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Influence of packaging material and storage time on physical, chemical and

microbiological properties of set yogurt: A comparative study between
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Journal of Engineering Science and Technology
Vol. 11, No. 10 (2016) 1437 - 1449
© School of Engineering, Taylor’s University

INFLUENCE OF PACKAGING MATERIAL AND STORAGE TIME

ON PHYSICAL, CHEMICAL AND MICROBIOLOGICAL
PROPERTIES OF SET YOGURT: A COMPARATIVE STUDY
BETWEEN MODIFIED BIODEGRADABLE POLY(LACTIC ACID)
AND POLYPROPYLENE
1 2
NAWADON PETCHWATTANA *, PHISUT NAKNAEN
1
Division of Polymer Materials Technology,
2
Division of Food Science and Nutrition,
Faculty of Agricultural Product Innovation and Technology,
Srinakharinwirot University, Sukhumvit 23, Wattana, Bangkok 10110 Thailand
*Corresponding Author: [email protected]

Abstract
The current paper investigates the influence of storage time and type of
polymeric packaging material on the chemical, physical and microbiological
properties of set yogurt. Firstly, poly(lactic acid) (PLA) was modified by using
a core-shell rubber (CSR) and an acrylic processing aid (PA) to produce PLA
with high toughness and good processability. Secondly, an appropriate
PLA/PA/CSR composition was selected and fabricated to yogurt cup. The
yogurt was stored in both modified PLA and polypropylene (PP) packages to
observe some physical, chemical and biological changes. Finally, the
biodegradation test was made on both packages and compared with that
cellulose. Experimental results revealed that adding 5wt% CSR gave PLA/PA
as tough as PP. Types of packaging material and storage time did not change
the color of yogurt. The number of lactic acid bacteria grew significantly after
they had been incubated for 6 days. The bacterial viability decreased
dramatically due to the increased acidity and the decreased pH. A positive
impact on the viability of bacterial growth was found when yogurt was stored in
modified PLA package. This made yogurt had more health benefits than stored
in PP package. The biodegradation test results indicated that the modified PLA
degraded at a rapid rate. It achieved approximately 50% biodegradation within
40 days which was comparable to the time required to degrade the cellulose,
whereas PP was non-biodegradable over the period studied. In summary,
substitution conventional PP by a novel modified PLA seems to be a better way
for both the health and the environment benefits.
Keywords: Biodegradable polymer; Food quality; Set yogurt; Biodegradation.

1437
1438 N. Petchwattana and P. Naknaen

Nomenclatures

a* Redness and Greenness

b* Yellowness and Blueness
L* Lightness
Tg Glass transition temperature, oC

Abbreviations
ANOVA Analysis of Variance
AR Acrylate rubber
ASTM American Society for Testing and Materials
CRD Completely Randomized Design
CSR Core-shell rubber
EGMA Poly(ethylene-glycidyl methacrylate)
ISO International Organization for Standardization
LDPE Low Density Polyethylene
MFI Melt Flow Index, g/10min
MRS de Man, Rogosa and Sharpe
OTR Oxygen Transmission Rate, ml/m224days
PA Processing aid
PCL poly(-caprolactone)
PET Poly(ethylene terephthalate)
PLA Poly(lactic acid)
PMMA Poly(methyl methacrylate)
PP Polypropylene
PS Polystyrene
RH Relative Humidity, %

1. Introduction
Nowadays, fossil based plastics have been extensively utilized as food packaging
materials due to their availability, processability, good mechanical and thermal
stabilities and low cost [1-2]. However, the disposal of petroleum based plastics
products also contribute significantly to environmental problems due to their non-
biodegradability [3-4].
For disposal issue, conventional petroleum based plastics are being substituted
with biodegradable and renewable materials [5-6]. Poly(lactic acid) (PLA) is an
example of the biodegradable materials which have drawn more attention from
both industries and research institutions [7-8]. Although PLA has been known for
more than a century but it has only been of commercial interest in recent years.
PLA offers numerous advantages such as biodegradability, recyclability,
renewability, commercial availability and good processability [8-10]. However,
brittleness, low resistance to impact and extremely low crystallization rate are the
major parameters restricting the use of PLA in many value-added applications [1,
8-10]. These have motivated many researchers to toughen PLA prior to utilize as
food packaging. Petchwattana et al. [4] toughened PLA with ultrafine fully
vulcanized acrylate rubber (AR). Adding 10wt% AR made PLA tougher with the

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

 Influence of Packaging Material and Storage Time on Physical, Chemical . . . . 1439

increased tensile elongation at break and impact strength by 40 and 4 times

respectively. Cabedo et al. [11] found an improvement in toughness and gas
barrier properties of the film prepared from PLA/poly(-caprolactone)
(PCL)/nanoclays hybrid composites. During storage, Koide and Shi [12] found
insignificant differences in the color, weight loss, hardness and ascorbic acid
content of the green peppers stored in PLA and low density polyethylene (LDPE).
However, PLA containers were found to be the cause of a faster wine quality loss
compared to poly(ethylene terephthalate) (PET) and glass due to its higher
moisture permeability [13].
This study aims to examine the possibility of using modified PLA as a
biodegradable package for set yogurt. The impact modified PLA and
polypropylene (PP) packages were fabricated through sheeting and
thermoforming process used for making yogurt cup. The performances of each
package were evaluated by means of biological, physical and chemical properties.
A set yogurt was selected as a food simulant for the PP and the modified PLA
cups. To extend the study, further evaluation was made by observing the
biodegradation of both packages and compared to cellulose.

2. Experimental Works
2.1. Materials
An extrusion/thermoforming grade of PLA (PLA2003D, NatureWorks LLC) was
used as a polymer matrix. Its melting range and density at room temperature were
160-170oC and 1.24g/cm3 respectively. Fine particles of core shell rubber (CSR)
(ParaloidTM BPM-515, Dow Chemical, USA) were applied to the PLA for
toughening purpose. The glass transition temperature (Tg) of the acrylic rubber
core and the poly(methyl methacrylate) (PMMA) shell were -40 and 105oC
respectively. The acrylic processing aid (PA) (ParaloidTM BPMS-260, Dow
Chemicals, USA) was applied to PLA for rheological modification purpose. Its
density and average particle size were 0.45g/cm3 and 200nm respectively. A
thermoformimg grade PP (Moplen HP748H, HMC Polymers PLC) was selected
as a conventional yogurt cup reference. Table 1 shows the blend formulations of
PLA, PA and CSR.

Table 1. Formulations of PLA, PA and CSR.

Formulation code Material compositions (wt%)
PLA CSR PA PP
PLA 100 0 0 0
PLAPA 99 0 1 0
PLAPACSR1 98 1 1 0
PLAPACSR3 96 3 1 0
PLAPACSR5 94 5 1 0
PLAPACSR7 92 7 1 0
PLAPACSR10 89 10 1 0
PP 0 0 0 100

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

1440 N. Petchwattana and P. Naknaen

2.2. Preparation of the modified PLA

The preparation of the impact modified PLA in the present study consisted of 3
experimental parts. The first was the dry-blending of PLA with 1wt% PA and various
CSR contents of 1.0, 3.0, 5.0, 7.0 and 10wt%. The PLA/CSR/PA compositions were
then melt-blended and pelletized by using a co-rotating twin screw extruder (Labtech,
LTE 20-40) and a pelletizer. The screw speed was set constantly at 100 rpm while the
barrel temperature was ranged from 155 to 185oC. Finally, they were injected by
using an injection molding machine (Manumold) for subsequent the mechanical tests.
The injection conditions were clearly indicated in Table 2.

Table 2. Injection molding conditions.

Designations Value
Barrel temperature Zone1/Zone2/Zone3 190/195/200 oC
Injection feed forward I pressure 40-80 bar
Injection feed forward II pressure 40-80 bar
Injection feed forward III pressure 40-80 bar
Holding pressure 20 bar
Mold clamping pressure 60 bar
Ejector pressure 40 bar
Mold temperature 45oC

2.3. Fabrication of modified PLA and PP yogurt cups

After selecting a desired PLA/CSR/PA composition from the mechanical test results
in Section 2.2, PLA CSR and PA were compounded by using an industrial-scaled
twin screw extruder and then pelletized to obtain the PLA compound pellets. PLA
compound and neat PP were then sheeted and thermoformed by using an industrial-
scaled sheet extruder equipped with chill rolls (Meaf, Sheetline 75-H34P) and
thermoforming machine (Cheng Met, CM F1000). The average thickness of the sheet
and the thermoformed yogurt cup was 450 and 250 µm respectively.

Fig. 1. Visual appearance of set yogurt stored in (a) PP and (b) modified PLA
packages.

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

 Influence of Packaging Material and Storage Time on Physical, Chemical . . . . 1441

2.4. Preparation of set yogurt and storage condition

The skimmed milk was inoculated with yogurt culture and incubated. After pre-
cooling at ambient temperature, the yogurt sample was filled in both the modified-
PLA and PP cups prior to seal with aluminium foil as illustrated in Fig. 1. The yogurt
was then incubated at 5oC for 18 days to observe the quality changes.

2.5. Testing and characterizations

2.5.1. Testing and characterizations of modified PLA and PP
A notched Izod impact test was evaluated by using an impact tester (Yasuda, 190)
in order to determine the energy required to break the test specimen upon impact
force following the procedure described in ASTM D 256. The oxygen
transmission rate (OTR) was evaluated in accord with ASTM D3985 by using a
gas permeation tester (Mocon OX-TRAN, 2/21). The temperature and relative
humidity (RH) of the test conditions were 25ºC and 0%RH respectively. The
samples were conditioned for 5 hours prior to test. The OTR determination was
performed until 10 values of the constant transmission rate were obtained. The
final transmission rate was averaged over the last 10 constant values for the
replicate samples. The melt flow index (MFI) was evaluated following the
process described by ASTM D 1238. The temperature and the piston load were
set at 190oC and 2.16kg respectively.

2.5.2. Testing of the food stimulant

Color measurement was carried out by using a Hunter Lab Colurflex colurimeter.
The colurimeter was adjusted for reflectance, illuminant D 65, and angle of 10 o.
Color measurement data was provided in accord with the CIE system in terms of
L* (lightness), a* (redness and greenness) and b* (yellowness and blueness).
The textural hardness measurement was applied on set yogurt at room
temperature by using a universal testing machine (Instron, 5966) equipped with a
1cm diameter stainless steel probe. It was employed to evaluate the hardness of
the set yogurt during storage in two different packaging materials. The cross-head
speed and the compression distance were set at 5 mm/min and 2.5 mm
respectively. A maximum load of ten replicate samples were averaged and
reported as the hardness.
The syneresis value of the yogurt was evaluated following the procedure
described by Keogh and O’Kennedy [14]. Twenty five grams of set yogurt were
prepared in a centrifuge cup and then centrifuged at 2500 rpm for 10 min at room
temperature. The transparent phase was then collected and measured. The
syneresis value was calculated following Eq. (1):

(1)

The acidity was estimated following the process described by Mistry and
Hassan [15]. The pH was determined by using a digital pH meter (Mettler Toledo,

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

1442 N. Petchwattana and P. Naknaen

FiveEasyTM pH). The lactic acid bacteria count was analyzed using the pour plate
technique on MRS agar and the plates were incubated at 37 oC for 2 days.

2.5.3. Biodegradability of packaging materials

The biodegradability of yogurt cup was estimated by measuring the carbon
dioxide content following the procedures described in ISO 14855 using a
Microbial Oxidative Degradation Analyzer (Saida FDS, MODA 6).

2.5.4. Statistical analysis

The experimental design was a completely randomized design (CRD). Data was
subjected to analysis of variance (ANOVA). The comparison of means was
carried out by Duncan’s multiple-range test.

Table 3. Mechanical properties of PP, PLA and modified PLA.

Formulation Impact strength MFI at 190oC OTR
code (J/m) (g/10 min) (ml/m224days)
PLA 24.10.12h 6.90.47a 58.20.64c
g d
PLAPA 25.70.19 2.10.60 n/a
PLAPACSR1 31.60.89f 2.41.31d n/a
PLAPACSR3 57.00.60e 2.30.05d n/a
PLAPACSR5 98.60.06c 2.91.50d 64.60.13b
PLAPACSR7 114.02.89b 4.30.43c n/a
a
PLAPACSR10 129.11.33 5.61.58b n/a
PP 97.60.47d 2.70.21d 19755.96a
For each column, means with the same letter do not differ significantly at p0.05
n/a = no data available

3. Results and Discussions

3.1. Physical and mechanical properties of packaging materials
It is widely known that the problems of brittleness and low impact resistance have
limited the use of PLA in many applications especially for food packaging [1, 8-
10]. A notched Izod impact test was employed to determine the impact resistance
of neat PLA and modified PLA and compared with that PP. With the presence of
1wt% PA, PLA showed slightly higher impact strength while the MFI was
dropped by more than threefold approaching the MFI value of PP. As shown in
Table 3, a substantial increment in the impact strength was observed in all
PLA/CSR/PA compositions. Adding 5wt% CSR to PLA/PA increased the impact
resistance higher than that observed in neat PLA and PP. This indicated that CSR
particles could absorb larger amounts of energy upon the impact force [4] than
that was observed in the neat PLA and PP. Numerous studies have reported a
drastic increment in the impact strength when an acrylic based toughening agent
was added to PLA. Petchwattana et al. [4] found a rise in the impact strength by
four times when AR was added to PLA at 10wt%. Oyama [16] reported a fourfold
increment in the impact strength when the poly(ethylene-glycidyl methacrylate)

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

 Influence of Packaging Material and Storage Time on Physical, Chemical . . . . 1443

(EGMA) was added to PLA. In term of mechanical performance and

processability, the PLA/CSR/PA blend could be used as a yogurt package as good
as the PP could.

Fig. 2. Change in lightness (L*) and yellowness (b*) of set yogurt during
storage in different packaging material (a) L* and (b) b*.

8
7 PP modified PLA
6
Lactic acid bacteria (x10 cfu/g)

5
6

1
0 3 6 9 12 15 18

Storage time (day)

Fig. 3. Viability of lactic acid bacteria in set yogurt during storage in two
different packaging material.

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

1444 N. Petchwattana and P. Naknaen

100
PP modified PLA
90

80

70

Syneresis (%) 60

50

40

30

20

10

0
0 3 6 9 12 15 18
Storage time (day)

Fig. 4. Syneresis of set yogurt during storage in different packaging material.

3.2. Changes in properties of set yogurt

Fig. 2 shows the color measurements of yogurt samples with various storage time
and packaging materials. The yogurts stored in both PP and modified PLA cups
showed undetectable difference in terms of lightness and yellowness. The L* and
b* values were observed at around 92 and 10 respectively. These values indicated
that the set yogurts were originally white and light yellow. Types of packaging
material and storage time did not show significant (P>0.05) effect on the lightness
and yellowness of the yogurt.
Changes in the number of viable lactic acid bacteria over the storage period
were also investigated and graphically presented in Fig. 3. The bacterial amounts
were influenced by the packaging material as well as storage time. Initially, the
total lactic acid bacteria in the yogurt were 1.50×10 6 and 1.53×106 cfu/g for the
PP and modified PLA packages respectively. After storage at 5 oC for 6 days, the
number of bacteria grew significantly (P<0.05) with time and reached their
maximum values at 4.4×106 and 5.6×106 cfu/g for the PP and modified PLA
respectively. Beyond 6 days, the counts decreased dramatically due to the
increased lactic acid concentration and the decreased pH which directly reduced
the survival of the bacteria. The number of bacteria declined after being stored for
18 days by around 1.1×106 and 1.4×106 cfu/g for the PP and PLA packages
respectively. These values were larger than the minimum amount of bacteria
required for the health benefits [17]. In comparison, the modified PLA exhibited
higher bacterial counts than PP at all durations of storage. This is due to the fact
that lactic acid bacteria were categorized as facultative bacteria which required
less oxygen for survival. Higher OTR of PP (see Table 3) was the cause of their
viability and growth restrictions under the excess oxygen environment. This could
be preliminary summarize that the yogurt stored in modified PLA package gave
more health benefits than PP package. In milk, Klaver and others [18] also found

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

 Influence of Packaging Material and Storage Time on Physical, Chemical . . . . 1445

a negative impact on the viability of bacteria such as Lactobacillus and

Bifidobacterium spp. under a high concentration of oxygen. Another investigation
compared the survival of bacteria in yogurt stored in polystyrene (PS) and glass
bottles. The authors reported that the yogurt contained in glass bottle had more
survival of L. acidophilus than that stored in PS over 35 days [19].
Syneresis is generally recognized as the shrinkage of the gel bringing the
liquid separation from the yogurt curd, which is an undesired property. It is
directly correlated to the acidity and is inversely related to the pH [20]. Fig. 4
shows the changes in the syneresis of set yogurt during incubation. As expected,
the degree of syneresis increased at all yogurt samples and all storage times. This
rise can be separated in two periods. Earlier than day 9, the syneresis increased at
very rapid rate leading the aggregation of casein micelles. After this period, the
syneresis still increased but at much lower rate. Generally, syneresis of yogurt
occurs when the use of high incubation temperature, excessive whey
protein/casein ratio, low solid content and physical mishandling during storage
[21]. In this case, higher lactic acid bacteria levels tended to increase the
hydrogen ion concentration during acidification and were the cause of casein
micelles aggregation. This made set yogurt stored in modified PLA package had
higher syneresis value than PP package.

Fig. 5. Change in chemical properties of set yogurt during storage in

different packaging material (a) pH and (b) acidity.

Generally, the pH of the set yogurt is directly affected by the lactic acid
bacterial activity and storage time. Fig. 5 shows the pH and the acidity of set
yogurt during storage. The initial pH of set yogurt was 5.26 and 5.24 for modified
PLA and PP packages respectively. These values were dropped significantly
(P<0.05) by storage time. This can be explained by the fact that lactic acid was

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

1446 N. Petchwattana and P. Naknaen

produced by bacteria during storage. This inversely related to the increased

acidity as illustrated in Fig. 5(b). Of the two packaging materials, modified PLA
package was found to have lower pH than that observed in PP package. This was
due to the higher production rate of lactic acid derived from the higher bacterial
counts as previously discussed.

17

PP modified PLA
15

13
Hardness (N)

11

3
0 3 6 9 12 15 18
Storage time (day)

Fig. 6. Hardness of set yogurt during storage in different packaging material.

Hardness is one of the parameters that affect the sensorial perceptions of

consumers [22]. Fig. 6 illustrates the relationship between the hardness and the
storage time of the yogurt stored in the different packaging materials. The
hardness of the set yogurt within the PP and modified PLA packages at the day 0
was 8.63 and 8.62 respectively. Set yogurts kept in both packaging material
showed significant increment in the hardness during storage period (P<0.05). This
hardening of yogurt gels could be explained through protein aggregates being
formed by the interaction between the denatured whey proteins and the casein
micelles via intermolecular disulfide bonds [22-23]. Compared to PP package, the
modified PLA package showed a greater hardness which occurred beyond day 3
due to the significant increment in syneresis as previously discussed in Fig. 4.

3.3. Biodegradability of packaging materials

As illustrated in Fig. 7, the biodegradability of each packaging material was
evaluated by collecting the amount of CO2 compared to cellulose samples. Of the
materials studied, cellulose started to degrade earlier than modified PLA and PP
packages. Cellulose reached its maximum biodegradation of around 80% within
40 days which was valid the reference material defined by ISO 14855. The
modified PLA package started to degrade at a very slow rate around the first 10
days. Beyond this period, the modified PLA package tended to degrade at a faster
rate and achieved approximately 50% biodegradation at around 40 days. The

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

 Influence of Packaging Material and Storage Time on Physical, Chemical . . . . 1447

modified PLA package continued to degrade and reached a degradation plateau at

60 days while the PP package did not show any biodegradation. The maximum
degree of biodegradation of the modified PLA was around 70%. This clearly
confirmed the biodegradability of the modified PLA package. A similar display of
biodegradation was observed in the PLA/starch blends. Petinakis et al. [24] found
that that the rate of biodegradation for the PLA/starch blends (more than 80%)
was higher compared to that of pure PLA. Liu et al. [25] indicated that there was
a higher degradation rate of PLA package after incorporating sugar beet pulp.
From the economic point of view, the modified PLA package is more expensive
than PP packages by around 40%. This limitation still limited the use of PLA for
the commodity products such as yogurt.

100
Cellulose PP modified PLA

80
Biodegradation (%)

60

40

20

0 20 40 60 80
Degradation time (Day)

Fig. 7. Biodegradation of PP and modified PLA packages compared to

cellulose.

4. Conclusions
Adding 5wt% CSR gave PLA/PA as tough as PP. Types of packaging material
and storage time did not change the color of set yogurt. The number of lactic acid
bacteria grew significantly after they had been incubated for 6 days. After that,
the bacterial viability was decreased dramatically due to the increased acidity and
the decreased pH. A positive impact on the viability of bacterial growth was
found when set yogurt was stored in modified PLA package. This made the
yogurt had more health benefits than stored in PP package. The biodegradation
test results indicated that the modified PLA package degraded at a rapid rate. It
achieved approximately 50% biodegradation at around 40 days which was
comparable to cellulose whereas PP package was non-biodegradable. In
summary, substitution conventional PP package by a novel modified PLA
package seems to be a better way for both the health and the environment
benefits.

Journal of Engineering Science and Technology October 2016, Vol. 11(10)

1448 N. Petchwattana and P. Naknaen

Acknowledgements
The authors acknowledge the research grant from Srinakharinwirot University
(Contract no. 117/2557) and supports from the National Innovation Agency
(NIA), Dairy Home Company Limited and Excel Packaging Company Limited.
Thanks are extended to Miss Koolrapas Bootpong and Miss Natchayathorn
Nattakornsoottanan for the preliminary study of the research.

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Journal of Engineering Science and Technology October 2016, Vol. 11(10)

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