Effects of Pullulan-Based Coatings Incorporating ɛ-Polylysine and Glutathione on the Preservation of Cowpeas (Vigna unguiculata L.) Postharvest

Abstract

Pullulan has a fine-coating-forming ability, ɛ-polylysine has an antibacterial activity, and glutathione has both a potent antioxidant activity and polyphenol-oxidase-inhibiting ability. This study explored the effects of pullulan-based coatings incorporating ɛ-polylysine and glutathione (1% pullulan + 0.2% ɛ-polylysine + 0.3% glutathione) on the preservation of cowpeas (Vigna unguiculata L.) during refrigerated storage. Pullulan-based coatings incorporating ɛ-polylysine and glutathione decreased the weight loss, decay and rust spot indices, respiratory rate and malondialdehyde by 49.01%, 60.38%, 91.09%, 69.09% and 49.23%, respectively, and increased soluble solid content by 34.21% compared with the control group after 15 days of refrigerated storage (p < 0.05). Results show that pullulan-based coatings incorporating ɛ-polylysine and glutathione treatment may be practical materials for the preservation of cowpeas during refrigerated storage.

1. Introduction

Cowpea (Vigna unguiculata L.), an annual entwining herbaceous plant under the subfamily Papilionidae, is a common vegetable variety in China. This species is rich in vitamins, plant protein and mineral elements and has a high economic value [1]. Cowpeas are a highly seasonal vegetable that is usually harvested in summer in high temperatures and humidity. After harvest, this vegetable is very intolerant to storage because of its crisp tissue, high water content and strong respiration [2]. Generally, the storage period of cowpeas at room temperature is 3 days. In a short period, dehydration, wilting, fading, rust spots and even rotting occur, causing major economic losses [3,4]. Therefore, the postharvest preservation of cowpea is a major problem for the healthy development of the cowpea industry, requiring the study of postharvest preservation technologies of cowpea.

Edible coating preservation technology can control moisture and solute migration, internal gas exchange, respiration, and oxidative reaction rates and improve the appearance and surface characteristics of fruits and vegetables [5,6]. Moreover, edible coatings serve as the carriers of food additives, such as preservatives, antioxidants, colorants and flavors [7]. Pullulan is a homogeneous polysaccharide with a fine coating-forming ability [8] and ɛ-polylysine is a broad-spectrum preservative used in the food industry [9]. Glutathione is an antioxidant that can suppress polyphenol oxidase (PPO) activity and browning [10,11].

Given their coating-forming ability and antibacterial and antioxidant activities, pullulan-based coatings incorporating ɛ-polylysine and glutathione may have antibacterial and antioxidant activities that can thus extend the shelf life of cowpea. Hence, further investigations into pullulan-based coatings incorporating ɛ-polylysine and glutathione are needed. Therefore, this study aims to investigate the preservative effects of pullulan-based coatings incorporating ɛ-polylysine and glutathione on cowpeas during refrigerated storage.

2. Methods and Materials

2.1. Materials

Cowpeas were obtained from a local agricultural product wholesale market in Haizhou, Lianyungang, China. Cowpeas with the same length, thickness and maturity and without mechanical damage, pests or diseases were selected, rinsed and drained naturally for use. Pullulan with a molecular weight of 3.1 × 10⁵ Da was obtained from Pharmacopoeia, Japan. ɛ-polylysine was obtained from Zhejiang Tongfa Biotechnology Co., Ltd., Ningbo, China. Glutathione was obtained from Zhejiang Shenyou Biotechnology Co., Ltd., Huzhou, China. All the other chemicals were of reagent grade.

2.2. Treatment of Cowpeas

Based on our previous study, dipping solutions were prepared as follows: pullulan was dissolved in purified water to obtain a solution with a concentration of 1% (w/v) to which 0.2% (w/v) ɛ-polylysine and 0.3% (w/v) glutathione were added. The cowpeas were soaked in the dipping solutions for 15 min at 4 °C, rinsed, drained naturally, sealed in 25 cm × 38 cm × 0.03 cm polyethylene fresh-keeping bags and placed on racks at −4 °C for 15 days in cold storage. Cowpeas without any treatment were used as a control.

2.3. Weight Loss

The weight of the cowpeas was assayed during refrigerated storage. The percent weight loss was calculated by weighing the cowpeas every 3 days.

2.4. Decay Index

The decay index was divided into five grades as follows: grade 0, no decay; grade 1, decayed area < 10%; grade 2, decayed area of 10%–25%; grade 3, decayed area of 25%–50%; and grade 4, decayed area > 50%. Decay index (%) = 100 × ∑[(decay grade × number of cowpeas in the grade)]/[(number of the highest grade × total number of cowpeas)] [12].

2.5. Rust Spot Index

The rust spot index was divided into five grades as follows: grade 0, no rust spots; grade 1, rust spots of 1%–25%; grade 2, rust spots of 26%–50%; grade 3, rust spots of 51%–75%; and grade 4, rust spots of 76% to 100%. Rust spot index (%) =100 × ∑[(rust spot grade × number of cowpeas in the grade)]/[(number of the highest grade × total number of cowpeas)] [12].

2.6. Soluble Solids

The cowpeas were homogenized, filtered and centrifuged to yield a supernatant, in which soluble solids were measured using a refractometer (WYA-2WAJ, Shanghai Lichen Instrument Technology Co., Ltd., Shanghai, China).

2.7. Respiratory Rate

The cowpeas (approximately 1 kg) were tightly sealed in a 1000 mL glass container at 25 °C for 2 h. The carbon dioxide content of the headspace was determined using a Trace 2000 GC and a Thermo mass spectrometer [13].

2.8. Malondialdehyde (MDA)

The MDA content of the cowpeas was assayed using thiobarbituric acid reactive substances methods. Approximately 2 g of cowpea tissue sample was extracted with a trichloroacetic acid solution. Then, the absorbance was measured at 450, 530 and 600 nm [14].

2.9. Statistical Analysis

All experiments were conducted in sextuplicate. All data are expressed in mean ± standard deviation (SD). Origin 7.0 statistical analysis software was used for data collation and analysis. Significant difference analysis was performed using the paired sample t-test.

3. Results

3.1. Weight Loss

As shown in Figure 1, the weight loss rate of cowpeas during refrigerated storage continuously increased with time. At the beginning of the six-day refrigerated storage, the weight loss rate of each group increased rapidly and then increased slowly from the 9th day of refrigerated storage.

3.2. Decay Index

As shown in Figure 2, the cowpeas in the control group started to decay from the 3rd day of refrigerated storage. The decay index increased steadily with time during refrigerated storage. After the 6th day, the decay index increased sharply and reached 53.14% on the 15th day. However, the decay index of the cowpeas in the treatment group increased slowly during the entire storage period, and the decay index was only 21.04% on the 15th day, which is much lower than that of the control group (p < 0.05).

3.3. Rust Spot Index

As shown in Figure 3, the cowpeas in the control group started to show rust spots from the 3rd day of refrigerated storage. The rust spot rate continued to increase with time during refrigerated storage. After the 6th day, the rust spot rate increased sharply, reaching 64.07% on the 15th day. However, the rust spot rate of the cowpeas in the treatment group increased slowly throughout the entire storage period, and the value was only 5.72% on the 15th day, which is much lower than that of the control group (p < 0.05).

3.4. Respiratory Rate

The peak respiratory intensity of the control group was observed on the 6th day, while that of the treatment group was delayed to the 9th day and is lower than that of the control group (p < 0.05, Figure 4). On the 15th day of storage, the respiration intensity of the cowpeas in the treatment group was 17.09 mg/kg·h, which is 69.09% lower than that of the control group (p ˂ 0.05), indicating that treatment with pullulan-based coatings incorporating ɛ-polylysine and glutathione significantly inhibited the respiration intensity of the cowpeas and reduced the nutrient consumption during refrigerated storage (p < 0.05).

3.5. Soluble Solids

Soluble solid contents in the cowpeas in the control group continuously decreased with time during refrigerated storage. After the 6th day, the soluble solid contents decreased sharply, reaching 3.81% on the 15th day. However, the soluble solid content of the cowpeas in the treatment group decreased slowly throughout the entire storage period with a value of 5.14% on the 15th day, which is much higher than that of the control group (Figure 5, p < 0.05).

3.6. MDA

As shown in Figure 6, the MDA content of the control group increased slowly in the first 9 days of refrigerated storage and then increased sharply after 9 days of refrigerated storage, while the MDA content of the treatment group increased slowly during refrigerated storage and at lower levels than those of the control group (p < 0.05).

4. Discussion

The edible part of the cowpeas was fresh and had tender pods, and the pods maintained a high moisture content, which is an indication of the preservation technology. After refrigerated storage, the treatment with pullulan-based coatings incorporating ɛ-polylysine and glutathione significantly inhibited the increase in the weight loss rate of the cowpeas and effectively maintained a high moisture content (p < 0.05). Pullulan solution treatment could form a dense protective layer on the surface of the cowpeas, thus effectively reducing the respiration, transpiration and water loss of the pod [8].

Glutathione has potent antioxidant activity and can suppress PPO activity and browning and thus decrease the rust spot rate [10,11]. The preservation treatment formed wax-like coatings on the cowpea surface, thus reducing the mechanical loss during refrigerated storage. In addition, the coatings blocked the passage between the outside world and the fruit cells, preventing infection with aerobic microorganisms such as fungi, which effectively prevented spoilage of the cowpeas during refrigerated storage. Moreover, ɛ-polylysine has broad-spectrum antibacterial activity, suppressing the growth of spoilage bacteria and preventing the spoilage of the cowpeas during refrigerated storage [9].

The browning of cowpeas after harvesting is a common phenomenon, and it often occurs in injured areas, especially the stem. After the harvest, the color of the cowpeas changed from dark green to light green or even yellow, resulting in rust spots. Glutathione has potent antioxidant activity and can suppress the PPO activity and browning, thus decreasing the rust spot rate [10,11].

Cowpea is a respiratory climacteric vegetable. As shown in Figure 4, the respiration intensity of the cowpeas during the whole storage period increased first and then decreased. The treatment with pullulan-based coatings incorporating ɛ-polylysine and glutathione affected spontaneous air conditioning and preservation. The coatings formed on the surface of the cowpeas, delaying their physiological metabolism and remarkably reducing the respiration intensity of the cowpeas, thereby inhibiting the process of other metabolic activities, reducing the consumption of nutrients and delaying the senescence of the cowpeas.

Soluble solids closely contribute to the taste and flavor of cowpeas. After the harvest, some sugar was degraded to carbon dioxide and water respiration, thus decreasing the soluble solid content in the cowpeas. The coatings that formed on the surface of the cowpeas reduced the physiological metabolism during refrigerated storage and inhibited the consumption of organic matter [12]. The treatment with pullulan-based coatings incorporating ɛ-polylysine and glutathione maintained a higher soluble solid content during the refrigerated storage of the cowpeas and maintained a higher nutritional quality of the cowpeas than the control group (p < 0.05).

Environmental stress and tissue aging can decrease the ability of the tissue to scavenge active oxygen and induce the production of many free radicals that then produce MDA, and the MDA content can reflect the degree of membrane damage. The treatment with pullulan-based coatings incorporating ɛ-polylysine and glutathione inhibited the respiration of the cowpeas during refrigerated storage, thereby inhibiting other physiological and biochemical activities in the cowpea cells, delaying the senescence of the cowpeas and inhibiting the accumulation of MDA. The coating treatment also reduced the weight loss rate of the cowpeas during refrigerated storage, thereby reducing the increase in cell membrane permeability. This indicated that the coating treatment can inhibit the occurrence of membrane lipid peroxidation to a certain extent, thus preventing the destruction of the cell structure and inhibiting cell senescence and death. Glutathione has potent antioxidant activity and can suppress membrane lipid peroxidation and thus inhibit the accumulation of MDA [10,11].

In conclusion, the treatment with pullulan-based coatings incorporating ɛ-polylysine and glutathione effectively decreased the weight loss, decay and rust spot indices, the respiratory rate, and the MDA content, and increased the soluble solids during refrigerated storage compared with the control group. Therefore, the treatment with pullulan-based coatings incorporating ɛ-polylysine and glutathione might be a promising method for extending the shelf life of cowpeas.