The

72

different concentrations of trans-Z-hexenal released from pellets and from sheets were
also caused by the fact that pellets were obtained using less processing (no casting
process was involved since no sheet was formed). It has been reported that ICs are
sensitive to external stimuli, such as temperature changes, shear forces, and the presence
of competing CD-binding molecules [91, 144]. Therefore, the exposure of the ICs to high
heat and relative humidity during processing caused a premature loss of the antimicrobial
compound encapsulated in the B-CD molecules for later release, and resulted in a reduced
antimicrobial activity. In agreement, Han et al (2003) reported that the antimicrobial

capacity of the polymer may change during processing [7].

 

Figure 22. Alternaria Solani in the absence (a) and presence (b) of antimicrobial pellets

after 7 days at 23°C.

73

The initial concentrations reached in the bioassay systems containing sheets or pellets
decreased during storage due to the absorption of the antimicrobial volatile by the culture

medium as has been reported above to happen for the trans-2-hexenal released from ICs.

 

 

 

 

 

8e-2 1
-0— Pellets
—a— h

6e-2 q S sets
5
'5
d
3 43'2 "
C
O
a
4.= 2e-2 )/ /
N
(I) 2864/ /(
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3 1e-6i

0 i 3 CJ

0 1 2 3 4 5 6 7 8
Days at 23°C

Figure 23. Evolution of trans-2-hexenal released from pellets (-O-) and sheets (-t:J-) in the
headspace of the bioassay systems containing Alternaria Solani cultures during 7 days of

storage at 23°C.

The release of antimicrobial compounds from a polymer can be regulated by controlling
the rate of wall solubility, the swelling of the wall material, pH effects, or changes in the
ionic strength of the surrounding medium [153]. More research is required to better
understand the effect of processing and/or the better incorporation of trans-2-hexenal in

the PLA matrix by using CDs.

74

3.3 Physical, mechanical, and barrier characterization of the antimicrobial sheet
The antimicrobial sheet was characterized by measuring its physical, mechanical, and
barrier properties. The neat PLA sheet was used as a control. The results are presented in
table 7. The DSC data shows slightly lower glass transition and melting temperatures for
the antimicrobial sheet than for the neat PLA sheet (approx. 1.5 and 2 °C for T3 and Tm,
respectively) but there were no statistical differences between both sheets. Byme et al.
(2009) observed no effect of the addition of several commercial masterbatches on the T8
and Tm of the PLA either [87]. The antimicrobial sheet and the neat PLA sheet showed
the same crystalline content in spite of B-CDS having been reported to play a role as
nucleating agents in PLA [128]. In contrast, Joo et a1. (2010) observed an effect on all the
thermal properties mentioned above when a masterbach formed from PLA containing
high B-CD content was blended with PLA [147]. These results may differ due to the
change in morphology of the B-CDS after the encapsulation of the antimicrobial as can be
observed in Figure 20, panels (a) and (c), or that the presence of the antimicrobial
compound affected the crystal growth during processing.PLA crystallinity has been also
reported to increase when the PLA has been blended with other molecules similar to CD3
like starch [88, 128-129].

The mechanical properties tensile strength (machine direction (MD) and cross-machine
direction (CD)), modulus of elasticity, and elongation at break of the neat PLA sheet and
the antimicrobial sheet are also presented in Table 7. There were significant differences
in the tensile strength and the modulus of elasticity but not in the elongation at break
between the neat PLA sheet and the antimicrobial sheet. The antimicrobial sheet showed

about 50% smaller values in these preperties which were caused by the filler effect of the

75

B-CDS in the PLA matrix as reported by Joo et a1. (2010). The presence of the B-CDS
produced the discontinuity between the PLA chains caused by the immiscibility between
PLA and B-CDS. Similar results have been reported also for blends of PLA and starch
[154]. The decrease in the tensile strength of PLA by adding a masterbatch has been
previously reported iny the literature. Byme et a1. [87] reported a reduction in 3-5% of
the tensile strength of the PLA by the addition of 4 different commercial masterbatches.
The water vapor permeability coefficient (WVPC) of the antimicrobial sheet and of the
neat PLA sheet was measured using permeation cells. The permeability coefficients were
calculated as 5.0i0.4><10'ls kg rn/m2 s Pa for the neat PLA sheet and 4.8d:0.7><10'l4 kg
rn/m2 3 Pa for the antimicrobial sheet. These WVPC are close to the values 2.4><10'15 kg
m/m2 5 Pa and 7.1 ><10'ls kg m/m2 5 Pa reported by Joo et a1. (2010) for neat PLA and for
PLA blended with B-CDS using a masterbatch. The WVPC of the neat PLA was lower
than the value 1.5~2.2><10'l4 kg m/m2 5 Pa reported in the literature for neat PLA at 10 to
37.8°C and 40 to 90% RH [76]. This might be due to differences in thickness, processing
history, and other parameters between these neat PLA samples. The permeability of the
antimicrobial sheet was higher than that of the neat PLA sheet due to the immiscibility
between phases and the resulting gaps. Similarly, it has been reported that water can
penetrate faster in PLA/starch blends than in neat PLA through the voids between phases
[79]. The antimicrobial sheet had a higher WVPC than the sheet resulting of blending
PLA with B-CDS using a masterbatch reported by Joo et a1. (2010), and this was caused
by less adhesion between [Cs and PLA than between B-CDS and PLA. This is due to

differences in structure between ICs and B-CDs as shown in figure 20. Further research

76

needs to be done for a better understanding of the effect of the volatile on the

characteristics of the polymeric matrix.

Table 7. Characterization of the neat PLA sheet and the antimicrobial sheet

 

 

 

 

 

 

 

 

 

 

 

 

Neat PLA Antimicrobial
Property Type sheets sheets
Tg 55.2;t2.0 a 53411.0 a
Thermal Tm 143.4:l:0.3 a 14l.6i0.8 a
%x 1.2d:0.9 a 1.7i1.5 a
Tensile strength (MPa)-MD 55.6:t4.5 a 17.6i2.2 b
Tensile strength h(MPa)-CD 54.1i4.6 a 16.8il .4 b
Elongation at break (%)-MD 1.0i0.2 a 0.6:t0.1 b
Mechanical
Elongation at break (%)-CD 0.8i0.2 a 0.5i0.4 b
Modulus of elasticity (GPa)-MD 10.0:t0.7 a 5 .4i0.7 b
Modulus of elasticity (GPa)-CD 10.1i0.5 a 5.7i0.4 b
. Water vapor permeability x y
Barrier (kg m/m2 5 Pa) 5.0i0.4 a 4.8i0.7 b
a and b indicate statistical differences(P<0.05) between neat PLA and antimicrobial
sheets.
X X 10-15
Y ><10'14

4. Conclusions

Enzyrnatically modified starch containing trapped trans-2-hexenal is an effective tool to
control major postharvest pathogens. Its incorporation in the PLA polymeric matrix
yields bio-based antimicrobial polymers. The addition of the antimicrobial didn’t affect

the physical pr0perties of the polymer matrix but did affect the mechanical, and barrier

77

ones. The exposure of the enzymatically modified starch containing trapped trans-2-
hexenal to high heat and relative humidity during processing caused a premature loss of
the antimicrobial compound, and resulted in a reduced antimicrobial activity of the

antimicrobial polymer.

Acknowledgement. The authors thank Atul Singla, Jin Shan, and DongHo Kang with
their help with analyzing and processing of the samples, and Carol Flegler and Abigail

Tirrell with their help with taking the scanning electron micrographics. The authors also

thank Drs. Phillip Wharton and William Kirk, from the Department of Plant Pathology at

MSU, for providing some of the fungal culture used in this study.

78

CHAPTER 5. CONCLUSIONS

A bio-based polymer made of PLA and B-CDS with the capacity to carry hydrophobic
molecules has been developed and characterized. Testing of mechanical, thermal, barrier,
optical, and morphological properties revealed that B-CDs and PLA are incompatible.
The higher the B-CD content, the higher the incompatibility due to the increase in size of
the B-CDs conglomerates, and the poor adhesion between phases. The use of a
masterbatch (PLA containing high B-CDL content) is an effective way to improve the
compatibility between PLA and B-CDS, and the properties of PLA/B-CDS blends. The use
of the masterbatch reduced the stiffness and brittleness of the PLA/B-CDS blend and
increased their barrier properties.

anymatically modified starch (B-CDS) containing trapped trans-2-hexenal (ICs of trans-
2-hexenal) is an effective tool to control major postharvest pathogens. Its effectiveness
has been confirmed against 5 main postharvest fungi. Its incorporation in the PLA
polymeric matrix yields bio-based antimicrobial polymers (pellets and sheets).
Antimicrobial pellets (30% of ICs) showed fungicidal activity against Alternaria Solani
and antimicrobial sheets delayed Alternaria Solani growth during day 3. The addition of
the antimicrobial didn’t affect the physical properties of the polymer matrix but did affect
the mechanical, and barrier ones. The exposure of the enzymatically modified starch
containing trapped trans-2-hexenal to high heat and relative humidity during processing
caused a premature loss of the antimicrobial compound, and resulted in a reduced

antimicrobial activity of the antimicrobial polymer (90%).

79

Future work

To improve the understanding of the encapsulation mechanism and of the
structure of the ICS (B-CD-trans-Z-hexenal-water) and to correlate these data with
the effect of humidity and temperature on the releasing process.

Release kinetics of the ICs of trans-2-hexenal, pellets, and sheets as a function of
temperature and relative humidity

Effect of the presence of the antimicrobial compound on the compostability of the
PLA/ B-CDS blends.

Shelf life studies of the antimicrobial pellets and sheets (the antimicrobial agent
remains above the critical inhibiting concentration).

To explore potential commercial applications of the new bio-based antimicrobial

materials (pellets and sheets).

80

10.

11.

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