CONSISTENCY OF VITAMIN A AND D3 OF FORTIFIED COW''''S MILK DURING PROCESSING, PACKAGING AND STORAGE

doi:10.21608/ejas.2019.104705

CONSISTENCY OF VITAMIN A AND D3 OF FORTIFIED COW''''S MILK DURING PROCESSING, PACKAGING AND STORAGE

Egyptian Journal of Applied Science

Article 18, Volume 34, Issue 9 - Serial Number 1, September and October 2019, Pages 278-293

Document Type: Original Article

DOI: 10.21608/ejas.2019.104705

Abstract

ABSTRACT
The impact of storage period and package light transmittance on retention of vitamin A and D3 content of fortified in pasteurized & sterilized cow''s milk, it can be indicated that packages without any light barrier properties like clear glass bottles are insufficient to protect the milk vitamins A and D3 under commercially relevant storage conditions. Due to the temperately short store ability of pasteurized milk, already the use of clear glass bottles led to a significant improvement so that after a 7day light-exposure period, losing 10% (90% retention) of the initial vitamin A and non-significant improvement of V D3 levels were losing 1.6% (98.4% retention), this means that vitamin D3 is more stability than vitamin A in light exposed under commercially relevant storage conditions and also package the milk in dark condition or high-pigmented bottle is better and led to more retention of both vitamin A and D3. In case of fortified sterilization milk protect from light exposed was necessary to obtain similar vitamins retentions of at least 88% under light expos and was been 94.1% in dark condition or protect bottles of vitamin A while vitamins D3 retentions reach 93.98% under light and 98.51% in dark of vitamins after a quite realistic light-exposure period of 60 days under commercially relevant storage conditions. However, it has to be considered that light-induced sensory changes in pasteurized milk and sterilization milk under commercially relevant storage conditions can only be excluded in light-tight packages.The stability of vitamin A and D3 was followed by its analysis in raw, pasteurized, and sterilized cow''s milk. Separation of vitamin A and D3 was done using C-18 column and isocratic elution of mobile phase consisting of 0.1 M acetic acid: acetonitrile (98: 2, v/v). Vitamin A and D3 were extracted from milk samples using 2.5 % solution of meta- phosphoric acid with extraction recoveries ranged 94 – 104 %. Confirmatory identification for presence of vitamin A and D3 in investigated milk samples were achieved using UV and MS data obtained from reference standard and sample at the same retention time.

Highlights

CONCLUSIONS

From such study it could be concluded that on the impact of storage period and package light transmition on retention of vitamin A and D3 content of fortified pasteurized & sterilized milk, the obtained results indicated that packages without any light barrier properties like clear glass bottles are insufficient to protect the milk after either pasteurization or sterilization under commercially relevant storage conditions for vitamins A and D3.

Keywords

Key Words: Vitamin A and D3; pasteurized milk; sterilization milk; fortifications

Full Text

Egypt. J. of Appl. Sci., 34 (9) 2019 278-293

CONSISTENCY OF VITAMIN A AND D3 OF FORTIFIED COW'S MILK DURING PROCESSING, PACKAGING AND STORAGE

Walid A. S. Gafour

Food Technology Research Institute, Agricultural Research Centre, Giza., Egypt.

Key Words: Vitamin A and D3, pasteurized milk, sterilization milk, fortifications.

ABSTRACT

The impact of storage period and package light transmittance on retention of vitamin A and D3 content of fortified in pasteurized & sterilized cow's milk, it can be indicated that packages without any light barrier properties like clear glass bottles are insufficient to protect the milk vitamins A and D3 under commercially relevant storage conditions. Due to the temperately short store ability of pasteurized milk, already the use of clear glass bottles led to a significant improvement so that after a 7day light-exposure period, losing 10% (90% retention) of the initial vitamin A and non-significant improvement of V D3 levels were losing 1.6% (98.4% retention), this means that vitamin D3 is more stability than vitamin A in light exposed under commercially relevant storage conditions and also package the milk in dark condition or high-pigmented bottle is better and led to more retention of both vitamin A and D3. In case of fortified sterilization milk protect from light exposed was necessary to obtain similar vitamins retentions of at least 88% under light expos and was been 94.1% in dark condition or protect bottles of vitamin A while vitamins D3 retentions reach 93.98% under light and 98.51% in dark of vitamins after a quite realistic light-exposure period of 60 days under commercially relevant storage conditions. However, it has to be considered that light-induced sensory changes in pasteurized milk and sterilization milk under commercially relevant storage conditions can only be excluded in light-tight packages.The stability of vitamin A and D3 was followed by its analysis in raw, pasteurized, and sterilized cow's milk. Separation of vitamin A and D3 was done using C-18 column and isocratic elution of mobile phase consisting of 0.1 M acetic acid: acetonitrile (98: 2, v/v). Vitamin A and D3 were extracted from milk samples using 2.5 % solution of meta- phosphoric acid with extraction recoveries ranged 94 – 104 %. Confirmatory identification for presence of vitamin A and D3 in investigated milk samples were achieved using UV and MS data obtained from reference standard and sample at the same retention time.

INTRODUCTION

Vitamins are defined as a group of complex organic micronutrients present in food and in nutritional supplements required for normal metabolism and function. Fat-soluble vitamins are essential for human health and play important roles in body functions, such as in vision (Vitamin A), calcium absorption (vitamin D), and antioxidant protection in cell membranes (vitamin E) (Morrissey and Hill, 2009). There are very important role of vitamins as antioxidants and their ability to prevent chronic diseases, especially those believed to have an oxidative stress component such as cardiovascular diseases and cancer (Lopes et al., 2003). The inadequate vitamin intake can cause metabolism disorders, both in humans and animals, with vitamins A and D being highly correlated with deficiency syndromes. Milk has been recognized as an important element in human diets because it provides a large variety of essential nutrients, such as lipids, proteins, AA, vitamins, and minerals, which are all required for the normal growth and development of mammals. Humans are a special case among mammals because after being deprived of their mother's milk, they keep on consuming milk and dairy products from other species, mainly from cows, buffaloes, goats, and sheep.

279 Egypt. J. of Appl. Sci., 34 (9) 2019

Vitamins are vital food ingredients for maintaining good health in humans; lack of a sufficient amount of any of them can cause serious diseases ( Riaz, et al., 2009). The human diet does not always contain the amount of vitamins needed for normal development and maintenance of body functions (Gomez & Jose, 2006).

For such reason, several food products are fortified with vitamins, mainly milk and milk products.Fluid milk products have been fortified with vitamins since the 1930’s when an industry wide program for vitamin D fortification was initiated in an effort to prevent infantile rickets, a bone disease of children related to vitamin D deficiency.As the addition of vitamin D to milk was becoming common practice, other nutrients were also being considered. This included vitamin A, which was added to milk in vitamin-mineral formulations manufactured in the 1940’s. Vitamin A shall be present in such quantity that each quart of the food contains not less than 2000 IU thereof within limits of Good Manufacturing Practices.” Vitamin D fortification at 400 IU/qt. remained optional for all products. Acceptable levels within limits of “Good Manufacturing Practices” were generally interpreted as the range of 80% to 120% of the label claim for both vitamin A and vitamin D. Determination of vitamin levels was required at least annually “in a laboratory acceptable to the regulatory agency,” though there were no established procedures to determine the competency of vitamin testing laboratories. (Newcomer and Murphy, 2001)

Vitamin A is a generic term referring to a variety of chemical substances showing vitamin A activity including retinol, retinal, retinoic acid, retinal esters, and pro-vitamin A carotenoids such as β-carotene. In dairy and animal food products, it is most often present as the retinal palmitate ester, which is hydrolyzed in the small intestine cells to the alcohol retinol. Retinol and its derivatives are found in animal tissues and dairy products, while β-carotene is present in foods of plant and animal origin. In the case of milk, the conversion of β-carotene in the feed to retinol may occur in the mammary gland of lactating dairy cow’s.

Egypt. J. of Appl. Sci., 34 (9) 2019 280

Vitamin A performs many functionsone is to prevent night blindness by enabling the retina of the eye to respond to dim lights. Vitamin A is also needed for normal growth, maintenance and function of epithelial cells, which include skin cells and the cells lining the respiratory, gastro-intestinal and reproductive tracts. Whole milk is a good source of vitamin A, though because it is a fat-soluble vitamin, its value is reduced in lower fat milks.(Newcomer and Murphy, 2001).

Vitamin D is a fat-soluble vitamin recognized for its importance in human health (Ceglia, 2009). Vitamin D is present in animal foods as cholecalciferol (vitamin D3). Ergocalciferol (vitamin D2) has plant origin, where it is converted from the pro-vitamin,ergosterol(Maweret al., 1998).Numerous studies have shown that the vitamin D status is far from optimal in many countries all over the world. The main reason for this is lack of sufficient solar Ultra Violet Blue. If one’s exposure to sunlight is limited, vitamin D deficiency may develop, and the need for supplementation, either as an oral supplement or through innovations in fortified food. There are limited dietary sources of vitamin D, including cod liver oil, fatty fish such as salmon, as well as small amounts found in egg yolks (Byrdwellet al., 2011). Vitamin D contents of whole milk provide only 1% of the daily value of vitamin D (Holden, 2009).

Vitamins are commonly used to supplement milk products, either to restore production losses, or to further enhance their availability to potentially at-risk groups (WHO/FAO, 2006).

Such potential losses during production are to be distinguished from those further accruing under post-production storage conditions.

Such inconsistencies between claimed and measured values in vitamin D content have been associated with chemical breakdown and/or processing effects (Renken&Warthesen, 1993). Vitamin D stability studies are limited and several contradictory results have been reported. Degradation of vitamins depends on specific conditions during the culinary process e.g., temperature, oxygen, light, moisture, pH and during heat treatments (Leskovaet al., 2006). Vitamin D3 was unstable to oxidation and light, but stable to acid & alkali, unstable to irradiation, moisture and trace mineralsCremin and Power (1985),(Charlton & Ewing,2007).The stability of VD3 is strongly dependent on the food in which it is present. (Upreti, et al 2002). Vitamin A&D3 appears to be stable in cheese during both short-term and long-term storage. Vitamin D3 was stable during extrusion, pelleting and is stable to oxidation, light and heat. Processing and cooking conditions cause variable losses of vitamins.Losses of V A&D3vary widely according to cooking method and type of food. The ability of a vitamin to retain its activity during storage and under chemical and physical stress refers to its stability. (Kazmi, et al., 2007; Wagner et al., 2008 and Riazet al. (2009).

281Egypt. J. of Appl. Sci., 34 (9) 2019

So the aim of this work is to determine the stability of vitamins A&D3 during processing, packaging and subsequent storage of fortified cow's milk during processing,packaging and storage, which will be beneficial to both the consumers and milk industry.

MATERIALS AND METHODS:

1. Milk samples

1.1. Raw Fresh Cow’s milk:

Raw Fresh Cow’s milk used in this study was obtain from silo milk one of selected private farm, which supply raw milk to deferent dairy company in Egypt. Samples of raw milk was collected in 100 ml plastic containers filled with no headspace and kept covered with aluminiumfoil until analyzed,the main raw milk quantity was divide into 2 potations regarding preparation heat treatments as following :

1.1.1. Pasteurized milk:

The milk was heated to 40°C, and vitamins A&D3 were fortified as (FDA 2007 standard) the treatment was stressed for 10 min for complete dissolution. After fortification of milk by vitamins A& D3, the samples were filled in 500 ml glass bottles with ISO thread Duran screwing cap with no headspace and kept covered with aluminium foil until analyzed, one of samples kept as (fortified raw milk for pasteurization) (control1) ,other samples were pasteurized at 63°C / 30 min. in a temperature controlled water bath and alkaline Phosphatase test used to verify pasteurization process is done in correctthen were grouped in two groups as follows :

1- Pasteurized glass bottle sample: in 500 ml in clear glass transparent bottles kept under light condition

2- Pasteurized glass bottle + aluminium foil layer: in 500 ml glass bottles kept covered and protected with aluminium foil as a dark conditions.

All samples were immediatelycooled to 4°C and were stored refrigerated (4 - 7°C) conditions, which was used to simulate retail conditions. Storage stability of vitamin A & D3 and loss (%) of vitamins in fortified milk during storage, the vitamins A&D3 retention were determined in 0, 5 and 7 days, respectively.

1.1.2.Sterilized milk:

The second part of raw milk was heated to 40°C, and vitamins A&D3 were added as (FDA 2007 standard) the treatment was stressed for 10 min for complete dissolution. After fortification milk by vitamins A& D3 the samples were filled in 500 ml and autoclave in glass bottles with ISO thread Duran screwing cap with 10% headspace and kept covered with aluminium foil until analyzed,one of samples kept as(fortified raw milk for sterilization) (control 2), other samples were autoclaved at 121°C / 15 min and 15 psi. Then were divided in two groups as follows:

1- Sterilized glass bottle sample: clear glass transparent bottles kept under light conditions

2- Sterilized glass bottle + aluminium foil layer: kept covered and protected with aluminium foil as a dark conditions.

Egypt. J. of Appl. Sci., 34 (9) 2019 282

All samples were immediately cooled to 4°C, and were stored (as commercial market retail UHT or Sterilized milk products) in period at room temperature (25°C) conditions. Storage stability of vitamin A & D3 and loss (%) of vitamins in fortified milk during storage, the vitamins A&D3 retention were determined at 0, 30and 60days, respectively.

Fortification of milk with vitamin A and D3:

Level of vitamin A& D3 was add on the bases of FDA(2007) as in table 1

Table (1) Fortification limits of vitamins A and D3 in milk as supplier recommendation

Pasteurized milk
105.60 gm/10000 Litter ( with 20% Process Loss )	2000 IU/Litter for pasteurized milk	Vitamin A Palmitate 250 CWS
50.40 gm/10000 litter ( with 20% Process Loss	400 IU/Litter for pasteurized milk	Vitamin D3 100 CWS
Sterilized Milk
114.4 gm/10000 Litter ( with 30% Process Loss	2000 IU/Litter for sterilized milk	Vitamin A Palmitate 250 CWS
54.60 gm/10000 litter ( with 30% Process Loss	400 IU/Litter for sterilized milk	Vitamin D3 100 CWS

Fortification limits of vitamins A and D3 in milk as standard of (FDA M-1-92-13, Nichols, 1992)

Chemical analysis:

Total solids (T.S), fat, protein and fiber contents of all milk samples were determined according to the method described by (AOAC, 2012), titratable acidity,pH values for milk was measured according to the methods of (BSI,1989), Alkaline Phosphatase was tested by using the novaLUM -Charm F-AP test is a simple one-step phosphatase method according to Indian Standard IS:8479 (Part-1)-1977.

Microbiological analysis:

Total bacterial count (TBC) of all the milk samples were estimated according to the method as described by (APHA, 2004), the coliforms were detected according to the methods of (BSI, 2010).

Statistical analysis

Data were subjected to analysis of variance of means vitamins A&D3 content using two-tailed( t tests) analysis where statistical differences among samples were noted(p < 0.05).

RESULTS AND DISCUSSION

1- Gross chemical composition;

Data of table (2) showed thegross chemical composition pH, Acidity % and Sp. gr of different types of milks fortified with Vitamin A and D3 of all milk samples during experimental period both pasteurized or sterilized fortified with vitamin A&D3 milkswhich were within standard range of fat, T.S, FNF%, pH, acidity and Sp.gr. which reflected in nutritional facts without any changes till expired date.

Table (2) Gross chemical composition, pH, Acidity % and Sp gr of different types of milks fortified with Vitamin A and D3

Milk type	pH	Temp	Fat %	Sp gr		Acidity %	TS %	SNF %
Raw Cow Milk	6.79 ± 0.15	5.07 ± 1.14	3.27 ± 1.77	1.0326 ± 0.01		0.14 ± 4.03	12.25 ± 0.24	9.05 ± 0.33
Raw milk fortified for Pasteurized Controlled 1	6.78 ± 0.23	5.20 ± 1.92	3.23 ± 1.79	1.0326 ± 0.02		0.14 ± 4.03	12.24 ± 0.42	9.04 ± 0.57
Raw milk fortified for Sterilized Controlled 2	6.77 ± 0.17	5.40 ± 1.85	3.23 ± 1.79	1.0325 ± 0.02		0.14 ± 4.03	12.22 ± 0.46	9.02 ± 0.62
Pasteurized treatment
*Pasteurized fortified Milk as light condition 0 day**	6.78 ± 0.09	5.00 ± 1.20	3.17 ± 1.80	1.0322 ± 0.0112		0.15 ± 0	12.03 ± 0.48	8.83 ± 0.65
*Pasteurized fortified milk as light conditions 5 day**	6.77 ± 0.09	5.00 ± 2.00	3.20 ± 3.13	1.0321 ± 0.0097		0.15 ± 0	12.13 ± 0.22	8.93 ± 0.30
*Pasteurized fortified milk as light conditions 7 day**	6.76 ± 0.23	5.90 ± 1.70	3.10 ± 1.80	1.0321 ± 0.0148		0.16 ± 0	12.14 ± 0.29	8.94 ± 0.39
*Pasteurized milk as dark conditions 0 day**	6.79 ± 0.17	5.30 ± 1.10	3.10 ± 1.80	1.0320 ± 0.0056		0.15 ± 0	12.11 ± 0.19	8.91 ± 0.26
*Pasteurized milk as dark conditions 5 day**	6.78 ± 0.17	5.00 ± 1.20	3.10 ± 1.84	1.0319 ± 0.0148		0.15 ± 0	12.08 ± 0.29	8.88 ± 0.40
*Pasteurized milk as dark conditions 7 day**	6.77 ± 0.17	5.10 ± 2.00	3.10 ± 1.80	1.0318 ± 0.0148		0.16 ± 0	12.06 ± 0.27	8.86 ± 0.36
Sterilized treatment
Sterilized fortified milk ( 0 day )	6.65 ± 0.15	25.9 ± 1.60	3.17 ± 1.82		1.0312± 0.0244	0.16 ± 0	11.86 ± 0.08	8.66 ± 0.12
Sterilized fortified milk (30 day )	6.64 ± 0.09	24.9 ± 1.20	3.13 ± 1.84		1.0308± 0.0194	0.163 ± 3.535	11.80 ± 0.47	8.60 ± 0.64
Sterilized fortified milk ( 60 day )	6.63 ± 0.20	25.27 ± 1.65	3.13 ± 1.84		1.0307± 0.0224	0.167 ± 3.464	11.79 ± 0.54	8.59 ± 0.68

* RSD = Relative Standard Deviation ,plus minus values are means ± RSD

* as light condition ( Clear glass bottle )

* as dark condition ( Glass bottle + aluminum foil layer)

Egypt. J. of Appl. Sci., 34 (9) 2019 284

Microbiological analysis:

Microbiological analysis (Coliform bacteria and Total Bacterial Count) table 3 was done for all fortified samples of this study based on therecent work suggests that vitamins may be deteriorate due to microbial activity such that vitamin analyses of aged milk samples will not reflect the actual level of fortification(Newcomer and Murphy, 2001). So, all testing was done on fresh samples. All milk samples are in good quality parameters during experimental period either for the pasteurised or sterilized samples.

3-Effect of heat treatments on vitamin A & D3 retention of milk:

From data of Table (3) we can conclude that the retention of vitamin A & D3 at different heat treatments was valued. 2 heat treatments which are generally used for milk processing were selected to evaluate retention of vitamin A & D3 in milk1) were pasteurization (63°C/30 min) and Sterilization (121°C/15 min at 15 psi) There was non-significant statically (p < 0.05) for losses and degradation of vitamin A during heat treatment by 0.54 % and 0.69% and the level of vitamin A content was 2009±1.06 and 2017±0.78 of pasteurized and sterilized fortified milk, respectively after heat treated whilesignificant differences (p < 0.05) degradation of level vitamin D3 content was 417.4 ±1.80 and 415.2±1.84 IU/L by loss 1.1% and 0.9% at the end of heat treatments of pasteurized and sterilized for fortified milk, respectively the same trend obtained by (Kaushik et al., 2104) .

Table (3) Microbiological analysis of different types of milks fortified withVitamin A and D

Milk Samples	Coliform CFU/ ml	Total Bacterial Count CFU/ ml
Raw Cow Milk	105 ± 1.65	64.100 ± 1.49
Raw milk fortified for Pasteurized (Control 1)	105 ± 1.65	64.117 ± 1.72
Raw milk fortified for Sterilized (Control 2 )	105 ± 1.65	64.000 ± 1.56
Pasteurized treatment
*Pasteurized fortified milk ( as light condition - 0 day)**	Nil	62 ± 5
*Pasteurized fortified milk ( as light condition - 5 day )**	Nil	77 ± 3
*Pasteurized fortified milk ( as light condition - 7 day )**	Nil	120 ± 2
*Pasteurized milk ( as dark condition - 0 day )**	Nil	62 ± 5
*Pasteurized milk ( as dark condition - 5 day )**	Nil	84 ± 4
*Pasteurized milk ( as dark condition - 7 day )**	Nil	135 ± 4
Sterilized treatments
Sterilized fortified milk ( 0 day )	Nil	Nil
Sterilized fortified milk (30 day )	Nil	Nil
Sterilized fortified milk ( 60 day )	Nil	Nil

* RSD = Relative Standard Deviation ,plus minus values are means ± RSD

* as light condition ( Clear glass bottle )

* as dark condition ( Glass bottle + aluminum foil layer )

In general the levels of vitamin A&D3 of each treatment pasteurized and sterilized milk was nearly of the targeted levels of fortification (vitamin A fortified milk samples (2000 IU/L) also, vitamin D3 (400 IU/L), the same trend was mentioned by(Newcomer and Murphy,2001).Also, FDA issued indicated that the acceptable levels of V (A&D) were plus or minus 20% of the label claims. The results indicated that approximately 20% of the milks were under-fortified while 27% of the milks tested were over-fortified for both vitamins A and D. Other studies supported these findings (Brown et al., 1992 and Holicket al., 1992) and also according to FDA recommendation that "fluid dairy products are allowed, not less than 100% and not more than 150% of the required values or label claims to be in compliance with good manufacturing practices.

285Egypt. J. of Appl. Sci., 34 (9) 2019

4 - Effect of storageperiod and package light transmittance on retention of vitamin A&D3 in Pasteurized Milk

The retention of vitamin A & D3 in both samples (pasteurized fortified glass bottle sample) in clear glass transparent bottles as light condition andPasteurized glass bottle protected by aluminium foil layer sample as a dark condition and stored under refrigerated conditions (4 - 7°C) was estimated in 0 day and after 5^th and 7^th day of pasteurization, respectively.

4.1. Vitamin A:

As shown in Table (4), vitamin A content at zero day was 2009±1.06 IU/L in both samples conditions (clear glass transparent bottles ( light) & ( glass bottle protected by aluminium foil layer sample(dark),after seven days storage the vitamin A (content) was significant losses (p < 0.05)1828 ± 1.19 and 1872± 1.64 IU/L by losing 10% & 8%,respectively.Gradually degradation and losses of vitamin A during storage from 0 time and after 5 days and 7days, respectively inboth light and dark conditions , the same trend was observed by (Chotyakulet. al., 2014) that the analysis of vitamin A(retinol &β-carotene) in milk samples showed losses generally increasing during the 14-day storage time. In most cases, short storage periods (up to 5 days) had no statistically significant effect (p > 0.05) whereas storage for 5–14 days led to significant losses (p < 0.05) of this vitamin and the loss after 14 days, was 15% &10 % in retinol &β-carotene, respectively.

On the other hand, the effect of light and dark condition was indicted that the effect of light intensity had a significant (p < 0.05) effect of vitamin A content for all samples exposed to(2000-2500 lux), More degradation of vitamin A level in light condition was noticeable than dark condition by 1920 IU/L & 1972 IU/L in 5days and was 1828 IU/L & 1872 IU/L in after 7 days, respectivelyand the final % loss of light conditions was 10 % and the loss was8% in dark condition at the end of storage 7 days, same results was obtained by (Gaylord et al., 1986), and Saffertet al. (2006) who found that in clear PET bottles a reduction of 22% of the initial content was observed for vitamin comparing of all pigment PET bottles the vitamin retention was significantly higher; the losses were 0–6% for vitamin A depending on the pigment level. Vitamin A may deteriorate gradually under normal storage conditions of milk. When milk is exposed to sunlight or fluorescent light, especially in transparent containers, vitamin A can be rapidly destroyed (Shipeet al, 1984).The destruction of vitamin A is dependent on the intensity and wave length of light, length of exposure (time) and the milk source. The use of amber or brown glass bottles, pigmented plastic containers formulated with specific light barriers and paper cartons has been shown to retard this destruction. Paper, especially with limited print or ink, is not exempt from light degradation, though it is generally more protective than the transparent plastics. Use of gold shields over fluorescent tubes has also been shown to help reduce losses by modifying the wavelength of the light(Newcomer and Murphy, 2001) also the same results was obtain by (Guneser and Karagul 2012; DeMan, 1981; Miller et al., 2007)and they stated that when milk is exposed to light, degradation in vitamin A. (Gaylord et al., 1986; Chapman et al., 1998; Whited et al., 2002).Investigated the loss of vitamin A in whole, 2% fat milk and skimmed milk exposed to fluorescent light with 2,200 lx intensity for 48 h at refrigerated temperatures. The researcher reported the loss of 32.3, 23.6, and 95.8% of vitamin A in whole milk, 2% fat milk, and skimmed milk, respectively, after 30h of exposure.

Egypt. J. of Appl. Sci., 34 (9) 2019 286

Table (4): The effect of heat treatment on vitamin A and D3 retention of different types of milks fortified with Vitamin A and D

Sample Vitamin	Raw milk	Raw milk fortified for Pasteurized	Raw milk fortified for Sterilized	Pasteurized fortified milk	Sterilized fortified milk
Vitamins content in milk ( IU/L ± RSD )
A	1363 ± 081	2020 ± 0.65	2031 ± 1.33	2009 ± 1.06	2017 ± 0.78
D3	20 ± 2.26	422 ± 1.94	419 ± 1.09	417.4 ± 1.80	415.2 ± 1.84

* RSD = Relative Standard Deviation ,plus minus values are means ± RSD

* To convert vitamin A value to treatment unit ( IU ) per liter , multiply by 1818 ( 1mg/L = 1818 IU/L )

* To convert vitamin D3 value to treatment unit ( IU ) per liter , multiply by 40 ( 1mg/L = 40 IU/L )

4.2. Vitamin D3:

Same trend which obtain in case of vitamin A data in table (5) Vitamin D3 content is more stable than vitamin A content during storage period 7 days and all samples recorded non-significant (p > 0.05)losses of vitamin D3 content from 417.4 ±1.80 IU/L in 0 day to 410.6±0.63 and 414.5±0.45 IU/L after 7 days in both package samples conditions light&dark, respectively by 1.6 % and 0.6% as a final loss%. These results were the same obtain byHanson and Metzge(2010) which clear that level of vitamin D at 0day of refrigerated storage was close to the targeted levels of fortification who observed (100 and 250 IU/serving). Additionally, there wasno significant (P >0.05) change in vitamin D duringthe 21-d storage period (Kaushik et al., 2104).

No significant difference observed for vitamin D2 content between 0, 3^rd, 5^th and after 7^th day of storage in pasteurised fortified milk, packed in glass bottles and stored under refrigerated conditions (4-7°C).Liu (2003) found that the effect of storage of vitamin D fortified milk in glass and plastic bottles and reported higher values of vitamin D in milk packed in glass bottles when compared to plastic bottles.

287 Egypt. J. of Appl. Sci., 34 (9) 2019

Table (5)Effect of storage period on vitamins A and D3 retention of pasteurized fortified milk with VA&D3in light and dark conditions

Vitamin		Storage period /day	0 Day	5 Days	7 Days	final % loss
		Sample
Vitamins content in milk ( IU/L ± RSD )
A	Clear glass bottle ( as light condition )		2009 ± 1.06	1920 ± 1.38	1828 ± 1.19	10%
	Glass bottle + aluminum foil layer (as dark condition )		2009 ± 1.06	1972 ± 0.70	1872.4 ± 1.64	8%
D3	Clear glass bottle ( as light condition )		2009 ± 1.06	1972 ± 0.70	1872.4 ± 1.64	8%
	Glass bottle + aluminum foil layer (as dark condition )		417.4 ± 1.80	415.7 ±1.08	414.5 ± 0.45	0.6%

* RSD = Relative Standard Deviation ,plus minus values are means ± RSD

* To convert vitamin A value to treatment unit ( IU ) per liter , multiply by 1818 ( 1mg/L = 1818 IU/L )

* To convert vitamin D3 value to treatment unit ( IU ) per liter , multiply by 40 ( 1mg/L = 40 IU/L )

Regarding to the effect of light exposed in comparing with dark condition was indicted that the effect of light exposed had a significant (p < 0.05) effect of vitamin D3 content for all samples exposed to(2000-2500 lux) , it was found more significant (p < 0.05) degradation in vitamin D3 level in light condition than dark condition by 412.3 IU/L & 1415.7 IU/L through 5days and was 410.6 IU/L & 414.5 IU/L in after 7 days, respectively and the final % loss in light condition was 1.6 % loss more than the dark condition 0.6 % at the end of storage 7 days ,(Renken and Warthesen 1993) reported a slight loss of vitamin D in fortified milk upon exposure to light.Cremin andPower (1985) and Kutsky (1981) reported that vitamin D was unstable to light conditions.

5. Effect of storage period and package light transmition on retention of vitamin A&D3 of sterilizated Milk:

The retention of vitamin A & D3 of both samples sterilized fortified sample in light condition and pasteurized sample in a dark condition and stored under 4°C was determined in 0 day, 30days and 60 days of sterilization, respectively.

5.1. Vitamin A:

Data of table (6) clear the effect of storage period and package light transmition on retention and loss of vitamin A in sterilized milk and could indicted significant decreased (p < 0.05) to 12 % and 5.9 % after 60 days in milk samples stored in light& dark conditions, respectively. Same trend was observed by (Chotyakulet. al., 2014)significant (p<0.05) losses vitamin A were found after 11 days and only 8% reduction after 31 daysin UHT milk.

Egypt. J. of Appl. Sci., 34 (9) 2019 288

Table (6) Effect of storage period on vitamins A and D3 retention of sterilized fortified milk with vitamins A&D3

Vitamin	Storage period /day	0 Day	30 Days	60 Days	final % loss
Vitamin	Sample	0 Day	30 Days	60 Days	final % loss
Vitamins content in milk ( IU/L ± RSD )
A	Clear glass bottle( as light condition )	2017 ± 0.78	1818 ±1.27	1774.96±1.31	12%
A	Glass bottle + aluminium foil layer(as dark condition)	2017 ± 0.78	1917 ±0.43	1898.8 ± 0.64	5.9%
D3	Clear glass bottle( as light condition )	415.2 ±1.56	405 ± 1.13	390.2 ± 1.34	6.02%
D3	Glass bottle + aluminium foil layer(as dark condition)	415.2 ±1.56	413 ± 0.87	409.02 ± 1.93	1.49%

* RSD = Relative Standard Deviation ,plus minus values are means ± RSD

* To convert vitamin A value to treatment unit ( IU ) per litter , multiply by 1818 ( 1mg/L = 1818 IU/L )

* To convert vitamin D3 value to treatment unit ( IU ) per litter , multiply by 40 ( 1mg/L = 40 IU/L )

On the other hand, the study of light exposed and package light transmaition on retention of vitamin A significant differences (p < 0.05) effect in vitamin A content for all samples exposed to (2000-2500 lux). The most pronounced effect of light was obtained for the completely transparent clear glass bottle samples comparing with that of dark condition and was indicted that the effect of light exposed had more significant (p < 0.05) degradation in vitamin A level in light conditions than dark conditions by 1818 IU/L & 1917 IU/L in 30days and was 1774IU/L & 1898.8 IU/L in after 60 days, respectively and the light exposed of fortified sterilization milk in clear glass led to loss 9.9 % after 30 days and reach to 12 % final after 60 days although be 4.96 % in 30 days and 5.9 % after 60 days in dark condition, respectively. From previous trend it could be concluded that oxygen in the absence of light was not a major factor in vitamin A degradation same trend was found by Saffertet al (2008)They reported a 66, 79 and 88% loss in vitamin A of milk stored in clear-transparent polyethylene terephthalate bottles after 12 wk of storage at 700, 1,700, and 3,000 lx at 2°C, respectively. Also, Saffertet al (2009) stated that in clear PET bottles, a reduction of 93%of the initial content was observed of vitamin A and 66% of vitamin D3, In all pigmented PET bottles, the vitamin retention was only slightly higher; the losses ranged between 70 and 90% depending on the pigmentation level. In the dark-stored ‘control’ sample, a 16% loss could be observed for vitamin A, while the level of vitamin D3 remained almost stable.

289 Egypt. J. of Appl. Sci., 34 (9) 2019

5.2. Vitamin D3:

Data in table(6) clear the effect of storage period and package light transmition on retention and loss of vitamin D3 in sterilized milk and could indicted significant decreased (p < 0.05) to 6.02 % and 1.49 % after 60 days in milk samples of clear glass transparent bottles (light) & glass bottle protected by aluminium foil layer sample(dark), respectively, when comparing the light-exposed samples clear glass transparent bottles (light) to the dark-stored & glass bottle protected by aluminium foil layer sample(dark), an impact of light on vitamin D3 stability is given. The loss in vitamin D3 level is higher in clear glass samples by 2.41 % after 30 day s and reach 6.02% after 60 days while glass bottle protected by aluminium foil layer sample(dark) was 0.48 % after 30 days and increased to be 1.49 % after 60 days of storage at room temperature and this observation did not show a clear dependency on the package light transmition as it was observed for vitamin A However, a difference could be observed between clear and in the dark-stored samples, and the initial vitaminD3 content was almost completely retained. Same trend result was found by Saffertet al (2008), Saffertet al (2009) when comparing light-exposed samples with the dark-stored control samples, the impact of light on the vitamin D3 stability seemed to be given. In pigmented PET bottles, light intensity was found to be more decisive than package light transmition for the degradation of vitamin D3 under light-exposed storage. For completely transparent PET bottles of the variant 1, such a dependency of vitamin D3 loss on light intensity could not be observed, and also they found vitamin D3 retention of 35% and PET bottles with the highest pigmentation levels where the vitamin D3 retention was lying at around 70%. In the dark-stored ‘control’ sample, the initial vitamin D3 content was almost completely retained

CONCLUSIONS

Egypt. J. of Appl. Sci., 34 (9) 2019 290

In case of fortified sterilized milk protection from light exposed necessary to obtain similar vitamins retentions of at least 94.1%and 98.51in dark condition forboth vitamin A & D3,respectively.

REFERENCES:

AOAC, (2012).Official methods of analysis. Association of Official Analytical Chemists 19^th ed., Published by AOAC International, Gaithersburg, Maryland, USA.

APHA, (2004).Standards methods for the examination of dairy products.17th ed., H. Michael Wehr and Joseph F. Frank, editors.American Public Health Association, Washington, DC, USA.

British standard Institutes (BSI) (2010)Determination of titratable acidity (Reference method) ISO, 6091.

Brown, D.P. ; D.K. Bandler ;K.T. Scolfield and S.C. Murphy(1992). Vitamin A levels in fluid milk from New York State dairy plants. J.Dairy Sci., 75 (suppl. 1):128.

Byrdwell, W.C. ;J.Exler ; S.E.Gebhardt ; J.M. Harnly ;J.M. Holden and R.L.Horst (2011). Liquid chromatography with ultraviolet and dual parallel mass spectrometric detection for analysis of vitamin D in retail fortified orange juice. Journal of Food Composition and Analysis, 24, 299e306.

Ceglia, L. (2009).Vitamin D and its role in skeletal muscle. Current Opinion in Clinical Nutrition and Metabolic Care, 12: 628e633, 9685.

Chapman, K.W. ;L.C. Rosenberry ; D.K. Bandler and K. J. Boor (1998). Light-oxidized flavor development and vitamin A degradation in chocolate milk. J. Food Sci., 63:930–934.

Charlton, S.J.andW.N. Ewing (2007).The vitamin directory. England: Context Products Ltd.

Chotyakul, N. ; C. Perez‑Lamela ; M. Pateiro‑Moure ; J. A. Saraiva and J.A. Torres (2104).Simultaneous HPLC–DAD quantification of vitamins A and E content in raw, pasteurized, and UHT cow's milk and their changes during storage, Eur .Food Res Techno., 238:535–547

Cremin, F.M.andP. Power (1985).Vitamins in bovine and human milks.In P. F. Fox (Ed.), Developments in dairy chemistryd3 (p. 346). Essex, England: Elsevier Applied Science Publishers.

database.http://www.nal.usda.gov/fnic/foodcomp/search/Accessed 16.05.11.

DeMan, J. M. (1981). Light-induced destruction of vitamin A in milk. J.Dairy Sci., 64:2031–2032.

Food and Drug Administration FDA (2008) Food Labeling Guide: Appendix B: Additional requirements for nutrient content claims.http://www.cfsan.fda.gov/∼dms/2lg-xb.html Accessed Feb., 2008.

291 Egypt. J. of Appl. Sci., 34 (9) 2019

Gaylord, A. M. ; J. J. Warthesen and D. E. Smith (1986). Influence of milk fat, milk solids, and light intensity on the light stability of vitaminA and riboflavin in lowfat milk. J. Dairy Sci., 69:2779–2784.

Gomez, M. and Jose (2006). The role of insulin-like growth factor I components in the regulation of vitamin D. Current Pharmaceutical Biotechnology, 7(2): 125-132.

Guneser O. and Y. Karagul(2012),Effect of ultraviolet light on water- and fat-soluble vitamins in cow and goat milk, Journal of Dairy Science., 95 (11):6230–6241

Hanson,A., and L.Metzge (2010) Evaluation of increased vitamin D fortification in high-temperature, short-time–processed 2% milk, UHT-processed 2% fat chocolate milk, and low-fat strawberry yogurt.J. Dairy Sci., 93:801–807

Holden, J. (2009).USDA national nutrient in processed dairy products.International Dairy Journal, 17, 753e759.

Holick, M.F. Q. Shao, W.W. Liu and T.C. Chen. 1992. The vitamin content of fortified milk and infant formula. NE J. of Med. 326:1178-1181.

Indian Standard IS: 8479 (Part-1) (1977) method for determination of Phosphatase activity in milk and milk

Kaushik R., Sachdeva, B., Arora, B. (2014) . Vitamin D2 stability in milk during processing, packaging and storageFood Science and Technology 56,421-426

Kazmi, S. A., Vieth, R., & Rousseau, D. (2007). Vitamin D3 fortification and quantification

Kutsky, R. J. (1981).Vitamin D.In Handbook of vitamins, minerals and hormones (pp.191-198). New York, NY: Van Nostrand Reinhold Co.

Leskova, E. ;J.Kubikova ;E.Kovacikova ;M. Kosicka ;J.Porubska and M.Kosicka (2006). Vitamin losses: retention during heat treatment and continual changes expressed by mathematical models. Journal of Food Composition and Analysis, 19: 252e276.

Liu, Y. (2003).Betalactoglobulincomplexed vitamin A and vitamin D in skim milk: Shelf life and bioavailability (Ph.D. thesis). Raleigh, N.C.: North Carolina State University.

Lopes, C. ; S. Casal ; B. Oliveira and H. Barros (2003) .Retinol, betacarotene, and alpha-tocopherol in heart disease.Pages 118–136 in Nutrition and Heart Disease: Causation and Prevention. R. R.Watson and V. R. Preedy, ed. CRC Press, Boca Raton, FL.

Mawer, E. B. ;G.Jones ;M. Davies ;P.E. Still ;V.Byford andN.J. Schroeder (1998).Unique 24-hydroxylated metabolites represent a significant pathway of metabolism of vitamin D2 in humans: 24-hydroxyvitamin D2 and 1, 24-dihydroxyvitamin D2 detectable in human serum. Journal of Clinical Endocrinology & Metabolism, 83(6); 2156e2166.

Egypt. J. of Appl. Sci., 34 (9) 2019 292

Miller, D. G. ; J. K. Jarvis and L. D. McBean(2007).Handbook of Dairy Food and Nutrition.3rd ed. CRC Press, Taylor &FrancisGroup, Boca Raton, FL.

Morrissey, P. A. and T. R. Hill (2009).Fat-soluble vitamins and vitamin C in milk and milk products. Pages 527–572 in Advanced dairy chemistry: Lactose, water, salts and minor constituents.Vol.3. 3rd ed. P. F. Fox and P. H. L. McSweeney, ed. Springer, New York, NY.

Newcomer Chris and Steven C. Murphy (2001). Guide line of fortification vitamin A&D in fluid milk

Renken, S. A., &Warthesen, J. J. (1993).Vitamin D stability in milk. Journal of Food Science, 58: 552e556.

Riaz, M.N. ;M.AsifandR. Ali (2009).Stability of vitamins during extrusion. Critical Reviews in Food Science and Nutrition, 49: 361-368.

Saffert, A. ; G. Pieper and J. Jetten(2006).Effect of package light transmittance on the vitamin content of pasteurized whole milk.Packag. Technol. Sci., 19:211–218.

Saffert, A. ; G. Pieper and J. Jetten(2008).Effect of package light transmittance on vitamin content of milk. Part 2: UHT whole milk. Packag. Technol. Sci., 21:47–55.

Saffert, A. ; G. Pieper and J. Jetten(2009).Effect of Package Light Transmittance on the Vitamin Content of Milk, Part 3: Fortified UHT Low-Fat Milk.Packag. Technol. Sci.; 22: 31–37

Shipe, W.F. ; G. F. Senyk and D. K. Bandler(1984). Relative protection of flavor and nutrients of milk from light induced changes by pigmented containers and light shields. J. Dairy Sci., Suppl., 1 (67):59.

Tanner, J.T. ;J. Smith ;P. Defibaugh ;G.Angyal ;M. Villalobus and M.P. Bueno(1988). Survey of vitamin content of fortified milk. Journal of the Association of Official Analytical Chemists, 71(3): 607- 610.

Upreti, P. ; V.V. Mistry andJ.J. Warthesen (2002).Estimation and fortification of vitamin D3 in pasteurized process cheese.Journal of Dairy Science, 85: 3173- 3181.

Wagner, D. ; G.Sidhom ;S.J.Whiting ;D. Rousseau andR. Veith (2008). The bioavailability of vitamin D from fortified cheeses and supplements is equivalent in adults. Journal of Nutrition, 138(7): 1365-1371.

293 Egypt. J. of Appl. Sci., 34 (9) 2019

Whited, L. J. ; B. H. Hammond ; K. W. Chapman and K. J. Boor(2002). Vitamin A degradation and light-oxidized flavor defects in milk. J. Dairy Sci., 85:351–354.

WHO/FAO.(2006). Guidelines on food fortification with micronutrients.World Health Organization and Food and Agriculture Organization of the United Nations.

ثبات فیتامین أ و د3 فی اللبن البقری المدعم أثناء التصنیع

و التعبئة و التخزین

ولید عبدالمتعال کافوری

معهد بحوث تکنولوحیا الاغذیة – مرکز البحوث الزراعیة – جیزة - مصر

من هذه الدراسة یمکن أن نستنتج تأثیر فترة التخزین ونفاذیة العبوة للضوء على الاحتفاظ بمحتوى فیتامین A و D3 المدعم فی الحلیب المبستر والمعقم ، حیث أشارت النتائج التی تم الحصول علیها إلى أن العبوات الشفافة غیر کافیة لحمایة فیتامینات الحلیب A و D3 فی ظروف التخزین التجاریة بعد البسترة أو التعقیم. کما أدى إستخدام العبوات الشفافة مع الحلیب المبسترإلی الإحتفاظ بنسبة 90٪ من فیتامین A والإحتفاظ بنسبة 98.4٪ من فیتامین D3 خلال التخزین فی الضوء لمدة 7 أیام، وهذا یعنی أن فیتامین D3 أکثر ثباتًا من فیتامین A فی الضوء تحت ظروف التخزین التجاریة، وکذلک تعبئة وتخزین الحلیب فی عبوات کرتونیة وغیر شفافة کان أفضل ویؤدی إلى المزید من الإحتفاظ بفیتامین A و D3 فی حالة حلیب المعقم طویل حیث بلغ الإحتفاظ فیتامین A بنسبة 88٪ فی الضوء وکانت 94.1٪ فی الظروف المظلمة بینما بلغت نسبة الإحتفاظ بفیتامینات 93.98%D3 تحت الضوء و 98.51 ٪ فی الظلام خلال فترة التخزین 60 یوما فی ظل ظروف التخزین التجاریة . ومع ذلک ، لا بد من اعتبار أن التغییرات الحسیة التی یسببها الضوء فی الحلیب المبستر وحلیب التعقیم فی ظل ظروف التخزین فی الاسواق تجاریا لا یمکن استبعادها إلا فی عبوات محکمة الغلق

References

REFERENCES:

AOAC, (2012).Official methods of analysis. Association of Official Analytical Chemists 19^th ed., Published by AOAC International, Gaithersburg, Maryland, USA.

APHA, (2004).Standards methods for the examination of dairy products.17th ed., H. Michael Wehr and Joseph F. Frank, editors.American Public Health Association, Washington, DC, USA.

British standard Institutes (BSI) (2010)Determination of titratable acidity (Reference method) ISO, 6091.

Brown, D.P. ; D.K. Bandler ;K.T. Scolfield and S.C. Murphy(1992). Vitamin A levels in fluid milk from New York State dairy plants. J.Dairy Sci., 75 (suppl. 1):128.

Ceglia, L. (2009).Vitamin D and its role in skeletal muscle. Current Opinion in Clinical Nutrition and Metabolic Care, 12: 628e633, 9685.

Chapman, K.W. ;L.C. Rosenberry ; D.K. Bandler and K. J. Boor (1998). Light-oxidized flavor development and vitamin A degradation in chocolate milk. J. Food Sci., 63:930–934.

Charlton, S.J.andW.N. Ewing (2007).The vitamin directory. England: Context Products Ltd.

Chotyakul, N. ; C. Perez‑Lamela ; M. Pateiro‑Moure ; J. A. Saraiva and J.A. Torres (2104).Simultaneous HPLC–DAD quantification of vitamins A and E content in raw, pasteurized, and UHT cow''s milk and their changes during storage, Eur .Food Res Techno., 238:535–547