P U B L I C A T I O N S CODON Italian Journal of Food Science, 2023; 35 (3): 1–16 ISSN 1120-1770 online, DOI 10.15586/ijfs.v35i3.2346 1 Quality changes during storage in Thai indigenous leafy vegetable, Liang leaves (Gnetum gnemon var. tenerum) after different preparation methods Sunisa Siripongvutikorn1*, Worapong Usawakesmanee1, Supachai Pisuchpen2, Nicha Khatcharin1, Chanonkarn Rujirapong1 1Centre of Excellence in Functional Foods and Gastronomy, Faculty of Agro-Industry Prince of Songkla University, Hat Yai, Songkhla, 90110, Thailand; 2Centre of Excellence in Bio-based Materials and Packaging Innovation, Faculty of Agro-Industry Prince of Songkla University, Hat Yai, Songkhla, 90110, Thailand *Corresponding Author: Sunisa Siripongvutikorn, Centre of Excellence in Functional Foods and Gastronomy, Faculty of Agro-Industry, Prince of Songkla University, Hat Yai, Songkhla, 90110, Thailand. Email: sunisa.s@psu.ac.th Received: 8 March 2023; Accepted: 19 June 2023; Published: 10 July 2023 © 2023 Codon Publications OPEN ACCESS ORIGINAL ARTICLE Abstract Liang or Gnetum gnemon var. tenerum, an indigenous southern vegetable has recently attracted increasing interest due to its high nutritional value, creamy taste and lack of smell. The leaves with or without stem are washed with chlorinated water at 100 ppm for 15 min, stored at 4°C and investigated for physiochemical, chemical and sensory evaluations over time. Total phenolic and flavonoid contents were higher in treatments with stems (P <  0.05). Washing significantly increased moisture content and water activity (aw) in all treatments (P < 0.05). In addition, washing resulted in significantly higher DPPH and ABTS activity (P < 0.05). However, washing and stem detach- ment had no effect on sensory and physicochemical qualities. The sensory score of the 8-days stored sample was comparable to the fresh one (Day 0). Keywords: antioxidant; Gnetum gnemon; preparation; quality; stem; washing Introduction Liang or Gnetum gnemon var. tenerum, a common sig- nature southern vegetable, has the potential to become a new economic plant with less or free from pesticide residue. Generally, Liang is grown in backyards or as a fence plant by people in Southern Thailand. Liang has a creamy, umami taste with less green flavour and is often grown as an intercrop between various economic plants such as rubber, palm oil, durian and orchard plants to maximise the use of space and increase income. The tenerum shrub variety is grown in Thailand, whereas in Malaysia and Indonesia, the gnemon variety is grown as a tree (Anisong et al., 2022). Preliminary tests showed that the tenerum variety produced leaves containing essential amino acids with high health benefits including antioxi- dant, antidiabetic, anti-inflammatory, anti-breast cancer and gut microbiota–enhancing effects (Suksanga et al., 2022) due to high protein and phytochemical compounds such as chlorophyll, beta-carotene, phenolic compounds, flavonoids and dietary fibre, both soluble and insoluble. After harvesting, the leaves are usually bunched with rubber bands or packaged in open bags for transport to local or fresh markets (Figure 1), while in supermarkets, packaging in sealed bags is usually applied (Figure  2). To serve the new generation of people who live busy lives, ready-to-cook or minimal process ingredients are required to meet their needs. In supermarkets, ready-to- cook leafy vegetables are usually packaged as leaves with- out stems. Liang leaves are eaten as a fresh vegetable or as a side dish with spicy foods. The leaves are also cooked and used in recipes for various menus (Suksanga et al., 2022). Liang 2 Italian Journal of Food Science, 2023; 35 (3) Siripongvutikorn S et al. to the liberation of substrates and enzymes from dam- aged or injured plant cells (Leveau and Lindow, 2001). Injured leaves lead to lower quality and shortened prod- uct shelf life (Ariffin et al., 2017). Physically damaged cells or wounds enhance both organic and inorganic nutrient release that accelerates microbial growth and chemical reactions (Aruscavage et al., 2008, 2010). Washing also increases moisture content, aw level as well as physical damage due to excessive forces during wash- ing and draining (FAO and WHO, 2003; Mulaosmanovic et al., 2021). However, no scientific information is avail- able on the preparation (washing and stem detachment) of leafy vegetables, particularly Liang. This is of significant interest for a new S-curve for Liang because of its con- sumer palatability, low chemical or pesticide content and high nutritional value. Therefore, quality changes in Liang due to the preparation process represent is of utter impor- tance. This study focused on the effect of stem detachment and washing on the physical, chemical and sensory qual- ities and antioxidant activity to develop a proper method for Liang leaf preparation in current commercial markets. Materials and Methods Leaf preparation and sampling Young Liang or Gnetum gnemon var. tenerum leaves (pae-salat) were collected from a farm. To avoid injury from weight and dense packing, 2 kg of bulky Liang was packed into a low-density polyethylene bag (LDPE) and sent to the laboratory within 24 h, as shown in Figure 4. Tropical leafy vegetables are usually stored above 4°C to avoid chilling injury. The sample was checked for visual damage, and old, torn and rotten leaves were removed before the stem was detached following hygienic prac- tice by wearing sanitation gloves. Leaves with and with- out stems  were soaked in chlorinated water at 100 ppm for 15  min, washed twice with running tap water to remove the chlorine residue and drained in a basket for 10 min with a controlled thickness of leaves overlay at not more than 1 cm. The Liang leaves were divided into four groups as follows: no washing with stem (NWS), no washing without stem (NWNS), washing with stem (WS) and washing without stem (WNS) (see Figure 5). leaves are typically washed before cooking to ensure hygiene and safety (Figure 3). Gardeners and merchants generally spray water on vegetables or soak them to remove dirt. This reduces plant temperature and controls weight loss. Low temperature and appropriate humidity (60–70%) could slow down the leaf deterioration rate after harvesting (de Frias et al., 2018). However, each preparation process causes cumulative physical damage (Mulaosmanovic et al., 2021), leading to chemical and microbiological spoilage (Ariffin et al., 2017). Wounds also increase biochemical and chemical reactions owing Figure 1. Flowchart of vegetable preparation from farm to market. Figure 2. Liang leaves bunched with a rubber band (A) and sealed in a plastic bag (B). (A) (B) Figure 3. Flowchart of the conventional washing process. Figure 4. Liang leaves received from the farm (A) Liang leaves packed in LDPE and (B) Liang leaves. (A) (B) Farm Vegetables Spray or soak with water Bunch with rubber band or pack in bag Market Wash under running tap or soak in water Leaves Remove rotten, stem and old stage Drain in basket Italian Journal of Food Science, 2023; 35 (3) 3 Quality changes of Liang leaves during storage Laboratory Inc., Virginia, USA) as described by Lee et al. (2022) and expressed as ΔE, as shown in Equation 2. � � � �E L a b� � �( ) ( ) ( )2 2 2 Where, ΔE = colour difference between standard (fresh produce) ΔL = difference between lightness (L*) standard (fresh produce) Δa = difference between redness-greenness (a*) stan- dard (fresh produce) Δb = difference between yellowness-blueness (b*) standard (fresh produce) pH, moisture content and a w The pH, moisture content and aw were measured using a pH meter (Sartorius- Sartorius AG, Docu-pH+ Meter, Goettingen, Germany), oven method (AOAC, 2000), and an aw analyser (Aqualab Pre., Decagon Devices Inc., Washington, USA) at predetermined times. Brix value Brix values were measured using a refractometer (Atago, Pen refractometer, Tokyo, Japan) (Thakulla et al., 2021). Chlorophyll content Chlorophyll content was measured by the colorimetric method at 400–700 nm, as described in AOAC Methods 940.03 662 and 646 (AOAC, 2000) for chlorophyll a and b, respectively. Finally, all four groups were stored at 4°C for 8 days, as presented in Figure 6. On Days 0, 4, 5, 6, 7 and 8, samples were collected for physical, chemical, quality and sensory evaluation. Physiochemical and chemical quality determination Colour change Colour changes were determined by CIE L*, a* and b* using a colorimeter (ColorFlex EZ, Hunter Associates Figure 5. The groups of Liang leaves used in this study including (A) no washing with stem (NWS); (B) no wash- ing without stem (NWNS); (C) washing with stem (WS) and (D) washing without stem (WNS). (A) (C) (D) (B) Figure 6. Flowchart of Liang leaves preparation. No washing No washing Draining Washing Washing Liang leaves No stem detachment Stem detachment No stem detachment Stem detachment Remove rotten, old and torn Storage 4 Italian Journal of Food Science, 2023; 35 (3) Siripongvutikorn S et al. incubated in the dark for 30 min at 30°C. Finally, the absorbance of the mixture was measured at 517 nm and reported as µg gallic acid equivalent/g DW using gallic acid as the standard at a concentration of 0.5–3.5 µg/ml (R2 = 0.9959). ABTS radical scavenging activity 2,2-Azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assay was determined as described by Arnao et  al. (2001). ABTS radical was generated by incubating 7.4 mM of ABTS solution in dark at 30°C for 12 h. The radical solution was then diluted to obtain an absorbance of 1.1 ± 0.02 at 734 nm. Then, 20 µl of sample extract was mixed with 280 µl of radical solution and kept in the dark for 2 h at 30°C. The absorbance of the mixture was mea- sured at 734 nm and reported as mg gallic acid equivalent g DW using gallic acid as the standard at a concentration of 5–30 µg/ml (R2 = 0.998). Ferric reducing antioxidant power (FRAP) assay The ferric–reducing antioxidant power (FRAP) assay was determined using the method of Benzie and Strain (1996). A freshly prepared FRAP solution contain- ing 300 mM acetate buffer pH 3.6, 10 mM TPTZ (2, 4, 6- tripyridyl-s-triazine) in 40 mM HCl and 20 mM FeCl3·6H2O (ratio 10:1:1) was warmed at 37°C for 30 min. Next, 15 µl of the sample extract was mixed with 285 µl of FRAP solution and incubated for 30 min at 37°C. The absorbance of the mixture was measured at 593 nm and reported as µg gallic acid equivalent/g DW using gallic acid as the standard at a concentration of 6–30 µg/ml (R2 = 0.9981). Sensory evaluation Before the sensory evaluation, all samples were steamed for 3 min, and eight attributes including appearance, colour, texture, odour, flavour, taste, overall acceptability and consumer acceptance were evaluated using a 9-point hedonic scale by 50 untrained panelists. Statistical analysis All quality parameters, except for sensory tests, were assessed using a completely randomized design (CRD), whereas the sensorial score was determined using a ran- domized complete block design (RCBD). Differences in mean values were tested using ANOVA with specific differences between groups or treatments assessed by Tukey’s test. Fibre content Fibre content was determined as described in AOAC Method 2009.01. Total phenolic content, total flavonoid content and antiox- idant activity Sample preparation and extraction Sample was extracted using the method described by Srisook et al. (2021) with some modifications, such as ethanol 90% for 24 h instead of 95% ethanol for 5 days. Liang leaves and 90% ethanol (v/v) at a ratio of 1:10 were mixed and stirred in the dark at 25°C for 24 h. The mixture was then separated by vacuum suction using a Buchner funnel and centrifuged at 4°C for 15 min at 12,000 rpm. An evaporator was used to vaporise the eth- anol and to obtain a concentrated sample. Total phenolic content (TPC) determination TPC was determined using the method described by Singleton and Rossi (1965) with some modifications. Briefly, 20 µl of sample extract was added to 96-well plates followed by 100 µl of 10% Folin reagent (v/v). After incubation in the dark at 30°C for 6 min, 7.5% Na2CO3 (anhydrous) (w/v) was added, and the mixture was incu- bated for another 30 min. The absorbance was measured at 765 nm using a microplate reader (Varioskan LUX, Thermo Scientific, Singapore). TPC content was reported as mg gallic acid equivalent/g DW using gallic acid as the standard at a concentration of 50–170 µg/ml (R2 = 0.999). Total flavonoid content (TFC) determination TFC was determined using the method described by Ha et al. (2020) with some modifications. Briefly, 100 µl of sample extract was mixed with 100 µl 2% AlCl3·6H2O (w/v) and incubated in the dark at 30°C for 60 min. The absorbance of the mixture was then measured at 420 nm and reported as mg quercetin equivalent/g DW using quercetin as the standard at a concentration of 10–50 µg/ml (R2 = 0.9963). DPPH radical scavenging activity 2,2-Diphenyl-1-picryl hydrazyl (DPPH) radical scaveng- ing activity was determined using the method described by Brand-Williams et al. (1995) with some modifica- tions. First, 100 µl of sample extract was mixed with 100 µl 0.2 mM DPPH in 95% ethanol. The sample was Italian Journal of Food Science, 2023; 35 (3) 5 Quality changes of Liang leaves during storage Brix value The Brix value is a measure of the soluble solids con- tent of a solution (Zoecklein et al., 2010). Brix val- ues of all treatments increased at fourth day of storage (Figure 9). An increase in Brix value indicates an increase in water-soluble substances such as sugar, soluble fibre, amino acids, salt and organic acids or a decrease in water in the solution system such as evaporation and respira- tion (Kusumiyati et al., 2020). Therefore, the increase in Brix value was related to the higher water-soluble solids retained in the leaf samples. The unwashed samples (NWS, NWNS) exhibited higher Brix values than the washed samples (WS, WNS) during storage for 5–8 days. Washing increased the water uptake of the samples. Brix values gradually decreased after storage for 4 days because of the higher utilisation of soluble solids particularly sugar, weak acids and min- erals from microbial growth and biochemical reactions over time. Decrease in Brix value by microbial utilisa- tion of soluble solids is also observed in yogurt, wort and beer (Adadi et al., 2017; Kim and Han, 2019). The sharp increase in Brix values on eighth day of storage indicated an increase in soluble solid compounds due to a softening process by microbial and biochemical autol- yses. Results revealed that the utilisation and produc- tion of such compounds occurred naturally, in parallel, as a result of the reaction. Therefore, when the former was lower than the latter, the increment increased rather than decreasing. Results and Discussion Physicochemical changes Moisture and a w Moisture content of all no washing treatments (NWS and NWNS) decreased at fourth day of storage, whereas the moisture content of all washing treatments (WS and WNS) increased and was significantly higher than that of the no washing treatments (Figure 7). At fourth day of storage, the aw of the no washing treatments (NWS and NWNS) remained constant, whereas the aw of the washing treatments (WS and WNS) increased (Figure 8). Throughout the study, the moisture content of WNS treatments was significantly higher than WS treatments because stem detachment in WNS treatments resulted in an increased surface area, particularly at the end of the petiole, leading to higher water uptake and weight reten- tion. Using LDPE plastic bags also prevented moisture loss, with moisture content reducing slightly until reach- ing equilibrium during 5 days of storage. aw of WS and WNS were higher than those of NWS and NWNS, indicating the effect of picking up water, as described in the moisture content determination. Thus, washing resulted in an increase in aw and moisture con- tent. Results showed the drawbacks of washing in terms of an increase in aw, an important parameter for micro- bial spoilage and biochemical reactions which lead to faster product deterioration. Figure 7. Moisture content of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differ- ences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. 6 Italian Journal of Food Science, 2023; 35 (3) Siripongvutikorn S et al. in buffering capacity. It is recognised that a buffer can resist changes in pH if it is sufficient to bind with added protons and the starting pH of the solution (Bobulescu, 2020). While acidic compounds can reduce pH by pro- viding hydrogen ions (H+), basic compounds increase pH by providing hydroxide ions (OH–) (Bartee et al., ND). Consequently, pH gradually increased until Day 7 of storage. At eighth day of storage, a decrease in the pH value The initial pH of the fresh leaf samples was 6.20 ± 0.03 (Figure 10), which is similar to the pH of approximately 6 as reported by Anisong et al. (2022). At the fourth day of storage, the pH of all treatments significantly decreased, indicating three features including reduc- tion of basic compounds, increase in acid and decrease Figure 8. The water activity of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different upper- case letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. Figure 9. Brix values of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase let- ters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. Italian Journal of Food Science, 2023; 35 (3) 7 Quality changes of Liang leaves during storage Fibre content The fibre content of Liang leaves is shown in Figure 11. The total dietary fibre content was approximately 31% (DW), which was slightly lower than the 36.3% DW as reported by Anisong et al. (2022). The fibre content remained constant during storage. The experimental pH of all treatments was observed. The reduction in pH in all treatments resulted in an unpleasant organic smell that was similar to the smell of sweet, fermented sticky rice (Khao mak), which may be due to fermentation by yeast and lactic acid bacteria (Mongkontanawat and Lertnimitmongkol, 2015) as well as anaerobic respira- tion (Toro and Manuel, 2015). Figure 10. pH of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indi- cate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. Figure 11. Fibre of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. 8 Italian Journal of Food Science, 2023; 35 (3) Siripongvutikorn S et al. confirmed the oscillation of chlorophyll content in rocket leaves stored in dark at 4°C with a relative humidity of 65 ± 4.5% for 2 days, depending on the day–night cycle (cir- cadian regulation). Interestingly, the chlorophyll content of Liang leaves in this study was higher than kale, which is considered as a high chlorophyll vegetable, at 136.18– 172.10 mg/100 g (Lal, 2014). These results indicated that Liang leaves could be used as an alternative source of chlorophyll. The results for total chlorophyll (Chl a/b) revealed that the chlorophyll a (Chl a) content in all treatments was higher than that of chlorophyll b (Chl b). The Chl a/b ratio in this study was 1.2, which is lower than that of most plant types (generally higher than 2) including trees, shrubs, herbs, conifers, broad-leaves, evergreen and deciduous (Li et al., 2018). The low Chl a/b ratio was due to oxidative stress, as Chl a is more prone to oxidative damage than Chl b (Kasajima, 2019). The Chl a/b ratio of healthy rice leaves was recorded at 3.5, and decreased to 1.5 after subjection to oxidative stress (Kasajima, 2019). Shade plants generally produce more chlorophyll to increase photosynthesis efficiency because of the absorp- tion of blue light in a low-light environment (Beneragana and Goto, 2010). This result was supported by Herrera et al. (2022), who reported that plants respond to shade by increasing the production of light-harvesting com- plexes by increasing Chl b. Shade-tolerant plants respond to low-light environments in diverse ways than normal plants by decreasing the Chl a/b ratio. Farmers usually plant Liang as an intercrop between rubber trees, and the plants adapt to the shaded environment by increasing the total chlorophyll content and Chl b, leading to a low Chl a/b ratio. TPC, TFC and antioxidant activity TPC and TFC Phenolic compounds are produced by chloroplasts to protect cells from damage caused by reactive oxygen species (ROS), a by-product of photosynthesis (Zhang et  al., 2018). Leaves are major photosynthetic organs, and green-leaf plants contain an abundance of phenolic compounds (Zhang et al., 2018). The TPC of Liang leaves at Day 0 was 4.32 mg/GAE DW lower than Malaysia Liang leaves which were 8.70 mg/GAE DW as reported by Wazir et al. (2011). The TPC and TFC values of Liang leaves are presented in Figures 13 and 14, respectively. Fluctuations in the TPC and TFC in each treatment were observed during storage. The results in this experiment concurred with TPC values in other vegetables and fruits after storage at 4°C, indicating that active plants or plant parts try to remain homeostatic for survival until the end of life (Hubert et al., 2017; Kim, 2015). Cold storage results corresponded to the fibre content of walnuts stored at 4°C (Zhang et al., 2017). During storage, the texture became tougher but the fibre content remained the same. Therefore, the total fibre content was not a good parameter for determining quality changes in Liang leaves, with soluble and insoluble fibres being bet- ter options. Details on parameters such as reducing and non-reducing sugar and cellulose contents require fur- ther investigation. Colour change ΔE values of Liang leaves are shown in Figure 12. The colour of the adaxial and abaxial surfaces and ground leaves in all treatments significantly changed during stor- age. After storage for 4 days, ΔE of the upper side of the treatments without stem (NWNS and WNS) was higher than that of treatments with stem (NWS and WS). ΔE of the adaxial surface of the WNS treatment was the highest, followed by that of WS, indicating that wash- ing had a greater effect on ΔE at 7 and 8 days of storage. Excess water or high moisture content and mechanical injury induced by washing and stem detachment led to biochemical reactions and increased microbial func- tions (Mulaosmanovic et al., 2021). Interestingly, ΔE values of the lower side of the stem detachment treat- ments (NWNS and WNS) were significantly higher than those of stem treatments (WS and NWS). Washing also resulted in a higher ΔE than no washing, suggesting that washing and stem detachment played significant roles in leaf colour. The ΔE values of ground WS and WNS gradually increased, while those of the others remained constant during storage. The highest ΔE in the ground sample was found in the NWS treatment, while the adaxial and abax- ial surfaces under the WNS treatment had the highest ΔE, indicating that grinding affected the ΔE value of the leaves. The difference between the ΔE values of the non- ground sample (adaxial and abaxial surfaces) and the ground sample was caused by the water after the sam- ples were ground. Therefore, the colour quality of stored leaves could be preserved using ground samples. Chlorophyll content At fourth day of storage, changes in chlorophyll content in the NWS treatment remained constant but not in the other treatments (NWNS, NWS and WS). See Table 1. Chlorophyll content in the NWS treatment decreased, whereas it increased in the WS treatment at eighth day of storage. The oscillation of chlorophyll content in this study may be due to the plant cell metabolism during storage (Mei et al., 2022). Larrinaga et al. (2019) Italian Journal of Food Science, 2023; 35 (3) 9 Quality changes of Liang leaves during storage Figure 12. Colour difference (ΔE) of adaxial surface (A), abaxial surface (B) and ground leaves (C) of Gnetum gnemon var. tenerum leaves after storage at temperature 4oC for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. (A) (B) (C) 10 Italian Journal of Food Science, 2023; 35 (3) Siripongvutikorn S et al. of wounds, requiring more energy and a curing agent to treat the damaged cells. The TPC content of all treatments increased or remained constant compared with that of the control (Day 0). The TFC content of the NWS and WS treatments was equal to that of the control (Day 0), whereas NWNS (sec- ond bar) and WNS (last bar) during storage for 8 days were lower on Day 0, indicating a loss of nutrition sup- ply from the stems. Detaching the stems can be linked induced phenolic production (polyphenolic phytoalex- ins) (Hubert et al., 2017). A decrease in TPC and TFC during 8 days of storage with stem detachment (NWNS and WNS) indicated a reduced availability of nutrients to produce phenolic compounds, in contrast to treatments with stems (NWS and WS) which provided nutrients from the stems to the leaves. An increase in phenolic compounds production in wounded, stressed plants was observed in sweet potato roots (Dovene et al., 2019). Stem detachment can also injure plant cells as a result Table 1. Total chlorophyll (Chl a+b), chlorophyll a (Chl a), chlorophyll b (Chl b) and the ratio of chlorophyll a to b (Chl a/b) of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Time Treatment Chl a + b (mg/g DW) Chl a (mg/g DW) Chl b (mg/g DW) Chl a/b D0 NWS 226.28 ± 22.25ABa 114.55 ± 7.46Ba 101.53 ± 6.71Ba 1.13 ± 0.10Aa NWNS 226.28 ± 22.25Aa 114.55 ± 7.46Aa 101.53 ± 6.71Aa 1.13 ± 0.10Aa WS 226.28 ± 22.25Aa 114.55 ± 7.46Aa 101.53 ± 6.71Aa 1.13 ± 0.10Ba WNS 226.28 ± 22.25Aa 114.55 ± 7.46Ba 101.53 ± 6.71Aa 1.13 ± 0.10Aa D4 NWS 254.46 ± 9.96Aa 138.41 ± 5.48Aa 116.05 ± 4.49Aa 1.19 ± 0.00Ac NWNS 188.16 ± 2.60Bb 103.61 ± 1.35ABb 84.55 ± 1.26Bb 1.23 ± 0.00Aab WS 172.60 ± 10.57Bb 95.01 ± 6.28Bb 77.60 ± 4.29Bb 1.22 ± 0.01ABb WNS 177.26 ± 1.47Bb 98.31 ± 1.00Cb 78.95 ± 0.47Bb 1.25 ± 0.01Aa D8 NWS 200.07 ± 2.55Bb 110.67 ± 1.38Bb 89.40 ± 1.19Cb 1.24 ± 0.00Ab NWNS 181.50 ± 5.16Bc 101.41 ± 2.82ABc 80.10 ± 2.45Bc 1.27 ± 0.02Aa WS 177.03 ± 6.56Bc 99.44 ± 4.01ABc 77.59 ± 2.56Bc 1.28 ± 0.01Aa WNS 229.74 ± 5.79Aa 126.56 ± 3.41Aa 103.18 ± 2.40Aa 1.23 ± 0.01Ab Remarks: NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). Figure 13. Total phenolic content of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate signifi- cant differences between treatments within each day (P < 0.05). NWS: no washing with the stem; NWNS: no washing without the stem; WS: washing with the stem; WNS: washing without the stem. Italian Journal of Food Science, 2023; 35 (3) 11 Quality changes of Liang leaves during storage antioxidants instead of very short-lived natural radicals such as hydroxyl (HO·), lipid alkyl (L·) and lipid peroxyl (LOO·) (Munteanu and Apetriel, 2021; Yeo and Shahidi, 2019). Ferric ion–reducing antioxidant power (FRAP) is a method for determining the electron transfer ability of antioxidants by reducing the colourless complex ferric ion (Fe3+) to blue ferrous complex (Fe2+) in an acidic environ- ment (pH 3.6) (Munteanu and Apetriel, 2021). Generally, the results revealed that WS treatment had the highest antioxidant capacity, followed by NWS and WNS treat- ments (Figures 15–17). Remarkably, NWS offered anti- oxidant capacity comparable to WNS treatments, even to the cutting of organs of living things; this not only causes injury but also requires more energy for recovery. Antioxidant compounds could be used to alleviate stress and control wounds (Comino-Sanz et al., 2021). Antioxidant activity (DPPH, ABTS and FRAP) DPPH and ABTS assays are used to stabilize 2,2- diphenyl-1-picrylhydrazyl (DPPH) or 2,2′- azinob is-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) to measure the ability of hydrogen and electron transfer of Figure 14. Total flavonoid content of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate signifi- cant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no wash- ing without the stem; WS means washing with the stem; WNS means washing without the stem. Figure 15. DPPH of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. 12 Italian Journal of Food Science, 2023; 35 (3) Siripongvutikorn S et al. capability of antioxidant in aqueous phase. As mentioned above, the FRAP value indicates the reducing power of the antioxidants to metal ions. The antioxidant activity in plants is generated by phenolic compounds and also other compounds such as vitamin C and pigments such as chlorophyll and carotenoids (Sarker et al., 2020), there- fore explaining why ABTS radical scavenging in this study was not well related to phenolic compounds because carotenoid, chlorophyll and vitamin C contained in Liang leaves were 3706 µg/100 g DW (Anisong  et  al.,  2022), though NWS contained lower TPC and TFC. The results indicated that injury from washing induced different groups of phenolic compounds with higher antioxidant ability in Liang leaves during storage (Pratyusha, 2021). The ABTS assay exhibited the highest antioxidant capac- ity, followed by FRAP and DPPH assays. The higher ABTS value compared to DPPH indicated strong polarity by donating electrons and H+. Pongsetkul et  al. (2023) also confirmed relation of ABTS assay and hydrogen donating Figure 16. ABTS of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. Figure 17. FRAP of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Different uppercase letters indicate significant differences within the same treatment group. Different lowercase letters indicate significant differences between treatments within each day (P < 0.05). NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. Italian Journal of Food Science, 2023; 35 (3) 13 Quality changes of Liang leaves during storage comparable to those of fresh Liang leaves (Day 0). The odour, texture and flavour of NWS treatments remained stable during the storage period. The sensory scores showed that the panelists accepted all treatments after 8 days of storage, even though there was an unpleasant organic smell starting on Day 5 of storage when the bag was opened. However, after cooking, the off-odour of 226.28 ± 22.25 mg/g DW and 2.71–5.25 mg/100 g DW (data in process for publication), respectively. Sensory evaluation The sensory qualities of treatments during storage for 8 days at 4°C are shown in Table 2. All treatments were Table 2. Sensory scores of Liang or Gnetum gnemon var. tenerum leaves after storage at 4°C for 8 days. Attributes Condition Time Day 0 Day 4 Day 5 Day 6 Day 7 Day 8 Appearance NWS 7.52±0.97ABa 7.30±1.13Ba 7.79±0.74Aa 7.45±0.97ABa 7.52±1.00ABa 7.79±0.82Aa NWNS 7.73±0.87Aa 7.87±0.86Aa 7.57±1.01Aa 7.67±0.92Aa 7.67±0.88Aa 7.57±0.86Aa WS 7.40±0.98Aa 7.51±0.95Aa 7.43±0.98Aa 7.60±0.88Aa 7.71±0.99Aa 7.43±1.07Aa WNS 7.83±0.79Aa 7.63±1.16Aa 7.77±0.82Aa 7.37±1.25Aa 7.57±0.90Aa 7.77±0.86Aa Color NWS 7.52±1.09Aa 7.36±0.99Aa 7.73±0.88Aa 7.45±0.90Aa 7.45±0.94Aa 7.73±0.80Aa NWNS 7.57±0.94Aa 7.77±0.77Aa 7.60±0.81Aa 7.50±0.94Aa 7.43±0.90Aa 7.47±0.97Aa WS 7.31±0.99Aa 7.54±0.89Aa 7.54±0.89Aa 7.57±0.92Aa 7.63±0.97Aa 7.66±0.94Aa WNS 7.63±0.85Aa 7.70±1.02Aa 7.53±0.94Aa 7.30±1.09Aa 7.70±0.84Aa 7.63±0.89Aa Odor NWS 7.21±1.17Aa 7.15±1.12Aa 7.28±0.96Aa 7.48±0.80Aa 7.39±1.06Aa 7.45±1.03Aa NWNS 7.23±0.97Aa 7.63±0.89Aa 7.20±0.92Aa 7.30±0.99Aa 7.10±1.24Aa 7.23±1.04Aa WS 7.26±1.22Aa 7.43±1.24Aa 7.17±0.89Aa 7.17±1.04Aa 7.20±0.99Aa 6.94±1.21Aa WNS 7.50±0.97ABa 7.53±0.82Aa 7.33±0.92ABCa 7.03±1.25BCa 7.17±0.99ABCa 6.97±1.33Ca Odor NWS 7.21±1.17Aa 7.15±1.12Aa 7.28±0.96Aa 7.48±0.80Aa 7.39±1.06Aa 7.45±1.03Aa NWNS 7.23±0.97Aa 7.63±0.89Aa 7.20±0.92Aa 7.30±0.99Aa 7.10±1.24Aa 7.23±1.04Aa WS 7.26±1.22Aa 7.43±1.24Aa 7.17±0.89Aa 7.17±1.04Aa 7.20±0.99Aa 6.94±1.21Aa WNS 7.50±0.97ABa 7.53±0.82Aa 7.33±0.92ABCa 7.03±1.25BCa 7.17±0.99ABCa 6.97±1.33Ca Texture NWS 7.45±1.23Aa 7.00±1.12Aa 7.30±1.33Aa 7.61±0.90Aa 7.15±1.39Aa 7.55±1.03Aa NWNS 7.47±0.82ABa 7.67±0.88Aa 7.30±0.92ABa 7.30±1.09ABa 7.00±1.17Ba 7.40±0.97ABa WS 7.37±1.06Aa 740±1.35Aa 7.11±1.13Aa 7.17±1.20Aa 7.29±1.05Aa 7.20±1.05Aa WNS 7.50±0.97Aa 7.57±0.94Aa 7.47±0.86Aa 7.17±1.23Aa 7.47±1.14Aa 7.10±1.37Aa Flavor NWS 7.00±1.22Aa 7.15±1.25Aa 7.30±0.98Aa 7.45±1.06Aa 7.18±1.13Aa 7.33±0.96Aa NWNS 7.23±1.01Aa 7.50±1.25Aa 7.13±1.43Aa 7.10±1.03Aa 7.10±1.24Aa 7.07±1.26Aa WS 7.11±1.21Aa 7.23±1.24Aa 7.03±1.04Aa 7.00±1.06Aa 7.20±1.02Aa 6.91±1.15Aa WNS 7.37±1.07ABa 7.63±0.81Aa 7.30±0.88ABa 7.07±1.23ABa 7.23±0.94AB 7.03±1.13Ba Taste NWS 7.06±1.14Aa 6.91±1.33Aa 7.27±0.88Aa 7.18±1.33Aa 7.12±1.32Aa 7.21±1.05Aa NWNS 7.20±1.00Aa 7.40±1.25Aa 7.00±1.58Aa 7.30±0.99Aa 6.80±1.42Aa 6.87±1.41Aa WS 7.26±1.38Aa 7.26±1.34Aa 7.06±1.11Aa 6.97±1.12Aa 7.14±1.26Aa 6.91±1.25Aa WNS 7.37±1.00Aa 7.30±1.39Aa 7.20±0.89Aa 6.97±1.19Aa 7.10±0.96Aa 7.17±1.12Aa Overall NWS 7.12±1.19Aa 6.94±1.25Aa 7.15±1.30Aa 7.30±1.05Aa 7.03±1.26Aa 7.55±0.90Aa NWNS 7.37±0.93Aa 7.50±1.20Aa 7.07±1.36Aa 7.23±1.01Aa 6.87±1.36Aa 7.13±1.20Aa WS 7.17±1.29Aa 7.26±1.15Aa 7.14±1.00Aa 6.97±1.15Aa 7.14±1.14Aa 7.03±1.12Aa WNS 7.40±1.04Aa 7.57±0.68Aa 7.37±0.96Aa 7.20±1.27Aa 7.30±0.95Aa 7.20±1.16Aa Acceptance NWS 75.76Fd 87.88Cc 81.82Dd 90.91Bc 75.76Ec 93.94Aa NWNS 90.00Bc 93.33Ab 86.67Cb 83.33Dd 93.33Aa 86.67Cd WS 94.29Aa 82.86Dd 85.71Cc 85.71Cc 88.57Bb 88.57Bc WNS 93.33Cb 100Aa 96.67Ba 100.00Aa 93.33Ca 90.00Db Remarks: NWS means no washing with the stem; NWNS means no washing without the stem; WS means washing with the stem; WNS means washing without the stem. 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Stem detachment and washing of Liang leaves did not affect the amount of fibre and chlorophyll after stor- age at 4°C for 8 days. Liang leaves without stems con- tained lower TPC and TFC contents; however, washing increased antioxidant capacity based on the DPPH and ABTS methods, while stem detachment did not cause significant differences. Liang leaves stored at 4°C for 5  days produced an acid-like odour during storage, but this unpleasant odour dissipated after steaming. Liang leaves maintained acceptable quality when stored at 4°C for at least 8 days, and microbial quality should be further examined in future studies. This study provides a starting point for the future development of commer- cial ready-to-eat and ready-to-cook Liang leaf products. However, postharvest techniques and storage conditions require more detailed investigations to extend the shelf life of Liang leaves. 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