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College of Food and Bioengineering, Hezhou College, Hezhou 542899, China

Abstract Prochloraz diluents (450×, 475×, 500×) were added with different concentrations of nano-titanium dioxide (TiO2) (0.8%, 1.0%) respectively to prepare complex fresh-keeping agents, which were applied in the refrigeration of Dioscorea alata Lirm. sp. With rot index, weight loss rate, browning degree (BD), total soluble solids (TSS) content, malondialdehyde (MDA) content, polyphenol oxidase (PPO) activity and peroxidase (POD) activity, DPPH· scavenging rate as evaluation indexes, the preservation effect of the prepared fresh-keeping agents on D. alata during the cold storage period was analyzed. The results showed that prochloraz added with nano-titanium dioxide significantly prolonged the storage life, reduced the rot rate and weight loss rate, and slowed the loss of pulp of D. alata. The preservation effect of complex fresh-keeping agent of prochloraz diluent (475×) and nano-titanium dioxide (1.0%) was better than that of other treatments.

Key words Dioscorea alata Lirm. sp., Prochloraz, Nano-titanium dioxide, Refrigeration

1 Introduction

DioscoreaalataLirm. sp., also known as Ziyu Huaishan or purple yam, is a plant native to Asia. The Se-enrichedD.alataproduced in Guangdong and Guangxi that have abundant natural resources and unique ecological environment such as abundant sunshine and abundant rainfall is favored by consumers[1]. According to the inspection and determination of the Vegetable and Fruit Quality Supervision and Inspection Center (Guangzhou) of the Ministry of Agriculture, the contents of starch, total sugar, zinc and other nutrients inD.alataare higher than those in Guihuai No.2 (a cultivar ofDioscoreaopposita); the content of anthocyanins inD.alatais 60 times that of Guihuai No.2, which has a health-care effect on human eyes; andD.alataalso contains ginsenoside, the main ingredient of ginseng[2].D.alatahas high water content and is not resistant o high humidity or low temperature. A study[3]has shown thatD.alatawill suffer from chilling damage when the storage temperature is lower than 7℃, and the tissue will be discolored and rotted[3], seriously affecting its commercial value as a fresh-selling good and making long-term storage and long-distance transportation more difficult.

Prochloraz, also known as Shibaoke or Pumeiling, can be used to treat and control diseases caused by microorganisms in oil crops, cereals, tropical and subtropical fruits, ornamental plants, vegetables and various economic crops and to control diseases during fruit storage after harvest[4]. Prochloraz was ever used to treat yam and chufa; and it was found that a certain concentration of prochloraz can inhibit the rapid growth of microorganisms on the surface of fruits and vegetables during storage, and it has a certain effect on prolonging the preservation period of fruits and vegetables[5-6]. Wu Zhonghongetal.[7]also treated guava with prochloraz and found that prochloraz can delay the shelf life of the fruit. Prochloraz has good compatibility, with obvious synergistic effect. The combination of prochloraz and chitosan has a good effect on fresh-keeping navel orange[4]. Nano-titanium dioxide (TiO2) is one of the most active nanomaterials used in organic material modification, with unique properties such as small particle size, high surface activity, good biocompatibility, and strong UV absorption, and characteristics of non-toxic, antibacterial and super-hydrophilic. There is little research on the addition of nano-titanium dioxide in the complex preservation of fruits and vegetables[8-10]. The application of prochloraz-nano-titanium dioxide complex in the preservation of fruits and vegetables has not been reported. In this study, under refrigeration conditions (10±2)℃, the synergistic effect of different concentrations of nano-titanium dioxide for prochloraz’s preservation effect forD.alatawas explored to prolong the sales period ofD.alataand provide reference for safe use of prochloraz.

2 Materials and instruments

The experimental materials includedD.alata(Hejie Huaishan, purchased in farmer’s field in Hejie Town, Hezhou City), prochloraz (25% EC, Jiangsu Yixing Xingnong Chemicals Co., Ltd.), nano-titanium dioxide (30 nm, analytically pure, Shanghai Maikun Chemical Co., Ltd.),etc. The used instruments and equipment included Mar-80 large capacity electric centrifuge, AR-124CN electronic analytical balance, VIS-723 visible spectrophotometer, 85-1 thermostatic magnetic stirrer, pHS-2C precision pH meter, Abel refractometer, laboratory small cold storage,etc.

3 Methods

3.1CompoundrefrigerationGrowth-uniform, disease-free, pest-free, damage-free, and maturity-uniformD.alataplants were selected randomly. They were cut into small pieces of about 20 cm for use. Through pre-experiment, the optimal dilution ratio of prochloraz (350-550 folds) and the optimal particle size of nano-titanium dioxide (0.6%-1.2%) were determined, and prochloraz and nano-titanium dioxide of optimal single treatment were subjected to complex treatment.

Prochloraz solutions with dilution ratio of 450 times, 475 times and 500 times were added with nano-titanium dioxide (0.8%, 1.0%), respectively to prepare prochloraz-nano-titanium dioxide complex fresh-keeping agents. The samples ofD.alatawere immersed into the fresh-keeping agents for 5 min, respectively. Subsequently, theD.alatasamples were taken out and air-dried. Then, they were placed into fresh-keeping bags (unsealed) and stored in the refrigerator (10±2)℃. The treatment with clear water was used as the blank control (CK). During the storage period (20 d), the rot index, weight loss rate, browning degree (BD), malondialdehyde (MDA) content, total soluble solids (TSS) content, DPPH· scavenging rate, polyphenol oxidase (PPO) activity and peroxidase (POD) activity of theD.alatasamples were observed and determined once every five days to comprehensively evaluate the preservation effect of each complex fresh-keeping agent.

3.2IndexdeterminationmethodsWeight loss rate was measured by weighing method. Browning degree (BD) was measured by extinction method, referring to the method of Xie Dongdietal[11]. PPO activity was determined by catechol method, referring to the method of Xiang Yang[12]. POD activity was determined by guaiacol method, referring to the method of Xiang Yang[12]. MDA content was determined by TBA colorimetric method, referring to the method of Song Muboetal[13-14]. TSS content was determined by Abel refractometer, referring to the method of Cao Jiankangetal[15]. DPPH· scavenging rate was determined by referring to the method of Larraurietal[16], and the results were expressed as a percentage. Rot index was measured by sensory method. According to the rot degree of surface ofD.alata, it was divided into five grades. Grade 0, there was no mildew on the surface; Grade 1, mildew area accounted for less than 1/4 of the total area; Grade 2, mildew area accounted for 1/4-1/2 of the total area; Grade 3, mildew area accounted for 1/2-3/4 of the total area; and Grade 4, mildew area accounted for 3/4-1 of the total area[5]. It was calculated according to the following formula:

4 Results and analysis

4.1Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentonweightlossrateofD.alataMoisture is an important factor in maintaining the freshness of fruits and vegetables. Early-maturingD.alatahas high water content. If there is too much water loss, the weight loss rate will be too large, which directly affects the shelf life ofD.alata. As shown in Fig.1, during the storage process, the weight loss rate ofD.alatain all the groups increased with the prolonging of preservation, indicating that the water loss inD.alatawas more serious during storage. The reason might be that the metabolism of cut fruits and vegetables or broken of fruits and vegetables was intensified, the membrane lipid peroxidation was strengthened, and the cell membrane permeability was changed, so that the moisture in the tissue was rapidly evaporated through the pores on the surface[12]. The study found that first of all, the surface ofD.alatawas wrinkled, and then the pulp was quickly softened and even rotted from the outside to the inside. The weight loss rates in the treatment groups were significantly lower than that of CK (P<0.05), suggesting that complex fresh-keeping treatment could effectively reduce the weight loss rate and prevent the loss of moisture inD.alata, thus prolonging the storage period. This might be caused by the high hydrophilicity of the nanomaterial. As shown in Fig.1, the weight loss rate of the CK group was larger, followed by that of the (1.0%+450 folds) treatment. The treatments of (1.0%+475 folds) and (0.8%+475 folds) were able to delay the increase of the weight loss rate and effectively inhibit the water dispersion loss ofD.alata. The weight loss rates in the two groups were significantly different from those in the CK group (P<0.01).

Fig.1Effectsofcomplexfresh-keepingtreatmentsonweightlossrateofDioscoreaalataLirm.sp.

4.2Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentonrotindexofD.alataDuring the post-harvest storage and cutting process, the components ofD.alatachange, and its resistance decreases, so that various physiological and invasive diseases are prone to occurring, causing rot, deterioration and nutrient loss ofD.alataand makingD.alatalose its value of goods[11]. As shown in Fig.2, the rot indexes in all the groups were tended to rise, and the rot index in the CK group was always the highest. The rot indexes ofD.alatain the complex fresh-keeping treatment groups were significantly lower than that of the CK group (P<0.05). On Day 20, the rot index of the (1.0%+475 folds) treatment group was 22.13%, but that in the CK group was as high as 55.02%. The reason was that the prochloraz-nano-titanium dioxide complex fresh-keeping agents destroyed the plasticity and fluidity of the cell membrane of microorganisms on the surface ofD.alataand made them lose their normal functions, resulting in inhibition of the growth of flora[5]. Among all the fresh-keeping treatment groups, the inhibition effect was very significant in the inhibition effect was very significant in the (0.8%+475 folds) treatment group and the (1.0%+475 folds) treatment group (P<0.01).

Fig.2Effectsofcomplexfresh-keepingtreatmentsonrotindexofDioscoreaalataLirm.sp.

4.3Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentonactivityofpolyphenoloxidase(PPO)ofD.alataPolyphenol oxidase (PPO) is a metalloproteinase that is widely distributed in nature. It is ubiquitous in organisms[11]. PPO catalyzes the oxidation of endogenous polyphenols in fruits and vegetables to produce melanin, which is the main cause of browning of damaged fruits and vegetables or fresh-cut fruits and vegetables[11]. The PPO activity of cutD.alatais strong, which is prone to causing browning and oxidation ofD.alata, seriously affecting the nutrition, flavor and appearance quality of the product[12]. As shown in Fig.3, during the storage period, the PPO activity ofD.alatain each group increased first and then decreased; and the PPO activity in the CK group was always at the highest level from the 5th d and reached the peak by the 10th d. In the complex fresh-keeping treatment groups, the PPO activities did not reach the peak by the 10th d, and they were much lower than that of the CK group (P<0.01). The PPO activity in the (1.0%+475 folds) treatment group was the lowest, and it was significantly different from that of the CK group throughout the storage period (P<0.01). The PPO activities in the (1.0%+475 folds) treatment group and the (0.8%+475 folds) treatment group almost showed no peak. It suggested that complex fresh-keeping treatments could inhibit the increase of PPO activity and increase the freshness ofD.alata. Among them, the treatment of (1.0%+475 folds) showed the best fresh-keeping effect.

Fig.3Effectsofcomplexfresh-keepingtreatmentsonPPOactivityofDioscoreaalataLirm.sp.

4.4Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentonactivityofperoxidase(POD)ofD.alataPlant contains a large amount of peroxidase (POD), and its activity is one of the indicators of ripening and aging of fruits and vegetables. It has a negative impact on the storage quality of fruits and vegetables[10]. As shown in Fig.4, the POD activity in all the groups increased first and then decreased. In the first five days, the change was slow; and by the 10th d, the POD activity of each group increased rapidly. The peak of POD activity of the CK group appeared earlier, on Day 10; and the maximum POD activities of the complex fresh-keeping treatment groups appeared around Day 15, and among them, the peak of POD activity of the (1.0%+475 folds) treatment group was not obvious. The POD activity in the CK group was at a high level throughout the storage period, followed by that in the (0.8%+450 folds) treatment group. The POD activities in the (1.0%+450 folds) treatment group and the (1.0%+475 folds) treatment group were lower, and they were significantly different from those in the CK group on Day 10, 15 and 20 (P<0.01). It suggested that complex fresh-keeping treatments effectively inhibited the increase of the activity of POD inD.alata.

Fig.4Effectsofcomplexfresh-keepingtreatmentsonPODactivityofDioscoreaalataLirm.sp.

4.5Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentonbrowningdegreeofD.alataThe absorbance of juice ofD.alatawas used to indicate the color status ofD.alataduring storage. The larger the absorbance value was, the more severe the browning was. As shown in Fig.5, splitting could cause browning inD.alata. During the storage period, with the extension of storage period, the browning degree ofD.alataincreased gradually overall. The prochloraz-nano-titanium dioxide complex fresh-keeping agents showed different degrees of inhibition on the increase of browning degree inD.alata, but the browning degree of the CK group was higher than those of the complex fresh-keeping treatment groups. After 10 d, the browning degree curves of the two treatment groups, (1.0%+475 folds) and (0.8%+475 folds), were flat compared with those in the remaining treatment groups, and the browning degrees in the two groups were significantly lower than that of the CK group (P<0.01). This was consistent with the results that the two treatments could inhibit the activities of browning enzymes PPO and POD. It indicated that complex fresh-keeping treatments could slow down the browning ofD.alata, which might because that the fresh-keeping agents inhibited the activities of PPO and POD, inhibited the oxidation of polyphenols and delayed the occurrence of browning ofD.alata.

Fig.5Effectsofcomplexfresh-keepingtreatmentsonBDvalueofDioscoreaalataLirm.sp.

4.6Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentoncontentofmalondialdehyde(MDA)ofD.alataMalondialdehyde (MDA) is one of the main products of membrane lipid peroxidation, and the change of its content is an important indicator to measure the post-harvest aging process of fruits and vegetables. When plants are exposed to environmental stress, as the free radicals are produced in large quantities, the cell membrane is peroxidized or delipidated and gradually degraded. As a result, a large amount of MDA is produced[13]. As shown in Fig.6, the MDA content in the CK group increased rapidly with the extension of storage period, indicating that ifD.alatawas not submitted to complex fresh-keeping treatment, MDA will be accumulated rapidly inD.alataas browning increased. This was consistent with the results that the rot index and weight loss rate increased rapidly after the 5th d of storage. Fig.6 showed that on Day 5, the MDA contents of the groups increased insignificantly (P>0.05). On Day 10, 15 and 20, the MDA content of the CK group increased rapidly. The MDA content growth curve of the (1.0%+475 folds) treatment group was flat compared to those of the other treatment groups, and the MDA content was significantly lower than that of the CK group (P<0.01). This might be because that the fresh-keeping agent effectively inhibited the POD activity, which reduced the ability ofD.alatato initiate membrane lipid peroxidation to destroy the membrane system, promotingD.alata’s effectively scavenging free radicals produced in the body, thereby delaying aging to a certain extent[15].

Fig.6Effectsofcomplexfresh-keepingtreatmentsonMDAcontentofDioscoreaalataLirm.sp.

4.7Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentoncontentoftotalsolublesolids(TSS)ofD.alataTotal soluble solids refer to a general term for all water-soluble compounds in plant tissues. The change of their content reflects the quality change ofD.alataduring storage to a certain extent. The smaller the change range was, the better the preservation effect was[12]. As shown in Fig.7, the total soluble solids contents of all the groups showed a downward trend during the storage. The reason might be that the respiration ofD.alatawas accelerated after being divided, leading to the change of intracellular substances[12]. Fig.7 showed that on Day 5, the decrease of TSS content in all the groups was unobvious; and on Day 10, 15 and 20, the TSS content in the CK group decreased rapidly. It suggested that with the aggravation of browning, rotting, and membrane lipid peroxidation, the loss of TSS content became more serious. During the whole storage period, the loss of TSS in the CK group was most significant, followed by that in the (0.8%+450 folds) treatment group. The loss of TSS in the (1.0%+475 folds) treatment group was smallest, and it significantly different from that in the CK group (P<0.01), suggesting that its preservation effect was the best. The complex fresh-keeping treatments reduced the metabolic intensity ofD.alata, slowing down the hydrolysis rate of the soluble matter, so that the quality ofD.alatawas relatively well maintained during a certain storage period.

Fig.7Effectsofcomplexfresh-keepingtreatmentsonTSScontentofDioscoreaalataLirm.sp.

4.8Effectofprochloraz-nano-titaniumdioxidecomplexfresh-keepingagentonscavengingrateofDPPH·ofD.alataThe DPPH method is a high stable and highly sensitive method for evaluating the antioxidant activity of plants[17]. A study[12]showed thatD.alatacontains polysaccharides, mucus and polyphenols, which have certain ability to scavenge DPPH·. As shown in Fig.8, as the storage time increased, the free radical scavenging rate inD.alatashowed a downward trend in all the groups. This was negatively correlated with the MDA content and rot index ofD.alataand positively correlated with TSS content ofD.alata. In the maturity process of plants, oxygen produces free radicals after a series of reactions in plant cells, allowing fruits and vegetables to mature and age rapidly, and the ability of the body to scavenge free radicals also drops rapidly. The more the free radicals are, the faster the maturity and rotting of fruits and vegetables are[12]. As shown in Fig.8, the decrease of DPPH· scavenging rate in the CK group was greater than that of each fresh-keeping treatment group. In the first 10 d, the differences were not obvious (P>0.05); and on Day 15, the differences became more significant (P<0.05). On Day 15 and 20, the free radical scavenging ability of the (1.0%+475 folds) treatment group was always higher than that of the CK group (P<0.01). It indicated that the complex fresh-keeping treatments had an inhibitory effect on the increase of free radicals inD.alata. The treatment of (1.0%+475 folds) could better maintain the antioxidant effect ofD.alata.

Fig.8Effectsofcomplexfresh-keepingtreatmentsonDPPH·scavengingrateofDioscoreaalataLirm.sp.

5 Conclusions

The experimental results showed that under refrigeration conditions (10 ± 2)℃, the prochloraz-nano-titanium dioxide complex fresh-keeping agents effectively inhibited the increase of weight loss rate and rot index, inhibited the PPO and POD activities and browning degree, reduced the accumulation of MDA, and slowed the loss of TSS, and better maintained the antioxidant capacity ofD.alata. Comprehensively considering the evaluation indexes, the preservation effect of the (1.0% +475 folds) treatment group was the best, followed by that of the (0.8% +475 folds) treatment group. The concentration of prochloraz was slightly lower than that in the previous studies[5-6]where prochloraz was added with additives to preserve fruits and vegetables. The combination of prochloraz and nano-titanium dioxide has a good preservation effect. The reasons are as follows. First, the combination of prochloraz and nano-titanium dioxide is beneficial to solve the bacteriostatic selectivity of prochloraz (prochloraz usually has excellent prevention and treatment effect on various diseases caused by ascomycetes and deuteromycetes[7]). Second, the advantages of nano-titanium dioxide such as high surface activity and good biocompatibility are fully exerted, thereby improving the adhesion and hydrophilicity of the fresh-keeping agent. Third, nano-titanium dioxide can strongly absorb ultraviolet light, denaturing microbial protein, thereby playing an antibacterial synergistic effect. Under the combined treatment of prochloraz and nano-titanium dioxide, the combination of 1.0% titanium dioxide and 475-fold-diluted prochloraz can achieve a good synergistic effect.