CHEMICAL AND ANTIOXIDANT PROPERTIES OF CULTIVATED AND WILD FRAGARIA AND RUBUS BERRIES

ABSTRACT

Content of individual sugars, organic acids, total phenolics (TPH), some important phenolic compounds (kaempferol, myricetin, quercetin, and ellagic acid) and total antioxidant capacity (TAC) in the fruits of strawberry, raspberry and blackberry were studied. A comparison was made between cultivars and wild relatives of each species (Fragaria vesca L., Rubus idaeus L. and Rubus fruticosus L.). The main sugars found were fructose and glucose both in the fruits of wild species and the studied cultivars. Citric acid was determined to be the major organic acid in most of tested berries with the exception of blackberry cultivars, where malic acid was dominant. The content of individual phenolic compounds varied among the wild species, as well as among the studied cultivars. Ellagic acid content was higher in F. vesca, R. idaeus and R. fruticosus (122.5 µg/g FW, 12.71 µg/g FW and 61.7 µg/g FW, respectively) than that obtained in analyzed cultivars. Overall, TPH expressed higher values in the wild strawberry and blackberry species in comparison to the studied cultivars, and consequently, the highest levels of TAC were recorded in F. vesca (5.78 mg asc/g FW), followed by R. fruticosus (4.95 mg asc/g FW).

PRACTICAL APPLICATIONS

In recent years, both wild and cultivated berries have become very attractive for consumers because of potentially beneficial phytochemicals contained in these fruits. Fruit nutritional quality can be described by standard quality parameters (sugars and organic acids), and the analysis of antioxidant capacity influenced by specific related compounds. The importance of flavonoids and other phenolics have been suggested to play a preventive role in the development of cancer and heart disease. A significant positive correlation observed in this study between total phenolics and total antioxidant capacity indicate the need for the use of wild species in the breeding programs of small fruits, especially strawberry and blackberry, in order to increase their nutritive value and the health benefits.     

6个草莓品种营养品质与抗氧化能力研究

本研究对6个草莓品种的质量参数(可溶性固形物、可滴定酸和硬度)和营养参数(总酚、类黄酮、花青素、抗坏血酸、VE和抗氧化能力)进行测定与分析,目的在于筛选具有较高营养价值的草莓遗传资源。研究表明,总酚是草莓抗氧化作用的重要物质基础,花青素与抗坏血酸是草莓抗氧化能力的主要组成参数。在6个草莓品种中,红太后的营养品质表现最好,是培育高含量生物活性物质新品种的重要遗传资源,然而基因型是决定草莓营养品质的重要因素。     

A Comparative Study of Phenolic Antioxidant Activity and Flavonoid Biosynthesis‐Related Gene Expression Between Summer and Winter Strawberry Cultivars

Strawberry (Fragaria ananassa Duch.) possesses good antioxidant properties. Phenolic compounds in strawberries, such as anthocyanins and ellagic acid, mainly act as antioxidants. This study aimed to compare the phenolic content and expression patterns of genes involved in flavonoid biosynthesis between summer and winter strawberry cultivars affected by seasonal variation, degree of ripeness, and genotype. Antioxidant activity and the total content of phenols and flavonoids decreased with fruit ripening. Most notably, summer strawberry cultivars showed higher antioxidant activity than winter cultivars. The expression patterns of flavonoid biosynthetic genes tested were cultivar‐dependent and were also affected by ripening. These results help us understand the nutritional and physiological characteristics of selected cultivars and provide a range of information for strawberry consumption.     

Main quality attributes and antioxidants in Hungarian sour cherries: identification of genotypes with enhanced functional properties

Sour cherry (Prunus cerasus L.) cultivars native to Hungary and some of them grown worldwide, display great phenotypic variability. Eleven sour cherry cultivars were analysed for their main quality attributes including fruit weight, soluble solid content, acidity, pH, specific sugars (glucose and fructose), antioxidant capacity as well as total polyphenolic (TPC), total anthocyanin (TMAC) and vitamin C contents. Results showed wide variation in both fruit quality and antioxidant parameters of Hungarian sour cherries. The anthocyanin contents varied from 11.3 to 93.5 mg/100 g. An amarelle‐type cultivar, ‘Pipacs 1’ showed the highest antioxidant capacity (21.85 mmol AA L^?1), TPC (49.04 mg GA L^?1) and vitamin C (8.98 mg/100 g) content. Key enzymes in anthocyanin biosynthesis were expressed in both yellow‐coloured flesh and red skin of ‘Pipacs 1’ fruits at all ripening stages. The detected diversity presents a choice that can satisfy different consumer preferences, and meet specific nutritional requirements.     

Ascorbate,not urate,modulates the plasma antioxidant capacity after strawberry intake

The consumption of antioxidant-rich foods has already been associated with acute significant increases in plasma total antioxidant capacity (TAC) in humans, but there is no consensus among different literature reports. Furthermore, the acute rise in plasma TAC observed after consumption of several non-berry fruits seems not to be necessarily related to the absorption of dietary antioxidants, such as micronutrients and phenolic substances, but is attributed to the relevant increase of endogenous urate in serum. In this study, we first compared the nutritional quality of strawberry fruits from different genotypes, all cultivated in the same experimental field. Significant genotype-to-genotype differences were observed in the antioxidant capacity and in the vitamin C, folate and phenolic contents of the fruits. Second, in order to investigate the individual effects of the selected strawberry genotypes on the post-prandial plasma antioxidant status in humans, we assessed the acute effects of a single dose of strawberries in healthy subjects. The variation of plasma TAC through the FRAP and TEAC assays, and any eventual changes in ascorbate and urate levels in serum, were measured during the three hours following the strawberry intake. The acute intake of strawberries resulted in a significant increase in plasma FRAP values in all the subjects, independently of the individual TAC baseline levels. Interestingly, the lowest increases in plasma TAC and in serum ascorbate levels were associated with consumption of the nutritionally-poorest cultivars. In addition, significant increases in ascorbate, but not in urate, concentrations were observed in serum. These findings suggested that the fructose-dependent hyperuremic effect, observed after the intake of non-berry fruits, is not responsible for the plasma antioxidant changes following strawberry consumption.     

Effect of ultrasonic treatment on enzyme activity and bioactives of strawberry puree

The aim of this study was to evaluate the impact of ultrasound (US) at different frequencies (20, 370, and 583 kHz) and power levels (35 and 48 W) on the residual activity (RA) of peroxidase (POD) and polyphenol oxidase (PPO) in strawberry puree. Total anthocyanin content (TAC), total phenolic content (TPC), ferric ion reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) were also assessed. Results were compared with untreated, thermally treated at 40 °C (control) and pasteurized (90 °C) strawberry puree. POD and PPO RA were significantly (P < 0.05) reduced, whilst there was a significant (P < 0.05) increase in TAC (5%–19%) in all US-treated samples in comparison with the untreated samples and the controls. US at 20 kHz (35 W) increased significantly (P < 0.05) TPC (9%) and FRAP (6%) in strawberry puree, whereas the effect of 583 kHz (48 W) on these parameters was insignificant (P > 0.05). Pasteurization inactivated POD and PPO, however, decreased dramatically TAC (14%), TPC (17%) and FRAP (9.5%) in strawberry puree. These findings suggest that US is a promising novel non-thermal food technology that can be tailored to improve the quality of strawberry puree by inactivating enzymes responsible for food deterioration whilst maintaining the content of bioactive compounds.     

2种草莓成熟过程中果实特征品质指标的变化

研究‘米拉’(‘Mira’)和‘哈尼’(‘Honeoye’)2个草莓品种的白色未熟期、粉色转熟期和红色成熟期3个阶段成熟过程中与风味、生理功能活性相关的特征品质指标变化。结果表明随着草莓成熟果实中总可溶性固形物含量、芳香性挥发成分、花色苷成分积累量有显著性上升,而总酸、总酚、总黄酮含量和抗氧化能力均有所下降。相关性和主成分分析结果表明,草莓成熟过程中总酚、总黄酮、总可溶性固形物和总酸含量均与总抗氧化-亚铁还原能力(ferric reducing-antioxidant power,FRAP)以及氧自由基吸收能力(oxygen radical absorption capacity,ORAC)的变化之间存在高相关性(r为0.859 1~0.994 7),而总花色苷含量与FRAP和ORAC之间的相关性仅为0.315 0和0.385 3。同时得出草莓果实的成熟度与芳香性挥发成分和花色苷组分积累量呈正相关性。     

Strawberry consumption improves plasma antioxidant status and erythrocyte resistance to oxidative haemolysis in humans

Significant increases in the plasma total antioxidant capacity (TAC) have already been reported after acute intake of strawberries. In addition, antihaemolitic effects of strawberry extracts have been recently demonstrated in vitro, revealing that part of the antioxidant properties of strawberry bioactive compounds could lie in their localisation within cell membranes. However, there is a lack of research evidence from in vivo protracted strawberry consumption studies. We carried out a 16-day pilot study where 12 healthy subjects ingested 500 g of antioxidants-rich strawberries daily, and we evaluated the potential effects of fruit consumption on biomarkers of plasma and cellular antioxidant status. A significant increase in fasting plasma TAC and in serum vitamin C concentrations were progressively observed during the period of strawberry supplementation. An enhanced resistance to haemolysis was also observed in both AAPH-treated and untreated erythrocytes, collected during and after the period of strawberry consumption. The results obtained in this work suggest that regular consumption of antioxidant-rich strawberries may exert an improvement on the plasma antioxidant status and an increase on the antihaemolitic defenses of human erythrocytes.     

Effects of temperature on the chemical composition and antioxidant activity of three strawberry cultivars

Three strawberry cultivars (Dover, Campineiro, and Oso Grande), grown in the same commercial plantation, were harvested at the ripe stage and stored at 6, 16 and 25 °C, for 6 days. During this period, chemical composition and antioxidant activity were evaluated. Results showed an increase in total soluble sugars, anthocyanin and vitamin C contents, indicating that a new biosynthesis had taken place during storage. Low temperature negatively affected anthocyanin and vitamin C accumulation, and positively affected soluble sugars, while flavonols, ellagic acid and total phenolic contents remained almost the same or even decreased at all temperatures. Despite differences in anthocyanin content between varieties and its increase during storage (higher with increasing temperature), there was no difference in the antioxidant activity between cultivars, which decreased after harvesting, independently of the temperature of storage. Variations in the proportion dehydroascorbic acid/ascorbic acid (DHA/AA) showed that there were differences between cultivars concerning adaptation of the fruit to low temperatures. The data obtained here indicate that cold storage is an effective way to maintain strawberry quality, but a compromise between sensorial and nutritional values can be achieved at 16 °C, for all the cultivars.     

Comparative analysis of the chemical composition and antioxidant activity of red (Psidium cattleianum) and yellow (Psidium cattleianum var. lucidum) strawberry guava fruit

Abstract: Strawberry guava (Psidium cattleianum Sabine) is a native fruit of Brazil widely consumed fresh and used in the food industry. In this context, the present study deals with the chemical characterization and the antioxidant activity of the red (Psidium cattleianum) and yellow (P. cattleianum var. lucidum Hort.) strawberry guava fruits, cultivars Irapuã and Ya‐Cy, respectively. Knowledge of chemical composition is fundamental to human nutrition, contributing to the quality of foods. Phenolic compounds in both fruits were analyzed by HPLC–DAD and the total flavonoid content was determined by the Folin–Ciocalteu assay. The antioxidant activity was evaluated by the total reactive antioxidant (TRAP) method. Psidium cattleianum presented a higher content of polyphenolic compounds than P. cattleianum var. lucidum (501.33 and 292.03 mg/100 g, respectively), with hyperoside being one of the major flavonoids identified for both cultivars. In addition to flavonoids, P. cattleianum presented an anthocyanin, identified as cyanidin. The antioxidant activity varied in a concentration‐dependent manner for both strawberry guava species. The volatile oils in fruits and fatty acids in seeds were quantified by GC‐EM. The analysis of the essential oil of yellow strawberry guava was compared with a previous study on the red cultivar, revealing β‐caryophyllene as the main component in both oils. The fatty acid composition was also quite similar and was especially characterized by the presence of unsaturated fatty acids (86.25% and 76%, respectively), among which linoleic acid as the most abundant. Practical Application: In this study, the chemical characterization and the antioxidant activity of the red (Psidium cattleianum) and yellow (P. cattleianum var. lucidum Hort.) strawberry guava fruits were investigated. This is important for potential application of strawberry guava as functional food. Moreover, it may be the experimental basis for further development and use in food industry.     

The effect of postharvest ripening on strawberry bioactive composition and antioxidant potential

BACKGROUND: The aim of the present work was to evaluate the phytochemical profile and the antioxidant activity of strawberry fruit (cv. Camarosa) upon postharvest ripening at room temperature (20 °C) and to correlate them with qualitative attributes. RESULTS: ‘Camarosa’ fruit retained an appreciable postharvest performance for up to 1 day shelf life; thereafter substantial quality deterioration was observed. An increased phenolic content, mainly attributed to hydroxycinnamic acids, flavonols and anthocyanins, was monitored after 3 days maintenance at 20 °C. The chromatographic fingerprint of athocyanins showed that pelargonidin‐3‐glucoside was the major anthocyanin, which increased with the increase of shelf life period, while cyanidin‐3‐glucoside and pelargonidin‐3‐rutinoside were found at lower concentrations. The potent radical scavenging activity, evaluated with four in vitro assays, showed a higher antioxidant capacity after 3 and 1 days of shelf life. In addition, the antioxidant effect of strawberry fruit extracts on lipid substrates and on an emulsion system showed a significant inhibition in the formation of conjugated diene hyperoxides. Interestingly, no direct correlation between the phytochemical profile and the quality attributes exist. CONCLUSION: ‘Camarosa’ fruit had acceptable quality attributes for fresh consumption and at the same time a high phytochemical content up to 1 day shelf life. The over‐ripe fruit continued to be an excellent reservoir of natural antioxidants, pointing to its potential use as food antioxidant and nutritional supplement. Copyright © 2011 Society of Chemical Industry     

Effect of natural biostimulants on yield and nutritional quality: an example of sweet yellow pepper (Capsicum annuum L.) plants

BACKGROUND: Modifications in growing techniques can affect the yield and nutritional quality of various cultivated plant species. Owing to its high nutritional value, pepper (Capsicum annuum L.) was used in this study as a model plant to investigate the effect of natural biostimulants on yield and fruit quality parameters under conditions of reduced fertilisation. RESULTS: A positive influence of biostimulant treatment on yield parameters was observed. The overall increase in the pigment content of leaves after biostimulant application agreed well with the higher total and commercial yields of treated pepper cultivars compared with their controls. The results showed that natural biostimulants had a positive effect on the vitamin C and total phenolic contents in pepper fruits during the hot summer season. The 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) and 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonate) (ABTS) antioxidant activities were also significantly higher (P < 0.05) in treated plants and correlated strongly with all measured quality parameters except total phenolic content. CONCLUSION: Generally, biostimulants improved the antioxidant activity, vitamin C and phenolic contents in fruits as well as the pigment content in leaves of treated compared with non‐treated pepper plants grown hydroponically. Thus the application of biostimulants could be considered as a good production strategy for obtaining high yields of nutritionally valuable vegetables with lower impact on the environment. Copyright © 2011 Society of Chemical Industry     

Cultivation approach for comparing the nutritional quality of two pepper cultivars grown under different agricultural regimes

The aim of this study was to assess the influence of the cropping system (organic or conventional) and the growth medium (soil or soilless) on the antioxidant content of peppers from Almuden and Quito cultivars. This cultivation study complements a previous farm study. For both cultivars, organic cultivation led to higher fruit lipophilic antioxidant content than conventional management but differences in the hydrophilic fraction were not observed. The effect of the growth medium on pepper composition depended on the studied cultivar. In Almuden, neither the hydrophilic nor the lipophilic fractions were affected by the growth medium, while in Quito most compounds showed higher values in soil-grown plants. Taking into account the results from the present and the previous farm study we conclude that the effect of organic vs. conventional cultivation on pepper quality can be attributed to the different nutritional regimes and other factors associated with the agricultural system. However, both aspects are not necessarily different in organic and conventional commercial crops. The effect of the growth medium (soil or soilless) on pepper quality depended on the cultivar: Almuden was not affected by the growth medium and Quito was seen to be less suitable for soilless cultivation.     

陕西安康地区10 个日本引种枇杷果实品质比较

通过果实品质比较分析建立陕西安康地区引种枇杷品质评价方法,为划分品种类型并筛选适宜当地枇杷产业发展的优良品种提供参考依据。对10 个日本引种枇杷果实的物性指标、营养指标及功能性指标进行测定,采用主成分分析和聚类分析方法对枇杷品质进行评价。结果表明：不同评价指标在10 个日本引种枇杷果实的检测结果中存在较大差异,但综合品质均优于当地品种‘麦后黄’。主成分分析提取出风味、抗氧化、加工和总酚4 项主成分因子,累计方差贡献率达到85.666%。根据主成分综合评价函数,综合排名前3 名的品种分别是‘ADVANCE’、‘津云’和‘CHAMPAGNE’。聚类分析将10 个日本引种枇杷聚为5 类,经初步分析,第1类具有一定的保健价值;第2类可考虑作为加工果汁、果醋类饮料的原料;第3类是适宜鲜食的品种。主成分分析和聚类分析方法相结合可综合评价枇杷果实品质,为其品种开发类型的选择及有效利用提供参考。     

High‐anthocyanin strawberries through cultivar selection

BACKGROUND: Diets high in fruit and vegetables are known to have significant health benefits. This is in part due to the presence of phytochemicals, which possess potential protective health benefits. This study focuses on the ability of strawberries to be bred for higher anthocyanin content. This is a major contributor to the characteristic colour and nutritional value of ripe strawberries, together with phenolic acids, ascorbic acid and total antioxidant capacity. RESULTS: Anthocyanins in five commercial strawberry cultivars and three breeding lines were assessed. This led to the discovery of one breeding line (BL 2006‐221) as an exceptional source of anthocyanins (～1 g kg^?1 fresh weight), with approximately double the levels of current commercial cultivars. Temperature was shown to influence anthocyanin extraction, with 40 °C being the best extraction temperature using the accelerated solvent extraction (ASE) method. Hue angle and anthocyanin concentration showed a good correlation (r² = 0.69). CONCLUSION: The new breeding line BL 2006‐221 has the potential to be used in the development of phytochemically rich strawberry cultivars. Using hue angle as a screening tool for total anthocyanin concentration and extraction of anthocyanins from strawberries by ASE at 40 °C would support such cultivar development. © 2012 Commonwealth of Australia     

Characterization of antioxidant compounds of red and white rice and changes in total antioxidant capacity during processing

Although most rice cultivars have whitish kernel, some varieties have a red testa. Aim of this work was to compare the total antioxidant capacity (TAC) and the antioxidant chemical composition (namely tocols, gamma-oryzanols, and polyphenols) of red and white rices. In addition, the effect of milling and cooking on antioxidants was investigated in both rices. Dehulled red rice showed a TAC more than three times greater than dehulled white rice and its high TAC was essentially characterized by the presence of proanthocyanidins (PA) and associated phenolics. Milling caused a significant loss of TAC, even if red rice maintained a higher TAC. Cooking caused a further loss of antioxidants, but when there was a full uptake of cooking water by the grains ("risotto") this loss was limited. Thus, the consumption of whole or partially milled rice cooked as risotto would be preferred to preserve its nutritional properties.     

Effect of conventional and organic production systems on the yield and quality of field tomato cultivars grown in Tunisia

BACKGROUND: The agricultural production system and cultivation practices are critical factors in determining yield and food nutritional quality. Controversy remains regarding whether or not organic foods have a nutritional advantage when compared with their conventionally produced counterparts. In this study the effect of conventional and organic production systems on yield, physicochemical and nutritional properties of four field tomato cultivars (Perfectpeel, Rio Grande, Hypeel 108 and Firenze) was determined. In the organic system, different fertiliser combination treatments were used. RESULTS: When production system data were combined, marketable yield was not affected by cultivar. When averaged across cultivars, organic yield was about 63% of conventional yield. The production system affected fruit soluble solids, pH, titratable acidity and firmness only in some cultivars. However, no effect was found on lycopene and total phenolics in all cultivars. In the organic system, fertiliser combination treatments affected tomato yield of Rio Grande and fruit quality of Firenze and Hypeel 108. A general trend of higher quality data was obtained by using mixed compost C2 combined with its extract and codahumus 20. CONCLUSION: The production system affects tomato yield and some fruit quality attributes. The effect on fruit quality varies among cultivars, thus highlighting the need to use a range of varieties in this kind of study. Copyright © 2009 Society of Chemical Industry     

Genotype and growing season influence blueberry antioxidant capacity and other quality attributes

Forty‐two blueberry cultivars (thirty‐six Vaccinium ashei, three V. ashei derivative hybrids and three northern highbush standards) were evaluated for their antioxidant capacities and other fruit quality attributes over two growing seasons. Total anthocyanins, phenolic content and antioxidant capacities varied substantially among species and cultivars. ‘Early May’ (V. ashei) had the highest amounts of anthocyanins, phenolics and antioxidant capacity. Cultivars from V. ashei hybrid derivatives had lower mean antioxidant components than those from northern highbush standards or V. ashei. The antioxidant capacity, as well as total anthocyanins and phenolics, had significant cultivar × year interactions. Correlation coefficient between years for total anthocyanins, total phenolics and oxygen radical absorbance capacity were high with values of 0.86, 0.81 and 0.93, respectively. Similar interactions were observed for soluble solids content (SSC), sugar, titratable acid and organic acids among cultivars both within and across the growing seasons. Correlation coefficients between years for SSC, fructose, glucose and sucrose were 0.78, 0.71, 0.83 and 0.96, respectively. Fructose and glucose were detected as two major sugars with sucrose as a minor constituent. ‘Clara’ contained the highest amounts of SSC and sugars, while ‘Satilla’ had the lowest. In general, V. ashei cultivars had a higher mean SSC and sugar contents than did the hybrid derivatives or the northern highbush standards. Cultivars of V. ashei contained higher malic acid than citric acid, whereas in hybrid derivatives and northern highbush cultivars, citric acid was the predominant organic acid. The diversity in the amount of these fruit quality attributes and antioxidant capacities presents a great opportunity for genetic improvement of blueberries through breeding programmes. The objective of the study was to identify blueberry cultivars with high antioxidant activity and good fruit quality, so they can be used as parents for future blueberry breeding programmes to develop new cultivars with higher antioxidant activity.     

Impact of selected factors – Cultivar,storage, cooking and baking on the content of anthocyanins in coloured-flesh potatoes

The impact of selected factors – cultivar, storage, cooking and baking on the content of total anthocyanins (TAC) in coloured-flesh potato cultivars has been studied. TAC ranged from 248.5 to 2257.8 mg kg⁻¹ dry matter (DM). TAC difference between cultivars was statistically significant. Cold storage (4 °C) influenced TAC differentially. In the Violette and Highland Burgundy Red cultivars TAC increased by 18.5% and 12.1% respectively, and in the Valfi cultivar it decreased by 33.9%. Baking increased TAC 3.34 times whereas cooking in boiled water increased it 4.22 times. Correlation between antioxidant activity (AOA) and TAC (r² = 0.659) has been found. The Violette, Vitelotte and Highland Burgundy Red cultivars with the highest TAC showed high AOA and the Shetland Black cultivar and the cultivars Salad Blue and Blue Congo with a “marbled” texture showed the lowest TAC and AOA. Individual anthocyanidins are fingerprints of colour-fleshed potato cultivars.     

A comparative study on different concentration methods of extracts obtained from two raspberries (Rubus idaeus L.) cultivars: evaluation of anthocyanins and phenolics contents and antioxidant activity

The effects of conventional (CV) and microwave (MW) heating on total soluble solid (TSS) content, total anthocyanin content (TAC), total phenolic content (TPC) and antioxidant activity of raspberry juice obtained from two cultivars of Amol (AM) and Siyahkal (SK) were scrutinised. Antioxidant activity of the juices was measured by 2,2 diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging capacity and expressed as EC₅₀ value. In the both methods, the concentration rate constant for the juices was significantly decreased by increasing operational pressure from 12 to 100 kPa (P < 0.05). A first‐order reaction kinetic model was successfully fitted for the degradation of monomeric anthocyanins at all operating pressures. The results also showed that thermal treatment of MW compared CV in both SK and AM cultivars caused a lower decrease in the TAC, TPC and antioxidant activity. Moreover, the changes in trend of the antioxidant activities due to the thermal treatment were positively correlated with the TPC (r = 0.74, P < 0.05) and TAC (r = 0.61, P < 0.05).