Enzymatically Produced Structured

Lipids for Infant Formula Use

Casimir C. Akoh
Department of Food Science and Technology
The University of Georgia, Athens, GA 30602

1
 Protein Carbohydrate

0.8-0.9 g/100 mL 6.9-7.3 g/100 mL

Whey protein:casein Lactose
(60:40) Oligosaccharides
Whey protein:
α-Lactalbumin
Serum albumin
Lactoferrin
Immunoglobulins
Lysozyme
Mature
Fat
Human
Others
3-5 g/100 mL
Milk
Water
98-99% triacylglycerols Vitamins
> 50% energy Minerals
Essential fatty acids (LA White cells
and ALA) Enzymes
Major fatty acids:
Oleic acid (28-44%)
Palmitic acid (15-25%)
Linoleic acid (11-25%)
Milk fat globules (Jenness, 1979; Riordan, 2005) 2
 Human Milk Fat (HMF) vs. Vegetable Oil

Human milk fat (“OPO”)

sn-1 U
U
60% sn-2 P Pancreatic lipase P U
Ca

sn-3 U
sn-2 monopalmitin

Poorly
Vegetable oil (“POP”) absorbed, hard
stools, and
loss of energy
sn-1 P P and calcium
Ca
sn-2 U Pancreatic lipase U
P
sn-3 P Calcium dipalmitate
“soap”
P = Palmitic acid; O = Oleic
U = Unsaturated fatty acids
(Lien, 1994; Carnielli et al., 1995; Lopez-Lopez et al., 2001) 3
Ca = Calcium
 Structured lipid
triacylglycerol

LCPUFA

Research goal

To explore the possibility of using structured lipids

(SLs) as fat ingredient in the delivery of functional
and physiological fatty acids in infant formulas
4
4
 SL Production – HMF Analogs
• Direct esterification: Glycerol + FA TAG + Water
• Acidolysis: TAG1 + FA1 TAG2 + FA2
• Alcoholysis: TAG1 + Alcohol1 TAG2 + Alcohol2
• Interesterification: TAG1 + TAG2 TAG3 + TAG4

Chemical Enzymatic

Both specific
Non-specific and non-
specific

Poor control
over the final Good control
product

Milder
Harsh
reaction
conditions 5
conditions
 Breast Milk
• Maternal milk: The gold standard of nutrition
for full term infants up to 6 months

• Lipids, nucleotides, oligosaccharides, probiotics,

immunoglobulins, whey proteins
 Growth and development
 Protection and immunity

• Compositional variations according to race, genetics, season,

birth term, physiology, lactation stage
6
 Palmitic Acid
• C16:0 (SFA)
• Second major FA in breast milk (~25%) (Innis, 2011)
• It constitutes >50 % by weight at the sn-2 position (Innis, 2011)

7
 Docosahexaenoic Acid (DHA)
• C22:6n-3 CH3
HO O

• Essential structural component of retinal, neural and other

cell membranes

• LC-PUFAs contribute to improved visual acuity, cognitive

development, immune responses, and motor functions (Heird
& Lapillonne, 2005; Innis & Friesen, 2008; Rudnicka et al., 2008)

8
 DHASCO®

• DHASCO® - DHA single cell oil

• Mixture of an oil extracted from the unicellular

algae Crypthecodinium cohnii and high oleic
sunflower oil

• 40-45% DHA by weight

• GRAS status by FDA

9

(http://www.accessdata.fda.gov/scripts/fcn/gras_notices/grn000080-1.pdf)
 Arachidonic Acid (ARA)
• C20:4n-6 CH3
HO O

• ARA is needed for brain growth and functional

development of infants

• Physiologically important in prenatal and postnatal life

• In the fetus and infant, the metabolic conversion of PUFA-

precursors to ARA and DHA is not sufficient to meet
adequate levels

10
 ARASCO®

• ARASCO® - ARA single cell oil

• Mixture of an oil extracted from the unicellular

fungus Mortierella alpina and high oleic sunflower
oil

• ~40% ARA by weight

• GRAS status by FDA

11

(http://www.accessdata.fda.gov/scripts/fcn/gras_notices/grn000080-1.pdf)
 Significance of n-3 LCPUFAs for
the Development of Infants
Diet

Benefits α-Linolenic acid (ALA)

C18:3 n-3
“pro-EPA”
Oxidative stability: ＞
Δ6-Desaturase EPA and DHA
Stearidonic acid (SDA)
C18:4 n-3
Sources
Eicosapentaenoic acid (EPA)
C20:5 n-3
Marine oil:
Δ6-Desaturase Fish oil (EPA, DHA)
Docosahexaenoic acid (DHA) Algal oil:
C22:6 n-3
DHASCO (DHA)
Plant oil:
SDASO (SDA),
Flaxseed oil (ALA)
(n-3 LCPUFAs: n-3 long-chain polyunsturated fatty acids) 12
(Simopoulos, 1991; Kidd, 2007; Lemke et al., 2010)
 SLs as HMF Analogs (SDA-Based)
Physical mixture of oils in SLs
traditional infant formula (mimic HMF)

P DHA SDA / DHA U

U DHA P P

P DHA DHA / SDA U

Vegetable Algal oil SLs
HMF
oil

P= Palmitic acid; U= Unsaturated fatty acids; SDA= Stearidonic acid; DHA= Docosahexaenoic acid

 A SL which mimics the unique structure of HMF as well as

containing DHA and SDA in the triacylglycerol backbone, may
maximize health benefits for infants
13
HMF Analogs (Olive Oil-Based)

14
 HMF Analogs: Olive Oil-Based
total sn-2
fatty acid SL1-1a SL1-2b SL2-1c SL2-2d SL1-1 SL1-2 SL2-1 SL2-2

C8:0 nde nd 0.50±0.00 a 0.60±0.00 a nd nd nd nd

C10:0 1.04±0.00 a 1.02±0.00 a 1.11±0.07 b 0.70±0.02 c nd nd nd nd
C12:0 0.21±0.00 a 0.18±0.00 a 0.44±0.00 b 0.42±0.00 b nd nd nd nd
C14:0 1.97±0.03 a 2.11±0.21 a 2.47±0.55 b 2.54±0.22 b nd 1.12±0.00 a 1.44±0.00 b 1.24±0.01 c
C16:0 36.69±2.12 a 44.23±2.87 b 32.23±1.78 c 38.07±1.93 d 52.67±3.07 a 58.25±2.65 b 51.23±2.84 a 55.34±2.22 c
C16:1n-7 1.03±0.07 a 0.87±0.01 b 0.85±0.00 b 0.84±0.00 b nd nd 0.87±0.00 a 0.73±0.00 b
C18:0 2.82±0.03 a 2.58±0.05 a 3.19±0.48 b 3.02±0.45 b nd nd 2.34±0.01 a 1.97±0.00 b
C18:1n-9
C18:2n-6
Fatty acid composition (mol%) of SLs
43.22±2.11 a
6.34±0.04 a
38.64±2.06 b
5.29±0.98 b
38.08±2.23 b
5.79±0.45 b
36.10±1.99 c
4.09±0.56 c
39.64±1.64 a
6.06±0.78 a
33.85±1.98 b
6.34±0.82 a
32.48±1.82 bc
5.38±0.79 b
31.50±1.83 c
4.91±0.57 c
C20:0 0.29±0.00 a 0.23±0.00 a 0.33±0.00 b 0.28±0.00 a nd nd nd nd
C18:3n-6 0.08±0.00 a 0.06±0.00 a 0.46±0.01 b 0.45±0.01 b nd nd nd nd
C18:3n-3 0.47±0.00 a 0.39±0.00 b 0.33±0.01 c 0.30±0.00 c nd nd nd nd
C22:0 0.16±0.00 a 0.12±0.00 b 0.28±0.00 c 0.27±0.02 c nd nd nd nd
C20:3n-3 0.16±0.00 a 0.11±0.00 b 0.55±0.00 c 0.54±0.01 c nd nd nd nd
C20:4n-6 3.67±0.21 a 2.97±0.11 b 8.23±0.96 c 7.95±0.33 d 2.25±0.02 a 1.09±0.03 b 5.29±0.22 c 5.13±0.21 c
C22:6n-3 1.53±0.05 a 1.39±0.72 b 4.71±1.01 c 4.60±0.29 c 0.18±0.00 a 0.23±0.00 b 2.41±0.01 c 2.75±0.03 d
minor6 0.33±0.00 a 0.30±0.03 a 0.52±0.02 b 0.53±0.00 b

aSL1-1, structured lipid synthesized using sequential design with substrate molar ratio 0.5:1:0.5 (TP:EVOO:AD). bSL1-2, structured lipid synthesized

using sequential design with substrate molar ratio 1:1:0.5 (TP:EVOO:AD). cSL2-1, structured lipid synthesized using one-port design with substrate
molar ratio 0.5:1:0.5 (TP:EVOO:AD). dSL2-2, structured lipid synthesized using one-port design with substrate molar ratio 1:1:0.5 (TP:EVOO:AD). 15
AD, ARASCO-FFA and DHASCO-FFA (2:1). end, not detected. 6Minor is the sum of C17:0, C20:1, C20:2, and C22:2. Each value is the mean of
triplicates ± standard deviation. Values with different letter in each row within total and sn-2 columns separately are significantly different at P ≤ 0.05.
 Relative (%) of TAG Molecular Species of EVOO and SLs
TAG EVOOa SL1-1b SL1-2c SL2-1d SL2-2e

OAO ndf 0.98±0.02 a 0.75±0.01 b 1.21±0.03 c 0.74±0.00 b

APA nd 2.56±0.69 a 1.78±0.08 b 6.11±1.04 c 5.24±0.28 d
OPD nd 1.40±0.11 a 1.59±0.04 b 2.36±0.83 c 2.14±0.19 c
ODO nd 0.33±0.00 a 0.36±0.01 a 1.20±0.04 b 0.98±0.00 c
LOL 6.59±1.21 nd nd 2.07±0.21 a 1.44±0.21 b
LPL 1.97±0.67 1.46±0.01 a 1.14±0.01 b 0.66±0.00 c 0.87±0.00 d
MPL nd 0.84±0.00 a 0.72±0.00 b 2.35±0.11 c 0.88±0.01 a
POLn nd 2.13±0.18 a 1.50±0.00 b 6.03±0.28 c 4.56±0.38 d
SMM nd nd nd 1.44±0.01 a 2.59±0.19 b
OOL 10.20±1.55 3.22±0.21 a 1.68±0.01 b 2.11±0.19 c 2.02±0.02 c
POL nd 6.28±1.01 a 4.68±0.33 b 6.08±2.03 a 4.34±0.27 b
PLP 0.74±0.01 2.53±0.46 a 3.42±0.19 b 2.32±0.79 a 2.37±0.68 a
PPM nd 1.12±0.06 a 1.11±0.07 a 0.88±0.04 ab 0.67±0.00 b
OOO 47.19±3.08 8.32±0.78 a 6.12±1.06 b 7.64±1.44 c 6.83±1.79 b
OPO 25.37±2.18 25.17±2.51 a 28.84±2.11 b 23.00±2.18 c 25.96±2.79 a
PPO 2.81±0.22 31.35±2.49 a 33.95±2.98 b 24.82±1.59 c 28.64±2.91 d
PPP nd 4.50±1.62 a 10.32±1.70 b 4.02±0.58 a 6.23±1.04 c
OOS 4.47±0.67 1.83±0.29 a 0.86±0.28 b 1.38±0.00 ac 1.15±0.00 c
POS 0.66±0.01 4.31±0.01 a 2.05±0.29 b 3.80±0.02 c 3.65±0.00 c
PPS nd 0.82±0.00 a 0.68±0.00 b 0.78±0.00 a 0.82±0.00 a
The fatty acids are not in regiospecific order. A, arachidonic acid. D, docosahexaenoic acid. L, linoleic acid. Ln, linolenic acid. M, myristic acid. O,oleic acid. P,
palmitic acid. S, stearic acid. aEVOO, extra virgin olive oil. bSL1-1, structured lipid synthesized using sequential design with substrate molar ratio 0.5:1:0.5
(TP:EVOO:AD). cSL1-2, structured lipid synthesized using sequential design with substrate molar ratio 1:1:0.5 (TP:EVOO:AD). dSL2-1, structured lipid
16
e
synthesized using one-port design with substrate molar ratio 0.5:1:0.5 (TP:EVOO:AD). SL2-2, structured lipid synthesized using one-port design with
substrate molar ratio 1:1:0.5 (TP:EVOO:AD). AD, ARASCO-FFA and DHASCO-FFA (2:1). fnd, not detected. Each value is the mean of triplicates ±
standard deviation. Values with different letter in each row are significantly different at P ≤ 0.05.
 Amaranth Oil-Based
Underutilized source: Amaranth oil

• Amaranth: pseudocereal from warm climates

– Fat content: 4-8.5%

• Fatty acid (FA) profile:

– Palmitic acid (16:0) ~ 18.6-23.4%
– Oleic acid (18:1) ~ 22.7-31.5%
– Linoleic acid (18:2) ~ 39.4-49.8%
– Linolenic acid (18:3) ~ 0.5-1.36%

• The sn-2 position of TAG contain…

Linoleic acid > Oleic acid > Palmitic acid

• Significant amounts of…

– Squalene: 4.2%
– Sterols: 834 mg/100 g oil
– Tocopherols and tocotrienols: 44 mg/100 g oil
HMF – Omega FAs

18
 HMF Analogs: Hazelnut Oil-Based

 SL resembling human milk fat (HMF) containing GLA

 SL resembling HMF containing EPA and DHA
 Lipozyme RM IM or Lipozyme TL IM as biocatalysts

 Substrates: Tripalmitin + Hazelnut oil FAs + GLA

OR
Tripalmitin + Fish oil fatty acids
 Hazelnut Oil-Based contd..

• Temp = 55 ºC, Time = 24 h, substrate molar ratio

= 14
Results:
• HMF analog enriched with EPA and DHA (45.5%
palmitic, 37.5% oleic, 4.4% linoleic, 6.2% EPA
and DHA)
• HMF analog enriched with GLA (10% GLA, 45%
oleic)
• Sn-2 position mostly occupied by palmitic acid
(>60%)
 Fatty acids Total fatty acid composition range (%)
Human milk fat Infant formula fat

Oleic acid C18:1 (n-9) 28.30 – 43.83 34.34 – 44.69

Palmitic acid C16:0 15.43 – 24.46 17.96 – 27.42
Linoleic acid C18:2 (n-6) 10.61 – 25.30 8.93 – 17.02
Stearic acid C18:0 4.60 – 8.13 3.05 – 6.72
Lauric acid C12:0 4.05 – 9.35 5.19 –12.64
Myristic acid C14:0 3.60 – 9.13 4.06 – 5.91
Alpha-linolenic acid (ALA) C18:3 (n-3) 0.41 – 1.68 0.67 – 2.83
Gamma-linolenic acid (GLA) C18:3 (n-6) 0.07 – 0.12 0.00 – 0.14
Arachidonic acid (ARA) C20:4 (n-6) 0.23 – 0.75 0.00 – 0.22
Docosahexaenoic acid (DHA) C22:6 (n-3) 0.15 – 0.56 0.00 – 0.20

López-López et al. 2002. Jensen 1999.

21
 Fatty acids % Fatty acid at the sn-2
Human milk fat Infant formula fat
Oleic acid C18:1 (n-9) 9.5 – 17.1 31.8 – 45.5
Palmitic acid C16:0 53.5– 57.1 1.2 – 19.4
Linoleic acid C18:2 (n-6) 3.7 – 8.4 18.7 – 25.5
Straarup et al. 2006

U sn-1
P sn-1

P sn-2 U sn-2

U sn-3
P sn-3

TAG TAG

Human milk fat Vegetable oil

22
 Lipase
 Betapol™ and InFat™, OPO
P sn-1 U sn-1
 Novel SLs should incorporate the
beneficial LCPUFAs (ARA, DHA, U sn-2 P sn-2

SDA, EPA, and GLA) U

P sn-3 sn-3

 SLs design TAG TAG

 Type of starting oil and acyl donor Vegetable oil Human milk fat
sn-1
 Type of lipase
sn-2
 sn-1, 3 specific lipase
 non-specific lipase sn-3

 Reaction conditions (time,

temperature, substrate molar ratio)
Vegetable oil

23
Long-chain polyunsaturated fatty acids
(LCPUFAs)
• Important in human development
– Components of membrane phospholipids
– Precursors for eicosanoids
– Ligands for membrane receptors in gene regulation (transcription factors)
• DHA (C22:6 n-3) - enriched in brain and retina phospholipids
• ARA (C20:4 n-6) - found in phospholipids throughout the body, a precursor
of eicosanoids
• Humans need dietary supply of these fatty acids
– Inability to form n-6 and n-3 fatty acids (lack of Δ-12 and Δ-15 desaturase
enzymes)
– Low capacity for de novo lipogenesis (desaturation and elongation of LA to ARA,
and ALA to EPA and DHA) Innis et al.,
2007

24
LCPUFAs
• GLA (C18:3 n-6) is a precursor of ARA (C20:4 n-6) and is
abundant in borage oil, black currant, and evening primrose
• Infants fed formula with 0.5 % GLA had higher ARA
concentrations in red blood cells(Jorgensen et al, 2006)

• EPA (C20:5 n-3) is essential for growth, development, and

intestinal absorption of fat-soluble vitamins in infants
• SDA (C18:4n-3) - Better conversion than ALA to EPA
• The greater the degree of unsaturation in fatty acids, the
more susceptible to lipid oxidation

• Microencapsulation and late addition of LCPUFAs are

recommended to avoid oxidizing conditions during
production

25
LCPUFAs
• Worldwide regulatory bodies support the addition of DHA and ARA to infant
formula to the levels found in breast milk for brain and retina development
• High blood plasma DHA (2.76%) in pregnant and lactating women who rely on
diet high in fish and seafood (Japan). Low plasma DHA (0.07%) in the
populations on diet high in vegetable protein (Sudan) (Jensen 1999)

Organization or Foundation % Fatty Acids*


DHA ARA
British Nutrition Foundation 0.4 0.4
Food and Agricultural Organization of the United
Nations/World Health Organization expert panel 0.35 0.7
Expert panel convened by the International Society for
0.35 0.5
the Study of Fatty Acids and Lipids

* Based on the median worldwide range of DHA and ARA concentration in breast milk
26
Method: Lipase reactions
Acidolysis reaction Interesterification reaction
R1 R1 R1 R1 R2
R1
lipase R1
+ 2 HOOC-R2 
R1 R2 R1 R1 + lipase
 R2 R2
 R1
R1 Free fatty R2 R2 
acids R1 R2
R2 R2

TAG FFA TAG TAG

• TAG hydrolases (EC 3.1.1.3) catalyze hydrolysis and

synthesis of TAG, diacylglycerols (DAG), and
monoacylglycerols (MAG)
• Modified lipid products are known as structured lipids
(SLs)

27
 Predicted value of % incorporation in PDA-SL (palm olein, DHA, ARA)
% Total DHA and ARA incorporation % Total incorporation of DHA and ARA at the sn-2 position

Molar ratio (mol/mol)

Molar ratio (mol/mol)

24 h 24 h

Temperature (˚C)

% Total DHA and ARA incorporation = 16.143 + 1.778*Sr +3.182*t-4.98*T2 +2.84*t2+1.218*Sr*T

% Total incorporation of DHA and ARA at the sn-2 position: = 7.858-0.992*T +2.011*t

Optimal conditions: 24 h, 60 °C, ratio of 18:1 (DHA and ARA mix:Palm olein)
Predicted % total incorporation of 23.10% DHA, ARA
Predicted % total incorporation of 10.28 % DHA, ARA at the sn-2 position

(Sr- substrate molar ratio, t-time, T-temperature)

Fatty acid profile comparison: PDA-SL, PDG-SL, TDA-SL, IFL, and human milk fat
PDA-SL PDG-SL TDA-SL
Human milk
Fatty acid Palm olein: Palm olein: FFA Tripalmitin: FFA IFLa
Palm olein fata
(% w/w) DHA-ARA DHASCO, GLA DHASCO- (Range, n=11)
(Range, n=36)
(1: 18) (2:1) ARASCO (1: 9)
Myristic acid 1.04±0.00 0.98±0.60 1.72±0.01 5.09±0.02 4.06-5.91 3.60 – 9.13

sn-2 myristic acid - 1.39±1.06 2.41±0.09 4.84±0.14 2.23-7.10 6.20 – 15.40

Palmitic acid 43.60±0.01 27.99±5.11 37.55±0.13 36.70±0.11 17.96-27.42 15.43 – 24.46

sn-2 palmitic
13.79±0.18 22.11±0.78 35.11±0.02 48.53±1.40 5.88-43.01 53.50 – 57.10
acid
Stearic acid 4.53±0.00 3.21±0.70 3.87±0.02 4.29±0.02 3.05-6.72 4.60 – 8.13

sn-2 stearic acid 0.87±0.03 4.13±1.49 3.55±0.17 4.03±0.03 0.56-2.38 1.60 – 4.90

Oleic acid 40.91±0.01 30.33±1.74 36.40±0.25 15.28±0.03 34.34-44.69 28.30 – 43.83

sn-2 oleic acid 66.38±0.12 44.24±0.26 33.99±1.05 9.82±0.12 26.33-52.37 9.50 – 17.10

Linoleic acid (LA) 9.92±0.01 9.05±3.45 10.09±0.09 2.89±0.02 8.93-18.43 10.61 – 25.30

sn-2 LA 18.96±0.15 10.96±0.13 10.14±0.16 1.83±0.01 8.14-26.69 3.70 – 8.40

ARA - 8.05±0.66 GLA 5.03±0.02 ARA 17.79±0.09 ND-0.35 0.23 – 0.75

sn-2 ARA - 5.47±0.29 GLA 5.43±0.90 9.73±0.13 ND-0.40 0.30– 0.70

DHA - 17.20±2.45 3.75±0.02 DHA 10.75±0.15 ND-0.20 0.15 – 0.56

sn-2 DHA - 11.72±0.21 2.25±0.10 4.80±0.03 ND 0.90 – 3.40

a Data from Lόpez-Lόpez et al., 2002. IFL: Fat extracted from commercial infant formulas.
29
 Analysis of Acidolysis Products of
SLs and DHA FFA (~40% DHA) from Small Scale
sample mole ratio time (h) total DHA (% mol) sn-2 PA (% mol)
NDHA 1:1 12 6.27 ± 0.74 50.68 ± 2.86
NDHA 1:2 12 9.41 ± 0.68 42.29 ± 1.86
NDHA 1:3 12 11.48 ± 1.07 44.46 ± 1.77
NDHA 1:1 24 10.27 ± 0.24 59.75 ± 2.13
NDHA 1:2 24 15.35 ± 0.52 48.62 ± 1.08
NDHA 1:3 24 14.77 ± 0.70 41.13 ± 0.35
LDHA 1:1 12 6.20 ± 2.42 50.15 ± 2.41
LDHA 1:2 12 9.36 ± 0.85 45.48 ± 1.91
LDHA 1:3 12 10.08 ± 0.19 45.65 ± 2.60
LDHA 1:1 24 10.06 ± 0.91 55.47 ± 6.98
LDHA 1:2 24 15.79 ± 1.88 43.03 ± 1.85
LDHA 1:3 24 16.31 ± 0.68 42.03 ± 1.02

NDHA: NSL enriched with DHA FFA (N = Novozym 435)

LDHA: LSL enriched with DHA FFA (L = Lipozyme)
 Analysis of Acidolysis Products
of SLs and GLA FFA (~70% GLA) from Small Scale
mole time
sample total GLA (% mol) sn-2 PA (% mol)
ratio (h)
NGLA 1:0.5 12 9.86 ± 1.24 54.46 ± 3.97
NGLA 1:1 12 12.33 ± 0.25 52.15 ± 0.87
NGLA 1:1.5 12 15.58 ± 0.94 45.49 ± 1.64
NGLA 1:0.5 24 12.80 ± 2.29 49.13 ± 2.78
NGLA 1:1 24 15.90 ± 1.06 48.97 ± 2.04
NGLA 1:1.5 24 19.65 ± 2.04 48.94 ± 1.42
LGLA 1:0.5 12 9.78 ± 1.02 51.74 ± 3.53
LGLA 1:1 12 13.45 ± 0.97 49.41 ± 1.78
LGLA 1:1.5 12 15.22 ± 1.61 46.08 ± 1.96
LGLA 1:0.5 24 11.90 ± 1.00 49.85 ± 1.37
LGLA 1:1 24 17.58 ± 0.66 46.23 ± 0.89
LGLA 1:1.5 24 22.68 ± 1.03 44.82 ± 0.77
NGLA: NSL enriched with GLA FFA
LGLA: LSL enriched with GLA FFA
Characterization of SLs and application in infant formulas
500 gram scale synthesis of
SLs

Acidolysis reaction in 1-L stir

batch reactor

Purification of SL (removal
of nonesterified products)
Short-path distillation,
Alkaline deacidification

SL physical and chemical

properties characterization
Melting and crystallization profile, FA positional,
TAG molecular species analyses

SL-containing infant
formula (powdered)
Wet-mixing/spray-drying vs. dry-blending
32
 Crystallization curve
Normalized Heat Flow Endo Down (W/g) (exothermic)

39.93 °C
Normalized Heat Flow Endo Down (W/g)

Tripalmitin

3.52 °C
-4.52 °C
Palm olein

1.5 °C 23.64 °C TDA-SL

-5.19 °C 2.85 °C 20.29 °C

PDG-SL
-5.19 °C
-26.65 °C -9.83 °C IFL

-60 -40 -20 0 20 40 60 80

TDA-SL: Tripalmitin and a mix of DHASCO and ARASCO FFAs

PDG-SL: Palm olein and a mix of DHASCO FFA, GLA, and Palmitic acids
IFL: Lipid extracted from a commercial infant formula 33
 Melting curve
(endothermic)
Normalized Heat Flow Endo Down (W/g)

Tripalmitin

52.84 °C

47.00 °C

66.03 °C

Palm olein

11.57 °C
5.53 °C
TDA-SL
36.36 °C
0.57 °C
PDG-SL
39.93°C
6.87 °C 22.97 °C
4.86 °C
IFL

-2.78 °C

-60 -40 -20 0 20 40 60 80

TDA-SL: Tripalmitin and a mix of DHASCO and ARASCO FFAs

PDG-SL: Palm olein and a mix of DHASCO FFA, GLA, and Palmitic acid
IFL: Lipid extracted from a commercial infant formula 34
IFL: Fat extracted from a commercial
infant formula

Analysis of
Palm olein Triacylglycerol
Molecular Species by
RP- HPLC

TDA-SL from tripalmitin, free fatty acids from

DHASCO® and ARASCO®

35
 Relative Percentages
TAG SLs modified from SDA
NGLA LGLA NDHA LDHA
StGSt 2.09 0.95 2.83 5.02 soybean oil, tripalmitin,
StLnLn/StGG 0.93 1.67 0.98 1.52 DHASCO, or GLA using non-
StLSt 1.73 2.32 0.59 1.28 specific (N), and sn-1,3
StPSt 4.37 4.97 8.39 12.22 specific lipozyme (L)
TAG Molecular Species

StOSt/LnGSt 6.81 6.14 10.38 14.35

LLG 1.19 0.81 ND ND
DPD ND ND 2.68 0.66
M , myristic acid
OPD ND ND 1.76 0.46
OOD ND ND 1.74 0.52
G, γ-linolenic acid
GGO 0.98 1.18 ND ND
D, docosahexaenoic
OLLn 3.42 4.85 1.29 1.08 acid
LnLP ND ND 1.42 1.18 L, linoleic acid
LnGSt 0.53 0.69 0.78 0.65 Ln, α-linolenic acid
OLL 0.39 0.58 1.37 1.74 O, oleic acid
LLP 8.17 8.59 6.55 6.52 P, palmitic acid
GLS 1.10 1.03 3.23 3.53 S, stearic acid
LnOP 2.37 1.89 1.93 1.95 St, stearidonic acid
OOL 4.52 5.76 0.39 0.29
PLO 0.92 1.02 4.57 0.99
PLP 11.88 11.88 12.51 6.42
PPM 0.73 0.44 2.30 2.25
POO 1.18 1.16 1.48 2.06
POP 6.89 7.38 9.68 10.88
PPP 37.75 34.78 23.15 23.94
PSS/PSO 2.07 1.92 ND 0.48
 Microencapsulation using Maillard reaction products
(MRPs) as encapsulant

Selection of microencapsulation method:

 Encapsulant matrix
MRPs encapsulation:
 natural ingredients, food graded
 Natural products from milk protein and food carbohydrate
ingredients
 Single-step formulation
 emulsify, build viscosity, and form gel
 Provides protection to the core oil from gastric conditions
 Effects of the encapsulant matrix to the bioavailability of
 Releases oil in the
encapsulated small intestine
components
 DoMethod of operation
not compromise the bioavailability of the encapsulated oil
Availability and cost , single-step formulation

37
MRPs microencapsulation steps
• Whey protein isolate (21 g) was reconstituted in
1. Preparation of 350 mL of water at 60°C
Maillard reaction • Corn syrup solids (42 g) was added to the mixture
products (MRPs) as • pH of the mixture was adjusted to 7.5
encapsulants • The mixture was heated in a water bath at 90°C for
30 min and cooled to 60°C before addition of oil

2. Addition of SL and • 21 g of TDA-SL or PDG-SL was dispersed into the

MRPs mixture using a benchtop homogenizer
preparation of oil-in- • Pre-emulsion was passed through a high-pressure
water (o/w) emulsion homogenizer in two steps at 35 MPa and 10 MPa

• Homogenized emulsion was held at 60°C and

3. Spray-drying spray-dried at inlet temperature of 180°C and
outlet temperature of 80°C

38
 Characterization of microencapsulated TDA- and PDG-SLsa

Product characteristics TDA-SL powder PDG-SL powder

Total oil (g/g of sample) 0.238±0.002 0.250±0.010

Free oil (g/g of sample) 0.024±0.002 0.024±0.001
Microencapsulation efficiency (%) 90.00±0.73 90.39±0.55
Moisture content (%) 1.78±0.09 1.96±0.03
Water activity, Aw 0.15±0.02 0.16±0.03
Hydroperoxide value, PV (mmol/kg oil) 20.22±0.65* 4.98±0.78*
TBARS (mmol/kg oil) 1.00±0.14 0.64±0.07
Oxidative onset temperatureb, OOT (°C) 225.67±1.15* 239.23±0.89*
Oxidative induction timec, OIT (min) 5.17±0.06* 11.60±0.00*

aMicroencapsulation was prepared using 1:1ratio of oil to protein and 25% oil load in powder. Average
values of at least triplicate measurements were reported. Asterisk indicates values with significant
difference (p < 0.05) between the two SL microcapsules. bOOT determined by DSC at a heating rate of
10°C/min. c OIT determined by DSC isothermally at 220 °C. Microencapsulation efficiency = [(total oil-free
oil)/total oil] x 100

39
Fatty acid composition of the starting oils (TDA-SL and PDG-SL)

Fatty acids TDA-SL PDG-SL

Total sn-2 Total sn-2
Lauric acid C12:0 1.94±0.01 3.00±0.13 0.53±0.00 0.65±0.08
Myristic acid C14:0 5.09±0.02 4.84±0.14 1.72±0.01 2.41±0.09
Palmitic acid C16:0 36.70±0.11 48.53±1.40 37.55±0.13 35.11±0.02
Stearic acid C18:0 4.29±0.02 4.03±0.03 3.87±0.02 3.55±0.17
Oleic acid C18:1 n-9 15.28±0.03 9.82±0.12 36.40±0.25 33.99±1.05
Linoleic acid C18:2 n-6 2.89±0.02 1.83±0.01 10.09±0.09 10.14±0.16
Gamma-linolenic acid C18:3 n-6 0.83±0.01 0.19±0.00 5.03±0.02 5.43±0.90
Arachidonic acid C20:4 n-6 17.69±0.09 9.73±0.13 - -
Docosahexaenoic acid C22:6 n-3 10.75±0.15 4.80±0.03 3.75±0.02 2.25±0.10

40
 Tocopherol content in starting oils (TDA-SL and PDG-SL)

60,00

50,00

40,00
Concentration (ppm)

30,00
TDA-SL
PDG-SL

20,00

10,00

0,00
alpha-T alpha-T3 beta-T gamma-T gamma-T3 delta-T delta-T3
Tocopherols

Tocopherol concentration (ppm) in TDA-SL and PDG-SL. T, tocopherol and T-3, tocotrienol
41
 Dispersibility of TDA-SL and PDG-SL powders in water
A B
35
3

30

Mean diameter (μm, d4,3)

25
2
% Obscuration

20

15

10 1 TDA-SL powder
TDA-SL powder
PDG-SL powder
5 PDG-SL powder

0
0
0 5 10 15
0 2 4 6 8 10 12
Time (min)
Time (min)

Influence of stirring time on obscuration of spray-dried TDA-SL and PDG-SL Mean droplet diameter (μm) measured as a function
powders. Obscuration was measured as a function of time after powders were of time after powders were added to the stirring cell
added to the stirring cell of a laser diffraction instrument. of laser diffraction instrument.

42
 Application of SLs in powdered infant formula
Wet-mixing/spray-drying Dry-blending

Preparation of liquid emulsion Microencapsulation of TDA-SL

Ingredient mixed in 800 mL, 50-60°C water TDA-SL was encapsulated in MRPs (1:1 protein: oil ratio,
Non-fat dried milk (20 g), whey protein isolate (10 g), lactose (31 25% oil load)
g), and maltodextrin (30 g) 30 g SL, 30 g whey protein isolate, and 60 g corn syrup
TDA-SL (30 g) and vitamin/mineral mix (3.9 g) solids

Pre-emulsion preparation Pre-emulsion, homogenization, and spray-

dried at 180°C
Homogenization (35 MPa and 10 MPa)

Pasteurization at 65°C, 30 min Dry-blending of all dried ingredients

Non-fat dried milk (20 g), whey protein isolate (10 g),
Spray-drying using two different temperatures lactose (31 g), maltodextrin (30 g), vitamin/mineral mix
(120°C and 180 °C) 3.9 g, and microencapsulated TDA-SL (120 g – 30 g SL, 30
g whey protein isolate, and 60 g corn syrup solids)

43
 Characterization of powdered infant formulas

Characteristics of Wet- Wet- Dry-blending Commercial

powdered infant mixing/spray- mixing/spray- infant formula
formulas drying at 120 drying at 180 °C
°C
Peroxide (μg/mg sample) 0.18±0.02b 0.37±0.02a 0.07±0.02c 0.06±0.03c
TBARS (μg/mg sample) 0.06±0.01b 0.11±0.01a 0.04±0.01c 0.05±0.01c
Color
L* lightness 94.59±2.46b 95.75±0.53b 98.83±0.49a 96.35±2.34a,b
a* -green, + magenta -2.28±0.08a -3.12±0.05b -3.80±0.08c -5.21±0.0.05d
b* -blue, + yellow 16.37±0.34b,c 15.30±0.79 c 17.71±0.55b 19.29±0.65a
C* saturation chroma 17.64±0.36 b,c 16.74±1.14c 18.83±0.33b 21.25±1.89a
h* hue 97.24±0.32c 100.30±0.76b 101.28±0.31b 103.89±1.04a

Mean±SD, n=6. Means with the same letter in the same row and category are not significantly differently
(p>0.05)
44
Mid-Summary: Production, characterization and
application of SLs in infant formulas
 Human milk fat analogs were successfully produced

 HMF analogs were composed of a variety of TAG molecular species

 Broad crystallization and melting temperature range were observed

 SLs completely melted at temperature close to the melting point of

human milk fat (below 38°C)

 Dry-blending formula with microencapsulated SL had a better

oxidative stability and color quality than the product from wet-
mixing/spray-drying

45
 Application Problems
Are tailor-made SLs oxidatively stable to allow their use
as ingredients
Oxidative stability: SLs << initial substrates
Loss of endogenous antioxidants, especially
tocopherols and tocotrienols
What is the reason for the loss

To apply these HMF analogs in real food systems (e.g.,

ready-to-feed infant formula, IF), consider:
Physical instability
Lipid oxidation

46
 Scaled-up Synthesis of SLs
(NSL & LDHA)
Interesterification
 Tripalmitin/SDASO (2:1, mol/mol) at 65 °C for 18 h catalyzed by 10% Novozym 435

P S SDA P SDA
Nonspecific
lipase
P ＋ SDA P ＋ SDA ＋ P …

P S SDA P S

Tripalmitin SDASO NSL

Acidolysis
 NSL/DHA (1:1, mol/mol) at 65 °C for 24 h catalyzed by 10% Lipozyme TL IM
SDA SDA / DHA
sn-1,3 specific
lipase S
P ＋ DHA P ＋
SDA

S DHA / SDA

NSL DHASCO LDHA

P= Palmitic acid; S= Saturated fatty acids; SDA= Stearidonic acid; DHA= Docosahexaenoic acid 47
(Teichert & Akoh, 2011)
 Fate of Vitamin E
Enzymatic reaction Short path distillation α-Tocopheryl oleate

M
Feed O

O
O

Roller
Conjugates of vitamin E
and fatty acids, which have
Heating no antioxidant activities in
vitro, were formed during
Vacuum
enzymatic acidolysis as
(Product) Residue
well as interesterification

Cooling

(Waste) Distillate

Oil Structured lipid Vitamin E

Lipase Free fatty acid Tocopheryl/tocotrienyl fatty acid ester

Zou, L., Akoh, C. C. 2013. Identification of tocopherols, tocotrienols, and their fatty acid esters in residues and
distillates of structured lipids purified by short-path distillation. J. Agric. Food Chem. 61: 238-246. 48
 GC-MS (EI) in Synchronous Scan/SIM Mode
 Full scan mode
 SIM mode

WasteNSL α-Tocopheryl
oleate from vit E
linoleate mixture

WasteLDHA

β (or γ)-Tocopheryl
palmitate from
WNSL

No. Tocopheryl/tocotrienyl ester

1 δ-tocopheryl myristate
2 β (or γ)-tocopheryl myristate
3 δ-tocopheryl palmitate
4 β (or γ)-tocopheryl palmitate
5 δ-tocopheryl oleate
6 δ-tocopheryl linoleate
7 α-tocopheryl palmitate α-Tocotrienyl
8 δ-tocopheryl stearate palmitate from
9 β (or γ)-tocopheryl oleate WLDHA
10 β (or γ)-tocopheryl linoleate
11 β (or γ)-tocopheryl stearate
12 α-tocotrienyl palmitate
13 α-tocopheryl oleate
14 α-tocopheryl linoleate
49
Tocopheryl and Tocotrienyl Esters Identified by GC-MS

None of the tocopheryl/tocotrienyl linolenate,

stearidonate, and docosahexaenoate were identified
It may be due to the preference of lipases, Lipozyme
TL IM and Novozym 435, for a specific fatty acid or
fatty acids with a certain chain length range and
unsaturation

50
 Application of HMF Analogs in Ready-to-Feed IF (O/W)

SDA / DHA

DHA / SDA

Hypothesis
 The physical and oxidative stabilities
of SL-based IF emulsion is highly
influenced by the type and
concentration of emulsifiers and
thickeners

51
 Thickeners Emulsifiers

Aqueous phase
(g/100 mL) (g/100 mL)

Carrageenan
(NFDM, WPC, lactose, micronutrient
Oil phase Lecithin
(0, 0.2, 0.4)
(0, 0.004, 0.02) (SLs & emulsifiers)
premix & thickeners) Monoacylglycerol (MAG)
Locust bean gum (LBG)
(0, 0.2, 0.4)
(0, 0.02, 0.1)

O/W emulsion preparation

(High-pressure homogenizer, 5000-7000 psi, 3 times)
Sterilization; Storage
(pH 6.8; 121 °C, 6 min); (Room temp., 28 days)
Experimental design &
Particle size
(Laser diffraction Optimization Lipid oxidation
particle size
analyzer) (RSM*) (PV; p-AnV; TOTOX)
(UV)

Optical Relative
analysis content of
(Turbiscan) DHA and SDA
Viscosity (GC)
(Rheometer)

52
(* Response surface methodology)
 Composition of SL-based IF emulsion
a
Ingredient Content Macronutrient contribution (g) Energy
(g) Lipid Protein Carbohydrate (kcal)
b
High heat nonfat dry milk 20.0 0.2 6.8 10.6 71.4
α-Lactalbumin enriched 8.8 1.1 6.9 0.1 37.9
c
whey protein concentrate
Structured lipid 33.2 33.2 0 0 298.8
Lactose 61.0 0 0 61 244.0
d
Micronutrient premix 3.9 0 0 0 0
e
Others 873.1 0 0 0 0
Total 1000 34.5 13.7 71.7 652
f
(47.6%) (8.4%) (44.0%)

The
a Energytotal
densityenergy density
for lipid, protein and is 65.2 kcal/100
carbohydrate is 9, 4, andmL, consisting
4 kcal/g, respectivelyof 5.3, 2.1, and 11.0 g/100
kcal lipid, protein and carbohydrate, respectively
b High heat nonfat dry milk contains 0.76% lipid, 34% protein, and 53% carbohydrate
c α-Lactalbumin enriched whey protein concentrate contains 12% lipid, 78% protein, and 1.5% carbohydrate
These
d The usagevalues
is 600 mgmeet the kcal
premix/100 nutrient requirements by both FDA regulation 21CFR107.100
and ESPGHAN recommended standards
e Others include lecithin, monoacylglycerol, LBG, carrageenan and deionized water, and their contributions to

macronutrient and energy are negligible

fEnergy contribution (%) from each macronutrient is given in parentheses 53
Application of HMF Analogues in Ready-to-Feed IF (O/W)

Lipid
SDA / DHA
oxidation

DHA / SDA

RH O2
Oxygen
Attack another scavengers
fatty acid
Metal ion chelators
ROOH
Fe2+ Reducing
agent
Fe3+
Inactivated by RO.
oil phase Inactivated by continuous
antioxidants phase antioxidants

Inactivated by surface
active antioxidants
Biopolymer
Oil phase interface Water phase
54
 Hypothesis
 The oxidative stability of SL-based IF emulsion is highly
influenced by the type and concentration of antioxidants

CH3
Antioxidant
HO

H3C O
CH3
O OH
O O
Lipid α-Tocopherol,
oxidation
HO OH
OH

Citric acid,
SL-based infant
formula emulsion β-Carotene,

OH CH2OH
CH OH CH2OOC(CH2)14CH3
O CH
O Oil O
O

HO OH H2O
HO OH

Ascorbic acid, Ascorbyl palmitate,

55
 Antioxidant Effectiveness
α-Tocopherol, ascorbic
acid, ascorbyl
palmitate, β-carotene, Lipid oxidation
citric acid and their (PV; p-AnV) (UV)
combinations

SL-based IF with antioxidants

(Individual, 0.005 or 0.02% of oil;
Mixture (1:1), 0.02% of oil)

Accelerated storage
(37 °C , 4 weeks)
SL-based IF without antioxidants
(Without N2 flushing, “control”;
with N2 flushing, “control_N2”)

Hexanal
(SPME-GC)

Commercial ready-to-feed IF
(“Reference”)
56
 Volatile Analysis by Dynamic Head Space GC-MS

1.8x107 5
The flavor volatiles probably derive from Maillard
reactions and lipid autoxidation during sterilization
processing and/or storage
6
1.2x107
Abundance

1 Due to the high level and a low odor threshold

19
(4.5 μg/kg in water), hexanal was chosen as a
6.0x10 6
23
marker to evaluate lipid oxidation in SL-based IF
3 12 22 during storage
11 15 21
4 7 8 10 13 14 18 20
2 9 17
16 24 25 26 27 28
0.0
0 5 10 15 20 25 30 35
Retention time (min)

Peak numbers correspond to 1, 2-ethylfuran; 2, pentanal; 3, dimethyl disulfide; 4, toluene; 5, hexanal; 6, 3,5-octadiene; 7, trans-2-hexenal; 8,
1-hexanol; 9, 1,3-trans-5-cis-octatriene; 10, 2-heptanone; 11, cis-4-heptenal; 12, heptanal; 13, 2-ethylphenol; 14, trans-2-heptenal; 15,
benzaldehyde; 16, dimethyl trisulfide; 17, 1-octen-3-ol; 18, 6-methyl-5-hepten-2-one; 19, 2-pentylfuran; 20, cis-2-(2-pentenyl)furan; 21,
octanal; 22, trans, trans-2,4-heptadienal; 23, D-limonene; 24, 3-octen-2-one; 25, trans-2-octenal; 26, 3,5-octadien-2-one; 27, nonanal; 28,
trans, trans-2,4-decadienal. 57
 Hexanal Analysis by SPME-GC

1.5x104

1.0x104
Hexanal Butyl acetate
Abundance

(Internal standard)

5.0x103

0.0

12.0 12.5 13.0 13.5

Retention time (min)

2 b
All these validated parameters
Lineality R LOQ Accuracy Precision demonstrated that the developed
(μg/mL) (LOD) Recovery Intra-day Inter-day SPME-GC method is reliable,
a
(ng) (%) (%) (%) sensitive, and convenient as a
routine technique to determine
0.001-10 0.9909 4(0.4) 112.8±0.1 1.4 4.0 hexanal in IF products

a Mean ±SD, n = 5. The spiked level was 1 μg/mL sample.

b RSD, n = 5. The spiked level was 1 μg/mL sample. 58
 Antioxidant Synergism
α-Tocopherol β-Carotene α-Tocopherol + β-carotene
3 3
A B
a a
Peroxide value (mmol/L)

p-Anisidine value
(absorbance/mL)
2 2
a a

1 1
b

0 0
0.02% 0.02%
Concentration Concentration
6 a C
a
The mixture of α-tocopherol and β-carotene
Hexanal (μg/mL)

b
4 showed a strong synergistic effect in inhibiting lipid
oxidation
2

Two possible mechanisms may be considered to

0 explain the synergism
A cycle is operated to regenerate tocopherols
0.02%
Concentration
from tocopheroxyl radicals by interaction with β-
carotene
α-Tocopherol protects β-carotene from being
autoxidized

59
 SL-Based IF with Optimized Formulation vs.
Commercial Ready-to-Feed IF
Differential scanning calorimetry (DSC) in an
isothermal mode at 80 °C
Comercial infant formula a
Fatty acid Commercial infant formula SL-based infant formula
Structured lipid-based infant formula
Exothermal

1.25 Total sn-2 Total sn-2

C8:0 3.3±0.0a 0.5±0.1a 0.2±0.0b ND
C10:0 2.4±0.0a 1.3±0.1a 0.4±0.0b ND
1.00 C12:0 16.6±0.0a 33.9±1.3a 1.2±0.0b 1.0±0.0b
OIT = 34.0 min C14:0 6.0±0.0a 4.2±0.1a 3.3±0.0b 3.2±0.0b
C16:0 10.9±0.0b 2.0±0.0b 54.5±0.1a 62.3±0.3a
Heat flow (mW/mg)

0.75 C18:0 3.7±0.0b 1.5±0.1b 9.2±0.0a 11.9±0.6a

OIT = 32.9 min C18:1n-9 33.5±0.1a 32.2±0.8a 8.5±0.2b 5.3±0.1b
C18:2n-6 20.3±0.0a 22.7±0.8a 7.5±0.0b 4.9±0.2b
C18:3n-6 0.1±0.0b ND 1.8±0.0a 1.5±0.0a
0.50
C18:3n-3 1.9±0.0b 1.4±0.1b 2.5±0.0a 1.8±0.0a
C18:4n-3 ND ND 6.2±0.0a 5.2±0.2a
C20:4n-6 0.2±0.0a ND ND ND
0.25
C22:6n-3 0.2±0.0b 0.1±0.1b 3.9±0.0a 2.3±0.1a

a Mean ±SD, n = 3. Values with different letter in the same row and
category (i.e., total or sn-2) are significantly different by Duncan’s
0.00
multiple-range test (p < 0.05). Unit, mol%.
10 20 30 40 50 60
Time (min)
 The shorter oxidation induction time (OIT) of SL-based IF compared to the commercial product is
possibly due to the differences in the composition, such as lipid unsaturation , type and level of
antioxidants, emulsifiers, and minerals

A good agreement of the result with PV, AV, and hexanal content suggests that DSC technique is
suitable for oxidation studies of liquid IF products 60
 Mini-Summary
 The efficacy of permitted compounds as antioxidants in SL-based IF
emulsion depends not only on their mechanism of action, but also on
polarity, concentration, oxidation time, method used to determine lipid
oxidation, and environmental conditions (e.g., headspace oxygen and
pH)
 A synergistic antioxidant effect was found between α-tocopherol and β-
carotene

 A high correlation between AV and hexanal content indicates the

goodness of hexanal analysis by SPME-GC as an indicator of flavor
deterioration in IF products
 The most effective antioxidant was ascorbyl palmitate at 0.005%

 Compared to the commercial ready-to-feed IF, our SL-based product was

slightly less stable on the basis of DSC measurement, but that was due
to the high incorporation of unsaturated fatty acids (e.g., DHA and SDA)

 Nitrogen flushing may be used as a supplemental technique to further

protect against oxidation
61
 Conclusions
A HMF analogue enriched with beneficial DHA and SDA was
successfully synthesized by enzymatic reactions and incorporated in a
ready-to-feed IF to benefit the development and growth of infants
 The SL contained 5.4 mol% DHA and 8.0 mol% SDA, with 57.0 mol%
palmitic acid esterified at sn-2 position
 Although tocopheryl and tocotrienyl fatty acid esters were formed during
interesterification and acidolysis, >50% of vitamin E isomers were lost into
distillates (wastes) during SPD, which contributed mostly to the rapid
oxidative deterioration of SLs in the recent and past studies
 The optimal conditions to achieve the highest physical and oxidative
stability of SL-based IF emulsion were 0.2 g/100 mL lecithin, 0.4 g/100 mL
monoacylglycerol, 0.045 g/100 mL LBG, 0.015 g/100 mL carrageenan, and
0.005% ascorbyl palmitate
Overall, this study has important implications for the successful use of
SLs for food formulations

62
Acknowledgements

Graduate and visiting

students over the years who
did the work in my laboratory

63