1. Introduction
Prevention of various diseases is possible by including fruits in our diet as they are an excellent source of minerals, vitamins, antioxidant components and other phytoconstituents [
1]. Generation of free radicals, which are triggering factors for several acute and chronic diseases, can be prohibited by the antioxidant potential of the fruits, thus promoting a healthier life [
2]. Elevated levels of plasma carotenoids and vitamin C are associated with the increased intake of fruits, which ultimately reduces the probability of diabetes, cardiovascular diseases, neurological disorders and cancer [
3,
4].
Citrus limon (L.) Burm. f., most commonly known as lemon, a yellow-colored edible fruit, is the third most widely produced representative of the Rutaceae family and hybrid of genus
Citrus—just after orange and mandarin—worldwide [
5].
Citrus limon can either be consumed as a fresh fruit, as beverage, as cooking material or for preservation purposes. Because of its tart flavor, it is most often used in manufacturing of beverages, desserts, ice creams, salad dressings, jams, jellies, pickles and in several kinds of vegetable and meat dishes [
6,
7].
Citrus limon is also a good supplier of a variety of phytoconstituents, including phenolics. Among these phenolics, eriocitrin, coumarins, flavonoids and limonoid glycosides are present in adequate concentrations [
8]. Consumption of
Citrus limon has shown reduction in the risk of several types of cancers, along with cardiovascular disorders [
7]. Organic compounds present in lemons are effective against asthma and can serve as antidepressants and stress relievers. Moreover, these also stimulate digestion and are effective in case of flu, fever, boils and in several kinds of ulcers, particularly mouth ulcers [
9]. In the past, when vitamin C was not discovered, the juice extracted from
Citrus limon fruit was used for treatment of scurvy [
10]. Additionally, the juice has also served as traditional medicine for the cure of hypertension, common cold, sore throat, chest pain and rheumatism [
11].
Cordial can be defined as sparkling, clear, or syrup concentrated fruit juice formed by the complete removal of pulp and other suspended particles, and needs to be diluted upon consumption [
12] (Yusof and Chiong).
Citrus limon is also cultivated in Pakistan and it occupies 6th position in terms of area and production [
13]. Due to more production and less utilization, the fruit obtain wasted. In order to overcome the losses, there is a need to convert them to some value-added products, such as squashes and cordials. The study focused on making cordial from lemons in order to meet the rising consumer demands regarding new value-added products.
During the last couple of years, consumers’ interest throughout the entire world has been changed regarding quality and safety of the food product [
14]. They demand minimally processed products that are not only healthy, but are also processed by means of safe preservation techniques, so that their quality and nutritional profile are not affected [
15].
In general, fruit juices are preserved by heating them near the boiling point of water, or slightly below it, for a set amount of time, to kill or inactivate deterioration-causing microbes and enzymes. [
16]. Although traditional heating methods ensure the safety and stability of the juices, they greatly affect the phytochemical profiles of the fruit juices, along with causing a decline in the physical and chemical properties, the nutritional profiling and the volatile compounds [
14,
17]. Similarly, chemical preservatives are other means to extend the shelf life of products, but these chemicals also produce several health complications in humans, particularly cancer, neurological dysfunction, asthma, hyperactivity and hypersensitivity, dermatitis, allergies and gastrointestinal and respiratory disorders [
18,
19,
20].
Due to disadvantages of thermal techniques and chemical preservatives, several novel non-thermal techniques have been in practice to enhance the shelf stability of processed products [
21,
22]. Microwaving, a novel technique, utilizes electromagnetic waves that heat the product by means of molecular interaction, as generated by the electromagnetic field. In this technique, there is a direct interaction of food particles with that of the incoming waves. As a result of this direct contact, penetration of heat is easier within the food, and thus volumetric heating takes place [
23,
24]. Microwaving also preserves the nutritional contents of products to a greater extent, such as the retention of vitamins, thus enhancing the quality and safety of products through the inactivation of microbes and enzymes within a short duration [
25,
26].
Keeping the above benefits in mind, the present research aimed to develop lemon cordial and to investigate the effect of the microwave technique for preserving lemon cordial’s physicochemical properties and microbial counts, and to determine the effect of microwave treatment on the shelf stability of lemon cordial at refrigerated temperature for a period of 90 days.
3. Results and Discussion
The impact of microwave treatments and storage on titratable acidity of lemon cordial retained at refrigerated temperature is illustrated in (
Table 3). Microwaving produced a highly significant impact (
p < 0.01) on titratable acidity, leading to a gradual reduction in the acidity of lemon cordial. The highest value for acidity was exhibited by T
0−, proclaiming a mean value of 0.38 ± 0.038%, while the lowest was observed in T
3, exhibiting a mean value of 0.32 ± 0.045%. The observations are in conformance with those obtained by Pandiselvam et al. [
35] and Bozkir et al. [
36], who reported the same decreasing tendency in the acidity after the application of microwave treatment on kalparasa and apple juice. They articulated that increased time and temperature during microwave treatment resulted in destruction of fermenting microbes, due to which the production of organic acids declines. Considering the impact of storage on the titratable acidity, it was noticed that titratable acidity increased gradually throughout the storage duration. The highest mean value of 0.41 ± 0.014% was observed at 90 days, while the lowest mean value of 0.29 ± 0.024% was examined at 0 days. These results are supported by the findings of Pandiselvam et al. [
35], who reported an increase in titratable acidity of microwave-treated kalparasa (coconut inflorescence sap) upon refrigeration storage and claimed that an increased production of organic acids during anaerobic fermentation is responsible for an increase in acidity. Similarly, Palanisamy et al. [
37] reported that increase in the acidity of noni fruit juice blended squash during storage was caused by the soluble proteins hydrolyzing to free amino acids, which ultimately resulted in an interlinkage of citric acid in squash. An increase in acidity during storage in mixed fruit squash and microwaved apple puree was also communicated by Jothi et al. [
38] and Picouet et al. [
39], respectively.
Microwave treatment and storage duration both produced a highly significant impact (
p ≤ 0.01) upon the pH of the lemon cordial (
Table 4). Gradual increase in the pH of the lemon cordial upon microwaving was observed. The highest pH value was expressed by T
3, with a mean value of 2.45 ± 0.050, while the lowest was observed in T
0−, exhibiting a mean value of 2.38 ± 0.046. A similar trend for pH was reported by Pandiselvam et al. [
35] in microwave-treated kalparasa, who documented that reduction in the contents of organic acids of kalparasa, as result of heating, increased the pH. Picouet et al. [
39] articulated that the pH of apple puree, when subjected to microwaving for 35 s at 652 W power, slightly enhanced from 3.2 to 3.3. Considering the impact of storage on the pH, it was noticed that it decreased throughout the storage duration. The highest mean value of 2.46 ± 0.027 was noticed at start of study while the lowest mean value of 2.35 ± 0.024 was observed at 90 days. The observation of the conducted study correlates with those proposed by Palanisamy et al. [
37], Yadav et al. [
40] and Jothi et al. [
38]. They claimed that the pH of noni fruit juice blended squash, guava mango RTS and squash and mixed fruit squash made from banana, papaya and carrot juice decreased during storage, which was attributed to the accumulation of acidic compounds—particularly lactic acid and acetic acid—because of the activity of microbes during natural fermentation process.
The influences of microwave treatment and storage on the total soluble solids (°brix) of lemon cordial at refrigerated temperature are illustrated in (
Table 5). The presented data clearly depicts that microwave as well as storage duration produced a highly significant impact (
p ≤ 0.01) upon the °brix of lemon cordial. A gradual increase in the °brix of lemon cordial was observed upon microwaving. The highest °brix were exhibited by T
3 expressing a mean value of 56.68 ± 2.612 °B while the lowest were observed in T
0−, exhibiting a mean value of 50.83 ± 0.755 °B. The observations of the study are supported by the findings of Sattar et al. [
41], Song et al. [
42] and Fazaeli et al. [
43], who stated an increment in total soluble solids of functional peach beverage, apple and black mulberry juice upon microwaving. They figured out that during microwaving, evaporation of water from the product takes place, and this phenomenon is directly linked to the increase in the internal temperature of juice. It leads to juice concentration and increase in total soluble solid contents. Considering the impact of storage, it was noticed that °brix of lemon cordial decreased throughout the storage duration. The highest mean value of 55.28 ± 3.499 °B was noticed at 0 days while the lowest mean value of 51.05 ± 1.746 °B was observed at 90 days. These findings are similar to those reported by Pandiselvam et al. [
35] and Yusof and Chiong [
12]. During storage sugars are utilized by the fermenting microbes, particularly yeast and acetic acid bacteria, as a source of nutrient, thus converting them into their respective acids and alcohol, along with the liberation of CO
2.
Data demonstrating the influence of microwave treatment and storage on total phenolic contents of lemon cordial during storage at refrigeration temperature is depicted in (
Table 6). Microwave treatment as well as storage duration produced a highly significant impact (
p ≤ 0.01) upon total phenolic contents of lemon cordial. Reduction in total phenolic contents of lemon cordial was observed upon the application of microwave. The highest values of total phenolics were exhibited by T
0+, expressing a mean value of 399.08 ± 67.745 mg GAE per 100 mL of cordial, while the lowest were observed in T
3, exhibiting a mean value of 346.42 ± 64.387 mg GAE per 100 mL of cordial. Findings of the conducted study were in line with those reported by Cheng et al. [
44] and Igual et al. [
45]. They observed reduction in total phenolics contents of
Citrus unshiu juice and grapefruit juice, respectively. According to de Souza et al. [
46] and Rasoulian et al. [
47], electromagnetic radiation generated by microwaves first liberates bound phenolics from their binding sites, then causes their cleavage due to increasing temperature, thus changing their composition and stability. In contrast to the microwave results, the storage data indicate that the total phenolic content increased considerably during storage. At 90 days, the highest mean value of 451.20 ± 26.940 mg GAE per 100 mL of cordial was recorded, whereas the lowest value of 281.00 ± 24.474 mg GAE per 100 mL of cordial was observed at 0 days. Increase in total phenolic contents of black mulberry juice was observed during storage at 3 different temperatures—5, 15 and 25 °C—by Jiang et al. [
48]. Pandiselvam et al. [
35] and Piljac-Zegarac et al. [
49] explained that there is a possibility some of the peroxidase may survive the pasteurization treatment, which might promote oxidation, thus elevating the total phenolic contents. Barba et al. [
50] figure out that during storage some products are formed because of Maillard reactions. These chemicals have antioxidant properties and, when combined with the Folin reagent, increase the concentration of total phenols.
Findings regarding the influence of microwave treatment and storage on the total flavonoids content of lemon cordial at refrigerated temperature are displayed in (
Table 7), which clearly depicts that the treatments and storage periods had a highly significant impact (
p ≤ 0.01) upon total flavonoid contents. Reduction in total flavonoid contents of lemon cordial were observed upon the application of microwave treatment. The highest values of total flavonoids were exhibited by T
0+, 178.70 ± 24.472 mg CE per 100 mL of cordial, while the lowest values were observed in T
3, exhibiting a mean value of 118.21 ± 24.078 mg CE per 100 mL of cordial. Decrease in total flavonoids after microwaving was also reported by Papoutsis et al. [
51], and Igual et al. [
45] in
Citrus limon L. pomace and grapefruit juice, respectively. They stated that higher power and long exposure time led to degradation of heat sensitive polyphenols. Cheng et al. [
44] communicated that water serves as a very important medium for absorbing microwave energy. At higher temperatures and long exposure time, evaporation of water becomes rapid, thus its ability to preserve the bioactive compounds is reduced, which, ultimately, drops their concentration. Therefore, microwave power, time, frequency and temperature play very critical roles in determining the concentration of these polyphenolic constituents. Storage also caused reduction in the total flavonoid contents of lemon cordial. The highest total flavonoid contents with mean value of 174.81 ± 23.349 mg CE per 100 mL of cordial were noticed at 0 days, while the lowest contents with mean value of 117.04 ± 21.678 mg CE per 100 mL of cordial was observed at 90 days. When total flavonoid contents were compared to total phenolic contents during storage, a discrepancy in the data was noticed, indicating that total flavonoid contents began to decline during storage. A 57% reduction in total flavonoid contents was observed when comparison was carried out at beginning and at the end of storage duration. Total flavonoid contents of microwave-treated functional peach beverage and sugarcane juice also declined during storage. Sattar et al. [
41] and Zia et al. [
24] stated that, during storage, generation of free radicals takes place to a greater extent, which ultimately reduces the total flavonoids contents.
Statistical results indicated highly significant impact (
p ≤ 0.01) of microwave treatment, as well as storage, on the antioxidant activity of lemon cordial stored at refrigerated temperature (
Table 8). Increment in total antioxidant activity of lemon cordial was observed upon the application of microwave. The highest value for total antioxidants was expressed by T
3, depicting a mean value of 1212.03 ± 716.5 µg equivalent of ascorbic acid per 100 mL of cordial, while the lowest was observed in T
0−, exhibiting a mean value of 837.73 ± 427.1 µg equivalent of ascorbic acid per 100 mL of cordial. Results of the conducted study coincide with the findings of Martins et al. [
52] and Zia et al. [
24], who proposed that total antioxidant potential of orange juicemilk beverage and sugarcane juice increased with application of microwaves, as the enzymes responsible for promoting oxidation are inhibited. Besides, microwaving also leads to greater extraction of antioxidant compounds from the product to which they are applied. Storage results revealed reduction in the total antioxidant activity of lemon cordial. The highest mean value of 1685.22 ± 264.73 µg equivalent of ascorbic acid per 100 mL of cordial for total antioxidants was noticed at 0 days, while the lowest mean value of 337.76 ± 27.03 µg equivalent of ascorbic acid per 100 mL of cordial was observed at 90 days. During storage, a huge reduction in the total antioxidant activity was observed. At the end of storage, a fourfold reduction in the antioxidant activity was recorded. Results are in conformance with the findings of Jiang et al. [
48], who stated that, during storage, loss in vitamin C and flavonoid contents triggers the reduction in the antioxidant activity of black mulberry juice. Additionally, onset of complex chemical reactions, particularly polymerization reactions, limits the availability of free hydroxyl groups, leading to diminished antioxidant potential.
At refrigerated temperature, the influence of microwave and storage on the total plate count of lemon cordial is presented in (
Table 9), which clearly depicts a highly significant impact (
p ≤ 0.01). Reduction in total plate count of lemon cordial was observed, upon the application of microwave treatment. Lowest values for microbial populations were expressed by T
0+ and T
3, exhibiting mean values of 1.72 ± 0.131 Log
10 CFU/mL and 1.75 ± 0.144 Log
10 CFU/mL of cordial, respectively, while highest were observed in T
0−, exhibiting a mean value of 1.83 ± 0.125 Log
10 CFU/mL of cordial. Observations of the conducted study correlate with investigations of Adulvitayakorn et al. [
53] and Li et al. [
54], who emphasized that microwaving causes cell lysis of bacteria due to coupling of electromagnetic energy. Storage results revealed an increment in the total plate count of lemon cordial. Lowest mean value of 1.58 ± 0.052 Log
10 CFU/mL of cordial for total plate count was noticed at 0 days, while highest mean value of 1.93 ± 0.039 Log
10 CFU/mL of cordial was observed at 90 days. Storage results contradict in a way that total plate count increased throughout storage. Pradhan et al. [
55] claimed that, during storage of microwave-treated sugarcane juice, enhancement in total plate count was noted.