Influence of the Extraction Process on the Characteristics of Romanian Mountain Walnut Oil

The objective of this work is to extract walnut oil using various processes in order to compare the influence on the nature of the components extracted, and thus identify the areas of potential use. We carried out the extractions by mechanical process, thanks to a press in reduced model provided with a worm. We obtained cold extracted oil whose characteristics slightly diverge from extra virgin oil found in shops in Romania, but its composition is similar. We were also able to extract by chemical process using two methods, Folch and Soxhlet. Commercially available table walnut oils are only cold extracted to avoid the presence of solvents. Those are difficult to remove and strongly oxidize the oil. Currently, consumers appreciate walnut oil for its taste and nutritional qualities. In nutrition, this oil is put forward for its composition rich in polyunsaturated fatty acids, which are needed for human body. Food supplements made from walnut oil are available today. For the moment, this is the only use of walnut oil. Indeed, there are some studies on other fields of application, but they remain in the field of research and nothing has yet been commercialized. In this present study, we compared the chemical and physical properties of cold-extracted oil with the solvent extraction of walnut kernel originating from the mountain region of Rumania. The cold extracted oil has a high content of polyunsaturated fatty acids (63%) and monounsaturated fatty acids (30%), a very low level of saturated fatty acid (7%) and no content of linolenic acid. The Soxhlet and Folch methods produced slightly different oils with increased amounts of minor components, which changes their characteristic. Even when solvent-extracted oils do not meet the standard criteria imposed by the Codex Alimentarius, they offer a possible use in the fields of food, cosmetics industries and biomedicine.

JrATHB-12 mediates JrMYB113 and JrMYB27 to control the anthocyanin levels in different types of red walnut

Red walnut has broad market prospects because it is richer in anthocyanins than ordinary walnut.However,the mechanism driving anthocyanin biosynthesis in red walnut is still unknown.We studied two types of red walnut,called red walnut 1(R1),with a red pericarp and seed coat,and red walnut 2(R2),with a red seed coat only.R1 mostly contained cyanidin-3-O-galactoside,while R2 contained a various amounts of cyanidin-3-Ogalactoside,cyanidin-3-O-arabinoside,and cyanidin-3-O-glucoside.The LDOX-2(LOC109007163)and LDOX-3(LOC109010746)genes,which encode leucoanthocyanidin dioxygenase/anthocyanidin synthase(LDOX/ANS),were preliminarily indicated as the crucial genes for anthocyanin biosynthesis in R1 and R2,respectively.The MYB differential genes analysis showed that MYB27 and MYB113 are specifically expressed in the red parts of R1 and R2,respectively,and they are regarded as candidate regulatory genes.Ectopic expression in Arabidopsis and transient injection in walnut showed that both MYB27 and MYB113 were located in the nucleus and promoted anthocyanin accumulation,while MYB27 promoted the expression of LDOX-2,and MYB113 promoted the expression of LDOX-3and UAGT-3.Yeast one-hybrid and electrophoretic mobility shift assays showed that MYB27 could only bind to the LDOX-2 promoter,while MYB113 could bind to the promoters of both LDOX-3 and UAGT-3.In addition,we also identified an HD-Zip transcription factor,ATHB-12,which is specifically expressed in the pericarp.After silencing the expression of ATHB-12,the R2 pericarp turned red,and MYB113 expression increased.Further experiments showed that ATHB-12 could specifically interact with MYB113 and bind to its promoter.This suggests that MYB27controls R1 coloration by regulating LDOX-2,while MYB113 controls R2 coloration by regulating LDOX-3 and UAGT-3,but ATHB-12 can specifically bind to and inhibit the MYB113 of the R2 pericarp so that it becomes unpigmented.This study reveals the anthocyanin biosynthetic mechanisms in two different types of red walnut and provides a scientific basis for the selection and breeding of red walnut varieties.

Transcriptome profiling of indole-3-butyric acid-induced adventitious root formation in softwood cuttings of walnut

Inducing adventitious root(AR)formation in mature walnut species(Juglans L.)is challenging.However,the AR formation of mature trees can be improved by rejuvenation.In rejuvenated cuttings,exogenous indole-3-butyric acid(IBA)is essential for AR formation,and the underlying mechanism is still not well understood.Therefore,we utilized transcriptome sequencing to investigate the mechanism of IBA-induced AR formation.Our results revealed that,in comparison to the control group,IBA treatment(9 mmol·L^(-1))significantly increased the endogenous indole-3-acetic acid(IAA)content,leading to an enhanced rooting rate.We performed RNA sequencing to identify differentially expressed genes(DEGs)between the IBA-treated and control(CK)groups at 1,2,3,and 5 days after cutting(DAC).The results showed that,compared to the control cuttings,there were 1539,889,785,and 984 up-regulated genes and 2791,2936,3017,and 1752 down-regulated genes,at 1,2,3,and 5 DAC,respectively.Analysis of RNA-seq data revealed that G-type ATP-binding cassette 36/37(ABCG36/37)and ATP-binding cassette subfamily D 1(ABCD1),associated with IBA transport,were down-regulated in the rejuvenation cuttings.In contrast,PIN-FORMED(PIN)and PINOID(PID),associated with auxin efflux,were up-regulated.We identified 49 auxin/indole-3-acetic acid(AUX/IAA)-encoding genes,including IAA1,IAA3,IAA5,IAA6,IAA8,IAA11,IAA12,IAA19,and IAA20,which were up-regulated at 1-5 DAC in the rejuvenated cuttings.This study highlights that the overexpression of JrWOX5/11 in poplar significantly enhance AR growth,as evidenced by increased root length,surface area,volume,and quantity.Moreover,the co-expression network analysis involving JrWOX11 and JrWOX5 in walnut cuttings elucidates complex genetic interactions,underscoring their pivotal role in the formation of AR.Our data supported the following molecular mechanism of IBA-induced adventitious root formation.Firstly,IBA is converted to free IAA in peroxisomes.Then,the highly concentrated IAA in the procambium and parenchyma cells induces WUSCHEL-related homeobox 11(WOX11)expression at two days.Finally,WOX11 acts redundantly to up-regulate WOX5,initiating the development of root primordia cells.

Exploration of cyclooxygenase-2 inhibitory peptides from walnut dreg proteins based on in silico and in vitro analysis

Walnut dreg protein hydrolysates(WDPHs)exhibit a variety of biological activities,however,the cyclooxygenase-2(COX-2)inhibitory peptide of WDPHs remain unclear.The aim of this study was to rapidly screen for such peptides in WDPHs through a combination of in silico and in vitro analysis.In total,1262 peptide sequences were observed by nano liquid chromatography/tandem mass spectrometry(nano LC-MS/MS)and 4 novel COX-2 inhibitory peptides(AGFP,FPGA,LFPD,and VGFP)were identified.Enzyme kinetic data indicated that AGFP,FPGA,and LFPD displayed mixed-type COX-2 inhibition,whereas VGFP was a non-competitive inhibitor.This is mainly because the peptides form hydrogen bonds and hydrophobic interactions with residues in the COX-2 active site.These results demonstrate that computer analysis combined with in vitro evaluation allows for rapid screening of COX-2 inhibitory peptides in walnut protein dregs.

Population genetic insights into the conservation of common walnut(Juglans regia)in Central Asia

The common walnut(Juglans regia)is one of the most economically important nut trees cultivated worldwide.Despite its importance,no comprehensive evaluation of walnut tree population genetics has been undertaken across the range where it originated,Central Asia.In this study,we investigated the genetic diversity and population structure of 1082 individuals from 46 populations across Central Asia.We found moderate genetic diversity of J.regia across Central Asia,with 46 populations clustered into three groups with a weak relationship between genetic and geographic distance.Our findings reveal that the western Himalaya might be the core region of common walnut genetic diversity in Central Asia and that,except for two populations in Gongliu Wild Walnut Valley,humans might have introduced walnut populations to Xinjiang,China.The observed distribution of the genetic landscape has probably been affected by historical climate fluctuation,breeding system,and prolonged anthropogenic activity.We propose the conservation of the core genetic diversity resources in the western Himalaya and pay special attention to populations from Gongliu in Xinjiang.These findings enhance our understanding of the genetic variation throughout the distribution range of J.regia in Central Asia,which will provide a key prerequisite for evidence-based conservation and management.

基于Walnut Street修正模型的短波信道仿真算法

传统短波信道Watterson模型只能模拟信道短期变化规律。为模拟短波信道在各种长度时间段内的变化,针对短波信道接收端信噪比统计特性,提出了一种基于信噪比重放的短波信道仿真算法。该算法以Walnut Street信道模型为基础,通过对实际观测数据进行分析处理,建立接收端信噪比变化的中期和长期变化统计模型,联合Watterson模型实现信道模拟。仿真结果表明,该算法可反映实际信噪比变化规律,为短波网络仿真提供精确的信道质量模拟。

Study on Adsorption of Hg(Ⅱ) by Chinese Walnut(Juglans mandshurica Maxim.) of Biomass Material

[Objective] The aim of the study was to make research on adsorption of Chinese walnut（Juglans mandshurica Maxim.） Shell（CWS） to Hg（Ⅱ） in water.[Method] Shells of Juglans mandshurica Maxim were used as biosorption to remove Hg（Ⅱ） in water solution to explore the influence to adsorption of Hg（Ⅱ） under different conditions,like pH solution,adsorption time,and Hg（Ⅱ）.[Result] The experimental results show that when absorptivity of Hg（Ⅱ） by CWS reached the highest,pH ranged within 5.0-6.0.The adsorptivity decreased as initial Hg（Ⅱ） concentrations increased.Fourier Transform Infrared Spectroscopy（FTIR） spectrum revealed some chemical groups of CWS may affect the adsorption of Hg（Ⅱ）,such as hydroxyl groups,methyl groups,aromatic methoxyl groups,unconjugated carbonyl,and typical aromatic ring,etc.Adsorption equation can be concluded considering the biosorption process relationship with Langmuir and Frendrich isotherm.[Conclusion] The study found that CW could be employed as a promising biosorption to remove Hg（Ⅱ） from aqueous environments.

Walnut Tree Grafting Experiments in Different Areas in Yunnan Province

Yunnan has an area of 2.88 million hm2 walnut trees. Due to freezing in- jury and rapid growth, however, there are a lot of iron walnuts or inferior-quality walnuts ready for grafting. In five years, we conducted experiments in Kunming （Yunnan Academy of Forestry）, Gongshan County, Gucheng City, Luliang County, and Ludian County by cleft grafting, veneer grafting, bark grafting and whip grafting. The grafting approaches should be selected according to grafting requirements, characteristics, and survival rate. For example, with a low survival rate, cleft grafting can be abandoned, and the rest approaches might be chosen as per practical demands.

Study on Technology and Formula of Walnut-Roxburgh Rose Beverage

[Objective] The study aimed to explore technology and formula of walnut- roxburgh rose beverage. [Method] With walnut and roxburgh rose as the main raw material, technology and formula of walnut-roxburgh rose beverage was study by or- thogonal test. In addition, effects of emulsion stabilizers were investigated. [Result] The great emulsifying effect could be achieved by composite emulsion stabilizer at 0.40%. The orthogonal test showed that the optimum formula of walnut-roxburgh rose beverage was composed of 24% walnut milk, 16% cili juice and 12% sugar, with pH of 4.0. The product with stable organization state, pleasant sweetness and sourness may be prepared. [Conclusion] The study laid the foundation for production of walnut-roxburgh rose beverage.

Study on Improvement of Walnut Bud Grafting Technology in the North

[Objective] This study was conducted to change the situation of high cost in walnut grafting by reducing seedling cost through technical means.[Method] Effects of different rootstock processing methods and rootstock pruning time on walnut growth and cost were studied by the square bud grafting method.[Result] The survival rate and cost under stumping were higher than those under no stumping,and the proportion of grafting-capable rootstocks under stumping was lower than that under no stumping by 9%.The cost differences between different rootstock time were remarkable.The cost for the stock pruning 15 d after grafting was 5 times as much as 1 d after grafting.[Conclusion] Direct grafting could achieve the effect of easy and convenient management,and the overall cost was lower though the survival rate was reduced to a limited extent.Sooner rootstock pruning resulted in a better effect.Pruning the rootstocks within 5 d after grafting not only could improve the quality of seedlings,but also could reduce the cost for seedling management.

Study on the Model of Excessive Staminate Catkin Thinning of Proterandrous Walnut Based on Quadratic Polynomial Regression Equation and BP Artificial Neural Network

The excessive staminate catkin thinning （emasculation） of proterandrous walnut is an important management measure for improving yield. To improve the excessive staminate catkin thinning efficiency, the model of quadratic polynomial regression equation and BP artificial neural network was developed. The effects of ethephon, gibberel in and mepiquat on shedding rate of staminate catkin of pro-terandrous walnut were investigated by modeling field test. Based on the modeling test results, the excessive staminate catkin thinning model of quadratic polynomial regression equation and BP artificial neural network was established, and it was validated by field test next year. The test data were divided into training set, vali-dation set and test set. The total 20 sets of data obtained from the modeling field test were randomly divided into training set （17） and validation set （3） by central composite design （quadric rotational regression test design）, and the data obtained from the next-year field test were divided into the test set. The topological struc-ture of BP artificial neural network was 3-5-1. The results showed that the pre-diction errors of BP neural network for samples from the validation set were 1.355 0%, 0.429 1% and 0.353 8%, respectively; the difference between the predicted value by the BP neural network and validated value by field test was 2.04%, and the difference between the predicted value by the regression equation and validated value by field test was 3.12%; the prediction accuracy of BP neural network was over 1.0% higher than that of regression equation. The effective combination of quadratic polynomial stepwise regression and BP artificial neural network wil not only help to determine the effect of independent parameter but also improve the prediction accuracy.

Studies on Powder Scattering Regularities of Male Flowers of Walnut in Xinjiang

[Objective] The aim was to find out the regularities of powder scattering about walnuts＇ male for improving walnut yield and quality. [Method] The powder was collected by the method of gravity glass slide, and the regulation of powders scattering of ＇Xinxin2＇ and ＂Wen185＇ which were the two main cultivated varieties of walnut in Xinjiang were studied. [Result] The results showed that the male inflo- rescence powder lasted for around 10 d, and centralized scatter powder was about 4 d, when walnut male at the daily mean temperature was from 20 to 24℃. Gen- erally, the large powder shedding amount was in east, south and central position of crown, in the range of 19 to 27 ℃, and the optimal temperature of powder scatter- ing was 20 to 23 ℃. [Conclusion] The pollens scattering regularities of walnut is closely related to temperature. With a favorable temperature, powder scattering per- forms outstandingly.

High Yield Cultivation Techniques of Two Fine Walnut Varieties, Qingxiang and Liaohe 1

Since the introduction of Qingxiang and Liaohe 1 in 2012, both varieties have shown characteristics of beautiful nuts appearance, high quality and high yield of nuts, strong disease resistance after six years regional varieties. Comparison shows that Qingxiang has strong tree vigor, making it suitable for loose planting, needs pollination tree, bears food late and shows very stable yield performance, while Liaohe 1 has medium tree vigor, bears food early and shows stable yield performance. Based on years of regional planting tests, suggestions were proposed to the high yield cultivation techniques of walnut.

New Walnut Cultivar “Ludian-dama No.1”

＂Ludian-dama No.1＂ is a late frost-avoiding new walnut varieties bred from seedling in cold region of Yunnan Province in the northeast of Yunnan Ludian County. The new variety had oval nut, circled base of fruit and lightly sharp top fruits with the average single fruit weight of 14.44 g, and the shell engraved a large shallow with wrinkle surface. The suture line was protuberant and tight, and the shell thickness was 0.89 mm. The inner fold wall had paper quality, and diaphragm was well-developed. The kernel rate was 51.2% with rough fat content of 74.67%, protein content of 18.5%. And it was easy to peel off the kernel, which was large and full with delicate taste. ＂Ludian-dama No.1＂ passed the examination and approval of the Forest Variety Approval Committee of Yunnan Province in December, 2014.

Adsorption of Cr(Ⅵ) in Water with Phosphoric Acid Modified and Ordinary Walnut Shells

A comparison between the effects of ordinary walnut shell and phosphoric acid modified walnut shell on adsorption of Cr（Ⅵ） was carried out. The experimental results showed that owing to larger surface void of modified walnut shell its adsorption of Cr（Ⅵ） was better. When the temperature was 35 ℃, adsorbent particle size was 1.0-1.6 mm, shaker shock rate was 200 r/min, and dosage of walnut shell was 0.80 g, the Cr（Ⅵ） removal rate reached 99.4%. The fitting of adsorption isotherm and kinetics model showed that, Langmuir isotherm model could reflect the adsorption process of modified walnut shell; and both the adsorption processes of ordinary and modified walnut shells accorded with the pseudo-second-order kinetic equations.

Toxicity and Antifeedant Activity of Extracts from Walnut Green Husk against Wheat Aphid

[Objective] The research aimed to study the toxicity and antifeedant activity of walnut green husk extract on wheat aphid. [Method] Using ultrasonic extraction, 5 kinds of different polarity solvents were used to prepare the extracts from walnut green husk. The toxicity and antifeedant activity on wheat aphid were determined, [ Result ] Walnut green husk extracts in the solvent of ethanol, acetone, ethyl acetate had higher toxicity and antifeedant activity on wheat aphid. When the concentration was 100 mg/ml, after 24 h, the toxicity and antifeedant activity of three kinds extracts on wheat aphid were greater than 84.35% and 66.98%, respectively. After 48 h, the toxicity and antifeedant activity were greater than 85.91% and 79.34%, respectively. Among 5 kinds extracts,ethanol extracts had the highest toxicity and antifeedant activity on wheat aphid. After 24 h, the corrected mortality rate was 93.64% and the antifeedant rate was 83.61%. After 48 h,the corrected mortality rate was 94.95% and antifeedant rate was 92.21%. Petroleum ether extract had the smallest toxicity and antifeedant activity on wheat aphid. After 24 h,the corrected mortality rate and antifeedant rate were 19.54% and 34.65%, respectively. After 48 h,the corrected mortality rate and antifeedant rate were 20.13% and 39. 03%, respectively. [ Conclsion] The extracts from walnut green husk with larger polarity solvent had better toxicity and higher antifeedant activity on wheat aphid.

Development of Walnut EST-SSR Markers and Primer Design

[Objective] This research aimed to develop walnut EST-SSR markers and design corresponding primers.[Method] 5213 EST sequences of walnut（Juglans regia Linn.） public online in NCBI were used for character analysis with bioinformatics methods,and primers were designed for the selected EST sequences by using Primer 3.0 software.[Result] 207 SSRs were obtained from the EST sequences,including 188 non-redundant sequences,the detection rate was 3.97% with an average distribution distance of 21.12 kb.Totally 92 types of repeat motifs were involved,which were mainly composed of dinucleotide and trinucleotide,accounting for 31.40% and 35.27% of the total number of repeat motifs,respectively.30 pairs of primers were initially selected from the 50 randomly-selected SSR primers by PCR amplification.[Conclusion] This research would lay foundations for the development of EST-SSR molecular markers in walnut and design of the targeted EST-SSR primers by mining and analyzing the SSR sites in walnut EST sequences.

Optimization of Conditions for the Extraction of Melanin from Walnut Husk

[Objective] This study was conducted optimize the conditions for the ex- traction of melanin from walnut husk. [Method] Melanin was isolated from walnut husk through alkali extraction and acid precipitation, and the factors that may influ- ence the extraction efficiency： temperature, duration, NaOH concentration and solid- liquid ratio were set at different levels. [Result] The optimal conditions for the ex- traction of melanin from walhut husk were temperature of 80 ℃, extraction duration of 120 min, NaOH concentration of 70 mol/L, and solid-liquid ratio of 1：20. [Conclu- sion] The optimal conditions for the extraction of melanin from walnut husk deter- mined in the present study will provide references for the development and utiliza- tion of natural melanin.

English and Black Walnut Phenolic Antioxidant Activity <i>in Vitro</i>and Following Human Nut Consumption

Background: Walnut consumption may reduce the risk of cardiovascular disease by providing antioxidant protection to low density lipoproteins (LDL). Aim: This study compared the phenolic profile and antioxidant activity of English ver- sus black walnuts. Methods: Nuts were extracted in methanol or acetone prior to analysis with HPLC/LC-MS-MS for phenolic identification and quantitation. The ability to prevent oxidation of LDL was examined in vitro using walnut extracts and ex vivo after walnut consumption for 28 days. Results: Flavonoids identified/quantified with HPLC/LC- MS-MS included the phenolic acids 5-caffeoylquinic acid, 3-caffeoylquinic acid (black walnut only), 4-caffeoylquinic acid, and the flavonol glycosides quercetin-3-rutinoside, quercetin-3-galactoside, quercetin-3-pentoside, quercetin-3- arabinoside, quercetin-3-rhamnoside, and the aglycone quercetin (English walnut only). Total phenolic yield of acetone extracts were 166.1 and 24.2 μg/g for English and black walnut respectively, and yield for methanol extracts were 147.6 and 4.1 μg/g for English and black walnut respectively. In vitro LDL oxidation by Cu++ with English walnut ex- tracts significantly extended oxidation lag-time (A234) in a dose dependent manner at 1.0 and 0.1 μg/ml and reduced TBARS formation (1.0 μg/ml). Black walnut extracts reduced TBARS significantly but had no effect on A234. Human consumption of English or black walnuts (30 g nuts/day) for 28 days resulted in no differences in LDL antioxidant ca- pacity (A234) between groups or within groups. Conclusion: This study suggests that the English walnuts have a pheno- lic profile and in vitro antioxidant capacity that is better than black walnuts, but that walnut consumption for 28 days does not improve LDL resistance to oxidation.

Chemical, Functional and Pasting Properties of Wheat (<i>Triticumspp</i>)-Walnut (<i>Juglansregia</i>) Flour

Walnut (Juglansregia) seeds are edible rich source of several essential nutrients which can be processed into flour for several purposes. Composite flour blends (wheat/walnut flour) in the ratio of 100:0, 90:10, 80:20, 70:30, and 50:50 were produced and analyzed for proximate composition, functional properties and pasting properties using standard techniques. Proximate composition results indicated increased level of protein and fat (12.17% - 25.70% and 2.40% - 37.57%) respectively while carbohydrate decreased (63% - 19.4%) with increasing level of substitution with walnut flour. Functional analysis result indicated a decreasing level of bulk density and water absorption capacity (0.78% - 0.52% and 5.17% - 4.03%) respectively while swelling capacity increased (2.00% - 7.53%) with increasing level of walnut flour substitution. Pasting properties data indicated a decreasing level of pasting viscosity (92.69 - 42.30), trough (59.19 - 39.60) RVU, breakdown (33.55 - 2.92) RVU, final viscosity (252.09 - 95.51) RVU, setback (192.85 - 55.93) RVU with increasing level of walnut flour substitution. Results showed that supplementing wheat flour with walnut flour considerably improved the protein and fat content of the flour, hence they can find useful application for making of pastries like cakes and other snacks.