短波紫外線(UV-C)對貯藏藍莓果實抗氧化活性的影響

Effect of ultraviolet-C on the change of antioxidant activity in stored blueberry fruit

XU Fangxu1，2， LIU Hongyuan3， HE Xi4， LIU Bojia4， LI Bingxin4， ZHANG Jinying4

（1. Experimental Teaching Center， Shenyang Normal University， Shenyang 110034， China;

2. Liaoning Key Laboratory of Cordyceps Militaris with Functional Value， Shenyang 110034， China;

3. College of Grain Science and Technology， Shenyang Normal University， Shenyang 110034， China;

4. College of Life Science， Shenyang Normal University， Shenyang 110034， China）

Abstract：The purpose of the study is to estimate the efficacy of ultraviolet-C（UV-C） on the change of antioxidant activity in blueberries at various cold storage time， shelf life and shelf temperature， which aims to provide a theoretical basis for finding a feasible method to improved the quality of blueberries. The results indicated that procyanidine content， total phenols content， total anthocyanin content， flavonoid content， and radical scavenging abilities in UV-C treated blueberries responded variously to storage time， shelf life， shelf temperature and their interactions. The antioxidant activity gradually decreased over the shelf life， especially for blueberries that have been in cold storage for a long time. UV-C irradiation treatment observably delayed the decline of procyanidine and total phenols content in samples. Low shelf temperature （5 ℃） effectively maintained high levels of radical scavenging abilities. In conclusion， storage time， shelf life and shelf temperature exerted different effects on the change of antioxidant activity in UV-C treated blueberries.

Key words：blueberry; ultraviolet-C（UV-C）; shelf life; antioxidant activity

CLC number：S663.9 Document code：A

doi：10.3969/j.issn.1673－5862.2023.01.009

摘 要：評估短波紫外線（Ultraviolet-C，（UV-C）在不同冷藏時間、貨架期和貯藏溫度下對藍莓抗氧化活性變化的影響，可以為提高藍莓品質的可行方法提供理論依據。結果表明，UV-C處理藍莓的原花青素含量、總酚含量、總花青素含量、類黃酮含量和自由基清除能力隨貯藏時間、貨架期、貯藏溫度及其相互作用而不同。 隨著貨架期的延長，抗氧化活性逐漸下降，特別是對于長期冷藏的藍莓，UV-C輻照顯著延緩了樣品中原花青素和總酚含量的下降，低溫（5 ℃）能有效維持高水平的自由基清除能力。總之，貯藏時間、貨架期和貯藏溫度對UV-C處理藍莓抗氧化活性的變化有不同程度的影響。

關鍵詞：藍莓; 短波紫外線; 貨架期; 抗氧化活性

A large number of studies have proved that the oxidation of free radicals in the body is an important cause of human aging， cancers and other problems［1］. Therefore， the supplement of antioxidant substances is conducive to scavenging free radicals in the body， which is good for human health［2］. Blueberry fruit are popular with people， because they are abundant in antioxidant substances such as procyanidine， phenols， anthocyanin and flavonoid［3］. Researches have shown that γ-aminobutyric acid stimulate the accumulation of flavonoids and phenolic compounds in postharvest blueberries［4－7］. Besides， Ultraviolet-B irradiation treatment increased anthocyanin and total phenols content in blueberries［8］ and fresh-cut carrots［9］. Ultraviolet-C（UV-C） is shortwave ultraviolet at wavelength of 200～280 nm， which has the advantages of no residue， safety and high efficiency［2］. UV-C irradiation is considered to reduce the infection of pathogenic bacteria［10］， improve the antioxidant［11］ and defense enzyme activity of fruits and vegetables［12］， such as peach［13］， apple［14］， grapefruit［15］， and tomato［11］. So the purpose of our present study is to estimate the efficacy of UV-C on the change of antioxidant activity， which aims to provide a theoretical basis for finding a feasible and efficient method to improve the quality of harvested blueberries.

1 Material and methods

1.1 Fruit and treatments

Blueberries （Vaccinium spp. Berkeley） were harvested at maturation stage 1（maturity 25%） from an orchard of Dandong， Liaoning Province， China. Blueberries were isolated with foam to avoid collisions and immediately transported to the laboratory of Shenyang Normal University. Undamaged samples of the same size （2±0.5） g and colour were selected.

Blueberries were randomly divided into two groups of 9000 g each for treatment in triplicate. One group was used for UV-C treatment and the other group was the untreated group. According to our previous results［16］， the UV-C irradiation treatment was 4 kJ·m-2 "for 3 min. The device was a UV-C emitting lamp （15 W， G15T8， Phillips， Netherlands） locating inside a metal cabinet （90×60×57 cm）. The irradiation intensity of 254 nm was measured with a UV radiometer and the distance from the light was 1 m. After UV-C treatment， both of the two groups of the samples were stored at 0 ℃ and 85±10% RH for cold storage.

After 0， 30， 60 and 90 days of refrigeration （according to our preliminary experiment）， the samples of the two groups were taken out and divided into four equal groups， which placed at 5， 10， 15， 20 ℃ for shelf life assessment， respectively. Three replicates （approx. 1 000 g each） from each shelf temperature were randomly taken on day 2， 4 and 8 during shelf life for measurement of antioxidant activity and mean value was reported.

1.2 Determination of procyanidine and total phenols content

Homogenized blueberries （1.5 g） were added with 15 mL of methyl alcohol （95%） and extracted by microwave at 80 ℃ for 45 min. Then 0.5 mL of supernate was diluted to 50 mL with 95% methyl alcohol. The samples were filtered by microporous membrane （0.25 μm） for further determination of high performance liquid chromatography（HPLC） （1 290 Infinity， Agilent Technologies， Santa Clara， USA）. The parameters were as follows： chromatographic column： Diamonsil C18（250 mm×4.6 mm， 5 μm）; mobile phase： methyl alcohol∶deionized water∶acetonitrile=75∶5∶20; flow velocity： 1.0 mL·min-1; UV detection wavelength： 280 nm; injection volume： 20 μL; column temperature： 25 ℃［17］.

Homogenized blueberries （2 g） were added with Folin-Ciocalteu reagent （0.2 mL） and mixed completely. Then 15% Na2CO3 （1 mL） was added after 5 min and the solution was stationary for 2 h. Whereafter， the absorbance at 760 nm was determined by a spectrophotometer （UV-4802， Unico， China）［15］.

1.3 Determination of total anthocyanin and flavonoid content

Homogenized blueberries （10 g） were added with ethyl alcohol， deionized water and hydrochloric acid （2∶1∶1） in a 50 mL colorimetric tube and extracted by ultrasonic wave for 30 min. Then the samples were hydrolyzed in boiling water for 1 h， whereafter cooled down to room temperature. The samples were mixed with the above extracting solution to volume， and the supernate was filtered by microporous membrane （0.25 μm） for further determination of HPLC［18］.

Samples （2 g） were homogenized and added to 5 mL of methyl alcohol containing 1% HCl， then extracted for 24 h at 4 ℃. 0.5 mL of extracting solution was attenuated with 25 mL distilled water， afterwards the absorbance at 325 nm was measured with a spectrophotometer （UV-4802， Unico， China）［19］.

1.4 Measurement of hydroxyl and DPPH radical scavenging ability

Hydroxyl radical scavenging ability assay kit was selected to determineHydroxyl radical scavenging ability（%）.

DPPH radical scavenging ability was calculated with the following formula： DPPH radical scavenging rate（%）=A0-（As-Ac）/A0× 100%. A0： absorbancy of 100 μL of distilled water and 3.0 mL of DPPH; As： absorbancy of 100 μL of sample solution and 3.0 mL of DPPH; Ac： absorbancy of 100 μL of sample solution and 3.0 mL of absolute methanol［20］.

1.5 Measurement of superoxide anion and ABTS radical scavenging ability

Superoxide anion radical scavenging ability was calculated with the following formula： superoxide anion radical scavenging rate（%）=（A0-A）/A0×100%. A0： autooxidation rate of pyrogallol; A： oxidation rate of sample solution［21］.

ABTS antioxidant capacity detection kit was selected to determine ABTS radical scavenging ability（%）.

2 Results and discussion

2.1 Antioxidant activity during cold storage

As shown from Fig.1 and Fig.2， application of UV-C irradiation played a positive role on increasing antioxidant content and activity of blueberries， which was consistent with our previous studies［22］. For example， UV-C irradiation treatment delayed the decrease of procyanidine， total phenols， total anthocyanin and flavonoid content in blueberries with the improvement of 25%， 13%， 13% and 10% comparing with the untreated samples on day 90 of cold storage， respectively （Fig.1）. Similar results were found in the study of Wu et al. and Nguyen et al.， they considered that UV-C treated blueberry fruit had higher level of total phenols during storage at low temperature［23－24］. Yang et al. also reported that UV-C irradiation treatment" promoted" the" accumulation" of" total" phenols and flavonoid content in blueberries at different maturation stages［4］. Yang et al. considered that postharvest UV irradiation significantly promoted anthocyanin biosynthesis and the transcripts of late biosynthetic genes in blueberries［25］.

After cold storage for 90 d， the content of procyanidine， total phenols， total anthocyanin， flavonoid and radical scavenging ability of hydroxyl， DPPH， superoxide anion， ABTS in control group went down， respectively. The decline of radical scavenging ability was likely attributed to the low level of antioxidant substances［12，26］. For UV-C irradiation treated blueberry fruit after cold storage for 90 d， the above antioxidant content（Fig.1） and activity（Fig.2） declined by 57.9%， 16.0%， 24.1%， 15.8%， 21.1%， 21.7%， 15.2% and 22.2%， respectively.

2.2 Procyanidine content during shelf life

Table 1 and Table 2 showed that procyanidine content in untreated or UV-C treated samples decreased with the extension of shelf life either for storage for 0 d or 60 d. After shelf storage for 8 d at 5， 10， 15 and 20 ℃， procyanidine content in UV-C treated blueberries after 0 d of cold storage were 8.2%， 17.5%， 25.0% and 12.0% higher than untreated samples， respectively. Thus it could be seen that the improved effect of UV-C irradiation on procyanidine content in later shelf life was better than that in the early shelf life. Besides， the influence of shelf temperature on procyanidine content was larger. Procyanidine content in blueberries could be maintained at a relatively high levels during the whole shelf life at low temperature storage（5 ℃）. Whereas， higher shelf temperature（20 ℃） promoted the accumulation of procyanidine content at earlier storage， but accelerated the decline at later storage， which was in agreement with the reported results of blueberries［27］.

2.3 Total phenols content during shelf life

As shown in Table 1 and Table 2， total phenols content in blueberry fruit treated with or without UV-C irradiation relaxedly decreased with the extension of shelf life. However， UV-C irradiation treatment increased total phenols content to some extent， which was similar to the results of grapes［28］， fresh-cut strawberries［29］， mushroom［30］， fresh-cut lotus root［31］， tomatoes［32］ and fresh-cut carrots［9］. The enhanced activities of phenylpropane-related enzymes might be the main reason why postharvest UV-C treatment promoted the accumulation of phenolic bioactive substances in blueberry fruit. In addition， as the shelf temperature went up， total phenols content in harvested and stored blueberries also decreased， but higher shelf temperature（20 ℃） was beneficial to the accumulation of phenol compounds at the early stage of storage. The present results was in line with the found in wounded carrots［33］ and blueberries［27］， but different from the conclusions in “Jonagold” apple［12］. One possible reason was that the effect of shelf temperature on total phenols content might be varied with the fruit variety and shelf life.

2.4 Total anthocyanin content during shelf life

Table 1 and Table 2 showed that total anthocyanin content in both untreated and UV-C treated blueberries continued to decline with longer shelf life. The degradation of anthocyanin in blueberry fruit during shelf life might be a results of impact by reactive oxygen species［12］. Moreover， after shelf storage for 8 d at 5， 10， 15 and 20 ℃， total anthocyanin content in UV-C treated blueberries were 25.4%， 32.4%， 58.2% and 65.9% higher than control， respectively， whereas the enhancement of UV-C were 28.8%， 27.7%， 63.5% and 70.9% after cold storage for 60 d， respectively. The data demonstrated that UV-C irradiation treatment played a better role in improving total anthocyanin content， especially at higher shelf temperature. Similar results were observed in strawberry［6］， blueberry［34］ and pomegranate juice［35］. The main reason for the increase of anthocyanin content in fruit treated with UV-C might be the up-regulation of the expression of structural genes and transcription factors of anthocyanin.

2.5 Flavonoid content during shelf life

As shown in Table 1 and Table 2， flavonoid content showed a descending trend with the extension of shelf life， and similar patterns were found in UV-C irradiation treated samples. However， application of UV-C irradiation dramatically increased the flavonoid content during the whole shelf life. After shelf storage for 8 d at 5， 10， 15 and 20 ℃， flavonoid content in UV-C treated blueberries were 17.5%， 14.2%， 37.1% and 33.9% higher than control， respectively， and the improvement of UV-C were 30.6%， 27.6%， 65.0% and 62.7% after cold storage for 60 d， respectively. Liu et al. also considered that application of UV-C resulted in an increase of flavonoids content in tomatoes during storage［32］， which was analogous with the results in pineapple and hawthorn［36－37］， respectively. The increase of flavonoid content might be attributed to their plant defense mechanism against oxidative damage of plant tissues by UV-C irradiation.

2.6 Radical scavenging abilities during shelf life

From Fig.3 and Fig.4， we concluded UV-C irradiation treatment significantly（plt;0.05） improved radical scavenging abilities of harvested and cold stored samples. Adetuyi et al. also found that UV-C irradiation caused an increase of DPPH and hydrogen peroxide scavenging abilities in fresh leaves of clerodenrum volubile［38］. Moreover， higher shelf temperature（20 ℃） significantly increased hydroxyl radical and DPPH radical scavenging abilities in the early stage of storage， but accelerated the decline of antioxidant activity in the later stage of storage and shortened the storage period of blueberries（Fig.4）. Nevertheless， low temperature（5 ℃） significantly（plt;0.05） maintained relatively high levels of hydroxyl radical and DPPH radical scavenging abilities in blueberry fruit， which was consistent with the results［39］.

3 Conclusions

Blueberries are abundant in antioxidant substances that scavenging free radicals. In our study， antioxidant activity of blueberries was affected by all factors and their interactions. Postharvest cold storage was beneficial to improve antioxidant activity and thereby extend the shelf life of blueberries. UV-C irradiation treatment observably enhanced procyanidine content and total phenols content in both harvested and stored samples. Low temperature effectively maintained high levels of radical scavenging abilities in blueberry fruit. Above all， UV-C treatment and low shelf temperature were contribute to improving antioxidant content and activity in blueberry fruit.

References

［ 1 ］FU L，LIU S Y，XU F X. Effect of treatment with MeJA on physiology and biochemistry in blueberry fruit［J］. J Shenyang Norm Univ（Nat Sci Ed）， 2017，35（4）：473－475.

［ 2 ］XU F X，LIU Y F，DONG S Z，et al. Effectiveness of treatment with salicylic acid on postharvest quality and ethylene receptor gene expression in apple fruit［J］. J Shenyang Norm Univ（Nat Sci Ed）， 2018，36（3）：275－278.

［ 3 ］XU F X，WANG S H，XU J，et al. Effects of combined aqueous chlorine dioxide and UV-C on shelf-life quality of blueberries［J］. Postharvest Biol Technol， 2016，117：125－131.

［ 4 ］YANG L，HOU Z X，YANG J F，et al. Effect of UV-C treatment on phenolic compounds and relevant enzymes activities of blueberry［J］. J Zhejiang Agric Univ， 2015，27（6）：955－960.

［ 5 ］CHUN H H，KANG J H，SONG K B. Effects of aqueous chlorine dioxide treatment and cold storage on microbial growth and quality of blueberries［J］. Korean Soc Appl Biol Chem， 2013，56（3）：309－315.

［ 6 ］WANG H，JIANG L，WANG H，et al. Effects of UV-C treatment on postharvest decay， antioxidant activity and phenylpropanoid metabolism in strawberry fruit［J］. Food Sci， 2015，36（10）：221－226.

［ 7 ］GE Y，DUAN B，LI C，et al. γ-Aminobutyric acid delays senescence of blueberry fruit by regulation of reactive oxygen species metabolism and phenylpropanoid pathway［J］. Sci Hortic， 2018，240：303－309.

［ 8 ］INOSTROZA-BLANCHETEAU C，REYES-DAZ M，ARELLANO A，et al. Effects of UV-B radiation on anatomical characteristics， phenolic compounds and gene expression of the phenylpropanoid pathway in highbush blueberry leaves［J］. Plant Physiol Biochem， 2014，85：85－95.

［ 9 ］FORMICA-OLIVEIRA A C，MARTNEZ-HERNNDEZ G B，DAZ-LPEZ V，et al. Effects of UV-B and UV-C combination on phenolic compounds biosynthesis in fresh-cut carrots［J］. Postharvest Biol Technol， 2017，127：99－104.

［10］SNCHEZ G J，CONTIGIANI E V，CORONEL M B，et al. Study of UV-C treatments on postharvest life of blueberries “ONeal” and correlation between structure and quality parameters［J］. Heliyon， 2021，7（6）：e07190.

［11］TIECHER A，PAULA L A，CHAVES F C，et al. UV-C effect on ethylene， polyamines and the regulation of tomato fruit ripening［J］. Postharvest Biol Tech， 2013，86：30－239.

［12］MA Y，BAN Q，SHI J，et al. 1-Methylcyclopropene（1-MCP）， storage time， and shelf life and temperature affect phenolic compounds and antioxidant activity of Jonagold apple［J］. Postharvest Biol Tech， 2019，150：71－79.

［13］ABDIPOUR M，HOSSEINIFARAHI M，NASERI N. Combination method of UV-B and UV-C prevents post-harvest decay and improves organoleptic quality of peach fruit［J］. Sci Hortic， 2019，256：108564.

［14］CHOWDHURY O J，XIE Y，DUAN Y，et al. UV-C treatment promotes quality of early ripening apple fruit by regulating malate metabolizing genes during postharvest storage［J］. PloS One， 2019，14（4）：1－17.

［15］OCHOA-VELASCO C E，SALCEDO-PEDRAZA C，HERNNDEZ-CARRANZA P，et al. Use of microbial models to evaluate the effect of UV-C light and trans-cinnamaldehyde on the native microbial load of grapefruit（Citrus×paradisi） juice［J］. Int J Food Microbiol， 2018，282：35－41.

［16］XU F X，LIU S Y. Control of ethylene activity in blueberry fruit by combined 1-methylcyclopropene（1-MCP） and UV-C irradiation［J］. Food Bioprocess Tech， 2017，10（6）：1695－1703.

［17］LIU L X，LIU Y，HE W P，et al. Determination of procyanidins in dried blueberries by HPLC［J］. Food Research and Development， 2016，37（17）：121－123.

［18］HU L，ZHONG L L，MAO J F，et al. Determination of anthocyanins in grains， vegetables and fruits by high performance liquid chromatography［J］. Chin J Anal Lab， 2012（12），31：43－47.

［19］XIE Y J，WANG Z M，WANG Q，et al. Assessment of the differences in physical， chemical and phytochemical properties of different blueberry cultivars harvested at different dates using principal component analysis［J］. Food Sci， 2017，38（23）：94－99.

［20］BRAND-WILLIAMS W，CUVELIER M E，BERSET C. Use of a free radical method to evaluate antioxidant activity［J］. LWT-Food Sci Technol， 1995，28（1）：25－30.

［21］FINKELSTEIN E，ROSEN G M，RAUCKMAN E J. Spin trapping of superoxide and hydroxyl radical： Practical aspects［J］. Arch Biochem Biophys， 1980，200（1）：1－16.

［22］WANG C Y，CHEN C T，WANG S Y. Changes of flavonoid content and antioxidant capacity in blueberries after illumination with UV-C［J］. Food Chem， 2009，117（3）：426－431.

［23］WU C C，YING Y Q，XU Y Y. Study on the effects of different physical treatments on storage quality of blueberries［J］. Agr Technol， 2017，37（8）：29.

［24］NGUYEN C T T，KIM J，YOO K S，et al. Effect of prestorage UV-A， UV-B， and UV-C radiation on fruit quality and anthocyanin of “Duke” blueberries during cold storage［J］. J Agric Food Chem， 2014，62（50）：12144－12151.

［25］YANG J，LI B，SHI W，GONG Z，et al. Transcriptional activation of anthocyanin biosynthesis in developing fruit of blueberries（Vaccinium corymbosum L.） by preharvest and postharvest UV irradiation［J］. J Agric Food Chem， 2018，66（42）：10931－10942.

［26］MA Y，HUANG H. Characterisation and comparison of phenols， flavonoids and isoflavones of soymilk and their correlations with antioxidant activity［J］. Int J Food Sci Technol， 2014，49（10）：2290－2298.

［27］KONG F Z，ZHI X T，JI Y Q. Effects of storage temperature on the antioxidant activity of blueberry［J］. J Anhui Agric Sci， 2016，44（32）：83－84.

［28］SHENG K L，ZHENG H H，SHUI S S，et al. Comparison of postharvest UV-B and UV-C treatments on table grape： Changes in phenolic compounds and their transcription of biosyntheticgenes during storage［J］. Postharvest Biol Technol， 2018，138：74－81.

［29］LI M，LI X，HAN C，et al. UV-C treatment maintains quality and enhances antioxidant capacity of fresh-cut strawberries［J］. Postharvest Biol Technol， 2019，156：110945.

［30］WU X，GUAN W，YAN R，et al. Effects of UV-C on antioxidant activity， total phenolics and main phenolic compounds of the melanin biosynthesis pathway in different tissues of button mushroom［J］. Postharvest Biol Technol， 2016，118： 51－58.

［31］WANG D，CHEN L，MA Y，et al. Effect of UV-C treatment on the quality of fresh-cut lotus（Nelumbo nucifera Gaertn.） root［J］. Food Chem， 2019，278：659－664.

［32］LIU C，ZHENG H，SHENG K，et al. Effects of postharvest UV-C irradiation on phenolic acids， flavonoids， and key phenylpropanoid pathway genes in tomato fruit［J］. Sci Hortic， 2018，241：107－114.

［33］HAN C，LI J，JIN P，et al. The effect of temperature on phenolic content in wounded carrots［J］. Food Chem， 2017，215：116－123.

［34］PERKINS-VEAZIE P，COLLINS J K，HOWARD L. Blueberry fruit response to postharvest application of ultraviolet radiation［J］. Postharvest Biol Technol， 2008，47（3）：280－285.

［35］PALA U，TOKLUCU A K. Effect of UV-C light on anthocyanin content and other quality parameters of pomegranate juice［J］. J Food Compos Anal， 2011，24（6）：790－795.

［36］SARI L K，SETHA S，NARADISORN M. Effect of UV-C irradiation on postharvest quality of “Phulae” pineapple［J］. Sci Hortic， 2016，213：314－320.

［37］VILA-SOSA R，VILA-CRISSTOMO E，REYES-ARCOS E A，et al. Effect of blue and UV-C irradiation on antioxidant compounds during storage of Hawthorn（Crataegus mexicana）［J］. Sci Hortic， 2017，217：102－106.

［38］ADETUYI F O，KARIGIDI K O，AKINTIMEHIN E S. Effect of postharvest UV-C treatments on the bioactive components， antioxidant and inhibitory properties of clerodendrum volubile leaves［J］. J Saudi Soc Agric Sci， 2020，19（1）：7－13.

［39］WANG F，LIU H，CHEN W R，et al. Effect of storage temperature on the active compounds and the antioxidant capacity of blueberry［J］. J Ningxia Univ（Nat Sci Ed）， 2011，32（2）：172－175.

Received date：2022－08－23

Supported：Project supported by National Innovation and Entrepreneurship Training Program Project for College Students， China（X202210166110X）; Key Project of Education Department of Liaoning Province， China（LJKZZ20220116）.

Biography：XU Fangxu（1986—）， female， was born in Xingcheng city of Liaoning province， associate professor of Shenyang Normal University， Doctor.