Comparison and effect of plain and calcium fortified yogurt on glycemic responses, anthropometrics and metabolic biomarkers

Abstract

Metabolic syndromes including obesity and diabetes are the most common health issues due to insulin resistance, disturbance in glucose homeostasis, lack of exercise, and improper diet. The current study was planned to evaluate the potential effects of regular diet with fortified yogurt on blood glycemia and anthropometric responses. Plain yogurt was procured from the local market, and then it was fortified with calcium. Furthermore, the subsequent effect of fortified yogurt on blood glucose, insulin, and anthropometric measurements was assessed at different time intervals. A total of 40 healthy females and males aged about 20 years with a normal BMI range (20–24.9 kg/m²) were recruited within the Government College University Faisalabad. Participants filled out the habits Performa, stress factors questionnaire, and activity questionnaire. Blood glucose (BG) and visual analogous scale (VAS) performs were also taken in the fasting stage and then assigned treatment was given. After 15, 30, 45, 60, 90, and 120 min intervals VAS and BG estimation was carried out. The results shows that fortified yogurt contained a higher amount of calcium. Likewise, a similar trend was observed for the desire to eat, a feeling of fullness, palatability, physical comfort, and overall acceptability. The results obtained from various analyses were statistically evaluated.

Keywords:

• Fortified yogurt
• metabolic syndromes
• blood glucose
• visual analogous scale
• calcium

1. Introduction

Metabolic Syndrome is a cluster of elevated blood pressure, hyperglycemia, excess abdominal fat, and unusual cholesterol levels that intensify the possibility of diabetes, obesity, and cardiovascular and neurological disorders [1]. In the history of Pakistan obesity, diabetes, and high blood pressure is considered the chief cause of death and morbidity [2,3].

Diabetes elucidates as a multifarious, long-lasting malady and fault in the body’s fitness to adjust glucose to energy [4]. It is estimated that the death rate is increasing day by day because of the high presence of chronic disorders and poor lifestyles. It is estimated that the number of diabetic patients will be up to 552 million worldwide by 2030 [5].

About 11% adult population is suffering from diabetes mellitus in Pakistan [6]. It is supposed that glucose homeostasis is primarily based on liver functioning by regulation of renin [7]. According to the glucostatic theory, a drop in blood glucose during the fasting state initiates hunger [8]. Adequate nutrition is the basic right of each human. The deficiency of vital nutrients or inaccessibility to food can be operationally defined as Malnutrition [9].

Mounting pieces of evidence have supported the beneficial impact of food fortification in the tackling of metabolic syndromes [10]. Recent studies related to food fortification with calcium salts showed a great deal in the prevention and treatment of metabolic disorders [11]. Some calcium supplements in the form of calcium salts like calcium citrate and calcium carbonate are used for fortification [12,13].

Yogurt is considered a fermented milk product prepared conventionally through milk to sour at 40 to 45 °C [14]. The consumption of yogurt should be 100 g/day [15]. In this study, our vision was to upsurge the consumption of yogurt for the prevention and management of the consequences of obesity and glycemic control.

2. Methodology

2.1. Subjects and sample size

For this purpose of nutritional study, a total number of 40 healthy young male and female individuals living in a privileged housing area of Faisalabad were determined through proper analysis and were divided into four groups to receive four treatments PY and CY, DY, and CDY. The age of the participants was up to 20 years with a normal BMI range (20–24.9 kg/m²) and these participants were engaged through advertisements in Government College University Faisalabad area or through university websites, notice boards, and social media.

Subjects were educated about the study. The consent form was signed and the techniques accepted for the study were permitted by the Human Research Ethics Committee of Government College University Faisalabad.

The research setting was Home and Food Sciences Government College University, Faisalabad. Blood samples for biochemical measurements and other anthropometric measurements were obtained during three sessions (0, 30 and 60 days). On the day of the session, subjects were asked to reach the trial room after 12 h of overnight fasting. Before the onset of each session, only water consumption was permitted for up to one hour. To evaluate the intervention, there were four groups and each group consisted of 10 members. These groups were coded accordingly PY, CY, DY, and CDY. The groups were given 20 packs of yogurt for 10 days with the amount of 125 g per pack. Treatment groups received 250 g yogurt per day (125 g in morning breakfast and 125 g in evening dinner). To reduce the variability within subjects each participant was asked to follow the same time and same day of the month for treatment. Participants were also educated to keep the same eating patterns from the day before each session.

2.2. Plain and calcium fortified yogurt development

Yogurt and calcium were purchased from the local market of Faisalabad. The samples were stored at refrigerator temperature (4 to 10 °C) for further studies. Two treatments i.e., plain yogurt (control; PY) and calcium-fortified yogurt (FY) were prepared according to the prescribed procedure below.

Fortification of yogurt was conducted by adding 520 mg calcium (calcium citrate) per 125 g (one serving) after fortification, while before fortification, yogurt contains 140 mg of calcium per serving. About two servings of 125 g yogurt having carbohydrates with the ratio of 33 g FY and 36 g CY were examined. The amount of protein in yogurt was estimated i.e., FY: 7.8 g; CY: 8.0 g, and the amount of energy was (FY: 232 kcal; CY: 248 kcal). While in the case of the control group amount of calcium was 280 mg per serving [16].

2.3. Yogurt analysis

The physicochemical and sensory evaluation of treatments prepared for prescribed parameters were conducted at 0, 30, and 60 days of storage at refrigerator temperature. The pH of plain yogurt and fortified yogurt samples were measured through a Cyber scan 510 digital pH meter. An appropriate amount of sample was taken in a beaker where the electrode of the pH meter was immersed and readings were recorded after standardizing the device with buffer solutions of pH 4.01 and 7.0. pH was measured to estimate the tolerability of fortification yogurt before conducting an efficacy trial.

The acidity of yogurt was analyzed by direct titration [17] of a 9-gram sample of yogurt with 0.1 N sodium hydro-oxides (NaOH). This solution was prepared by dissolving 4 g sodium hydroxide in a 9-gram yogurt sample. The indicator was prepared when 1 g phenolphthalein was dissolved in ethyl alcohol (95%v/v) to find its final volume

For acidity determination, the 9 mg yogurt sample was taken in a beaker and 2–3 drops of phenolphthalein solution were added to it. Then sample solution was titrated against N/10 NaOH till its light pink color appeared for a duration of 30 s [18]. The percentage of acidity of the yogurt sample was calculated as follows: $$\text{Acidity}\left(\%\right)=\frac{0.009\times \text{volume of N}/10\text{ NaOH used}\left(\text{mg}\right)}{\text{Weight of sample}\left(\mathrm{g}\right)}\times 100$$

To measure the inorganic matter (minerals) of treatments, the ash content in yogurt samples was determined by the explosion of solid materials in an electric muffle furnace described in AACC Method No. 08-01 (AACC, 2000) (Model: MF-1/02, PCSIR, Pakistan). The samples were weighed in a crucible and charred gently over a low flame [19]. The crucible is then placed in muffle at 550 °C for 5–6 h. After cooling, the ash in the desiccator was calculated as: $$\%\text{Ash}=\frac{\text{Weight of ash}\left(\mathrm{g}\right)}{\text{Weight of original sample}\left(\mathrm{g}\right)}\times 100$$

The nitrogen content in the yogurt sample was estimated following AACC Method No. 46-10 using Kjeldahl’s apparatus (Model: Technik GmbH D-40599, Behr Labor, Germany). A 2 g sample of plain yogurt and a digestion tablet was added to a flask for digestion (copper sulfate: potassium sulfate: ferrous sulfate with a ratio of 5: 94: 1 and 30 mL concentration of sulphuric acid. Then the flask was removed, chilled, and shifted material into a 250 mL volumetric flask. The volume is up to 250 mL with distilled water. A 0 mL from 250 mL was shifted to a volumetric flask and 10 mL alkali was added until made alkaline. It was distilled in a 10 mL (4% boric acid) solution by using methyl red as an indicator. The titration was conducted by using an N/10 sulphuric acid solution [20] $$\begin{array}{l}\text{Nitrogen}\left(\%\right)=\frac{0.{\text{1N H}}_2{\text{SO}}_4\text{ used}\times 0.0014\times 250\times 100}{\text{Sample weight}\times \text{volume of aliquot sample}}\\ \text{Crude protein}\left(\%\right)=\%\mathrm{N}2\times 6.25\end{array}$$

2.4. Microbiological analysis

2.4.1. Total plate count

The saline solution was prepared by using NaCl with a quantity of 8.9 g/L for sample dilution. The plate count agar was prepared according to the method [21] and it was autoclaved at 121 °C for 15 min.

The seven sterilized test tubes were labeled as 10⁻¹, 10⁻², 10⁻³… 10⁻⁷. Then, 9 mL of normal saline was transferred into each test tube. About 1 g of yogurt sample was shifted into the first test tube and mixed. After that sample was transferred from the first tube to the next one and mixed thoroughly. Likewise, the sample was shifted from the second tube to the next one, and by use of this technique all samples were shifted to the other five tubes, and serial dilutions were prepared. A serial dilution for each sample was recommended by the method of Cappuccino [22]. The dilutions were as follows (Table 1):

Table 1. No. of dilutions.

Tube No.	1	2	3	4	5	6	7
Dilution	1:10	1:100	1:1000	1:10000	1:100000	1:1000000	1:10000000
Vol. of yogurt samples/ mL	0.1 (10^–1)	0.01 (10^–2)	0.001 (10^–3)	0.0001 (10^–4)	0.00001 (10^–5)	0.000001 (10^–6)	0.0000001 (10^–7)

From each dilution, 1 mL of contents was shifted on the surface of count agar plates and then incubated at 37 °C for 24 h. The average number of colonies was counted from dilutions which showed the size of colonies between the ranges from 30 to 300 with the help of the colony counter.

Total Plate Count = Average number of colonies × dilution factor/volume factor

2.5. Hematological analysis

2.5.1. Blood glucose

According to American Diabetes Association, the normal range of fasting blood sugar level is 100 to 125 mg/dl and random blood sugar level is 140 to 199 mg/dl. The standard protocol to measure blood glucose was after a 10–12-hour overnight fast. Blood samples were collected to check the fasting sugar levels. For this purpose, the sample was collected by prick-in finger. Before drawing blood, the area was sterilized with spirit, and the band was tied around the upper arm to swell the veins with blood. After the blood collection, a drop of blood was placed on a glucometer to measure blood glucose (Roche Diagnostics, Mannheim, Germany). Similarly, blood samples were collected every two months to find out the effects of overall hematological examination [23].

2.5.2. Insulin resistance

After twelve hours of fasting blood samples were collected and stored at room temperature for 30–60 min. The sample was centrifuged to collect serum at 2000 g and transferred into micro tubes in aliquots at 280 °C until the day of analysis. Previously start of the efficiency study, a blood sample of subjects was collected to achieve baseline values. Fasting serum insulin was assessed through immune radiometric assay (IRMA) (Biosource, Dorest, Belgium) and a gamma-counter system (Gamma I; Genesys). The blood samples were analyzed in every group at respective intervals on the 0, 30th and 60th day.

2.5.3. Insulin, HOMA-IR

The blood sample collected and evaluated through the homeostasis model of insulin resistance was calculated by using the following formula [24]

HOMA-IR = [fasting insulin (mU/L) × FSG (mmol/L)]/22.5 (28).

2.5.4. HbA1C

The calorimetric method was used to analyze glycated hemoglobin after the separation of ion exchange chromatography (Biosystem, Barcelona, Spain). HbA1c was measured by commercially available sticks according to the technique [25].

2.5.5. OGTT

The blood sample was analyzed in every group at respective intervals while fasting blood glucose concentration and oral glucose tolerance tests were measured by an enzymatic method (Roche COBAS INTEGRA 400 plus, USA). The blood samples were collected after an overnight fast in the morning and stored at −70^° C until analysis. Fasting blood samples were collected after oral intake of 7 g glucose [Trutol, 10 fluid ounces (296 mL) dextrose beverage, Nerl Diagnostics, Rhode Island, USA]. To measure OGTT, the patient submitted 5 or more samples for at least 2–3 h at the interval of half an hour.

2.5.6. Serum lipid profiles

In protocols of lipid profiles, the serum cholesterol level of the subject was measured by using the method CHOD-PAP [26]. High-density lipoprotein was determined through technique [27] and Triglycerides in form of liquid triglycerides were determined by (GPO-PAP) method [28]. The sample was analyzed in each group at respective intervals at zero time and again after 60 days. Statistical analysis was done using SPSS 8.1 to analyze the data for demonstrating the fortification of yogurt with calcium and vitamin D exerting more favorable effects than the non-fortified yogurt. Analysis of variance (ANOVA) with adjustment of Tukey’s test was used to determine the differences between time and type of yogurt. The chi-square test was used to determine the ratio between FY vs. PY. There were statistically significant results with a p-value < .05.

3. Results

The current research aimed to prove the fortified yogurt for its consumer acceptability and evaluated obesity and glycemic management responses through short-term human efficacy trials by involving healthy male and female subjects. At the start (0 day), mid (30 day), and end (60 day) of the study, the anthropometric measurements, fasting insulin level, fasting blood glucose (FBG), oral glucose tolerance test (OGTT), serum calcium and lipid profile from blood collected were assessed. Similarly, blood samples were assessed for hematological analysis.

Table 2 describes the total cholesterol analysis. Results indicate mean value and standard deviation cholesterol.

Table 2. Total cholesterol analysis.

Variables	0	30	60	Mean
Cholesterol	5.65 ± 0.69	4.60 ± 0.58	5.50 ± 0.54	5.25 ± 0.6
LDL	3.59 ± 0.76	3.37 ± 0.66	3.25 ± 0.54	3.40 ± 0.65
HDL	1.02 ± 0.25	0.77 ± 0.20	0.55 ± 0.09	0.78 ± 0.18
Triglycerides	2.24 ± 0.80	2.02 ± 0.70	1.78 ± 0.65	2.01 ± 0.71

Results in Table 3 indicate the difference regarding tests between PY and CY.

Table 3. Analysis of variance regarding tests between PY and CY.

Source	Df (Degree of freedom)	SS (Sum of Squares)	MS Mean-square)	F (F-statistic)	P (p-value)
BMI	2	1.599	.799	.899	.410
Blood Glucose	117	40274.617	20137.308	276.781	.000
Insulin Resistance	119	14964.717	7482.358	224.976	.000
HOMA_IR		2.885	1.443	9.835	.000
HbA1c		2.217	1.108	1.003	.370
OGTT		2526.050	1263.025	9.035	.000
Calcium		129.672	64.836	65.340	.000

Table 4 shows the physio-chemical analysis of yogurt at 0 days 30 days and 60 days respectively (Table 5).

Table 4. Physiochemical analysis of Yogurt.

Components	0	30	60
pH	4.5 ± 0.1	4.0 ± 0.1	3.7 ± 0.1
Acidity	4.2 ± 0.1	4.5 ± 0.1	4.9 ± 0.11
Crude protein	3.21 ± 0.21	4.22 ± 0.22	5.66 ± 0.24
Crude fiber	1.33 ± 0.23	1.42 ± 0.29	1.55 ± 0.32
Ash	0.82 ± 0.06	0.59 ± 0.05	0.39 ± 0.04
Syneresis	24.0 ± 4.25	42.0 ± 4.45	59.0 ± 3.65
Water holding capacity	82.0 ± 69.0	88.0 ± 70.0	92.5 ± 75.6
Viscosity	72.60 ± 0.14	69.60 ± 0.11	64.60 ± 0.08

Values are means ± standard deviation (N = 40).

Table 5. Micro biological analysis.

Storage period	Shop verbal count (10^10 c.f.u/mL)
0	2.72
30	2.60
60	2.32
Mean	2.30 ± 0.30

Results indicate the mean of Blood glucose levels for PY and CY is 80.7 ± 3.18 and 94.4 ± 10.6, insulin resistance levels for PY and CY is 30.2 ± 4.9 and 30.8 ± 5.6, for HOMA-IR levels of PY and CY is 1.43 ± 0.36 and 1.38 ± 0.41 respectively. HbA1c levels of the PY (yogurt plain) control group value is 4.26 ± 1.0 and the CY (yogurt and calcium) value is 4.1 ± 0.61, OGTT value for PY and CY is 82.3 ± 7.5 and 81.0 ± 8.7 respectively and WHR value of PY and CY is2.50 ± 0.97 and 2.04 ± 3.8.

4. Discussion

According to researchers overweight and obesity, ranges have been increasing from 10 to 25% and 2 to 10% respectively among them, and reported that in the United States (U.S.) 30% of adult children are considered obese [29]. Probiotics are used at the time of the invention of microbes and fermented milk products were produced in the era of Egyptian hieroglyphs [30]. In the 1800s, scientists observed the health effects of fermented milk products but the reason remains unseen. A scientist named Louis Pasteur work on it and discovered bacteria and yeast that are responsible for fermentation but he couldn’t connect microbes to obvious health effects [31]. Results indicate the means for Blood glucose levels, resistance levels, HOMA-IR levels, HbA1c levels, OGTT, WHR, and body fat in fortified yogurt [32]. Yogurt is a composite physiological smoothy fat that facilitates the postnatal adaptation of a baby through digestive maturation simultaneously [33]. Developing scourges demonstrated that endless infections and way of life ailments have turned out to be not kidding difficulties the around the world [34]. Diabetes and stoutness are head-inclining elements to permit cardiovascular (CVD) sickness in people [35].

Metabolic disorders and micronutrient deficiencies result in numerous health-related malfunctions. Metabolic syndrome is considered havoc for developing economies and Pakistan is also considered in the list of developing countries. While in the history of Pakistan obesity, diabetes, and hypertension are considered the leading cause of death and morbidity. Universally, it is considered that natural dietary sources of calcium existence are in shortage so need to fulfill their optimal level by fortification [36].

Micronutrient deficiencies have emerged as a leading problem in the growth and performance of individuals. Recently, the scientific community is in the quest to find rational approaches to reduce the associated severity. Pakistan is also facing serious issues related to micronutrient deficiencies with special reference to Iron, Vitamin A, Zinc, and Calcium. Because of this hidden hunger, the highest rate of mortality among infants in Pakistan with a severe incidence of Stunting, wasting and being underweight.

Results indicate the difference regarding tests between PY and CY. Recent studies related to the fortification of food with calcium salts have a great deal of consideration because of the role of calcium in the prevention and treatment of metabolic disorders like bone problems and high blood pressure. Amongst many approaches currently, food fortification of yogurt is assumed a real interest of the scientific community. Numerous scientific expeditions have illustrated the success of fortification to curtail micronutrient deficiencies regarding calcium and vitamin D. Food fortification is defined as the addition of deficient nutrients in the staple diet to improve the status of specific nutrient deficiencies in a population. Fortification involves the addition of a specific amount of nutrient-rich premix that contains the required vitamins and minerals that are lost during processing [37]. Some calcium supplements are used for fortification in the form of calcium salts like calcium citrate and calcium carbonates [38].

1993 Hong Kong conference recommends that RDA calcium intake for teenagers, adults [39], and women are at the ratio of 1200:1000:1500 milligrams respectively [40]. Yogurt is considered a good source of calcium and its components such as lactate [41] citrate and phosphopeptides with a ratio of 1:0:8 that’ play important role in calcium absorption [42]. Research regarding calcium intake with aging shows that the intake of calcium is lower than its recommendation [43] because of the low capacity of the intestine to absorb calcium.

5. Conclusion

Based on the results of our study, it can be concluded that the consumption of calcium-fortified yogurt resulted in significantly lower glycemic responses compared to plain yogurt. This finding suggests that calcium fortification may be a beneficial strategy to help regulate blood sugar levels in individuals at risk of developing metabolic disorders. Additionally, we found that both plain and calcium-fortified yogurt consumption resulted in significant improvements in various anthropometric measures, such as body weight, BMI, and waist circumference. These results suggest that incorporating yogurt into one’s diet may have positive effects on weight management and overall body composition. Overall, our study provides evidence that the incorporation of yogurt, particularly calcium-fortified yogurt, into the diet may have beneficial effects on glycemic responses, anthropometrics, and certain metabolic biomarkers. However, further research is needed to fully understand the underlying mechanisms and long-term effects of yogurt consumption on these outcomes.

Disclosure statement

No potential conflict of interest was reported by the author(s).