EFFECT OF NANOENCAPSULATION OF PURIFIED POLYPHENOLIC POWDER ON ENCAPSULATION EFFICIENCY, STORAGE AND BAKING STABILITY
A THESIS SUBMITTED TO
THE GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES OF
MIDDLE EAST TECHNICAL UNIVERSITY
BY
ALEXANDRU LUCA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF MASTER OF SCIENCE IN
FOOD ENGINEERING
JULY 2012
Approval of the thesis:
EFFECT OF NANOENCAPSULATION OF PURIFIED POLYPHENOLIC POWDER ON ENCAPSULATION EFFICIENCY, STORAGE AND BAKING
STABILITY
submitted by ALEXANDRU LUCA in partial fulfillment of the requirements for the degree of Master of Science in Food Engineering Department, Middle East Technical University by,
Prof. Dr. Canan Özgen ______________________
Dean Graduate School of Natural and Applied Sciences Prof. Dr. Alev Bayındırlı ______________________
Head of Department, Food Engineering
Prof. Dr. Gülüm Şumnu ______________________
Supervisor, Food Engineering Dept., METU
Prof. Dr. Serpil Şahin ______________________
Co-Supervisor, Food Engineering Dept., METU
Examining Committee Members:
Prof. Dr. Vasıf Hasırcı ______________________
Dept. of Biological Sciences , METU
Prof. Dr. Gülüm Şumnu ______________________
Food Engineering Dept., METU
Prof. Dr. Serpil Şahin ______________________
Food Engineering Dept., METU
Prof. Dr. Esra M. Yener ______________________
Food Engineering Dept., METU
Assist. Prof. Dr. Özge Şakıyan Demirkol ______________________
Food Engineering Dept., Ankara University
Date: 03.07.2012
iii
I hereby declare that all information in this document has been obtained and presented in the accordance with academic rules and ethical conduct. I also declare that, as required by these rules and conduct, I have fully cited and referenced all materials and results that are not original to this work.
Name, Last Name: Alexandru Luca Signature:
iv ABSTRACT
EFFECT OF NANOENCAPSULATION OF PURIFIED POLYPHENOLIC POWDER ON ENCAPSULATION EFFICIENCY, STORAGE AND BAKING
STABILITY
Luca, Alexandru
M.Sc., Department of Food Engineering Supervisor: Prof. Dr. Gülüm Şumnu Co-supervisor: Prof. Dr. Serpil Şahin
July 2012, 151 pages
The primary objective of this study was to obtain nano-emulsion containing polyphenolic compounds extracted from sour cherry pomace and to investigate the effect of degritting of polyphenolic concentrates on the encapsulation efficiency and particle size distribution of capsules and emulsions. It was also aimed to study storage and baking stability of the capsules. Extracted polyphenolic concentrate was degritted at 10,000 rpm for 2 min. Purification reduced Sauter mean diameter (D[32]) of concentrated extract from 5.76 μm to 0.41 μm. Unpurified and purified concentrates were freeze dried for 48 h to obtain extracted phenolic powder (EPP) and purified extracted phenolic powder (PEPP), respectively. Powders were entrapped in two types of coating materials which contain 10% maltodextrin (MD) or 8% MD-2% gum arabic (GA). Samples were prepared by ultrasonication (160 W, 50% pulse) for 20 min.
v
Emulsions containing EPP had D[32] of 1.65 and 1.61 μm when they were entrapped in 10% MD and 8% MD-2% GA coating material solutions, respectively. It was possible to obtain nano-emulsions when purification step was performed. Emulsions prepared with PEPP and coated with 10% MD and 8% MD-2% GA had D[32] of 0.396 and 0.334 μm, respectively. Encapsulation efficiency of the capsules increased significantly from 86.07-88.45% to 98.01-98.29% by means of degritting (p≤0.001). Loss of total phenolic content during storage at 43% and 85% relative humidities was smaller for encapsulated powders when compared to powders not entrapped in coating material. In addition, encapsulation significantly increased retention of phenolic compounds from 15.1-22.2%
to 30.4-30.7% during baking (p≤0.05).
Keywords: degritting, encapsulation, nano-emulsion, sour cherry pomace, ultrasonication
vi ÖZ
ARINDIRILMIŞ POLİFENOLİK TOZUN NANOENKAPSÜLASYONUNUN KAPLAMA VERİMİ, SAKLAMA VE PİŞİRME SIRASINDAKİ
STABİLİTELERİ ÜZERİNE ETKİSİ
Luca, Alexandru
Yüksek Lisans, Gıda Mühendisliği Bölümü Tez Yöneticisi: Prof. Dr. Gülüm Şumnu Ortak Tez Yöneticisi: Prof. Dr. Serpil Şahin
Temmuz 2012, 151 sayfa
Bu çalışmanın asıl amacı, vişne posasından özütlenmiş polifenolik maddeler içeren nano-emülsiyonların elde edilmesi ve konsantre edilmiş polifenoliklerin arındırılmalarının kaplama verimi, kapsüllerin ve emülsiyonların parçacık boyutu dağılımları üzerine etkilerinin araştırılmasıdır. Bu çalışmada ayrıca, kapsüllerin saklama ve pişirme sırasındaki dayanıklılıklarının çalışılması da amaçlanmıştır. Özütlenmiş konsantre polifenolikler, 10000 dev/dak hızda 2 dakika boyunca arındırılmıştır.
Arındırma işlemi, konsantre edilmiş özütün Sauter ortalama çapını (D[32]) 5.76 µm’den 0.41 µm’ye düşürmüştür. Fenolik özüt (FÖ) ve arındırılmış fenolik özüt (AFÖ) elde etmek için, sırasıyla arındırılmamış ve arındırılmış konsantre özütler, 48 saat boyunca dondurmalı kurutucuda kurutulmuştur. Elde edilen toz ürünler, %10 maltodekstrin (MD) veya %8 MD-%2 Arap zamkı (AZ) olmak üzere iki tip kaplama malzemesi
vii
kullanılarak hapsedilmiştir. Örnekler 20 dakika boyunca ultrasonikasyon (160W, %50 vurgu) metodu uygulanarak hazırlanmıştır. FÖ içeren emülsiyonların D[32]’si %10 MD ve %8 MD-%2 AZ içeren kaplama maddesi kullanıldığında sırasıyla 1.65 ve 1.61 µm olmuştur. Arındırma işlemi uygulandığında nano-emülsiyonların elde edilmesi mümkün olmuştur. AFÖ ile hazırlanan ve 10% MD ve %8MD-%2 AZ ile kaplanan emülsiyonların D[32] değerleri sırasıyla 0.396 ve 0.334 µm’dir. Arındırma işlemi ile, kapsüllerin kaplama verimi %86.07-88.45’den %98.01-98.29’ye önemli derecede artmıştır (p≤0.001). %43 ve %85 bağıl neme sahip ortamlarda saklanan kaplanmış tozların toplam fenolik madde kayıpları kaplanmamışlara göre daha az olmuştur. Ayrıca, kaplama işlemi, pişirme sırasında fenolik maddelerin dayanıklılıklarını istatistiksel olarak önemli derecede %15.1-22.2’den %30.4-30.7’ye arttırmıştır (p≤0.05).
Anahtar kelimeler: arındırma, enkapsülasyon, nano-emülsiyon, ultrasonikasyon, vişne posası
viii To my family
ix
ACKNOWLEDGEMENT
I am heartily thankful to my supervisor, Prof. Dr. Gülüm Şumnu and co-supervisor, Prof. Dr. Serpil Şahin for their assistive suggestions, support, help, and guidance throughout my thesis.
I am grateful to Prof. Dr. Vasıf Hasırcı for his valuable assistance throughout this study.
I would like to thank to my project friend Betül Çilek for her help and assistance in laboratory and in normal life.
I am grateful to Dr. Ibrahim Çam (Central Laboratory) for his valuable support in particle size measurements
I would also like to thank to Res. Assist. Cengiz Tan and Res. Assist. Serkan Yılmaz for their help in performing SEM assays.
I am grateful to Hazal Turasan for her help in translations.
Also I am heartily thankful to Ministry of Education of Turkey for their financial support during my undergraduate and graduate studies.
Finally, I would like to thank The Scientific and Technological Council of Turkey (TÜBITAK 1100071) for the financial support during this study.
x
TABLE OF CONTENTS
ABSTRACT ... iv
ÖZ ... vi
ACKNOWLEDGEMENT ... ix
TABLE OF CONTENTS ... x
LIST OF FIGURES ... xiii
LIST OF TABLES ... xv
CHAPTERS 1. INTRODUCTION ... 1
1.1 Antioxidants ... 1
1.1.1 Antioxidants used as food additives ... 2
1.1.2 Health benefits ... 4
1.2 Phenolic compounds ... 4
1.2.1 Naturally occurring phenolic compounds ... 5
1.2.2 Phenolic compounds from residual sources ... 7
1.3 Encapsulation ... 9
1.3.1 Encapsulation technologies ... 9
1.3.2 High-energy ultrasonication method ... 12
1.3.3 Encapsulating materials ... 13
1.3.3.1 Maltodextrin ... 15
1.3.3.2 Gum arabic ... 16
1.3.4 Encapsulation of food ingredients containing polyphenols... 18
1.4 Aim of this study ... 19
2. MATERIALS AND METHODS... 21
2.1 Materials ... 21
xi
2.1.1 Supplying of samples ... 21
2.1.2 Reagents ... 21
2.2 Extraction of polyphenolic compounds ... 22
2.3 Degritting of extracted phenolic concentrate ... 23
2.4 Production of phenolic powders ... 23
2.5 Preparation of Emulsions and Capsules ... 24
2.6 Preparation of cake ... 25
2.7 Storage of phenolic powder and encapsulated phenolic powder ... 26
2.8 Physical analysis... 27
2.8.1 Particle size analysis ... 27
2.8.2 Color analysis ... 27
2.8.2.1 Color measurement of phenolic powder and encapsulated samples ... 28
2.8.2.2 Determination of color of crumb and crust of cakes ... 28
2.8.3 Specific volume of cake ... 29
2.8.4 Crumb texture ... 30
2.8.5 Hygroscopicity ... 30
2.9 Chemical analysis ... 31
2.9.1 Reducing sugar content ... 31
2.9.2 Total phenolic content ... 32
2.9.3 Surface phenolic content and encapsulation efficiency of capsules ... 34
2.9.4 Total antioxidant activity of phenolic powders, capsules and cakes ... 34
2.10 Surface morphology of phenolic powders and capsules ... 35
2.11 Sensory analysis of cakes ... 36
2.12 Statistical analysis ... 36
3. RESULTS AND DISCUSSION ... 37
3.1 Effect of degritting on physical and chemical properties of polyphenolic powders and capsules ... 37
3.1.1 Effect of degritting on particle size of extracted concentrates ... 37 3.1.2 Effect of degritting of concentrates on particle size distribution of emulsions40
xii
3.1.3 Effect of degritting of concentrates on particle size distribution of capsules .. 43
3.1.4 Effect of degritting of concentrates on color of phenolic powder and capsules ... 45
3.1.5 Effect of degritting of concentrates on total antioxidant activity of polyphenolic powders and capsules ... 46
3.1.6 Effect of degritting of concentrates on encapsulation efficiency ... 47
3.1.7 Effects of degritting of concentrates on surface morphology of polyphenolic powders and capsules ... 50
3.2 Effect of degritting of concentrates on hygroscopicity and storage stability of capsules and phenolic powders ... 52
3.2.1 Hygroscopicity of capsules and phenolic powders ... 52
3.2.2 Storage stability of capsules and phenolic powders... 54
3.3 Incorporation of polyphenolic powders and capsules into cakes ... 60
3.3.1 Baking stability of polyphenolic powders and capsules ... 61
3.3.2 Quality of cakes ... 62
3.3.2.1 Color of crumb and crust of cake ... 62
3.3.2.2 Texture of cakes ... 63
3.3.2.3 Volume of cakes ... 64
3.3.2.4 Sensory analysis of cakes ... 65
4. CONCLUSION ... 67
5. RECOMMENDATIONS ... 69
REFERENCES ... 70
APPENDICES A1 CALIBRATION CURVES ... 79
A2 SENSORY ANALYSIS ... 81
B STATISTICAL ANALYSES ... 83
C PICTURES OF PHENOLIC POWDERS AND CAPSULES ... 151
xiii
LIST OF FIGURES
FIGURES
Fig. 1.1 Classification of starch hydrolysates based on the dextrose equivalent (DE) value (Wandrey et al., 2010). ... 155 Fig. 2.1 Flow chart of the processes in the preparation of extracted phenolic powders . 22 Fig. 3.1 Effect of degritting on particle size distribution of concentrated polyphenolic extracts: P1, P2 and P3. ... 38 Fig. 3.2 Particle size distribution of micro-emulsions (containing EPP) prepared with 10% MD (solid line) and 8% MD-2% GA (dotted line), nano-emulsions (containing PEPP) prepared with 10% MD (dashed line) and 8% MD-2% GA (dash dotted line) ... 42 Fig. 3.3 Particle size distribution of capsules prepared from micro-emulsion (solid line) and nano-emulsion (dashed line) ... 44 Fig. 3.4 Encapsulation efficiency of capsules prepared with different coating materials and powder types. Bars with different letters (a & b) are significantly different (p ≤ 0.05) ... 48 Fig. 3.5 Scanning electron micrographs of polyphenolic powders: EPP (A) and PEPP (B) ... 50 Fig. 3.6 Scanning electron micrographs of encapsulated phenolic powders with formulation of 10% MD-EPP (A), 10% MD-PEPP (B), 8% MD-2% GA-EPP (C) and 8% MD-2% GA-PEPP (D) ... 51 Fig. 3.7 TPC loss of phenolic powders and capsules during storage at 43% RH [(♦):
EPPb*, (■): PEPPa, (▲): encapsulated EPPc and (●): encapsulated PEPPc.*
Samples having different letters (a, b & c) are significantly different (p ≤ 0.05).]
... 55
xiv
Fig. 3.8 TPC loss of phenolic powders and capsules during storage at 85% RH [(♦):
EPPb*, (■): PEPPa, (▲): encapsulated EPPc and (●): encapsulated PEPPb. * Samples having different letters (a, b & c) are significantly different (p ≤ 0.05).]
... 56
Fig. 3.9 TAA loss of phenolic powders and capsules during storage at 43% RH [(♦): EPPb*, (■): PEPPa, (▲): encapsulated EPPd and (●): encapsulated PEPPc. * Samples having different letters (a, b, c & d) are significantly different (p ≤ 0.05).] ... 58
Fig. 3.10 TAA loss of phenolic powders and capsules during storage at 85% RH [(♦): EPPb*, (■): PEPPa, (▲): encapsulated EPPc and (●): encapsulated PEPPb. * Samples having different letters (a, b & c) are significantly different (p ≤ 0.05).] ... 59
Fig. A.1 Calibration curve used in TPC analysis ... 79
Fig. A.2 Calibration curve used in the SPC analysis ... 80
Fig. A.3 Calibration curve used in TAA analysis ... 80
Fig. A.4 Sensory analysis evaluation sheet of cake prepared with capsules containing 8% MD, 2% GA and EPP (394), control cake (716) and cake prepared with capsules containing 8% MD, 2% GA and PEPP (528) ... 81
Fig. C.1 Picture of phenolic powders and capsules ... 151
xv
LIST OF TABLES
TABLES
Table 1.1 Food additives and condition of use in food categories (Anonymous, 2008) ... 3
Table 1.2 Total phenolic content of selected fruits and vegetables ... 6
Table 1.3 Total antioxidant capacity of selected fruits and vegetables ... 7
Table 1.4 Microencapsulating materials overview and their sources (Wandrey et al., 2010) ... 14
Table 2.1 Composition of cake batters ... 25
Table 3.1 Influence of angular velocity on purification of dispersion ... 39
Table 3.2 Particle size analysis results of emulsions prepared with different coating materials and powder types ... 40
Table 3.3 Color measurements of polyphenolic powders and capsules ... 46
Table 3.4 Total antioxidant activity data for capsules prepared from EPP and PEPP with different MD and GA concentrations ... 47
Table 3.5 Surface phenolic content data for capsules prepared from EPP and PEPP with different MD and GA concentrations ... 49
Table 3.6 Hygroscopicity of polyphenolic powders and capsules at 43% and 85% RH 53 Table 3.7 Loss of TPC after storage for 91 and 45 days at 43% and 85% RH ... 57
Table 3.8 Loss of TAA after storage for 91 and 45 days at 43% and 85% RH... 60
Table 3.9 Retention of TPC and TAA after baking at 175 °C for 22 min ... 61
Table 3.10 ∆E* values of crumb and crust of cakes ... 63
Table 3.11 Texture measurements of cakes ... 64
Table 3.12 Specific volume of cakes ... 64
Table 3.13 Sensory analysis of cakes prepared with capsules... 65
Table A.1 Data of panelist scores ... 82
xvi
Table B.1 D[32] values of particles dispersed in the extracted concentrates ... 83
Table B.2 Span of particle size distribution of polyphenolic concentrates ... 85
Table B.3 Specific surface area of particles dispersed in the polyphenolic concentrates ... 87
Table B.4 D[32] values of particles dispersed in the emulsions ... 89
Table B.5 Span of particle size distribution of the emulsions ... 91
Table B.6 Specific surface area values of particles dispersed in the emulsions ... 93
Table B.7 L* values of capsules ... 95
Table B.8 a* values of capsules ... 96
Table B.9 b* values of capsules ... 97
Table B.10 ∆E* values of capsules ... 98
Table B.11 TAA of capsules ... 99
Table B.12 Encapsulation efficiency of capsules ... 101
Table B.13 SPC of capsules... 103
Table B.14 Hygroscopicity values of capsules and phenolic powders at 43% RH ... 105
Table B.15 Hygroscopicity values of capsules and phenolic powders at 85% RH ... 107
Table B.16 TPC loss of capsules and phenolic powders during storage at 43% RH.... 109
Table B.17 TPC loss of capsules and phenolic powders during storage at 85% RH.... 112
Table B.18 TPC loss of capsules and phenolic powders after storage for 91 days at 43% RH ... 115
Table B.19 TPC loss of capsules and phenolic powders after storage for 45 days at 85% RH ... 117
Table B.20 TAA loss of capsules and phenolic powders during storage at 43% RH ... 119
Table B.21 TAA loss of capsules and phenolic powders during storage at 85% RH ... 122
Table B.22 TAA loss of capsules and phenolic powders after storage for 91 days at 43% RH ... 125
Table B.23 TAA loss of capsules and phenolic powders after storage for 45 days at 85% RH ... 127
Table B.24 Retention of TPC after baking at 175 °C for 22 min ... 129
xvii
Table B.25 Retention of TAA after baking at 175 °C for 22 min ... 131
Table B.26 ∆E* values of crumb of cakes... 133
Table B.27 ∆E* values of crust of cakes ... 135
Table B.28 Hardness of crumb of cakes ... 137
Table B.29 Gumminess of crumb of cakes... 139
Table B.30 Chewiness of crumb of cakes ... 141
Table B.31 Specific volume of cakes ... 143
Table B.32 Sensory analysis results for flavor of cakes... 145
Table B.33 Sensory analysis results for color of cakes ... 147
Table B.34 Sensory analysis results for texture of cakes ... 149
1 CHAPTER 1
INTRODUCTION
1.1 Antioxidants
Antioxidants are essential components found in most of the food products. Their main role is to provide protection against the oxidation of lipids. Antioxidants can be classified into two groups: primary (chain-breaking) antioxidants, which can convert lipid radicals to more stable product, and secondary (preventing) antioxidants, which can prevent autoxidation by different mechanisms (Ingold, 1968). Prevention of autoxidation of lipid molecule by rapid donation of hydrogen atom mostly occurs according to the reactions (1) or (2).
ROO ∙ + AH → ROOH + A ∙ (1)
RO ∙ + AH ⇋ ROH + A ∙ (2)
Primary antioxidant should be able to donate rapidly hydrogen atom to a lipid radical and to form a stable radical derived from it or this new formed radical should be converted to other stable product. Most commonly used primary antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertiary butyl
2
hydroquinone (TBHQ), gallates, and tocopherols. Secondary antioxidants cannot prevent autoxidation of lipids directly, but perform this by various mechanisms. They may combine to metal ions, scavenge oxygen, quench singlet oxygen, decompose hydrogen peroxide in stable product or absorb UV light.
There are many studies performed on the toxicology of antioxidants. For instance, BHT was found to be carcinogenic when it was given in the diet to mice (Clapp et al., 1974), BHA has an effect on rodent forestomach hyperplasia when it was fed to rats (Ito et al., 1983) etc. Toxicological effects and synthetic origin of these antioxidants decrease the acceptability of the products which contain them, because consumers prefer organic food or food products which contain natural food additives. Therefore, recently there is an increased interest in the replacement of synthetic antioxidants by natural ones. High interest to natural antioxidants also arises from their beneficial effects on the human health.
1.1.1 Antioxidants used as food additives
Regulation No 1333/2008 of the European Parliament and of the Council lays down rules on food additives to improve the functionality on the market, protection of human health, protection of consumer, including protection of consumer interests, fair food trading, and if appropriate, the protection of the environment. Part E of this regulation lists authorized food additives and conditions of use in the food categories (Anonymous, 2008). Some of synthetic and natural antioxidants mentioned in this part are summarized in Table 1.1.
3
Table 1.1 Food additives and condition of use in food categories (Anonymous, 2008)
Name E-number Maximum
level (mg/l or mg/kg)
Food categories
Ascorbic acid E 300 quantum
satis
Fats, oils, fruits, vegetables, flours, pasta, bread, fish, fruit juices, fruit nectars, beer Alpha-tocopherol E 307 100 Food for young children
quantum satis
Fats, oils
Citric acid E 330 500 Cocoa and Chocolate products
3000 Fruit juices 5000 Fruit nectars quantum
satis
Fats, oils, fruits, vegetables, pasta, fish, food for young children, beer
Potassium citrates E 332 quantum satis
Fats, oils, fruits, vegetables Gallates, TBHQ,
BHA
E 310-320 25 Dehydrated potatoes
200 Fats and oils for professional manufacture of heat treated foods, fat and oil
emulsions, seasonings, condiments, soups, broths, sauces, processed nuts, precooked cereals, cake mixes, dehydrated meat, cereals-based snacks
400 Chewing gum, food supplements
BHT E 321 100 Fats and oils for professional manufacture
of heat treated foods, fat and oil emulsions Extract of rosemary E 392 30 Vegetable oils
50 Fish oil, algal oil, soups, broths, snacks 100 Sauces, dried sausages, mustard 150 Dehydrated meat
200 Nut butters, dehydrated potatoes products, chewing gum, cake mixes, processed eggs and egg products, seasonings, condiments, processed nuts
400 Food supplements
4 1.1.2 Health benefits
Antioxidants do not only prevent oxidation of lipids but also are beneficial for human health. They can prevent damage of cell by neutralizing free radicals and oxidizing agents, thus preventing development of cancer. Lycopene intake caused a significant reduction in the pancreatic cancer risk and an inverse association was observed between pancreatic cancer risk and dietary β-carotene intake among those who never smoked (Nkondjok et al., 2005). Consumption of carrots on average at least once a day among women had a 35% reduction of breast cancer risk when compared to the consumption of less than once a month. Therefore, it was suggested that α- or β- carotene may decrease breast cancer risk (Tamimi et al., 2005). In another study an inverse association between intake of carotenoids and risk of lung cancer has been shown (Ziegler et al., 1996). Apart from prevention of cancer, antioxidants were found to improve cardiovascular health (Vita, 2005; Keen et al., 2005). In addition, inverse associations have been observed between intake of antioxidants and cataract and chronic obstructive pulmonary disease, including asthma, bronchitis and wheezing (Brown et al., 1999; Hatch, 1995; Schwartz & Weiss, 1990).
1.2 Phenolic compounds
Phenolic compounds affect the organoleptic and sensory attributes of fruits and vegetables (Alasalvar et al., 2001; Serra & Ventura, 1997). There is a wide range of phenolic compounds occurring in the nature, from which tannins, flavonoids and phenolic acids are major dietary compounds (King & Young, 1999). Various polyphenols from natural sources presenting antioxidant properties were proposed for the prevention of autoxidation of lipids (Hagerman et al., 1998). Antioxidant activity of fruit and vegetables is dependent not only on vitamin C, tocopherol or β-carotene
5
content, but also on phenolic compounds content. For instance, flavonoids which are abundant in the diet of human demonstrated high antioxidant capacity (Bors et al., 1990; Hanasaki et al., 1994). Therefore, it is important to measure total antioxidant capacity of natural products.
1.2.1 Naturally occurring phenolic compounds
Toxicological effect of synthetic antioxidants and health benefits of natural phenolic compounds resulted in the increased number of studies on antioxidant capacity and total phenolic content of natural sources (fruits, vegetables, beverages, etc.). Among the fruits banana, mango and papaya have lower total phenolic content when compared to apple, blackberry, red grape or strawberry (Table 1.2). It can also be seen from the Table 1.2 that some vegetables have high total phenolic content. In addition, fruit juices, tea, coffee and especially red wines are rich in phenolic compounds (Minussi et al, 2003; Lakenbrink et al., 2000; Gardner et al., 2000).
6
Table 1.2 Total phenolic content of selected fruits and vegetables Fruit/vegetable
Total phenolic content (mg gallic acid equivalents/
100 g fresh weight)
Reference
Apple 296±6.4 Sun et al. (2002)
Banana 90±3.2 Sun et al. (2002)
Blackberry 417-555 Sellappan et al. (2002)
Broccoli 289±6.1 Sikora et al. (2008)
Brussels sprouts 331±33.2 Sikora et al. (2008)
Cauliflower 144±3.3 Sikora et al. (2008)
Litchi 28±1.7 Luximon-Ramma et al. (2003)
Mango 56±2.1 Luximon-Ramma et al. (2003)
Papaya 57±4.1 Luximon-Ramma et al. (2003)
Peach 84±0.7 Sun et al. (2002)
Red grape 201±2.9 Sun et al. (2002)
Strawberry 160±1.2 Sun et al. (2002)
The other important attribute is the antioxidant activity of the compounds found in the natural sources. This parameter shows how active as antioxidants are these substances.
In Table 1.3, the total antioxidant capacities of some fruits and vegetables are given. It can be seen that cruciferous vegetables (broccoli, cauliflower, Brussel sprouts), orange and red grape contains more active compounds compared to apple, banana or tomato. It can also be seen that antioxidant capacity is not always correlated with the content of total phenolic compounds. For instance, total phenolic content of apple is approximately three times greater than that of banana (Table 1.2), but their antioxidant capacities are almost the same (Table 1.3).
7
Table 1.3 Total antioxidant capacity of selected fruits and vegetables Fruit/vegetable Total antioxidant capacity
(µM trolox/g fresh weight) Reference
Apple 2.2±0.35 Wang et al. (1996)
Banana 2.2±0.19 Wang et al. (1996)
Broccoli 26.2±1.37 Sikora et al. (2008)
Brussels sprouts 32.1±1.71 Sikora et al. (2008)
Cauliflower 20.9±0.55 Sikora et al. (2008)
Orange 7.5±1.01 Wang et al. (1996)
Red grape 7.4±0.48 Wang et al. (1996)
Strawberry 15.4±2.38 Wang et al. (1996)
Tomato 1.89±0.12 Wang et al. (1996)
White grape 4.5±1.06 Wang et al. (1996)
Processes such as cooking, blanching, canning and freezing reduce total phenolic content and cause decrease in total antioxidant capacity of the product (Sikora et al., 2008; Scalzo et al., 2004; Chaovanalikit & Wrolstad, 2004).
1.2.2 Phenolic compounds from residual sources
Processing of fresh natural materials and fabrication of target products lead to the production of many by-products, which are actually wastes of food industry. Even at home, skin of orange, banana, onion and potato, seeds of cherry, apricot and peach are thrown out. The fact that fresh natural materials are rich in phenolic compounds makes it obvious that residues of them will also contain phenolics. Many studies have been performed on the phenolic content of wastes of food industry.
Wine by-products are of great interest due to the fact that grapes and wine are good sources of phenolic compounds, which were shown in previous section. Yi et al. (2009) reported that grape pomace powder from the production of “Cabernet Sauvignon” and
8
“Royal Rouge” wines contained 250±2.6 and 480±2.8 mg gallic acid equivalents (GAE)/100 g fresh weight (FW) phenolics and 131±2.1 and 302±3.2 mg cyanidin 3- rutinoside equivalent/100 g FW anthocyanins with antioxidant capacity of 52.8±0.3 and 79.0±0.1 µM trolox equivalent/100 g FW, respectively. Alonso et al. (2002) showed that by-products of different wines contained polyphenols such as: gallic acid, furfural, p-OH-phenethyl alcohol, catechin, vanillic acid, syringic acid, epicatechin, caftaric acid, trans-coutaric acid, clorogenic acid, and trans-p-coumaric acid. Similar phenolic compounds were reported in the study by Lafka et al. (2007).
Garcia et al. (2009) showed that apple pomace from the cider industry contained 3.9- 13.9 g GAE/kg dry matter (DM) with 4.3-12.5 g ascorbic acid equivalents/kg DM antioxidant capacity. Sixty phenolic compounds were positively identified by Sanchez- Rabaneda et al. (2004) in apple pomace; phloridzin, chlorogenic acid, quercetin and quercetin glycosides were emphasized among the identified components.
Bayberry pomace was found to have total phenolic content of 27.7-47.4 mg GAE/g DM with 78.5-100.6 mg trolox equivalents/g DM antioxidant capacity (Zhou et al., 2009).
Sojka & Krol (2009) reported that industrial seedless black currant pomace had total phenolic content of 1855.5-2285.6 mg (-)epicatechin equivalents/100 g pomace and antioxidant capacity of 93.3-126.5 µM trolox equivalents/g pomace.
During extraction process depending on the solvent some soluble compounds other than phenolics can also be extracted. Manto Negro red grape (Vitis vinifera) pomace was found to contain 3.27±0.10% (dry matter) of soluble sugar, 6.20±0.30% of soluble pectins and 74.5±2.43% of total dietary fibre (Llobera & Canellas, 2007). Grape pomace powder from the production of “Cabernet Sauvignon” and “Royal Rouge”
wines contained 5.32 and 6.03 g/100 g FW of soluble sugars and 56.9 and 55.4 g/100 g FW of total dietary fiber, respectively (Yi et al., 2009).
9 1.3 Encapsulation
Encapsulation is a technology used in food industry to coat small particles of ingredients or even whole material (nuts, confectionary products, etc.).
Microencapsulation is one of the technologies of encapsulation. Used by the food industry for more than 70 years, microencapsulation can be defined as a process of sealed packaging by coating material (wall material) of miniature substances (solid, liquid, or even gaseous materials) in microcapsules or by embedding them in a homogeneous or heterogeneous matrix from which coated material (core material) can be further released under specific environmental conditions and at the specific rate (Desai & Park, 2005). Reduction of the susceptibility to environmental factors such as, light, moisture or oxygen, controlled release of the core material to the outside environment, easier handling, masking of the taste of core material, and dilution of core material in coating material when it should be used in small amounts are the main reasons for the application of microencapsulation in food industry (Shahidi & Han, 1993).
1.3.1 Encapsulation technologies
Many different types of microcapsules are produced using wide range of coating materials and different microencapsulation processes such as: spray drying, spray chilling, spray cooling, air suspension coating, fluidized bed coating, coacervation, emulsion, nanoparticles, extrusion, freeze drying, centrifugal extrusion, and liposome entrapment (Augustin & Hemar, 2009; Desai & Park, 2005; Gibbs et al., 1999; Gouin, 2004, Shahidi & Han, 1993).
Spray drying is a well-established method used in different sectors of food industry. It is most commonly used in microencapsulation due to its low cost and its ability to
10
convert fluid material into solid or semi-solid material in a single step. The basic process of microencapsulation by spray drying involves dissolving of core ingredient in the dispersion of coating matrix. Contact with hot air causes rapid moisture loss in the droplets. In addition, temperature of the droplets remains relatively low, so high temperatures of air do not affect products’ quality negatively (Augustin & Hemar, 2009; Roustapour et al., 2009). However, operating under high temperatures can cause inactivation and damage of some heat-sensitive food components like vitamins and enzymes (Yoshii et al., 2008).
Freeze drying is another method used for the microencapsulation for almost all heat- sensitive materials. This method is also known as lyophilization or cryodesiccation.
The principle of operation is based on the direct sublimation from the solid state to the gas phase at low temperature and pressure. When compared to spray drying, freeze drying is more expensive and time consuming process. Microencapsulation by freeze drying is performed by dissolving core material in the coating material matrix which is then lyophilized. The resulting dry product is then grinded to a fine powder containing particles with uncertain forms. Freeze drying can be used in the encapsulation of water- soluble essences, aromas and drugs (Desai & Park, 2005). Freeze drying results in the formation of more porous particles when compared to spray drying (Augustin &
Hemar, 2009).
Emulsions are used to deliver lipophilic food components (carotenes, tocopherols, etc.).
However, water phase of the emulsion can be also involved in the delivery of water- soluble food components (Augustin & Hemar, 2009). Nano-emulsions are formed under treatment by high-energy input such as ultrasonication or by high-pressure homogenization and microfluidization. Nano-emulsions with particle size range of 50 to 200 nm are transparent while emulsions with particle size up to 500 nm have milky appearance (Tadros et al., 2004). It has been shown that particle size distribution of emulsion is important parameter for encapsulation efficiency and it also defines
11
stability, color and rheology of the emulsion (Jafari et al., 2007a). Low-energy emulsification and high-energy or high-pressure homogenization are two methods used in the production of nano-emulsions. Phase inversion temperature technique is an example of low-energy emulsification. Low-energy emulsification methods have several disadvantages, such as careful control of temperature, large amount of surfactant requirement and restricted application to large-scale industrial productions (Seekkuarachchi et al., 2006). On the other hand, high-energy emulsification methods, such as ultrasonication and microfluidization have several advantages and are more applicable because of ability to control particle size distribution and to prepare emulsions with different types of materials (Seekkuarachchi et al., 2006).
Generally, nano- term is added to a technology or process in which the material is at the 10-9 meter scale in size. Nanotechnology is a developing interdisciplinary field in which knowledge from different sciences, including physics, chemistry and engineering, are brought to one area. It is also studied and applied in food science. Unique and novel physical, chemical and biological properties which are offered by nanoscale structures caused an increased interest of the application of nanotechnology in the encapsulation of food ingredients. Nanoencapsulation has been proposed as a different type of encapsulation. Nano-emulsions and nanocapsules are the products of this novel encapsulation technology. The advantages of nanoencapsulation are ease of handling, enhanced stability and bioavailability, change in flavor character, and moisture- and pH-triggered controlled release (Neethirajan & Jayas, 2011). Recently, only few studies on the nanoencapsulation are present in the literature. Semo et al. (2007) showed that casein micelle can be used in the nanoencapsulation of hydrophobic food ingredients such as vitamin D2 for potential enrichment of low- or non-fat food products. β- carotene encapsulated in biopolymer ultrafine fibers of edible zein prolamine by means of electrospinning presented a remarkably good protection against oxidation when exposed to UV-Visible irradiation (Fernandez et al., 2009). Sunflower oil in water nano-emulsions prepared by high pressure homogenization (5 passes at 300 MPa) had
12
mean particle size distribution in the range of 100-200 nm (Donsi et al., 2012). In the same study, antimicrobial activities of carvacrol, D-limonene and trans- cinnamaldehyde were found to be dependent on the formulation nanoemulsion-based delivery systems.
1.3.2 High-energy ultrasonication method
Ultrasound waves are sound waves beyond the audible frequency range. When ultrasound wave passes through liquid medium which contains dissolved gas it causes a phenomenon known as acoustic cavitation, which leads to the generation of physical forces including microjets, shear forces, shock waves and turbulence (Chandrapala et al., 2012). In addition, friction between the probe, medium and container’s walls leads to the heat generation. There are many applications of ultrasound in different areas of food industry. McClements (1995) described that ultrasound can be used in the detection of presence/absence, thickness, level and foreign body, in the measurement of flow rate and temperature, and in determination of microstructures. In a recent review by Chandrapala et al. (2012) other types of applications, such as emulsification, filtration, viscosity modification, diary systems and tenderization are described.
Ultrasonication is successfully applied in the production of micro- and nano-emulsions.
Sound waves with low frequencies (e.g. 20 kHz) are able to form strong shear forces, but waves with higher frequencies generate weaker shear forces. Therefore, emulsions are produced only by ultrasound waves with high intensity and low frequency (16-100 kHz) (Chandrapala et al., 2012). There are many parameters which affect the emulsification process including position of the ultrasonic probe in the relation to the liquid-liquid interface, viscosity of the continuous phase, ultrasonic power and processing time (Behrend & Schubert, 2000; Cucheval & Chow, 2008; Jafari et al., 2006; Jafari et al., 2007a).
13
After sonication of whey protein dispersions at 20 kHz and 750 W, it was observed that particle size distribution curve shifted to the left for more diluted dispersions. It was also reported that longer time of ultrasound treatment resulted in the formation of smaller particles when compared to short time processing (Gordon & Pilosof, 2010).
In another study by Leong et al. (2009) it was shown that ultrasonication (400 W, 20 min) of triglyceride oils in water resulted in the production of transparent nano- emulsion with mean particle size less than 40 nm.
Among the key parameters of emulsification using ultrasound, special attention should be paid to the optimization of applied power. Kentish et al., (2008) performed a study in which they showed that droplet particle size decreased with increasing power then particle size reached its minimum at an intermediate power application and after that increased at higher power levels. The same phenomenon was also observed and described as “over-processing” in the studies performed by Desrumaux & Marcand (2002) and Jafari et al. (2006).
1.3.3 Encapsulating materials
There are many different types of encapsulating/entrapping materials used in encapsulation of food ingredients. They can be used separately or in the mixture of two or more different materials. Coating material used for the encapsulation should meet the required criteria, such as compatibility with the food product, mechanical strength, appropriate thermal or dissolution release, and appropriate particle size (Gharsallaoui et al., 2007). Wall materials can be composed of sugars, gums, proteins, natural and modified polysaccharides, lipids and synthetic polymers (Gharsallaoui et al., 2007;
Gibbs et al., 1999). Generally, encapsulating materials used in food industry are
14
considered as biomolecules. There are different sources of biomolecules, such as plants, animals, and microorganisms. Overview of coating materials with their origins is given in the Table 1.4. The most commonly used materials such as maltodextrins and gum arabic belong to the polysaccharides category (Wandrey et al., 2010).
Table 1.4 Microencapsulating materials overview and their sources (Wandrey et al., 2010)
Origin Carbohydrate
polymer
Protein Lipid
Plant Starch Gluten (corn) Fatty acids/alcohols
- Derivatives Isolates (pea, soy) Glycerides
Cellulose Waxes
- Derivatives Phospholipids
Plant exudates - Gum arabic - Gum karaya - Mesquite gum Plant extracts - Galactomannans - Soluble soybean Polysaccharides
Marine Carrageenan
Alginate
Microbial/animal Xanthan Caseins Fatty acids/alcohols
Gellan Whey proteins Glycerides
Dextran Gelatin Waxes
Chitosan Phospholipids (Shellac)
15 1.3.3.1 Maltodextrin
Maltodextrin is a functional derivative of starch. It is formed by hydrolysis of starch (usually corn or potato starch in US and wheat starch in Europe) by acid, enzyme, or acid/enzyme combinations (Wandrey et al., 2010). Dextrose equivalent (DE) of maltodextrin is less than 20. DE term is defined as a measure of reducing power of a starch derivative compared with D-glucose on a dry-weight basis and is higher for greater extent of starch hydrolysis (Wang & Wang, 2000). Starch and some starch derivatives with their DE are shown in the Fig.1.1.
Fig. 1.1 Classification of starch hydrolysates based on the dextrose equivalent (DE) value (Wandrey et al., 2010).
16
Maltodextrins are cold-water soluble, easily digestible, creamy white powders which are tasteless or moderately sweet. Klinkesorn et al. (2004) studied stability and rheology of oil-in-water emulsions which contained maltodextrins with different DE.
They found that, minimum amount of maltodextrin required to promote rapid creaming decreased as the DE of maltodextrin decreased. They also reported that, at high concentrations maltodextrins with lower DE cause higher relative viscosity. Wang &
Wang (2000) reported DE values of 8.2, 5.9 and 14.2 for commercial corn, potato and rice maltodextrins, respectively. They emphasized that, high concentrations of high molecular weight saccharides (low DE) in maltodextrins contributed to higher viscosity and freezing temperature, less water sorption and greater tendency to retrogradation.
Maltodextrin with 10 DE had higher phenolic retention when compared to 20 DE in the study performed on encapsulation of açai pulp by spray drying (Tonon et al., 2009).
Cactus pear (Opuntia streptacantha) was encapsulated by spray drying using two types of commercial maltodextrin with DE of 10 and 20, and better binder properties were reported for maltodextrin 10 DE when compared to maltodextrin 20 DE which resulted in greater capacity of retention of vitamin C after encapsulation (Rodriguez-Hernandez et al., 2005). Microencapsulated powders of cloudberry (Rubus chamaemorus) phenolics prepared with maltodextrin 5-8 DE were remarkably better in the encapsulation yield and efficiency when compared to powders which contained maltodextrin 18.5 DE (Laine et al., 2008).
1.3.3.2 Gum arabic
Gum arabic (GA) is naturally occurring, edible and gummy exudate collected from the stems and branches of Acacia senegal and, to a lesser extent, from Acacia seyal that is rich in non-viscous soluble fiber (Ali et al., 2009; Yadav et al., 2007). Gum arabic is composed of arabinogalactan oligosaccharides, polysaccharides, and glycoproteins and
17
its chemical composition is dependent and may vary due to the factors such as source, climate, season, age of trees, rainfall, time of exudation, etc. (Wandrey et al., 2010). It is colorless, odorless and tasteless and does not add any odor, taste or color to the product it is added. Gum arabic is both cold- and hot-water soluble and it is a good emulsifying agent. Gum arabic is used as a stabilizing, emulsifying and thickening agent in the food industry (in beverages, candy, confections, etc.) and it is also used in pharmaceutical, textile, cosmetics, pottery and lithography industries (Verbeken et al., 2003).
Shiga et al. (2001) reported that blending of gum arabic and cyclodextrin in the feed liquid in the encapsulation of flavors by spray drying resulted in an increase of flavor retention. Microcapsules of cinnamon oleoresin obtained by spray drying and prepared with gum arabic:maltodextrin:modified starch (4:1:1) were spherical and had smooth surface, whereas microcapsules prepared from gum arabic had some dents and capsules prepared from maltodextrin and modified starch were broken and not complete (Vaidya et al., 2006). Better emulsion stability and better stability against environmental factors such as NaCl concentration and thermal treatments has been reported for the soybean- stabilized oil-in-water emulsion in the presence of gum arabic (Wang et al., 2011).
Gum arabic was successfully used in the production by spray drying of açai powder and particles produced with it exhibited the lowest mean diameter when compared to maltodextrin and tapioca starch (Tonon et al., 2009). It was also used as a carrier agent in the encapsulation by microfluidization and spray drying of red rasberry (Rubus idaeus) puree (Syamaladevi et al., 2012). Maltodextrin and gum arabic were used separately as carrier agents in the production of lemon juice powders (Martinelli et al., 2007).
18
1.3.4 Encapsulation of food ingredients containing polyphenols
Many studies on the encapsulation of phenolic compounds from natural sources have been performed over the last decade. Generally, core materials in these studies were homogenized natural sources or extracts from them.
Tonon et al. (2009) prepared açai powder from açai (Euterpe oleraceae Mart.) pulp by spray drying. In this study maltodextrin (10 DE and 20 DE), gum arabic and tapioca starch were used as carrier agents and powders produced with maltodextrin and gum arabic exhibited high retention of polyphenols and antioxidant capacity during storage at 40 °C for 15 days at two different relative humidities (32.8% and 84.3%).
Bioactive compounds from cactus pear (Opuntia ficus-indica) pulp were microencapsulated by spray drying using maltodextrin 10 DE and inulin as coating materials by Saenz et al. (2009). In this study, encapsulation yield of betacyanin and indicaxanthin reached values above 98% and 92%, respectively, whereas, phenolic compound encapsulation yield values ranged from 23% to 81%. Produced powder was suggested to be used as a red colorant and as a functional food due to its high antioxidant content.
Sanchez et al. (2011) dissolved maltodextrin (10 DE) in wine to 20% concentration (total weight basis) and freeze dried prepared mixture to obtain “wine powder”. Total phenolic content of “wine powder” made from red wine Cabernet Sauvignon remained essentially constant during the storage period.
Gum arabic and maltodextrin (25 DE) were added directly in different combinations to the concentrated immature acerola juice to give a total of 50% soluble solids and then this mixture was spray dried (Righetto & Netto, 2005). Critical water activity range, in
19
which physical transformations were observed in the encapsulated samples, was 0.33- 0.43, depending on the storage temperature.
In the encapsulation of Elderberry (Sambucus nigra L.) juice by spray drying five different wall materials were tested: maltodextrin (4-7 DE), gum acacia, isolated soya protein, soya protein powder, and soya milk powder (Murugesan & Orsat, 2011). All wall materials were added at the total juice solids to coating material ratio of 5:1, 5:2, 5:3, 5:4 and 1:1 (weight basis). The highest total phenolic content was found to be 48.1 mg GAE/g for powder produced with gum acacia at 1:1 ratio. In general, powders with gum acacia had higher phenolic content when compared to powders with maltodextrin and all other soya products. In addition, gum acacia followed by maltodextrin showed the best results in phenolic content retention during storage at different conditions and different packaging materials.
Cooking stability in pasta of microencapsulated garcina cowa fruit extract produced by spray drying with whey protein isolate as a carrier agent was studied by Pillai et al.
(2012). It was reported that pasta prepared with powder with 1.5:1 core-to-coating ratio which was spray dried at 90 °C outlet temperature exhibited the highest antioxidant activity when compared to powder with 1:1 core-to-coating ratio and to powders produced at 105 °C outlet temperature. There is no explanation regarded to the change of total antioxidant capacity due to the cooking process.
1.4 Aim of this study
In the last decade many studies have been performed on the possible sources of natural antioxidants, since there is a demand in different industries towards the replacement of synthetic antioxidants by natural ones. However, the factors such as loss of quality during storage and processing, unpleasant taste, and in some cases, off-color decrease
20
the effectiveness of the utilization of natural antioxidants in food products.
Encapsulation of these functional food ingredients can improve their quality attributes and mask unwanted features. In addition, food ingredients which are encapsulated in particles or dispersed in emulsions with particle size less than 200 nm can be released under control depending on the pH or temperature of the environment and bioavailability of the coating material.
There is a lack of study on the encapsulation of phenolic materials from the residual sources. In addition, there are limited researches on nano-emulsion containing food ingredients other than oils. The primary aim of this study was to develop a method to obtain nano-emulsion containing dry polyphenolic powder extracted from sour cherry pomace. It was also aimed to investigate the effect of degritting of the extract of sour cherry pomace on the encapsulation efficiency and particle size distribution of the emulsions. It was important to obtain capsules of both unpurified and degritted phenolic powders. In addition, surface morphology, color and particle size of capsules obtained at the optimum conditions were studied.
Another objective of this research was to compare storage stability of capsules prepared from micro- and nano-emulsions. There are limited studies on the baking or cooking stability of capsules of phenolic powders. Effect of encapsulation on retention of phenolic compounds and total antioxidant capacity of phenolic powders and capsules during processing at high temperatures was investigated by incorporating them into cakes. Quality of cakes containing capsules and degritted phenolic powders were also compared.
21 CHAPTER 2
MATERIALS AND METHODS
2.1 Materials
2.1.1 Supplying of samples
Sour cherry pomace was supplied by Karmey Fruit Juice Factory (Karaman, Turkey).
Seeds, stems and other foreign materials were removed from pomace by screening.
Fine pomace was stored in a refrigerator (D 8340 SM; Beko, Istanbul, Turkey) at 4 °C until the extraction.
2.1.2 Reagents
Maltodextrin (MD) (DE=4–7), gum arabic (GA), potassium carbonate, potassium chloride, Folin-Ciocalteu’s phenol reagent, sodium carbonate, 1,1-diphenyl-2- picrylhydrazyl (DPPH), ethanol (absolute), potassium sodium tartrate tetrahydrate, methanol G CHROMASOLV®, CuSO4.5H2O, gallic acid, glucose, potassium hydroxide, acetic acid (100%) were purchased from Sigma-Aldrich Chemical Co. (St.
Louis, MO, USA). Ingredients used in the production of the cake were obtained from the local commercial markets.
22 2.2 Extraction of polyphenolic compounds
Extraction of phenolic compounds (Fig.2.1) from sour cherry pomace was performed by conventional maceration method in shaking water bath (GFL, GFL Gesellschaft für Labortechnik mbH, Burgwedel, Germany) at 30 °C and 70 rpm for 24 h. In maceration 20 g of pomace and 400 ml ethanol:water (1:1, v/v) solvent were used (Simsek, 2010).
At the end of maceration process large particles of pomace were removed by filter cloth followed by vacuum filtration. Clear extract was then concentrated by a vacuum rotary evaporator (Heidolph Laborota 4000 efficient; Heidolph Instruments GmbH & Co, Schwabach, Germany) at 40 °C in order to remove ethanol and sufficient amount of water.
Fig. 2.1 Flow chart of the processes in the preparation of extracted phenolic powders
23 2.3 Degritting of extracted phenolic concentrate
Firstly, concentrates were purified in SIGMA 2–16PK centrifuge (Sigma Laborzentrifugen GmbH; Osterode am Harz, Germany) at different angular velocities in order to define appropriate operating conditions (Fig.2.1). For this purpose, 7.5 ml of concentrate was centrifuged at 5000 and 10000 rpm angular velocities for 2 min.
Supernatant was accurately collected and used for particle size analysis. As shown in the Fig.2.1 three samples analyzed were P1 (unpurified), P2 (purified at 5000 rpm) and P3 (purified at 10000 rpm).
According to the results of particle size analysis degritting of 7.5 ml of concentrate at 10000 rpm for 2 min was selected as operating parameter and was used in the production of purified extracted phenolic powder (PEPP).
2.4 Production of phenolic powders
In order to investigate the effects of degritting, control powder was produced from unpurified concentrate which was frozen at –20 °C in a deep freeze (D 8340 SM; Beko, Istanbul, Turkey) and then dried (Christ Alpha 1–2 LD plus; Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) at –52 °C and 0.075 mbar for 48 h (Fig.2.1). At the end of freeze drying, samples were manually ground to obtain homogeneous extracted phenolic powder (EPP). PEPP was prepared from purified concentrate by freezing, freeze drying and then grinding as in the case of EPP (Fig.2.1). Both powders were stored in a deep freeze at –20 °C and kept until utilization.
24 2.5 Preparation of emulsions and capsules
Maltodextrin (MD) solution was prepared one day prior to emulsification by dissolving MD in distilled water and kept in shaking water bath at 27 °C for 24 h. Gum arabic (GA) was dissolved in distilled water and stirred for 30 min at 50 °C using magnetic stirrer (MR 3001K; Heidolph Instruments GmbH & Co, Schwabach, Germany) 2 h prior to encapsulation. In addition, GA solution which was used in the preparation of emulsions containing PEPP was centrifuged at 10000 rpm for 2 min and filtered through 0.45–µm filter (Gema Medical S. L.; Barcelona, Spain). Total soluble solid content of coating material solution was 10% (w/w) and it was prepared with two different formulations composed of: a) 10% (w/w) MD and 90% distilled water, b) 8%
MD, 2% GA and 90% distilled water.
All emulsions were prepared in two stages: a) pre-emulsions were obtained by mixing dry phenolic powder with coating material solution at the ratio of 1:20 (core-to- coating), and by stirring at 4000 rpm for 5 min using high-speed blender (IKA Works Co, Rawang, Selangor, Malaysia). b) Pre-emulsions (21 g) were further homogenized by ultrasonication (Sonic Ruptor 400; OMNI International, Kennesaw, GA, USA) for 20 min (160 W, 50% pulse, 20 kHz). Ultrasonic homogenizer was equipped with titanium 5/32" stepped micro tip with diameter of 3.8 mm and length of 255.8 mm. In order to prevent overheating of the emulsions during ultrasonication, they were placed in a water bath at 4 °C.
Emulsions were frozen in deep freezer at –20 °C and then dried under vacuum at –52
°C and 0.075 mbar for 48 h. Dry matrices of encapsulated phenolics were manually crushed using a glass rod and stored at –20 °C until analysis or utilization.
25 2.6 Preparation of cake
Two types of cake batters were used for the preparation of cakes for different analyses.
Cakes were formulated with sugar for quality analysis. Sugar content has a negative effect on the analysis of total phenolic content (Waterhouse, 2002). Therefore, cakes containing encapsulated phenolic powders used in the measurement of baking stability of phenolic compounds were prepared from batters with no sugar content. The compositions of cake mixes are given in the Table 2.1.
Table 2.1 Composition of cake batters
Ingredients
Cake batter With sugar
(% flour weight basis)
Without sugar (% flour weight basis)
Sugar 100 -
Flour 100 100
Margarine 25 25
Milk powder 12 12
Egg powder 9 9
Salt 3 3
Baking powder 5 5
Water 90 90
Firstly, sugar was mixed with egg white powder at 85 rpm for 1 min by using home type mixer (Kitchen Aid, 5K45SS; Benton Harbor, MI, USA) (in the preparation of batter for cake without sugar this step was omitted). Then, margarine melt was added and mixing continued at the same speed for 1 min. All remaining dry ingredients and water were added simultaneously and mixed at 85 rpm for 1 min followed by mixing at 140 rpm for 1 min and one more mixing at 85 rpm for 2 min until the smooth cake
26
batter was obtained. 500 mg of encapsulated phenolic powder or phenolic powder (EPP, PEPP) was added to 100 g of batter. Then batter was further mixed at 85 rpm for 1 min. Encapsulated phenolic powders added to batter were of two types: a) prepared with 8% MD, 2% GA and EPP, b) prepared with 8% MD, 2% GA and PEPP.
Cake batters of 100 g were placed in glass pans lined with wax paper and baked at 175
°C for 22 min in a preheated conventional electrical oven (Arçelik 9411 FT; Arçelik, Istanbul, Turkey). Six cakes were baked at a time. After baking, cakes were removed from the pans and cooled for 1 h at room temperature. Control cake was prepared under the same conditions and contained neither phenolic powder nor encapsulated phenolic powder. Each experiment was duplicated.
2.7 Storage of phenolic powder and encapsulated phenolic powder
Phenolic powders (EPP and PEPP) and encapsulated phenolic powders (prepared with 8% MD, 2% GA and EPP; 8% MD, 2% GA and PEPP) were used in the evaluation of storage stability. Samples were stored in two desiccators with different relative humidities (RH) at room temperature (21±2.0 °C). Saturated aqueous solutions of potassium carbonate and potassium chloride were used to obtain 43% and 85% RH, respectively (Greenspan, 1977). Before placing the sample, each desiccator was kept closed overnight to achieve equilibrium. Storage stability analyses were performed once in 5 days for samples stored at 85% RH and once in 10 days for samples stored at 43% RH. The parameters analyzed were total phenolic content, total antioxidant activity and hygroscopicity.
27 2.8 Physical analysis
2.8.1 Particle size analysis
Particle size analysis was performed for extracted concentrates, emulsions and encapsulated phenolic powders by the laser light scattering method using Mastersizer 2000 (Malvern Instruments, Worcestershire, UK). The mean diameter of the particles was expressed as Sauter mean diameter (D[32]) and calculated with the following formula:
𝐷[32] = 𝑛𝑖𝑑𝑖3 𝑛𝑖𝑑𝑖2 (3)
where, ni stands for the frequency of occurrence of particles in size class i, and di for mean diameter (µm) of these particles.
Span of the particle size distributions was calculated with the following formula:
𝑆𝑝𝑎𝑛 = 𝑑 𝑣,90 −𝑑(𝑣,10)
𝑑(𝑣,50) (4)
where, d(v,10), d(v,50), and d(v,90) are the diameters at 10%, 50%, and 90%
cumulative volume, respectively (Elversson et al., 2003). The instrument also reported the specific surface area (m2/g) of the particles.
2.8.2 Color analysis
Color analysis was performed for phenolic powders, capsules, crumb and crust of cakes.
28
2.8.2.1 Color measurement of phenolic powder and encapsulated samples
Color determination was performed in CIE (L*, a*, b*) color space by using a UV- 2450 UV-Vis spectrophotometer (Shimadzu Co., Kyoto, Japan). It was measured as reflected color from the surface of capsules or powders. Illuminant type C (2° standard observer) was used in this analysis. Mean of 3 determinations was calculated for L*
(darkness/whiteness), a* (greenness/redness) and b* (blueness/yellowness) parameters of each sample. The difference in the color (∆E*) with the reference sample (barium sulfate) was calculated for each sample using the following formula:
∆𝐸∗= ∆𝐿∗2+ ∆𝑎∗2+ ∆𝑏∗2 (5)
where, ∆L*, ∆a* and ∆b* are the differences between the color values of reference and sample.
2.8.2.2 Determination of color of crumb and crust of cakes
Color reflected from the crumb and crust of the cake was measured separately in CIE (L*, a*, b*) color space by using Minolta color reader (CR-10; Japan). The instrument was standardized each time by a white sample. Duplicate readings were performed from different position and mean value of ∆E* calculated by the instrument was recorded. Color measurement was performed only for cakes which contained sugar.
