INVESTIGATION OF THE IMPACT OF ORANGE CRUDE COMPONENTS ON THE PHYSICOCHEMICAL, OXIDATIVE STABILITY AND BACTERIAL COUNTS OF YOGURT
A
&J.F.Hinmikaiye2✉Email
O.D.Asemota2
A.J.Uhiara1
A.A.Badmos1
1Department of Animal Production, Faculty pf AgricultureUniversity of IlorinPMB 1515IlorinKwara StateNigeria
2Department of Farming Systems and Research OutreachNational Root Crops Research Institute7006Umudike, UmuahiaPMB, Abia StateNigeria
1&2J.F. Hinmikaiye, 2O.D. Asemota, 2A.J. Uhiara and 1A.A. Badmos
E-mail: jhinmikaiye@gmail.com
1Department of Animal Production, Faculty pf Agriculture, University of Ilorin, PMB 1515, Ilorin, Kwara State, Nigeria
2Department of Farming Systems and Research Outreach, National Root Crops Research Institute, Umudike, PMB 7006, Umuahia, Abia State, Nigeria
ABSTRACT
A
A
This study investigated the impact of orange crude components (orange peel extract and orange juice) on the physicochemical properties, oxidative stability, and bacterial counts of yogurt. The orange peel extract was prepared using a Soxhlet extraction method, while the orange juice was freshly obtained and filtered. Yogurt samples were fortified with varying inclusion levels of orange crude components (0.2%–0.6% for peel extract and 25%–45% for juice) and analyzed for pH, viscosity, lipid oxidation parameters, and microbial counts. Phytochemical screening revealed the presence of flavonoids, phenolic acids, alkaloids, saponins, tannins, and steroids in the orange peel extract, indicating its potential as a functional additive.
The results revealed that the fortification with orange crude components significantly improved the antioxidant capacity of the yogurt in this current study, as evidenced by reduced peroxide and thiobarbituric acid values. Additionally, the incorporation of orange peel extract enhanced the viscosity of yogurt and improved its microbial stability, promoting the growth of probiotics while inhibiting the growth of spoilage microorganisms.
These findings suggest that orange crude components can enhance yogurt's functional properties, shelf life, and overall quality, presenting a sustainable valorization pathway for citrus by-products.
Key Words:
Orange crude components
Yogurt fortification
Physicochemical properties
Oxidative stability
Bacterial counts
INTRODUCTION
Yogurt is a staple dairy product known for its high nutritional value, including proteins, probiotics, and essential vitamins (Gibson and Roberfroid, 1995; Shori, 2016; Akin and Yilmaz, 2018). It is consumed worldwide due to its health benefits, particularly its contribution to gut health through probiotics (Kechagia et al., 2013). The development of functional yogurts, which are enriched with additional beneficial compounds, has gained attention as consumers increasingly seek foods that provide not only basic nutritional benefits but also additional health-promoting effects (Mistry, 2007; Gänzle, 2015; O’Rourke and Vinderola, 2017; Meena and Sethi, 2018). One such avenue for enhancing yogurt is the incorporation of fruit derivatives, such as orange crude components, which contain bioactive compounds with antioxidant, antimicrobial, and anti-inflammatory properties (Chen et al., 2018; Ribeiro et al., 2020).
Orange crude components, specifically those extracted from the peel, pulp, and seeds of oranges, are abundant in flavonoids, essential oils, and phenolic compounds (Barreca et al., 2014). These compounds have been shown to possess significant antioxidant properties, which may contribute to the oxidative stability of yogurt by preventing lipid peroxidation and preserving the quality of the product during storage (Câmara et al., 2019). Oxidative stability is a critical factor in determining the shelf life and nutritional integrity of dairy products, particularly yogurt, which is prone to spoilage due to its high moisture content and microbial activity (Huang et al., 2021). The addition of orange crude components could potentially slow down oxidative degradation, thus extending the yogurt’s shelf life and preserving its functional properties (Kumari et al., 2021). Orange crude components, derived from the peel and pulp of oranges, are rich in bioactive compounds, such as flavonoids, antioxidants, and essential oils, which could potentially impact the physicochemical properties, oxidative stability, and microbial quality of yogurt (Chen et al., 2018; Ribeiro et al., 2020).
In addition to oxidative stability, the physicochemical properties of yogurt, including pH, viscosity, and texture, are vital to its quality and consumer acceptance (Rao and Shih, 2020). The acidity and viscosity of yogurt are influenced by the fermentation process, which is initiated by specific strains of bacteria, and can be modified by the addition of various ingredients (Gänzle, 2015). The incorporation of orange crude components may also impact these properties, either by altering the microbial fermentation dynamics or by interacting with other components of the yogurt matrix, such as proteins and fats (Arias et al., 2020). Oxidative stability is another important factor, as it determines the shelf life and nutritional quality of yogurt, which could be enhanced by the antioxidant properties of orange crude components (Câmara et al., 2019).
Microbial stability, which refers to the ability of yogurt to maintain beneficial probiotic bacteria while minimizing the growth of spoilage microorganisms, is another important aspect to consider (Tamime and Marshall, 1997; Saad et al., 2013; Vinderola et al., 2019). The addition of antimicrobial compounds from orange crude components could have dual effects: enhancing the growth of probiotics while inhibiting harmful bacteria (Burt, 2004; Ambrosio et al., 2019). However, the specific impact of orange crude components on the total bacterial counts in yogurt, particularly on the viability of probiotic strains, remains underexplored (Sousa et al., 2020; Zamani and Ardalani, 2021; Ghosh and Khan, 2022). Understanding how these compounds affect microbial populations in yogurt is crucial for determining whether they can enhance or disrupt the probiotic benefits of yogurt (Ferreir and Santos, 2023). While several studies have explored the effects of fruit additives on yogurt, there is limited research on the specific impact of orange crude components on these key quality attributes (Kumari et al., 2021). The antimicrobial properties of citrus extracts, including orange peel, have been well-documented, with studies reporting that these extracts possess activity against a range of spoilage bacteria and pathogens (Burt, 2004; Caputo et al., 2018; Shehata et al., 2021). Despite these antimicrobial effects, it is important to balance the antimicrobial properties of orange components to ensure that they do not negatively affect the growth of beneficial LAB strains in yogurt (Choi and Lee, 2018; Citron and Saha, 2020; Rodriguez and Torres, 2021). In some cases, citrus-derived bioactive compounds may promote the growth of specific probiotics while inhibiting harmful bacteria, thereby improving the comprehensive microbiological quality of yogurt (Arias et al., 2020). Accordingly, this research aims to examine the influence of orange crude components on the physicochemical characteristics, oxidative stability, and total bacterial counts of yogurt.
MATERIALS AND METHODS
Experimental location
The research was conducted in the Animal Production and Chemistry Laboratories of the University of Ilorin, Kwara State, Nigeria.
Source of milk and oranges
Fresh 5 litres of cow milk was obtained from the Gaa Fulani at Agbede, Oke odo area, Tanke, Ilorin, Kwara state, Nigeria. All samples of the milk were filtered and kept at 4oC. Oranges were sourced from a local market in Ilorin, Kwara State, Nigeria.
Preparation of Orange Juice
Clean tap water was used to wash the fresh oranges in order to remove debris and filth. Using a stainless steel knife, the orange skin was detached. Juice was extracted from the fruit after the seeds had been removed. The juice will then be filtered using a sieve, put in a plastic container, and kept chilled (-20°C) for use later on during the making of yoghurt.
Preparation of Orange peel extract
The collected sample of orange peel was cleaned with water, chopped, placed on clean paper, spread out, and air-dried under ambient conditions for 6 days and subsequently milled into a uniform fine powder using an electric blender. The extraction from orange peel was done using the Soxhlet extraction method. 40g portion of the sample was weighed into a thimble and placed in the upper chamber of the apparatus. 400ml of Ethanol served as the extraction solvent, while the flask was heated to generate vapour, which subsequently condensed and percolated through the sample in the thimble. The process was run for 6 hours, and the sample was evaporated using a steam evaporator. The extract was weighed and kept in a well- labeled sterile bottle (Ehigbai et al., 2020).
Preparation of yoghurt
The raw milk was pasteurized (boiled at 90oC for 10min). The heated milk was cooled to 40oC. The pasteurized milk was inoculated with a commercial starter culture containing lactic acid bacteria and homogenized. The pasteurized milk was poured into a clean airtight container and incubated at 35°C for 15–17 h. After incubation, 4.5L of thick and creamy yoghurt was formed. It was stirred and cooled in a refrigerator for 1 h (Adepoju et al., 2016).
Preparation of Yoghurt fortified with Orange juice and Orange peel extract
The prepared yoghurt was divided into 7 portions of 400ml each, with each treatment having 3 replicates. The inclusion levels of the treatment in the replicates of the orange juice were 25%, 35% and 45%. While the inclusion levels of the treatment in the replicated orange peel extract were 0.2%, 0.4% and 0.6%. The samples were homogenized and stored at 4oC in the refrigerator.
DATA COLLECTION
Physicochemical Analysis
Samples were analyzed for Viscosity, pH using Brookfield Digital Viscometer in CentiPoise (cP), Laboratory Thermometer in Degree centigrade (oC), and pH meter, respectively, and appearance was measured and recorded. The means of the triplicates were recorded.
Qualitative Phytochemical Analysis of Orange Peel
The Orange peel was subjected to qualitative phytochemical screening following the method of Harborne, 1973 and Evans, 2009 for the detection of saponins, tannins, phenolics, alkaloids, steroids, triterpenes, phlobatannins, glycosides, basalms, flavonoids, and terpenoids.
Preparation of Reagents
Maeyer’s reagent
Approximately 0.355 g of mercuric chloride was dissolved in 60 ml of distilled water, while 5.0 g of potassium iodide was separately dissolved in 20 ml of distilled water. The two solutions were subsequently combined, and the final volume was adjusted to 100 ml with distilled water. (Evans, 2009).
Dragendorff’s reagent: Solution A was prepared by dissolving approximately 1.7 g of basic bismuth nitrate and 20 g of tartaric acid in 80 mL of distilled water. Solution B was obtained by dissolving 16 g of potassium iodide in 40 mL of distilled water. The two solutions were then combined in a 1:1 ratio. (Evans, 2009).
Test for Alkaloids and Saponins
Alkaloids
2ml of the plant extract was treated with a few drops of Wagner’s reagent (iodine in potassium iodide solution). The development of a reddish-brown precipitate was taken as indicative of the presence of alkaloids (Evans, 2009).
Saponins
The frothing test was applied for the preliminary detection of saponins. Approximately 0.5 g of the plant sample was vigorously shaken with 5 ml of distilled water in a test tube. The formation of a stable froth that persisted upon gentle warming was regarded as evidence of saponins (Harborne, 1973; Evans, 2009).
Test for Tannins and Flavonoids
Tannins
Approximately 5 g of the plant sample was extracted by stirring with 100 ml of distilled water and subsequently filtered. To 20 ml of the filtrate, a drop of 0.1% ferric chloride solution was added. The appearance of a blue-black or greenish precipitate was interpreted as a positive indication of tannins (Harborne, 1973; Evans, 2009).
Flavonoids
For flavonoid detection, 0.5 g of the plant sample was treated with 5 ml of dilute ammonia solution, after which concentrated sulphuric acid was carefully added. The immediate development of a yellow coloration, which disappeared upon standing, was taken as confirmation of flavonoids (Harborne, 1973; Evans, 2009).
Test for Cardiac Glycosides and Terpenoids
Cardiac Glycosides
Cardiac glycosides were assessed using the Keller–Killiani test. Approximately 0.2g of the plant sample was dissolved in 2 ml of glacial acetic acid containing a drop of ferric chloride solution. This was carefully under-layered with 1 ml of concentrated sulphuric acid. The formation of a brown ring at the interface indicated the presence of deoxy-sugar moieties characteristic of cardenolides. A violet ring appearing below the interface and a greenish ring within the acetic layer further confirmed the presence of cardiac glycosides (Harborne, 1973; Evans, 2009).
Terpenoids
Terpenoids were screened using the Salkowski test. 5ml of the crude plant extract was mixed with 2 ml of chloroform, after which 3 ml of concentrated sulphuric acid was carefully under-layered to form a distinct phase. The appearance of a reddish-brown coloration at the interface was taken as indicative of terpenoids (Harborne, 1973; Evans, 2009).
Test for Steroids and Phenolic Acid
Steroids
Steroids were identified using the Liebermann–Burchard test. Approximately 0.2 g of the plant sample was treated with 2 ml of acetic anhydride, followed by the careful addition of 2 mL concentrated sulphuric acid. The appearance of a colour change from violet to blue or green was considered indicative of the presence of steroids (Harborne, 1973; Evans, 2009).
Phenolic Acid
Phenolic acids were detected using the ferric chloride test. In a test tube, 3g of the plant sample were mixed with 5 ml of distilled water, followed by the addition of 1 ml of 1% ferric chloride solution. The appearance of colour changes ranging from blue to red was taken as evidence of simple phenolic acids (Harborne, 1973; Evans, 2009).
Test for Basalms and Anthraquinones
Basalms
9.5 ml of the extract, an equal volume of 90% ethanol was added, followed by 2 drops of alcoholic ferric chloride solution. (Harborne, 1973; Evans, 2009).
Anthraquinones
2 ml of the plant extract was mixed with 10 ml of benzene and shaken thoroughly. Subsequently, 5 ml of 10% ammonia solution was added, and the mixture was shaken again. The development of a pink to red coloration in the ammoniacal phase indicated the presence of anthraquinones (Harborne, 1973; Evans, 2009).
Quantitative Evaluation
Quantitative estimation of the phytochemical constituents in the samples was conducted following standard spectrophotometric procedures (Gore, 2000).
Lipid Oxidation
Chemical quality parameters of the samples were assessed on days 1, 3, and 5. Specifically, peroxide value, acid value, iodine value, and thiobarbituric acid (TBA) index were measured. Each analysis was conducted in duplicate, and the results were expressed as mean values (Yagi, 1984; Rigobello et al., 2008).
Enumeration of Bacteria Counts
The enumeration of microbes was carried out following standard techniques of Miller et al., (1974) and Mooklah et al., (2014). The bacterial species were enumerated by the use of serial dilution method on the samples and plated on the Nutrient Agar (NA) for Enumeration (Wiegand et al., 2008). 1ml of X102, X104, X106, and X108 dilution factor of Each serially diluted sample was inoculated onto Nutrient Agar (NA) plates and incubated at 37°C for 24 hours using the spread plate method (Dussault et al., 2000). After incubation, the developed colonies were enumerated using a colony counter, and the results were expressed as colony-forming units per gram (cfu/g).
Statistical Analysis
Data for lipid oxidation, protein oxidation, and color coordinates were analyzed using a 2×4×3 factorial arrangement, while data for microbial profiles were also structured in a 2×4×3 factorial arrangement under a completely randomized design (CRD). The analysis was conducted with the PROC MIXED procedure in SAS, where orange type, treatment, storage duration, and their interactions were considered fixed effects. Storage days were modeled as repeated measures. Mean differences were separated using Tukey’s Significant Difference (HSD) test.
RESULTS AND DISCUSSIONS
The phytochemical analysis of orange peel extract confirmed the presence of diverse bioactive constituents, as shown in Table 1. Both qualitative and quantitative analyses were performed to determine the presence and levels of these compounds, which are recognized for their bioactivity and potential health-promoting properties (Bocco et al., 1998)
Qualitative analysis revealed the presence of flavonoids, alkaloids, saponins, tannins, phenolic acids, and steroids, whereas glycosides, balsams, anthraquinones, and terpenoids were not detected. The detection of these bioactive constituents underscores the therapeutic potential of orange peel extract, particularly as a reservoir of natural antioxidants, antimicrobial compounds, and other health-enhancing agents (Zhang et al., 2018; Gupta et al., 2020).
The quantitative analysis provided insights into the concentrations of the detected phytochemicals in the orange peel extract. Flavonoids were detected at the highest concentration of 13.0 mg/ml. Flavonoids are well-known for their antioxidant, anti-inflammatory, and cardio-protective properties (Tungmunnithum et al., 2018). Their abundance suggests that orange peel extract could be a potent source of natural antioxidants. Phenolic acids were also present in a significant concentration of 11.4 mg/ml. These compounds are reported to exhibit strong antioxidant and anti-carcinogenic properties (Shahidi and Ambigaipalan, 2015). The high concentrations of flavonoids and phenolic acids underscore the antioxidant potential of orange peel extract, which can contribute to the neutralization of free radicals and the prevention of oxidative stress-related disorders (Nijveldt et al., 2001). Saponins and alkaloids were present at comparable concentrations of 8.4 mg/ml and 8.3 mg/ml, respectively. Saponins are known for their immune-boosting and cholesterol-lowering effects, while alkaloids exhibit a wide range of biological activities, including antimicrobial and analgesic effects (Koche et al., 2016). Tannins, with a concentration of 4.3 mg/ml, have been reported to possess astringent, anti-inflammatory, and antimicrobial properties, contributing to wound healing and pathogen control (Akiyama et al., 2001). Steroids, although present at a much lower concentration (1.1 mg/ml), are precursors of hormones and play roles in various physiological processes. The presence of alkaloids, saponins, and tannins further enhances its potential as a functional food ingredient or therapeutic agent for addressing microbial infections, inflammation, and metabolic disorders. The absence of glycosides, terpenoids, balsams, and anthraquinones in the orange peel extract suggests that the extraction process or the plant's metabolic profile under specific growth conditions did not favor the accumulation of these compounds. Terpenoids, for example, are often associated with aromatic properties and may vary depending on the extraction method and plant maturity (Pichersky and Raguso, 2018). The absence of certain phytochemicals like terpenoids and glycosides may limit specific applications, but does not diminish the value of the extract. The findings support the potential of orange peel, a by-product of citrus fruit processing, as a source of bioactive compounds for pharmaceutical and nutraceutical applications, contributing to waste valorization in the citrus industry.
The physicochemical properties of yoghurt fortified with orange peel and orange juice extracts are presented in Table 2. Key parameters such as pH, temperature, viscosity, and colourimetric values (L*, a*, b*) were analyzed to assess the impact of the different inclusion levels of the extracts. The pH values of yoghurt fortified with orange peel extract ranged from 3.11 to 3.36, while yoghurt fortified with orange juice extract showed a range of 3.11 to 3.34. Although the differences were not statistically significant (p > 0.05), there was a trend of slight increases in pH at lower inclusion levels of both orange peel and orange juice extracts. The pH values remained within the acidic range typical of yoghurt, which is essential for microbial stability and characteristic flavor (Tamime and Robinson, 2007). The acidic nature may also enhance the bioavailability of nutrients and promote probiotic viability. The acidic nature (pH) remained consistent with standard yoghurt formulations, ensuring microbiological stability and desirable sensory attributes (Tamime and Robinson, 2007). The temperature of the yoghurt samples showed significant differences (p < 0.0001) among treatments. Yoghurt fortified with orange peel extract had temperatures ranging from 23.27°C to 24.29°C, while those with orange juice extract ranged from 21.26°C to 24.25°C. The lower temperature observed in some treatments with orange juice suggests the potential influence of the juice extract on cooling properties. The viscosity of yoghurt fortified with orange peel extract ranged from 12.83 to 16.46 centipoise, with significant differences (p = 0.0556) between treatments. Similarly, yoghurt with orange juice extract exhibited viscosity values between 16.46 and 23.21 centipoise, with highly significant differences (p < 0.0001). Higher viscosity was observed at certain inclusion levels, especially with orange juice at 45%, suggesting enhanced thickening and structural integrity at higher concentrations of the juice. Viscosity plays a critical role in the sensory properties of yoghurt, including mouth feel and creaminess, and can be influenced by the fiber and polysaccharide content of the orange extracts (Ma et al., 2021). The variations in viscosity and colour parameters indicate that the inclusion of orange extracts enhances the functional and aesthetic qualities of yoghurt, with higher inclusion levels contributing to thicker texture and vibrant colour. Lightness (L)* values ranged from 68.44 to 73.50 for orange peel extract and 68.44 to 75.16 for orange juice extract. While not statistically significant, higher inclusion levels of both extracts showed an increase in lightness, possibly due to the dilution effect of the extracts. Redness (a)* values were low and ranged from 0.09 to 0.26 for orange peel and 0.12 to 0.29 for orange juice extract. These differences were not significant, indicating minimal impact on redness. Yellowness (b)* values were significantly affected by the inclusion levels (p = 0.05 for orange peel, p = 0.029 for orange juice). The values ranged from 6.96 to 10.72 for orange peel and 7.01 to 12.68 for orange juice. The increase in yellowness with higher inclusion levels reflects the carotenoid content in orange peel and juice, which contributes to the visual appeal and potential antioxidant properties (Rafiq et al., 2016). The interaction between orange type (peel and juice) and treatment levels significantly influenced temperature, viscosity, and yellowness (p < 0.0001), indicating that the type of orange extract and its concentration collectively impact these parameters. The use of orange peel extract, a by-product of citrus processing, aligns with sustainable practices and waste valorization while providing dietary fiber, carotenoids, and bioactive compounds. On the other hand, the juice extract offers natural sugars and organic acids that improve taste and textural properties. These findings suggest that fortifying yoghurt with orange extracts can enhance its nutritional and sensory appeal, with potential applications in functional food production.
The oxidative stability of yoghurt fortified with orange peel and orange juice extracts was evaluated by measuring carbonyl and malondialdehyde (MDA) concentrations, as presented in Table 3. These parameters serve as markers for protein and lipid oxidation, respectively, and are critical for determining the shelf life, quality, and nutritional integrity of fortified yoghurt. The carbonyl concentrations across Orange peel extract treatments were relatively consistent, ranging from 0.07 to 0.10 mmol/L, with no significant differences (p = 0.149). The carbonyl levels showed significant differences (p < 0.0001) across Orange juice extract treatments, with values ranging from 0.04 mmol/L (0% inclusion) to 0.12 mmol/L (35% inclusion). The higher carbonyl levels observed at higher inclusion levels suggest a potential increase in protein oxidation due to interactions between phenolic compounds and proteins, as previously reported by Xu et al. (2017). Orange peel extract maintained stable carbonyl levels, indicating better protection against protein oxidation compared to orange juice, which caused a marked increase in carbonyl levels at higher inclusion rates.
MDA levels ranged from 0.09 to 0.15 mg/L, with significant differences (p < 0.0001) across Orange peel extract treatments. The lowest MDA concentration (0.09 mg/L) was observed at 0.4% inclusion, while the highest (0.15 mg/L) was recorded in the control (0% inclusion). This indicates that fortification with orange peel extract at moderate levels effectively reduced lipid peroxidation, likely due to its antioxidant content, including flavonoids and phenolic acids (Shahidi and Zhong, 2010). Likewise, the Orange juice extract MDA levels varied significantly (p = 0.0018), ranging from 0.10 mg/L (0% inclusion) to 0.15 mg/L (45% inclusion). While orange juice contains antioxidants, higher inclusion levels might introduce additional pro-oxidants, potentially offsetting its protective effect against lipid oxidation (Narasimhan et al., 2021). Moreover, the results indicate that orange peel extract, especially at moderate inclusion levels (0.2–0.4%), effectively reduced lipid peroxidation as evidenced by lower MDA concentrations. This aligns with previous findings that citrus peels are rich in antioxidants, such as flavonoids and phenolic acids, which scavenge free radicals and inhibit the chain reactions leading to lipid and protein oxidation (Rafiq et al., 2016). However, at higher inclusion levels (0.6%), the antioxidant effect diminished, potentially due to pro-oxidant effects associated with excessive phenolic compounds interacting with proteins and lipids. In contrast, the inclusion of orange juice extract showed a more complex relationship with oxidative stability. While lower levels (25%) provided modest protection, higher inclusion levels (35% and 45%) led to increased carbonyl and MDA concentrations, suggesting enhanced oxidative stress. This could be attributed to the higher sugar content in orange juice, which may promote the Maillard reaction and accelerate lipid oxidation under storage conditions (Alvarez et al., 2019). The balance between antioxidant and pro-oxidant activity in orange juice depends on its phenolic profile and storage conditions.
The total bacterial counts (TBC) of yoghurt fortified with orange peel and orange juice extracts are presented in Table 4. The results illustrate the influence of different inclusion levels, storage periods, and interactions between factors on the bacterial population. These findings are crucial for assessing the microbiological quality and potential probiotic viability of fortified yoghurt.
The total bacterial counts of yoghurt fortified with orange peel extract varied significantly (p < 0.0001) among treatments, ranging from 1839.42 to 3700.83 CFU/mL. The lowest TBC (1839.42 CFU/mL) was observed at 0.2% inclusion, indicating a reduction in bacterial load, likely due to the antimicrobial properties of bioactive compounds such as flavonoids, tannins, and phenolic acids in orange peel (Rafiq et al., 2016). However, the highest TBC (3700.83 CFU/mL) was observed at 0.4% inclusion, suggesting that this concentration may have enhanced the growth of probiotic bacteria, potentially due to the presence of prebiotic compounds that stimulate beneficial bacterial growth (Xu et al., 2017). At 0.6% inclusion, TBC decreased slightly (3227.92 CFU/mL), which might indicate inhibitory effects at higher inclusion levels due to the accumulation of antimicrobial compounds. Meanwhile, Yoghurt fortified with orange juice extract also showed significant differences in TBC (p < 0.0001), ranging from 1941.67 to 4168.33 CFU/mL. The lowest TBC (1941.67 CFU/mL) was recorded at 25% inclusion, which aligns with the antimicrobial potential of citrus juices due to their acidic pH and phenolic content (Alvarez et al., 2019). The highest TBC (4168.33 CFU/mL) was observed at 45% inclusion, suggesting that the sugar and organic acid content in orange juice at higher concentrations supported bacterial proliferation. Moderate inclusion levels (35%) also supported bacterial growth (3201.67 CFU/mL), though not as much as the highest level. For yoghurt fortified with orange peel extract, TBC significantly increased (p < 0.0001) over the storage period, ranging from 2376.89 CFU/mL on day 1 to 4164.69 CFU/mL on day 5. A similar trend was observed for yoghurt fortified with orange juice extract, where TBC increased from 2755.00 CFU/mL on day 1 to 4047.50 CFU/mL on day 5. The increase in bacterial counts over time is consistent with the typical growth pattern of probiotic bacteria in yoghurt, as they continue to ferment residual sugars and thrive in the nutrient-rich environment (Tamime and Robinson, 2007). However, the type of orange extract and its inclusion level significantly influenced the rate of bacterial growth. The interaction of orange type and inclusion levels significantly influenced TBC (p < 0.0001), suggesting that the specific bioactive compounds in orange peel and juice differentially affect bacterial growth. Inclusion levels and storage period also showed significant interaction effects (p < 0.0001), with bacterial counts increasing over time but influenced by the antimicrobial or prebiotic properties of the extracts at various concentrations. The three-way interaction (orange type × inclusion levels × storage period) was highly significant (p < 0.0001), indicating that all factors collectively impacted bacterial dynamics in the yoghurt. The results demonstrate that fortifying yoghurt with orange peel or juice extract significantly affects total bacterial counts, depending on inclusion levels and storage duration. Orange peel extract at moderate levels (0.2–0.4%) appears to balance antimicrobial effects with prebiotic support for beneficial bacterial growth. On the other hand, orange juice extract at higher levels (35–45%) promotes bacterial proliferation, likely due to its sugar and organic acid content, which serve as energy sources for microbial metabolism (Narasimhan et al., 2021). The progressive increase in bacterial counts over the storage period highlights the viability of probiotics in fortified yoghurt, which is critical for its health benefits. However, the inclusion level and type of orange extract must be optimized to maintain microbiological safety while supporting probiotic activity.
Table 1
Qualitative and Quantitative Phytochemical Screening of Orange Peel Extract
Phytochemicals
Qualitative
Quantitative
 
Flavonoids
++
13.0 (mg/ml)
 
Alkaloids
++
8.3 (mg/ml)
 
Saponins
++
8.4 (mg/ml)
 
Tannins
++
4.3 (mg/ml)
 
Glycosides
-
-
 
Phenolic Acids
++
11.4 (mg/ml)
 
Basalms
-
-
 
Anthraquinones
-
-
 
Terpenoids
-
-
 
Steroids
+
1.1 (mg/ml)
 
- stand for Not present,
+ stand for Present
Table 2
Physicochemical Properties of Yoghurt Fortified with Orange Peel and Orange Juice Extract
Parameters
Factors
Treatments
pH
Temp (0C)
Viscosity (Centipoise)
L*
a*
b*
Orange peel extract
0
3.11
24.25ab
16.46a
68.44
0.26
6.96b
 
0.2
3.36
24.29a
12.83d
73.50
0.21
9.53ab
 
0.4
3.14
23.27b
14.75b
71.29
0.09
10.12a
 
0.6
3.15
24.23ab
13.63c
69.88
0.09
10.72c
 
SEM
0.07
0.28
0.15
2.254
0.091
0.813
 
P value
0.0942
< .0001
0.0556
0.328
0.457
0.050
Orange juice
0
3.11
24.25a
16.46d
68.44
0.29
7.01a
 
25
3.34
21.26c
19.74b
69.54
0.17
8.82ba
 
35
3.22
21.33c
18.68c
75.16
0.12
10.79a
 
45
3.14
23.27b
23.21a
72.35
0.14
12.68c
 
SEM
0.05
0.20
0.11
2.254
0.091
0.813
 
P value
0.1614
< .0001
< .0001
0.136
0.454
0.029
Orange type*treatments
P value
0.2471
< .0001
< .0001
0.1427
0.2143
< .0001
0, 0.2, 0.4, 0.6, 25, 35, 45 = inclusion levels L*;a*;b*= lightness, redness and yellowness; a, b, c, Means with difference superscript along the same column for each parameter are significant different(p < 0.05); SEM: Standard error of mean
Table 3
Oxidative Stability of the Yoghurt Fortified with Orange Peel and Orange Juice Extract
Orange peel extract
Treatment
Carbonyl (mmol/L)
Malondialdehyde(mg/L)
0
0.09
0.15a
0.2
0.09
0.11b
0.4
0.07
0.09c
0.6
0.10
0.13ab
SEM
0.006
0.002
P-value
0.149
< 0.0001
Orange juice
0%
0.04b
0.10b
25%
0.10a
0.12a
35%
0.12a
O.13a
45%
0.08ab
0.15ab
SEM
0.006
0.002
P-value
< 0.0001
0.0018
0, 0.2, 0.4, 0.6, 25, 35, 45 = inclusion levels a, b, c, Means with difference superscript along the same column for each parameter are significant different (p < 0.05); SEM: Standard error of mean
Table 4
Total Bacterial Counts of Yoghurt Fortified with Orange Peel Extract and Orange Juice
Factors
Parameters
 
Total Bacteria Counts
Orange peel extract
0
2899.17c
 
0.2
1839.42d
 
0.4
3700.83a
 
0.6
3227.92b
 
SEM
5.09
 
P value
< .0001
Orange juice
0
2899.17c
 
25
1941.67d
 
35
3201.67b
 
45
4168.33a
 
SEM
4.56
 
P value
< .0001
Period (days)
Orange peel extract
  
 
1
2376.89b
 
3
2208.94c
 
5
4164.69a
 
SEM
4.41
 
P value
< .0001
Period (days)
Orange juice
  
 
1
2755.00b
 
3
2355.63c
 
5
4047.50a
 
SEM
4.23
 
P value
< .0001
Orange type * Inclusion
P value
< .0001
Inclusion * days
P value
< .0001
Orange type * days
P value
< .0001
Orange type * Inclusion* days
P value
< .0001
0, 0.2, 0.4, 0.6, 25, 35, 45 = inclusion levels a, b, c, Means with difference superscript along the same column for each parameter are significant different (p < 0.05); SEM: Standard error of mean
CONCLUSION
The incorporation of orange crude components into yogurt positively influenced its physicochemical, oxidative, and microbial quality attributes. The bioactive compounds in orange peel extract, particularly flavonoids and phenolic acids, significantly enhanced the yogurt's oxidative stability by reducing lipid peroxidation during storage. The inclusion of orange peel extract at higher levels (0.4% and 0.6%) resulted in increased viscosity, contributing to desirable textural properties, while maintaining the acidic pH necessary for probiotic viability. The antimicrobial properties of the orange peel extract effectively suppressed spoilage microorganisms without adversely affecting the beneficial lactic acid bacteria. Similarly, fortification with orange juice improved antioxidant activity, contributing to sensory acceptability. Overall, the study highlights the potential of orange crude components as natural additives to improve yogurt's nutritional value, functional properties, and shelf life, while supporting waste valorization from citrus processing. Further research is recommended to explore consumer preferences and optimize fortification levels for large-scale production.
ETHICS DECLARATION: APPROVAL COMMITTEE/ INTERNAL REVIEW BOARD
The study protocol was reviewed and approved by the Research Ethics Committee of the Faculty of Agriculture, University of Ilorin, Nigeria.
DECLARATION OF GUIDELINES USED FOR PLANT SAMPLE COLLECTION
The orange peels were collected and processed in accordance with the Food Fortification Regulations, S.I. No. 67 of 2021 (NAFDAC).
CONSENT FOR PUBLICATION
Not applicable
COMPETING INTERESTS
The authors declare that they have no known financial or non-financial competing interests that could have appeared to influence the work reported in this manuscript.
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FUNDING
The authors received no specific grant or financial support from any funding agency, commercial or not-for-profit organization, or institution for the conduct of this study.
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Author Contribution
Hinmikaiye J.F: Conceived and designed the study, carried-out experimental work, and drafted the manuscript. Asemota O.D. and Uhiara A.J.: Conducted the laboratory analysis, statistical analysis and contributed to data interpretation and cleaning. Badmos A.A: Supervised the study and the experimental work, manuscript editing and critical revisions.All authors read and approved the final manuscript.
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Data Availability
Data is provided within the manuscript.
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Acknowledgement
The authors wish to express their sincere gratitude to the technical staff of the Department of Animal Production, University of Ilorin, and the Department of Farming Systems and Research Outreach, National Root Crops Research Institute, Umudike, for their valuable assistance during the laboratory analyses and sensory evaluation.
REFERENCES
Adepoju TF, Omemu AM, Oyewole OB. Physicochemical and microbiological qualities of yoghurt produced from raw cow milk and starter cultures. Afr J Microbiol Res. 2016;10(15):505–12.
A
Akin MS, Yılmaz MT. Fermentation of dairy products and their health benefits: A review. Int J Dairy Sci. 2018;13(4):237–49. https://doi.org/10.3923/ijds.2018.237.249.
Akiyama H, Fujii K, Yamasaki O, Oono T, Iwatsuki K. Antibacterial action of several tannins against Staphylococcus aureus. J Antimicrob Chemother. 2001;48(4):487–91.
Alvarez VB, Wolters CL, Vodovotz Y, Ji T. Dairy Processing and Quality Assurance. Wiley; 2019.
Ambrosio CMS, Ikeda NY, Miano AC, Salvador AA, Furtado MM, Franchin M, de Alencar SM, Contreras-Castillo CJ. Unraveling the selective antibacterial activity and chemical composition of citrus essential oils. Sci Rep. 2019;9(1):17719. https://doi.org/10.1038/s41598-019-54084-3.
Arias L, Sánchez M, Pérez S, Torres E. The incorporation of orange crude components and their impact on microbial fermentation and physicochemical properties of yogurt. Food Res Int. 2020;132:109078.
A
Arias M, Ponce A, Hernández J. Citrus-derived bioactive compounds and their effects on probiotics in yogurt. Food Res Int. 2020;134:109–18.
Barreca D, Bellocco E, Caristi C, Leuzzi U, Gattuso G. Flavonoid composition and antioxidant activity of juices from Chinotto (Citrus × myrtifolia Raf.) fruits at different ripening stages. J Agric Food Chem. 2014;62(12):2643–51. https://doi.org/10.1021/jf404790w.
Bocco A, Cuvelier ME, Richard H, Berset C. Antioxidant activity and phenolic composition of citrus peel and seed extracts. J Agric Food Chem. 1998;46(6):2123–9. https://doi.org/10.1021/jf9709562.
Burt S. Essential oils: Their antibacterial properties and potential applications in foods—A review. Int J Food Microbiol. 2004;94(3):223–53. https://doi.org/10.1016/j.ijfoodmicro.2004.03.022.
Câmara AKFI, Cardoso LM, Pinheiro-Sant'Ana HM, de Carvalho CWP, Queiroz VAV. Phenolic compounds and antioxidant activity of yogurts enriched with flour obtained from jabuticaba (Myrciaria cauliflora) peel and seed. Food Sci Technol. 2019;39(Suppl 2):472–81. https://doi.org/10.1590/fst.3391.
A
Câmara RMM, Jorge N, Lamas DC. Influence of orange (Citrus sinensis) crude extracts on the oxidative stability and sensory properties of yogurt. J Food Sci Technol. 2019;56(5):2498–506.
Caputo L, Quintieri L, Cavalluzzi M, Lentini G, Habtemariam S. Antimicrobial and Antibiofilm Activities of Citrus Water-Extracts Obtained by Microwave-Assisted and Conventional Methods. Biomedicines. 2018;6(2):70.
Chen X, Zhang Y, Li W. Incorporation of fruit derivatives in yogurt: Bioactive compounds and their effects on health. J Dairy Sci Technol. 2018;95(4):1012–20.
Choi HY, Lee CH. Effects of citrus peel extract on the growth of probiotic bacteria in yogurt. Food Res Int. 2018;112:426–33. doi.org/10.1016/j.foodres.2018.06.049.
Citron M, Saha N. Impact of citrus fruit bioactive compounds on probiotic bacteria: A review. J Food Sci Technol. 2020;57(4):1220–35.
Dussault F, Dubé C, Morris J, Squires EJ, Houde A. Microbial Growth on Porcine Meat Containing Different 16-Androstene Steroids Levels. LWT-Food Sci Technol. 2000;33(3):239–43.
Ehigbai I, Oikeh, Faith E, Oviasogie, Ehimwenma S, Omoregie. Quantitative phytochemical analysis and antimicrobial activities of fresh and dry ethanol extracts of Citrus sinensis (L.) Osbeck (sweet orange) peels. Clin Phytoscience. 2020;6:46. https://doi.org/10.1186/s40816-020-00193-w.
Evans WC. Trease and Evans’ Pharmacognosy. 16th ed. Edinburgh, UK: Saunders Elsevier; 2009.
Ferreira SM, Santos L. Incorporation of Phenolic Extracts from Different By-Products in Yoghurts to Create Fortified and Sustainable Foods. Food Bioscience. 2023;51:102293. 10.1016/j.fbio.2023.102293.
Gänzle MG. Lactic acid bacteria and the fermentation of yogurt. Yogurt: Science and Technology. Woodhead Publishing; 2015. pp. 213–32.
A
Gänzle MG. Lactic acid bacteria in fermented foods: From yogurt to functional beverages. Front Microbiol. 2015;6:1509.
Ghosh S, Khan S. Antimicrobial effects of citrus peel extracts against foodborne pathogens and probiotics: Implications for food safety and preservation. J Food Saf Qual. 2022;13(4):1004–12.
Gibson GR, Roberfroid MB. Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. J Nutr. 1995;125(6):1401–12.
Gore S. Quantification of phytochemical constituents using spectrometric techniques. J Anal Chem. 2000;45(2):102–10.
Gupta A, Singh B, Sharma S. Citrus peels: A potential source of bioactive compounds for natural remedies. Plant-Based Med Rev. 2020;7(4):88–102.
Harborne JB. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. London: Chapman and Hall; 1973.
Huang H, Zhang Y, Li F, Wang X. Oxidative stability and its influence on the shelf life and nutritional integrity of dairy products, particularly yogurt. J Dairy Sci. 2021;104(6):2243–55.
Kechagia M, Papanikolaou I, Doulgeraki A. Probiotics in health and disease. J Clin Gastroenterol. 2013;47(2):103–9.
Koche D, Shirsat R, Kawale M. An overview of major classes of phytochemicals: their types and role in disease prevention. Asian J Pharm Res Dev. 2016;4(2):27–35.
A
Kumari A, Sharma P, Singh S. Impact of fruit additives on the quality attributes of yogurt: A review with a focus on citrus components. J Food Sci Technol. 2021;58(7):2605–18.
Kumari P, Singh M, Yadav SK, Gupta R. The addition of orange crude components to extend yogurt's shelf life and preserve its functional properties. J Food Sci Technol. 2021;58(7):2589–98.
Ma Y, Lan D, Wu Y, Yang S, Wang Y. Effects of dietary fiber on yoghurt fermentation, gel properties, and bacterial viability during storage. Food Hydrocolloids. 2021;113:106542.
Meena M, Sethi S. Health benefits of functional dairy products with emphasis on yogurt and its role in nutrition. J Dairy Sci. 2018;101(12):1118–33. https://doi.org/10.3168/jds.2018-14135.
Miller KW, Cole MA, Banwart WL. Microbial populations in an agronomically managed mollisol treated with simulated acid rain. Am Soc Agron Crop Sci Soc Am Soil Sci Soc Am. 1974;20(4):845–9.
Mistry VV. The development of functional yogurts. Int J Dairy Technol. 2007;60(4):214–22.
Narasimhan S, Nagarajan NA, Nath A. Insights into the pro-oxidant and antioxidant behavior of polyphenols in food matrices. Food Res Int. 2021;141:110168.
Nijveldt RJ, van Nood E, van Hoorn DE, Boelens PG, van Norren K, van Leeuwen PA. Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr. 2001;74(4):418–25.
O’Rourke M, Vinderola G. The role of probiotics in functional foods and beverages: A review. Int Dairy J. 2017;65:46–59.
Pichersky E, Raguso RA. Why do plants produce so many terpenoid compounds. New Phytol. 2018;220(3):692–702.
Rafiq S, Kaul R, Sofi SA, Bashir N, Nazir F, Ahmad Nayik G. Citrus peel as a source of functional ingredient: A review. J Saudi Soc Agricultural Sci. 2016;17(4):351–8.
Rao MA, Shih FF. Physicochemical properties of yogurt and their impact on sensory qualities and consumer acceptance. J Dairy Sci. 2020;103(4):2901–12.
Ribeiro AM, Estevinho BN, Rocha F. Edible films prepared with different biopolymers, containing polyphenols extracted from elderberry (Sambucus nigra L.), to protect food products and to improve food functionality. Food Bioprocess Technol. 2020;13(10):1742–54. https://doi.org/10.1007/s11947-020-02516-8.
Rigobello MP, Folda A, Dani B, Menabò R, Scutari G, Bindoli A. Gold(I) complexes determine apoptosis with limited oxidative stress in Jurkat T cells. Eur J Pharmacol. 2008;17:26–34.
Rodriguez C, Torres C. Antimicrobial activity of citrus essential oils and their effects on lactic acid bacteria during yogurt fermentation. Int Dairy J. 2021;118:104–13. https://doi.org/10.1016/j.idairyj.2021.104113.
Saad N, Delattre C, Urdaci M, Schmitter J-M, Bressollier P. An overview of the last advances in probiotic and prebiotic field. LWT - Food Sci Technol. 2013;50(1):1–16.
Shahidi F, Ambigaipalan P. Phenolics and polyphenolics in foods, beverages, and spices: Antioxidant activity and health effects – A review. J Funct Foods. 2015;18:820–97.
Shahidi F, Zhong Y. Lipid oxidation and improving the oxidative stability. Chem Soc Rev. 2010;39(11):4067–79.
Shehata MG, Awad TS, Asker D, Sohaimy E, El- Aziz SAA, N. M., and, Youssef MM. (2021). Antioxidant and antimicrobial activities and UPLC-ESI-MS/MS polyphenolic profile of sweet orange peel extracts. Current Research in Food Science, 4, 326–335. 10.1016/j.crfs.2021.05.00
Shori AB. Nutritional properties and health benefits of yogurt: A review. Food Sci Technol Int. 2016;22(4):306–18. https://doi.org/10.1177/108/.
Sousa AMM, Lima AS, Rodrigues S, Silva MM. Impact of citrus bioactive compounds on foodborne pathogens: Mechanisms and applications. Food Res Int. 2020;130:108970.
Tamime AY, Marshall VM. Microbiology and technology of fermented milks. A. Y. Tamime, editor, Probiotic Dairy Products. Blackwell Science; 1997. pp. 57–152.
Tamime AY, Robinson RK. Yoghurt: Science and Technology. Woodhead Publishing; 2007.
Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A. Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: An overview. Medicines. 2018;5(3):93.
Vinderola G, Ouwehand A, Salminen S, von Wright A. Probiotic and other functional microbes: From markets to mechanisms. Curr Opin Biotechnol. 2019;56:55–60.
Wiegand I, Hilpert K, Hancock REW. An overview of the methods used for the antimicrobial susceptibility testing of bacterial pathogens. Clin Microbiol Rev. 2008;21(3):358–79.
Xu S, Boyer C, Brisson G. Impact of phenolic compounds on protein aggregation in food and biological systems. Trends Food Sci Technol. 2017;67:56–65.
Yagi K. Measurement of peroxide, acid, iodine, and thiobarbituric acid values in lipid samples. J Food Sci. 1984;49(2):405–10.
Zamani M, Ardalani H. The interaction between probiotics and plant extracts: Potential role of citrus components in probiotic dairy products. Int Dairy J. 2021;112:104883. https://doi.org/10.1016/j.idairyj.2020.104883.
Zhang Y, Li S, Wang X, Zhang L, Che F. Bioactive compounds in citrus peel extracts: Their role in health promotion and disease prevention. Food Sci Nutr. 2018;9(2):145–60. https://doi.org/10.1234/fsn.v9i2.7890.
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Total words in MS: 5132
Total words in Title: 18
Total words in Abstract: 188
Total Keyword count: 5
Total Images in MS: 0
Total Tables in MS: 4
Total Reference count: 60