Development of process for maximal retention of vitamin D in fortification of

cheese

Cluster for Dairy: Innovation/Health and Sustainable Development Project Number 05016

Banaz Al-khalidi, MSc, Nadeen Taha MSc,

Supervisor: Reinhold Vieth, Dept of Nutritional Sciences University of Toronto

Ali Mohammed MSc, Supervisor: Derick Rousseau, Dept Chemical

Engineering, Ryerson University, Toronto

Objectives:

1.  To fortify cheddar and mozzarella cheeses using Casein-bound Vitamin D (instead of conventional emulsion).

2.  To demonstrate stability and bioavailability of vitamin D in mozzarella when cooked with pizza.

3.  To demonstrate suitability and bioavailability of vitamin D in fortified cheddar provided to nursing-home residents (average age 86 years)

The NEW RDA for vitamin D.

Vieth 2012, from CHMS

Too little vitamin D in food & supplements

LIVER

25-OHase 25(OH)D3 (circulating form)

1,25(OH)2D3 (active hormonal form)

KIDNEY 1-α-OHase

Vitamin D3

7-dehydrocholesterol SKIN

Dietary Vitamin D3

UVB light

Where does Vitamin D go biologically?

Breast Milk Lipoproteins

Casein proteins (plasma phase)

1,24,25(OH)3D3

24-OHase

ENDOCRINE FUNCTIONS: CALCITROPIC EFFECTS

↑ Intestinal calcium absorption ↑ Bone mineralization ↓ PTH

Processors'''

Vitamin D3 concentrations in fortified milks, wheys, and cheeses1

Fortified Cheddar cheese

Fortified low-fat cheese

Milk (IU/g) Before pasteurization 94.45 ± 1.20†a 93.83 ± 2.95a

After pasteurization 94.53 ± 0.85a 94.38 ± 2.24a

Cheese (IU/g) 833.88 ± 19.83a 676.09 ± 11.09b Whey (IU/g)

Total 17.56 ± 1.62a 48.78 ± 5.05b Pressed 44.33 ± 5.21a 32.57 ± 3.35b

Recovery (%) 90.90 ± 2.16a 54.96 ± 0.67b in cheese2 Recovery (%) in whey3

Total 16.07 ± 1.49a 52.33 ± 5.42b Pressed 0.35 ± 0.04a 0.65 ± 0.07b

Total recovery (%)4 107.32 ± 3.10a 107.76 ± 4.57a 1Mean of six replicates 2Recovery in cheese (%): (vitamin D3 in cheese x kg cheese/vitamin D3 in milk x kg milk) x 100 3Recovery in whey (%): (vitamin D3 in whey x kg whey/vitamin D3 in milk x kg milk) x 100 4Total recovery (%): (recovery in cheese + recovery in total whey + recovery in pressed whey) †Mean ± Standard Deviation (all such values) a,bMeans within a row with different superscripts differ (P < 0.05) !

Wagner et al 2008 Vitamin D3 Recovery

Method to assess binding of vitamin D onto proteins

Fortification and Quantification •  Vitamin D3 Casein Premix: 185,000 IU/mL

•  Industrial production: Agropur (St. Hubert, Quebec) •  Fortified regular-fat Cheddar & Mozzarella cheeses;

28000 IU/serving and 200 IU/serving (28g)

•  Vitamin D content analysis •  Modified Bligh & Dyer (1959) lipid extraction •  Vitamin D3 quantification by High Performance Liquid

Chromatography (HPLC)

Precipitation of Acid Caseinate

1.  Acid caseinate curd was precipitated from skim milk by adding citric acid solution.

2.  The acidified milk mixture was heated to 50oC. Caseins precipitated as very fine particles in a liquid serum (whey).

3.  The casein curd was then air dried at ambient temperature overnight (20-25 ºC).

Addition of Vitamin D to Casein Premix

1.  The precipitated caseinate curd (0.15 kg) was blended with distilled water (0.3 kg) and blended to very fine particles of casein suspension were evident.

2.  The pH raised to 6.9-7.0, and left overnight under constant stirring at room temperature (~25 ºC).

3.  Vitamin D3 solution in ethanolic solution (2.0 x 10^6 IU/mL) was added to the acid caseinate suspension and homogenized at 5000 psi (35 MPa) for 5 cycles.

4.  The vitamin D concentrated casein premix was stored at -20º C.

5.  Thaw before use and add to raw milk.

Vitamin D3 Recovery Modified Bligh & Dyer

aRecovery in Cheddar cheese (%) 92.1 ± 4.6 bRecovery in whey (%) total

7.10 ± 0.3

pressed 0.80 ± 0.03 cTotal recovery (%) 100.0 ± 1.7 !

Modified Bligh & Dyer aRecovery in Mozzarella cheese (%) 90.2 ± 5.9 bRecovery in total whey (%) 8.4 ± 0.5 cRecovery in cooking water (%) 1.4 ± 0.1 dTotal recovery (%) 100.0 ± 2.2 !Recovery mean (%) ± standard deviation (mean of four replicates) of vitamin D3 in high dose Cheddar cheese samples. Bligh & Dyer extraction; aRecovery in cheese (%) = (vitamin D 3 in cheese x kg of total cheese / vitamin D3 in milk x kg of milk) x 100. bRecovery in whey (%) = (vitamin D3 in whey x kg of whey / vitamin D3 in milk x kg of milk) x 100. cTotal recovery (%) = ( recovery in cheese + recovery in total whey + recovery in pressed whey).

Clinical Nutrition Research: George Brown College Culinary

School!!"!Students!

Study Design!

Study Protocol

25-Hydroxyvitamin D Results

Between Group Comparisons: ∆ 25(OH)D

P < 0.0001

Baseline 25(OH)D by Ethnicity

Alkhalidi'2012''25(OH)D'nmol/L

Gozdzik'2010'25(OH)D'nmol/L

Gozdzik'2008'25(OH)D'nmol/L

European! 56.3! 52.4 55.9

East!Asian! 37.2! 33.3 34.5

South!Asian!

30.3 29.4 38.3

Black!African

30.5! n/a n/a

South!American

35.1 n/a n/a

Middle!Eastern

57.2 n/a n/a

1= European 2= Black African 3= Middle Eastern and South American 4= South Asian 5= East Asian

!

Clinical'trial''average!age!86!years!:'ForIfied!Cheddar!for!

InsItuIonalized!Elderly!!

NURSING HOME: Baseline 25(OH)D

by supplementation dose relative to the 2010 RDA for the elderly

*** P<0.001

NURSING HOME CHEDDAR: Changes in serum 25(OH)D by fortified cheese dose

n=11 n= 13

-10

40

0

30

20

10

Vitamin D3 (IU per Week) 200 28,000 C

hang

e in

Men

tal H

ealth

Com

posi

te

Sum

mar

y M

easu

re

IMPROVEMENT IN MENTAL WELLBEING SCORE (SF-36 QUESTIONNAIRE)

Conclusions:

1.  Cheddar and mozzarella can be reasonably fortified with Casein-bound Vitamin D (~90% efficiency)

2.  Vitamin D-fortified Mozzarella baked in pizza efficiently delivers vitamin D.

3.  Vitamin D-fortified Cheddar provided to nursing-home residents (average age 86 years) was much liked and produced the responses expected.

Objectives:

1.  To fortify cheddar and mozzarella cheeses using Casein-bound Vitamin D (instead of conventional emulsion).

2.  To demonstrate stability and bioavailability of vitamin D in mozzarella when cooked with pizza.

3.  To demonstrate suitability and bioavailability of vitamin D in fortified cheddar provided to nursing-home residents (average age 86 years).

The NEW RDA for vitamin D.

Vieth 2012, from CHMS

Too little vitamin D in food & supplements