ARTICLE   Open Access    

Comparative evaluation of the impact of processing methods in determining the levels of health promoting chemical constituents and quality of green tea

  • # Authors contributed equally: Biplab Adhikary, Bishwapran Kashyap

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  • The first step of green tea manufacture involves enzyme deactivation by heat application. The present study investigated the effects of various fixing and processing methods viz. steam-roasting (S-6), pan-firing (P-8), blanching (B-2), and CTC cuts after steam-roasting (S-CTC), on the bio-chemical profiles and organoleptic quality of green teas processed differently into orthodox and CTC types, from region-specific tea cultivars, suitable for green tea production under agroclimatic condition of Dooars, West Bengal, India. Differences in fixing method and processing style showed notable variation (p ≤ 0.05) in the chemical quality indicators of green tea viz. Total catechin, polyphenol, flavonoid, and water extract content among the differently processed green teas. The most significant finding of the study revealed that when B-2 is employed for deactivation, it resulted in a substantial reduction (47%−52%) of caffeine levels without affecting the catechins content and antioxidant potential of green tea samples when compared to S-6 and P-8 methods. Interestingly, our results demonstrated significantly higher water extract values (42.19% dry weight) in green CTC teas and lower values in B-2 green tea samples (33.67%), as compared to S-8 and P-8 green teas, which received better taster ratings (≥ 7). These findings have highlighted the role of processing method and the impact of fixing technique in determining the contents of health-promoting attributes and taste quality of green teas, thus providing diverse choices to tea producers and consumers to opt for specific green tea products and expediting the need to further explore its commercial application in the nutraceutical and pharmaceutical industry (Supplemental Fig. S1).
  • The process of degradation and spoilage occurs naturally in all fruits and vegetables. It can happen as soon as the nutrients are removed. During ripening and senescence, these vegetables are at risk of developing decay. A significant portion of the vegetables and fruits produced worldwide are lost due to spoilage[1]. Various microbes capable of causing infections and microbiological decay in fruits and vegetables can contaminate the food at any point from farm to table. Microorganisms such as molds, yeasts, and both beneficial and harmful bacteria contribute to the spoilage of fruits and vegetables. While parasites pose a potential threat to food safety, their impact on the sensory qualities of produce is minimal, and they are not commonly associated with the deterioration of whole or freshly cut vegetables[2]. Maintenance of the microbiological integrity of fresh vegetables and fruits during commercial production and distribution processes is challenging, as produce retains metabolic activity while moving from the time of maturity to the period reaching senescence, and total degeneration. When purchasing fresh fruits, consumers typically evaluate their quality based on appearance and freshness[3]. Coating, a technique involving the application of a protective layer on fruits or vegetables, serves to inhibit microbial intrusion and reduce decay. These coatings, applicable through rubbing, spraying, or immersion, employ environmentally friendly ingredients[4]. In the realm of fresh produce, edible coatings have traditionally served to mitigate harm to vegetable cells, achieving prolonged shelf life by diminishing moisture content, solute dispersion, gas exchange, aerobic respiration, oxidative reactions, and visible disorders[5]. This technology applies not only to whole and freshly cut fruits and vegetables but also to nuts, seeds, and cheese. The simplicity of application and the use of eco-friendly components make these coatings a viable solution for enhancing food preservation and safety in the fresh produce sector[6].

    Belonging to the Cucurbitaceae family, the pointed gourd (Trichosanthes dioica Roxb.), often referred to as the king of gourds, is renowned for its enhanced nutritional and therapeutic properties, particularly in regulating blood sugar and total cholesterol levels. Native to the Indian subcontinent, these gourds are initially classified as non-climacteric but exhibit climacteric behaviour post-harvest, marked by an increased respiration rate. Traditional storage methods prove inefficient as the fruits quickly deteriorate, displaying symptoms such as shrivelling, skin yellowing, hard seed development, and susceptibility to fungal infections[7]. Pointed gourds experience substantial moisture loss (8%–9%) from their initial weight, leading to pronounced surface wrinkling, and they exhibit a very short shelf life of 3–4 d under standard storage conditions. Rapid chlorophyll depletion results in the pulp and skin turning yellow, making such gourds less appealing to consumers. In response, traders and retailers resort to the use of potentially harmful chemicals to enhance the fruits appearance and extend its shelf life. Solutions containing copper sulphate and malachite green are usually applied to mask the undesirable yellow color of the fruit[8].

    A vital economic crop and commonly used spice, the chilli plant, scientifically known as Capsicum annuum L. and belonging to the Solanaceae family, faces various postharvest challenges. Notably, its short shelf life, high perishability, and susceptibility to fungal diseases result in quality deterioration, chilling damage when stored below 7 °C, and rapid weight loss leading to shrivelling. Despite these challenges, chilli remains a globally significant cash crop, highly valued for its widespread use as a spice[9]. India holds a central position in influencing the global chili market, contributing around 36% of the world's chili production, surpassing 1.4 million tons. Remarkably, India is the foremost chilli exporter globally, claiming a 25% share in international trade and exporting 0.209 million tons, solidifying its dominance in the global chilli landscape[10]. Challenges arise from the innate non-climacteric nature of green chillies. Their vulnerability to microbial infections is exacerbated by the elevated moisture content, ranging from 60% to 85% at harvest. Losses in the chilli supply chain predominantly result from moisture content (15%–25%), field spoilage (1%–10%), transportation from fields to factories (6%–10%), and losses during assembly for distribution (2.5%–5%). Addressing these factors is crucial for mitigating losses and ensuring the efficiency of the chilli supply chain[11].

    An edible coating is a type of barrier that helps to maintain the quality and freshness of vegetables and fruits by preventing oxidation, dehydration, and microbial contamination[12,13]. One possible strategy to lessen postharvest deterioration and maintain quality during storage appears to be the application of a coating that is edible. It enhances the physical attributes and flavor of fruits and vegetables by adding essential oils. Due to their antioxidant, antimicrobial, and flavour-enhancing properties, the addition of essential oils in these coatings has gained widespread attention[14]. In addition to being able to enhance the appearance and flavor of fruits and vegetables, essential oils can also help preserve them by providing natural compounds such as eugenol[15]. The essential goal of consumable coating is to restore or strengthen the natural barricade where it has been eliminated by transporting and cleaning. Additionally, it does not give the product any unfavourable qualities and can be taken without any harm to health. Fruit respiration rate is slowed down, water loss is prevented, texture and flavour are preserved, and fragrance compounds, moisture exchange, and partial barriers to CO2 and O2 are all partially blocked by edible coatings[16].

    By the ingredients utilized during preparation, edible coatings are divided into three categories: (i) lipids like waxes, acylglycerol, and fatty acids; (ii) proteins or polysaccharides, and (iii) composites[17,18]. Lipid coatings have superior water vapour barrier qualities because they are hydrophobic. Lipid materials are often used to improve the appearance of fruits and vegetables even though they are not able to polymerize on their own. Examples consist of waxes and natural resins (gum), essential oils of citrus fruits (camphor), and animal and vegetable oils (coconut, peanut, palm, cacao, butter, fatty acids)[19]. Proteins have more flexible structures than lipids and polysaccharides, which allows them to create bonds in a variety of locations and provide films with better mechanical properties. The dietary quality of the coated foodstuffs is improved by these protein-based films, which also function as efficient coatings. This group includes plant-based proteins like corn, soy, wheat, cottonseed, rice, and peanut as well as proteins like casein, whey protein, gelatine, and egg albumin[20]. Polysaccharide coatings are susceptible to significant moisture absorption due to their hydrophilic properties. They do, however, stick effectively to the cross sections of vegetables and fruits and have minimal permeability to gases[21]. This category includes natural gums (acacia, gum arabic, guar, etc.)[22,23]. Composite-based multicomponent edible coats are also used in this regard. Composite coatings are made up of lipid-based compounds, proteins, and polysaccharides. It can be utilized to decrease the gas permeation and increase the strength and water vapour resistance[24]. Composites can be divided into the following two groups by combined entities and bilayer materials. Double-layer composites are prepared using two layers linked with the same or different coating materials. Examples of these coating materials are protein/protein, polysaccharides + protein, lipid + lipid, lipid + polysaccharides, etc.[25].

    Chitin and chitosan are biopolymers that offer a vast array of structural alterations through chemical and mechanical processes, resulting in the creation of unique properties, functions, and applications. The entire family of linear heteropolysaccharides that are soluble in acid is referred to as chitosan The degree of N-deacetylation has not been used to establish a clear nomenclature border between chitin and chitosan[26,27]. Due to its versatile qualities and numerous applications, chitosan is highly desirable in food packaging. Chitosan finds extensive use in both the food industry and agriculture among its many other applications[28]. In most organic solvents, chitin remains insoluble, whereas chitosan can easily be dissolved in acidic solutions that are diluted with a pH of less than 6.0. The amino groups' presence suggests that chitosan's charged state and other characteristics are significantly changed by pH.

    The complex substance known as beeswax is secreted in liquid form by unique wax glands in the abdomen of immature worker bees, which are between the ages of 12 and 18 d. The material that makes up a honeycomb's structure is beeswax, which is secreted by the bees to construct the structures that hold honey. Beeswax is found in beauty products due to its rich hydrophobic protective properties. Furthermore, beeswax is utilized in the food sector. Pure beeswax is nearly white when it is secreted by the bee; it only takes on a variable, intense yellowish color upon contact with honey and pollen, and after approximately four years, it turns brown due to the presence of the cocoon[29].

    The eugenol present in clove trees and other phenolic compounds extracted from floral buds (Syzygium aromaticum L.) demonstrates notable antibacterial and antioxidant properties. This essential oil, known for its efficacy against significant foodborne pathogens[30], possesses anti-free radicals and metal chelating capabilities, functioning effectively as a bactericide. Despite its powerful attributes, the strong and distinct odour of clove oil has restricted its applications in the food industry. To address this limitation, encapsulation has been proposed as a viable strategy for mitigating the potent smell associated with clove oil[31].

    Recent studies have shown using Carnauba wax (1.0% and 0.50%), the sensory quality of pointed gourd treated with Carnauba wax was initially good to very good but deteriorated significantly by the 6th day of storage which offered benefits such as delayed ripening and reduced water loss, their efficacy in maintaining sensory quality and extending shelf life appears limited[32]. In another study involving coatings developed with starch, ethylenediaminetetraacetic acid (EDTA), and sodium alginate, the composite coatings were effective in extending the shelf life of green chillies[33]. However, the weight loss in coated samples (5.60%−6.90%) was still relatively high compared to the uncoated samples (12.35%)[34]. Priyadarshi et al.[34] has studied the effect of chitosan/citric acid-based packaging film pouches on the shelf-life of green chilli. The authors have shown that the shelf life can be slightly improved or affected by the application of an active packaging system. Therefore, a better alternative is proposed, the combination of chitosan with essential oil and beeswax with essential oil could be a better alternative to Carnauba wax and composite coatings developed with starch, EDTA, and sodium alginate or chitosan/citric acid.

    This research work presents and discusses the chitosan and beeswax-based functional coating to understand the possible applications of these coatings to evaluate the quality and freshness of pointed gourd and green chilli in real-time. These coatings offer enhanced antimicrobial properties, reduced weight loss, and better preservation of sensory quality, making them more effective in extending the shelf life of green chillies and pointed gourds.

    Chitosan (75%–85 % deacetylated) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Glycerol was obtained from Loba Chemie (Mumbai, India). Beeswax and essential oils were purchased from the local market, Phagwara, Punjab, India. Green chillies of equal size, colour, and maturity were purchased from the nearby market, Moga, Punjab, India. Pointed gourds commonly called parwals were also bought from the local market, Phagwara, Punjab, India. The green chillies were chosen based on their uniformity in size, color (green), and maturity (fully ripe), while the pointed gourds were selected for consistency in size, color (green), and maturity (medium stage of ripening). Any other reagents used in this study were of analytical grade and used without any purification.

    The experimental requirements consisted of fresh pointed gourd and green chillies, they were carefully selected for their physical condition, devoid of injuries, and exhibiting uniformity in color, size, and maturity. To make sure that optimal hygiene of the experimental fruits was followed and therefore a washing process was initiated utilizing sodium hypochlorite as a chlorine-based cleaner. This process involved immersing the fruits in chlorine-infused water for 10 min. Subsequently, the washed fruits were left to air dry under a fan, ensuring a thorough drying process. The coating application involved immersing the prepared fruits in the respective coating solutions for 5 min. Following each immersion, the fruits were extracted from the solution and allowed to air dry for an additional 10 min post-coating application, both the coated and uncoated samples were systematically arranged on plastic trays and stored at an ambient temperature of 25 ± 2 °C and a relative humidity of 70%−80%.

    The selection of samples was done with uniform colour, size, and maturity and devoid of any injuries. Then the sample was washed properly for 5−7 min. Washed fruits are then allowed to air dry completely. The selected green chillies and pointed gourd were divided into five groups according to the type of coating (M1 = uncoated fruits, M2 = coated with only chitosan, M3 = coated with chitosan and clove essential oil, M4 = coated with only beeswax, and M5 = coated with beeswax and clove essential oil). Each group contains three green chillies and three pointed gourds. Both chillies and pointed gourd were cleaned with water for 5−7 min and air dried before applying the edible coating. The samples were immersed in the coating solution for 5 min and then were kept for air drying for 10 min. The same procedure was repeated three times for both chillies and the pointed gourd sample.

    There were five treatments: M1 (control sample), M2 (only chitosan coating 2%), M3 (chitosan 2% + 0.1% (w/w) clove essential oil), M4 (only beeswax coating), and M5 (beeswax + 0.1% (w/w) clove essential oil).

    Chitosan coating (M2) - prepared by taking 2 g chitosan and dissolving it in 0.5% acetic acid in distilled water. This was then placed on a magnetic stirrer for 8−10 h. The pH was adjusted to 5.60 by adding 50% citric acid. Then 0.2 mL glycerol was added to act as a plasticizer.

    Chitosan incorporated with clove essential oil coating (M3) – chitosan solution - prepared by taking 2 g chitosan and dissolving it in 0.5% acetic acid in distilled water. This was then placed on a magnetic stirrer for 8−10 h. Then the pH was adjusted to 5.60 due to the addition of 50% citric acid. Then 0.2 mL glycerol was added. Essential oil solution - prepared by combining 1 mL of clove essential oil, 0.5 mL of Tween-20, and 8.5 mL distilled water in a test tube. 1 mL of this essential oil solution was then added to the previously prepared chitosan solution and thoroughly mixed for 15−20 min on a magnetic stirrer.

    Beeswax coating (M4) – prepared by taking 20 g beeswax, melting it at 55−60 °C and then filtering it. 80 mL of edible oil (rice bran oil) and 0.2 mL glycerol were dissolved in melted beeswax and then thoroughly mixing and used for coating.

    Beeswax incorporated with clove essential oil coating (M5) – prepared by taking 20 g beeswax and melting it at 55−60 °C and then filtering it. 80 mL of edible oil (rice bran oil) and 0.2 mL of glycerol were added along with melted beeswax. The solution was then stirred until homogenous. Clove essential oil solution. One mL of this essential oil solution was added to the beeswax solution and mixed thoroughly.

    The evaluation of scavenging activity for the blended sample was carried out using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. DPPH solution was prepared by mixing 4 mg of DPPH in 100 mL of methanol. A 50 mg aliquot of the sample solution was blended with a 10 mL DPPH solution, and absorbance values were recorded post 30-min incubation in the dark. The baseline values were established at 517 nm, the specific wavelength for the DPPH assay. Methanol was used as a blank and only the DPPH solution was referred to as control. The quantification of sample scavenging activity was achieved through a designated equation, and the entire experiment was executed in replicates to ensure the accuracy and reliability of the results. This methodology provides insights into the antioxidant potential of the blended sample through DPPH radical scavenging assessment[35].

    Freeradicalscavengingactivity(%)=AcAsAc×100

    where, AC and AS were the absorbances of DPPH of the control and sample, respectively.

    The application of four distinct coatings on green chilli and pointed gourd surfaces was executed utilizing the immersion method to enhance the adhesion and retention of the coatings. Following each dipping of the chilli and pointed gourd, the residual coating material was allowed to drip off, and this process was iterated three times. Subsequently, the coated pointed gourds and chilli were air-dried until complete desiccation. The dried specimens were then stored under ambient conditions with a temperature of 25 ± 2 °C for subsequent physiochemical analysis by established research protocols.

    The soil on the pointed gourd and green chillies was removed by washing with tap water. To assess the efficiency of the coating solution in preservation, samples were immersed in the solution containing the coating material. Uncoated samples were used as a reference for comparison. The samples were kept in different petri plates and were placed under ambient temperature, with continuous monitoring and recording[36]. Over an 8-d and 15-d storage-life test period an ambient temperature of 25 ± 2 °C and a relative humidity of 70%−80%. The changes in skin color, texture, and weight loss were observed for both coated and uncoated pointed gourds as well as for the chillies. The initial weight of the fruits was recorded at the start of the experiment, and subsequent weights were measured.

    Throughout the research duration, sensory characteristics were meticulously evaluated using a 9-point Hedonic scale. Various aspects, including physical attributes, texture, and overall acceptance, were scrutinized by panellists. For the sensory analysis, a panel of four trained evaluators was assembled, comprising three male teachers and one female teacher. The panellists were selected from the faculty members of the college conducting the research, representing a diverse age range. The ages of the panellists varied between 30 and 50 years. As the sensory analysis was repeated at fixed intervals the faculty members were familiar with the experimental protocol and provided valuable insights into the changes observed in the appearance and colour of the pointed gourds and green chillies. This approach ensured rigorous and reliable sensory evaluations. The scale, ranging from extremely liked (9) to extremely disliked (1), provided a nuanced assessment of the sensory attributes. This comprehensive approach enabled a detailed exploration of the subjective preferences and responses to the sensory qualities of the subject under investigation[37].

    The assessment of the storage impact on the 3-pointed gourd and green chillies involved weighing both samples at the commencement and conclusion of each storage interval. The total weight loss during storage was calculated by determining the difference between the initial and final weights of the fruit. To quantify this loss, percentages were computed based on the fresh weight of the fruit. This methodology provides insights into the perishability and stability of three pointed gourd and green chillies over time, aiding in understanding their post-harvest behaviour and potential shelf life[38].

    Lossinweight(%)=(Initialwt.Finalwt.)(Initialwt.)×100%

    The coating formulation was successfully prepared using chitosan and beeswax alone and in combination with clove essential oils. The developed formulation was applied for coating applications. Shelf-life analysis of pointed gourds and green chillies was performed by coating them with an edible coating made from chitosan, beeswax, chitosan with clove essential oil, beeswax with essential oil and one control sample with no coating. The results are displayed in Tables 16. Tables 1 & 2 shows the sensory analysis and weight loss for pointed gourd and Tables 3 & 4 show the sensory analysis and weight loss for green chillies, while the effect of coating on pointed gourd and green chillies are presented in Tables 5 & 6 respectively. M1 in the tables represents a sample without any coating to assess the impact of edible coating on raw fruit, M2 represents the coating with chitosan, M3 represents coating with chitosan and clove essential oil, M4 represents coating with beeswax and M5 represents coating with beeswax and clove essential oil. Pointed gourd samples were analyzed for 8 d and the observation was made every 2 d. The samples were analyzed based on sensory analysis and weight loss. The sensory evaluation was based on the changes in the color and the overall appearance of the pointed gourd

    Table 1.  Representing sensory evaluation of pointed gourds.
    Treatments Sensory properties (days in storage)
    2 4 6 8
    M1 7.67 ± 0.82 5.67 ± 0.47 3.33 ± 0.47 1.33 ± 0.47
    M2 8.33 ± 0.47 7.67 ± 0.47 5.67 ± 0.47 5.33 ± 0.47
    M3 8.67 ± 0.47 8.33 ± 0.47 7.63 ± 0.47 7.07 ± 0.47
    M4 7.67 ± 0.47 7.33 ± 0.47 6.3 ± 0.47 4.67 ± 0.47
    M5 8.33 ± 0.47 7.67 ± 0.47 7.21 ± 0.47 6.84 ± 0.82
     | Show Table
    DownLoad: CSV
    Table 2.  Representing weight loss of pointed gourds during storage.
    Treatments Physiological loss in weight (%) (days in starage)
    2 4 6 8
    M1 12.71 ± 2.91 18.57 ± 2.68 22.11 ± 1.18 24.72 ± 0.21
    M2 1.70 ± 0.16 3.40 ± 0.32 5.91 ± 0.45 7.60 ± 0.26
    M3 0.05 ± 0.01 0.25 ± 0.11 0.59 ± 0.20 0.82 ± 0.33
    M4 2.39 ± 0.21 5.46 ± 0.48 9.97 ± 1.10 11.3 ± 0.78
    M5 0.89 ± 0.30 1.76 ± 0.52 3.82 ± 0.41 4.67 ± 0.31
     | Show Table
    DownLoad: CSV
    Table 3.  Representing sensory evaluation of green chillies.
    Treatments Sensory properties (days in storage)
    3 6 9 12 15
    M1 7.67 ± 0.47 6.3 ± 0.47 4.67 ± 0.47 2.67 ± 0.94 1.67 ± 0.47
    M2 8.33 ± 0.47 7.67 ± 0.47 6.33 ± 0.94 6.00 ± 1.41 5.00 ± 0.82
    M3 8.67 ± 0.47 8.33 ± 0.47 8.00 ± 0.82 7.67 ± 0.47 7.33 ± 0.47
    M4 7.67 ± 0.47 6.84 ± 0.47 5.33 ± 0.47 5.00 ± 0.81 4.66 ± 0.47
    M5 8.67 ± 0.47 8.33 ± 0.47 7.67 ± 0.47 7.33 ± 0.47 6.67 ± 0.47
     | Show Table
    DownLoad: CSV
    Table 4.  Representing weight loss of green chillies during storage.
    Treatments Physiological loss in weight (%) (days in storage)
    3 6 9 12 15
    M1 3.26 ± 0.73 4.37 ± 1.2 6.36 ± 0.92 11.67 ± 1.3 15.34 ± 1.10
    M2 0.92 ± 0.31 1.23 ± 0.35 3.86 ± 0.75 5.45 ± 0.23 8.45 ± 1.20
    M3 0.40 ± 0.21 0.96 ± 0.23 1.40 ± 0.46 1.95 ± 0.74 2.30 ± 0.36
    M4 1.81 ± 0.73 2.62 ± 0.47 4.77 ± 0.18 5.11 ± 0.54 6.21 ± 0.27
    M5 0.69 ± 0.19 0.98 ± 0.23 3.21 ± 0.46 3.40 ± 0.36 4.47 ± 0.63
     | Show Table
    DownLoad: CSV
    Table 5.  Visual records of pointed gourd coated and uncoated for 8 d.
    Days M1 M2 M3 M4 M5
    2
    4
    6
    8
     | Show Table
    DownLoad: CSV
    Table 6.  Visual records of green chillies coated and uncoated for 15 d.
    Days M1 M2 M3 M4 M5
    3
    6
    9
    12
    15
     | Show Table
    DownLoad: CSV

    Based on the obtained results and per the evaluation it can be concluded that there was a minimum change in the pointed gourd and chillies sample coated with chitosan + clove essential oil which is followed by pointed gourd and chillies coated with beeswax + clove essential oil. The control sample showed the maximum change in the overall appearance in both cases. Physiological losses in weight increase gradually in all the treatments with the advancement of the storage period. The loss in weight in uncoated pointed gourd was observed from 12.71% to 24.72%. The pointed gourd sample coated with chitosan and chitosan + clove essential oil showed an increase in weight loss from 1.7% to 7.60%, and 0.05% to 0.82% respectively. The pointed gourd coated with beeswax and beeswax + clove essential oil showed an increment of weight loss from 2.39% to 11.3%, and from 0.89% to 4.67% respectively. Only limited work has been done on pointed gourd coating and the Carnauba wax (1.0% and 0.50%) coating on the pointed gourd showed earlier some promising results[32]. The sensory quality of pointed gourd treated with Carnauba wax was found to be primarily appealing, but the shelf life declined meaningfully after storage for 6 d which indicates although the coating is beneficial for delaying ripening and reducing water loss, its usefulness in preserving sensory quality and extending shelf life still appears restricted.

    The sample of fresh green chillies was analyzed for 15 d and the observation was made every 3 d and analysis was done based on sensory analysis and weight loss. The sensory evaluation focused on color and overall appearance changes in the chillies. The findings suggest minimal alterations in the chilli sample coated with chitosan + clove essential oil, followed closely by chillies coated with beeswax + clove essential oil. In contrast, the control sample exhibited the most significant changes in overall appearance. Notably, the chitosan-only coating displayed better color retention compared to the sole beeswax coating. These results underscore the effectiveness of combined coatings, particularly with chitosan and clove essential oil, in preserving the visual attributes of chillies, highlighting their potential for enhancing the shelf life and consumer appeal of the produce.

    During storage at room temperature, changes in weight loss were observed in both the control and coated green chillies samples. Uncoated green chillies experienced a notable increase in weight loss, escalating from 3.26% to 15.34%. In contrast, chillies coated with chitosan and chitosan + clove essential oil showed a rise from 0.92% to 8.45%, and 0.40% to 2.30%, respectively. Additionally, chillies coated with beeswax and beeswax + clove essential oil demonstrated an increase in weight loss from 1.81% to 6.21% and from 0.69% to 4.47%. These findings indicate the potential of coatings, particularly chitosan and beeswax, in mitigating weight loss during storage, contributing to the preservation of green chillies. A similar result was reported in the case of alginate/carboxymethyl cellulose/starch-based coating for green chilli preservation[39]. The application of an edible coating of green chilli significantly enhanced the shelf life by reducing moisture loss. The color and texture of the coated chilli were superior compared to the uncoated counterpart. Similarly, coatings were developed using starch, EDTA, and sodium alginate, and used in maintaining the shelf life of green chillies[33]. The authors showed that the weight loss in coated chillies samples (5.60%−6.90%) was still relatively high compared to the uncoated samples (12.35%). The same authors also studied the shellac-based surface coating on green chillies in combination with modified atmosphere packaging (MAP)[40]. The coated chillies exhibited longer shelf life compared to the uncoated counterpart and the use of MAP in combination with coating further extended the shelf life of chilli. Thus, the combined treatment could be very effective in preserving the shelf life of chilli. The effect of edible gum arabic and chitosan-based coating on green chillies has also been recently studied[41]. The obtained results showed that the application of the edible coating on chilli significantly improved the shelf life by maintaining the respiration rate and vitamin C content. In another work, chitosan/citric acid film was used to make pouches for green chilli packaging[34]. The biopolymer-made pouches used for green chilli packaging showed some effect on the color and shelf life of chilli due to the presence of citric acid but the effect was not pronounced due to the lack of presence of a strong functional ingredient in the packaging system. The presence of essential oil in the current packaging system and direct coating application showed an overall better effect in improving the shelf-life of chilli.

    DPPH analysis was carried out for the control sample, chitosan-coated sample, chitosan with clove essential oil-coated sample, and clove essential oil sample. No antioxidant activity was observed in the control, whereas in the chitosan-coated sample, it was found to be 17.3% ± 0.1%, in the case of the chitosan with clove essential oil-coated sample it was found to be 31.4% ± 1.0%, while in the case of clove essential oil 67.1% ± 1.5%. Therefore, it can be concluded that the control sample shows no radical scavenging activity since it has no added antioxidant coating. The chitosan-coated sample exhibits moderate antioxidant activity compared to the control as chitosan is known for its antioxidant action owing to the presence of function hydroxyl and amine group at carbon number six and two respectively[42]. The chitosan with clove essential oil-coated sample shows a much higher antioxidant activity compared to the control and chitosan-coated samples but less than only clove essential oil. The clove essential oil-coated sample shows the highest antioxidant activity among all three tested samples which is presumably due to the presence of strong antioxidant compounds such as eugenol, monoterpenes, propanoids, caryophyllene, etc.[43,44].

    Coating is an effective method for the improvement of the shelf life of fruits and vegetables. Chitosan, known for its biodegradability and environmental friendliness, boasts potent antimicrobial properties that make it a superior choice for various applications including food packaging. Beeswax, a natural and renewable resource offers remarkable water-repellent qualities, providing surfaces with robust protection against moisture and physical damage. Combining chitosan with clove essential oil results in a coating with heightened antimicrobial activity, offering broader protection against diverse microorganisms while enhancing the flavour and aroma of coated products. Similarly, blending beeswax with clove essential oil yields a coating with enhanced antimicrobial efficacy that may improve adhesion, and a delightful sensory experience, making it an excellent choice for preserving the freshness and quality of various products.

    The edible coating can be used in the shelf-life extension of pointed guard and green chillies. Weight loss, visible decay, and visual surface color change of the tested food were evaluated for 15 d. The results showed that the coated samples experienced more self-life and less weight loss than the control sample. The samples coated with chitosan + clove essential oil showed minimum changes in the appearance making it a suitable combination for shelf-life extension. After chitosan + clove essential oil, the samples coated with beeswax + clove essential oil showed minimum changes in the samples. Pointed gourd and green chilli samples coated with chitosan and beeswax displayed little variation in the physical appearance and made them fall behind the samples coated with beeswax + clove essential oil respectively. Hence inference can be drawn that chitosan with clove essential oil and beeswax with clove essential oil are the most potent methods for shelf-life extension in the case of pointed gourd and green chillies. Still, there is scope as well as the need for further improvement in edible functional coating. The work presented here is a preliminary study and thus needs further research for the practical implementation of this work for active food packaging purposes.

    The authors confirm contribution to the paper as follows: conceptualization, validation: Vidyarthi EV, Khela RK, Thakur M, Roy S; methodology: Vidyarthi E, Khela RK, Thakur M; writing—original draft preparation: Vidyarthi EV, Khela RK, Thakur M; writing—review and editing, visualization, supervision: Roy S. All authors have read and agreed to the published version of the manuscript.

    All data generated or analyzed during this study are included in this published article.

  • The authors declare that they have no conflict of interest.

  • Supplemental Table S1 Standard calibration curve equation and R2 (coefficient of determination) values used for analysis of biochemical parameters.
    Supplemental Fig. S1 Graphical abstract.
    Supplemental Fig. S2 HPLC chromatograms of mixed standard solution and green tea extracts processed by different methods.
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  • Cite this article

    Adhikary B, Kashyap B, Kanrar B, Gogoi RC, Varghese S, et al. 2024. Comparative evaluation of the impact of processing methods in determining the levels of health promoting chemical constituents and quality of green tea. Beverage Plant Research 4: e027 doi: 10.48130/bpr-0024-0016
    Adhikary B, Kashyap B, Kanrar B, Gogoi RC, Varghese S, et al. 2024. Comparative evaluation of the impact of processing methods in determining the levels of health promoting chemical constituents and quality of green tea. Beverage Plant Research 4: e027 doi: 10.48130/bpr-0024-0016

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Comparative evaluation of the impact of processing methods in determining the levels of health promoting chemical constituents and quality of green tea

Beverage Plant Research  4 Article number: e027  (2024)  |  Cite this article

Abstract: The first step of green tea manufacture involves enzyme deactivation by heat application. The present study investigated the effects of various fixing and processing methods viz. steam-roasting (S-6), pan-firing (P-8), blanching (B-2), and CTC cuts after steam-roasting (S-CTC), on the bio-chemical profiles and organoleptic quality of green teas processed differently into orthodox and CTC types, from region-specific tea cultivars, suitable for green tea production under agroclimatic condition of Dooars, West Bengal, India. Differences in fixing method and processing style showed notable variation (p ≤ 0.05) in the chemical quality indicators of green tea viz. Total catechin, polyphenol, flavonoid, and water extract content among the differently processed green teas. The most significant finding of the study revealed that when B-2 is employed for deactivation, it resulted in a substantial reduction (47%−52%) of caffeine levels without affecting the catechins content and antioxidant potential of green tea samples when compared to S-6 and P-8 methods. Interestingly, our results demonstrated significantly higher water extract values (42.19% dry weight) in green CTC teas and lower values in B-2 green tea samples (33.67%), as compared to S-8 and P-8 green teas, which received better taster ratings (≥ 7). These findings have highlighted the role of processing method and the impact of fixing technique in determining the contents of health-promoting attributes and taste quality of green teas, thus providing diverse choices to tea producers and consumers to opt for specific green tea products and expediting the need to further explore its commercial application in the nutraceutical and pharmaceutical industry (Supplemental Fig. S1).

    • Green tea is an un-aerated product made from the tender shoots of the tea plant (Camellia sinensis L.). Unlike black tea, the processing technique employed for green tea production omits the oxidation stage by fixing enzymes, allowing the tea to remain green in color which leads to an assortment of external and inner qualities[1]. Green tea production employs both Chinese and Japanese-style processing methods. Steaming is preferred in Japan and India, whereas the pan-firing method is popular in China and Korea. It has been previously reported that differences in processing methods impart distinguishable changes in the biochemical profile and taste quality of green teas[2]. Green tea processed in orthodox forms requires hand or machine-assisted rolling of deactivated tea leaves to obtain relatively bigger-sized tea particles, but in the CTC method, the fixing step is followed by complete maceration of the tea leaves to produce smaller-sized granular particles. Green tea fixing methods like steaming, pan-firing, and blanching have developed over the years depending on cultural variability, market acceptability, and end-user preference[3]. The fixing stage of enzyme deactivation along with the rolling step, in which leaves are curled and twisted, are considered important technical parameters to attain desirable quality in green tea. Monitoring the initial fixing step at elevated temperatures during green tea production, is crucial to avoid catechin oxidation that is responsible for the astringency and bitterness of green teas, during storage[4]. It has been reported that over-fixing increases the amino acid content due to rapid protein hydrolysis, scorches the leaf and results in a smoky taste and a higher ratio of broken leaf whereas under-fixing results in the browning of green teas upon storage due to residual enzyme activities[2]. Research studies suggest that bigger-sized orthodox green teas retain more moisture and are more likely to be oxidized, reducing their storage life and extraction efficiency, whereas, in contrast, the CTC teas' increased surface area causes smaller particles to interact with water more during brewing, which increases the extraction efficiency of the final brew and therefore has been found to be suitable for ready-to-drink dip tea sachets[5]. Production of green CTC tea has been shown to be a simple, better recovery and less time-consuming method[6], wherein, the teas exhibited bright green colour and more cuppage. The blending and mixing of herbs for desirable flavors become easier with the smaller-sized CTC tea granules. Ready-to-drink flavoured CTC green tea variants are becoming popular nowadays and present competitive and customer-appealing products in the market. Few researchers have reported that the type and quality of the green tea reflected by its chemical composition and the soluble solids extracted during infusion not only depend upon genetic character and temperature of infusion but are also affected by multifarious biochemical changes within the tea leaves during fixing as well as harvesting and subsequent processing conditions[3,7]. Primary leaf biochemicals imparting the taste characteristics and health-attributes of green tea include catechin polyphenols, caffeine, free amino acids, chlorophyll and other compounds jointly or separately[8]. The water-soluble extracts of green tea are the main compounds responsible for bioactivity and the associated medicinal benefits[9].

      Scientific investigations on tea biochemicals in recent years have justified the ancient belief of health promoting benefits of green tea consumption[10], which is gaining popularity among the wider community. Increased mass media propaganda on the medicinal health-promoting effects linked with green tea consumption has created a lucrative market potential. As a popular non-alcoholic beverage, green tea products with diverse physical and chemical quality parameters would be equally beneficial to tea manufacturers and consumers.

      Ensuing the futuristic trend of the Indian tea industry, there is a scope for Dooars tea estates to divert attention to these alternative ways of green tea processing as well as its promotion to capture the domestic market domain. Recent findings have generated information on region-specific green tea suitable cultivars[11], however, a detailed scientific study in terms of biochemical constituents and organoleptic taste profiles of differentially fixed and processed orthodox and CTC green teas has not been addressed under Dooars agroclimatic conditions. Therefore, the present study was conducted to investigate the impact of various fixing methods viz. steaming, pan-firing, and blanching for orthodox and CTC green teas, in determining the anti-oxidative and health-promoting biochemical profiles, by keeping minimal variations in cultivars, plucking, and other technical aspects of process-parameters.

    • All the chemicals used for this study were of analytical grade. Miniature manufacturing was carried out using a mini-CTC machine (Mesco Equipment) with a 10 tpi (tooth-per-inch) roller. UV-VIS spectrophotometer (Cary Bio 50) was used for biochemical analysis. HPLC analyses (Agilent 1260 infinity UPLC) for the estimation of catechin fractions and caffeine were carried out.

    • The Dooars region is situated in the Himalayan foothills and has loamy to sandy loam type and silt clay type of soil. Tea cultivars used in the study were grown in the trial plot of our center, situated at 26°54' North Latitude and 88°54′ East Longitude with an elevation of 226.60 m. Long-term weather data showed a daily temperature range of 18.7 to 28.8 °C, humidity levels of 91.5% (morning) and 64.2% (evening), 5.72 h average sunshine, and approximately 3,800 mm of annual rainfall. Monsoon commences from May-June and lasts until September, followed by cold winters. During the period of our study from July to September, the maximum and minimum temperatures were 31.3 to 32.3 °C and 20.5 to 21.8 °C respectively with corresponding precipitation recorded between 867.7 to 1,058.2 mm. The experimental plot received the balanced fertilizers of nitrogen, phosphorus, and potassium in two splits at a ratio of 110:25:110, as recommended for tea cultivation in this region[12].

    • Locally grown tea cultivars viz. Tocklai Vegetative (TV)-9, TV-20, Teenali (TA)-17/1/54, and Tocklai Stock (TS)-462 of similar age from our experimental plots were used for green tea processing. These cultivars were selected based on previous studies on the suitability of these cultivars for green tea production[11,13]. Leaf quality of 50%−60% fineness, was maintained. Three sets of green tea samples were processed by different methods, on three separate occasions, during July, August, and September respectively, coinciding with monsoon flush, and subsequently analyzed for chemical and sensory parameters.

    • The leaf samples in equal proportions from each cultivar were mixed and then divided into four parts of 0.5 kg each. Green tea processing of the orthodox and CTC-type was done in a miniature factory set-up by various methods, which included conventional steam-roasting, pan-firing, blanching, and steam-roasting followed by 2 cut CTC, with minor simplified modifications as follows.

    • Green tea was processed by steaming in a perforated chamber at 105 ± 5 °C for 6−8 min, depending on leaf standard. The steamed leaves were surface dried and cooled by blowing air for 15 min, rolled in a peizzy roller for 30 min and finally dried in a cabinet drier at 110 °C for 40 min[13].

    • Fresh leaves were deactivated by pan-firing in an electric panner at 250 ± 10 °C for 8−10 min, depending on leaf standard, cooled by blowing air for 15 min, rolled in a peizzy roller for 30 min and finally dried in a cabinet drier at 110 °C for 40 min[11].

    • Fresh leaves were fixed by dipping in boiling distilled water for 1−2 min depending on leaf standard. The subsequent procedure was similar to the conventional steam-roasting method.

    • Fresh leaves were deactivated by steaming in a perforated chamber at 105 ± 5 °C for 6−8 min, depending on leaf standard. The steamed leaves were surface dried and cooled by blowing air for 15 min, rolled in a peizzy roller for 30 min prior to cutting in a mini-CTC machine for twice (2-cut). Final drying was carried out in a cabinet drier at 110 °C for 40 min.

    • Extraction of green tea catechins was carried out following the ISO method[14]. Samples were finely grounded in a mortar-pestle, and 0.2 g samples were taken in graded tubes. To the sample, 5 mL of 70% (v/v) pre-heated (70 °C) methanol/water extraction mixture was added and mixed thoroughly on a vortex mixer (Remi). The tubes were put in a water bath at 70 °C (10 min), followed by mixing in a vortex mixer after 5 and 10 min, respectively, and then cooled to room temperature, before centrifugation (3,500 rpm, 10 min) (Remi). After decanting the supernatant in a graduated tube, the residue was extracted a second time with 5 mL of extraction mixture. The extracts were then mixed and concentrated to a volume of 10 mL using the methanol/water extraction mixture. For catechin analysis by HPLC, 1 mL of sample extract was mixed with 4 mL of stabilizing solution (10% v/v acetonitrile with 500 μg/mL EDTA and ascorbic acid), and filtered through a 0.45 μm filter and injected.

      Individual catechins, gallic acid and caffeine was analyzed using Agilent 1260 infinity series HPLC equipped with Zorbax Eclipsed plus Phenyl-Hexyl column (4.6 mm × 250 mm, 5 μm) and Agilent Zorbax Eclipsed Plus Phenyl-Hexyl guard column (4.6 × 12.5 mm, 5 μm), in accordance with the ISO method[14]. Different ratios of water, acetonitrile, and acetic acid solvents were employed for mobile phase A (89:9:2) and mobile phase B (18:2:80). Both the mobile phases received an addition of EDTA (20 μg/mL). The injection volume was 20 μl. Flow rate, column temperature and detection wavelength were set to 1 mL/min, 35 ± 0.5 °C, and 278 nm respectively. A binary gradient condition was initiated with 100% mobile phase A for 10 min, followed by a linear gradient over a period of 15 min to 68% mobile phase A and 32% mobile phase B, which was run and maintained for 10 min. The system was then reset to 100% mobile phase A and given 10 min to equilibrate before the next injection. Catechin and caffeine peaks were identified by comparing retention times from sample chromatograms with those obtained from the mixed standard solutions under the same chromatographic conditions.

      Quantification of individual catechins, gallic acid and caffeine was done by using the Relative Response Factors (RRFs) values for catechins and gallic acid with respect to caffeine and comparing the peak area of individual components to the caffeine calibration graph (Supplemental Table S1) as described[14].

    • Folin-Ciocalteau reagent was used to estimate the total polyphenol content by the ISO method[15]. Approximately, 0.2 g of well-ground green tea powder was extracted using a methanol-water (70−30) mixture, and the extract was then diluted to 100 times with water. To 1 mL of the diluted extract, 5 mL (10 % v/v) Folin-Ciocalteau reagent was added, followed by 4 mL of Sodium Carbonate solution (7.5% w/v), which was thoroughly mixed. The mixture was left to stand at room temperature for 1 h. The result of the absorbance measurement at 760 nm was reported as a weight percentage by the gallic acid standard calibration curve values (Supplemental Table S1).

    • TFC was estimated following the method described by Akbay et al.[16]. Powdered green tea sample (0.2 g) was extracted with methanol-water (70−30) mixture. One mL of this extract was diluted with 3 mL of methanol. To this diluted solution 0.2 ml of Aluminium Chloride (AlCl3, 1 M), 0.2 mL potassium acetate solution (10% w/v) and 5.6 mL distilled water were added and mixed well. Similarly, a sample blank was prepared by replacing AlCl3 with water. The absorbance reading was taken at 415 nm and the result was obtained by quercetin standard calibration curve values (Supplemental Table S1).

    • The water extract content in green tea samples was determined using the ISO method[17], with minor modifications, to evaluate the effect of process type in determining the water-soluble extract content. Briefly, the non-grounded orthodox and CTC green tea samples (2.0 g) were put in a 250 mL flask, and 200 mL hot distilled water was added and refluxed over low heat for 1 h, rotating the flask occasionally. The flask was repeatedly washed out with hot distilled water transferring all the insoluble residue into a pre-weighed crucible. Finally, the residue was washed with 200 mL of hot water. The residue was dried by suction. The crucible and its contents were heated in an oven at 103 ± 2 °C, for 16 h. The dessicator-cooled crucible with its content was weighed. The result was expressed as a weight% on a dry mass basis, as mentioned in ISO 9768:1994[17].

    • ISO 3103:2109[18] was followed in the preparation process of tea infusion and liquor. This method entails extracting soluble substances from 2.8 ± 0.2 g of dried tea leaves contained in a porcelain cup using 150 ml of freshly heated water, that is covered with a lid for 4−5 min for brewing. The liquor is then poured into the bowl through the serration in the lid cover to keep the infused leaf in the pot. The lid was removed and then inverted and the infused leaf was placed on it. The tea taster used a randomized method for the tasting, concealing the sample details in accordance with ISO 3163[19] and examined the infused leaf and liquor organoleptic qualities. Once the tea is cool enough, a tea taster uses a large spoon to noisily slurp the liquor into the lips, which guarantees a consistent taste profile by allowing adequate oxygen and tea to travel through the tongue's taste receptors[20]. The liquid was spit subsequently in a spittoon, before the next sample. The parameters of tea quality (color, strength, briskness, and brightness), manufacturing defect parameters, and field-related agro-practices have been described by FAO[21]. After taking these findings into account, the taster evaluated the tea's organoleptic quality in Hedonic scale ratings ranging from 0 to 10[11,22].

    • The DPPH antioxidant activity assay was carried out following a previously reported method[23], with slight modifications. Briefly, the powdered tea sample (0.2 g) was extracted with methanol-water (70−30) mixture. DPPH solution (0.1 mM in methanol, 4 mL) was added to 2 mL each of the different concentration of the extract (2.5, 5, 7.5, 10, 12.5, 15, and 20 μg/mL). The reaction mixture was incubated at room temperature for 30 min and absorbance readings were taken at 517 nm, with ascorbic acid (100 μg/mL) as positive control and methanol as blank. The inhibition ratio (i.e., the concentration of the tea extract required to reduce the absorbance of DPPH by 50%) of the sample was obtained using the following equation:

      Inhibition ratio (%) = {(Ac – As)/Ac} × 100; where, Ac = Absorbance of the control and As = Absorbance of the sample

      A plot of inhibition ratio against concentration gives a straight line from which IC50 was calculated.

    • The FRAP assay was performed following a previously reported method[24], with some modifications. Preparation of FRAP reagent was carried out by mixing 10 mM 2,4,6-Tripyridyl-S-triazine (TPTZ) in 40 mM HCl with 20 mM Ferric Chloride (FeCl3) in 300 mM acetate buffer. For the assay, 200 μL of tea extract, 1.8 mL of water and 4 mL FRAP reagent was mixed and the solution was incubated in the dark for 30 min. Then, the absorbance was taken at 593 nm using water as blank. The result was obtained by comparison with the Ferrous Sulphate (FeSO4) standard graph.

    • The data of each experimental analysis that was performed in triplicate was analyzed by one-way analysis of Variance (ANOVA). Mean values of the biochemical parameters were separated using Duncan's multiple test range (p ≤ 0.05). All values are represented as mean ± standard error (SE). Statistical analysis and correlations among the biochemical quality parameters were calculated by Pearson's correlation coefficient test using SPSS software (version 16.0., SPSS < Chicago, IL, USA).

    • The content of individual catechin fractions and total catechins, caffeine, gallic acid in green tea samples processed differently are presented in Table 1 and Fig. 1 respectively.

      Table 1.  Content (% dry weight) of non-gallated catechins viz. catechin +(C), epicatechin (EC) and epigallocatechin (EGC), gallated catechins viz. epicatechin gallate (ECG) and epigallocatechin gallate (EGCG), in green tea samples processed by different methods.

      Process type Catechin fractions Total catechins (TC)
      C EC EGC EGCG ECG
      S-6 0.99 ± 0.07 1.30 ± 0.06 3.87 ± 0.33 10.49 ± 0.57 2.84 ± 0.25 19.48 ± 0.44ab
      P-8 0.93 ± 0.03 1.46 ± 0.08 4.35 ± 0.31 10.84 ± 0.50 2.70 ± 0.14 20.26 ± 0.47b
      B-2 0.95 ± 0.05 1.39 ± 0.07 4.47 ± 0.40 10.79 ± 0.34 2.74 ± 0.25 20.33 ± 0.27b
      S-CTC 0.94 ± 0.05 1.43 ± 0.07 3.91 ± 0.21 9.84 ± 0.53 2.58 ± 0.21 18.69 ± 0.42a
      All values are represented as mean ± SE. Values within a column with different letters are significantly different by ANOVA with Duncan multiple test range at p < 0.05. C, (+)-catechin; EC, (−)-epicatechin; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; ECG, (−)-epicatechin gallate; TC, total catechins. S-6, steam-roasting for 6 min; P-8, panning for 8 min; B-2, blanching for 2 min; S-CTC, steam-roasting for 2 min followed by two CTC cuts.

      Figure 1. 

      Content (% dry weight) of gallic acid and caffeine of green tea samples processed by different methods. Values are represented as mean ± SE. Different lowercase letters on top of the bars indicate significant difference by ANOVA with Duncan multiple test range at p < 0.05; S-6, steam-roasting for 6 min; P-8, pan-firing for 8 min; B-2, blanching for 2 min; S-CTC, steam-roasting for 2 min followed by two CTC cuts.

      The data indicates that the processing method exhibits a significant impact on the chemical composition of green tea samples, especially caffeine. The content of epigallocatechin was found to be marginally higher in pan-fired and blanched-green teas than in steamed orthodox and CTC-mode processing. Although insignificant, the amounts of epigallocatechin gallate and epicatechin gallate were found to be notably higher in S-6, P-8, and B-2 as compared to S-CTC samples. However, significant differences were observed between the total catechin contents of S-CTC type green tea as compared to other process-type green teas, which may be attributed to the loss of gallated catechin fractions viz. epigallocatechin gallate and epicatechin gallate due to frictional heat generation at the crushing-tearing and curling step, that is congruent with a previous report that the level of green tea catechins is reduced due to epimerization and degradation during processing[25], and storage conditions such as temperature and relative humidity[2]. Contrary to our findings, higher total catechin content for CTC green tea than orthodox type has been detailed[26,27], the difference can be attributed to the diversity in tea cultivars, agroclimatic variation in the study and smaller size of CTC granules enhancing the extraction efficiency. It was noted that deactivation by blanching (B-2) reduced the caffeine content by 40%−50% as compared to other green tea processing types viz. S-6, P-8 and S-CTC respectively (Supplemental Fig. S2). Representative HPLC chromatographs of the mixed standards and green tea samples processed by different methods is mentioned in Supplemental Fig. S2.

      Our finding of significantly lower caffeine content of B-2 orthodox green tea (1.94%) as compared to other green tea types, is consistent with a previous report[27], where it was conferred that blanching tea leaves for 3 min eliminated 83% of the caffeine while retaining 95% of the catechins. It has been reported that the solubility of caffeine is very low in water at room temperature (2.2% w/w), however, in boiling water, the solubility of caffeine increases greatly (66.7% w/w). Specific removal of a significant amount of caffeine as compared to catechins, from fresh tea leaves during blanching, may be attributed to the higher solubility of caffeine in hot water and its lower molecular weight (21.7 g·L−1, 194.2 kDa) than the catechins (~ 5 g·L−1, 290−458 kDa), that allows caffeine molecules to diffuse through the cell membrane and hence, during the blanching step, a large amount of caffeine goes out of the leaf resulting in lower caffeine content in the green tea[28]. The results also indicated significant variation (p ≤ 0.05) in the gallic acid content of S-6 and P-8 orthodox green teas that could have occurred due to wet and dry mode of heat application and resulted in greater loss of gallic acid in steaming by wet-heat as compared to pan-firing.

      The combination of catechins with caffeine and gallic acid is often associated with green tea taste. Although caffeine intake has some proven health benefits, higher intake of caffeine can have a negative impact on the human central nervous system and is therefore contraindicated for children and pregnant women[29,30]. Moreover, studies have linked consumption of caffeine containing beverages with irritation of the gastrointestinal tract and sleeplessness[31]. There is persistent market demand for decaffeinated versions of tea and coffee beverages, therefore, caffeine reduction in the green tea manufacturing process is often desirable. Based on these findings, it can be suggested that fixation by blanching is one of the simple, non-toxic, and low-cost processes of decaffeinating green tea without removal of the catechin antioxidants.

    • The TPC and TFC in green teas processed by different methods is presented in Fig. 2. Significant variation in polyphenol content was observed for green tea samples processed by different fixing-methods and types.

      Figure 2. 

      Content (% dry weight) of total polyphenol, total flavonoid and water extract of green tea samples processed by different methods. Values are represented as mean ± SE. Different lowercase letters on top of the bars indicate significant difference by ANOVA with Duncan multiple test range at p < 0.05; TPC, Total Polyphenol Content; TFC, Total Flavonoid Content; WE, Water Extract; S-6, steam-roasting for 6 min; P-8, pan-firing for 8 min; B-2, blanching for 2 min; S-CTC, steam-roasting for 2 min followed by two CTC cuts.

      The conventional steam-roasting process (S-6) of orthodox green tea production retained the maximum polyphenols. Green teas (S-6, P-8, and S-CTC) exhibited higher polyphenol content (23.65%, 22.97%, and 22.50%) as compared to B-2 (21.90%). Similar data was obtained for TFC, wherein significant differences (p ≤ 0.05) were obtained for S-CTC and B-2 type green tea. The extraction rate of green tea polyphenols is influenced by the shape, size of tea leaves, and degree of destruction during the fixing of leaves and, usually the longer time of fixation during the steaming and panning method causes more destruction of leaves yielding more small particles compared to the shorter duration blanching process. The bi-directional rolling after fixation step aids the juices to spread out in steam-roasted green teas as compared to other methods. The CTC step after steaming leads to the loss of polyphenolic compounds because of prolonged processing[1]. Our results on differences of TPC and TFC content due to process-variation are in congruence with a previous finding[32], where the researchers inferred that thermal treatment by blanching resulted in transformation, as well as, loss of phenolic and flavonoid compounds due to leaching in water and therefore reduction in the phenolic and flavonoid content. Likewise, variation in polyphenol levels in the water infused extracts of green tea due to processing has also been described[33].

      Green tea polyphenols and flavonoids are key compounds conferring the antioxidative and therapeutic properties of tea consumption and also impart astringency and bitter taste to green tea infusion. The water-soluble polyphenols and flavonoids have the potential used singly or in combination with other active principles in the food, pharmaceutical, and cosmetic industries[10]. The health value of green tea beverages is determined, among others, by the content of polyphenolic substances, therefore, in summary, the conventional methods of steam-roasting and pan-firing are best suited to conserve polyphenols and flavonoids in green tea products.

    • Water extract content is a quality indicator that constitutes the phenolics, alkaloids, amino acids, and many minor water-soluble substances extracted from the tea samples which determines the quality and cuppage of the tea, and is employed in the tea industry[17]. Data presented in Fig. 2, show the average water extract contents to be 38.52%, 39.60%, 33.67%, and 42.19% for S-6, P-8, B-2, and S-CTC type- green teas respectively.

      Based on the current findings, all the analyzed green tea samples complied with the ISO requirement with regard to WE content, implying the presence of adequate extractable substances. However, significant variation was observed between orthodox and CTC type green tea samples. The WE content was higher in case of S-6, 2-CTC (42.19%) and lower in B-2 type processed orthodox green teas (33.67%). It has been reported that the water extract of tea depends on tea and water ratio, temperature of the tea brew, type, and size of made tea particles[34]. Our findings emphasized that the CTC cut facilitated smaller particle size thereby presenting a larger surface area of tea granules exposed to water and enabling effective extraction of soluble constituents in water during brewing as compared to the orthodox tea type, whereas, the soluble solid content of B-2 process was lower compared to other fixing methods which can be attributed to the draining-out of water-soluble components during the blanching process[35].

      The benefit of CTC-type green teas consisting of fannings and dust grades is that these are readily packaged in tea bags for easy marketability and a lesser time is required for brewing out the extractable bioactive compounds, than the leafy orthodox green teas, however, the smaller-sized particles of CTC green teas are more influenced by oxidative processes during storage than whole tea leaves, because a larger surface area is exposed to oxygen and light[7].

      The TQS of steamed (S-6) and pan-fired green teas (P-8) showed significant differences with blanched (B-2) and steamed-CTC (S-CTC) type with lesser bitterness components in orthodox green tea liquor than CTC type (Fig. 3), that indicates the preference of tea tasters towards conventionally processed orthodox type green teas[11,13]. Representative pictures of dry leaf, infused leaf and liquor appearance of green teas processed differently are shown in Fig. 4.

      Figure 3. 

      Taster Quality Score (TQS) of green tea samples processed by different methods. Values are represented as mean ± SE. Different lowercase letters on top of the bars indicate significant difference by ANOVA with Duncan multiple test range at p < 0.05; TQS, Tasters' Quality Scores. S-6, steam-roasting for 6 min; P-8, pan-firing for 8 min; B-2, blanching for 2 min; S-CTC, steam-roasting for 2 min followed by two CTC cuts.

      Figure 4. 

      Representative pictures of dry leaf appearance, infused leaf and liquor colour of green tea processed by different processing methods, used for organoleptic evaluation.

    • The antioxidant activity serves as an indicator of the proportion of antioxidant substances in green tea. Since it is not always possible to characterize the antioxidant potential of tea by a single assay because the majority of naturally occurring antioxidants found in tea have multiple functions, hence, we used the DPPH and FRAP assays in the current study to describe the antioxidant activity of green tea samples processed in different ways. Under the conditions described in this manuscript, no significant differences were observed in the IC50 values of green tea samples processed by different methods (Fig. 5).

      Figure 5. 

      Anti-oxidant activity in terms of DPPH and FRAP assay values of green tea samples processed by different methods. Values are represented as mean ± SE. Different lowercase letters on top of the bars indicate significant difference by ANOVA with Duncan multiple test range at p < 0.05; DPPH, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging ability; FRAP, Ferric reducing antioxidant power; S-6, steam-roasting for 6 min; P-8, pan-firing for 8 min; B-2, blanching for 2 min; S-CTC, steam-roasting for 2 min followed by two CTC cuts.

      The B-2 and P-8 type processed green teas exhibited the maximum and minimum IC50 value of 18.21 and 16.34 μg/ml respectively in the DPPH assay, which may be attributed to the loss of water-soluble antioxidants during blanching and negligible loss during deactivation by dry heat application or pan-firing. Likewise, in the FRAP assay, the values of S-6 and P-8 green teas were significantly higher than in B-2 and S-CTC methods, wherein, the variance noted in the FRAP values can primarily be attributed to the loss of anti-oxidative polyphenols during the blanching and CTC steps of green tea processing[23].

    • Pearson correlation was used to evaluate the relationship among the various chemical quality parameters and tasting scores for the green teas processed by four different methods (Table 2), wherein, a strong positively significant correlation was obtained between EGC and EC, TC and also between C and ECG, TFC. Additionally, caffeine content was positively and significantly correlated with TPC, WE, TQS and likewise, TFC with EC; TC with EGCG; TQS with ECG, WE also displayed significant positive correlation. However, a negative correlation was observed between EGC and C, ECG, WE, TQS; C and EGCG, TC; EC, ECG with EGCG; TC with ECC, TFC, WE, TQS as reported earlier[36]. These results confirm the above discussed results that some of the green tea chemical constituents are correlated to the different methods of green tea processing.

      Table 2.  Correlations between gallic acid (GA), epigallocatechin (EGC), catechin (C), caffeine (CFF), epicatechin (EC), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), total catechin (TC), total polyphenol content (TPC), total flavonoid content (TFC), water extract (WE) and tasters' quality score (TQS) in green tea samples processed by different methods.

      GA EGC C CAFF EC EGCG ECG TC TPC TFC WE TQS
      GA 1
      EGC 0.018 1
      C 0.099 −0.529** 1
      CAFF 0.090 −0.170 −0.118 1
      EC 0.035 0.510** 0.073 −0.186 1
      EGCG −0.021 0.093 −0.669** 0.269* −0.514** 1
      ECG 0.030 −0.679** 0.767** −0.100 −0.044 −0.430** 1
      TC 0.021 0.523** −0.630** 0.084 −0.051 0.809** −0.409** 1
      TPC −0.167 −0.073 −0.067 0.514** −0.088 0.175 0.035 0.137 1
      TFC 0.158 0.241* 0.437** −0.034 0.612** −0.755** 0.147 −0.443** −0.097 1
      WE 0.141 −0.495** 0.273* 0.514** −0.072 −0.326* 0.302* −0.556** 0.149 0.272* 1
      TQS 0.126 −0.483** 0.243* 0.399** −0.079 −0.216 0.364** −0.400** 0.173 0.137 0.856** 1
      **, Correlation is significant at the 0.01 level (1-tailed). *, Correlation is significant at the 0.05 level (1-tailed). GA, gallic acid; EGC, (−)-epigallocatechin; C, (+)-catechin; CAFF, caffeine; EC, (−)-epicatechin; EGCG, (−)-epigallocatechin gallate; ECG, (−)-epicatechin gallate; TC, total catechins; TPC, total polyphenol content; FC, flavonoid content; WE, eater extract; TQS, Tasters' Quality Score. The direction and magnitude of correlation between variables was quantified by the correlation coefficient r. One-tailed p value: *, p < 0.05; **, p < 0.01.
    • The findings of the current study provide practical information about the role of different processing methodologies on green tea quality, and the composition of health-promoting constituents in green tea extract and also establish a trend in which the level of these green tea biochemicals is governed. The phytochemicals and antioxidative properties of orthodox green teas were significantly higher than the CTC type. It is reported that the blanching method of deactivation results in the production of green tea with a significantly lesser amount of caffeine compared to steaming and pan-firing methods. Moreover, a significant increase in the content of water extract was observed in CTC-green tea compared to the orthodox type. However, the organoleptic evaluation by tasters preferred the orthodox green teas processed by steam and pan-firing to blanched and CTC-green teas. The detailed scientific and comparative study on the variation of chemical constituents due to processing differences of green tea provides relevant information for consumers and professionals from the tea and pharmaceutical industry. Further work on calibration of the green tea process parameters to obtain desirable biochemicals of therapeutic value, under large-scale settings, can be attempted.

    • The authors confirm contribution to the paper as follows: Adhikary B and Kashyap B contributed equally to this work. Conceptualization, data curation, writing - original draft: Adhikary B, Kashyap B; funding acquisition: Adhikary B, Kashyap B, Babu A; project administration, Supervision: Adhikary B, Varghese S, Babu A; investigation, validation: Kashyap B, Gogoi RC; resources: Adhikary B, Kanrar B, Babu A; methodology: Kashyap B, Kanrar B; formal analysis: Kashyap B; writing - review & editing: Adhikary B, Kashyap B, Kanrar B, Gogoi RC, Varghese S, Babu A. All authors reviewed and approved the final manuscript.

    • All data analyzed during this study are included in the published article and its electronic supplementary information files.

      • This work was supported by a research grant from the Department of Science and Technology and Biotechnology- Government of West Bengal vide Research Grant, Ref: 936 (Sanc.) ST/P/S&T/1G-18/2016 dated 10/01/2017.

      • The authors declare that they have no conflict of interest.

      • # Authors contributed equally: Biplab Adhikary, Bishwapran Kashyap

      • Supplemental Table S1 Standard calibration curve equation and R2 (coefficient of determination) values used for analysis of biochemical parameters.
      • Supplemental Fig. S1 Graphical abstract.
      • Supplemental Fig. S2 HPLC chromatograms of mixed standard solution and green tea extracts processed by different methods.
      • Copyright: © 2024 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (2) References (36)
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    Adhikary B, Kashyap B, Kanrar B, Gogoi RC, Varghese S, et al. 2024. Comparative evaluation of the impact of processing methods in determining the levels of health promoting chemical constituents and quality of green tea. Beverage Plant Research 4: e027 doi: 10.48130/bpr-0024-0016
    Adhikary B, Kashyap B, Kanrar B, Gogoi RC, Varghese S, et al. 2024. Comparative evaluation of the impact of processing methods in determining the levels of health promoting chemical constituents and quality of green tea. Beverage Plant Research 4: e027 doi: 10.48130/bpr-0024-0016

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