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Natural sweeteners: health benefits of stevia

Аннотация
Stevia rebaudiana (Bertoni), a perennial shrub, is the sweetest plant belonging to the Asteraceae family. Stevia leaves are an excellent source of diterpene glycosides stevioside, rebaudioside A-F, dulcoside, and steviolbioside, which are responsible for sweetness and have been utilized commercially for sugar substitution in foods, beverages, and medicines. To the best of our knowledge, a large number of studies have been carried out on composition, health implications, and safety of steviol glycosides. However, commercial production of stevia-incorporated food products needs further research in order to meet the huge global demand. Stevia-incorporated products possess better sweetening potency and maximum consumer acceptability, when compared with other sugar substitutes. Hence, the current research attempts to review the health promoting effects of stevia with special emphasis on its application in the food system. The paper majorly features 1) the anti-hyperglycemic, anti-hypertensive, anti-caries, anti-inflammatory, and anti-cancer benefits of stevia, 2) value-added stevia-incorporated products, e.g. bakery, dairy, and beverages, 3) the effect of incorporation of stevia on physicochemical, rheological, and nutritional food properties, 4) the current status and regulatory perspective of utilizing stevia at national and international level. Due to legislative actions and growing consumer awareness, public interest in natural sweeteners has significantly increased. Since the use of artificial sweetener has recently been questioned, the data the present article provides will be useful for consumers and manufacturers that seek an alternative.
Ключевые слова
Stevia rebaudiana; sugar substitutes, health benefits, value addition, product characteristics, consumer acceptability
ВВЕДЕНИЕ

One out of the six basic taste sensations in humans is sweetness. Honey, coconut sugar, blackstrap molasses, table sugar, agave, high fructose, corn syrup, maple syrup and other natural sweeteners contain glucose, fructose, and sucrose as their primary constituents. However, sweeteners obtained from natural sources possess a high caloric value, which may lead to obesity, diabetes, and cardiovascular diseases. There has been a gradual rise in the number of diabetic patients all over the world. India has become the diabetic capital of the world with about 72 million cases of diabetes in 2017 (International Diabetes Federation, Diabetic Atlas, 2017). Due to the growing health awareness, there has been a huge demand for sugar substitutes that would provide lesser or no calories and possess better sweetening potency. There is a variety of artificial zero-calorie sweeteners on the market, e.g. saccharin, aspartame, acesulfame potassium, cyclamates, etc. However, artificial sugar substitutes became associated with health complications, and the use of these artificial sugar substitutes has subsequently been restricted. Thus, there is a continuous search for high intensity low- calorie or non-caloric sweeteners of natural origin that are safe for consumption. Stevia, which plays an important role as a non-nutritive natural sweetener, emerged as a safe sugar substitute that does not pose any threat to human health [1].

РЕЗУЛЬТАТЫ И ИХ ОБСУЖДЕНИЕ

Stevia rebaudiana (Bertoni) is a perennial shrub belonging to Asteraceae family, native to Paraguay. Out of 230, only two species – rebaudiana and phlebophylla – produce sweet steviol glycosides [2]. Stevia leaves contain eleven diterpene glycosides, such as stevioside, rebaudioside A-F, dulcoside, etc. Stevioside and rebaudioside are the sweetest glycosides present in stevia leaves, which are 250–300 times sweeter than sucrose, and are chemically and thermally stable. Stevioside and rebaudioside obtained from stevia leaves have been utilized commercially in Japan, South America, China, and Korea to sweeten various foods. Dietary supplements containing stevia extracts have been utilized in USA extensively [3]. Market stevia products contain such steviol glycosides as stevioside or rebaudioside A [4].

Health benefits. Stevia is known to provide a wide variety of health benefits (Fig. 1). The leaves possess functional properties superior to those of many other high potency sweeteners.  Hence,  stevia  is  likely to become the ultimate natural sweetener in the food industry.

Fig. 1. Health benefits of stevia on various organs.

Anti-hyperglycemic. According to WHO global report on diabetes (2016), as many as 422 million adults were suffering from diabetes all over the world, and diabetes-related deaths accounted for 1.5 million. Diabetes mellitus is one of the major metabolic diseases characterized by hyperglycemia. It is a chronic disease resulting either from defects in insulin secretion of β-cells of pancreatic islets (islets of Langerhans) or from the response to insulin, or combination of both. Type 1 diabetes mellitus is juvenile-onset, or insulin independent diabetes mellitus, in which the exact etiology of the disease is unknown, while  in  type  2  diabetes  the  risk to predisposal depends upon metabolic and the genetic factors.

Traditionally, the extract obtained from stevia leaves proved its effectiveness in treatment of diabetes [5, 6]. There is a stimulation of insulin secretion from β cells of islets of Langerhans and INS-1 cells by direct action of stevioside and steviol [7, 8]. The antihyperglycemic effect of stevioside was investigated in type 2 diabetic Goto-Kakizaki rats, and it was concluded that the hypoglycemic action of stevioside was due to increased secretion of insulin and induction of genes of glycolytic pathway [9].

A limited number of human studies have been reported to depict the mechanism of stevioside (Fig. 2). A study of acute effects of stevioside was conducted on twelve type 2 diabetic patients. It was observed that there was an average 18% decline in the post-prandial blood glucose levels of the diabetic patients after supplementing the standard test meal with 1 g stevioside. However, the slight increase in the insulin levels was not statistically significant [10]. The effect of steviol glycosides on insulin sensitivity and glucose metabolism have been elucidated [11]. In diabetic rats, intake of steviol glycosides resulted in a decrease in blood glucose levels, along with a decrease in the rate of gluconeogenesis and reduction in insulin resistance. Similar findings suggest that oral administration of medium polar extract of stevia leaves at 200 and 400 mg/kg body weight basis for 10 days in alloxan- induced diabetic rats resulted in delayed but significant antihyperglycemic effect without producing hypoglycemia, along with lesser body weight loss in contrast to standard positive control drug glibenclamide [12].

Fig. 2. The possible mechanism of anti-hyperglycemic action of stevia.

There was a research on the blood glucose lowering effects of rebaudioside A on the activity of carbohydrate metabolizing enzymes in induced diabetic rats [13]. An increase in the rate of glycolysis and reduction in gluconeogenesis produced a significant antihyperglycemic effect. Another study featured the antihyperglycemic mechanism of stevia. It was found that stevia (400 mg/kg) reduced blood glucose levels better than pioglitazone (10 mg/kg), which can be used to control blood sugar levels in diabetics. There was an elevation in insulin levels due to the impact of stevia on the pancreatic tissue, and the valuable anti- hyperglycemic action was exerted via PPARϒ dependent mechanism and its antioxidant property [14].

Anti-hypertensive. Persistent elevation of systolic blood pressure (≥ 140 mm Hg), or diastolic blood pressure (≥ 90 mm Hg), or both is the principal cause for developing cardiovascular diseases, which are associated with high mortality rates globally. In primary hypertension, etiology is unknown and accounts for 90% of the hypertension cases. By contrast, secondary hypertension is known, and it affects less than 10% of the hypertensive population. It is precipitated by another medical condition affecting kidneys, arteries, heart, or endocrine system.

An early investigation conducted on rats showed that stevia extract at doses greater than those used for the purpose of sweetening resulted in vasodilation and a lower mean arterial pressure in hypertensive rats [15]. A reduction in blood pressure was elucidated upon studying the effectiveness of stevioside (250 mg three times a day for a period of 3 months) in human subjects suffering from mild or moderate primary hypertension [16]. The mechanism by which stevioside produces vasodilatory effect is analogous to that of verapamil, an antihypertensive drug which acts by blocking calcium channels in myocardial and arterial smooth muscle cells (Fig. 3). The antihypertensive effect of stevioside was antagonized by administration of indomethacin (parostaglandin synthesis inhibitor), suggesting stevioside produces reduction in mean arterial pressure via prostaglandin activity [17]. In the study, to evaluate the effect of stevia on renal function, it was reported that steviol increased the excretion of sodium and potassium along with glomerular filtration rate in a dose dependent manner. It acted as a diuretic agent by preferably affecting the Na+-glucose coupled cotransporter in proximal convulated tubule of kidney [18]. The hemodynamic effects of rebaudioside A were investigated in human trial. No significant changes in mean arterial pressure or heart rate were observed after daily intake of 1000 mg/kg body weight Rebaudioside A in healthy individuals who were normotensive or having low normal mean arterial pressure [19].

Fig. 3. Antihypertensive action of stevioside. Inhibition of Ca2+ influx in vascular smooth muscle resulting in vasodilation and hence reduction in systemic vascular resistance.

Anti-caries. Dental caries is a widespread chronic disease in humans and affects oral health. Dental caries may lead to the development of painful sensation in tooth, infections, oral and pharyngeal cancers, oral tissue lesions, and the outcome may be life-threatening if untreated, as in case of cavernous sinus thrombosis and Ludwig’s angina.

Routine consumption of caloric sweeteners, such as sucrose, results in ingestion of carbohydrates, which boosts the amount of harmful microbes in the oral cavity. That may ultimately result in plaque and gingivitis [20]. Stevia is a non-caloric sweetener that possesses antimicrobial properties benefiting oral health via prevention of dental caries. There are 57.82% less chances of developing a plaque with consumption of stevia, compared to sucrose recorded by aid of Silness-Loe plaque index [21]. A study on the cariogenic potential of commercial sweeteners conducted on artificial enamel proved that stevia- incorporated products exhibited antimicrobial activity and showed the least potential to act as cariogenic in contrast to sucrose and other commercial artificial sweeteners [22].

Anti-inflammatory. Inflammatory bowel disease, or IBD, is a group of chronic diseases that involve the inflammation of the alimentary tract. It includes Crohn’s disease (inflammation of the entire lining of alimentary canal) and ulcerative colitis (inflammation of the innermost lining of intestine). Ample evidences are available showing that stevioside acts as an anti- inflammatory in vivo, as well as in vitro. Stevia stem extract was suggested to act as a gastroprotective since it reduced histamine-induced gastric abnormality in rainbow trout [23]. The suggested mechanism of action was inhibition of smooth muscle contraction by blocking the calcium channel. The active substance in the stem extract was stevioside, which was potentially responsible for decreasing the acid secretion caused by histamine and inhibiting the action of pepsin [24]. Similar findings were reported when the anti- ulcerogenic activity of stevia was examined by oral administration of stevia extracts leading to a significant reduction in the free acidity, which resulted in inhibition of gastric lesions [25].

Anti-cancer. Cancer can be defined as a group of diseases involving the abnormal proliferation of cells, which is associated with high mortality rate. The anticancer effect of stevioside, isosteviol, and the derivatives obtained from isosteviol upon microbial transformation was evaluated. All the components were found to be potent in inhibiting Epstein-Barr virus early antigen (EBV-EA) while the highest potency was exhibited by the derivatives obtained by microbial transformation [26].

The ent-kaurene diterpene glycosides isolated from Stevia rebaudiana were investigated for anti- inflammatory activity against inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), and it was found to suppress inflammation along with a significant inhibitory effect on the tumour formation [27, 28]. The methanolic and ethanolic extract of stevioside exhibited anti-cancer potential against the Caco cell line [29]. Stevioside exhibited in vitro anticancer activity against MCF-7 cells, which are most commonly used for breast cancer studies in humans. The suggested mechanism of antibreast cancer activity was enhanced expression of proteins participating in apoptotic pathway [30].

Value-added products prepared with incorporation of stevia. Value-added products are prepared for enhancing the value of food items through the addition of ingredients, processing or packaging. Value-added food products are more attractive and usable by the consumer than the original commodity. Commercially, leaves of stevia are exploited directly or after processing in preparation of various value-added products. As a functional food ingredient, stevia has been partially or fully incorporated into baked, dairy, confectionery products, etc. The present review features the commercial use of stevia in various food products and its effect on various properties.

Bakery products. Sweetened food items, such as cakes, cookies, muffins, and biscuits, are the major contributors to sugar intake globally [31]. Sucrose is a crucial ingredient in baked goods. It contributes not only to the taste but also to the characteristic texture and structure [32]. However, excessive consumption of sugar leads to acute elevation in postprandial glucose levels [33]. There is an increased risk of obesity, diabetes, dental caries, and coronary heart disease associated with regular consumption of high amounts of sugar-containing foods.

The use of stevia in baked products is suitable since it maintains its stability throughout the baking process and can be heated up to 200°C [34]. The non-nutritive high intensity sweeteners contribute to the sweet taste of the product, but the maintenance of texture, colour, and flavour is also crucial. Hence, whenever stevia is used as a partial replacement of sugar, other additives, such as bulking agents, hydrocolloids, proteins, etc., should be added to compensate for the loss of texture.

To evaluate the physical properties of muffins sweetened with stevia suggested a study that muffins with 25% of the sucrose replaced by steviol glycosides were ranked best in terms of browning index, texture, cooking yield, and sensory acceptance. Sucrose replacement greater than 50% had a negative impact on the quality characteristics of muffins [35]. Low glycemic index muffins were made by adding stevia and cocoa dietary fibre. The replacement of 20% sucrose with stevia along with substitution of cocoa powder with cocoa fibre did not produce any negative impact on the quality attributes and consumer acceptability [36]. Functional cookies prepared by 20% replacement with defatted soy flour and powder obtained from stevia leaves were ranked best for all the sensory characteristics [37].

Dairy products. Milk and milk-based products are a vital component of functional foods. Dairy processors are investigating novel technologies to sweeten their products without adding more calories, since people are averted from the consumption of intensely sweetened dairy-based products. There has been a continual search for natural low-calorie alternatives to sweeten dairy products that would reduce the sugar content while maintaining the texture, body and mouthfeel. Stevia has emerged as a suitable choice for dairy products since it preserves its stability, when subjected to heat treatment.

One of the most popular frozen dairy products is ice cream. Sugar influences its texture, viscosity, and freezing point and, hence, plays a significant role in determining the consumer acceptability. The most popular ice cream sweetener is sucrose because of its cost effectiveness and consumer acceptability. However, stevia and other non-nutritive sweeteners are gaining popularity due to health hazards associated with sucrose. The replacement of sugar with stevia in ice cream and kulfi can lead to a significant decrease in the caloric value. Studies revealed that ice cream mixtures in which sucrose had been partially replaced with stevia had better sensory scores than those with stevia only [38, 39].

Yogurt is one of the best-known foods that contain probiotics. Sensory analysis of strawberry flavoured yogurt with stevia elucidated that yogurt with a mixture of stevia and sucrose had the best sensory profile [40]. Stevia exhibited a synergistic sweetening effect when used in combination with other sweeteners to sweeten strawberry flavoured yogurt [41].

Flavoured milk supplies vital nutrients equivalent to plain milk. Studies have revealed that flavoured milk is preferred for consumption by children and adults [42]. However, flavoured milk contains a high quantity of sugar, which has been linked to promotion of obesity amongst children and adults [43, 44]. The use of stevia is an appealing alternative in order to reduce the caloric value of flavoured milk. It is of primary importance to maintain the acceptance of the product by the consumer while decreasing the sugar content. In the study [45], the perception of sweetness intensity in skim chocolate milk prepared by addition of extracts of stevia and monk fruit was defermind. It was established that flavoured milk containing stevia had a maximum consumer acceptability.

Beverages. The primary function of stevia is sweetening, but it may modify the flavour in certain cases.

Peach juice was formulated with a blend of stevia (160 mg/L) and sucrose (56 g/L), which led to 25% reduction in calories without affecting the sensory attributes of the product compared to the control sample containing 9% sucrose [46]. Orange juice is one of the most popular non-carbonated beverages globally since it contains high amounts of vitamin C, carotenoids, folic acid, flavonoids, etc. It acts as an antioxidant preventing the damage caused by free radicals to tissues and also decreases the chances of heart disease and cancer [47, 48]. Low calorie orange nectar and orange juice were formulated with the addition of stevia [49, 50]. Other beverages, such as mango nectar and passion fruit juice, were developed with partial replacement of sucrose with stevia and evaluated for their sensory as well as physicochemical parameters [51, 52].

Effect of incorporation of stevia on different properties. Physicochemical properties. Physical properties of food material are its measurable and quantifiable characteristics. They are used to describe matter without altering its composition. They elucidate the unique way a food material will react to physical treatments – thermal, optical, electromagnetic, mechanical, etc. Knowing these properties helps design optimum operation parameters and equipment to ensure the quality and safety of foods.

Some important physicochemical parameters for evaluation of ice cream are overrun ratio, first melting point, last melting point, and fat destabilization. The addition of stevia in ice cream resulted in a higher first melting time being the longest for the sample containing stevia and cocoa. Table 1 shows that the last melting time for ice cream containing cocoa with sucrose and cocoa with stevia was lower as compared to plain ice cream with sucrose and plain ice cream with stevia [38]. The overrun ratio for ice cream with stevia and cocoa was found to be the highest, while the overrun ratio was the lowest for plain ice cream with stevia [38]. Similarly, the overrun ratio of soft ice cream increased when stevia was added [39]. Fat destabilization was reported to be slightly higher in ice cream containing stevia [38]. Increasing levels of sugar replacement with stevia in kulfi resulted in a decrease in melting rate [53]. In case of juices, the important physicochemical properties to be evaluated are °Brix and titratable acidity.

Table 1. Effect of stevia on physicochemical properties of various food products.

Rheological and textural properties. Texture may be referred to as a collection of physical attributes that emerge from structural makeup of food. They are perceptible via sense of touch and associated with disintegration, distortion, and flowability under the influence of force. Some important textural properties in foods are hardness, cohesiveness, springiness (elasticity), adhesiveness, chewiness, gumminess, resilience fracturability, stringiness, and initial modulus.

Viscosity (the resistance to flow) is an important evaluation parameter for ice cream mix. As given in Table 2, a decrease in viscosity of ice cream was observed when nothing but stevia was added. However, the ice cream sample containing both stevia and cocoa possessed the highest viscosity amongst all other ice cream samples. With a gradual increase in stevia concentration in ice cream, the viscosity was relatively lowered, compared to the sample containing sucrose only [39]. A disaccharide produces the solution with high osmolality due to its hydrophilicity and high solubility, as well as its ability to form hydrogen bonds [54].

Table 2. Effect of stevia on rheological and textural properties of various food products.

Viscosity of the low calorie nectar (sugar content reduced to 70% compared to the control sample) was found to be slightly higher than that in the blank sample, which was possibly due to incorporation of 0.03% pectin in the sample [49]. Similar results were seen in the case of mango nectar, in which the sample sweetened with stevia exhibited a higher viscosity compared to nectar sweetened with other sweeteners [51]. The critical textural variables for muffins are firmness, springiness, and cohesiveness. The texture profile analysis (TPA) of muffins revealed that with the increase in replacement of sucrose with stevia, a significant increase in hardness and springiness was observed [36]. The possible reason for this was the decrease in the amount of sucrose in the muffins since sucrose played a significant role in governing the texture [55]. Reduction in the amount of sucrose led to an increase in porosity. However, when sucrose was used in combination with stevia, the value of porosity was slightly lower [36]. In a functional yogurt cake, an increase in firmness and hardness after adding ground stevia leaves has been reported [56]. When bittersweet chocolate was combined with rebaudioside A, which is the sweetest component present in stevia leaves, it resulted in an increase in hardness of low fat chocolate [57].

Nutritional properties. Excessive consumption of food containing high content of sugar and saturated fats is one of the most critical nutritional issues mankind have to face in the current scenario. It has been linked to serious health problems. Health awareness is growing nowadays, and this has led to a significant rise in demand for low-calorie/fat products. Many low- calorie food products have been developed with the addition of dietary fibre or low calorie sweeteners as the consumers are striving to choose healthier food alternatives (Table 3).

Table 3. Effect of stevia on nutritional properties of various food products.

Significant reduction of glycemic index and caloric value was reported in ice cream prepared with addition of stevia, which propounds the utilization of stevia as a substitute for sugar and a healthy alternative for people who are predisposed to diabetes [39]. Kulfi samples prepared with incorporation of stevia showed a decrease in calorific value but a significant increase in fat, ash and protein content, due to a decrease in total solids, which resulted from partial replacement of sugar with stevia [53]. Similar results were reported for herbal kulfi that contained both stevia and a mixture of herbs including Foeniculum vulgare, Ocimum sanctum, and Piper betle [58]. A hot water extract of stevia leaves was used to produce a functional yoghurt cake.

There was a reduction in calories by 35.72% as compared to a regular yoghurt cake. The biological evaluation of yoghurt cake in diabetic rats revealed that there was no significant change in blood levels of cholesterol, alkaline phosphatase, bilirubin, glucose, creatinine, and triglycerides. However, the level of urea and Aspartate transaminase (AST), as well as HDL cholesterol, decreased slightly. Lower doses did not produce any effect on serum glucose, AST, ALT, total cholesterol, protein, and triglycerides, but higher doses produced a slight elevation in these parameters [56]. Similarly, muffins prepared by partial replacement of sugar with steviol glycosides showed a decrease in caloric value [32, 36]. The incorporation of stevia into muffins led to a prolonged release of reducing sugars during the digestion of starch in vitro, thus, reducing the predicted glycaemic response [32]. A decrease in energy value was noted when sucrose free chocolates were prepared with the addition of stevia [59].

Sensory properties. Sensory properties of a food product are regarded as one of the most crucial attributes since they are most noticeable by the consumer [60]. Sensory evaluation refers to the scientific technique of invoking, computing, analysing, and interpreting the responses by the perception of senses [61]. The palatability of the product has become the primary criteria for consumers nowadays, whereas such parameters as nutritional value and the wholesomeness of the product are secondary. Therefore, to ensure market success, a product must have the desired sensorial characteristics, i.e. taste, flavour, aroma, mouthfeel, aftertaste, and textural parameters.

As described in Table 4, kulfi prepared with addition of stevia indicated maximum overall acceptability, whereas no significant effect was observed on the body, texture, and flavour of the kulfi if the level of replacement of sugar with stevia was less than 50%. However, a negative impact on the textural properties, as well as flavour, was observed when the level of sugar replacement with stevia exceeded 50% [53].

Table 4. Effect of stevia on sensory properties of various food products.

Current status and future perspective of utilizing sweeteners at national and international front. The US Food and Drug Administration regulate sweeteners as food additives. Food additives must be approved by the FDA, which publishes a Generally Recognized as Safe (GRAS) list of additives. To date, the FDA has not been presented with scientific information that would support a change in conclusions about the safety of the five approved artificial sweeteners (saccharin, aspartame, sucralose, neotame, and acesulfame potassium). The safe conclusions are based on a detailed review of a large body of information, including hundreds of toxicological and clinical studies. Steviol glycoside has been used as a non-nutritive low calorie sweetener in beverages, teas, and medicines in Japan, China, South America, and Korea. Many international beverage industries use stevia as a sweetener in different fruit juice drinks. The incorporation of stevia into such bakery products as puddings and cakes is highly suitable since stevioside is required in minute quantities to sweeten the product. It neither ferments nor exhibits any browning reaction during cooking, which broadens the field of application in baking and makes it possible to enhance the quality, decrease the calories, and increase the shelf-life. Stevia can be used as a sugar substitute in confectionery to sweeten chewing gums, candies, mints, chocolates, etc. Besides, stevia can be extensively used to sweeten various products, e.g. ice-cream, chocolates, fruit drinks, biscuits, soft drinks, yoghurt, biscuits, beverages like tea, and coffee.

The joint FAO/WHO expert committee on food additives (JECFA, 2008) stated that steviol glycosides were safe for use in foodstuffs and beverages. JECFA also suggested the acceptable daily intake (ADI) of 0–4 mg/kg body weight of steviol glycosides, which is equivalent to 0–10 mg/kg stevioside [62]. Currently, the largest market for production and application of stevia and its glycosides is in China and Japan. In 2009, the use of rebaudioside A was permitted in France. In European Union, the approval for use of steviol glycosides as food additive was granted by European Commission in 2011.

In India, the use of steviol glycosides was permitted by FSSAI in a notification issued in 2015 for a variety of products, including yoghurts, carbonated beverages, jams, fruit nectars, dairy based desserts, ready-to-eat cereals, etc.


ВЫВОДЫ

Diterpene glycosides obtained from Stevia rebaudiana can be used to sweeten various foods and beverages without increment in calories. Apart from sweet contents, the other constituents of Stevia rebaudiana exert various health benefits, such as anti- hyperglycemic, anti-cancer, hepatoprotective, anti- hypertensive, anti-caries, antioxidant, and antimicrobial. Low-calorie stevia-incorporated products are rich in antioxidants, amino acids, and certain vitamins. They possess many other therapeutic properties in that they are anti-diabetic, anti- hypertensive, antitumor, antiulcer, etc.

КОНФЛИКТ ИНТЕРЕСОВ

The authors declare that there are no conflicts of interest related to this article.

БЛАГОДАРНОСТИ

The authors would like to thank the Department of Food Technology and Nutrition, Lovely Professional University, for providing all the necessary resources.

ФИНАНСИРОВАНИЕ

The authors received no specific funding for this work.

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