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2018 Vol. 33, No. 10

Crop Science
Development of Thermo-Sensitive Male Sterile Rice with CRISPR/Cas9 Technology
WU Ming-ji, LIN Yan, LIU Hua-qing, CHEN Jian-min, FU Yan-ping, YANG Shao-hua, WANG Feng
2018, 33(10): 1011-1015. doi: 10.19303/j.issn.1008-0384.2018.10.001
Abstract:
Breeding of photoperiod or thermo-sensitive male sterile line is the key for the development of two-line hybrid rice. The allele of tms5 is the major TGMS genetic resource for two-line hybrid rice breeding in China. Targeted editing on TMS5 of fertile rice using CRISPR/Cas-9 technology could effectively create thermo-sensitive genic male sterile rice. This study used the approach to edit the TMS5 gene successfully. The CRISPR/Cas-9 vector of TMS502 was constructed, then the T-DNA was transformed into GH89. Ten transgenic plants were thus regenerated, with 6 of them containing either the insertion or deletion mutations. The T0 plant of tms5-l was a homozygous mutant, and tms 5-4 a bi-allelic mutant. Both of them displayed thermo-sensitive male sterile phenotype. There were 4 homozygous mutant genotypes in the T1 generation of the non-transgenic plants. The mutants showed the same characteristics as shown in the T0 generation without any new mutations. Male fertility of T2 generation of tms5 mutants was tested in natural high temperature environment and in phytotrons at 3 temperature levels. The results indicated that the critical sterility-inducing temperature of the tms5 sterile lines was approximately 24 C, which satisfied the requirements for two-line male sterile line breeding. The successful conversion of GH89 line into a two-line male sterile rice using the gene editing technology suggested that CRISPR/Cas-9 could be applied to facilitate the development of two line hybrid rice breeding in the future.
Principal Component Analysis and Cluster Analysis of 39 Spring Soybean Germplasm Resources
LIN Wen-lei, LÜ Mei-qin, LI Ming-song, KANG Rong-rong, ZENG Hong-ying, YAO Wen, CAI Jin-ling
2018, 33(10): 1016-1022. doi: 10.19303/j.issn.1008-0384.2018.10.002
Abstract:
7 major agronomic traits of 39 soybean germplasm resources were analyzed with the method of principal component analysis(PCA), and two principal component factors were obtained. The first principal component(MF1) was positively correlated with plant height, bottom pod height and the number of main stem segments. The second principal component(MF2) was positively correlated with the number of grains per plant and the grain weight per plant, and negatively correlated with the number of 100-grain weight and effective branches. The cluster analysis were studied through the integrated principal component values and the agronomic trait normalization data. 39 germplasms were divided into 3 major categories and 2 sub-categories through the former method, and divided into 3 major categories and 4 sub-categories through the latter method. By comparison, the analysis results of the two clustering methods were generally consistent.
Horticultural Science
Agronomic Effects of Recommended NPK Fertilization on Vegetable Farming
ZHANG Ming-qing, LI Juan, ZHANG Zan-de, YAO Bao-quan, XU Wen-jiang, SHEN Jin-quan
2018, 33(10): 1023-1029. doi: 10.19303/j.issn.1008-0384.2018.10.003
Abstract:
To promote proper fertilization for vegetable farming in open fields, the application rates of NPK fertilizers were studied according to their agronomic benefits on the plants. The 290 field experiments conducted in recent years on leaf mustard (Brassica juncea), Chinese cabbage (Brassica rapa), headed cabbage (Brassica oleracea), lettuce, broccoli, green soy bean, areca taro (Colocasia esculenta), boilen taro (Colocasia esculenta L. Schott.), and carrots allowed a classification on soil fertility using the systematic cluster analysis and significance differentiation test, as well as a proposed recommendation on the fertilization using the ternary non-structural fertilizer efficiency model. The coefficients of variation on vegetable yields were 26.0%-60.2% for the reference and 13.2%-50.3% for a balanced fertilization indicating significant differences on the effect of soil fertility among the various vegetables. For the further experimentation, 4 levels of fertility for the headed cabbages and 3 levels for the remaining 8 vegetables were applied to ensure a significance on the fertilization effect. It was found that all 28 ternary fertilizer efficiency models obtained were typical, and therefore, 9 formulas for the fertilization based on agronomic effect were established. In the following field tests, 14 of them confirmed that the recommended NPK fertilization resulted in an average increase on yield of boilen taros, headed cabbages, green soy beans, and areca taros ranged from 6.8% to 10.6%with a net increasing on revenue of 692 Yuan to 3 834 Yuan per hm2 over the conventional fertilization.
Genetic Diversity of Vegetable Daylily Germplasms in China
ZHENG Jia-zhen, LI He-ping, LAI Zheng-feng, HONG Jian-ji, WU Song-hai
2018, 33(10): 1030-1038. doi: 10.19303/j.issn.1008-0384.2018.10.004
Abstract:
The genetic diversity of 28 vegetable daylily germplasms in China were studied based on 19 morphological characteristics as well as the start codon targeted polymorphism(SCoT) markers of the plants. The agronomic analysis showed that the variations on 10 quantitative indices among the germplasms were large with the variation coefficients ranging from 14.5% to 37.2%. The highest coefficient was found on tiller number, followed by leaf width at 35.5%, while leaf length being the lowest. The results obtained from the molecular marker clustering were similar, and both methods classified the germplasms into two classes.However, the SCoT technique clustered the 3 newly introduced germplasms into Class Ⅰ (red flower) and could satisfactorily distinguish Wu Yue Hua from Liu Yue Hua while the morphological approach failed to do so. It appeared that the utilization of SCoT markers was faster, more sensitive and accurate than that of the agronomic properties for the identification of the daylily cultivars among various germplasms.
Secondary Metabolites in Leaves of Purple-leaf Plum and Red-leaf Peach
WANG Xiao-an, ZHOU Dan-rong, PAN Shao-lin, FANG Zhi-zhen, JIANG Cui-cui, LIN Yan-juan, YE Xin-fu
2018, 33(10): 1039-1042. doi: 10.19303/j.issn.1008-0384.2018.10.005
Abstract:
Contents of secondary metabolites, such as anthocyanins, chlorophyll, flavonoids, and soluble sugars, as well as the POD and PAL activities in the leaves of purple-leaf plum and red-leaf peach during growing stages were determined. The results showed that the changes in the contents of anthocyanins, chlorophyll, flavonoids, and total soluble sugars, as well as the anthocyanins/chlorophyll ratio, of both varieties were characterized by a decline-rise-decline pattern. The anthocyanin content peaked in September and April. Whereas, the activities of POD and PAL decreased initially and followed by an increase. The anthocyanin content and POD and PAL activities significantly correlated to the soluble sugar content in the leaves. However, no significant correlation between chlorophyll or flavonoids and the enzymatic activities was observed.
Effect of Substrates Containing Hypsizygus marmoreus Discards on Growth of Lettuce
WANG Tao, LEI Jin-gui, CHEN Yong-kuai, LIN Bi-ying, HE Jin-feng, HUANG Yu-yan
2018, 33(10): 1043-1048. doi: 10.19303/j.issn.1008-0384.2018.10.006
Abstract:
Effect of Hypsizygus marmoreus discards in a peator vermiculite culture substrate on the growth of lettuce (CX012) was studied in a greenhouse. Five mixing ratios of the composite substrate were formulated for the seedling cultivation. The results showed that, when a 3:1 volumetric ratio of mushroom discards and vermiculite medium was used, the seedlings performed significantly superioron leaf size, plant height, stem girth, soluble sugars and chlorophyll contents. The effects were extremely significantly different from those of control.
Effect of Restricted Rooting on Greenhouse Cultivation of Nectarine
WANG Peng, HAN Juan, GUO Shu-mei, NIU Zhen-fu
2018, 33(10): 1049-1053. doi: 10.19303/j.issn.1008-0384.2018.10.007
Abstract:
Effect of cultivation of nectarine with restricted rooting in greenhouse on the plant characteristics was studied. Three treatments on root growth, i.e., circular root-restriction (TA), bilateral root-restriction (TB), and container root-restriction (TC) along with a cultivation without restriction (CK), were imposed on the plants of two cultivars, Freestone Sweet and Zhongyou 13.The results showed that the restricted rooting treatments had a limiting effect on the plant height or crown width. However, they resulted in higher soluble solids and chlorophyll contents and a greater leaf stomatal conductance of the plants than those of CK. The number of new shoots, percent branch formation, and fruit setting rate of the treated plants were lower than those of CK. Compared with CK, TA and TB produced more flowers per branch, while TC significantly reduced the length, thickness and area of the leaves. In all, TB significantly restricted the extension on the plant height and crown width of the two cultivars but maintained similar measurements on the length, width, thickness and area of leaves to those of CK.It enhanced the flower bud differentiation, while exerted no significant effect on the fruit setting rate, yet increased the number of fruits as compared to CK.Thus, the bilateral root-restriction appeared to be an appropriate maneuver on rooting to improve nectarine plant growth in a greenhouse.
Animal Science
SYBR Green Ⅰ RT-qPCR Assay for Mycoplasma ovipneumoniae Detection
LIN Yu-sheng, JIANG Jin-xiu, ZHANG Jing-peng, YOU Wei, HU Qi-lin
2018, 33(10): 1054-1058. doi: 10.19303/j.issn.1008-0384.2018.10.008
Abstract:
A SYBR Green Ⅰ RT-qPCR assay was established for detecting the pathogenic Mycoplasma ovipneumoniae in sheep.Using Beacon Designer 7.9 software, a pair of specific primers based on the P80 gene of the M.ovipneumoniae strain Y-98 in Genbank (accession number. No.KM435069.1) was designed for the experiment. The result showed that the new methodology could specifically detect the target pathogen but not any others from sheep or goats. The detection limit was 10 copies·μL-1, and the Ct of intra-and inter-variation all below 2%. The assay method was subsequently applied on 96 clinical samples to yield a positive rate of 67.7% (i.e., 65 out of 96), which was as sensitive as TaqMan RT-PCR. It suggested that the newly established methodology was high on sensitivity, specificity and reproducibility, and applicable for the diagnosis and epidemiologic studies on M.ovipneumoniae.
Application of Fermented Silage for Pigs at Growing Stage
CHEN Bing-dian, ZHOU Wen-yi, LI Yong-ning, LIU Jing, CHEN Xin-zhu
2018, 33(10): 1059-1062. doi: 10.19303/j.issn.1008-0384.2018.10.009
Abstract:
The applicability of a fermented silage material for raising pigs at growing stage was studied. One-hundred-twenty healthy Duroc×Landrance×Rongchang crossbred piglets with an average body weight of 25.37±0.74 kg were randomly divided into 4 groups. Each triplicate group consisted of 10 piglets, which were fed with feed containing 0% (Control), 0.5% (T1), 1.0% (T2) or 2% (T3) of the fermented silage for 7 days in the pre-testing and 30 days in the experimentation feeding with a duplicate run. The results showed that the piglets in T2, as compared to Control, significantly improved the growth performance (P < 0.05) with an increase on the average daily weight gain by 17.66% and a reduction on the feed conversion ratio by 14.61% (P < 0.05), as well as an increase on the Lactobacillus count by 21.26% and a decline on Escherichia coli by 16.02%in the rectum of the piglets (P < 0.05). Meanwhile, the apparent digestibility on total energy, dry matters, crude protein, and crude fiber for the piglets also improved significantly (P < 0.05). It was concluded that a 1% addition of the fermented silage to the feed could enhance the growth performance, desirably regulate the intestinal flora, and improve the absorption and metabolism of nutrients for the piglets.
Resources and Environmental Science
Effects of Fertilization on Carbon Emission and Iron Content in Soil at Jasmine Flower Garden in Fuzhou
LIU Xu-yang, CHEN Si-cong, CHEN Xiao-xuan, CHEN You-yang, JIN Qiang, WANG Wei-qi
2018, 33(10): 1063-1070. doi: 10.19303/j.issn.1008-0384.2018.10.010
Abstract:
Effects of fertilizer application on the carbon emission and iron content in soil of a jasmine flower garden were investigated at Difengjiang Jasmine Garden in Fuzhou. Based on the application normally used by the trade, 4 levels of fertilization were implemented, i.e., control (C), normal (N), of N (H), and 2x of N (D). The results showed that the average hourly CO2 fluxes at various lots were(430.88±142.06) mg·m-2 for C, (473.08±52.18) mg·m-2 for H, (435.23±61.21) mg·m-2 for N, and (478.75±118.64) mg·m-2 for D. The average CH4 emission rateswere (38.84±9.69) μg·m-2·h-1 for C, (16.77±10.71) μg·m-2·h-1 for H, (4.11±4.79) μg·m-2·h-1 for N, and(11.92±2.27) μg·m-2·h-1 for D. Thus, the CO2 flux increased by 9.7% for H, 1.01% for N, and 11.10% for D over control, while the CH4 flux reduced by 56.82% for H, 89.41% for N, and 69.30% for D over control. The average total Fe contents were(9.11±0.08) g·kg-1 for C, (7.87±0.09) g·kg-1 for H, (9.37±0.25) g·kg-1 for N, and (8.90±0.21) g·kg-1 for D; theaveraged Fe2+ content, (1.08±0.01) g·kg-1 for C, (1.08±0.07) g·kg-1 for H, (1.01±0.09) g·kg-1 for N, and(1.09±0.04) g·kg-1 for D; and, while the average Fe3+ content, (8.04±0.08) g·kg-1 for C, (6.80±0.15) g·kg-1 for H, (8.37±0.19) g·kg-1 for N, and(7.82±0.22) g·kg-1 for D. The total Fe contents decreased by 13.61% for H and 2.30% for D, while increased by 2.85% for N over control. The Fe2+ content decreased by 6.48% for N; and, Fe3+ contents increased by 4.10% for N, and decreased by 15.42% for H and 2.73% for D over control. A correlation analysis indicated that the carbon emission fromthe lots treated with C, N, and D significantly inversely correlated with the Fe3+ and total Fe contentsat(P < 0.01), whereas H, at P < 0.05.
Nitrous Oxide Emission Estimation of Fujian Agricultural Activities Based on the IPCC Methods
WANG Cheng-ji, LI Yan-chun, LIU Cen-wei, WANG Yi-xiang, LUO Tao, HUNG Yi-bin
2018, 33(10): 1071-1077. doi: 10.19303/j.issn.1008-0384.2018.10.011
Abstract:
Farmland and livestock operations are the major sources of environmental nitrous oxide (N2O) emission generated from agricultural activities. Using the IPCC method, emission data were estimated and analyzed for 2005, 2010 and 2015 aiming to mitigate the pollution in Fujian. The N2O emissions from the agricultural activities in the province were 34 100 t in 2005, 33 500 t in 2010, and 32 900 t in 2015. Compared to 2005, they were 3.52% in 2015 and 1.76% in 2010 less, and the reductions on farmland emission were 1.67% in 2015 and 1.00% in 2010, while those on livestock emission, 16.67% in 2015 and 7.14% in 2010. It appeared that the N2O emission from farmlands was affected by the crops grown, soil conditions and fertilization practices on the land as well as the climate and resident population in the area, with nitrogen fertilizer application being the pivotal factor. For the livestock industry, optimizing husbandry structure, expanding operation scale, selecting appropriate breed, adopting advanced technology, and/or upgrading waste treatment program could benefit the pollution abatement effort.
Spatiotemporal Distributions of Nitrifying and Denitrifying Bacteria in Soil of Minjiang Estuary Wetland
CHEN Li-hua, LÜ Xin, LIU Lan-ying, HUANG Wei, LI Yue-ren
2018, 33(10): 1078-1083. doi: 10.19303/j.issn.1008-0384.2018.10.012
Abstract:
Nitrifying and denitrifying bacteria are the microorganisms that carry out the two major nitrogen removal processes in nature. The spatial and temporal characteristics of these microorganisms in soil on Minjiang estuary wetland were studied using the method of most probable number. The count on the nitrifying bacteria was shown a decreasing trend with increasing depth on vertical distribution, whereas, that of the denitrifying bacteria the opposite. In terms of seasonal changes, both bacteria were greater in numbers in spring and autumn than in summer. The Pearson's correlation analysis showed no significant correlation between the bacterial counts and nitrogen contents (i.e., TN, NO3--N and NH4+-N). However, the count of nitrifying bacteria significantly correlated with that of denitrifying bacteria(P < 0.01), and NO3--N inversely correlated with NH4+-N (P < 0.05).
Effect of Nitrogen Fertilizers on Pb and Cd Uptake of Rice Plants and Associated Food Safety Lssues
LIU Lan-ying, HUANG Wei, CHEN Li-hua, LÜ Xin, TU Jie-feng, SHANGGUAN Liang, XIE Ya-xing
2018, 33(10): 1084-1089. doi: 10.19303/j.issn.1008-0384.2018.10.013
Abstract:
Effect of nitrogen fertilizers on the uptakes of Pb and Cd by rice plants was studied. A pot experiment was conducted using amide, ammonium or nitrate nitrogen fertilizer to determine the responding soil properties, heavy metals bioavailability in rhizosphere soils, and metal uptakes by the plants. The heavy metal contaminations in different parts of a rice plant were evaluated by the pollution indices, and the food safety risk by an assessment model. The results showed that the pH in soil was lowered by the application of ammonium fertilizer, while raised by that of nitrate, as compared to amide. Among the three different nitrogen fertilizers, ammonium introduced significantly less Pb and Cd in the soil, as well as in the rice plants. The risk assessment on the rice did not raise a concern on Pb with ammonium but suggested its use to minimize Cd contamination.
Distribution Characteristics of Soil Physical and Chemical Properties Under Different Vegetations in Red Soil Erosion Area
ZHAO Zhuang, ZOU Shuang-quan, HUANG Zhao-chang, WANG Xiao-peng, HU Kun, ZHU Qiao-lian, TONG Chen-xiao, MAO Yan-ling
2018, 33(10): 1090-1096. doi: 10.19303/j.issn.1008-0384.2018.10.014
Abstract:
The soil collected from 6 types of vegetation inclued Podocarpus macrophyllus, Yulania denudate, Acer palmatum 'Atropurpureum', Camellia japonica, Armeniaca mume, Nageia nagi were studied and their soil physical and chemical properties were analyzed, in order to study the distribution characteristics of soil physical and chemical properties during the restoration process of ecosystem in eroded red soil region. The results showed that (1) Soil bulk density had a significant or extremely significant level of negative correlation with maximum water holding capacity, capillary water holding capacity, minimum field water holding capacity, non-capillary pores and total pores. Camellia japonica had the largest bulk density, poor soil porosity and water holding capacity, and the soil bulk density of Armeniaca mume and Nageia nagi cypress was small, and the soil pore and water holding capacity were better. (2) The soil pH ranged from 4.60 to 5.20, which was acidic. Nageia nagi soil had the lowest pH and the strongest acidity. The 0-20 cm soil layer of Armeniaca mume had the highest pH and the weak acidity. (3) The vegetation had an effect on the accumulation of soil nutrients. The soil organic matter content decreased with the increase of soil depth, and the range was 2.27-20.02 g·kg-1. There was a significant positive correlation between total nitrogen and organic matter content. Yulania denudata soil showed higher phosphorus content, and available potassium was mainly concentrated in surface soil. (4) Analysis of soil comprehensive fertility index values showed that the soil quality collected from Yulania denudata was the highest, and the recovery effect was also the best. The physical and chemical properties of soil collected from 6 types of vegetation showed obvious differences. Yulania denudata was the dominant vegetation variety to improve soil structure and enhance soil fertility.
Utilization of Municipal Sludge Passivated by Coal Ash on Yellow Garden Soil
WANG Shu-min, CHEN Neng-hai, LIU Xiang-guo, FU Ying, LIU Hong
2018, 33(10): 1097-1103. doi: 10.19303/j.issn.1008-0384.2018.10.015
Abstract:
Utilization of the ash generated from coal-fired power plants to reduce heavy metal content in municipal sludge was investigated.By using the coal ash to passivate heavy metals in the sludge to be added to yellow garden soil for a pot experiment, the effect on the growth of Ruellia Brittoniana was monitored. The results showed that the coal ash significantly reduced the heavy metals, especially available Fe and Mn, in the sludge. The passivation effect was most significant when the ratio of municipal sludge to coal ash was 1 to 2 in an 8-hour treatment. By comparison, the phosphor-gypsum addition showed no significant passivating effect on the sludge. The coal ash passivated sludge significantly increased the contents of nitrogen, phosphorus, potassium, and microelements in the gardensoil. Meanwhile the plants grown on the composite soil significantly increased on total nitrogen. Among all mixing ratios, the mixture of equal parts of soil and the passivated sludge allowed the greatest amount of dry matters accumulated in both the above-and underground parts of R.brittoniana, and consequently, was deemed appropriate for urban gardening applications.
Selection of Carrier for Dried Bacillus natto
CHEN Long-jun, LIN Chen-qiang, ZHANG Hui, CHEN Ji-chen
2018, 33(10): 1104-1107. doi: 10.19303/j.issn.1008-0384.2018.10.016
Abstract:
Preparation of a dried Bacillus natto product was studied. The carrier material, mixing ratio, and drying temperature for the making were evaluated according to the survival rate of B. natto in the dried product. The optimal process was determined to be stirring the bacterial solution with quartz monzonite as the adsorbent carrier in a mixing ratio of 1:1 at 150 r·min-1 and 37℃ for 60 min in a container prior to dehydration at 40℃. The dried product had a bacterial count of 1.09×109 CFU·g-1. After 6-month storage at 25℃, the count decreased by 20.2% to 2.9×108 CFU·g-1. Inclusion of wheat bran, soymeal and/or corn meal in the mix would provide nutrients for B. natto resulting in aprolonged shelf life of the dried product.
Food Science
Effects of Different Drying Methods on the Nutritional and Bioactive Components of Radix Puerariae Residue
LAI Pu-fu, LI Yi-bin, WU Li, WENG Min-jie, TANG Bao-sha, CHEN Jun-chen
2018, 33(10): 1108-1112. doi: 10.19303/j.issn.1008-0384.2018.10.017
Abstract:
In this study, the major nutritional component, bioactive component and amino acid composition of Radix puerariae residue were monitored after hot air drying and vacuum freeze drying to unravel the effects of different drying methods on the nutritional and bioactive component. The results were as followed:the starch content of Radix puerariae residue in vacuum freeze drying (53.6%) was significantly higher than that in hot air drying (47.0%), but the protein, reducing sugar and ash content were significantly lower than those in hot air drying, no significant difference of the fat, sucrose, total flavonoids, puerarin and polysaccharides content was found between hot air drying and vacuum freeze drying. And the dietary fiber, crude fiber, lignin, total amino acids, essential amino acids, non-essential amino acids contents of Radix puerariae residue in hot air drying were significantly higher than those in vacuum freeze drying. In comprehensive consideration of determination results of various nutritional and bioactive composition of Radix puerariae residue, hot air drying is suitable for processing Radix puerariae residue in order to keep its quality with the cost of production considered.
Improvements on Melt-Resistance and Sensory Quality of Yogurt Ice Cream by Using Xanthan Gum, FJAT-10151-DTJZ
ZHENG Mei-Xia, ZHU Yu-Jing, LIU Bo, CHEN Zheng, HUANG Su-fang
2018, 33(10): 1113-1118. doi: 10.19303/j.issn.1008-0384.2018.10.018
Abstract:
The overrun, melt-resistance, hardness, dynamic viscoelasticity, microstructure, and sensory quality of the yogurt ice cream products emulsified with a commercial or FJAT-10151-DTJZ xanthan gum were compared. FJAT-10151-DTJZ was extracted in our lab for the experiment. Both ice cream products were pseudoplastic exhibiting the characteristic shear-thinning property. However, the product using FJAT-10151-DTJZ increased the resistances to melting and overrun, retarded ice crystal formation, and improved the smoothness and softness in texture.
Optimized Extraction of Functional Ingredients from Callicarpa nudiflora Hook.et Arn.by Box-Behnken Design
HUANG Mei, YU Fu-lai, TAN Xiang-jie, CHEN Zhen-xia, PANG Yu-xin, HU Xuan, LI Wei
2018, 33(10): 1119-1124. doi: 10.19303/j.issn.1008-0384.2018.10.019
Abstract:
To optimize the extraction of functional ingredients from the leaves of Callicarpa nudiflora Hook. et Arn., an experiment based on the Box-Behnken design was conducted. The extraction rate and contents of functional ingredients including flavonoids, phenolic acids, and saponins in the resulting extracts were weighed against the method, duration, cycle, material to solvent ratio, and ethanol concentration of the process. The weighted value for evaluation was calculated by the formula of(dry weight extract 0.7+ combined weight of functional ingredients 0.3). The optimized conditions included the application of ultrasound for 42 min, a material:solvent ratio of 1:17, the use of 62% ethanol for solvent, and two extraction cycles to arrive at a weighted value of 119.27, which was close to the predicted value of 120.47. The established extraction process was simple and applicable with desirable reproducibility and predictability.