Evaluation of accelerated shelf life testing of UHT milk

93 Pages·2016·2.63 MB·English

by  Anne Vuholm Sunds

Evaluation of accelerated shelf
life testing of UHT milk
Master of Science thesis - 60 ECTS
Anne Vuholm Sunds
Student No. 20113104
Master thesis 2016
Molecular Nutrition and Food Technology - Aarhus University
Title: Evaluation of accelerated shelf life testing of UHT milk

Project period: 3th August 2015 to 1st July 2016
Defence: 8th July

Written by: Anne Vuholm Sunds
Student number: 20113104
Education: MSc in Molecular Nutrition and Food Technology

Internal supervisor:
Lotte Bach Larsen, Professor
Department of Food Science, Faculty of Science and Technology

External supervisor:
Valentin Maximilian Rauh, Research Scientist
Arla Strategic Innovation Centre, Ingredients and Milk Science

Project location:
Aarhus University Arla Strategic Innovation Centre
Department of Food Science Ingredients and Milk Science
Blichers Allé 20 Rørdrumvej 2
8830 Tjele 8220 Brabrand
Denmark Denmark

Number of pages: 85
Preface and acknowledgements

This master thesis project is a result of 11 months of work at Arla Strategic Innovation Centre and
the Faculty of Science and Technology, Department of Food Science at Aarhus University, in the
period from August 2015 to July 2016.
My greatest thank goes to my university supervisor Lotte Bach Larsen and my supervisor at Arla
Valentin Maximilian Rauh. Lotte, thank you for your great support and scientific guidance, I am
very glad to have had you as my supervisor. Valentin, thank you for the opportunity to work with
you and your colleagues at Arla, it has been a pleasure and thank you for your great guidance and
proofreading of manuscripts.
I also owe a huge thank to the laboratory technicians at Arla for the help with equipment and data
analysis. Mona Slyngborg and Betina Mikkelsen I appreciate all your help with the functionality
analyses, and for your support. Jan Breinholt Carlsen and Lene Buhelt Johansen, thank you very
much for your help with GC-MS and HPLC. Thank you to Gitte Hald Kristiansen and Ida Sørensen
for your assistance with the quantification of furosine.
Thanks to my fellow master student Lina Berg for great discussions, support and motivational pep
talks, and thank you to my family and friends for their encouragement and support.

Aarhus University, Department of Food Science, July 2016

Anne Vuholm Sunds
Abstract

Development of UHT dairy products requires time consuming and resource intensive shelf life
tests. Thus, a valid accelerated shelf life test would be of high value in the development of new
products. This thesis provides a quantification of chemical and physical changes in commercial
UHT milk stored at different temperatures, with the aim of establishing a valid setup to accelerate
shelf life development. The temperatures selected were; 10 °C, 20 °C, 30 °C, 40 °C and 50 °C as
well as three temperature cycles. The skimmed and full fat UHT milk samples were analysed during
a storage period of 24 weeks. This included chemical analyses of the three phases of the Maillard
reaction (MR) as well as the lipid oxidation. The initial stage of the MR was analysed by a
quantification of furosine using high performance liquid chromatography (HPLC). The intermediate
stage of the MR was analysed by fluorescence spectroscopy and gas chromatography-mass
spectroscopy (GC-MS). Finally, the late stage of the MR was analysed with colour measurements.
An evaluation of physical destabilization was conducted with focus on gravitational separation, in
form of creaming and sedimentation. Physical changes were analysed using; optical stability
analyzers, evaluation of protein and fat distribution in; top, middle and bottom fractions, as well as
analysis of fat globule size distribution.
Exposure to elevated temperatures accelerated both chemical and physical changes over the storage
period. The chemical changes revealed data possible to describe with kinetic models. Formation of
furosine followed a first order reaction kinetic, whereas fluorescence and colour changes followed a
zero order reaction kinetic. Additionally, all three stages of the MR fitted into the Arrhenius
equation. Following corresponding Q values were obtained; for the initial MR of 1.5 to 2.3, for
10
the intermediate MR of 3.9 to 10.9 and for the late MR of 2.8 to 6.
The acceleration of physical changes varied between the applied methods, where different rates of
creaming and sedimentation were observed. No changes in fat globule size distribution were found,
which may indicate that other parameters are affecting the creaming rate, possibly by viscosity and
density changes. Development of the three temperature cycles varied between chemical and
physical parameters analysed. This was illustrating that it is only slightly possible to delay the MR
once it has started, even when exposed to lower storage temperatures. On the other hand, physical
parameters followed the average temperature. For future accelerated shelf life tests, a prediction of
shelf life seems to be possible within the temperature range of 20 °C to 30 °C. These findings are
based on the Arrhenius plots obtained in the present study.
Sammendrag

Udviklingen af UHT mejeri produkter, resulterer i tids- og ressourcekrævende holdbarhedstests.
Derfor vil en valid accelereret holdbarhedstest, være af stor værdi i udviklingen af nye produkter.
Denne specialeafhandling vil give en kvantificering af kemiske og fysiske ændringer i kommerciel
skummet og sød UHT mælk, oplagret ved forskellige temperaturer. Formålet med studiet er at
etablere en gyldig opsætning for acceleration af de processer der har indflydelse på holdbarheden.
De udvalgte temperaturer var; 10 °C, 20 °C, 30 °C, 40 °C og 50 °C så vel som tre temperaturcykler.
UHT mælkeprøverne blev analyseret gennem oplagringsperioden på 24 uger. Dette inkluderede
kemiske analyser af de tre stadier af Maillard reaktionen (MR) samt af lipidoxidationen. Det
indledende stadie af MR´en blev analyseret ved en kvantificering af furosin, ved brug af højtydende
væskekromatografi (HPLC). Det intermediære stadie af MR´en blev analyseret ved fluorescens
spektroskopi og gaskromatografi-massespektrometri (GC-MS) og det sidste stadie af MR´en blev
analyseret ved farvemåling. En evaluering af fysisk destabilisering blev udført med fokus på
tyngdeseparation, i form af dannelse af fløde på overfladen og sedimentation af protein. Fysiske
ændringer blev analyseret ved optiske stabilitetsanalyser, evaluering af protein- og fedtfordeling i
top, midte og bund, samt analyse af fordelingen af fedtkuglestørrelser.
Både kemiske og fysiske ændringer i mælken accelererede over oplagringsperioden ved udsættelse
for forhøjede temperaturer. Det var muligt at beskrive data fra de kemiske analyser med kinetiske
modeller. Dannelse af furosin fulgte en første ordens reaktionskinetik, mens fluorescens- og
farveændringerne fulgte en nulte ordens reaktionskinetik. Desuden passede alle tre stadier af MR´en
ind i Arrhenius ligningen. Følgende korresponderende Q værdier blev fundet: 1,5 til 2,3 for den
10
indledende MR, 3,9 til 10,9 for den intermediære MR og 2,8 til 6 for den sene MR.
Accelerationen af fysiske ændringer varierede mellem de benyttede metoder, hvor forskellige rater
af flødedannelse og sedimentation blev observeret. Der blev ikke observeret nogen ændringer i
fordelingen af fedtkuglestørrelse, hvilket kan indikere at andre parametre har indflydelse på den
observerede flødedannelse, muligvis på grund af ændringer i viskositet og densitet. Udviklingen af
de tre temperaturcykler varierede mellem de kemiske og fysiske parametre der blev analyseret.
Dette illustrerer at det kun delvist er muligt at forsinke MR´en når først den er startet, selv ved
udsættelse for lavere oplagringstemperaturer. De fysiske parametre fulgte derimod gennemsnits-
temperaturen. En forudsigelse af holdbarheden synes at være mulig mellem 20 °C og 30 °C, for
accelererede holdbarhedstests i fremtiden. Disse resultater er baseret på Arrhenius graferne fra de
kemiske analyser.
Abbreviations

AGE: Advanced glycation end-products
CN: Casein
DAD: Diode array detector

DHS: Dynamic headspace sampling
DTE: Dithioerythritol
DLVO: Deyaguin-Landau-Verwey-Overbeek
ESL: Extended shelf-life

FT-IR: Fourier transform infrared spectroscopy
GC-MS: Gas chromatography–mass spectrometry
HMF: Hydroxymethylfurfural
LA-transformation: Lobry de Bruyn-van Ekenstein-transformation
LC-MS: Liquid chromatography–mass spectrometry
MR: Maillard reaction
MRP: Maillard reaction product
MFG: Milk fat globule
MFGM: Milk fat globule membrane
PCA: Principal component analysis
RP-HPLC: Reversed phase-high pressure liquid chromatography
SIM: Selected ion monitoring
SLS: Static light scattering
SPME: Solid phase micro-extraction
TAG: Triacylglyceride
TIC: Total ion current
UHT: Ultra high temperature
α-La: α-Lactalbumin
β-Lg: β-Lactoglobulin
Table of content

1. Aim and hypothesis……………………………………………………………………………… 1
2. Outline of the thesis………………………………………………………………………………2
3. Introduction……………………………………………………………………………………… 4
3.1 Milk…………………………………………………………………………………………………….. 4
3.1.1 Carbohydrates………………………………………………………………………………………………... 4
3.1.2 Proteins……………………………………………………………………………………………………….. 5
3.1.2.1 Analysis of protein composition by HPLC……………………………………………………………… 7
3.1.3 Lipids………………………………………………………………………………………………………….. 8
3.2 UHT milk………………………………………………………………………………………………. 8
3.3 Changes induced by UHT treatment…………………………………………………………………. 11
3.4 Enzymatic hydrolysis………………………………………………………………………………… 13
3.5 Chemical changes in UHT milk during storage………………………………………………………. 14
3.5.1 Maillard reaction…………………………………………………………………………………………… 15
3.5.2 Lipid oxidation……………………………………………………………………………………………… 19
3.6 Physical changes in UHT milk during storage……………………………………………………….. 21
4. Material and methods…………………………………………………………………………. 25
4.1 Milk samples and treatments…………………………………………………………………………. 25
4.2 Analysis of chemical changes………………………………………………………………………… 25
4.2.1 Peptide analysis by HPLC………………………………………………………………………………… 26
4.2.2 Initial Maillard reactions…………………………………………………………………………………. 26
4.2.3 Intermediate Maillard reactions and lipid oxidation………………………………………………….. 27
4.2.4 Late Maillard reactions……………………………………………………………………………………. 28
4.2.5 Protein composition………………………………………………………………………………………... 28
4.3 Analysis of physical changes…………………………………………………………………………. 29
4.3.1 Physical destabilization…………………………………………………………………………………… 29
4.3.2 Protein and fat content……………………………………………………………………………………. 31
4.3.3 Fat globule size distribution………………………………………………………………………………. 31
4.4 Data analysis………………………………………………………………………………………….. 32
5. Results…………………………………………………………………………………………... 33
5.1 Chemical changes…………………………………………………………………………………….. 33
5.1.1 Enzymatic hydrolysis………………………………………………………………………………………. 33
5.1.2 Initial Maillard reactions…………………………………………………………………………………. 34
5.1.3 Intermediate Maillard reactions and lipid oxidation………………………………………………….. 38
5.1.4 Late Maillard reactions…………………………………………………………………………………… 46
5.1.5 Protein composition………………………………………………………………………………………... 49
5.2 Physical changes……………………………………………………………………………………… 50
5.2.1 Physical destabilization…………………………………………………………………………………… 51
5.2.2 Protein and fat content…………………………………………………………………………………….. 53
5.2.3 Fat globule size distribution……………………………………………………………………………… 55
5.3 Principal component analysis…………………………………………………………………………. 56
6. Discussion………………………………………………………………………………………. 57
6.1 Chemical changes…………………………………………………………………………………….. 57
6.2 Physical changes……………………………………………………………………………………… 67
6.3 Comparison of accelerated parameters……………………………………………………………….. 70
7. Conclusion……………………………………………………………………………………… 71
8. Perspectives…………………………………………………………………………………….. 73
9. List of references………………………………………………………………………………. 74
10. Appendix……………………………………………………………………………………… 84
1. Aim and hypothesis
Consumers demand high quality dairy products with good sensory attributes and commercial
sterility throughout shelf life. To guarantee these properties heat treatment is almost always applied
to dairy products today (Chavan et al., 2011; Lewis and Deeth, 2008). The main aim of heat
treatment is to inactivate undesired factors of the raw milk, such as pathogenic and spoilage
microorganisms and enzymes. On the other hand it is desired to preserve functional, nutritional and
organoleptic properties, by prevention of undesired heat induced chemical changes (Lewis and
Deeth, 2008; Singh and Waungana, 2001). In this perspective the choice of heat treatment is a
balance between preferences. Different heat treatments are applied to commercial milk products,
mainly high temperature short time (HTST) pasteurization (72 ºC, 15 sec), extended shelf-life
(ESL) (130-145, <1 sec) and ultra-high temperature (UHT) (135-150 ºC, 1-10 sec) (Walstra et al.,
2006). The market for milk treated at UHT is growing worldwide, today these products are found in
most countries, especially in Asia, Europe and South America (Bimbo et al., 2016; Jansson, 2014a).
In addition to prolonged shelf life, UHT processing is beneficial due to low energy costs and
elimination of cooling conditions during distribution and storage (Chavan et al., 2011). The
reported shelf life of UHT dairy products stored at ambient temperatures is between 6-9 months
(Bimbo et al., 2016; Richards et al., 2014). During processing and storage the UHT milk is
subjected to considerable chemical and physical changes, affecting the consumer acceptability and
hence the shelf life of the product. Possible undesirable effects include loss of nutrients, browning,
emulsion instability, age gelation and formation of off-flavours. Flavour changes are a major shelf
life limiting factor in UHT milk, mainly due to the Maillard reaction (MR), but possibly also lipid
oxidation or hydrolysis depending on the UHT treatment (Richards et al., 2014). The MR can be
affected by several factors including temperature, time, pH, water activity, type of sugar etc. (Oliver
et al., 2006). Physical destabilization is another major factor, which can result in creaming of fat
and/or sedimentation of protein (Calvo and de la Hoz, 1992; Chavan et al., 2011).

Food manufacturing today meets high expectations in the development of new products within short
time (Hough et al., 2006). The long shelf life of UHT dairy products result in very expensive and
time consuming shelf life tests in the development of new products. From this perspective
accelerated shelf life tests are highly valuable, with a significant reduction of time from product
development to market (Richards et al., 2014). An accelerated shelf life test can be performed by
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exposing the product to storage conditions with an accelerating effect on physical, chemical or
microbial changes. The accelerating factors depend on the specific product and the normal storage
conditions. Often changes in temperature, humidity or water activity are applied to accelerate shelf
life (Hough et al., 2006; Richards et al., 2014). Exposing the product to such a controlled
environment makes it possible to increase the deterioration rate and hence predict the shelf life
(Richards et al, 2014). Previous studies have attempted to accelerate the shelf life of milk, but
mainly with a focus on sensory attributes (Hough et al., 2006), proteolysis (Button et al., 2011) or
single components from the Maillard reaction (Richards et al., 2014). A valuable tool in the
development of UHT milk would therefore be a valid shelf life test accelerating both chemical and
physical changes, to give a more complete estimate of the predicted shelf life.

Hence the aim of this study is:
 To give a quantification of physico-chemical changes depending on storage conditions, and
hereby to establish a valid setup to accelerate shelf life development.

The hypothesis of this study is that:
 It is possible to establish a system for accelerated shelf life testing of UHT milk by
exposure to elevated storage temperatures including temperature cycles.
 Such an accelerated shelf life test can be used in prediction of shelf life from
characterisation of chemical and physical changes.
 A valid accelerated shelf life test for prediction of shelf life of UHT milk is possible.

To test these hypotheses, conventional skimmed and full fat indirect UHT milk were exposed to
five different storage temperatures and three temperature cycles, over a period of 24 weeks.
2. Outline of the thesis
This master thesis gives a presentation of the results obtained in relation to existing knowledge
within the field. To test the hypothesis two commercial UHT milk products were subjected to
different storage temperatures, in order to accelerate the shelf life development over a period of
24 weeks. The selected milk types were skimmed and full fat commercial UHT milk products, from
Arla Foods produced in Pronsfeld, Germany. The accelerating factors used were elevated storage
temperatures, temperature cycling and centrifugation with the use of Lumifuge. Storage
temperatures selected for the study were 10 °C, 20 °C, 30 °C, 40 °C and 50 °C, representing slightly
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