Evaluating the behaviour of probiotic

Lactobacillus plantarum 299v in non-dairy oat

based yogurt using two different packaging
materials
Divya Mohan
DIVISION OF PACKAGING LOGISTICS | DEPARTMENT OF DESIGN SCIENCES
FACULTY OF ENGINEERING LTH | LUND UNIVERSITY
2017

MASTER THESIS
This Master’s thesis has been done within the Erasmus Mundus
Master Course FIPDes, Food Innovation and Product Design.

FIPDes has been funded with support from the European

Commission. This publication reflects the views only of the
author, and the Commission cannot be held responsible for any
use which may be made of the information contained therein.
Evaluating the behaviour of probiotic
Lactobacillus plantarum 299v in
non-dairy oat based yogurt using two
different packaging materials

Divya Mohan
Evaluating the behaviour of probiotic Lactobacillus
plantarum 299v in non-dairy oat based yogurt using two
different packaging materials

Copyright © 2017 Divya Mohan

Published by
Division of Packaging Logistics
Department of Design Sciences
Faculty of Engineering LTH, Lund University
P.O. Box 118, SE-221 00 Lund, Sweden

Subject: Food Packaging Design (MTTM01)

Division: Packaging Logistics
Supervisor: Dr. Annika Olsson
Co-supervisor: Dr. Jenny Schelin
Examiner: Dr. Erik Andersson

This Master´s thesis has been done within the Erasmus Mundus Master
Course FIPDes, Food Innovation and Product Design.

www.fipdes.eu

ISBN 978-91-7753-363-4
Abstract

There is an upsurge of consumer interest in functional foods, especially probiotics.

Alongside there is a global rise in the ‘vegan’ market. However, there is
insufficient research and development in the field of non-dairy probiotic food
formulations. Development of probiotic oat-based yogurt, called oatgurt that is
manufactured by Oatly AB, was studied by incorporating Lactobacillus plantarum
299v strain, which maintains the products’ vegan label. The two important factors
that could affect probiotic bacterial strain viability and oatgurt’s physicochemical
properties include the step of strain incorporation and presence of oxygen. The
probiotic strain was incorporated into two different food matrices; fresh oatgurt
(incorporated before fermentation) and commercial oatgurt (incorporated after
fermentation) maintained at 8°C# in# an# incubator# for# 8# weeks. The effect of
oxygen was evaluated by comparing polypropylene (PP) and glass as packaging
materials for the two food matrices. In both food matrices, the viability of the
strain in PP cups (~1 mm thickness), which has an oxygen transmission rate of
150-200 ml/m2.day.atm, was similar to the viability obtained in glass jars, which is
impermeable to oxygen. The presence of probiotic strain in oatgurt# resulted in a
gradual reduction in pH over time in both packaging materials. Glass had
comparatively superior effect on maintaining oatgurt colour stability than PP
(p<0.05), which was perceptible only after close observation even at week 8. The
overall comparative analysis showed that PP cups could be effectively used as
packaging units for probiotic oatgurt. Sensory evaluation and pilot scale
experimentation of the resulting probiotic oatgurt remains necessary to confirm
commercial product stability.

Keywords: oatgurt; Lactobacillus plantarum 299v; probiotic; polypropylene;

glass.
Preface

‘Go basic!’ When I started this project my view of science and research always
focused on finding innovative and alternative solutions. With a new food product
to study, my entire attention was on finding the most exciting solution. As I started
working on my project plan I soon realized when the basic questions were not
answered, every other research, no matter how well done cannot show you the
complete picture!
This is my attempt to answer a ‘basic’ question and further advance research on
non-dairy probiotic food products.

I would like to dedicate my work to my mom, dad and brother.

Lund, June 2017

Divya Mohan
Acknowledgments

I would firstly like to thank all my family, professors and friends who have shaped
my curiosity for science that led me to this project. Without your continuous
inspiration I would not have the zeal to explore and experiment.
I want to express my deep gratitude to Dr. Jenny Schelin who enriched my
knowledge and constantly supported me through out my journey during my thesis.
Thank you for being so kind and for teaching me how to meticulously plan and
execute a project. I am very grateful to Dr. Annika Olsson for her continuous
encouragement and inspiration. Thank you for all your valuable lessons that taught
me how to focus and pragmatically approach a question. They made my thesis
experience manifold richer.
I would like to thank Oatly for giving me an opportunity to work on their product.
I especially want to thank Dr. Karin Petersson for always encouraging me and
being open to ideas and innovation. Without your support and help I would not
have been able to successfully complete this project.
I want to thank Probi for trusting me with their product and helping me with my
analysis. I specially want to thank Anna Andrys and Fabian Skarvad for sharing
their ideas and knowledge with me that helped me design my experimental plan.
I am also grateful to Dr. Sören Vang Andersen who helped me understand the
world of statistics and numbers. Thank you for teaching me the importance of
presenting effective and reliable results.
I would like to thank Dr. Erik Andersson whose encouragement and positivity
helped me throughout my study at Lund University. Thank you for believing in me
and helping me at every step during my study.
I would like to thank Christer Larsson for his constant help and always having a
solution. I would also like to extend my gratitude to the entire Applied
Microbiology and Packaging Logistics divisions at Lund University for their
support during my study.
I would like to dedicate this work to my FIPDes family who have enriched my
experience away from home.
Lund, June 2017
Divya Mohan
Executive Summary

Introduction
Oatly AB, a Swedish food company founded in the 1990s, produces vegan milk-
product alternatives made from oats. It caters to the appeal for veganism and
vegan products, which is on a rise. This shift of food preference goes beyond a
niche group who avoid animal meat for ethical purposes, towards consumers
looking for a cleaner and healthier diet (Lea et al. 2006). With increased consumer
awareness in recent years, food products are not only being consumed to satisfy
hunger and basic nutritional requirements but also to enhance the quality of
physical and mental wellbeing (Siro et al. 2008). This has paved way for the
concept of ‘functional foods’, which include ingredients with additional health
benefits or that can support specific body functions that conventional nutrition
models do not address (Buttriss 2000; Menrad 2003). Probiotics represent a major
segment of this functional food market (Granato et al. 2010).
Probiotics are live microorganisms that are natural inhabitants of the human
gastrointestinal tract. The viability of probiotic strains in dairy products like yogurt
has been studied extensively, however the research on non-dairy food matrices is
limited. Among probiotic dairy products, yogurt has a wider consumer market
(Siró et al. 2008) and hence, Oatly’s spoonable oat based yogurt called ‘oatgurt’
was selected as the test product for the experiment (Figure 1). When probiotic
strain is incorporated in certain quantities into food matrices and ingested, they can
potentially improve the health of the host especially by contributing to intestinal
microbial balance (FAO/WHO 2002; Grajek et al. 2004). The survivability and
functionality of the probiotic culture is strain specific and depends on several
factors including method of incorporation, temperature (Mokarram et al. 2009),
pH, composition of the food matrix and level of available oxygen (Tripathi & Giri
2014).

Figure 1. Oatly’s vanilla/blueberry oatgurt in polypropylene cup.

Objectives
This study aimed to investigate the behaviour of a probiotic strain obtained from
Probi AB, Lactobacillus plantarum 299v (L. plantarum 299v), in Oatly’s non-
dairy oat-based yogurt called ‘oatgurt’. The food product selected was Oatly’s
blueberry/vanilla oatgurt (Figure 1).
! To study the viability of L. plantarum 299v in oatgurt
! To evaluate the change in the physicochemical properties (pH and colour) of
the probiotic oatgurt during storage.
! To evaluate if incorporating L. plantarum 299v into oatgurt packed in
polypropylene (PP) cups would be viable for Oatly
The two main influencing factors included how the strain was incorporated into
the oatgurt and presence of oxygen in the packaging units.

Materials and Method

The viability of L. plantarum 299v (Probi AB, 2017) and changes in the oatgurts’
physicochemical properties (pH and colour) was studied with respect to the two
main influencing factors. Each factor had two levels that was analysed (Table 1).
Factor 1: Step of probiotic strain incorporation into the oatgurt
Fresh Oatgurt: L. plantarum 299v was added along with starter culture
and fermented together. This was prepared without other additives like the
stabilizers and fruit.
Commercial Oatgurt: L. plantarum 299v was added directly into ready-
to-eat flavoured finished vanilla/blueberry oatgurt, post fermentation and
cooling.
Factor 2: Presence of oxygen in packaging unit during storage
Polypropylene cups: The packaging unit currently used at Oatly are 1mm
thickness polypropylene (PP) cups. They have an oxygen transmission
rate (OTR) of around 150-200 ml/m2.day.atm (Buntinx et al. 2014).
Thermosealable aluminium foil was used to seal the cup. Analysis of the
gas in the headspace of sealed PP cups over time showed a gradually
increase in O2%.
Glass jars: To evaluate the effect of oxygen that is present in the PP cups,
glass, which is impermeable to oxygen (Jayamanne & Adams 2004) was
 used as a packaging unit for comparative analysis. Glass jars were sealed
using heal sealed metal screw caps that maintained anaerobic condition
inside the unit.

Commercial Commercial Glass

Polypropylene (PPcomm) Fresh Polypropylene
(PPfr) 125g and 25g Fresh Glass (Gfr) 125g
125g and 25g (Gcomm) 125g and 25g

Figure 2 Manually packed commercial and fresh oatgurt samples in PP cups and glass jars.

The four samples were: Commercial oatgurt in PP cup, PPcomm; commercial

oatgurt in glass jar, Gcomm; fresh oatgurt in PP cup, PPfr; and fresh oatgurt in glass
jar, Gfr.The samples were prepared and packaged manually (Figure 2). They were
stored at 8°C in a closed incubator with no light. Different tests (Table 1) were
carried out for every sample in triplicates once a week for a period of eight weeks
and analysed.
Table 1 Experimental design with two different packaging materials and food matrices.

Packaging
Material Polypropylene Glass

Food Matrix

Commercial Oatgurt Viability pH O2 Colour Viability pH O2 Colour

Fresh Oatgurt** Viability pH O2 - Viability pH O2 -

** prepared without additives (colour, flavour, stabilisers

Viability of L. plantarum 299v: To study the viability of the four samples,
standard spread plate method was used (NMKL 140). The samples at different
dilutions were spread on solid MRS agar and incubated at 37°C for 48 hours
followed by the enumeration of the colony forming units (cfu).
Oatgurt pH: The change in pH over time was evaluated to understand probiotic
strain behaviour in all four samples.
Colour Stability: The colour stability was measured using Spectrophotometer-
CM Food (Konica Minolta Colorimeter, Japan). L*a*b colour system was used
and the change in visual perception, ΔE* was calculated.
Results and Discussion
As a food matrix, the oatgurt manufactured by Oatly AB appeared to be suitable
for probiotic strain incorporation. It is rich in beta-glucan that is known to be a
prebiotic (Mårtensson et al. 2002) and has a pH of 4.2 that most probiotic strains
are tolerant to.
Viability: In all four samples, PPcomm, Gcomm, PPfr and Gfr, the strain viability from
an initial concentration of 1.14±0.16*108cfu/mL remained above recommended
dosage of 107 cfu/mL well after the storage period. There was however, a
statistically significant difference in strain viability between fresh and commercial
oatgurt from week 2 of analysis. The increased stress during fermentation during
processing and presence of starter culture in the fresh oatgurt sample could explain
the comparatively lower stability in viability of the probiotic strain in fresh
sample. With the resulting data it was not possible to establish a significant effect
that the packaging material had on the viability.
pH: The reduction in pH of the samples could mainly be attributed to the probiotic
strain activity, as the pH of the control sample declined at a much slower rate (pH
4.26 to pH 4.01). There was a statistically significant reduction in pH over time in
fresh and commercial samples; however, the effect of packaging material on the
pH could not be established. Although the strain viability is not affected by the pH
decline, there could be an influence on product flavour (taste). The fresh oatgurt
had a comparatively low initial (start) pH and therefore the subsequent decline
could have a bigger impact on product sensory properties.
Colour Stability: The change in colour in the vanilla/blueberry oatgurt
specifically was a concern for the manufacturer. The presence of L. plantarum
299v in the product did not have an obvious affect on the product colour. The
packaging material, however, seemed to have an impact on the change in colour,
or change in visual perception (ΔE*) of the product. The colour of week 0 sample
was considered as the reference colour that was accepted by the manufacturer
(desired value).
There was a steeper increase in total change in colour of oatgurt in PP compared to
glass. In PPcomm after week 5, ΔE* was above 2, indicating that the change in
colour compared to desired reference would be perceptible at a glance. Till week 5
the change in colour in PP cups was only perceptible through close observation.
Conclusion and Future Recommendations
The study presents the possibility of commercially incorporating probiotic strain L.
plantarum 299v into Oatly’s oat-based yogurt (oatgurt) and packaging it in PP
cups. Oatly’s oatgurt was able to sustain L. plantarum 299v stably for over eight
weeks when stored in cold chain. The current packaging material used for the
commercially available non-probiotic oatgurt, PP, could be used for probiotic
oatgurt as well since it sustains the viability of L. plantarum 299v over the
incubation period well above recommended dosage. The colour of the oatgurt
could be improved with a packaging material having higher oxygen barrier,
however PP could ensure acceptable colour stability for atleast five weeks of
storage.
Table 2 Overall conclusions of experimental results.

Packaging
Material Polypropylene Glass

Food Matrix

Commercial Oatgurt Viability pH O2 Colour Viability pH O2 Colour

Fresh Oatgurt** Viability pH O2 - Viability pH O2 -

Note: Green – Favourable; Yellow – Acceptable; Red - Unacceptable

In order to further advance the development of this probiotic product, sensory
analysis for appearance and change in flavour should be conducted. The organic
acid profile due to the incorporation of L. plantarum 299v should be studied to
understand its metabolic activity in this food matrix. Pilot scale experiments are
required to ensure viability of the bacteria is not compromised because of other
process and production related factors. With limited research on probiotic non-
dairy food products, there is a need for several studies to establish concrete
commercial product development strategies.
Table of Contents

List of acronyms and abbreviations 13#

1 Introduction 14#
1.1 Purpose 15#
1.2 Limitations/Focus 16#
2 Literature Review 17#
2.1 Functional Foods 17#
2.2 Non-Dairy Probiotic Food Products 18#
2.3 Commercial Probiotic Strains 19#
2.4 Factors Affecting Viability of Probiotics (Oatly’s Oatgurt as a delivery
vehicle) 22#
2.5 Packaging for Probiotics 23#
3 Research Methodology 25#
4 Materials and Methods 27#
4.1 Pre-experimental Design 27#
4.2 Sample Preparation 29#
4.3 Experimental Design 31#
4.4 Probiotic Viability Test 32#
4.4.1 Plate count method (PCM). 32#
4.4.2 Flow Cytometry (FCM) 32#
4.5 pH (Acidity Analysis) 33#
4.6 Headspace Gas Analysis 34#
4.7 Colour Stability 34#
4.8 Statistical Analysis of Data 36#
5 Results 37#
 5.1 Viability of L. plantarum 299v 37#
5.2 pH 39#
5.3 O2% in PPcomm and PPfr Headspace 40#
5.4 Colour Stability of Commercial Oatgurt 41#
6 Discussion 45#
7 Conclusion and Future Scope 48#
References 49#
Appendix A Material and Method 57#
A.1 Packaging Materials 57#
A.2 Experimental Materials 58#
A.2.1 Colorimeter Equipment 58#
A.2.2 Steps of Incorporation 58#
Appendix B Supplementary Results 59#
B.1 Viability Analysis of Freeze Dried Probiotic Powder 59#
B.2 FCM Analysis Results 60#
List of acronyms and abbreviations

cfu Colony Forming Unit

FCM Flow Cytometry
Gcomm Commercial oatgurt in glass jars
Gfr Fresh oatgurt in glass jars
GRAS Generally regarded as safe
L. plantarum 299v Lactobacillus plantarum 299v
MRS Agar de Man, Rogosa and Sharpe Agar
OTR Oxygen transmission rate
PCM Plate Count Method
PP Polypropylene
PPcomm Commercial oatgurt in polypropylene cup
PPfr Fresh oatgurt in polypropylene cup
RT Room temperature
TBC Total Bacterial Count

13
1 Introduction

Oatly AB, a Swedish food company founded in the 1990s, produces vegan milk-
product alternatives made from oats. It caters to the appeal for veganism and
vegan products, which is on a rise. This shift of food preference goes beyond a
niche group who avoid animal meat for ethical purposes, towards consumers
looking for a cleaner and healthier diet (Lea et al. 2006). With increased consumer
awareness in recent years, food products are not only being consumed to satisfy
hunger and basic nutritional requirements but also to enhance the quality of
physical and mental wellbeing (Siro et al. 2008). This has paved way for the
concept of ‘functional foods’, which include ingredients with additional health
benefits or that can support specific body functions that conventional nutrition
models do not address (Buttriss 2000; Menrad 2003). Probiotics represent a major
segment of this functional food market (Granato et al. 2010).

Probiotics are live microorganisms that are natural inhabitants of the human
gastrointestinal tract. When incorporated in certain quantities into food matrices
and ingested, they can potentially improve the health of the host especially by
contributing to intestinal microbial balance (FAO/WHO 2002; Grajek et al. 2004).
The survivability and functionality of the probiotic culture is strain specific and
depends on several factors including method of incorporation, temperature
(Mokarram et al. 2009), pH, composition of the food matrix and level of available
oxygen (Tripathi & Giri 2014). The viability of probiotic strains in dairy products
like yogurt has been studied extensively, however the research on non-dairy food
matrices is limited.

Among probiotic dairy products, yogurt has a wider consumer market (Siró et al.
2008) and hence, Oatly’s spoonable oat based yogurt called ‘oatgurt’ was selected
as the test product for the experiment. The vanilla/ blueberry flavour was chosen
as the manufacturers wanted to study the product colour stability over time. Due to
lack of research on probiotic incorporation in oatgurt, two different steps at which
the strain could be incorporated was studied (before fermentation and after
fermentation). The effect of the presence of oxygen during storage can influence
the viability and product physicochemical properties. The primary packaging unit
currently used at Oatly, polypropylene cup, that has an oxygen transmission rate
(OTR) of around 150-200 mL/m2.day.atm (Buntinx et al. 2014) was evaluated.

14
This is the packaging that is in direct contact with the product (Hellström & Saghir
2006). In order to evaluate the effect of oxygen, the change in viability of the
strain should be compared with a material with no oxygen transmission. Glass,
although impractical for commercial use, is impermeable to oxygen and easily
available and was therefore selected for this comparative study. In addition to
probiotic viability, the effect of permeability to oxygen of the packaging material
on product physicochemical properties like pH and colour was analysed.

1.1 Purpose
This study aimed to investigate the behaviour of a probiotic strain obtained from
Probi AB, Lactobacillus plantarum 299v (L. plantarum 299v), in Oatly’s non-
dairy oat-based yogurt called ‘oatgurt’. The viability of the strain in an oatgurt and
change in the physicochemical properties (pH and colour) of the oatgurt during
storage was studied. The factors that could influence these aspects included how
the strain was incorporated into the oatgurt and presence of oxygen in the
packaging units. The food product selected was Oatly’s blueberry/vanilla oatgurt
(Figure 1.1b), which is sold as a twinpack (Figure 1.1a).
The process step when the probiotic strain is incorporated into the product may
influence its behaviour. Of the two steps selected for probiotic incorporation, one
was called the ‘commercial’ oatgurt sample in which L. plantarum 299v was
added directly into ready-to-eat flavoured finished oatgurt, post fermentation. As
an alternative, the probiotic strain was added along with starter culture and
fermented together. This was called the ‘fresh’ oatgurt sample. In fresh oatgurt, the
frozen probiotic strain may thrive better due to more time for adaptation during the
fermentation process or its viability may suffer due to stress and competition due
to the presence of the starter culture.

(a) (b)

Figure 1.1 Oatly’s twin pack blueberry/vanilla oatgurt. (a) 2 oatgurts cups sold as one twin pack
unit with cardboard secondary packaging; (b) oatgurt packed in polypropylene cup (current primary
packaging material).

15
The effect of the presence of oxygen in the packaging unit was evaluated
comparing polypropylene (PP), which has an oxygen transmission rate (OTR) of
150-200 ml/m2.day.atm and glass that allows no oxygen transmission (Jayamanne
& Adams 2004a). If the current oatgurt packaging material (PP) is able to match
the functionality of glass, Oatly can use PP without having to invest in a new
packaging material even for their potential range of probiotic oatgurt.
To our knowledge there has been no study conducted to evaluate the viability and
growth behaviour of any probiotic strain in oatgurt. The effect of the oxygen
permeability into a product-packaging unit on the viability and behaviour of the
probiotic strain L. plantarum 299v has also not been explored.

1.2 Limitations/Focus

The study focussed on the current primary packaging material (PP) and glass with
respect to the oxygen transmission rates and not chemical structure or
environmental considerations. Other physical parameters like strength and
flexibility of the packaging materials were not considered as influencing factors.
Other packaging levels were not considered for this study and therefore will not be
discussed. The samples were stored in controlled temperature with no light
exposure, eliminating the influence of those factors on the experimental results.
The study was centred on the viability of probiotics in the oatgurt including the
physicochemical changes like the pH and colour. The sensorial changes and
consumer acceptance was not evaluated.

16
2 Literature Review

2.1 Functional Foods

More than 2,500 years ago Hippocratic stated “let food be thy medicine and
medicine be thy food”, presenting the relationship between our diet and health
(Spyropoulou et al. 2016). Foods or food ingredients that are able to provide health
benefits beyond basic nutrition and increase consumer physical and mental
wellbeing are defined as functional foods by the academia and food industries (Da
Cruz et al. 2007; Menrad 2003). This concept was first introduced in Japan in the
mid-1980s and since then there has been an upsurge of consumer interest in the
health enhancing abilities of these food products (Hasler 1998). Functional food
plays a role in promoting overall health and not towards curing diseases (Sanders
1998). Foods with such properties are becoming more readily accepted by the
industry and can be used as marketing tools (Nematollahi et al. 2016). The
labelling laws for functional foods depend on the country legislation. The label
‘probiotics’, for example, is allowed in certain countries including the United
States but is banned in Europe by the European Food Safety Authority (Crane
2015).
Functional foods are associated with different mode of operations including
vitamin and mineral fortification, cholesterol reduction, dietary fibre, probiotics,
prebiotics and symbiotics, antioxidants, phytochemicals, and herbs and botanicals
(Roberfroid 2000; Hironaka et al. 2011; Alzamora et al. 2005). In the European
market, functional foods that improve the intestinal microbial balance and activity
has been gaining a lot of interest (Alzamora et al. 2005). The microbial activity in
the human gut is essential in maintaining metabolic and immune functions of the
host (Cencic & Chingwaru 2010). The equilibrium of the gut microbiome can be
affected by aging, type of food consumed, stress, use of antibiotics and other
health conditions (Andrade et al. 2012). The increased consumption of processed
food can further lower the chances of colonisation by certain types of bacteria in
the gut. This imbalance can cause diarrhoea for the host, mainly affecting the
elderly and also weaken the immunity of the host (Andrade et al. 2012). The main
ways to sustain the population of viable bacteria in the gut is by ensuring
consumption of sufficient daily nutrition without external stresses or by
continuous ingestion of high viable population of probiotics (Mortazavian et al.
2012). It is not possible to permanently establish a probiotic organism in the gut
and therefore multiple doses are required (Sanders 2008).
17
Probiotics have been defined in several ways over the past century. The definition
used today is “probiotics are live microorganisms, administrated in certain
quantities that confer health benefits to the host” (FAO/WHO 2002). The general
attributes of a probiotic strain includes being naturally present in the hosts’
gastrointestinal tract, surviving the passage through the stomach and retaining its
functionality in the intestine (Andrade et al. 2012). There are also some strains
used as probiotics for humans that are not isolated from humans (Sanders 2008).
Probiotic are generally consumed by adding the culture concentrate into a food, or
is directly present in ready-to-eat food products, or via dietary supplements like
powders, capsules or tablets. A widespread and efficient system to ingest
probiotics is via commonly consumed food products (Mortazavian et al. 2012).
With a present consumer trend of convenience and health, probiotic functional
food products are being extensively consumed (Granato et al. 2010). Additionally,
probiotics have been used for a long time as natural components in fermented
foods and has a positive association with health among consumers (Forssten et al.
2011). Hence, probiotic food products have gained popularity in the market.

2.2 Non-Dairy Probiotic Food Products

Probiotic food products have been frequently cited as having health benefits
including cholesterol level reduction, stimulating antibody production and
phagocytic activity against pathogens in the gut and other tissues, anti-
carcinogenic effect and reducing the symptom of Irritable Bowel Syndrome (IBS).
The effect is strain specific (Fiorentini et al. 2011). Probiotics have been used
mostly in dairy food products as they have proven to be efficient delivery vehicles
for live bacteria (Andrade et al. 2012; De Vuyst 2000). Consumers are used to its
presence in this type of product both in fluid milk and fermented dairy products
(Pimentel et al. 2015). It also does not require a significant change in the
manufacturing process or technology. There is a global rise of probiotic use,
however, recently there is a reduction in dairy applications in Europe due to
inflexibility in dairy regulatory laws and increasing dairy stock maintenance costs
(Reid 2015). Moreover there is an evolving trend of vegetarianism and reduction
in the consumption of dairy for ethical reasons. There is also a prevalence of
lactose intolerance or allergy to milk proteins in many populations around the
world (Nematollahi et al. 2016). Considering these factors, food producers are
exploring non-dairy probiotic products that can be suitable alternatives that also
offers variety in the market (Pimentel et al. 2015; Nematollahi et al. 2016). Fruit
juices, desserts and cereal products have been found to be appropriate non-dairy
media for incorporation of probiotics (Granato et al. 2010). Close to 78% of
probiotic sales are through yogurt (Panthari & Kharkwal 2017), indicating that
development of non-dairy yogurt alternatives could be a profitable option for the

18
food industry. The non-dairy probiotic yogurt products are mostly artisanal,
generally small-scale and are mainly produced and sold in the USA (Table 2.1).
Table 2.1 Commercial non-dairy probiotic yogurts.

Country Packaging
Product Company Culture(s) Reference
Manufactured Material
L. casei, S.
The Hain thermophiles, L.
Dream™ (’dreamplant
USA Celestial PP rhamnosus, L
Yogurt based’ 2017)
Group, Inc delbrueckii lactis
and bulgaricus
('Silk
Silk Dairy Live and active
USA Silk PP Yogurts’
Free Yogurt cultures.
2017)
Bifidobacteria, L.
Almond milk Plastic- ('kite hill
acidophilus, S.
Artisanal USA kite hill cardboard Yogurt’
thermophiles, L
Yogurt combination 2017)
bulgaricus
L. acidophilus, S.
Nancy’s thermophiles, L ('Nancy’s
Cultured Soy USA Nancy’s HDPE bulgaricus, L. Yogurt’
Yogurt rhamnosus, L. 2017)
casei, B. lactis
('Original
Living
Original hemp
USA Harvest PP Active cultures
Hemp yogurt yogurt’
Tempt
2017)

2.3 Commercial Probiotic Strains

One of the initial steps in the process of developing non-dairy probiotic products is
selecting a suitable probiotic strain (Figure 2.1). Probiotic strains that are used in
the food industry should have well documented health benefits and possess certain
technological properties. It includes being safe for consumption by the host,
genetically stable, mass producible, be stable in the food product and not
negatively influence product flavour (Forssten et al. 2011). Apart from these
factors, the cost also influences the selection process of probiotic strains
(Mortazavian et al. 2012). In a vegan food product it is essential that the strain be
produced in dairy-free cultivation conditions (Figure 2.1). O’Soy vanilla, a soy
based yogurt that used active culture that was cultivated on milk received huge
consumer displeasure (’thevegblog’ 2007).

19
Figure 2.1 Non-dairy probiotic product development process (Granato et al. 2010)

20
The bacterial strains commonly used as probiotics in the food industry belong to
Lactobacillus and Bifidobacterium genera (Andersson et al. 2001). Bacteria from
both genera have been used for decades in food and are considered as GRAS
(generally regarded as safe). Many of them are isolated from human faecal matter
maximising their compatibility to the human intestine (Mortazavian et al. 2012).
Probiotic strains of the Lactobacillus species are generally more robust and
technologically compatible for usage in food production (Lee et al. 2009). They
also demonstrate comparatively higher resistance to low pH, which is essential to
survive the acidic environment of the stomach.
Viability is the number of live microbial cells per gram or mL of a food product.
For a food product to be considered a probiotic or to have the stated/claimed
health efficiency, the viability or minimum number of probiotic bacteria at the
time of consumption (date of minimum durability) should be 107 colony forming
units (cfu) per mL (or gram) of the product (Beena Divya et al. 2012). The
recommended inoculation level is at least 108 or 109 cfu/ mL to account for the loss
of bacteria that may occur till and after consumption of the product (Talwalkar et
al. 2004). The survival of the bacterial strain(s) in the food matrix and throughout
the time of storage is essential. The commonly used probiotics in the Lactobacillus
genera are strains of L. rhamnosus, L. rueteri, L. casei and L. acidophilus (Cencic
& Chingwaru 2010). Recently however, new strains with high stability and health
benefits have been emerging both in different food matrices and as individual or
mixed microbial cultures. All the benefits, chemical stability and adaptability to a
food matrix is strain specific, hence choosing the appropriate probiotic strain is the
first prerequisite for product development (Varga-Visi & Pápai 2015).
L. plantarum 299v, a versatile lactic acid bacterium patented by Probi AB, has
been successfully used commercially in non-dairy food matrices ('ProViva’ 2012).
The L. plantarum 299v strain is found in environments that are protein rich. It is a
rod shaped, gram positive, aerotolerant strain (Molin 2015). This strain is able to
resist the low pH passage of the stomach and also the bile acids. It has also been
found to be able to colonize the entire gastrointestinal tract (Nematollahi et al.
2016). It is a well documented and researched strain in a variety of food
environments, with its complete genome sequenced (de Vries et al. 2006). It can
ferment different types of carbohydrates, making it more adaptable (Molin 2015).
There have been clinical trials positioning this probiotic as benefitting the host by
reducing bloating, abdominal pains, improvement of the IBS symptoms and
normalizing stool frequency (’Probi’ 2016). A clinical study also demonstrated the
ability of this strain to induce a pro-inflammatory response and increase immune
alertness in intestinal epithelial cells (Cammarota et al. 2009). Considering all
these factors, along with its vegan label, it is a suitable and promising choice for a
new probiotic non-dairy formulation. Its natural environment is either anaerobic or
microaerobic. In the absence of oxygen, L. plantarum produces D- and L-
configurations of lactate. In aerobic conditions the lactate is further converted to
acetate (Kleerebezem et al. 2003). The effect of this aerobic metabolism could

21
influence the flavour and other characteristics of the food product. Additionally, in
aerobic conditions, at the early stages L. plantarum have reported to have growth
stagnation (Stevens et al. 2008). It is documented to have maintained viability for
about one month when refrigerated in fruit juices having pH <2.8-3.4 (Molin
2001).

2.4 Factors Affecting Viability of Probiotics (Oatly’s

Oatgurt as a delivery vehicle)
There are three main factors that influence the stability of incorporated probiotic
strains in a food matrix: production factors, formulating factors (food matrix) and
physical factors. First, the strain has to be able to sustain the production process
and manufacturing conditions that the product goes through (Mortazavian et al.
2012). The stage at which the strain is incorporated into the product is detrimental.
If the probiotic is added along with the starter culture, it should be able to remain
functional throughout the fermentation process at both the incubation temperature
and along the cooling rate (Andrade et al. 2012). The scale of production can also
impact probiotic survival; therefore, choosing a robust strain is vital.
The matrix and product selected influences the probiotic stability, which are the
formulating factors. The chemical composition of the food with respect to the
carbon source available, nitrogen, minerals, growth promoters, inhibitors, salt and
sugar content can alter the bacterial performance (Mortazavian & Cruz 2011;
Granato et al. 2010). Including prebiotics in the matrix, for example, can aid the
viability of the probiotic strain (Varga-Visi & Pápai 2015). It is important that the
probiotic survives and is benefited from the food matrix, but does not affect its
flavour adversely (Andrade et al. 2012). During strain selection and product
formulation, the researcher should check if any ingredients of the product have an
inhibitory activity on the probiotic strain.
Apart from sustaining the production process, the strain should also stay viable
throughout the storage period till the time of consumption. This depends on the
conditions of the food matrix the strain is added into. Oatly AB produces a non-
dairy yogurt alternative called oatgurt that is available in different flavours ('Oatly’
2017). It is an oat-based product rich in beta-glucan. Beta-glucan is considered a
prebiotic that aids the survival of probiotic bacterial strains (Mårtensson et al.
2002). The important physical factors of the matrix that could affect the probiotic
strain include water activity, pH, oxygen content and temperature during storage
(Heller 2001). The oatgurt has high water activity (>0.95), therefore, will have a
short shelf life (4-6 weeks) (Forssten et al. 2011). The recommended storage
condition of the oatgurt by the manufacturer is at 4-8°C. Such low temperatures
tend to favour the survivability of probiotic strains and restrict post-acidification
(Anadón et al. 2016). The temperature can be controlled as this product is
22
generally transported and stored in cold chain. The pH of oatgurt is around 4.27,
and L. plantarum 299v is known to be resistant to even lower pH.
In the industry several approaches have been used to reduce the impact of O2 on
the probiotic strain viability. The effect of the presence of O2 in the packaging unit
and in the food matrix depends on the strain used. Addition of O2-scavengers,
encapsulation of the probiotics and varying production methods to lower O2
incorporation are some of the techniques used (Macbean 2009). The packaging
container used during storage can also be an important barrier to control the
permeation of O2 into the product. This can, hence, effect on the viability of the
probiotic based on its characteristics (Talwalkar & Kailasapathy 2004).

2.5 Packaging for Probiotics

The important factors that the packaging of a probiotic product can impact include
presence of oxygen and light (Varga-Visi & Pápai 2015). The selection of the
packaging should be done keeping in mind shelf life and product stability (Figure
2.1). Since the oatgurt is stored after production for several weeks, it is liable to
oxygen permeation (Talwalkar et al. 2004). The packaging material can limit this
phenomenon. Oxygen permeation could lead to a negative effect on probiotic cells
viability or change in its metabolic pathway and activity (Kleerebezem et al.
2003). It can also affect the physicochemical characteristics of the product. Studies
have shown that adding probiotics did not significantly influence physicochemical
properties like colour (Kailasapathy 2006), but the presence of oxygen could have
an impact on it. The use of appropriate packaging, that acts as a barrier against the
external environment, could ensure maintenance of physicochemical quality and
bacterial viability throughout the shelf life (Da Cruz et al. 2007).
A challenge for most probiotic food product developers is maintaining the
probiotic level during storage till the end of storage time to guarantee therapeutic
results. Studies have shown that packaging material with lower oxygen
permeability rates have higher stability in probiotic bacteria count (Da Cruz et al.
2007). The type and thickness of the packaging material are important features to
be considered during selection. This further affects the gas and light permeability
(Korbekandi et al. 2011). Most probiotic products commercially available are
stored in plastic containers, which may be a problem because of high oxygen
permeability depending on the probiotic and food matrix used (Pimentel et al.
2015). Conversely, glass containers have practically no gas permeability
(Jayamanne & Adams 2004a) but poses other challenges in food production such
as risk of breakage and high material and logistics cost (Da Cruz et al. 2007). The
sealing technique used in glass jars, however, may allow permeability of gases.
The screw cap is considered to be a closure with the best barrier to oxygen (Poças
et al. 2010). Apart from the method of sealing, the packaging technique, that is if it

23
has been vacuum packed or involves a modified atmosphere packaging, can alter
stability of the probiotic strain (Korbekandi et al. 2011). Comparing glass and
plastic, which have contrasting oxygen permeability rates would be an effective
method to understand the influence of the presence of oxygen on the viability of
the probiotic strain. In previous studies comparing a lactic acid probiotic bacterial
strain stability in dairy yogurt in glass and high density polyethylene (HDPE), it
was found that glass bottles demonstrated the best results (Dave & Shah 1997).
Another study with Bifidobacteria in fermented buffalo milk packaged in clay,
plastic and glass jars demonstrated the comparative superiority of glass in
sustaining the viability of the strain (Jayamanne & Adams 2004a). L. plantarum
299v is microaerobic and can switch to an aerobic metabolism, but the study of the
dependence of its viability on packaging material (varying OTRs) has not been
carried out. The change in behaviour that could result from this dependence when
incorporated into a food matrix as well is not researched.

24
3 Research Methodology

The study was carried out to evaluate the influence of packaging materials with
different oxygen transmission rates and incorporation methods on the survivability
of probiotic strain L. plantarum 299v in non-dairy, spoonable blueberry/vanilla
flavoured oat-based yogurt (oatgurt). Oatly AB is exploring the development of
vegan, oat-based probiotic food products. Research on non-dairy food products as
carriers for probiotic bacteria is limited compared to its dairy counterparts (Molin
2001). L. plantarum 299v (Probi AB 2017) was selected as the probiotic bacterial
strain as it maintains the vegan label of oatgurt. It is also an extensively researched
and documented probiotic strain.
The viability and behaviour of L. plantarum 299v (Probi AB, 2017) was studied
by incorporating it at two alternative stages of processing and in two packaging
materials with differing oxygen transmission rates. Additionally, the effect of the
packaging material and its interaction with the product and outer atmosphere on
commercial oatgurt colour stability was investigated. To understand the behaviour
of the bacterial strain the product pH and gas in the headspace were monitored.
This work mainly shows the effect of using PP and glass as packaging materials
on survival of L. plantarum 299v strain in Oatly’s vanilla/blueberry oatgurt and on
its physicochemical properties (colour, stability and pH).
The framework of the experimental tests was based on the literature studies. The
aim was to obtain information about previous literature on different aspects of the
study. Journals, books and company specification sheets were used. The focus was
on:
i. Research on probiotic food products.
ii. Probiotic strain requirements in non-dairy products.
iii. Factors affecting survival of probiotic bacteria in food matrix.
iv. Probiotic food and packaging material interaction.
v. Experimental tests reported to evaluate the viability of probiotic strains in
yogurt.
vi. Consumer acceptability to non-dairy probiotic foods.
vii. Influence of material oxygen transmission rate on food characteristics.

25
The application and product development specialist at Probi AB, Anna Andrys,
was consulted prior designing the experimental plan. The results of the previous
experiments carried out at Probi with the probiotic were discussed in order to
avoid repetition and explore different approaches.
Since there is limited research on probiotic stability in non-dairy food matrices in
different packaging material, it was difficult to correlate literature data with no
practical evidence. Oatly has not yet developed a probiotic product; hence,
experimental data was crucial for understanding the behaviour of the food matrix.
The efficiency and viability of probiotics is specific to the strain and will vary
depending on the product selected. With a detailed experimental design, several
aspects of the research could be validated.
Laboratory experiments were performed to evaluate the performance of the two
packaging materials (PP and glass) over a period of eight weeks in controlled
temperature environment (Figure 3.1). The evaluation was done once a week for
eight weeks as an extended shelf life analysis. The expected shelf life was 4-5
weeks. The microbiological behaviour and physicochemical parameters were
tested and analysed. The trials were performed in triplicates to eliminate bias and
experimental errors.

Sampling Frequency Sample Volume Length

• 1 time/week • 25g for viability and pH • 8 weeks at 8°C#

experimentation
• 3 biological replications/test • Expected shelf life: 4-5
• 125g for color and gas weeks
analysis

Figure 3.1. Conditions followed for experimental set-up and trials.

26
4 Materials and Methods

4.1 Pre-experimental Design

Viability test for probiotic freeze-dried powder: Sealed aluminium sachet
with freeze-dried L. plantarum 299v powder was received from Probi AB. Freeze
dried powder was suspended in sterile saline solution. Ten times serial dilutions of
the sample were prepared and spread on de Man, Rogosa and Sharpe (MRS) agar
plates (Merck, Germany) in duplicates using standard spread plate method
(NMKL 140). The plates were incubated in 37°C and monitored to determine the
time required for the colonies to develop. After 48 hours of incubation time the
colonies on the plates were enumerated using plate count method (PCM) (Annex
Figure B.1). The mean value for each dilution was multiplied by the respective
dilution factor to determine the cfu/mL (Table 4.1).
For a product to be considered a probiotic it should have a probiotic strain
concentration of atleast 107 cfu/mL of product. In the study the start concentration
was around 108 cfu/mL and the change with time was evaluated. To reach 108 cfu
of probiotic bacteria /mL of oatgurt sample, 0.2 g of freeze dried powder needed to
be added to 1 litre (kg) of sample as predetermined at Probi AB and confirmed
from the viability test.
Table 4.1 L. plantarum freeze dried powder viability results using PCM.

Plate 1 Plate 2 Mean Mean

Dilution (cfu) (cfu) (cfu/0.1 mL) (cfu/1 mL)
105 884 1010 947*105 0.9*109
6 6
10 100 109 105*10 1*109
107 10 10 10*107 1*109
Mean - 1*109

Glass jar sealing: 156 mL glass jars (56 mm diameter, 85 mm height) were
received from Oatly AB as one of the packaging unit for analysis. The glass jars
are impermeable to oxygen but the method of sealing/closing the jar could alter the
condition inside the packaging unit. The glass jars were provided with heat seal
metal caps (Annex Figure A.1). The glass jar was heat sealed in two different

27
ways. The first method involved heat-sealing the screw cap directly to the glass jar
(Figure 4.1a) and the second had an additional layer of heat seal aluminium foil
between the jar and cap (Figure 4,1b). The caps were heated directly after
screwing it on to the jar using a clothes iron at the highest temperature setting
(~150°C) (Schneider, 2010) and evaluated for different exposure times.
Anaerotest® strip (Merck, Darmstadt, Germany), was used as an indicator for
absence of O2 in the sealed glass jar. The change of colour of the strip from blue to
white indicated anaerobic environment in the jar (Figure 4.1a). The strip reversed
colour back to blue within 20 minutes of exposure to aerobic condition. The glass
jars that were directly heat-sealed for 10 seconds at ~150°C maintained anaerobic
condition without exchange of O2 with the atmosphere for eight weeks. There was
a change in strip colour back to blue for the jar with the additional aluminium
layer indicating non-hermeticity (Figure 4.1b). Therefore, all glass jars used in the
experiments were hermetically sealed by screwing the metal cap on directly and
exposing it to ~150°C (Schneider, 2010) for 10 seconds.

(a) (b)

Figure 4.1 Anaerotest® strip (Merck, Darmstadt, Germany), indicator to evaluate anaerobicity
inside the glass jar packaging. (a) Strip remaining white indicating anaerobic condition maintained
in glass jar closed with heat sealed screw cap; (b) Colour of the strip reversed back to blue colour
indicating aerobic condition in glass jar closed with screw cap having aluminium foil barrier.

PP cups sealing: 150 mL PP cups, having 1mm thickness are currently used as
the packaging unit for the non-probiotic oatgurt at Oatly AB. These cups are
closed using thermosealable aluminium foil (Annex Figure A.2). The foil
thickness was 38±3.04 µm (Oatly AB, 2017). The cups and the foil were received
from Oatly AB. To check for leakage, the cups were half filled with water and
sealed using a clothes iron at the highest temperature setting (~150°C) (Schneider,
2010) and evaluated for different exposure times. The sealed cups were held
upside down for 60 seconds to carry out leakage detection tests. After several trials
the sealing condition was determined to be direct heating at ~150°C for 20
seconds.
28
4.2 Sample Preparation
Fresh Oatgurt Preparation and Packaging: ‘Fresh’ oatgurt was prepared in
the product development laboratory at Probi AB, Sweden. Probi has standardized
the method. 12 L of 0.5% fat pasteurised Oatly ecologic oat drink was added into a
sterilized 12 L culture vessel (fermenter). Fermentation was carried out in a double
jacketed fermenter with 0.52 g of commercially available yogurt starter culture
used by Oatly (Danisco Yo-Mix 511 LY0300 DCV) along with 1 mL of defrosted
L. plantarum 299v culture pellet (2.4 g of probiotic culture). The pH and
temperature was monitored using probes. The resulting product, which did not
contain the additives (stabilizer, fruit concentrate, sugars) present in the
commercial oatgurt, was termed as ‘fresh oatgurt’ for this study. The fresh oatgurt
was cooled to 4°C before packaging.
25 mL of the fresh oatgurt was added (by weighing) into PP cups and glass jars as
the sample to be used for testing pH and viability. 125 mL of this fresh oatgurt was
added into PP cups for headspace gas analysis. The cup/jar was partially closed
with the foil or cap and flushed with nitrogen (N2) gas using a syringe probe
(Figure 4.2). The cup/jar was then sealed as described in the preparatory setup and
stored at 8°C. The entire process was carried out in the sterile bench. Biological
triplicates of 25 g and 125 g samples were prepared to test for 9 weeks (week 0 + 8
weeks). The sampling was performed once per week.
Commercial Oatgurt Packaging: The commercially available oatgurt
(vanilla/blueberry flavour) was received from Oatly AB. 125g and 25g of oatgurt
were added into both PP cups and glass jars.
The results obtained from the preparatory experiments resulted in 108 cfu/mL
when 0.2 g of freeze-dried probiotic powder was suspended in 1 L (1000g) of
saline. Therefore, 0.025 g of freeze-dried probiotic powder should be added to 125
g of the oatgurt sample. To simplify the process of adding a small quantity of
probiotic to every sample packaging unit (PP cup and glass jar) a stock solution
was prepared. 4 g of the probiotic powder was added to 160 mL of saline. The
powder was suspended in saline by vortexing the tube till it completely dissolved.
This stock solution was also plated using standard spread-plate method on MRS
plates to verify the concentration of the probiotic bacteria (NMKL 140, 2007).
1 mL and 0.2 mL of the probiotic stock solution was pipetted into the 125g and
25g cups/jars respectively and stirred thoroughly. The cup/jar was partially closed
with the foil or cap and flushed with nitrogen (N2) gas using a syringe probe. The
cup/jar was then sealed as described in the preparatory setup and stored at 8°C.

29
Control samples (without probiotic) were also prepared as described without the
addition of the probiotic and sealed. The entire process was carried out in the
sterile bench (Figure 4.3). Biological triplicates of 25 g and 125 g samples were
prepared to test for 9 weeks (week 0 + 8 weeks) along with 9 control samples
each. The sampling was done once per week.

N2 Flush Check

Weigh Heat Seal

Figure 4.2 Process of packaging fresh oatgurt. 25 g and 125 g of fresh oatgurt was weighed and
packed in PP cups. 25 mL fresh oatgurt was weighed and packed in glass jars.

Add probiotic
& mix Heat Seal

Weigh N2 Flush Check

Figure 4.3 Process of packaging commercial oatgurt. 25 g and 125 g of commercial oatgurt was
weighed and packed in PP cups and glass jars

Storage of Packaging Units: The temperature and relative humidity of the

atmosphere a package is kept in can impact oxygen permeability (Da Cruz et al.
2007); therefore both PP cups and glass jars must be stored in the same conditions
to effectively compare the results. The manufacturer expected to store the
probiotic oatgurts at 4-6°C. All prepared samples were stored in 8°C cold
incubator (Termaks, Norway). The recommended temperature for storage of
oatgurt is a maximum of 8°C and this was selected for the study. The packaging
units were not exposed to light, eliminating its influence in the study.

30
4.3 Experimental Design
The experiment was a 22 factorial design (Table 4.2). The two factors were the
packaging material and stage of incorporation. The packaging materials used were
PP and glass. The PP cup used was ~1mm thick with an OTR of around 150-200
mL/m2.day.atm (Buntinx et al. 2014). The glass jars did not allow oxygen
permeation. The experimental results from two different packaging materials
could demonstrate the influence of the presence of oxygen inside the packaging
unit on product characteristics. The method of incorporation included using
commercial and fresh samples. The strain was incorporated during production by
adding probiotic stain along with the oatgurt starter culture followed by
fermentation; this was called ‘fresh oatgurt’ (Annex Figure A.5). The other
method was to incorporate the strain after production to the ready-to-eat finished
product post fermentation called ‘commercial oatgurt’ (Annex Figure A.4).
Table 4.2 Experimental design with two different packaging materials and food matrices.

Packaging
Material Polypropylene Glass

Food Matrix

Commercial Oatgurt Viability pH O2 Colour Viability pH O2 Colour

Fresh Oatgurt** Viability pH O2 - Viability pH O2 -

** prepared without additives (colour, flavour, stabilisers

The combination of sample used were: Commercial oatgurt in PP cup, PPcomm;

commercial oatgurt in glass jar, Gcomm; fresh oatgurt in PP cup, PPfr; and fresh
oatgurt in glass jar, Gfr (Figure 4.4). Commercial oatgurt without probiotic strain
was prepared as control samples and packed in PP (PPC) and glass (GC).

Commercial Commercial Glass

Polypropylene (PPcomm) Fresh Polypropylene
(PPfr) 125g and 25g Fresh Glass (Gfr) 125g
125g and 25g (Gcomm) 125g and 25g

Figure 4.4 Manually packed commercial and fresh oatgurt samples in PP cups and glass jars.

31
The viability of L. plantarum 299v and change in pH was tested in all the samples
(25g of sample/ packaging unit). The headspace O2% was measured in the PP
cups (PPfr and PPcomm) (125g of sample/ packaging unit). The anaerobicity of the
glass jars was evaluated during the pre-experimental setup. The colour stability of
the commercial oatgurt (125g of sample/ packaging unit) over time was measured
(PPcomm and Gcomm), as the fresh oatgurt did not contain the colour pigment. All
experiments were conducted in biological triplicates and once every week for 8
weeks.
As an additional study, the effect of temperature on colour commercial oatgurt
control samples that were packaged at Oatly were stored at room temperature (RT)
and analysed.

4.4 Probiotic Viability Test

4.4.1 Plate count method (PCM).

25 g of fresh and commercial oatgurt in sealed PP cups and glass jars were used as
experimental samples. The four samples, PPcomm, Gcomm, PPfr and Gfr were analysed
at each time point (once per week).
100µL of fresh oatgurt was pipetted into 900µL of saline in a 2mL eppendorf tube,
followed by 10x serial dilution. The commercial product was denser and therefore
was first diluted with 25 mL saline and mixed into a homogeneous mixture. 200µL
of this mix was added to 800µL of saline for the first dilution. Transferring 100µL
from the first dilution to 900µL of saline continued the 10x serial dilution of the
commercial samples.
The 10-6, 10-7 and 10-8 dilutions of the samples were plated using standard spread
plate method. Biological triplicates of every sample and technical duplicates of
each dilution were plated using standard spread plate method (NMKL 2007). The
commercial samples were free of the starter culture, therefore plated on solid MRS
media. The fresh oatgurt sample had to be plated on solid MRS media
supplemented with vancomycin (Sigma Aldrich Co., St. Louis, Mo.), an antibiotic,
to suppress the growth of starter culture colonies ('Probi’ 2016). The plates were
incubated at 37°C incubators for 48 hours and counted to determine the total
colony forming units (cfu) per mL of sample.

4.4.2 Flow Cytometry (FCM)

The standard plate count techniques accounts only for the viable bacteria that are
able to grow and form colonies. It was therefore possible to underestimate the
32
actual total bacterial count (TBC) (Cassoli et al. 2007). To determine if the results
of PCM corresponds to the total viable cell count, a dual staining technique that
detects membrane integrity was carried out with two fluorophores (Gatza et al.
2013). BD Accuri™ C6 personal flow cytometer was used for detection and
measurement according to the Eawag (2013) method (Gatza et al. 2013). It is a
comparatively rapid and powerful method that uses less resources (Cassoli et al.
2007). SYBR® Green I (Sigma Aldrich Co.) is a green fluorescent nucleic acid
stain that enters live and dead cells (Stiefel et al. 2015) and is detected by one of
the flow cytometer fluorescent detectors (FL1). Propidium iodide (PI) (Sigma
Aldrich Co.) is a red fluorescent nucleic acid stain that is unable to penetrate intact
plasma membranes (healthy cells). The FL3 detector in the flow cytometer
measures the red light. PI was added to differentiate between live and
dead/damaged cells. By co-staining the sample, the cells with both stains get
detected by both detectors (Annex Figure B.3). These results were plotted (Annex
figure B.3) and the instrument automatically quantified the cell viability.
A mixture of SYBR® Green I (1X final concentration) and PI (0.3mM final
concentration) was added to the 100 times diluted oatgurt sample. Sample
preparation for FCM was done by adding 6 µL (SYBR Green I + PI), 489 µL
deionised water and 5 µL sample (100X) in 150 mL eppendorf tubes. The samples
were incubated at 37°C for 10 minutes and analysed. The control commercial
sample was run unstained in the instrument to detect the background. The four
samples (PPcomm, Gcomm, PPfr and Gfr) at 10-2 dilutions in 1.5 mL eppendorf tubes
were stained with the dual flurophores and analysed in a similar manner. The
results obtained were analysed to compare it with PCM. The experiment was set
up only at week 4 and week 8 as it took time to standardise the method.

4.5 pH (Acidity Analysis)

The sample used for the viability test (remaining) was also used to measure pH
using a pH meter (Mettler-Toledo™ FE20 Benchtop, Switzerland). Based on the
week 0 sample pH values, the pH meter was calibrated between pH 2 and 4 while
measuring fresh oatgurt samples and between pH 4 and 7 for commercial oatgurt
and control. Biological triplicates of all samples were measured once every week
for 8 weeks.

33
4.6 Headspace Gas Analysis

The O2 content in the headspace of sealed 125 mL PPcomm and PPfr samples was
determined using Dansensor CheckMate® 9900 O2/CO2 (PBI Dansensor A/S,
Ringsted, Denmark). A small volume of the headspace gas was drawn through a
sampling probe needle introduced to the PP cup by piercing the aluminium foil.
This was an intrusive method to analyse headspace in the packaging unit. The
instrument determined the O2% and CO2% in the withdrawn headspace gas. The
measurement was done once a week for 8 weeks.

4.7 Colour Stability

The colour stability was measured using the portable Spectrophotometer-CM Food
(Konica Minolta Colorimeter, Japan). The spectrophotometer was equipped with a
MAV target mask with an 8 mm opening for wet and viscous samples. L*a*b
system was selected at D65 illumination and a 10° standard observation angle
(Konica Minolta, Japan): L* value is a measure of luminosity (between 0 for black
and 100 for white), a* value is a measure for redness (positive value) or greenness
(negative value) and b* value is a measure for yellowness (positive value) or
blueness (negative value) (Valencia 2011) (Figure 4.5). Zero calibration by
measuring the surrounding and white calibration using the supplied white
calibration plate (Konica Minolta, Japan) was done at the beginning of sample
measurements.

Figure 4.5 CIE LAB colour space used by the Spectrophotometer (’ColorCodeHEx’ 2017). This
model was used to detect change in colour of the oatgurt samples using their L*, a* and b* values.

34
Commercial oatgurt samples stored in PP cup and glass jars were tested for colour
stability once a week. 125 mL of the sample was transferred from the packaging
unit to an opaque black plastic container, 5.5 cm in height and diameter (Annex
Figure A.3). The portable instrument was held perpendicularly to the sample cup
and the readings were carried out. The measurement was done thrice for each of
the biological triplicates of every sample. The L*, a* and b* values were obtained
and analysed using SpectraMagic™ NX, colour data software.
The total colour difference or change in visual perception, ΔE*, was calculated
using the formulae (Equation 4.1) ('Konica Minolta' 2017).

ΔE* = [ΔL*2 + Δa*2 + Δb*2]1/2 (4.1)

ΔL*is the difference between the absolute L* value of the sample and a reference.
Similarly Δa* and Δb* was calculated by finding the difference between the
absolute a* and b* values of the sample and reference respectively. The ΔE*
values of the probiotic and control samples were calculated with respect to two
different reference values. For statistical comparison the pre-set target colour (L*=
87.99, a*=-3.06 and b*=15.13) configured in the instrument was used as the
reference. These values indicated the colour of all samples including week 0
across incubation period with respect to the instrumental target coordinates. This
was used to find if there was a variation in colour with time.
The other reference used was the average absolute coordinates values of the
commercial control sample (week 0), which was considered as the ‘desired’ value
(L*=58.55, a*=9.83 and b*= 0.07). ΔE* values of all samples with respect to this
reference value was calculated to evaluate the deviation of the sample colour from
desired colour over the eight weeks incubation period. The higher the value of
ΔE* of the sample, more the deviation or difference in colour (Konica Minolta
2017). When ΔE* is <1, the change in colour is not perceptible through human
eyes; when the value is between 1 and 2, the change in perceptible through close
observation; and if the value is between 2 and 10, the change is perceptible in a
glance (Schuessler 2016). The qualitative analysis of this difference was done by
analysing the Δb* values of the samples compared to ‘desired’ value (b*sample –
b*desired). An increase in the value of Δb* towards the positive scale indicated the
sample to be less blue and more yellow compared to the selected reference sample.
A separate experiment was set up with commercial oatgurt in PP (control), stored
at RT to evaluate the influence of temperature on colour. The evaluation was
carried out only on weeks 2 and 4.

35
4.8 Statistical Analysis of Data
Statistical data analysis with 95% confidence interval was performed using the
GraphPad software (Prism 7, 2017). The two factors were the matrices L.
plantarum was incorporated into (fresh or commercial) and type of packaging (PP
or glass). There were two levels within each factor that were compared against
each other. Viability, pH and change in colour perception with respect to
instrument target reference value were analysed using Analysis of Variance
(ANOVA).

36
5 Results

The results obtained from eight weeks experimentation on fresh and commercial
oatgurt in PP cups and glass jars (PPcomm, Gcomm, PPfr and Gfr) were documented
and analysed.

5.1 Viability of L. plantarum 299v

The samples that were plated using standard spread plate method and incubated at
37°C for 48 hours were enumerated. Each week the incubated MRS agar plates at
different dilutions containing 10-300 colonies were selected and documented as
cfu per mL of oatgurt sample. The viability of probiotic L. plantarum 299v
inoculated into the commercial oatgurt in PP and glass remained well above the
required recommended dosage (107 cfu/mL) (Figure 5.1). The average starting
concentration at week 0 was 1.14±0.16 and 1.10±0.20 *108 cfu/mL of commercial
oatgurt in PP and glass respectively. After eight weeks the concentration went
down to 0.58±0.07 and 0.59±0.08 *108 cfu/mL commercial oatgurt in PP and glass
respectively. There was a significant decrease in viability compared to week 0,
only after seven weeks of incubation in both packaging materials (p< 0.05). In
fresh oatgurt also the inoculated probiotic remained above recommended dosage
but reduced significantly after two weeks of incubation (p< 0.05) compared to
week 0. The average starting concentration at week 0 was 1.13±0.15 and 1.10±0.2
*108 cfu/mL of fresh oatgurt in PP and glass respectively. The starting
concentrations were similar to that of the commercial oatgurt samples. After eight
weeks the concentration reduced to 0.38±0.09 and 0.30±0.08 *108 cfu/mL of fresh
oatgurt in PP and glass respectively.
It was not possible to establish a significant effect of the packaging material, and
hence presence of O2, on the viability of the probiotic strain. However, the stage of
incorporation (food matrix) of L. plantarum did have a significant effect on
viability (p< 0.05). The viability remained comparatively more stable when the
probiotic strain was added to the finished commercial product (Figure 5.3).

37
 2.00

1.50
*10^8 cfu/mL

1.00

0.50

0.00
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
PPcomm 1.14 1.41 1.29 1.64 1.44 1.26 1.05 0.77 0.58
Gcomm 1.10 0.89 1.09 1.15 1.10 1.01 0.79 0.71 0.59

Figure 5.1 Viability of L. plantarum 299v in commercial oatgurt sample. The average probiotic
cfu/mL of commercial oatgurt using PCM for samples incubated for eight weeks in PP cups and
glass jars.

2.00

1.50
*10^8 cfu/mL

1.00

0.50

0.00
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8
PPfr 1.13 0.98 0.84 0.44 0.56 0.64 0.22 0.26 0.14
Gfr 1.10 0.88 1.03 0.51 0.69 0.46 0.38 0.38 0.30

Figure 5.2 Viability of L. plantarum 299v in fresh oatgurt sample. The average probiotic cfu/mL
of fresh oatgurt using PCM for samples incubated for eight weeks in PP cups and glass jars.

2.00

1.50
*10^8 cfu/mL

1.00

0.50

0.00
Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

PPcomm Gcomm PPfr Gfr

Figure 5.3 Viability of L. plantarum 299v in commercial and fresh oatgurt sample. The average
probiotic cfu/mL of sample using PCM for samples incubated for eight weeks in PP cups and glass
jars.

38
 1.0E+10

1.0E+08

1.0E+06
cfu/mL

Week 4 (FCM)
1.0E+04 Week 4 (PCM)
Week 8 (FCM)
1.0E+02
Week 8 (PCM)
1.0E+00
PPcomm Gcomm PPfr Gfr
Oatgurt Sample

Figure 5.4 Comparing FCM and PCM results of L. plantarum viability on week 2 and week 4
for PPcomm, Gcomm, PPfr and Gfr samples.

The viability (live cells) was measured in all samples on week 4 and 8 using FCM
and compared with the number of cfu/mL using PCM. The results of FCM did not
deviate greatly from PCM (Figure 5.4). The FCM results were relatively higher
than the PCM (Annex Table B.1), which could be accounted as the cells that were
unable to form colonies.

5.2 pH
The pH values of the control, commercial oatgurt and fresh oatgurt were measured
once a week over eight weeks of incubation. There was a significant difference
(p< 0.05) between the pH of commercial product control samples and product
samples containing probiotic right from week 1. This ascertained that the drop in
pH was caused by the activity of the probiotic strain. In the commercial and fresh
oatgurt it was not possible to establish a significant effect of packaging material on
the pH change. There was a statistically significant difference between using glass
and PP at week 3 and week 7 in the commercial oatgurt and week 5 and week 6 in
fresh oatgurt. However, this was not sufficient data to ascertain a difference
caused by packaging material. The time of storage had a significant effect (p<
0.05) from week 1 of incubation in all samples (PPcomm, Gcomm, PPfr and Gfr). There
was a gradually decreasing trend in pH with time. The change in pH of the
commercial product, PPcomm and Gcomm was from pH 4.2±0.01 to pH 3.75±0.02 and
3.82±0.03 respectively. In the fresh product, PPfr and Gfr, the pH declined from
3.78±0.01 to 3.44±0.01. The starting pH at week 0 for commercial and fresh
samples were different, therefore there was an obvious significant difference with
time based on method of incorporation (Figure 5.5).

39
 4.4

4.2

3.8
pH

3.6

3.4

3.2
Week Week Week Week Week Week Week Week Week
0 1 2 3 4 5 6 7 8
PPcomm 4.27 4.14 4.12 4 3.81 3.86 3.77 3.75 3.75
Gcomm 4.27 4.18 4.19 4.1 3.81 3.83 3.83 3.83 3.82
Control PPcomm 4.26 4.2 4.25 4.23 4.13 4.09 4.05 4.05 4.01
Control Gcomm 4.26 4.22 4.25 4.29 4.05 4.09 4.09 4.07 4.05
PPfr 3.78 3.71 3.71 3.57 3.58 3.51 3.49 3.44 3.44
Gfr 3.79 3.72 3.72 3.57 3.58 3.47 3.44 3.44 3.44

Figure 5.5 Sample pH measured over eight weeks of incubation. The control commercial samples
(grey) were plotted against commercial probiotic sample to analyse the cause for change in pH.

5.3 O2% in PPcomm and PPfr Headspace

Headspace Oxygen %

15.00#
12.00#
9.00#
Oxygen %

6.00#
3.00#
0.00#
Week#1# Week#2# Week#3# Week#4# Week#5# Week#6# Week#7# Week#8#
PPcomm# 3.84# 5.25# 4.56# 4.90# 5.27# 5.76# 5.94# 6.28#
PPfr# 7.51# 9.07# 9.83# 10.85# 10.67# 11.21# 13.03# 13.30#

Figure 5.6 O2% in PP cup headspace using Dansensor CheckMate® 9900 O2/CO2. The results
were susceptible to errors as it was an intrusive method to measure headspace gas.

The O2% in the headspace of the PP cups over 8 weeks of storage was analysed.
The plotted graph (Figure 5.6) showed an increasing trend, which could be
indicative of the permeability of the packaging material. The glass packages
maintained close to an anaerobic environment that was determined during the pre-

40
experimental setup. The instrument used was intrusive and obtained results could
be susceptible to a sub-optimal way of performing the measurements or due to
handling error. Non-intrusive oxygen analysing methods might have provided
more accurate outcome and the glass jar anaerobicity could also be asserted.

5.4 Colour Stability of Commercial Oatgurt

The commercial oatgurt colour was measured using a handheld
spectrophotometer. It detected a peak at around 480 to 550 nm (Figure 5.7), which
corresponds to the typical absorption bandwidth of anthocyanin (Routray & Orsat
2011). Anthocyanin is the naturally occurring blue colour pigment in blueberries.
The absolute L*, a* and b* coordinate values of the samples from week 0 over
eight weeks were measured. The change in visual perception, ΔE*, was calculated
using the absolute coordinates and instrumental reference point (Figure 5.8). There
was a significant difference between the ΔE* values of the oatgurt sample in PP
and glass from week 2 (p< 0.05). Compared to week 0, a significant change was
observed after week 2 in the PP cup and after week 6 in the glass jars. The
interaction between the packaging material and time, therefore, had a significant
effect on colour (p< 0.05).

Instrument target value

(zero and white
calibration)

Detected sample values

Figure 5.7 Detection spectrum for blueberry/vanilla oatgurt sample using Spectrophotometer.
Peak detected at around 480 to 550 nm, corresponding to anthocyanin (blue colour pigment)
absorption wavelength. .

41
 38.00

36.00
ΔE*

34.00

32.00
0 1 2 3 4 5 6 7 8
Week Number
dE*GC Gcomm dE*PPC PPcomm

Figure 5.8 Change in visual perception of colour with respect to instrumental target reference
over eight weeks of incubation at 8°C. The ΔE* was calculated using the target reference value
(L*= 87.99, a*=-3.06 and b*=15.13) for control (PPC and GC) and probiotic commercial sample.

The presence of the probiotic strain, L. plantarum 299v, in the product did not
seem to have an impact on the visual perception of colour. There was no
significant difference in ΔE* values between the control sample and Gcomm with
probiotic strain till week 6 (Figure 5.8). In PP cups a significant difference after
week 3 of incubation was observed between control and probiotic sample. A clear
relation between the presence of the probiotic and change in ΔE* values could not
be established with measured results.

3.00

2.50

2.00
Gcomm
ΔE*

1.50
PPcomm
1.00

0.50

0.00
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8

Figure 5.9 Deviation of sample colour from desired colour over eight weeks of incubation at
8°C. The change in visual perception was calculated using average colour coordinates of week 0
control sample referred to as the desired colour coordinates (L*=58.55, a*=9.83 and b*= 0.07) as the
reference.

The average value of week 0 control colour coordinates was considered as the
reference colour (Figure 5.11a) accepted by the manufacturer (desired value). The
values measured for all samples were calculated using the ‘desired’ coordinates as
the reference to compare deviation of the product colour from the desired colour.
There was a steeper increase in total change in colour of oatgurt in PP compared to
glass (Figure 5.9). The ΔE* value for Gcomm remained below 2 even at week 8. In
PPcomm after week 5 the value increases beyond 2, indicating that the change in

42
colour would be perceptible at a glance. Upto week 3 the change in colour is not
perceptible through human eyes when in glass jar, however after week 2 it is
perceptible for PP. The Δb* values (Table 5.1) of the samples increased and
shifted towards more positive values indicating decrease in blue appearance and
increase in yellowness of the product over time.
Table 5.1 Sample Δb* (blueness-yellowness indicator) values across time.

Incubation PPcomm Gcomm

Period

Week 1 !0.01±0.01 !0.03±0.13

Week 2 0.27±0.09 0.44±0.09
Week 3 0.18±0.20 0.31±0.08
Week 4 0.27±0.12 0.42±0.02
Week 5 0.07±0.20 0.25±0.13
Week 6 0.42±0.15 0.61±0.13
Week 7 0.71±0.13 0.81±0.07
Week 8 0.87±0.14 0.74±0.11
Note: Values calculated using ‘desired’ reference coordinate (Average b* week 0); b*sample-b*desired.

The additional study results showed the influence of temperature on colour. The
evaluation was done on week 2 and week 4 of incubation at RT. The coordinates
were calculated using ‘desired’ value as the reference. The deviation from desired
value was calculated. The results were compared with the values of oatgurt stored
at 8°C. The total colour difference was significantly higher, that was observed
both in the instrumental values (Figure 5.10) and visual observation (Figure
5.11b). The colour right at week 2 is perceptible at a glance when the oatgurt is
stored in RT.

43
 6.00

4.00

ΔE*

2.00

0.00
Week 2 Week 4
8°C 0.72 1.52
RT 4.27 4.9

Figure 5.10 Deviation of sample colour incubated at 8°C.and RT at week 2 and week 4 from
week 0 desired reference value.

(a) (b)

Figure 5.11 Visual appearance of commercial oatgurt sample. The samples analysed were (a)
commercial control sample at week 0 stored at 8°C and (b) commercial control sample at week 2
stored at RT.

44
6 Discussion

As a food matrix, the oatgurt manufactured by Oatly AB appeared to be suitable

for probiotic strain incorporation. It is rich in beta-glucan that is known to be a
prebiotic (Mårtensson et al. 2002) and has a pH of 4.2 that most probiotic strains
are tolerant to. Oatgurt is currently packed in ~1mm thick PP cups closed with
thermosealable aluminium foil. The packaging unit could be susceptible to O2
permeation due to the material properties. An increase in the O2% in the headspace
of the PP cups over time (Figure 5.5) filled with the commercial and fresh oatgurt
samples (PPcomm and PPfr) ascertained this occurrence. The impact of the presence
of O2 was not found to be adverse on the viability of selected probiotic strain, L.
plantarum 299v (Figure 5.5). In all four samples, PPcomm, Gcomm, PPfr and Gfr, the
strain viability from an initial concentration of 1.14±0.16*108cfu/mL remained
above recommended dosage of 107 cfu/mL well after the storage period. This was
the required concentration for a food product to be considered a probiotic. There
was however, a statistically significant difference in strain viability between fresh
and commercial oatgurt from week 2 of analysis. The increased stress during
fermentation during processing and presence of starter culture in the fresh oatgurt
sample could explain the comparatively lower stability in viability of the probiotic
strain in fresh sample. With the resulting data it was not possible to establish a
significant effect that the packaging material had on the viability. The tolerance
towards presence of O2 could be explained by the microaerophilic nature of L.
plantarum 299v. The FCM results did not deviate greatly from that of PCM,
indicating the prospect of using FCM for future viability studies of L. plantarum
299v. This would significantly reduce the time and labour required to conduct
conventional plate viability counts. The flow cytometer needed to be optimized for
the oatgurt sample and L. plantarum 299v bacteria. This process took time;
therefore, it was not possible to get results for week 0. This information could
have improved the analysis by providing a reference to compare the week 4 and
week 8 results.
The strain viability was also not influenced by the decrease in pH over time in
both food matrices. This result agrees with the studies that show L. plantarum
299v stable in food matrix at pH as low as 2.8 (Molin 2001). The reduction in pH
of the samples can mainly be attributed to the probiotic strain activity, as the pH of
the control sample declined at a much slower rate (pH 4.26 to pH 4.01). There is a
statistically significant reduction in the pH over time in fresh and commercial
sample, however the effect of packaging material on the pH could not be
established. Although the strain viability is not affected by the pH decline, there
45
could be an influence on product flavour (taste). The increasing acidity of fresh
oatgurt that had a comparatively low initial (start) pH could have a bigger impact
on product sensory properties. During commercial production, the fresh oatgurt
would be supplemented with additives like stabilizers and sweeteners. This would
potentially increase its pH, and could considerably reduce sensory impact.
However, adding the probiotic strain at the end of the production line (commercial
oatgurt sample) could potentially reduce the change required in the production and
processing conditions. This could benefit the manufacturer during commercial
production.
The change in colour in the vanilla/blueberry oatgurt specifically was a concern
for the manufacturer. The packaging material seemed to have an impact on the
change in colour, or change in visual perception (ΔE*) of the product. Statistically
there was a significant difference from week 2 of incubation between the two
packaging materials. The Gcomm samples were comparatively more consistent with
the week 0 ΔE* value. The change mostly occurred in the form of increasing Δb*
values compared to the desired reference value. This increase in Δb* values of the
sample meant that the product was becoming less blue and showing more
yellowness according to the L*a*b* colour format. Visually it was perceived as
changing from bluish-purple to a dull bluish-grey colour.
The presence of L. plantarum 299v in the product did not have an obvious affect
on the product colour. The resulting colorimetry results comparing the control and
oatgurt with probiotic was not sufficient to establish a relationship between the
colour and presence of probiotic strain. On direct visual inspection, the difference
in colour between sample stored in PP and glass was not obvious. However, there
was a clear difference, both with the instrumental data and visual inspection,
between the samples that were stored at 8°C and RT (21°C). The colour seemed to
be greatly influenced by the temperature of the atmosphere compared to presence
of oxygen that permeated through the packaging material. This change in colour
could also be affected by growth of other organisms that are generally supressed at
cold chain temperatures.
Although glass as a packaging material demonstrated better results in terms of
colour stability over time, it is not suitable for commercial usage. Glass also
allows light, which could also affect the product physicochemical properties. The
influence of light, however, was controlled in this study. The weight and fragility
of glass compared to PP, along with the high cost limits its application in the food
industry. An alternative would be selecting a packaging material with improved
oxygen barrier properties or adding an additional layer like ethylene vinyl alcohol
(EVOH) to the current PP cup. This could reduce the change in colour perception,
while being practical in terms of handling and logistics.
Using packaging with improved oxygen barrier properties compared to PP could
improve blueberry/vanilla oatgurt colour positively (closer to desired value), but
under controlled conditions (cold chain and no light) PP demonstrated results

46
similar to glass. The material used did not affect probiotic strain viability and pH
in this study. The results obtained are specific for L. plantarum 299v and the
incorporation methods that were used. Jayamanne & Adams (2004b) demonstrated
glass to be superior while using bifidobacteria strain as the probiotic. A study with
Lactobacillus paracasei conducted by Pimentel et al. (2015) also concluded glass
to be better at maintaining viability of that particular strain. This indicates that the
results are exceedingly dependent on the selected strain. However, similar to the
results found in this study, Pimentel et al. (2015) concluded that the packaging
material (plastic or glass) did not affect physiochemical properties (colour and pH)
of probiotic apple juice. The results obtained from the study indicate that Oatly
can continue using PP for their probiotic oatgurt.

47
7 Conclusion and Future Scope

The study presents the possibility of commercially incorporating probiotic strain L.

plantarum 299v into Oatly’s oat-based yogurt (oatgurt) and packaging it in PP
cups. Oatly’s oatgurt was able to sustain L. plantarum 299v stably for over eight
weeks when stored in cold chain. The current packaging material used for the
commercially available non-probiotic oatgurt, PP, could be used for probiotic
oatgurt as well since it sustains the viability of L. plantarum 299v over the
incubation period well above recommended dosage. Adding the strain to the
finished oatgurt would be a more viable option for the manufacturer in terms of
processing method. The viability and pH of the fresh oatgurt in this study is
comparatively less favourable than commercial oatgurt. The colour of the oatgurt
could be improved with a packaging material having higher oxygen barrier,
however PP could ensure acceptable colour stability for atleast five weeks of
storage.
Table 7.1 Overall conclusion of experimental results.

Packaging
Material Polypropylene Glass

Food Matrix

Commercial Oatgurt Viability pH O2 Colour Viability pH O2 Colour

Fresh Oatgurt** Viability pH O2 - Viability pH O2 -

Note: Green – Favourable; Yellow – Acceptable; Red - Unacceptable

In order to further advance the development of this probiotic product, sensory
analysis for appearance and change in flavour should be conducted. The organic
acid profile due to the incorporation of L. plantarum 299v should be studied to
understand its metabolic activity in this food matrix. Pilot scale experiments are
required to ensure viability of the bacteria is not compromised because of other
process and production related factors. Literature study indicates oat-based
medium to be well suitable for the incorporation of L. plantarum 299v, paving
way for probiotic product development with other Oatly food products. With
limited research on probiotic non-dairy food products, there is a need for several
studies to establish concrete commercial product development strategies.

48
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Appendix A Material and Method

A.1 Packaging Materials

Figure A.1 Glass jar with heat seal metal cap.

Figure A.2 Polypropylene cups (current packaging at Oatly) with thermosealable aluminium
foil.

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A.2 Experimental Materials

A.2.1 Colorimeter Equipment

Sample

Measure

Figure A.3 Commercial oatgurt sample transferred into black plastic container for colorimeter
measurement.

A.2.2 Steps of Incorporation

Weigh &
add in Add L. Test Evaluate
Oatly plantarum Seal and Incubate (once a and
Commercial Oatgurt glass & Label at 8°C
299v week) Analyze
PP

Figure A.4 Commercial oatgurt sample formulation and packaging process.

Starter
Weigh & add Seal Test Evaluate
culture + L. Incubate
Fresh Oat milk Fermentation in glass & and (once a and
pantarum at 8°C
PP Label week) Analyze
299v

Figure A.5 Fresh oatgurt sample formulation and packaging process.

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Appendix B Supplementary Results

B.1 Viability Analysis of Freeze Dried Probiotic Powder

Figure B.1 106 dilution of freeze-dried probiotic

powder stock plated using standard spread plate
method on solid MRS agar. L. plantarum colonies
are reported to be large, creamy and white-
yellowish colour (Johansson et al. 1998)

Figure B.2 Microscopic image of rod shaped

Lactobacillus plantarum cells scooped from
obtained colonies on MRS plate.

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B.2 FCM Analysis Results

Table B.1 FCM and PCM results for viability of commercial and fresh oatgurt.

Week 4 Week 8
Sample
PCM FCM PCM FCM
5.77E+07±6.81 7.31E+07±1.48
1.44E+08±4.52 2.66E+08±7.
PPcomm E+06 E+07
E+07 7E+06
5.93E+07±8.14 5.71E+07±6.48
1.10E+08±1.37 1.88E+08±3.
Gcomm E+06 E+06
E+07 37E+07
1.43E+07±4.04
5.57E+07±1.88 5.2E+08 ±5.7 5.98E+07±1.69
PPfr E+06
E+07 E+07 E+06
3.03E+07±7.51 6.57E+07±8.95
6.87E+07±5.13 5.06E+08±6.
Gfr E+06 E+06
E+06 7E+07

The graphs obtained from FCM demonstrated the events that were detected by the
flow cytometer having the green or red fluorescent stains. Figure B.3a is the graph
resulting from the unstained oatgurt sample. The background was defined using
this sample. Figure B.3b is the control sample without probiotic. The resulting
graph represents the dead region. The live region was defined using the probiotic
stock solution. Figure B.3c, B.3d, B.3e and B.3f are the results of PPcomm,
Gcomm, PPfr and Gfr respectively. The coloured dots on the graph represent the
detected events (cells). Red colour dot is a single event and as the number of
events increase in the same position the increasing intensity is represented by
different (colours red<orange<yellow<green<blue). Since it was not possible to
analyse week 0 samples, this study using FCM is not complete. However, the
results at week 4 and week 8 of the samples are comparable with PCM. This is a
good indication of using this technique for quicker results.

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 5
10

alive
4
10
FL1(529)-Log_Height

3
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dead

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(a)
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(b)

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alive
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3
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dead

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1
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(c)
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alive
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3
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dead

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 5
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alive
4
10
FL1(529)-Log_Height

3
10

dead

2
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1
10

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10 0 1 2 3 4 5
10 10 10 10 10 10
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(e)
5
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10
FL1(529)-Log_Height

3
10

dead

2
10

1
10

0
10 0 1 2 3 4 5
10 10 10 10 10 10
FL3(620)-Log_Height

(f)
Figure B.3 Viability assessment results using FCM (a) Unstrained control oatgurt
(background); (b) stained control oaygurt; (c) PPcomm; (d) Gcomm; (e) PPfr and (f)Gfr. The
different colours in the graph represent the intensity or number of events that occur in a particular
position. The red dots are single events, while it changes to yellow, orange, green and blue with
increasing number of events that occur in the same position.

63