Evaluating the effect of vitamin D₃ fortification on physicochemical and sensory properties of yogurt

Abstract

More than one billion people around the globe are suffering from severe to moderate vitamin D deficiency. In Pakistan, 53.5% people suffer from vitamin D deficiency. The purpose of this study is to develop vitamin D fortified yogurt with two varying concentrations of vitamin D₃, aiming to observe how these concentrations impact the physicochemical and sensory characteristics of the fortified products. A total of 750 mL of pasteurized milk was divided into three equal batches. For T₁ and T₂, 50 µL and 66.67 µL of emulsified vitamin D₃ were added at 50 °C, respectively. Yogurt samples were prepared in triplicates and underwent storage tests, including proximate analysis, pH, viscosity, and vitamin D₃ stability at 4 ± 1 °C. During the first week of storage, the viscosity of yogurt increased but later it started decreasing because of syneresis. T₁ (8.1) and T₂ (8.2) obtained a better overall sensory score using a 9-point hedonic scale as compared to T₁ (7.9) During the 28-day storage period, the vitamin D₃ content significantly decreased (P < 0.05) in T₁ and T₂. By the end of the 28-day period, in T₁ and T₂, the vitamin D₃ content decreased from 15.0 and 20.32 to 13.78 and 17.67 respectively. The results of the current study demonstrate that the fortification of yogurt with vitamin D₃had no detrimental effect on the physicochemical as well as the organoleptic properties of yogurt. Although the vitamin D₃ content decreased during storage, it could be preserved if fortified yogurt is stored in opaque containers.

Keywords:

• Fortification
• vitamin D
• pasteurized milk
• yogurt
• storage
• viscosity and syneresis

Reviewing Editor:

• M. Luisa Escudero-Gilete, Universidad de Sevilla, Spain

Subjects:

• Food Additives & Ingredients
• Food Chemistry
• Food Engineering

Introduction

Vitamin D deficiency has become one of the major health concerns around the globe. There are more than one billion people suffering from moderate or severe vitamin D deficiency worldwide (Holick, 2017). Sunlight is one of the major source of vitamin D, as ultraviolet (UV) rays especially UVB rays coming from sunlight, helps in the synthesis of vitamin D from its precursor, 7-dehydrocholesterol (provitamin D₃), into previtamin D₃ (Björn et al., 2023; Neville et al., 2021). Although Pakistan is blessed with ample sunshine, more than half of the population, i.e., 53.5% of Pakistanis, suffer from vitamin D deficiency (Riaz et al., 2016). Several underlying personal, social, and physiological factors, such as the application of sunscreens, type of clothing and time of the day play a significant role in hindering the conversion of 7-dehydroxycholesterol to previtamin D3, ultimately leading to vitamin D deficiency (Maeda et al., 2013). Vitamin D deficiency causes numerous skeletal and non-skeletal diseases in people. Vitamin D deficiency reduces intestinal calcium absorption, resulting in decreased bone mineral density (Lips, 2006). It is the main culprit behind rickets in children (Chibuzor et al., 2020), osteoporosis, and an increased risk of bone and hip fractures in postmenopausal women (Kanis et al., 2019). Furthermore, vitamin D deficiency is also associated with various non-skeletal diseases such as inflammatory bowel disease (IBD), gestational diabetes mellitus (GDM) in women (Kim et al., 2020), as well as diabetes mellitus, cancer, and cardiovascular disease (Autier et al., 2014). According to National Academics of Sciences, Engineering and Medicine (NASEM) serum hydroxyviatmin D 25(OH)D of more than 50 nmol/L (20 ng/mL) is considered as optimal for proper bone health. But according the Endocrine Society serum 25(OH)D levels of more than 75 nmol/L (30 ng/mL) is considered as optimal (Holick et al., 2011). To achieve these optimal serum vitamin D levels, in 2011, the Institute of Medicine (IOM) raised up the recommended dietary allowance (RDA) for vitamin D for adults, from 400 IU to 600 IU and individuals who are 70 years or older are advised to consume 800 IU vitamin D (Institute of Medicine, 2011). There are very few dietary sources which are naturally rich in vitamin D e.g., egg yolk which typically contains about 1.3-2.9 µg of vitamin D per 100 grams, fatty fish is another good source, with a vitamin D content ranging from 5–25 µg per 100 grams and certain wild varieties of mushrooms can provide significant amounts of vitamin D, with levels ranging from 21.1–58.7 µg per 100 grams (Benedik, 2021). Synthesis of vitamin D in the skin (a significant) source of vitamin D has its own limitations because of social and cultural factors (Hussain et al., 2014). Though, fatty fish is indeed an excellent source of vitamin D but fish consumption (1.9 Kg per capita) in Pakistan is generally low (Qasim et al., 2020). Hence, there is a desperate need for a variety of vitamin D-fortified foods to increase the overall dietary intake of vitamin D in people. While countries like the USA, UK, and Canada offer various options for vitamin D-fortified foods and beverages (Wilson et al., 2017). It would be best to have different vitamin D fortified foods options to target different population. Yogurt, a fermented dairy product, has been consumed for many years and is widely accepted by masses which makes it an outstanding choice for vitamin D₃ fortification as it is rich in calcium (Gasparri et al., 2019). Furthermore, Lactobacillus plays a crucial role in the breakdown of lactose during milk fermentation. This process leads to the production of fermented dairy products that are far better tolerated by individuals with lactose intolerance (Savaiano, 2014).

Food fortification is considered a more sustainable and consumer-friendly approach compared to vitamin D supplementation. Pakistan has successfully mitigated the rate of goiter by adopting the approach of iodine-fortified salt (Khattak et al., 2017). Therefore, it is necessary for public health policy makers to make policies for vitamin D fortification in Pakistan. Considering the dairy consumption habits of the people fortifying yogurt with vitamin D would be an excellent option, as it is suitable for individuals who are partially or completely lactose intolerant thus increasing the options of vitamin D fortified products in market. Vitamin D fortified products have proven to be an excellent way to increase vitamin D intake in masses. In a clinical trial the group of women which were given vitamin D fortified cheese had better bone mineral density as compared to the group of women which were given unfortified cheese (Bonjour et al., 2009). Another controlled trial on postmenopausal women demonstrated that there was a dose dependent improvement in serum 25(OH)D levels. Different studies have investigated the fortification of yogurt with different doses of vitamin D3, ranging from 400 IU to as high as 2000 IU (Gasparri et al., 2019). However, there is a scarcity of research specifically focused on yogurt fortified with 800 IU of vitamin D3. Given that the IOM recommends 800 IU of vitamin D for elderly people, it is crucial to design studies which specifically address the nutritional requirements of the geriatric population. Such research outcomes will not only aid a broad understanding of the health needs of geriatrics but also contribute to the development of health-friendly products tailored to meet their explicit nutritional needs. The purpose of this study is to create vitamin D-fortified products with two varying concentrations of vitamin D3, aiming to observe how these concentrations impact the physicochemical and sensory characteristics of the fortified products.

Materials and methods

Whole milk with a fat content of 3.9 g/100 mL was purchased from a local dairy of Rawalakot. Emulsified vitamin D₃, reagents, and chemicals were obtained from Merck & Co (Lahore, Pakistan). The yogurt culture, consisting of Lactobacillus delbrueckii (subsp. Bulgaricus) and Streptococcus salivarius (subsp. Thermophilus), was purchased from The American Type Culture Collection (ATCC, Lahore). All the chemicals and reagents used were of chemical grade, except for the emulsified vitamin D₃, which was food grade.

720 mL milk was homogenized according to the methodology developed by Dhankhar (2014) and 0.07% (0.504 g) gelatin was added to it. It was pasteurized at 85 °C for 15 minutes to ensure the elimination of all pathogenic bacteria. After pasteurization, milk was divided equally into three portions i.e., control (T₀), Treatment 1 (T₁), and Treatment 2 (T₂) and it was allowed to cool down to 50 °C for fortification. At 50 °C, 50 µL and 66.7 µL (20 µg) emulsified vitamin D₃ was vigorously mixed into T₁ and T₂, respectively. Milk was further cooled to 43 °C in a water bath for inoculation with the starter culture while being stirred continuously. Each batch of milk (T₀, T₁ and T₂) was inoculated with a 2% starter culture containing an equal proportion of Lactobacillus bulgaricus and Streptococcus thermophillus, and it was incubated at 43 °C for 6 hours until its pH reduced to 4.6 and a firm gel was formed. Once the yogurt was set, it was cooled and stored at 4 °C for 28 days. All the yogurt samples were made in triplicates, stored at 4 ± 1 °C were subjected to qualitative and quantitative analysis.

Proximate analysis (ash, proteins, fats and moisture) of yogurt samples was analyzed using the method developed by AOAC (2005). Protein content was determined using the Kjeldahl method with a conversion factor of 6.25. Moisture content was quantified using a Precisa moisture analyzer. Ash and fats were measured using a muffle furnace and the solvent extraction method, respectively. Yogurt samples were subjected to pH analysis every 7^th day for four weeks using a benchtop digital pH meter (PH-2005), following the method designed by AOAC (2016). The pH meter was calibrated daily with buffer solutions having pH 4.00 and pH 7.00. The viscosity of yogurt was analyzed using an IMADA texture analyzer. A disk probe was employed to measure the viscosity, with a compression speed of 1.0 m/s and a compression force of 1.0 N.

Sensory evaluation

Sensory evaluation of each treatment of yogurt was conducted using a 9-point hedonic scale (Khalid et al., 2022). 30 semi-trained members were asked to evaluate the yogurt samples, rating their perception on a scale from 1 to 9, with 9 indicating the highest level of liking. Each participant received a 15 g yogurt sample in a white bowl, along with a spoon, and water was provided as a palate cleanser. The yogurt was evaluated for taste, texture, aroma, appearance, and overall acceptance.

Vitamin D₃ quantification

Vitamin D₃ content in T₁ and T₂ was analyzed every week for 28 days. The quantification of Vitamin D₃ in yogurt samples was accomplished using reverse-phase HPLC with an ultraviolet (UV) detector. The mobile phase utilized for analyzing vitamin D3 consisted of a mixture of methanol and acetonitrile (80:20), following a modified methodology developed by Franke et al. (2013) Kazmi et al. (2007). A 20 g yogurt sample was saponified in an alcoholic solution of potassium hydroxide for 24 hours. To prevent oxidation, ascorbic acid (vitamin C) was added to the solution. The saponified yogurt sample, along with 15 mL of 80% potassium hydroxide (KOH) solution, was placed in a dark bottle. Then, 50 mL of absolute ethanol was added to the solution. Additionally, 3 g of ascorbic acid was mixed into the solution to protect against the oxidation of vitamin D in the sample. This solution was kept at room temperature in a dark location for 24 hours. The saponified samples were then extracted with 3 mL of hexane to extract the vitamin D. The solvent was evaporated under nitrogen. After the solvent evaporation, the residues were dissolved in 1 mL of methanol. This methanol solution was filtered through a filter paper with a pore size of 0.45 µm and injected into the HPLC system for analysis.

The chromatographic analysis was done with the help of HPLC equipped with an UV-detector. The separation of analytes was done on C-18 column. The mobile phases used for analysis consist of methanol and acetonitrile with the ratio of 80:20 (v/v). Injection volume was 20 µL and flow rate of mobile phase was 1.2 mL/min. Results were recorded and saved in the system (Figure 1).

Figure 1. Chromatogram of Vitamin D₃ on HPLC.

Chromatogram of vitamin D3 is observed at a retention time of 6.9 minutes when using a mobile phase composed of 80% methanol and 20% acetonitrile. The injection volume for the sample is 20µL, and the flow rate of the mobile phase is set at 1.2 mL/min.

Statistical analysis

Data were analyzed using IBM SPSS version 26. Descriptive statistics including mean and standard deviation was used to summarize the results of product analysis and sensory evaluation. One way ANOVA and Duncan’s test with a confidence interval 95% was applied on proximate analysis, viscosity, pH and vitamin D₃ stability in yogurt samples during storage period.

Results and discussion

Ash, protein, and moisture content of T₁ and T2 did not change significantly compared to the control (Table 1). However, there was no significant change in proteins, fats, ash and moisture content in the control and vitamin D₃ fortified yogurt samples of T₁ and T₂. Moisture content in all yogurt samples and fats in the control yogurt were in harmony with results reported by Saeed et al. (2021). According the Codex Alimentarius standards established in 2003, fermented milk products, including yogurt, are expected to contain less than 2.7 grams of fats. All the milk samples under investigation in this study adhered precisely to the specified standard. Ash represents minerals in yogurt samples. The emulsified vitamin D₃ which was used for fortification lacked both minerals and proteins. This could be the reason behind its negligible impact on the protein and mineral content of T1 and T2 in comparison to T0. Ash in all yogurt samples was similar to the results reported by Amadarshanie et al. (2022), and total proteins in control, product 1, and product 2 are similar to the results reported by Chandan (2017).

Table 1. Ash, protein, fat and moisture content in control, treatment 1 and treatment 2.

Proximate Analysis %	Control	Treatment 1	Treatment 2
	Non-fortified	600 I.U. vitamin D3	800 I.U. vitamin D3
Ash	0.81 ± 0.003^NS	0.81 ± 0.009^NS	0.82 ± 0.09^NS
Fat	3.27 ± 0.006^NS	3.32 ± 0.043 ^NS	3.35 ± 0.057 ^NS
Protein	3.9 ± 0.068^NS	3.5 ± 0.323^NS	3.7 ± 0.176^NS
Moisture	82.7 ± 0.003^NS	82.1 ± 0.215^NS	83.4 ± 0.094^NS

Proximate analysis (mean ± SEM) of control, T₁ and T₂. NS" stands for "no significant difference" between the means for a specific parameter among the samples.

The pH (Figure 2) of all yogurt samples decreased over time due to microbial growth, as bacteria consumed lactose, producing lactic acid through fermentation, leading to increased acidity and curdling. The fermentation process is the reason behind decreasing pH of yogurt over time. Similar findings were reported by Medina et al. (2023) and Peng at al. (2009).

Figure 2. pH of yogurt samples during storage.

pH of yogurt samples during storage time of 28 days.

The viscosity of control, T₁ and T₂ increased during first week of storage period at 4 °C, as depicted in Figure 3. This increase in viscosity during early days of storage can be attributed to the addition of the stabilizer, gelatin similar to the results reported by Arioui et al. (2018) as it could cause protein-protein rearrangement which results in increase viscosity over time. It also may be a result of the starter culture, Streptococcus thermophilus. Streptococcus thermophilus is known to produce exopolysaccharides during the process of lactic acid fermentation. These exopolysaccharides have the ability to bind with the casein present in milk, thereby contributing to the viscosity of the yogurt (Guzel-Seydim et al., 2005). The production of exopolysaccharide from S. theromphilus and protein restructuring, facilitated by gelatin, effectively mitigates syneresis during yogurt production, resulting in an enhanced yogurt viscosity. After 7^th day viscosity of yogurt samples i.e. control, T₁ and T₂ was observed to be declining which could possibly because of syneresis as the water holding capacity of yogurt declined over time (Guénard-Lampron et al., 2020).

Figure 3. Viscosity of yogurt samples during storage.

Viscosity of yogurt samples over storage time period (28 days) at 4 °C.

It was evaluated from sensory analysis (Figure 4) that vitamin D fortification had no detrimental effect on sensory attributes i.e. taste, texture, aroma, appearance and overall acceptance. Possible reason could be that the emulsified vitamin D, when added in an optimal amount, does not surpass the sensory threshold and therefore does not disrupt the sensory characteristics of the yogurt. These findings were similar to the findings of the findings of Kiani et al. (2021) and Aghdasian et al. (2022).

Figure 4. Sensory analysis of yogurt samples.

The effect of vitamin D₃ fortification on sensory attributes of yogurt i.e. texture, taste, aroma, appearance and overall acceptance.

The vitamin D3 content was analyzed weekly, and the results clearly showed a significant decline (P < 0.05) in yogurt samples during storage, as illustrated in Figure 5. The decrease in vitamin D levels in fortified dairy products during storage has also been reported by Loewen et al. (2018). The stability of vitamin D in fortified products is highly influenced by factors such as packaging, air, light, and temperature (Zareie et al., 2021). These findings align with the results reported by Jafari et al. (2016), which indicate that vitamin D losses are greater in transparent packaging compared to opaque packaging, as vitamin D is susceptible to oxidation by light.

Figure 5. Vitamin D₃ stability in yogurt samples.

Stability of Vitamin D₃ in yogurt fortified yogurt samples.

Conclusion

Fortifying yogurt with 15 µg and 20 µg of vitamin D3 did not significantly alter the protein, ash, moisture, or fat content of the yogurt. However, the pH of the yogurt samples decreased over time, resulting in increased acidity. Notably, fortifying with vitamin D3 did not negatively impact the sensory aspects of the yogurt. T₁ showed the most favorable scores compared to the control and T₂. It’s worth mentioning that the stability of vitamin D3 in fortified yogurt declined over the storage period. This degradation could be mitigated by storing the yogurt in opaque containers. This study holds significance for policymakers, public health nutritionists, and food technologists, providing insights to formulate vitamin D fortification recommendations for the government. Importantly, it can contribute to elevating overall serum 25(OH)D levels in the general population. Fortifying yogurt with 20 µg of vitamin D₃ can significantly increase serum 25(OH)D levels, particularly benefiting the elderly population, especially those who are 71 or above. Public health experts and the dairy industry should collaborate to establish essential food regulations for the fortification of vitamin D. This collaborative effort is essential for achieving and maintaining optimal serum vitamin D levels, ultimately reducing the prevalence of skeletal and non-skeletal diseases linked to vitamin D deficiency.

Authors contribution

S.S made significant contributions to conducting the research and played a primary role in preparing this article. Z.K contributed in write up and provided all the facilities to carry out research under her supervision. I.H and M.F.R played a crucial role in interpreting the results and augmenting the manuscript. F.K, E.Z. F.A.K. and M.A.B contributed to the statistical analysis and quantification of vitamin D3 using HPLC. M.F.R. he helped edit and review the manuscript. A. A. Z, and F.A. provided valuable assistance in yogurt development.

Acknowledgment

We are thankful to the digital library of University of Poonch for providing access to research data. The authors are also grateful to Times Institute, Multan, Pakistan, for helping in providing resources for literature.

Disclosure statement

I/we hereby declare that this research work has not been published elsewhere, nor is it under consideration for any other publication.

Data availability statement

Data is contained within the article.

Additional information

Funding

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant No. 6212].

Notes on contributors

Saneela Saleem

Saneela Saleem Department of Food Science and Technology, Faculty of Agriculture, University of Poonch Rawalakot (UPR), Azad Jammu and Kashmir, Pakistan.

Zahra Khan

Zahra Khan Dr Zahra Khan is a Senior lecturer in Nutrition and Health Science in the Faculty of Engineering and Science with diverse experience in clinical nutrition, public health nutrition, research, and teaching. At the University of Veterinary and Animal Sciences Lahore, she was appointed as a lecturer in the Department of Food Science and Human Nutrition, where she played a pioneering role and dedicated herself to advancing the discipline. Before joining the University of Greenwich, Dr Khan worked as Assistant Professor at A’Sharqiyah University Oman.

Imtiaz Hussain

Imtiaz Hussain Department of Food Science and Technology, Faculty of Agriculture, University of Poonch Rawalakot (UPR), Azad Jammu and Kashmir, Pakistan.

Faran Khan

Faran Khan Department of Clinical Services, School of Health Sciences, University of Management and Technology, Lahore, Pakistan.

Fahad Al-Asmari

Fahad Al-Asmari Dr. Fahad Al-Asmari. He specializes in food microbiology, preservation, and food safety. His research focuses on the manufacturing food development framework. His current research endeavors focus on reducing post-harvest losses by adding nutritional value to food waste and extending perishable food products’ shelf-life utilizing natural preservatives and non-thermal preservative methods. This approach not only enhances the nutritional profile of these foods but also leads to the creation of sustainable, value-added products to feed people suffering from hunger worldwide. Dr. Al-Asmari’s commitment extends beyond the laboratory and academia. He is passionate about engaging with local communities to improve the public concept of food security and environmental sustainability. He believes in the transformative power of his work to generate positive socio-economic impacts. Dr. Al-Asmari is keen to make a tangible difference in society.

Faima Atta Khan

Faima Atta Khan University Institute of Diet and Nutritional Sciences, Faculty of Allied Health Sciences, The University of Lahore, Sargodha, Pakistan.

Alyan Ali Zafar

Alyan Ali Zafar Department of Food Science and Technology, Faculty of Agriculture, University of Poonch Rawalakot (UPR), Azad Jammu and Kashmir, Pakistan.

Muhammad Abdul Rahim

Muhammad Abdul Rahim Dr. Muhammad Abdul Rahim completed his Doctorate (Food Science and Technology) in from Government College University of Faisalabad (GCUF), Punjab, Pakistan. Dr. Muhammad Abdul Rahim has expertise in spray drying, microencapsulation, lipids chemistry, extrusion technology, sensory evaluation and food process engineering. Dr. Muhammad Abdul Rahim has attended several International Conferences (held nationally and locally) as Invited and Keynote Speaker. Currently, Dr. Muhammad Abdul Rahim is working as an Assistant Professor in the Department of Food Science & Nutrition at Times Institute, Multan, Punjab, Pakistan.

Zongo Eliasse

Zongo Eliasse Laboratoire de Recherche et d’Enseignement en Santé et Biotechnologies Animales, Université Nazi BONI, Bobo Dioulasso, Burkina Faso.

Mohamed Fawzy Ramadan

Mohamed Fawzy Ramadan Dr. Mohamed Fawzy Ramadan is a Professor of Biochemistry and Food Chemistry at Umm Al-Qura University, Saudi Arabia. He obtained his PhD in food chemistry from Berlin University of Technology in 2004. His areas of interest include food chemistry, food science, nutrition, plant molecular biology, biochemistry, physiology, and biotechnology with a specialization in food chemical safety, sensory evaluation, and functional food.