Development, physicochemical, and sensory analysis of moringa oleifera l. powder added buffalo milk yoghurt with pharmacological potentialDownload PDF Download PDF ArticleOpen accessPublished: 27 August 2025Farzana Siddique1,Salman Ahmad1,Ashiq Hussain1,Nida Firdous2,Rizwan Nisar1,Muhammad Bilal3,Ayesha Najam4,Abdullah Salik5,Muhammad Zia1 &…Abdeen Elsiddig Elkhedir6 Scientific Reports volume 15, Article number: 31519 (2025) Cite this articleSubjectsBiotechnologyPlant sciencesAbstractYoghurt is a one of the commonly consumed nutritious dairy products, which lacks certain bioactive compounds that are present in abundance in many plants. While, a significant range of phytonutrients are present in the leaves of Moringa oleifera. Thus, the purpose of this work is to develop a functional yoghurt, which could promote the health of the consumers to its bioactive components. In the present work, the buffalo milk yoghurt supplemented with Moringa oleifera leaves powder [at 0% (T0), 0.2% (T1), 0.4% (T2), 0.6% (T3) and 0.8% (T4)] was prepared to check the variations in the physicochemical, bioactive, and antioxidant properties, during 21 days of refrigerated storage. The results showed that the fat content was decreased from T0 (4.42%), to T4 (3.37%), and decreased from 4.40 to 4.38% during storage period of 21 days. The highest protein content was observed in T4 (4.15%), while the lowest in T0 (3.61%), and this protein content also deceased during storage. The moisture content was high in T0 and lowest in T4, while increased in all samples during the storage. The pH and total plate count decreased significantly from T0 to T4, whereas with the storage the pH decreased and total plate count was increased. A significant increase in the TPC, TFC and antioxidant activity of yoghurt samples was observed, with increasing the level of incorporation of Moringa oleifera leaves powder, as T4 presented the highest values for these parameters. However, as storage days passed from 0 to 21, the TPC, TFC and antioxidant activity of all the samples was significantly decreased. Minerals analysis also revealed a significant increase in the Ca, K, Na, Fe and Zn content of the yoghurt, as a result of increasing the level of Moringa oleifera leaves powder, as T4 was found to be having the highest amounts of these minerals. Current study concludes that Moringa oleifera leaves powder could be an excellent fortification ingredient for the development of functional yoghurt, which due to the presence of bioactive compounds could provide pharmacological benefits.IntroductionBecause of the critical relationship between the diet and human wellbeing, consumers worldwide pay close attention to the choice of right food and its nutritional composition1,2,3. Natural antioxidant-rich foods are becoming increasingly popular around the world. Natural antioxidants from plant sources are used to supplement an extensive foods and dairy items’ range4,5,6,7,8. Milk is usually used in the development of various food products9. The structure of milk is an important, since it affects the consistency of the finished product. The relative proportion of milk components vary greatly between the dairy animals based on their types, breeds, practices of milking, lactation stage, season, climate, and the feed given. While buffalo milk is considered as a good source of fat and other nutrients10,11. Because of its special nutritional profile, buffalo milk is receiving increased scientific interest and ventures in many countries. It’s high in cholesterol, protein, lactose, minerals, and vitamins12. For human diet milk is considered as healthy and essential food13. It is a gift from nature as it contains almost all food components in proper proportion, which are essential for human health14. Various physical forms like cheese, butter, concentrated milk, fermented milk, buttermilk, ice cream, yoghurt and yoghurt like products, utilize milk and its components15. Other dairy products besides milk have been made and consumed for centuries all over the world. Dairy products have a substantial impact on human health, as various studies have looked at both the overall product and individual components to determine this health promoting potential16. Dairy products account for about 25–30% of an individual’s total diet17.Yoghurt is considered as an ideal food because of its splendid aromatic flavor and thick creamy consistency. The product is called as yoghurt with the specified use of bacterial cultures and presence of high bacterial content18. Consumption of yoghurt is increasing all around the globe however aroma, flavor and consistency of the yoghurt vary from area to area, and these factors depend on various factors19. Moisture content is high in yoghurt and its texture is affected by the presence of good quality proteins, stabilizers, fruits and total solids. Due to the therapeutic properties of yoghurt, it is produced and consumed all over the globe20. However, scientists are always in search to modify the physicochemical and bioactive contents of the yoghurt in a way that it could become healthier and more functional. For this purpose, at different times different plant materials, rich in bioactives have been tried to be added in the yoghurt formulations.Moringa is genus of flowering plant of family Moringaceae. Total 13 species combine to form the genus Moringa. Moringa oleifera is the general genus Moringa specie and is found in numerous tropical and subtropical areas. This plant has a high potential for people, particularly those living in under develop and developing countries suffering from malnutrition, poverty, poor health, unemployment and separation in international trade. As reported in many studies, the Moringa oleifera leaves have significantly high content of protein and iron. The leaves of this plant are rich in B complex vitamins, iron, potassium and calcium21,22. Due to the substantial number of flavonoids, phenolics, carotenoids and ascorbic acid, Moringa oleifera leaves are considered as great source of antioxidants23,24,25. The use of plants and their byproducts in dairy products has been gaining importance and several trials have been found in the literature aiming to improve the nutritional and antioxidant abilities of the dairy products7,8. Yoghurt fortified with Moringa oleifera have showed promise as a functional food with higher antioxidant qualities and a better nutritional profile, according to an analysis by Azis et al.26. Higher concentrations, however, could result in unfavorable sensory qualities as astringency and greenish coloring, which could influence customer approval. In a recent study, Getsemani et al.27 investigated the incorporation of Moringa oleifera seeds in yoghurt, and changes in microbial and antioxidant properties were observed. In another relevant study, Sari et al.28 studied the effect of Moringa oleifera leaves extracts on the physicochemical and antioxidant quality of the frozen yoghurt, however, the authors have not performed sensory analysis. In a study by Shokery et al.29, ten bio-stirred set type yoghurt samples were prepared with different concentrations of Moringa oleifera leaves powder. The scientists verified that adding more Moringa oleifera powder to probiotic yoghurt greatly improves the symbiotic condition, offers health advantages, and is regarded as a crucial prebiotic component for the probiotic adjunct strains. Powdered Moringa oleifera leaves are a useful addition to food products and can be used as a fortifier in any edible product. It’s a great plant food with health benefits because it’s a good source of minerals, vitamins, phenolics, proteins, unsaturated fats, and folates30,31. The acceptance of the finished product is adversely affected when large concentrations of plant powders or extracts, for example of Moringa oleifera, are added to the yoghurt. These effects include greenish colorations, herbal flavors, and changes in the physicochemical qualities26. However, at the same time the high concentrations of these powders and extracts also enhance the functional characteristics of fortified yoghurts, through provision of different bioactive components. Therefore, it is vital to investigate what concentration of dried Moringa oleifera leaves powder would prove optimum to develop the yoghurt that may conform the standards of dairy foods but having high nutritional and antioxidant contents. Thus, Moringa oleifera leaf powder could be considered as appropriate contender to be used in the development of yoghurt due to its significant amount of nutrients and bioactive components. Therefore, the purpose of this study was to utilize dried Moringa oleifera leaf powder at different concentrations in the buffalo milk, to develop functional yoghurt with high nutritional and antioxidant contents. Further, the developed yoghurt formulations were stored for 21 days at refrigerated temperature to assess the changes occurred in different quality parameters, during 7 days of interval.Materials and methodsThe plant materials used in this work are cultivated and no permission was required for their use. Further, national/International guidelines were followed for the use of plant materials.Procurement of raw materialsThis research was conducted at IFSN, UOS, Sargodha, Pakistan, during the year 2022-23. For the research project, the raw materials were purchased from the local market of Sargodha, Pakistan. Yoghurt starter cultures (Lactobacillus bulgaricus and Streptococcus thermophilus) of CHR-Hansen, Denmark were purchased from Alpha scientific store, Sargodha, Pakistan. Moringa leaves of specie Moringa oleifera were carefully selected from local market and brought to the laboratory for further experiments. Full fat fresh buffalo milk was collected from Sadiq dairy farm Sargodha. After collection, milk was homogenized (55 °C, 20 Mpa) and pasteurized (65 °C, 15 min) for further use to develop yoghurt for the research work. All the chemical and reagents were of analytical grade and were procured from Aladdin chemicals Shanghai China and Sigma Aldrich Germany.Procedure for moringa leaves powder manufacturingAfter collection of leaves, they were washed with tap water and dried in a microwave oven (Model R-3556 M, 2450 MHz, Sharp Electronics Ltd., UK) using 800 W power, at 60 °C, as was guided by Hussain et al.4. After drying, these leaves were grounded to form fine powder with 50 mesh size, by using stainless steel grinding machine and 0.5 mm diameter plastic sieve. The final powder was carefully packed and stored in polythene pouches under standard laboratory conditions.Analysis of moringa leaves powderMoringa oleifera leaves powder was analyzed for its proximate composition following the procedures detailed by AOAC32. Briefly, ash of Moringa oleifera leaves powders was examined according to AOAC32 method No. 923.03, moisture was determined following the method No. 925.10, For protein content Kjeldahl’s method was used, for fat contents Soxhlet method was used, and fiber contents were determined by method No 962.09. Whereas nitrogen free extract (NFE) was determined by difference method by using the formula as; [100 - (fat% + protein% + ash% + moisture%)].Analysis of milk and yoghurtDetermination of fat contentFat concentration of milk and yoghurt was evaluated by Gerber method as reported by Gurd et al.33. Briefly explaining, sulphuric acid (10 mL) was poured into butyrometer and then 10.94 mL of sample was also slowly mixed in the acid. Sample was added slowly into butyrometer followed by 1 mM of isoamyl alcohol. Thorough mixing and centrifugation of the contents of butyrometer was done at 1100 rpm for 5 min. Butyrometer was relocated to a water bath for about 3 min at 65 °C and percentage of fat was noted directly from scale of butyrometer.Determination of protein contentAs previously described for Moringa oleifera leaves powder, the protein content of milk and yoghurt was determined using the Kjeldahl’s method, following the protocols given in method No. 991.20 of AOAC32,.Determination of total solids (TS)TS of milk were determined by method No. 925.23 given by AOAC32. Briefly explaining a known amount of milk samples was placed in crucible and relocated in hot air oven for drying. The content of total solids in milk was experimented by drying the milk to constant weight at 105 °C overnight using a laboratory scale heating oven, and adopting the below given formula (Eq. 1);$$\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{s}\text{o}\text{l}\text{i}\text{d}\text{s}\:\left(\text{\%}\right)=\:\frac{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{d}\text{r}\text{i}\text{e}\text{d}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\left(\text{g}\right)}{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\times\:100$$Determination of solids not fat (SNF)The SNF content in milk was calculated by the difference in TS and fat content of the milk, as has been given in the formula below (Eq. 2);$$\:\text{S}\text{N}\text{F}\:\left(\text{\%}\right)\hspace{0.17em}=\hspace{0.17em}\text{T}\text{S}\:\text{o}\text{f}\:\text{m}\text{i}\text{l}\text{k}\:\text{a}\text{f}\text{t}\text{e}\text{r}\:\text{d}\text{r}\text{y}\text{i}\text{n}\text{g}\: -\text{o}\text{r}\text{i}\text{g}\text{i}\text{n}\text{a}\text{l}\:\text{f}\text{a}\text{t}\:\text{i}\text{n}\:\text{t}\text{h}\text{e}\:\text{m}\text{i}\text{l}\text{k}$$Determination of pHElectronic digital pH meter (Inolab WTW Series 720) was used to determine the pH of milk. Calibration of the pH meter was done by the buffer solution of pH 4 and 7. Electrodes of pH meter were immersed into the milk sample taken in a beaker to determine pH, following the procedure provided by AOAC32,, and taking guidelines from the study of Fatima et al.34.For pH determination of yoghurt, the sample was lightly stirred for brief period of time and then pH probe was inserted in the sample and reading was taken. All yoghurt samples were tested for pH with the same technique using same apparatus. After incubation and on 1 st day intervals, pH of each of the sample was determined and recorded. The pH values of all the samples stored at 4 °C were also examined after 7, 14 and 21 days of interval, according to the guidelines of AOAC32,.Determination of titratable acidityMethod No. 947.05 given in AOAC32, was used to determine the titratable acidity of the buffalo milk sample. Briefly, 2–3 drops of phenolphthalein were added to titration flask including 9 mL f milk sample. The sample comprising indicator was titrated against 0.1 N NaOH until the appearance of light pink color. Volume of 0.1 N NaOH used was noted to evaluate the acidity of sample in terms of lactic acid by utilizing formula given below (Eq. 3);Similarly, according to the procedure given in AOAC32, the determination of titratable acidity of yoghurt was also carried out for each treatment. In a beaker, 10 mL of a thoroughly blended, homogenous yoghurt sample was diluted with 20 mL of distilled water. As an indication, two to three drops of phenolphthalein solution were then added. This was titrated against a solution of 0.1 N sodium hydroxide until a light pink color appeared, which might last for around 30 s. The percent of titratable acidity was measured following the below given formula (Eq. 3);$$\:\text{\%}\:\text{T}\text{i}\text{t}\text{r}\text{a}\text{b}\text{l}\text{e}\:\text{a}\text{c}\text{i}\text{d}\text{i}\text{t}\text{y}=\:\frac{0.009\:\times\:\text{v}\text{o}\text{l}.\:\text{o}\text{f}\:\text{N}\text{a}\text{O}\text{H}\:\text{u}\text{s}\text{e}\text{d}}{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\times\:100$$Manufacturing of yoghurtYoghurt with and without Moringa oleifera leaves powder was prepared by using same concentration of bacterial culture and different percentages of Moringa oleifera leaves powder. Control sample was prepared by 1 L of buffalo milk, and using 2% sugar and 2.5% yoghurt culture in the same 1:1 ratio. Milk was heated to destroy harmful microorganisms. The incubation temperature (45 °C) and time (6 h) was same for all samples. Milk was cooled to about to 4 °C to control the growth of micro-organisms and enzymatic activities. The whole process of yoghurt manufacturing was in accordance to the guidelines provided by Ahari and Massoud35,. Different treatments of yoghurt having different concentrations of the Moringa oleifera leaves powder have been shown in Table 1, and a graphical representation of this work is shown in Fig. 1.Table 1 Treatment Plan.Full size tableFig. 1Graphical representation of the work.Full size imageAnalysis of yoghurtAll proximate analysis, physicochemical analysis, antioxidant potential, and mineral analysis of yoghurt were performed after 0, 7, 14, and 21 days, during the storage.Determination of TPCThe TPC, TFC, and antioxidant capacity of the yoghurts were assessed using an extraction method according to Gomes et al.36. To do that, 8 mL of ethanol was combined with 2 g of each sample. The combination was vortexed for one min, and then it was immersed in an ultrasonic bath for five min. The same process was repeated twice. Last but not least, the samples were centrifuged for 20 min at 3000 rpm in order to analyze the TPC and TFC further. After that, the supernatant was gathered and stored in the dark at 4 °C. The Folin–Ciocalteu technique was used to calculate the TPC of the yoghurt extracts. First, 20 µL of each extract was mixed with 1580 µL of water and 100 µL of Folin-Ciocalteu reagent. After 3–6 min, 300 µL of sodium carbonate was added, and the mixture was left to rest at room temperature in the dark for two hr. The absorbance at 750 nm was ultimately measured using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, EUA). Three measurements were made for each. The data were expressed in mg gallic acid equivalents (GAE)/g after a calibration curve for gallic acid was created.Determination of TFCThe TFC of the yoghurt formulations was assessed using the aluminium chloride colorimetric technique in compliance with the instructions provided by Hussain et al.37. The absorbance at 510 nm was measured using the same spectrophotometer that was previously reported for TPC, and a standard curve was created using catechin. The findings were given as mg catechin equivalents (CE)/g. Every experiment was run three times in order to get the mean data.Antioxidant activity (DPPH assay)The antioxidant activity of yoghurt samples was determined using the method of Brand-Williams et al.38, with slight modifications. Briefly, divided into 200 µL aliquots, each sample was mixed with 1.0 mL of 0.1 mM DPPH in ethanol. The reaction mixture was thoroughly shaken and then allowed to react at room temperature for 20 min. The residual DPPH free radical was identified by measuring the absorbance at 517 nm using a spectrophotometer (UNICO SQ2802S, UNICO Co. Suite E Dayton, New Jersey, USA) and comparing the results to trolox blanks. The control was a 0.1 mM DPPH solution without extract, while the blank was pure methanol. Each measurement was performed three times, and the percentage of antioxidant activity was the output.Minerals analysisIn 100 mL digestion flask, 1 mL of yoghurt was mixed with 10 mL of concentrated nitric acid. Heating was done for the time period of 20 min. Afterwards; cooling was done at room temperature. Then 5 mL of perchloric acid (Panreac quimica SA) was added and heating was done vigorously till the appearance of white fumes, and reduction of the sample to 2–3 mL. By adding 50 mL re-distilled water the final volume was made. Mineral’s concentration was determined by atomic absorption spectrophotometric as reported by Hussain et al.37. The concentration of minerals was calculated by using graphite atomic absorption spectrophotometry with an Analyst 800 (Perkin Elmer, USA). The maximum absorbance was pursued by the adjustment of the cathode lamp at specific wave lengths and slit. For graphite furnace measurements, inert gas used was argon. Graphite tubes coated pyrolytically with a platform was used. Single element hollow lamps were used for calculation of measurements like integrated absorbance peak areas. The results were expressed as mg/kg.Microbial analysis (total plate count)The sample was prepared by using normal saline solution as by mixing 8.9 g sodium chloride in 1 L of water, the saline solution was prepared. Afterwards, media was prepared by taking 15 g plate count agar, which was weighed and dissolved in distilled water (1 L), and then sterilized for 15 min at 121 °C. The method of media preparation was adopted from the study of Mada et al.39. Samples were prepared by taking 9 mL of prepared normal saline, which was poured into sterilized test tubes for the preparation of dilution blank. Micropipette ware was used for inoculation, and only one tip was used for one milk sample. A fresh tip dipped half an inch into the liquid, sucked up and down the sample ten times to mix it thoroughly. Each 1 mL sample was taken and relocated to next dilution blank. The tip was discarded and the process was recurring till the requisite number of dilutions was obtained. Afterwards, on the surface of separate nutrient agar plates, 1 mL concentration from every dilution was spread and shifted well. These plates were incubated for 24 h at 37 °C. Counting of colonies of average numbers was done from those dilutions and total plate count was determined using below given formula (Eq. 4);$$\:Total\:Plate\:Count\:\left(TPC\right)=\:Average\:numbers\:of\:colonies\:\times\:dilution\: factor/{volume} \:TPC\:was\:expressed\:as\:CFU/ml$$Sensory evaluationUsing a 9-point hedonic scale, the sensory qualities of yoghurt were assessed for texture, appearance, color, flavor, and general acceptability during the course of a 21-day storage period. This approach was followed by Rafique et al.40. The finished product’s quality and accessibility were assessed by semi-trained panelists from the University of Sargodha’s Institute of Food Science and Nutrition. The panelists were provided with brief recommendations regarding the procedures and technique of sensory evaluation. The evaluation was conducted at room temperature and under standard lighting. There were thirty male and twenty-five female participants in the sensory panel, aged thirty to forty. A score of one signified extreme poverty; two very poor; three poor; four below fair and above fair; five as fair; six below fair and below poor; seven good; eight very good; and nine excellent. In order to facilitate evaluation, code numbers were allocated to the yoghurt samples placed in the disposable plates, and panelists were given bottles of distilled water to rinse and neutralize. Sensory evaluation performa has been provided at the end of document, as Appendix 1.Statistical analysisResults, which were obtained from various parameters were evaluated statistically by using TWO-WAY Analysis of Variance Technique (ANOVA) under least significant difference (LSD) test as selected by Steel et al.41 to analyze the effects on acceptability and quality of yoghurt statistically, by Statistix 8.1 software (Analytical software Tllahassee, FL, USA). LSD pair wise comparison was completed at a significance level of p ≤ 0.05 and p