Effect of ultrasonic on the chemical composition of stirred yoghurt supplemented with orange peel powder

Heat treatments in dairy processing exert undesirable effects on the final product. Alternatively, a high-intensity ultrasonic technique was applied to process stirred yoghurt supplemented with orange peel powder (OPP). For this reason, OPP was added to the fresh milk at concentrations of 0.0, 1, and 2%, and then the mixture was subjected to ultrasound waves (24 kHz at 45 ± 2 °C for 30 min) before being incubated with the starter culture w/v%. Analysis of fresh and stored samples (7 and 14 days) showed that ultrasonic-treated milk (UTM) had a longer fermentation time than the heat-treated sample (HTM). No significant differences in total solid content, protein, ash, and volatile fatty acids were observed between UTM and HTM samples, while slight differences due to the addition of OPP were detected. Treating milk with ultrasound waves improved the processed yogurt’s apparent viscosity and water-holding capacity; however, adding OPP resulted in minor reverse effects. Total flavonoid and phenolic contents and antioxidant activity increased by adding OPP and were significantly higher in yoghurt made from UTM than in HTM. Overall, the application of ultrasonication in processing OPP- supplemented yoghurt resulted in better chemical and physical properties and maintained bioactive antioxidant components compared to yogurt manufactured from HTM.


Introduction
Yogurt is a fermented milk product rich in numerous vitamins and minerals, such as calcium, potassium, magnesium, and vitamin B12.It is also rich in probiotic bacteria, increasing its health benefits and nutritional value [1,2].It also and aromatics that may be affected by temperature, different storage conditions, and exposure of the product to light and oxygen [11].Ultrasound is an excellent alternative to studying, as it is a good alternative to applying heat, which may affect vitamins and other antioxidants [12].Polyphenols are among the most effective antioxidant compounds because of their diverse chemical composition, some simple and some complex [13,14], which have a significant role in cancer reduction and prevention because of their scavenging properties [15].Polyphenols can be preserved when treated with sound waves below 40 kHz and temperatures below 50 °C.However, sound waves above 40 kHz form free hydroxyl radicals, which decompose polyphenols, especially in high water content [16].Ultrasound also affects fat when frequencies are from 16 to 100 kHz, where shear forces act as an alternative to the homogenization process, and the size of fat globules decreases [17].
The lipid granule membrane is broken and disrupted, and the lipid granule becomes tightly coated with proteins, which depend on temperature and energy density [18].They also inhibit bacteria and enzymes, so they preserve and prolong the final product, which may affect its quality [19].These waves can also be combined with heat, called the thermosonication treatments.This increases the possibility of killing organisms associated with the food product and inhibits the activity of proteases and lipases [20].
Residual orange peel (OP) is discarded from the food industry, although its use saves costs when added to food processing.OP is distinguished by its high nutritional value, which helps us to use it in many food industries, especially as it is a biodegradable substance et al. [21].It is industrial waste rich in many different nutrients that make it used in medicine because of its effect on the health of the colon, lungs, and liver.They are rich in many other nutrients that make them used as food supplements.It is also rich in antioxidants such as ascorbic acid, phenolic compounds, flavonoids, and pectin, with antimicrobial properties that inhibit bacterial or fungal microorganisms [22,23].Also, it contains high levels of thiamine, niacin, pyridoxine, phosphorus, calcium, iron, magnesium, potassium, and soluble and insoluble dietary fiber [24,25].
The main objective of this research is to preserve the essential compounds that are affected by the pasteurization process, such as antioxidants, antimicrobials, and peptides, by replacing the pasteurization process with an ultrasound technique.

Materials and methods
Fresh cow's milk from Sakha Research Station of the Agricultural Research Center (ARC) was used.Milk's chemical composition was as follows: 4% fat, total protein 3.55%, ash 0.74 and total solid 12.23%, and acidity was recorded as 0.18%.
The yogurt culture used in the manufacture consists of Streptococcus thermophilus & Lactobacillus delbrueeckii sub ssp.bulgaricus (DVS) was gained from Hansen Copenhagen Laboratories, Denmark.The gelatin used in this study was from Misr Food Additives Company (MIFAD), Egypt.Egyptian Baladi oranges were collected from the market in Egypt.

Ultrasonic-treated milk (UTM)
Ultrasound homogenizer 4710 Series (Parmer Instrument Co., Chicago, Illinois 60,648, USA) was used for mixing the materials.The treatments were treated at a frequency of 24 kHz for 30 min at a temperature of 45 ± 2 °C.The used prop was immersed at a distance of 2.5 cm).

Fabrication of orange peel powder (OPP)
The oranges were carefully selected (intact, equal in size, and with no fungal contamination or cracks on the outside).They are washed with running tap water, and the edible parts are carefully separated.The peel of orange was carefully peeled and then cut into small pieces of equal size.Then it was dried using a pneumatic oven (Shellab-Model 1350FX-Made in the USA) at 40 ± 2 °C for approximately 48 h.The dried peels were ground into a fine powder using a mechanical laboratory mill, and the resulting flour was sieved using a 35 mesh (0.425 mm) sieve.Then the bags are packed in polyethylene and stored at 4 ± 1 °C until needed.A chemical analysis of (OPP) was carried out, and the results were as follows: fat 2.54%, total protein 16.38%, ash content 5.44, total solids 98.34%, total phenolic comet al. pounds 1106 (mg gallic acid equivalent /100 g) and flavonoids 601 (mg rutin /100 g).

Stirred yogurt manufacture
Fresh cow's milk has been divided into two parts.
In all previous treatments, a 2% yogurt starter was added as an active mixture starter and packed in sterile plastic cups with a capacity of 100 ml.Then the treatments were incubated at 42 °C until reaching pH 4.8, then cooled at 4 °C.Then the samples were kept for 15 h, and the analyzes were conducted for 14 days.Three replicates were used for each treatment.Before completing the analysis, the gel was broken by stirring it 10 times clockwise and 10 times counterclockwise using a glass rod.

Physicochemical analysis
Total solids (TS), total protein (TP), fat contents, ash, and titratable acidity lactic acid were identified by the earlier study [27].According to a recent approach, a direct distillation method determined total volatile fatty acid (TVFA) content [28].At the same time, the pH value was computed by a JENCO pH meter model 1671, USA.

Antioxidant activity I) Free radical removal activity (DPPH):
The DPPH scanning activity of the samples was tested as followed by [29].Yogurt extract samples were mixed in a 1:1 ratio (v/v) with 0.1 mM DPPH dissolved in methanol.For 30 min, the mixture was incubated in the dark at room temperature (R.T).Deionized water was used as the blank instead of the sample.Scavenging potency was detected as follows: Scavenging activity (%) = (A0 -AS) / A0 × 100 where A0 and As are the absorbances at 517 nm for the blank and the samples, respectively.II) Ferric reducing power) FRAP(: FRAP was evaluated based on the previous study with some modifications [30].The sample (0.5 ml) had an equal volume of 0.2 M phosphate buffer saline (pH 6.6) and potassium ferricyanide of 1%.The mixture was then incubated at 50 °C for 20 min.Afterward, 1 ml of TCA (10%) was added, followed by 0.15 ml of ferric chloride (0.1%).The solution was left at R.T. for 10 min and then detected spectrophotometrically at 700 nm.The blank sample was a similar mixture using deionized water instead of the piece.The increase in the absorbance indicated the reduced power.

Determination of total phenolic and flavonoid
I) Total phenolic: total phenolic contents were identified by the Folin-Ciocalteu method, explained as follows: 200 µl of the sample extract (1 mg/mL) was made up to 3 mL with distilled water; Mix thoroughly for 3 min with 0.5 ml of Folin-Ciocalteu reagent; Then 2 ml of 20% sodium carbonate (w/v) were added.The mixture was placed in the dark for an hour, and the absorbance was measured at 650 nm.The total phenol content was computed from a calibration curve.The findings were communicated as mg of gallic acid equivalent per gram dry weight [31].II) The total flavonoid: The total flavonoid content of the yogurt sample extract was estimated by the aluminum chloride chromatography method with modifications [31].50 µl of the crude extract (1 mg/mL ethanol) was made up to 1 mL of methanol and then mixed with 4 mL of water, then distilled 0.3 mL of 5% NaNO 2 solution; Then 0.3 ml of 10% AlCl 3 was added after 5 min of incubation and waited 6 min.Then, 2 mL of 1 moL/L NaOH solution was added, and the final volume was brought to 10 mL using distilled water.Then waited for 15 min, and absorbance was measured at 510 nm.The total flavonoid content was computed from the titration curve, and the result was illustrated as the equivalent of mg rutin per gram dry weight.

Apparent viscosity and water-holding capacity (WHC)
A Brookfield digital instrument Viscometer (model DV-II, Canada) fitted with four spindles was used to measure the apparent viscosity at 10 ± 2 °C.FHTB.Samples were subjected to select shear rates ranging from 3.0 to 50.0 S1 of the bullish curve.Apparent viscosity was expressed in pascals (Pa).The yogurt's water holding capacity (WHC) was centrifuged at 4500 rpm for 30 min at 10 °C for a five-gram yogurt sample, and the WHC was calculated as follows [32]: where W 1 is the weight of whey after centrifugation, and W 2 is the weight of yogurt samples.

Sensory evaluation
The sensory properties of the prepared stirred Yoghurt is mainly evaluated based on the senses Scheme presented by Fresh and stored samples, which were assessed by five denaturation and the peptides, which increases the fermentation time [35].

Chemical and physicochemical analysis
Table 1 shows that the highest TS content was recorded when OPP was used at a concentration of 2%, where HT3 treatment was recorded at 13.82% and UT3 treatment at 13.56% in 14 days.This is due to the composition of the OPP, which contains 98.34% TS.The differences between treatments were significant when using OPP but insignificant using either heat or ultrasonication [18].
Increasing the concentration of OPP increased the TP on day 14.The results on day 14 were 5.29, 5.34, 5.52, 5.31, 5.33, and 5.41% in the HT1, HT2, HT3, UT1, UT2, and UT3 treatments, respectively Table 1.This is due to the composition of OPP, which contains a TP of 16.38%.However, TP was not affected when heat treatment was compared to the ultrasound technique, and no significant differences were seen when comparing them [18].But ultrasound plays a vital role in reducing the size of protein and fat globules at 24 kHz [36,37].
The addition of OPP affected Ash content and TVFA Table 1, with results in the case of ash on the 14th day being 0.71, 0.77, 0.79, 0.71, 0.78, and 0.78%.While in the case of TVFA, they were 0.21, 0.29, 0.29, 0.21, 0.28, and 0.3 (ML 0.1 N NaOH/100 g) in HT1 and HT2, HT3, UT1, UT2, and UT3 in the same order.This may be due to the composition of the OPP.There was no significant difference between heat treatment and sonication.
Results are recorded in Table 1.Acidity increased with increasing concentration of OPP.This is due to the presence of citric and ascorbic acid in OPP.On day 14, were scored 0.21, 0.29, 0.29, 0.21, 0.28, and 0.3 in HT1, HT2, HT3, UT1, UT2, and UT3, respectively.While the acidity in yoghurt develops and increases with storage due to the activity of microorganisms that continue to produce lactic acid [34].The acidity of yoghurt was not affected by sonication and heat treatment.

Antioxidant activity
DPPH and FRAP assays commonly detect antioxidant activity based on stabilizing free radicals by donating hydrogen or electrons [38].
Figure 2 shows that using a concentration of 2% of OPP gave the best DPPH assay results, as the treatment HT3 recorded 22.7, 22.17, and 21.87% in both fresh and stored at 7 days and 14 days, respectively.During the treatment, UT3 recorded 23.98%And 23.49, and 23.36%in the case of fresh and stored at 7 and 14 days, respectively.It also turned arbitrators from the researchers of the Dairy Chemistry Department at the Animal Production Research Institute of the Agricultural Research Center specializing in sensory arbitration [33].

Statistical analysis
Triplicate measurements were taken for all parameters, and results were presented as means of standard errors.The SPSS statistical software package for analysis of variance and Duncan's test (SPSS 20 for Windows, SPSS INC., IBM, New York) was used.

Fermentation time
The effect of the ultrasound technique and the addition of OPP on the fermentation time was studied.There was a noticeable effect when adding OPP on the fermentation time, as shown in Fig. 1.With an increase in the concentration of OPP, the pH decreased, possibly due to the rise in the acidity arising from the presence of citric and ascorbic acid in OPP [34].This was evident both with heat treatment and with the use of ultrasound technique.The following scores were recorded at 180 min: 4.7, 4.6, 4.6, 5.1, 5.0, and 4.9 in treatments HT1, HT2, HT3, UT1, UT2, and UT3, respectively.
The sonication technique Fig. 1 extended the fermentation time, as it took 210 min to reach pH 4.6, while pH 4.6 was reached in the case of heat treatment at 180 min.This may be due to the effect of heat treatment on the protein Fig. 1 Fermentation time for stirred yogurt.*Heat treatment was used for treatments of HT1, HT2 and HT3 with addition 0.0, 1%, and 2% OPP, respectively.While ultrasonic technic was used for UT1, UT2, and UT3 with additional 0.0, 1%, and 2% OPP, respectively from food materials.In addition, the absence of heat treatment also greatly contributed to this respect [39].

Determination of total phenol and flavonoid content
The total phenolic and Flavonoid content of the treatment UT3 recorded the best results of 32.36, 30.43, and 30.43 mg GAE/g and 11.87, 11.72, and 11.42 mg QUE/g in the fresh and stored yogurt at 7 and 14 days in order Fig. 3, which confirmed that the use of 2% of OPP was the best concentration and indicator of the efficiency of the sonication technique.This may be due to the richness of OPP with flavonoids, as mentioned above [40].out that the sound waves treatment recorded better results than the thermal treatment.
Figure 2 shows that using a concentration of 2% of OPP gave the best results for FRAP, as the results recorded 0.335, 0.388, 0.445, 0.347, 0.401, and 0.456 in the HT1, HT2, HT3, UT1, UT2, and UT3 in order.The results also showed the efficiency of the sonication technique as an alternative to the pasteurization process.
The increased antioxidant activity caused by the addition of OPP was due to its content of polyphenols and flavonoids.Yogurt manufactured from ultrasonic-treated milk has a higher content of antioxidant compounds since ultrasonication enhances the release of phenols and flavonoids 0.01 Heat treatment was used for treatments of HT1, HT2, HT3, and OPP added at the rate of 0.0, 1, and 2%, respectively.While ultrasonic technic was used for UT1, UT2, and UT3, adding 0.0, 1, and 2% OPP, respectively.Averages (a, b,….etc.)within the same row with different superscripts differ significantly (P < 0.05).Fig. 2 DPPH and ferric-reducing ability on stirred yoghurt.*See legend to Fig. 1 for details sonication technique.The previous results agreed with the results of [35,44], which confirmed that the sonication technique in treatment is almost similar to what was done in this research, which led to the improvement of the rheological properties, which attributed this improvement to the length of the fermentation period in the ultrasonic-treated products.This is in contrast to treatments that used the heat method where the temperature was raised to 90 °C, which affected the protein decomposition and thus affected the fermentation time.Also, this improvement in the rheological properties, whether viscosity or WHC, may be because exposure to sonication affects the lipid granules as sonication acts as an alternative to homogenization and has a vital role in causing significant changes in the lipid granule membrane, which affects the binding of the lipid granules with each other or with casein mesylate [45].

Sensory evaluation
As shown in Table 2, the sensory evaluation results showed that the arbitrators preferred the treatment UT3, in which OPP was used at a concentration of 2%, and the sonication technique.The results of the treatment UT3 for fresh and at 7 and 14 days were recorded as 48.21, 47.3, and 46.34, in order.The improvement in the taste, as explained by [35], may be because the heat treatment leads to denitrification of both β-lactoglobulin and α-lactalbumin and the SH groups responsible for the cooked taste increase, which does not happen with the sonication technique.The whey-off was also improved by increasing the concentration of OPP and using sonication.Also, the clarity of color in the samples used with the sonication technique gave an illusory impression of increased attention.This may be due to the role of the sonication technique in reducing the component size [36].The opinion of the arbitrators has not changed when storing transactions.On the contrary, they confirmed the Ultrasound increases the number of total phenols obtained using waves less than 40 kHz and temperatures below 50 °C.Also, high waves work on forming free radicals, which decompose polyphenols, especially when water is present [16].Also, the vitamin C available in the OPP is affected by heat treatments, so it becomes unstable [41].Therefore, sonication is suitable for preserving the phenolic compounds and vitamin C. The heat treatment also results in the deuteration of milk protein, which is rich in milk peptides, which have an essential role as antioxidants [42].

Apparent viscosity and water-holding capacity (WHC)
When studying the apparent viscosity, the results recorded clear differences between the treatments.At shear rate (S − 1 ) 3, the scores recorded as in Fig. 4 for HT1, HT2, HT3, UT1, UT2, and UT3 treatments are 3050, 4600, 5830, 3240, 4890, and 6150, respectively, while the same as for the previous treatments recorded at 14 days 2010, 3530, 4730, 2210, 3840, and 4920, respectively.The apparent viscosity increases with increasing concentration of OPP and the application of sonication technique while decreasing with storage.Yogurt is a complex food whose rheological behavior is affected by sugars, fats, and water; it is also affected by vegetable fibers [43].
Denaturation of the protein in yogurt by ultrasound may significantly increase wave-treated yogurt's consistency and apparent viscosity.The sound waves also affect the micelles of casein and the size of the fat globules, which may help improve the apparent viscosity [35].
The results of the water holding capacity were recorded in Fig. 5 42 for the treatments HT1, HT2, HT3, UT1, UT2, and UT3, respectively.This indicates the improvement by increasing the concentration of OPP and using the same previous results.The results showed the extent to which the arbitrators preferred the products manufactured with the sonication technique and using OPP at a concentration of 2% due to the improvement of the taste and flavor, and texture improvement.

Conclusion
Presently, interest in food products with high nutritional value, low costs, chemical-free, and a good flavor for the consumer has increased.This makes the product a strong competitor in markets all over the world.Orange peel powder is an industrial waste that can be added to stirred yogurt.It is rich in antioxidants, which are affected by heat treatments.Therefore, sonication technology was used as an alternative to heat treatment.The study showed that using orange peel powder at a rate of 2% with the application of the sonication technique works to obtain stirred yogurt rich in antioxidants and with good rheological and sensory properties.It can be applied to get a good competitive product in the markets.This study sheds light on the utilization of industrial waste while preserving its nutritional value through the alternative sonication technique of heat treatment and the homogenized process.The future perspective of this study is to try to track the change of antioxidants when changing different ultrasound frequencies.

Funding Not applicable.
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Fig. 3
Fig. 3 Total phenolic content and total flavonoids on stirred yoghurt.*See legend to Fig. 1 for details.

Table 2
Sensory evaluation of stirred yogurt See legend to Table 1 for details.