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

Others

Water Vitamins Minerals White cells Enzymes

2

Fat

3-5 g/100 mL 98-99% triacylglycerols > 50% energy Essential fatty acids (LA and ALA) Major fatty acids: Oleic acid (28-44%) Palmitic acid (15-25%) Linoleic acid (11-25%) Milk fat globules

Protein

0.8-0.9 g/100 mL Whey protein:casein (60:40) Whey protein: α-Lactalbumin Serum albumin Lactoferrin Immunoglobulins Lysozyme Mature

Human Milk

Carbohydrate

6.9-7.3 g/100 mL Lactose Oligosaccharides

(Jenness, 1979; Riordan, 2005)

Human Milk Fat (HMF) vs. Vegetable Oil

3

Human milk fat (“OPO”) U

U

P Pancreatic lipase

P = Palmitic acid; O = Oleic U = Unsaturated fatty acids Ca = Calcium

P

P

U

Vegetable oil (“POP”)

U

U P

sn-2 monopalmitin

U

Calcium dipalmitate “soap”

Ca

P

P Ca

Poorly absorbed, hard

stools, and loss of energy and calcium

(Lien, 1994; Carnielli et al., 1995; Lopez-Lopez et al., 2001)

sn-1

60% sn-2

sn-3

Pancreatic lipase

sn-1

sn-2

sn-3

4

To explore the possibility of using structured lipids (SLs) as fat ingredient in the delivery of functional and physiological fatty acids in infant formulas

Research goal

4

LCPUFA

P

Structured lipid triacylglycerol

U

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

5

Chemical

Non-specific

Poor control over the final

product

Harsh conditions

Enzymatic

Both specific and non-specific

Good control

Milder reaction

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

• 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

CH3

OOH

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

• 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

CH3

OOH

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)

Diet

Sources Marine oil: Fish oil (EPA, DHA) Algal oil: DHASCO (DHA) Plant oil: SDASO (SDA), Flaxseed oil (ALA)

Benefits

Significance of n-3 LCPUFAs for the Development of Infants

α-Linolenic acid (ALA) C18:3 n-3

Stearidonic acid (SDA) C18:4 n-3

Eicosapentaenoic acid (EPA) C20:5 n-3

Docosahexaenoic acid (DHA) C22:6 n-3

Δ6-Desaturase

Δ6-Desaturase

12

“pro-EPA” Oxidative stability: ＞ EPA and DHA

(n-3 LCPUFAs: n-3 long-chain polyunsturated fatty acids) (Simopoulos, 1991; Kidd, 2007; Lemke et al., 2010)

SLs as HMF Analogs (SDA-Based)

Physical mixture of oils in traditional infant formula

P

P

U

DHA

DHA

DHA

Vegetable oil

Algal oil

SLs (mimic HMF)

P

SDA

DHA

DHA

SDA

P

U

U

SLs HMF

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

Fatty acid composition (mol%) of SLs

15

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 43.22±2.11 a 38.64±2.06 b 38.08±2.23 b 36.10±1.99 c 39.64±1.64 a 33.85±1.98 b 32.48±1.82 bc 31.50±1.83 c

C18:2n-6 6.34±0.04 a 5.29±0.98 b 5.79±0.45 b 4.09±0.56 c 6.06±0.78 a 6.34±0.82 a 5.38±0.79 b 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). 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

16

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 synthesized using one-port design with substrate molar ratio 0.5:1:0.5 (TP:EVOO:AD). eSL2-2, 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…

• Significant amounts of… – Squalene: 4.2% – Sterols: 834 mg/100 g oil – Tocopherols and tocotrienols: 44 mg/100 g oil

Linoleic acid > Oleic acid > Palmitic acid

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HMF – Omega FAs

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

22

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

U

P

sn-1

sn-2

sn-3

TAG

Human milk fat

U

P

P

sn-1

sn-2

sn-3

TAG

Vegetable oil

23

sn-1

sn-2

sn-3

Vegetable oil

Lipase

U

U

P sn-1

sn-2

sn-3

TAG

Human milk fat

U

P

P

sn-1

sn-2

sn-3

TAG

Vegetable oil

Betapol™ and InFat™, OPO

Novel SLs should incorporate the beneficial LCPUFAs (ARA, DHA, SDA, EPA, and GLA)

SLs design Type of starting oil and acyl donor Type of lipase sn-1, 3 specific lipase non-specific lipase

Reaction conditions (time, temperature, substrate molar ratio)

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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

25

• 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

LCPUFAs

26

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 the Study of Fatty Acids and Lipids 0.35 0.5

* Based on the median worldwide range of DHA and ARA concentration in breast milk

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Method: Lipase reactions

• 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)

R1

R1

R1

2 HOOC-R2 Free fatty acids

+

R1

R2

R2

R1

R1

R2

Acidolysis reaction

lipase R1

R1

R1

+

R1

R2

R2

R1

R1

R2

Interesterification reaction

lipase

R2

R2

R2

TAG TAG TAG FFA

24 h

% 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

Predicted value of % incorporation in PDA-SL (palm olein, DHA, ARA)

Mol

ar ra

tio (m

ol/m

ol)

24 h

Temperature (˚C)

% Total DHA and ARA incorporation % Total incorporation of DHA and ARA at the sn-2 position

Mol

ar ra

tio (m

ol/m

ol)

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

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Fatty acid (% w/w) Palm olein

PDA-SL Palm olein: DHA-ARA

(1: 18)

PDG-SL Palm olein: FFA DHASCO, GLA

(2:1)

TDA-SL Tripalmitin: FFA

DHASCO-ARASCO (1: 9)

IFLa

(Range, n=11)

Human milk fata

(Range, n=36)

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 acid 13.79±0.18 22.11±0.78 35.11±0.02 48.53±1.40 5.88-43.01 53.50 – 57.10

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

Fatty acid profile comparison: PDA-SL, PDG-SL, TDA-SL, IFL, and human milk fat

a Data from Lόpez-Lόpez et al., 2002. IFL: Fat extracted from commercial infant formulas.

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

sample mole ratio

time (h)

total GLA (% mol) sn-2 PA (% mol)

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

32

Characterization of SLs and application in infant formulas

500 gram scale synthesis of SLs

Purification of SL (removal of nonesterified products)

SL physical and chemical properties characterization

SL-containing infant formula (powdered)

Acidolysis reaction in 1-L stir batch reactor

Short-path distillation, Alkaline deacidification

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

Wet-mixing/spray-drying vs. dry-blending

Nor

mal

ized

Heat

Flo

w E

ndo

Dow

n (W

/g)

Crystallization curve (exothermic)

-60 -40 -20 0 20 40 60 80

-5.19 °C 2.85 °C 20.29 °C PDG-SL

23.64 °C 1.5 °C TDA-SL

39.93 °C

Tripalmitin

-26.65 °C -9.83 °C -5.19 °C

IFL

-4.52 °C 3.52 °C

Palm olein

33

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

Nor

mal

ized

Heat

Flo

w E

ndo

Dow

n (W

/g)

-60 -40 -20 0 20 40 60 80

TDA-SL

PDG-SL

Palm olein

IFL

-2.78 °C

Nor

mal

ized

Heat

Flo

w E

ndo

Dow

n (W

/g)

Melting curve (endothermic)

Tripalmitin

47.00 °C

52.84 °C

66.03 °C

0.57 °C 36.36 °C

22.97 °C 4.86 °C

6.87 °C 39.93°C

5.53 °C 11.57 °C

34

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

Analysis of Triacylglycerol

Molecular Species by RP- HPLC

IFL: Fat extracted from a commercial infant formula

Palm olein

TDA-SL from tripalmitin, free fatty acids from DHASCO® and ARASCO®

35

TAG Relative Percentages NGLA LGLA NDHA LDHA

StGSt 2.09 0.95 2.83 5.02 StLnLn/StGG 0.93 1.67 0.98 1.52

StLSt 1.73 2.32 0.59 1.28 StPSt 4.37 4.97 8.39 12.22

StOSt/LnGSt 6.81 6.14 10.38 14.35 LLG 1.19 0.81 ND ND DPD ND ND 2.68 0.66 OPD ND ND 1.76 0.46 OOD ND ND 1.74 0.52 GGO 0.98 1.18 ND ND OLLn 3.42 4.85 1.29 1.08 LnLP ND ND 1.42 1.18 LnGSt 0.53 0.69 0.78 0.65 OLL 0.39 0.58 1.37 1.74 LLP 8.17 8.59 6.55 6.52 GLS 1.10 1.03 3.23 3.53

LnOP 2.37 1.89 1.93 1.95 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

M , myristic acid G, γ-linolenic acid D, docosahexaenoic acid L, linoleic acid Ln, α-linolenic acid O, oleic acid P, palmitic acid S, stearic acid St, stearidonic acid

TAG

Mol

ecul

ar S

peci

es

SLs modified from SDA soybean oil, tripalmitin, DHASCO, or GLA using non-specific (N), and sn-1,3 specific lipozyme (L)

37

Selection of microencapsulation method: Encapsulant matrix

natural ingredients, food graded ingredients emulsify, build viscosity, and form gel

Effects of the encapsulant matrix to the bioavailability of encapsulated components

Method of operation Availability and cost , single-step formulation

MRPs encapsulation:

Natural products from milk protein and food carbohydrate

Single-step formulation

Provides protection to the core oil from gastric conditions

Releases oil in the small intestine

Do not compromise the bioavailability of the encapsulated oil

Microencapsulation using Maillard reaction products (MRPs) as encapsulant

38

• Whey protein isolate (21 g) was reconstituted in 350 mL of water at 60°C

• Corn syrup solids (42 g) was added to the mixture • pH of the mixture was adjusted to 7.5 • The mixture was heated in a water bath at 90°C for

30 min and cooled to 60°C before addition of oil

1. Preparation of Maillard reaction

products (MRPs) as encapsulants

• 21 g of TDA-SL or PDG-SL was dispersed into the MRPs mixture using a benchtop homogenizer

• Pre-emulsion was passed through a high-pressure homogenizer in two steps at 35 MPa and 10 MPa

2. Addition of SL and preparation of oil-in-water (o/w) emulsion

• Homogenized emulsion was held at 60°C and spray-dried at inlet temperature of 180°C and outlet temperature of 80°C

3. Spray-drying

MRPs microencapsulation steps

39

Characterization of microencapsulated TDA- and PDG-SLsa

Product characteristics TDA-SL powder PDG-SL powder

Total oil (g/g of sample)

Free oil (g/g of sample)

Microencapsulation efficiency (%)

Moisture content (%)

Water activity, Aw

Hydroperoxide value, PV (mmol/kg oil)

TBARS (mmol/kg oil)

Oxidative onset temperatureb, OOT (°C)

Oxidative induction timec, OIT (min)

0.238±0.002

0.024±0.002

90.00±0.73

1.78±0.09

0.15±0.02

20.22±0.65*

1.00±0.14

225.67±1.15*

5.17±0.06*

0.250±0.010

0.024±0.001

90.39±0.55

1.96±0.03

0.16±0.03

4.98±0.78*

0.64±0.07

239.23±0.89*

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

40

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

Myristic acid C14:0

Palmitic acid C16:0

Stearic acid C18:0

Oleic acid C18:1 n-9

Linoleic acid C18:2 n-6

Gamma-linolenic acid C18:3 n-6

Arachidonic acid C20:4 n-6

Docosahexaenoic acid C22:6 n-3

1.94±0.01

5.09±0.02

36.70±0.11

4.29±0.02

15.28±0.03

2.89±0.02

0.83±0.01

17.69±0.09

10.75±0.15

3.00±0.13

4.84±0.14

48.53±1.40

4.03±0.03

9.82±0.12

1.83±0.01

0.19±0.00

9.73±0.13

4.80±0.03

0.53±0.00

1.72±0.01

37.55±0.13

3.87±0.02

36.40±0.25

10.09±0.09

5.03±0.02

-

3.75±0.02

0.65±0.08

2.41±0.09

35.11±0.02

3.55±0.17

33.99±1.05

10.14±0.16

5.43±0.90

-

2.25±0.10

41

0,00

10,00

20,00

30,00

40,00

50,00

60,00

alpha-T alpha-T3 beta-T gamma-T gamma-T3 delta-T delta-T3

Conc

entr

atio

n (p

pm)

Tocopherols

TDA-SL

PDG-SL

Tocopherol concentration (ppm) in TDA-SL and PDG-SL. T, tocopherol and T-3, tocotrienol

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

42

0

5

10

15

20

25

30

35

0 5 10 15

% O

bscu

ratio

n

Time (min)

TDA-SL powder

PDG-SL powder

0

1

2

3

0 2 4 6 8 10 12 M

ean

diam

eter

(μm

, d4,

3)

Time (min)

TDA-SL powder

PDG-SL powder

Mean droplet diameter (μm) measured as a function of time after powders were added to the stirring cell of laser diffraction instrument.

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

Dispersibility of TDA-SL and PDG-SL powders in water

A B

43

Application of SLs in powdered infant formula

Preparation of liquid emulsion Ingredient mixed in 800 mL, 50-60°C water Non-fat dried milk (20 g), whey protein isolate (10 g), lactose (31 g), and maltodextrin (30 g) TDA-SL (30 g) and vitamin/mineral mix (3.9 g)

Pre-emulsion preparation Homogenization (35 MPa and 10 MPa)

Pasteurization at 65°C, 30 min Spray-drying using two different temperatures (120°C and 180 °C)

Microencapsulation of TDA-SL TDA-SL was encapsulated in MRPs (1:1 protein: oil ratio, 25% oil load) 30 g SL, 30 g whey protein isolate, and 60 g corn syrup solids

Pre-emulsion, homogenization, and spray-dried at 180°C

Dry-blending of all dried ingredients Non-fat dried milk (20 g), whey protein isolate (10 g), lactose (31 g), maltodextrin (30 g), vitamin/mineral mix 3.9 g, and microencapsulated TDA-SL (120 g – 30 g SL, 30 g whey protein isolate, and 60 g corn syrup solids)

Wet-mixing/spray-drying Dry-blending

44

Characteristics of

powdered infant

formulas

Wet-

mixing/spray-

drying at 120

°C

Wet-

mixing/spray-

drying at 180 °C

Dry-blending Commercial

infant formula

Peroxide (μg/mg sample)

TBARS (μg/mg sample)

Color

L* lightness

a* -green, + magenta

b* -blue, + yellow

C* saturation chroma

h* hue

0.18±0.02b

0.06±0.01b

94.59±2.46b

-2.28±0.08a

16.37±0.34b,c

17.64±0.36 b,c

97.24±0.32c

0.37±0.02a

0.11±0.01a

95.75±0.53b

-3.12±0.05b

15.30±0.79 c

16.74±1.14c

100.30±0.76b

0.07±0.02c

0.04±0.01c

98.83±0.49a

-3.80±0.08c

17.71±0.55b

18.83±0.33b

101.28±0.31b

0.06±0.03c

0.05±0.01c

96.35±2.34a,b

-5.21±0.0.05d

19.29±0.65a

21.25±1.89a

103.89±1.04a

Characterization of powdered infant formulas

Mean±SD, n=6. Means with the same letter in the same row and category are not significantly differently (p>0.05)

45

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

Mid-Summary: Production, characterization and application of SLs in infant formulas

46

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

Application Problems

To apply these HMF analogs in real food systems (e.g., ready-to-feed infant formula, IF), consider: Physical instability Lipid oxidation

Scaled-up Synthesis of SLs (NSL & LDHA)

Interesterification

47

P

P

P Nonspecific

lipase ＋

S

S

P ＋ ＋ SDA

SDA

SDA

SDA

P

P

… P

SDA

S

Tripalmitin NSL

Tripalmitin/SDASO (2:1, mol/mol) at 65 °C for 18 h catalyzed by 10% Novozym 435

NSL

Acidolysis NSL/DHA (1:1, mol/mol) at 65 °C for 24 h catalyzed by 10% Lipozyme TL IM

LDHA

SDASO

sn-1,3 specific lipase

＋ P DHA

SDA

DHA

＋ P

SDA

S

S

SDA

DHA

SDA

/

/

P= Palmitic acid; S= Saturated fatty acids; SDA= Stearidonic acid; DHA= Docosahexaenoic acid

DHASCO

(Teichert & Akoh, 2011)

M

Distillate

Residue

Feed

Heating

Cooling

Vacuum

Roller

α-Tocopheryl oleate Enzymatic reaction Short path distillation

O

O

O

Lipase Free fatty acid Tocopheryl/tocotrienyl fatty acid ester Oil Vitamin E Structured lipid

48

Conjugates of vitamin E and fatty acids, which have no antioxidant activities in vitro, were formed during

enzymatic acidolysis as well as interesterification

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.

Fate of Vitamin E

(Product)

(Waste)

Full scan mode

α-Tocopheryl oleate from vit E linoleate mixture

β (or γ)-Tocopheryl palmitate from

WNSL

α-Tocotrienyl palmitate from

WLDHA

GC-MS (EI) in Synchronous Scan/SIM Mode

49

WasteNSL

WasteLDHA

SIM mode

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 8 δ-tocopheryl stearate 9 β (or γ)-tocopheryl oleate 10 β (or γ)-tocopheryl linoleate 11 β (or γ)-tocopheryl stearate 12 α-tocotrienyl palmitate 13 α-tocopheryl oleate 14 α-tocopheryl linoleate

50

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

51

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

P

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

Experimental design & Optimization

(RSM*)

52

Aqueous phase (NFDM, WPC, lactose, micronutrient

premix & thickeners)

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)

Particle size (Laser diffraction

particle size analyzer)

Optical analysis (Turbiscan) Viscosity

(Rheometer)

Lipid oxidation (PV; p-AnV; TOTOX)

(UV)

Relative content of

DHA and SDA (GC)

Oil phase (SLs & emulsifiers)

(* Response surface methodology)

Lecithin (0, 0.2, 0.4)

Monoacylglycerol (MAG) (0, 0.2, 0.4) Locust bean gum (LBG)

(0, 0.02, 0.1)

Carrageenan (0, 0.004, 0.02)

Emulsifiers (g/100 mL)

Thickeners (g/100 mL)

53

a Energy density for lipid, protein and carbohydrate is 9, 4, and 4 kcal/g, 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 d The usage is 600 mg premix/100 kcal e Others include lecithin, monoacylglycerol, LBG, carrageenan and deionized water, and their contributions to macronutrient and energy are negligible f Energy contribution (%) from each macronutrient is given in parentheses

Composition of SL-based IF emulsion

Ingredient Content Macronutrient contribution (g) Energy a (g) Lipid Protein Carbohydrate (kcal)

High heat nonfat dry milk b 20.0 0.2 6.8 10.6 71.4

α-Lactalbumin enriched whey protein concentrate c

8.8 1.1 6.9 0.1 37.9

Structured lipid 33.2 33.2 0 0 298.8 Lactose 61.0 0 0 61 244.0 Micronutrient premix d 3.9 0 0 0 0 Others e 873.1 0 0 0 0 Total 1000 34.5 13.7 71.7 652

(47.6%) f (8.4%) (44.0%)

The total energy density is 65.2 kcal/100 mL, consisting of 5.3, 2.1, and 11.0 g/100 kcal lipid, protein and carbohydrate, respectively These values meet the nutrient requirements by both FDA regulation 21CFR107.100 and ESPGHAN recommended standards

54

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

RH

ROOH

RO.

O2

Fe2+

Fe3+

Attack another fatty acid

Inactivated by oil phase antioxidants

Inactivated by surface active antioxidants

Inactivated by continuous phase antioxidants

Oil phase Biopolymer

interface Water phase

Reducing agent

Oxygen scavengers

Metal ion chelators

P

SDA

DHA

DHA

SDA

/

/

Lipid oxidation

SL-based infant formula emulsion

H2O

Oil

Lipid oxidation

O

CH3

HO

H3C

CH3

OO

HO OH

CHCH2OHOH

OO

HO OH

CHCH2OOC(CH2)14CH3OH

HO OH

O O

OH

OHO

Antioxidant

Citric acid,

Ascorbic acid, Ascorbyl palmitate,

α-Tocopherol,

β-Carotene,

55

Hypothesis The oxidative stability of SL-based IF emulsion is highly

influenced by the type and concentration of antioxidants

Commercial ready-to-feed IF (“Reference”)

SL-based IF without antioxidants (Without N2 flushing, “control”; with N2 flushing, “control_N2”)

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)

α-Tocopherol, ascorbic acid, ascorbyl

palmitate, β-carotene, citric acid and their

combinations

Antioxidant Effectiveness

56

Lipid oxidation (PV; p-AnV) (UV)

Hexanal (SPME-GC)

Volatile Analysis by Dynamic Head Space GC-MS

57

0 5 10 15 20 25 30 350.0

6.0x106

1.2x107

1.8x107

2827262524

23222120

19

181716

151413

1211

987

6

5

4

3

2

1

10

Abu

ndan

ce

Retention time (min)

The flavor volatiles probably derive from Maillard reactions and lipid autoxidation during sterilization processing and/or storage Due to the high level and a low odor threshold (4.5 μg/kg in water), hexanal was chosen as a marker to evaluate lipid oxidation in SL-based IF during storage

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.

Hexanal Analysis by SPME-GC

58

12.0 12.5 13.0 13.5

0.0

5.0x103

1.0x104

1.5x104

Butyl acetate(Internal standard)

Abu

ndan

ce

Retention time (min)

Hexanal

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.

All these validated parameters demonstrated that the developed SPME-GC method is reliable, sensitive, and convenient as a routine technique to determine hexanal in IF products

Lineality (μg/mL)

R2

LOQ

(LOD) (ng)

Accuracy

Precision b

Recovery (%) a

Intra-day (%)

Inter-day (%)

0.001-10

0.9909

4(0.4)

112.8±0.1

1.4

4.0

59

The mixture of α-tocopherol and β-carotene showed a strong synergistic effect in inhibiting lipid oxidation

Two possible mechanisms may be considered to explain the synergism

A cycle is operated to regenerate tocopherols from tocopheroxyl radicals by interaction with β-carotene

α-Tocopherol protects β-carotene from being autoxidized

Antioxidant Synergism

0

1

2

3

b

a

a

α-Tocopherol β-Carotene α-Tocopherol + β-carotenePe

roxi

de v

alue

(mm

ol/L

)

0.02%0

1

2

3BA

Concentration

ConcentrationConcentration

b

a

a

p-A

nisi

dine

val

ue(a

bsor

banc

e/m

L)

0.02%

0

2

4

6 C

b

aa

Hex

anal

(μg/

mL)

0.02%

60

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

10 20 30 40 50 600.00

0.25

0.50

0.75

1.00

1.25

OIT = 32.9 min

Hea

t flo

w (m

W/m

g)

Time (min)

Comercial infant formula Structured lipid-based infant formula

Exot

herm

al

OIT = 34.0 min

SL-Based IF with Optimized Formulation vs. Commercial Ready-to-Feed IF

Differential scanning calorimetry (DSC) in an isothermal mode at 80 °C

Fatty acid a Commercial infant formula SL-based infant formula 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 C12:0 16.6±0.0a 33.9±1.3a 1.2±0.0b 1.0±0.0b 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 C18:0 3.7±0.0b 1.5±0.1b 9.2±0.0a 11.9±0.6a 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 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 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 multiple-range test (p < 0.05). Unit, mol%.

61

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

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

63

Graduate and visiting students over the years who did the work in my laboratory