Vitamin-A enriched yogurt through fortification of pumpkin (Cucurbita maxima): A potential alternative for preventing blindness in children

Abstract

Vitamin-A deficiency associated with night blindness in children is a global health problem that could be prevented or reduced by promoting the intake of β-carotene in food. The fortification of β-carotene in yogurt using pumpkin flesh (PF) could be a very cost-effective public health intervention. The current study aimed to analyze the proximate, functional, microbial, textural, and sensory properties of yogurt fortified with PF to evaluate its suitability as a β-carotene enriched food. The research was conducted with three treatments, control set-type yogurt (CSY) with no PF, 15% PF-fortified set-type yogurt (15PFSY), and 20% PF-fortified set-type yogurts (20PFSY) followed by pumpkin pie spice and ground pumpkin seed to improve consumer acceptability. The fortified yogurt with 20PFSY and 15PFSY contained a higher amount of β-carotene, protein, fiber, and ash, and lower carbohydrate, fat, and energy in compression with CSY, which might attract health-conscious people. In addition, viable bacterial count, firmness, consistency, cohesiveness, and viscosity index were found better in fortified yogurt. Based on reports of sensory panellists, 15PFSY achieved a significantly (p < 0.01) highest overall acceptability than 20PFSY and CSY. These findings suggest that pumpkin-fortified yogurt could be used widely as a nutrient-enriched fermented food. In addition, as a β-carotene (vitamin-A) fortified yogurt, it could be a potential alternative to prevent or reduce blindness in children with minimal cost.

1. Introduction

Food fortification is a very cost-effective process to enrich the nutritional quality of food considering the public health perspective [1]. Generally, the process of food fortification is done via altering an original recipe by adding ingredients to improve the product's characteristics, sensory, and nutritional profile [[2], [3], [4]]. Based on FAO, fortification is the purposeful addition of one or more micronutrients to a food to increase the nutritional quality with minimal public health risk [5]. In consequence, food fortification using natural resources (fruits, vegetables, cereal, etc.) is the best way to improve the overall nutritional quality of food with no or negligible side effects [[6], [7], [8]]. Also, the incorporation of a perishable agricultural product helps to reduce post-harvest loss and improves the higher nutrient profile and bioavailability [[9], [10], [11]]. Indeed, there is a growing demand for formulated dairy items due to lactose intolerance, and allergy to milk protein and cholesterol [[12], [13], [14], [15]]. The fortification of widely consumed yogurt might effectively prevent or reduce diseases associated with nutritional deficiencies and a variety of physiological disorders.

Vitamin-A deficiency (VAD), a nutritional disorder, responsible for blindness in children has been considered a severe public health concern, particularly in developing nations [5,16]. The most prevalent cause of avoidable blindness in children is severe VAD, which is known as xeropthalmia [17]. Inadequate consumption of β-carotene (precursor of vitamin A) results in low serum retinol levels, attributed to a variety of physiological disorders [18]. Reports revealed that around 44–50% of preschool children (>5 years) have been impacted by severe VAD in the south Asian region, while one-third of the global death rate in Bangladesh and India is due to malnutrition and VAD [19,20]. In addition, about 2% of preschool children are affected by night blindness and about 6% of children (<5 years) die in Africa due to VAD [21,22]. As of 2020, three out of every five children would require vitamin A supplementation, whereas the highest prevalence of VAD was reported in Sub-Saharan Africa, followed by South Asia [23]. Since the national vitamin-A supplementation program faces a variety of challenges in developing countries, consuming vitamin-A bio-fortified food from available plant sources like pumpkin would be a sustainable way to meet the nutrient demand.

Pumpkin belongs to the Cucurbitaceae family is a nutrient-enriched vegetable, widely grown in most of the countries with minimal cost [[24], [25], [26]]. It contains β-carotene precursor, which is converted to vitamin-A in the human body and plays a significant role in preventing some chronic diseases [[27], [28], [29]]. Besides, it has numerous medicinal and health-promoting properties, including antioxidant, anti-carcinogenic, anti-diabetic, anti-inflammatory, immunity-boosting, obesity reducing, eyesight protection and many more [30,31]. Pumpkin flesh is an excellent source of different bioactive compounds, vitamins, minerals, carotene, and dietary fiber [26,32], whereas seeds are high in vitamin E, particularly tocopherols which can deter prostate cancer and slow the progress of hypertension, arthritis, and kidney stones [33,34]. Because of the abundant source of β-carotene and other micronutrients, the utilization of pumpkin and its similar items has rapidly increased over the years [35,36]. As socio-economic determinants lead the developing nations to rely on plant sources for meeting vitamin-A requirements, pumpkin-rich yogurt could be an effective functional food to fight against VAD in long run.

Globally, yogurt is the most popular fermented dairy food, which contains almost all essential nutrients, easily digestible protein and a good source of probiotics [7,[37], [38], [39]]. It helps to prevent gastrointestinal disorders, improves teeth and bones, reduces obesity, and boosts immunity [[40], [41], [42]]. Nowadays, the incorporation of fruits or vegetables in yogurt is a new trend in modern food industries, due to their high demand by consumers concerning taste and nutrient intake [[43], [44], [45]] It also helps to intake fruits and vegetables, particularly for those who do not prefer to consume cooked vegetables [46,47]. Though some research on pumpkin powder production and its use in bread [[48], [49], [50]], cake [51], ice cream [46], and some other products [36,52,53] are available in the literature, but very limited research focused on the fortification of pumpkin in yogurt [[54], [55], [56]]. Therefore, this study is aimed to develop a PF-fortified βcarotene-rich yogurt to improve the nutritional proposition and quality which might be an option to prevent or reduce blindness in children through food intake.

2. Materials and method

2.1. Raw materials

Full cream milk (FCM), mature pumpkin fruit, non-sugar whole milk (NSWM), pumpkin seed (PS), starter culture (Lactobacillus acidophilus), pectin, stevia, and other ingredients were obtained from Mohammadpur town hall market, Dhaka, Bangladesh. Pumpkin pie spice (PPS) and pumpkin flesh (PF) were prepared in the laboratory condition of the Department of Agricultural Engineering, Sher-e-Bangla Agricultural University, Bangladesh.

2.2. Preparation of pumpkin pie spice

Freshly purchased matured pumpkin (Cucurbita maxima) was peeled, chopped into small cubes, and washed with chlorinated water (50 ppm sodium hypochlorite) followed by distilled water. The small cubes were steamed for 15 min in an autoclave, cooled at room temperature, blended for 2 min and stored in a refrigerator at 4 ± 1 °C until use. For PPS preparation, four spices e.g. cinnamon, ginger, cloves, and nutmeg were grounded, mixed with pumpkin at the ratio of 4:2:1:0.5 and ground for 2 min.

2.3. Preparation of pumpkin-fortified yogurt

Varied quantities (Control, 15% and 20%) of PF with other ingredients, including PPS, were heated at 95 °C for 5 min (Fig. S1), cooled immediately to 40 °C, added 0.4% starter to the mixture, and filled into glass cups for incubation at 40 °C for 8 h. Then, the prepared yogurt cups were stored in a refrigerator at 4 ± 1 °C for 4 weeks. Three treatments were applied including 15% PF-fortified set-type yogurt (15 PFSY), 20% PF-fortified set-type yogurt (20 PFSY) and control set-type yogurt (CSY), respectively (Table S1). The experiment was conducted following the completely randomized design (CRD) with three (n = 3) replication.

2.4. Determination of proximate composition

The prepared pumpkin-fortified yogurts were analyzed for proximate composition including moisture, protein, fat, fiber, and ash content as per official methods of AOAC [57] following references [52,58]. All analyses were carried out in triplicate (n=3). Carbohydrate content was obtained mathematically, subtracting the sum of all other compositions from 100 [59]. According to the standard formula, the energy content of the yogurt samples was calculated by multiplying the protein content by 4, carbohydrate content by 4, and fat content by 9 [60].

2.5. Determination of β-carotene content

The β-carotene content of yogurt samples was determined by the UV photodiode-assisted high-performance liquid chromatography (HPLC method) (Waters 2695, Milford, USA) as described in Ref. [61] with slight modification. The flow rate and ingestion volume of the mobile phase (methanol and t-butyl methyl ether, 8:2, v/v) were 0.8 mL min⁻¹ and 20 μl, respectively. The analysis was carried out for 1 h at 25 °C. All analyses are performed in triplicate (n = 3).

2.6. Determination of pH kinetics

In the study pH values of yogurt samples during incubation and storage were determined by a digital pH meter (Orion, USA). Each trial was repeated three times (n=3), and the mean value was taken.

2.7. Determination of viable bacteria

The total viable bacterial count was evaluated by the standard pour plate count procedure using dehydrated plate count agar [54,62]. The culture media was prepared following the manufacturer's instructions. The required glassware was sterilized using a hot air oven at 170 °C for 2 h, whereas an autoclave was used to obtain sterilized reagent-grade water and pipette tips. 1 mL of homogenous yogurt was serially diluted in a 9 mL ringer solution to prepare an eight-fold dilution. Then 1 mL sample was transferred into a sterile duplicate plate having about 20 mL of the growth medium, mixed properly, and incubated at 32 °C for 48 h. The number of viable bacteria was counted in colony-forming units per gram (cfu/g).

2.8. Determination of textural parameters

The textural profile (firmness, cohesiveness, consistency, and viscosity index) of yogurt samples was measured by the TA-XT texture meter (Stable Micro Systems, UK) following the procedure described in Ref. [63]. 35 mm (Ø) compression disk with A/BE attachment was used to compress the sample within 50 mm (internal Ø) cylinder at 1 mm/s pretest and 10 mm/s post-test speed. The samples' firmness, cohesiveness, consistency, and viscosity index were obtained from the Texture Exponent software (version 4.0.9.0).

2.9. Sensory analysis of yogurts

The sensory assessment was performed following standard protocols (ISO 6658:1985) using freshly prepared yogurt samples. Each sample was coded with randomly selected 3 digits and provided to the 25 semi-trained panelists (13 males aged 18 to 35 and 12 females aged 18 to 22). Before the sensory evaluation, the purpose of the test and test procedure were described and requested to record individual scores in the supplied form. Members rinsed their mouths with water between responses while different spoons were used for each sample. Each sample was assessed for color, flavor, texture, and overall acceptability on a 9-point hedonic scale (1 = dislike extremely, and 9 = like extremely). By distributing a questionnaire to the assessors, additional suggestions on quality development were also recorded. The method of sensory assessment was reviewed and approved by the Department of Agricultural Engineering, SAU, Bangladesh. Furthermore, all participants involved in this study for sensory evaluation have given their written consent for participation of our research.

2.10. Statistical analysis

The single factor analysis of variance (ANOVA) was performed to determine a significant difference between yogurt treatments, followed by Tukey's HSD test using the SPSS v.18.0 program. The obtained result was presented as mean ± standard deviation (SD). The formulated yogurts' kinetics and storage stability study followed a random sampling method. In all cases, the average value (n = 3) was considered for each presented data. Calculation and graphical works were performed by Microsoft Excel (version 2016).

3. Results

3.1. Proximate and functional composition of fortified yogurt

The proximate and functional composition of fortified yogurt including moisture, protein, fat, fiber, ash, total carbohydrate, β-carotene, and energy contents were determined and displayed in Table 1. A significant difference (p < 0.05) was observed in proximate and functional parameters in 15 PFSY and 20 PFSY compared to the control treatment (CSY).

Table 1.

Nutritional composition (mean ± SD) of yogurts fortified and unfortified with pumpkin flesh.

Parameters	CSY	15PFSY	20PFSY
Moisture (%)	83.37 ± 0.64c	85.22 ± 0.28b	86.68a ± 0.36
Protein (%)	3.72c ± 0.01	3.77a ± 0.02	3.75b ± 0.01
Fat (%)	3.92a ± 0.01	3.65b ± 0.01	3.56c ± 0.01
Fiber (%)	ND	0.57a ± 0.01	0.59a ± 0
Ash (%)	0.64c ± 0	0.72b ± 0.0	0.74a ± 0.01
Carbohydrate (%)	7.25a ± 0.50	6.07b ± 0.25	4.68c ± 0.36
β-carotene (mg/100 g)	ND	0.94b ± 0.01	0.98a ± 0
Energya (Kcal/100 g)	79.14a ± 1.97	72.21b ± 1.13	65.77c ± 1.46

Mean with different superscripts in a row indicates a significant difference (p < 0.05); ND: Not detected.

^aCalculated value.

3.1.1. Moisture content

The percentage of moisture content of fortified yogurt was increased with increasing the quantity of pumpkin flesh (PF). A significant (p ≤ 0.05) variation in the moisture content was observed due to the application of different amounts of PF compared to the control treatment (Table 1). The percentage of moisture content was a maximum of 86.68 ± 0.36% in 20 PFSY followed by 85.22 ± 0.28% in 15 PFSY and was a minimum of 83.37 ± 0.64% in CSY.

3.1.2. β–carotene content

A gradual increase in β–carotene content was observed in all pumpkin-fortified yogurt compared to the control treatment (Table 1). An increasing amount of β–carotene content in pumpkin-fortified yogurt is a common phenomenon, as ripe pumpkin contains 11.2 ± 0.007 mg/100 g of β–carotene. This occurs mostly in fortified yogurt due to the progressive boost in β–carotene-enriched PF in yogurt. In the present investigation, to some extent, significant variation (p ≤ 0.05) was also found in relation to β–carotene content of fortified yogurt at different treatments containing different doses of PF. Among the different treatments, in control treatment (CSY), the content of β–carotene was absent (not detected) as compared to yogurt in other treatments fortified with PF. The highest level of β–carotene in fortified yogurt was 0.98^a ± 0.0 mg/100 g in 20 PFSY, whereas the lowest level (0.94^b ± 0.01 mg/100 g) was observed in 15 PFSY (Table 1). Though, β–carotene in pumpkin-fortified yogurt is a bit lesser compared with its content in ripened pumpkin (11.2 ± 0.007 mg/100 g).

3.1.3. Carbohydrate, protein and fat contents

Nowadays low carbohydrate diet is the first preference of health-conscious people. In current research, the percentage of carbohydrate content in fortified yogurt decreased with an increasing quantity of PF. From Table 1, a significant (p ≤ 0.05) variation in the percentage of carbohydrate content was noticed due to the application of different treatments compared to the control. The percentage of carbohydrate content was maximum in CSY (control treatment) (7.25 ± 0.50%) followed by 6.07 ± 0.25% in 15 PFSY, whereas it was minimum of 4.68c ± 0.36% in 20 PFSY (Table 1).

Protein is an important part of a healthy diet to build and repair muscles and bones and make hormones and enzymes. In this experiment, we also determined the percentage of protein in fortified yogurt with ranged from 3.72 to 3.77% (Table 1). The highest protein content was measured in 15 PFSY (3.77 ± 0.02%) and the lowest was found in CSY (3.72 ± 0.01%). It was found that the protein value in 15 PFSY was significantly higher than the 20 PFSY.

A significant (p < 0.05) decreasing trend in fat content was observed in fortified yogurt with an increase in the amount of PF (Table 1). This is a common phenomenon of a decreasing amount of fat content with the addition of PE, whereas higher fat content is in the control treatment (CSY). The highest percentage of fat (3.92a ±0.01%) was retained in yogurt with control treatment (CSY), in contrast, the lowest fat content (3.56c ± 0.01%) was recorded in 20 PFSY.

3.1.4. Fiber, ash and energy contents

A significant (p < 0.05) increasing trend in fiber and ash contents was observed in pumpkin-fortified yogurts (20PFSY and 15PFSY), whereas a decreasing trend was obtained for energy contents compared to control treatment (CSY) (Table 1). The fiber contents of 15PFSY and 20PFSY were 0.57% and 0.59%, respectively, and no dietary fiber was detected in CSY. On the other hand, the ash content of yogurt varied from 0.64 to 0.74% (Table 1). The maximum ash content in 20PFSY (0.74 ± 0.01%) was due to higher PF add-ons over 15PFSY (0.72 ± 0.0) and the least content was in CSY (0.64c ± 0.0). Pumpkin-fortified yogurt had a significantly (p < 0.05) higher amount of ash compared to control treatment (CSY). The calculated gross energy from 100 g of yogurt varied from 65.77 to 79.14 Kcal. The highest value was obtained in CSY (79.14 ± 1.97 Kcal/100 g), and the lowest value was obtained in 20 PFSY (65.77 ± 1.46 Kcal/100 g).

3.2. Kinetics of yogurt pH

3.2.1. Changes in pH during incubation

In present study, the pH of all treated (15 PFSY and 20 PFSY) and untreated (CSY) yogurts was reduced to below 5.0 after 8 h of incubation (Fig. 1). The faster pH drops within the first 3 h of fermentation were observed in all yogurts, whereas a very subtle difference was recorded in the last 2 h. The lower pH value was observed in yogurt with higher PF, which affects bacterial growth.

Fig. 1.

pH kinetics of yogurts fortified and unfortified with pumpkin flesh during incubation.

3.2.2. Changes in pH during storage

A significant difference (p < 0.05) in pH content was noticed in treated (15 PFSY and 20 PFSY) and untreated (CSY) yogurts during 28 days of storage (Fig. 2a). The highest pH changes (5.29 to 4.56) were recorded in 20 PFSY, followed by 15 PFSY (5.14 to 4.66) and CSY (4.95 to 4.76). The lowest pH values were found on the 14th day of storage in all treated (15 PFSY and 20 PFSY) and untreated (CSY) yogurts. After 2 weeks of refrigerated storage, pH contents rose in all samples, recorded highest on 28 days of storage and those values were near the initial values.

Fig. 2.

Storage stability study of pumpkin flesh enriched yogurt: pH change (a) and total bacterial count change (b).

3.3. Viable bacterial count in yogurt

The total viable bacterial count in treated (15 PFSY and 20 PFSY) and untreated (CSY) yogurts was accomplished and displayed in Fig. 2b. It was observed that pumpkin flesh has a significant (p < 0.05) influence on the bacterial load of yogurts. Similar to pH change, the total number of bacteria decreased up to 14 days of the storage period, followed by substantial increases and counted highest on the 28th day of storage. The change in bacterial number was most significant in 20 PFSY (Log 1.21 cfu/g) followed by 15 PFSY (Log 1.19 cfu/g) and CSY (Log 0.6 cfu/g).

3.4. Effect of PF on textural properties of yogurt

The comparison of textural analysis of yogurts enriched with different proportions of PF is presented in Table 2. All the studied parameters of the textural profile were significantly increased (p < 0.05) with the higher PF add-on. The storage study showed a substantial increase in firmness, consistency and viscosity index up to 14 days of storage regardless of treatments. However, these parameters then started to decrease until the 28th day of storage.

Table 2.

Comparison of the textural profile of yogurts fortified and unfortified with pumpkin flesh.

Sample)	Storage time (day)	Firmness (g)	Consistency (g.s)	Cohesiveness (g)	Viscosity Index (g.s)
CSY	0	33.56b ± 0.31	1022.45b ± 1.08	56.23a ± 0.41	106.32b ± 0.34
	14	36.43a ± 0.45	1026.32a ± 1.56	55.54a ± 0.23	112.56a ± 0.31
	28	34.12b ± 0.04	1023.14b ± 1.54	53.12b ± 0.32	104.76a ± 0.21
15PFSY	0	43.85a ± 0.43	1035.45b ± 0.11	67.47c ± 0.09	112.43b ± 0.25
	14	46.63a ± 0.09	1044.34a ± 1.03	65.11a ± 0.87	118.09a ± 0.32
	28	44.53b ± 0.19	1038.09b ± 1.01	61.87b ± 0.65	113.98b ± 0.53
20PFSY	0	45.42c ± 0.81	1056.62c ± 1.05	74 .45a ± 0.56	123.34b ± 0.61
	14	50.40a ± 0.98	1067.34a ± 2.41	71.45b ± 0.67	134.26c ± 0.67
	28	45.98b ± 1.69	1059.89b ± 2.32	68.65c ± 0.97	125.64b ± 0.91

Values represent mean ± SD. For each sample, different superscript letters in the column indicate a significant difference at the 5% level.

3.4.1. Firmness and consistency of yogurt

The maximum firmness was measured in 20 PFSY samples followed by 15 PFSY and CSY. The highest firmness in 20 PFSY on the 14th day of storage. Consistency plays a vital role in consumer preference, which indicated the strength of the internal bond of food. PF is a good source of dietary fiber with high water holding capacity, resulting in higher consistency and viscosity index of the yogurt. The consistency profile of yogurt treatments followed a similar trend like firmness during four weeks of storage.

3.4.2. Cohesiveness and viscosity index

The cohesiveness of yogurt also indicates internal strength, which is an important textural parameter to judge yogurt quality. Similar to firmness and consistency, 20 PFSY showed the highest cohesiveness, followed by 15 PFSY and CSY during the entire storage period. The viscosity index is a measure of temperature influence on the product's viscosity. The viscosity index of the yogurts increased with the addition of pumpkin flesh. Fortified yogurt with 20 PFSY showed the highest viscosity index (123.34 ± 0.61), followed by 15 PFSY (112.43 ± 0.25) and CSY (106.32 ± 0.34) at the beginning of the storage. Like firmness and consistency, the viscosity index followed a similar increasing trend until the 14th day of storage and then dropped onwards.

3.5. Effect of PF on sensory properties of yogurt

Mean scores of color, flavor, texture, and overall acceptability of yogurts were conducted based on the sensory evaluation form (Form S1) presented in Table 3. As expected, the color preference of yogurt samples increased with the increase in PF content. The Post-ANOVA study showed that flavor and textural preference were strongest for 15PFSY, followed by 20PFSY and CSY respectively. Tukey's HSD test revealed a significant difference (p < 0.01) in the overall acceptability of yogurt treatments. It was observed that the overall acceptability of 15PFSY is significantly higher than 20PFSY and CSY. Thus, the best-perceived yogurt was 15PFSY, followed by 20PFSY and CSY.

Table 3.

Sensory evaluation of yogurts fortified and unfortified with pumpkin flesh.

Sample	Sensory Parameters
	Color	Flavor	Texture	Overall acceptability
CSY	5.84c ± 0.90	5.56b ± 1.12	6.08c ± 1.22	5.44c ± 0.82
15 PFSY	7.84ab ± 0.62	6.92a ± 1.53	7.48a ± 1.08	7.56a ± 0.58
20 PFSY	7.92a ± 0.82	5.60b ± 1.04	6.12bc ± 0.83	5.56bc ± 1.26

Scores are given as Mean ± SD. Mean with the different superscript letters in the same column indicate a significant difference (p < 0.01).

4. Discussion

The moisture content is considered one of the key attributes in determining the storage life and quality of fermented food. In general, higher moisture content supports microbial growth and reduces the shelf-life and quality of yogurt [37]. In this study higher amount of moisture with more pumpkin flesh (PF) add-ons may be due to water present in PF. Therefore, it could be suggested that extra PF-containing yogurt has higher moisture and relatively lower shelf life.

On the other hand, β-carotene is an indicator of vitamin-A, whereas more β-carotene content indicates more vitamin-A supplements through the food product. In this study, an increased amount of β-carotene in higher PF-contained yogurt is supported by Ref. [64]. However, lower β-carotene in yogurt samples compared to PF due to the thermal treatment followed by oxidative and non-oxidative reactions occurred during yogurt preparation [65]. In the current research, for the first time, we determined β-carotene content in pumpkin-incorporated yogurt since no published data is available in the literature.

Carbohydrates, protein, and fat are important considerations for the consumption of healthy food items. In this study, carbohydrate reduces with the increase of PF add-ons due to the lower carbohydrate content of PF. The higher protein content in 15 PFSY compared to 20 PFSY is due to the formulations, whereas 20 PFSY contained more non-sugar whole milk (NSWM) and PF than 15 PFSY. Yogurt has higher solid content with PF add-ons but has no significant effect on protein reported in the previous study, whereas an increase of 0.5% PF was employed [54]. We know the fat content of formulated products depends on the fat content of raw materials. In this study, decreasing fat content with an increase of PF was due to having a lower percentage of full cream milk in the formulation. Moreover, 0.5% of PF add-ons do not influence the fat content of reduced-fat set-type yogurt reported in reference [54]. Thus, it is suggested that PF helped to reduce carbohydrates and fat and increase protein content in fortified yogurt.

The slightly higher fiber content in pumpkin-fortified yogurts was attributable to higher PF as it is a good source of dietary fiber [59]. The fiber in food helps to reduce obesity, cholesterol, and blood sugar levels and prevent constipation [66]. The increasing trend of ash contents in the samples is due to the presence of different minerals, like K, Fe, Mg, Mn etc., in the PF [33]. The variation in energy content in yogurt was due to the interpretation of carbohydrate, protein, and fat contents in different formulations.

pH is a good indicator of bacterial growth and yogurt's shelf life. During incubation, lower pH with higher PF add-ons indicates favorable bacterial growth conditions. A study reported a pH drop to 4.6 within 11 h of fermentation from the initial pH of 6.7 in Zea mays incorporated yogurt [67]. The incorporation of vegetables significantly impacts yogurt's fermentation time, as reported in Refs. [47,68]. The most remarkable pH changes were in 20 PFSY followed by 15 PFSY during the storage stability study, whereas the lowest pH values were found on all samples' on 14th day of storage. The addition of plant fiber showed that fiber has a significant effect on yogurt's pH drop, while the lowest pH was observed on 14th day of storage, then started to increase again and measured to be near to the initial pH on 28th day of storage [67,68]. No significant effect on pH drop during 14 days of storage was observed in reduced set-type yogurts with 0.5% pumpkin flesh incorporation [54]. The increase in pH after 14 days of storage is attributed to the lactic acid-producing bacteria, which was described in previous studies [11,38,68].

The bacterial load in any food item is related to the product history and its shelf life. Improved bacterial load with higher PF yogurt in this study is aligned with previous studies. Storage studies of different vegetables incorporated in yogurt showed that phenolic compounds, dietary fiber, and organic acids support lactic acid-producing bacteria [69]. A higher amount of added PF in yogurt leads to a higher bacterial count as it supports the growth of a few types of bacteria, including streptococci [54,63]. A study reported that log 7.69 ± 0.26 cfu/mL of initial bacterial counts in fresh yogurt fermented with lactobacillus, which was recorded to log 9.48 ± 0.14 to log 10.34 ± 0.36 cfu/mL at the end of 28 days storage period [70]. A storage study on 14 different fermented dairy items reported an increase of 107 to 108 cfu/mL at the end of 30 days storage period [71]. However, the variation of bacterial count during storage study varies with the strain used in fermentation [70]. The increasing counts of total viable bacteria after 14 days of storage is mainly due to the increased S. thermophilus reported for many fermented dairy products [44,53,72].

The textural feature of yogurt is a crucial criterion for judging its quality. Firmness or hardness is yogurt's most common texture assessment parameter, defined as the force required to ensure certain deformation [69]. In this study, the highest firmness in 20 PFSY on 14th day of storage was due to higher bacterial growth [73] and denser protein aggregation [54]. The addition of PF decreases voids' number and size, leading to a more filament structure in the yogurt [54]. It was reported that pumpkin flesh's pectin showed a strong interaction with milk protein and provided high firmness to the yogurt [74]. Also, fortified yogurts with carrots, green peas, and zucchini increased the fermented dairy products' firmness, consistency, and viscosity [43,75]. The addition of flaxseed increases the firmness of probiotic yogurt reported in Ref. [73]. However, the hardness of yogurt depends on the protein and fat content of ingredients used, heat treatment, stature culture, and thickening agent [43,69,73]. The decrease in yogurt's firmness after 14 days of storage is due to a weaker protein network affected by higher acidity and microbial load reported in Refs. [59,61,62]. The increased consistency in 20 PFSY over 15 PFSY and CSY is due to higher PF fiber and pectin content [74]. The incorporation of vegetable fiber improves yogurt consistency, also reported in Ref. [69]. Additionally, the increment in yogurt cohesiveness by incorporated vegetables was also reported in Refs. [43,69], which confirms our observation as well. The influence of PF on the viscosity index during storage study is the same as the influence on firmness, discussed earlier in this section. The fiber and pectin content of pumpkin flesh significantly influences the viscosity index of dairy products [75]. The higher viscosity index of yogurt with adding pumpkin, carrot, and broccoli was reported in Refs. [43,69].

In most cases, personal preference for food items is decided by sensory qualities, but these attributes also serve a more practical purpose by influencing food selection and consumption patterns. Even though different features are considered for the sensory assessment of food, the overall sensory score is the main parameter to judge sensory acceptance of formulated food. This study reported lower overall acceptability with higher PF add-ons, while an optimum amount is preferred over the control sample. The lower acceptability of 20 PFSY compared to 15 PFSY might be because of excessive pumpkin flavor, which is not preferred by most adolescents [76,77].

5. Conclusion

Yogurt has long been a staple of the fermented food spectrum for fortifying functional ingredients, transforming dairy technology into health and well-being through consumption. Adding pumpkin-fortified yogurt increases the β-carotene, a functional ingredient to fight against vitamin A deficiency in humans. The addition of PF has a significant (p < 0.05) influence on the moisture and proximate composition and functional properties of yogurt. The fortified yogurt with 20 PFSY and 15 PFSY contained a greater amount of β-carotene, protein, fiber, and ash, and lower carbohydrate, fat, and energy compared to CSY. Besides, viable bacterial count, firmness, consistency, cohesiveness, and viscosity index were found better in fortified yogurts. Based on the evaluation of sensory panellists, 15 PFSY achieved a significantly (p < 0.01) highest overall acceptability than 20 PFSY and CSY. Therefore, the current research reveals that incorporating PF into yogurt significantly improves its functional, proximate, and sensory properties, particularly β-carotene content, which could be a very cost-effective alternative to prevent blindness in children.

6. Limitations of the study

The influence of pectin on microbial growth and bioavailability of β-carotene in the human body need to be investigated. In addition, instrumental color, flavor analysis, and micro-structure analysis could help to explain sensory data and textural parameters of fortified yogurt.

Author contributions

Md. Wadud Ahmed: Conceived and designed the experiments; Performed the experiments; Contributed reagents, materials, analysis tools or data; Analyzed and interpreted the data, Wrote the paper.

Md. Sirajul Islam Khan, Aney Parven, Md Harunur Rashid: Analyzed and interpreted the data; Wrote the paper.

Islam Md Meftaul: Contributed reagents, materials, analysis tools or data; Analyzed and interpreted the data, Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement

Data will be made available on request.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: