Sulfite gold plating bath composition and plating condition.
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Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 179 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 252 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\n'}],latestNews:[{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"}]},book:{item:{type:"book",id:"3205",leadTitle:null,fullTitle:"Design of Experiments - Applications",title:"Design of Experiments",subtitle:"Applications",reviewType:"peer-reviewed",abstract:"This book is a research publication that covers original research on developments within the Design of Experiments - Applications field of study. 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Milk processing allows the preservation of milk for days, weeks, or months and helps to reduce food-borne illness.",isbn:"978-1-78985-730-6",printIsbn:"978-1-78985-729-0",pdfIsbn:"978-1-78985-919-5",doi:"10.5772/intechopen.73442",price:119,priceEur:129,priceUsd:155,slug:"milk-production-processing-and-marketing",numberOfPages:202,isOpenForSubmission:!1,hash:"d0b383fbc5e2a2fcc9da5bd58766529d",bookSignature:"Khalid Javed",publishedDate:"July 17th 2019",coverURL:"https://cdn.intechopen.com/books/images_new/6911.jpg",keywords:null,numberOfDownloads:6071,numberOfWosCitations:0,numberOfCrossrefCitations:1,numberOfDimensionsCitations:3,numberOfTotalCitations:4,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"July 12th 2018",dateEndSecondStepPublish:"August 2nd 2018",dateEndThirdStepPublish:"October 1st 2018",dateEndFourthStepPublish:"December 20th 2018",dateEndFifthStepPublish:"February 18th 2019",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:"Edited by",kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"136829",title:"Dr.",name:"Khalid",middleName:null,surname:"Javed",slug:"khalid-javed",fullName:"Khalid Javed",profilePictureURL:"https://mts.intechopen.com/storage/users/136829/images/system/136829.jpeg",biography:"Dr. Khalid Javed is a Professor of Animal Breeding and Genetics in the Department of Livestock Production at the University of Veterinary and Animal Sciences, Lahore. He graduated in Animal Husbandry from University of Agriculture, Faisalabad in 1982. He earned his Master’s and Doctorate degrees in Animal Breeding and Genetics from University of Agriculture, Faisalabad. He joined Government of Punjab, Livestock and Dairy Development as Veterinary Officer in 1983 and remained engaged in research in different capacities. Dr. Khalid conducted research, trainings and teaching in the fields of Animal Breeding, Population/Quantitative Genetics, and Statistical Genetics. He analyzed the production data of various livestock species (e.g., cattle, buffalo, sheep, goats, chicken) to characterize the phenotypic and genetic structure related to different traits of economic importance and subsequent selection. Moreover, he has been engaged in inter-disciplinary collaborative research with colleagues from various academic and research institutes to study the genetic, breeding, management and environmental factors affecting productivity of livestock species. He joined University of Veterinary and Animal Sciences during 2003 as Assistant Professor where he was later selected and appointed as Associate Professor and Professor, in 2006 and 2011 respectively. His research focus is on selection and breeding of large and small ruminants. He also supervises and evaluates postgraduate research to ensure successful and timely completion of the projects focusing on genetic improvement, enhancing breeding efficiency and production enhancement of farm animals. In addition, he participates and conducts trainings, workshops, conferences and seminars, and writes scientific publications to disseminate knowledge and techniques to the researchers and livestock producers about various areas of animal husbandry for improving behaviour, health, growth, fertility and production of livestock. He has more than 200 publications/research articles published and is working as Senior Editor of an internationally recognized Journal of Animal and Plant Sciences-JAPS.",institutionString:"University of Veterinary and Animal Sciences",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"2",institution:{name:"University of Veterinary and Animal Sciences",institutionURL:null,country:{name:"Pakistan"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"326",title:"Food Industry",slug:"food-industry"}],chapters:[{id:"65652",title:"Current Standing and Future Challenges of Dairying in Pakistan: A Status Update",slug:"current-standing-and-future-challenges-of-dairying-in-pakistan-a-status-update",totalDownloads:1359,totalCrossrefCites:0,authors:[{id:"270832",title:"Dr.",name:"Muhammad Naeem",surname:"Tahir",slug:"muhammad-naeem-tahir",fullName:"Muhammad Naeem 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Feynman spoke “There is Plenty of Room at the Bottom” [1]. This talk is given on 26 December 1959, at the annual meeting of the American Physical Society (APS) at the California Institute of Technology, reconsidered the importance and possibility of micro and nanotechnology in recent years. Microelectromechanical systems (MEMS) shows sensors and devices fabricated based on semiconductor microfabrication technology [2]. MEMS are usually used in pressure sensors, inkjet printers, microfluidics, etc. In recent years, product integration has increased, various problems are occurring. Electric contacts are also no exception, and here we discuss new gold plating that provides a solution to the problem. It is widely used as a contact material.
Gold was used by Chinese and Egyptians of ancient times (at least ca 3000 BC) [3, 4, 5]. For many years, gold based materials have received great attention from people, due to the good conductor, high chemical stability, unique optical and processable properties [6]. Electrodeposition technology is a long established technique for synthesizing metals on conductive substrates. The properties of the deposited film are simply controlled by their morphologies (grain size, shape, roughness, brightness). Moreover, the deposited structure depends on process parameters such as the composition, temperature and pH of electrolyte, the magnitude of applied current densities, substrate. Advances in equipment and creations of nanomaterials could carry out new technological progress, a large duty ratio with a pulse overvoltage became possible and new composite fillers (for example, carbon nanotubes: CNTs) appeared. Moreover, environmental considerations have become more important as Sustainable Development Goals (SDGs). SDGs were adopted at the United Nations Summit in September 2015 and are the goals set by the 193 member countries to achieve in the 15 years from 2016 to 2030. For the global environment and workers, friendly manufacturing methods have become more important.
In this chapter, two nanostructured golds (hard pure-gold plating and gold-CNT composite plating) are discussed. Additionally, the synthesis and characteristics of electrostatic deposition films with properties using environmentally friendly sulfite bath are discussed.
In recent years, there has been a demand for miniaturization to various parts due to an increase in degree of integration of electronic parts. Electric signal probes for inspecting semiconductor package parts have the same demand, and the terminal pitch is gradually narrowed. As a result, the mechanical contact pressure of the probe was lowered and its contact resistance increased. Technical problems have been occurred such as the device stability, and heat generation of the electrical contacts for inspecting the electronic parts. For example, at a contact pressure of about 0.5 N, the contact resistance of the probe is 30 mΩ, whereas at a contact pressure of about 0.15 N, the contact resistance reaches 100 mΩ. This Joule heating value is approximately three times as high as the original. Therefore, there is a demand for a new plating film on the probe surface which does not increase the contact resistance even with a low contact force, while maintaining the environment/durability equal to or higher than the conventional level.
Generally, the gold used as the contact point is a soft metal. However, gold plating for inspection probes has a moderate hardness and durability improved by adding trace amount of Co. In this conventional gold plating film, the specific resistance value is higher than that of pure gold, and the contact resistance associated with this value is also increased. Therefore, we focused on hard pure-gold plating technique without alloying [7]. There are reports [8, 9, 10] that can control the grain size toward small by pulse plating that repeats ON/OFF of current. The plating bath and the pulse electrodeposition method were devised to decrease the crystal size. In metals, according to Hall-Petch law [6, 11], metals are hardened by reducing crystal size. The obtained hard pure-gold plating film was applied to the probe surface [12] and its electrical contact characteristics were evaluated. Furthermore, in consideration of recent SDGs correspondence, the non-cyanide base bath has been used for a friendly environmental society.
There is a report [13] in which a sulfite complex is used for a non-cyanic plating bath. In this section, the base sulfite bath was used as a non-cyan gold plating bath. Table 1 shows the composition of the plating bath and the plating conditions.
Sodium gold sulfite | 0.05 M |
Sodium sulfite | 0.5 M |
2,2′-bipyridyl | 100 ppm |
Additive | Small quantity |
Bath temperature | 60°C |
Bath volume | 100 ml |
Average current density | 50 A/m2 |
Film thickness | 5 μm (efficiency 100%) |
Substrate | Amorphous Ni-P alloy film |
Anode | Pt-plated Ti mesh |
Agitation | Magnetic stirring |
Sulfite gold plating bath composition and plating condition.
The condition of rectangular pulse current was optimized at the average current density of 50 A/m2, the pulse period of 100 ms, and the duty ratio of 0.1. The Ni-P plated film was used as an experimental substrate (amorphous alloy as a base film) with thickness of about 3 μm on Cu plate. A plating bath manufactured by Meltex was used for plating the base film. The substrate used for the experiment was insulated with a masking tape excepting the deposition surface (10 mm × 20 mm), alkaline degreasing and acid treatment were carried out as plating pretreatment. Further, for comparison, a hard gold plating film with Co-content was similarly prepared, which was plated on the probe, used a commercially cyan bath (by Meltex: Auronal 44 BC). The characteristics of the plated films were evaluated in terms of surface morphology, Vickers hardness, X-ray diffraction (XRD), and specific resistance value, by a field emission scanning electron microscope (FE-SEM: S-4100, Hitachi), a dynamic indentation tester (DUH-201, SHIMADZU), X-ray diffractometer (RINT2200V, Rigaku), a four probe method, respectively.
Figure 1 shows the photograph of the probe with both ends moved (spring movable stroke of 0.65 mm). A prototype was fabricated by plating the developed plating film on the movable part at both ends of the probe (Cu alloy, and the Ni-P plating as base) shown in Figure 1.
Photograph of the probe with both ends moved (spring movable stroke of 0.65 mm).
The repeated durability of the contact resistance was compared and evaluated for the prototype probes (using the same parts) of the current plating film and the developed plating film. Both gold plates were used for the contact resistance measurement of the probe, and a four-terminal method was used for the electric circuit measurement. Here, the measurement was performed at room temperature (RT, the temperature: 24°C, the humidity: 50% [14]) with the probe stroke of 0.65 mm, the load of about 0.24 N, and the measuring current of 10 mA.
The three types of electrodeposited films, (a) a hard gold plating film with Co-containing (AuCo film) from a cyanide bath, (b) a direct current (DC) gold plating film (DC-Au film) from an original sulfite bath, (c) a pulsed current plating film from the same original sulfite bath (PC-Au film), were prepared and observed the surface morphology by FE-SEM are shown in Figure 2, respectively.
Surface SEM images of various Au plating films, (a) AuCo film, (b) DC-Au film, (c) PC-Au film.
Compared with the surface morphology of AuCo film from Figure 2(a), it can be confirmed that the DC-Au film has larger angular crystals (Figure 2(b)). The surface of PC-Au film (Figure 2(c)) shows the small crystals without corners by comparing with that of DC-Au film (Figure 2(b)).
Figure 3 shows the average value, the maximum value, and the minimum value of Hv obtained from three kinds of plated film surfaces by measuring 10 times at RT using a dynamic indentation tester. Here, the measurement conditions using a diamond indenter were carried out at the test load of 10 gf, the load speed of 0.675 gf/s, and the holding time of 20 s.
Vickers hardness (the average, maximum and minimum value) of various Au plating films; (a) AuCo film, (b) DC-Au film, (c) PC-Au film.
From this result, (c) PC-Au film is about twice as hard as (b) DC-Au film, which is harder. The Hv value is close to that of commercially available (a) AuCo film. It is presumed that the improved hardness of PC-Au film is due to the crystal size. Next, the crystal structure of the plating film was investigated by XRD.
Figure 4 shows the results of XRD measurement of the three plating films, (a) AuCo film, (b) DC-Au film, and (c) PC-Au film, using Cu tube (CuKα1: wavelength 1.5405 Å), at 40 kV and 20 mA.
X-ray diffraction patterns of various Au films; (a) AuCo film, (b) DC-Au film, (c) PC-Au film.
From Figure 4, gold peaks of face-centered cubic (FCC) crystal were confirmed in all the samples. The crystallite size Dhkl of the (hkl) face (Miller Index) was calculated by the Scherrer equation of the following Eq. (1) [15].
The apparent crystallite size (Da) was determined using a weighted average of the crystallite sizes from the XRD peaks on each face. In this calculation, the following Eq. (2) was used.
As a result, the values of the crystallite size (Da) from (a) AuCo, (b) DC-Au, and (c) PC-Au films were (a) 19.1 nm, (b) 28.9 nm, and (c) 17.0 nm, respectively. In the original bath, (c) PC-Au film obtained by the pulse electrodeposition has wider peak and smaller Da than that of (b) DC-Au film obtained by the direct current electrodeposition. The reason for these results is related to the fact that the critical radius rc of the crystal nucleus according to the following Eq. (3) is decreased by pulse electrodeposition with a large overvoltage (η) [16].
It is possible to form small crystal nucleus in this case. Here, γ is the intrinsic surface energy, V is the atomic capacity (the volume occupied by 1 g atom in the solid state), z is the valence, F is the Faraday constant, and η is the overvoltage. In general, for metals, the Hall-Petch rule expressed by the following Eq. (4) is established with mechanical yield strength σ [6, 11].
Here, σ0 is the intrinsic yield strength (independent of crystal size), k is the constant depending on materials, and d is the crystal size. For the metal film, a proportional relationship is established between the yield strength and the hardness Hv. Paying attention to Eq. (4), the smaller the crystal size is, the harder the metal material is.
Various samples were prepared by changing pulse conditions in the same method, and the relationship between grain size (Da) from XRD and Hv was as shown in Figure 5 (Hall-Petch rule).
Relationship between crystallite size (grain size Da) from XRD and Vickers hardness Hv; (a) AuCo film, (b) DC-Au film, (c) PC-Au film.
From the results in Figure 5, although the Hv of the pulsed electrodeposition film increases as the crystallite size decreases, the plot tends to decrease from around the critical point (Da−1/2 = 0.24). The reason for this is presumed to be the result of the inverse Hall-Petch rule [17, 18, 19] that the self-weight collapse occurs in this gold electrodeposition film when the crystallite size becomes too small. Therefore, the condition around Da−1/2 = 0.22 (D = 20.7 nm) was selected to form the plated film to obtain appropriate hardness for probe application.
In general, the four probe method of the following Eq. (5) is used as a standard method for measuring the resistivity ρ at RT [20]. Here, the variable t is the plating thickness, and the value (V/I) indicates the resistance value between the measuring terminals.
In order to measure the resistivity, the sample plated films were peeled. The resistivity ρ was calculated by the four probe method using the tungsten probe at intervals of 1 mm, and the results are shown in Table 2 (reference description: crystallite size (Da), average value of Hv). The plating thickness is obtained by averaging five points as measured by the fluorescent X-ray meter (SFT 3200, SII).
(a) AuCo | (b) DC-Au | (c) PC-Au | |
---|---|---|---|
Average of measured resistance values (mΩ) | 4.03 | 2.6 | 3.2 |
Deviation of measured resistance value (mΩ) | 0.11 | 0.06 | 0.92 |
Average plating thickness (μm) | 4.4 | 3.95 | 3.44 |
Specific resistivity ρ (10−6 mΩ cm) | 8.04 | 4.65 | 4.98 |
Average value of Vickers hardness (Hv) | 211 | 97 | 199 |
Apparent crystallite size Da (nm) | 19.1 | 28.9 | 17 |
Specific resistivity ρ of samples; (a) AuCo film, (b) DC-Au film, (c) PC-Au film (reference description: crystallite size Da, average value of Vickers hardness).
Compared to the resistivity of the commercially available (a) AuCo film, those of both (b) DC-Au film and (c) PC-Au film from the original pure gold plating bath were about 3 × 10−6 mΩ cm lower.
In a system in which elastic deformation is predominant, such as 0.1 N < P < 100 N (P is the contact load), the contact resistance R is expressed by the empirical formula of the following Eq. (6) [21, 22]. Here, ρ1 and ρ2 are the resistivity values of two materials in contact, H is the Brinell hardness, and P is the contact load.
It is suggested that the contact resistance can be lowered by the decrease of the resistivity proportional to its value. Utilizing this, the pulsed hard pure gold plating film was applied to the parts of the probe. Using two types of plated film probes, (a) AuCo film (conventional product) from commercial cyanide bath and (c) PC-Au film (fabricated product) from original bath, the results of the durability comparison during the contact resistance of 100,000 times are shown in Figure 6. In Figure 6, the vertical axis represents the contact resistance value [mΩ] of the probe; (a) AuCo plating and (c) PC-Au plating, and the horizontal axis represents every 1000 durability measurement times. The bar is maximum and minimum values at every 1000 times measurement, and the average values were plotted.
The durability about the contact resistance of 100,000 times; the probe of (a) AuCo plating and (c) PC-Au plating.
Calculating the average value of the contact resistance, (a) the resistance of the conventional product probe was 32.6 mΩ (deviation 2.07), and (c) that of the fabricated product probe was 21.6 mΩ (deviation 0.80). This fabricated probe has an advantage that the contact resistance value decreases by 11 mΩ and its variation is low, and improvement of electrical contact characteristics could be realized.
Carbon materials have attracted attention for a long time due to their characteristic structural, electronic, thermal, chemical, mechanical properties [23]. Since the discovery of carbon nanotube (CNT) [24, 25], great number of fundamental and technological research on CNT has been developed [26]. CNT is characterized by small diameter and high aspect ratio with outstanding mechanical strength, flexibility, high electrical conductivity and chemical stability [27].
For instance, material-CNT composites as a lightweight and high strength material have been actively studied [28, 29, 30, 31, 32]. Recently, CNT’s application of seawater desalination has progressed in Shinshu University and has been drawing attention [33]. In general, the hydrophilic property at the interface and the difference in specific gravity between a metal matrix and filled CNTs make it difficult to fabricate a metal-CNT composite film with uniformly distributed CNTs and a good tribological behavior. Metal-CNT composite films have been fabricated [34, 35, 36, 37], using various types of metals (Ni, Cu and Ag) by composite plating [38, 39, 40, 41, 42, 43, 44]. However, the pH levels of plating baths, being on the acidic side, are low. There are very few reports about the fabrication of a CNT composite plating film under the basic condition. Moreover, recently used industrial electric contacts, for example, a probe for inspecting parts of a semiconductor package, have encountered a drawback due to their adhesion and increase in resistivity after repeated use. Thus, a non-adhesive plating material for connector application is strongly required.
In this section, to develop a new composite material for next-generation electric contact applications, an Au-CNT composite plating film was fabricated by electrodeposition [40]. An effective additive was added into a non-cyanide CNT plating solution, affording the formation of an Au-CNT composite film in a basic solvent by an environmentally friendly method.
A vapor-grown carbon fiber (VGCF; Showa Denko) which is one type of multiwalled CNTs, was used in this study. An Au-CNT plating bath was prepared by adding 0.05 M Na3Au(SO3)2, 0.5 M Na2SO3, 100 pp. 2,2′-dipyridyl, and 0.2 g/l VGCF into ion-exchanged water. Moreover, 0.1 g/l trimethyl stearyl ammonium chloride (C21H46ClN) was added into the above solution. The mixture was stirred using an ultrasonic agitator (28 kHz) for 2 h. The volume of the plating bath was 100 cm3. The plating was performed at 60°C and pH 8 using a magnetic stirring agitator. The current density was 50 A/m2 and a Pt-plated Ti mesh was used as the anode [8]. A copper (Cu) board with an exposed area of 200 mm2 (10 × 20 mm2) was used as the substrate. The Cu substrate was plated using a Ni-P amorphous alloyed layer with a thickness of 3 mm. A Ni-P plating bath is a commercially available plating solution (Meltex). Before plating, the substrate was pretreated by alkali degreasing and acid treatment. For comparison, an Au plating film was prepared using an Au plating bath without adding CNTs. FE-SEM equipped with energy-dispersive X-ray spectroscope (EDX; JED-2300F, JEOL) was used to study the surface morphology and CNT content of the fabricated plating films. Hv was measured using a dynamic hardness tester (DUH-201, Shimadzu). A diamond indenter was vertically employed with a test load of 0.098 N, a load speed of 6.6 mN/s, and a hold time of 20 s at RT. The intrinsic resistance of the fabricated plating films was determined by a four-point probe method. Tungsten (W) probes were used with a probe spacing of 1 mm. Tribological properties were measured using a ball-on-plate-type reciprocating friction abrasion test machine (MMS-2419, Nissho-EW). A brass ball (8 mm in diameter) plated with 4.5 μm Ni-P and 1 μm hard-Au layers, was used as the counter surface. The test was repeatedly conducted in 50 cycles at a load of 0.5 N, a sliding length of 2 mm, a sliding speed of 0.5 mm/s at RT. The test was performed under ambient conditions without any lubricants. During the test, the friction coefficient was measured continuously using a load tester.
The fabricated Au-CNT composite film appeared to be relatively black. Figure 7(a) and (b) shows SEM images of the fabricated Au-CNT composite film and the Au film, respectively.
SEM images of the surface sample; (a) the fabricated Au-CNT composite film, (b) Au film.
CNTs were tangled with the Au matrix and protruded from the matrix surface. The surface morphology of the Au-CNT composite film was relatively rough owing to the existence of many voids. It was previously reported that, in the composite film, metal is easily separated at the CNT apex and defects of CNTs. Before CNTs are entirely coated, the plating process is promoted at the apex or defects of CNTs, resulting in the formation of voids in the deposited film [34]. The result of EDX analysis indicated that the content of CNTs in the Au plating film was about 4 mass %. In the plating process, CNTs could not be homogeneously dissolved in the solution without C21H46ClN even if it had been mechanically stirred for 48 h, and consequently, the plating bath could not be prepared. From the above results, C21H46ClN was found to be an effective additive for Au-CNT composite film fabrication using a non-cyanide bath, which is an environmentally friendly method.
The mechanism of the eutectoid composition of the Au-CNT composite film is assumed to be based on the deposition of CNTs by electrophoresis. Normally, the zeta potential of CNTs decreases and easily becomes negative when the solution becomes basic [45, 46]. Although CNTs are modified by N-doping or heat treatment, their zeta potential becomes negative at a basic pH of 8 [47, 48]. Since the CNTs repulsed by the cathode during reduction react, it is difficult to combine CNTs in a basic solution. At this stage, although the role of the additive has not yet been clearly elucidated, the results obtained in this study apparently show that the additive is important for Au-CNT composite film fabrication.
Figure 8 shows the VGCF zeta potential of 0.05 g/L under various potential of hydrogen (pH) in three solutions; (a) conventional dispersant of 0.5 mM in water, (b) fabricated dispersant (C21H46ClN) of 0.5 mM in water, (c) plating solution diluted 20 times.
The VGCF zeta potential of 0.05 g/L under various potential of hydrogen (pH) in three solutions; (a) conventional dispersant of 0.0005 M in water, (b) fabricated dispersant (C21H46ClN) of 0.0005 M in water, and (c) plating solution diluted 20 times.
The hydrophobicity of the additive is likely to effectively provide a positive zeta potential for CNTs. This was also evidenced in the plating process. The zeta potential of CNTs was found to be positive in the solution, and consequently, CNTs with a positive zeta potential were attracted toward the plating cathode.
Table 3 shows the Hv and resistivity of the Au-CNT composite film and the Au film, respectively. The Hv is the average measured at 10 points. In general, to measure the Hv accurately, film thickness is required to be 10 times higher than the depth of the indenter pressed into the film. Thus, it should be noted that the measured value includes the substrate effect.
Vickers hardness (Hv) | Resistivity (10−6 mΩ cm) | |
---|---|---|
Au-CNT composite film | 133 | 5.63 |
Au plating film | 95 | 4.65 |
The Vickers hardness and resistivity of the Au-CNT composite film and the Au film.
The Hv of the Au-CNT composite film and the Au film were Hv 133 and Hv 95, respectively. The fabricated Au-CNT composite film was harder than the Au film by 1.4 times.
For intrinsic resistance measurement, while the film thickness is sufficiently small compared with the probe spacing, the intrinsic resistivity ρ can be expressed by ρ = (πt/ln2)/(V/I) [20], where value t is the average film thickness measured at five points by X-ray fluorescence analysis (SFT3200, SII), and (V/I) is the average resistance of the thin film measured at three points. The resistivity of the Au-CNT composite film and the Au film were 5.6 and 4.7 × 10−6 mΩ cm, respectively. The fabricated Au-CNT composite film showed a higher resistivity than the Au film by 1.2 times. The voids and the interface between Au and CNTs are likely attributed to the Vickers hardness and the resistivity.
Figure 9 shows the friction coefficients of the Au-CNT composite film and the Au film, as a function of sliding length against Sn ball of 8 mm diameter.
The friction coefficient of (a) the Au-CNT composite film and (b) the Au film, as a function of sliding length against Sn ball of 8 mm diameter.
In the case of the Au-CNT composite film, the friction coefficient gradually decreased toward the sliding length. After the test, at a total sliding length of 200 mm (50 repeated cycles), the friction coefficient was 0.28. On the other hand, in the case of the Au film, the friction coefficient increased to 0.58 and gradually decreased at a sliding length of 130 mm. The Au-CNT composite film showed a lower friction coefficient than the Au film.
Figure 10(a) and (b) shows SEM images of the Au-CNT composite film and the Au film after the wear test, respectively.
SEM images of (a) the Au-CNT composite film and (b) the Au film after the wear test.
The worn area of (b) the Au film was relatively large compared with that of (a) the Au-CNT composite film. The track with a width of approximately 200 mm was found on the surface of the Au film. The entire surface of the worn area of the Au film was damaged, while that of the Au-CNT composite film was partly damaged. CNTs still remained in the worn area of the Au-CNT composite film, lying transversely [Figure 10(a) inset]. Adhesive wear is one of the friction phenomena. The smaller the surface area, the weaker the force of the adhesion shear [49]. The small contact area and transversely lying CNTs seem to contribute to the low friction coefficient.
To summarize, a pure-gold plated film with nano-order crystals was fabricated by devising a pulse electrolysis method using a non-cyanide bath. As a result of confirming their structure with FE-SEM or XRD, although refinement of this crystal showed an increase in hardness according to Hall-Petch rule, this law did not hold in the range smaller than around Da = 20 nm, and on the contrary it showed a decrease in hardness. By controlling a crystallite size (around Da = 21 nm) where the hardness does not decrease, the fabricated pure gold plating film without alloying maintains a moderate hardness (Hv = 199: Hv = 210 with AuCo film), its specific resistance (5 × 10−6 mΩ cm: 8 × 10−6 mΩ cm with AuCo film) than the conventional hard gold plating film. By applying the developed plating film to the probe, the contact resistance value of the repeated test could be reduced by about 11 mΩ, be achieving high performance with low variation. Conventionally, non-cyanic plating baths have lower life and stability compared to cyanide baths, and this is also a problem in the developed plating baths. However, compared to conventional AuCo plating, the fabricated hard pure gold plating has superiority in environmentality and workability with non-cyan, reliability of electric contact (reduction in contact resistance, stability of repeated variation).
In summary of the second film, an Au-CNT composite plating film was fabricated by electrodeposition. A non-cyanide Au-CNT composite plating film was successfully formed by adding an effective additive, at alkaline environment. This was presumed to relate the zeta potential. The Au-CNT composite film is advantageous over the Au plating film in terms of a high hardness and a low friction coefficient. Thus, the Au-CNT composite film along with the desired method and the precise control of the content and orientation of CNTs are expected to make CNTs as promising material of sliding electric contacts, such as connectors.
In view of the application of contact materials used for MEMS, two methods of gold plating were introduced. In the society where miniaturization is advancing, the role of electrical contacts has also grown. Gold as a contact material will become more important. As described above, the advantage and potential of the new gold plating were confirmed, but there is still room for improvement in terms of performance. In addition, issues such as economical cost, safety of the use side, system construction for sustainable society formation (recycling) remain. Technological progress in the future is strongly expected.
This work was supported by MEXT Cluster brochure (Nagano Prefecture region). We express our appreciation to Prof. Morinobu Endo of Shinshu University for his great support and fruitful discussion. We also thank to Dr. Kenji Takeuchi and Dr. Noboru Akuzawa of Shinshu University, Dr. Winadda Wongwiriyapan of King Mongkut’s Institute of Technology Ladkrabang, Prof. Feng Wang of Beijing University of Chemical Technology, for their kind advices.
Pests and diseases have always had repercussions directly on losses of crops and livestock products and indirectly over the income decreases due to insufficient harvests of commercial crops. Chemical pesticides are used in an excessive way and without prior technical assistance to pest distribute control, which instead of solving the problem, has produced strong damage to agricultural and livestock productivity, as well as important environmental effects with implications to public health [1]. Pesticides are chemical substances designed to prevent, delay, repel or fight any pests [2].
In [3], it was mentioned that Mexico ranked fifth in the world in the use of 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) in agricultural programs and fourth place for its use in public health. As many as 69,545 ton of DDT were used in health campaigns for the control of malaria and agricultural activities, from 1957 onward, DDT was applied every 6 months indoors and outdoors with a coverage of 2 g/m2, and almost 1000 ton DDT/y were used in agricultural areas [4]. Indeed, DDT was used in Mexico until the year 2000, and DDT and its metabolites have been found in the environment [5] as well as in human tissues [5, 6], breast milk [7], raw cow’s milk and bovine meat [8, 9]. DDT is a very stable organochlorine pesticide that is almost completely metabolized, but small percentage remains as o,p´-DDT, while the most of its concentration is transformed into p,p´-DDE, which is characterized for its poor solubility in water and high affinity for lipids. It is considered a pollutant of high persistence due to its half-life of up to 15 years in the environment [10]. This pesticide persistence is responsible of the wild flora and fauna deterioration, as well as the contamination of soil, water table, continental, and coastal waters. Besides, pesticides can be incorporate into pasture, vegetables, and edible animals, which when consumed, act as transporters facilitating its accumulation in living organisms [11, 12].
In the state of Chiapas, México, [13] found high levels of total DDT in outdoor soil samples that ranged from 0.002 to 27 mg kg−1, while the levels found by [4] in Chihuahua, México, ranged from 0.001 to 0.788 mg kg−1. Taking into account the guideline for total DDT in residential soil of 0.7 mg kg−1 in Canada [14], the soil samples from Chiapas had levels higher than the guideline. The high levels of OCs observed may be due to ongoing usage as well as the emission of old residues from soil. Soils are an important sink and source for persistent organic pollutants to the atmosphere. In many of the cases, the contamination of soil with pesticides is due to its incorrect storage, either by leakage of corroded tanks containing liquid pesticides or by aerial dissemination of powder pesticide. However, when pesticides infiltrate the soil, their dissemination depends on the nature of the pesticide, as well as the composition, moisture, pH, and temperature [12, 15]. Because of that, a small portion of spilled pesticides can generate a high soil contamination. Moreover, soil pesticides infiltration can cause their introduction and distribution in the food chain, accumulating successively on each ecological niche until reaching lethal doses for some constituent organisms of the chain, or until reaching high levels of the trophic network [16].
This problem is aggravated due to the excessive use of pesticides in the agricultural sector, the absence of technological remediation and the lack of safety interval (waiting period) for the harvest of agricultural products, which has significant impacts on public health [17]. Some of the toxic effects of DDT and its metabolite identified in mammalian have been alterations in fetal development and adverse effects on testicular function (semen, sperm, and sperm motility decrease) due to the mimetization or antagonism of reproductive hormones [18, 19]. Thus, DDT metabolites and DDT are considered endocrine disruptors, with estrogenic properties related to several types of estrogen-dependent cancers, such as breast cancer; therefore, the use of this pesticide was prohibited on the decade of the 1970s in most countries [20]. Nevertheless, pesticides remain in the environment (persistence); therefore, the pesticides have been widely distributed, and their traces can be detected in all areas of the environment (air, water, and soil) [21, 22]; therefore, current tendency is focused on natural sources for biological pesticide control. On the other hand, the indiscriminate and uncontrolled application of synthetic pesticide besides to accumulate residues in the environment and, in some cases, in living beings, has caused resistance in some pest. The first case reported of DDT resistance occurred in 1947, and since then, it has increased alarmingly, and it has been estimated that there are currently around 489 species of pest resistant to 400 different pesticides in the world [23]. The irrational use of synthetic pesticide has produced genotypic and phenotypic changes in many species, generating resistance to the action of most of them, including inorganics, DDT, cyclodienes, organophosphates, carbamates, pyrethroids, juvenile hormone analogues, avermectins, neonicotinoids, and antimicrobial [22, 23, 24].
Though the use of pesticides has offered significant economic benefits by enhancing the production and yield of food and fibers and the prevention of vector-borne diseases, evidence suggests that their use has adversely affected the health of human populations and the environment [21]. Because of this problem, several researchers are focused their studies on the identification of new natural sources containing active metabolites that could be used on the control of pest [25, 26].
The development of essential oils (EOs) as plant protection products is especially suited to organic farming as well as to integrated pest management. They are natural in origin and biodegradable, have diverse physiological targets within insects, and, consequently, may delay the evolution of insect resistance [27]. EOs act as fumigants, pesticides, repellents, and antifeeds that could affect some biological parameters such as grown rate, biological cycle, and reproduction [26]. One of the most widely analyzed oils is neem (Azadirachta indica) which has a toxic effect over several pests and it is a potential alternative to the synthetic pesticide [28]. Neem oil active compounds are azadirachtin, salannin, nimbin, and their respective analogues; being the azadirachtin the most abundant compound [29]. Azadirachtin acts on the immature stages of the insects avoiding their molt or maturation from larva to pupa and generating mutations in the development of different essential parts for their survival, because it affects their ability to oviposit in mature stage and hatch during the larval stage [30]. Its effect is reinforced by the action from the rest of limonoides, such as salannin and nimbin, which have repellent and antifeeder effects over many insects [31]. Although the concentration of azadirachtin is sufficient and its location well established in the seed, its extraction presents some problems, because it is soluble in polar organic solvents, but slightly soluble in water, besides is photosensitive and thermolabile, which conditions its activity in the oil.
Among the methods used for the extraction of azadirachtin, it could be mentioned the cold extrusion in a mechanical press, maceration, and percolation with the use of organic solvents. Each of the proposed methods stimulates the extraction of azadirachtin but in different proportions. Various researchers have suggested that the high variability in the extraction of azadirachtin from neem depends on several factors as age of the tree, region of its production, stage of fruit development, availability of the internal portion of the seed, storage conditions of the seed, methods and solvents used for its extraction, and the particle size [32, 33, 34, 35]. However, one aspect that has not been taken into account is the physical barrier exerted by the cell wall of the seed that directly affects the availability and extraction of azadirachtin and other acaricidal compounds. This problem has been overcome with great success in the plant extract industry through the application of cellulases or preparations with multiple enzymatic activities (cellulases, hemicellulases, and pectinases) [34, 36, 37].
The assisted extraction by cellulolytic enzymes has proven to be a viable and feasible tool to obtain bioactive metabolites from plants, due to its effect on lignocellulosic structures of its cell wall, which increase the yield of oils, pigments, flavorings, and aromas extracted in comparison with traditional extraction methods [38]. Due to the advantages of the use of cellulolytic enzymes for the production of bioactive metabolites from plants and the inherent need to develop sustainable and environmentally friendly alternatives that avoid the resistance phenomenon, the present study had the purpose of evaluating the use of food-grade enzyme preparation on the hydrolysis of the neem seed to obtain extracts with higher concentrations of azadirachtin, but without the use of solvents.
The study was divided into two stages: (1) determination of optimum activity conditions of four enzyme preparations and (2) evaluation of the azadirachtin release kinetics under optimal conditions of enzyme preparation activities. The enzyme preparations used in this study were Crystalzyme PML-MX, Cellulase 17600, Crystalzyme Cran, and Crystalzyme 100XL, and their optimum pH and temperature conditions were identified by using dehydrated and pulverized neem seed. Then, four volumes (1, 2, 3, and 4 mL) of each enzyme were tested to determine the optimum enzyme concentration to neem seed hydrolysis.
Evaluations of azadirachtin release kinetics were conducted at optimum pH and temperature for each enzyme preparation, and the maximum time required for the azadirachtin extraction was determined.
Neem seeds were provided by a local producer from Jamapa, Veracruz during June, 2017. One kilogram of 110–120 day old neem seeds (green-yellow coloration) was collected. Harvested seeds were washed and cut in half to extract the cotyledon extraction, which was stored by in freezing at −20°C for 24 h before using.
Each preparation enzymatic activity was measured by filter paper test [39], where the activity was defined as filter paper units per enzyme milliliter (FPU.mL−1). After FPU.mL−1 determination, the effect of the enzyme concentration on the hydrolysis of the neem seed cellulose structures and the azadirachtin release was evaluated. Neem seed hydrolysis was indirectly determined by the quantification of reducing sugar released during the enzymatic reaction. Total protein was estimated with [40] bovine serum albumin (BSA) as standard, and enzymatic activity (EA) for each enzymatic preparation was defined as changes in absorbance in 0.001 of reducing sugars mg protein−1 min−1.
The optimal conditions of pH and temperature were established based on the enzymatic hydrolysis of the neem seed and the release of azadirachtin. To determine the optimum pH of the enzymatic preparations, 1 g of the dehydrated seed was homogenized with 19 mL of phosphate buffer (3.0, 3.5, 4.0, 4.5, 5.0, and 5.5) and was conditioned at 50°C for 5 min and after 1 mL of the corresponding enzyme preparation was added. The enzymatic reaction was carried out for 2 h and was stopped by immersion in ice water. Subsequently, 1 mL of the sample was taken for reducing sugar determination and 1 mL for azadirachtin quantification by HPLC [41]. Once the optimum pH of each enzyme preparation was identified, the effect of the temperature at 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70°C was evaluated. Additionally, one extract without enzyme added was prepared as control, and the concentrations of reducing sugars released were subtracted from the values obtained in each of the enzymatic treatments. Furthermore, enzyme:neem seed (dry base) ratio was evaluated under optimal conditions of pH and temperature, and for this, 2 g of neem seeds (wet base) were used and 0.5, 1.0, 2.0, and 4 mL of each of the enzyme preparations were tested.
To determine kinetics of azadirachtin release, 100 g of neem seed (wet base) was homogenized with phosphate buffer at the optimum pH previously identify (1:10, w v−1) ratio for 3 min using an Ultra Turray homogenizer, T-25 basic (IKA®, Wilmington, NC). Five extracts were elaborated, one for each enzyme preparation and one without enzyme (control), and were incubated at optimum temperatures for each one. An aliquot was taken at 0, 2, 4, 6, 12, 18, and 24 h for the determination of azadirachtin. All samples were analyzed by triplicate.
Azadirachtin quantification was carried out by the HPLC technique proposed by [41], using a binary HPLC system (Waters 1525) and a photodiode detector (Waters 2996). The analytical separation was carried out with a Nova-Pak C18 column of 4 μm (3.9 × 150 mm) SUM (Waters® Milford, MA). Neem samples were centrifuged and diluted with acetonitrile (1:1, v v−1) before analyzing by HPLC. The samples were filtered through acrodiscs (Millipore) of 0.22 μm, and 20 μl was injected into the column. The flow rate was set at 1 mL min−1, the mobile phase was acetonitrile: water (40:60, v v−1), and azadirachtin was read at 217 nm in a retention time of 3.1 min.
The optimal conditions of pH, temperature, and the enzyme:substrate ratio were analyzed by means of analysis of variance (ANOVA) at a level of significance of p < 0.05, and Tukey’s tests were used to evaluate the differences between means with the statistical program Minitab 17.3. The kinetics of release of reducing sugars and azadirachtin were analyzed by a first-order empirical equation Yi = Ye (1−e−kt), in which the equilibrium concentrations (Ye) and its release rate constant (k) were determined, and the results were analyzed by ANOVA (p < 0.05) to determine differences between treatments.
In order to achieve the optimum cellulolytic activity of the enzyme preparation, filter paper units (FPU.mL−1) of each one were first determined. Crystalzyme PML-MX and Cellulase 17600 L showed the highest cellulolytic activities with 6.96 and 5.60 FPU.mL−1, respectively. This result is probably due to the presence of cellulases in these enzymatic preparations, since the determination of the activity is carried out with filter paper, which is formed by cellulose [42], while the enzymatic preparations, Crystalzyme 100XL and Crystalzyme Cran, showed lower activities with 2.65 and 2.38 FPU mL−1, respectively, and do not contain cellulases (Table 1). These results were used as reference to define the amount of enzyme needed to hydrolyze the neem seed cellulolytic structures and extract the highest concentration of azadirachtin.
Enzyme preparations | Cellulolytic activity | Activity (FPU mL−1) |
---|---|---|
Crystalzyme PML-MX | Pectinase, endoglucanase, exoglucanase, hemicellulase | 6.96 |
Crystalzyme Cran | Pectinase | 2.38 |
Crystalzyme 100XL | Pectinase and arabinase | 2.65 |
Cellulase 17600 | Endoglucanase, exoglucanase, β-glucosidase, pectinase and arabinoxylanase | 5.6 |
Enzymatic activities of the food-grade enzyme preparations.
Determination of the optimal reaction conditions was carried out at 50°C, taking into account that temperature is indicated by the enzyme preparation supplier as the optimum. However, in the evaluations, dehydrated neem seed was used instead of filter paper or crystalline cellulose. Figure 1 shows the determinations of the optimum pH of each enzyme preparations, and the results indicate that all enzyme preparations perform the hydrolysis of the neem seed under very similar pH conditions regardless of the combinations of cellulolytic activity they contain. Optimal pH of the enzymatic preparations was 5.0 for Crystalzyme Cran and 4.5 for Crystalzyme PML-MX, Cellulase and Crystalzyme 100XL. Of the four enzyme preparations, Crystalzyme PML-MX exhibited the highest hydrolysis of the neem seed with 2.2114 (± 0.1879) mg reducing sugars mL−1extract. A second reaction was carried out adjusting each hydrolysis to the optimum pH of each enzyme, and the effect of the temperature in the range of 25–70°C was evaluated (Figure 2).
Optimum pH of food-grade enzyme preparations: (A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme Cran, and (D) Crystalzyme 100XL.
Optimum temperature of food-grade enzyme preparations: (A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme Cran, and (D) Crystalzyme 100XL.
Cellulase 17600L, Crystalzyme Cran, and Crystalzyme 100XL shown higher activity at 50°C, while to Crystalzyme PML-MX was at 45°C. These conditions are within the limit considered by [34] to maintain the neem extracts without the loss of azadirachtin. However, it is necessary to evaluate these conditions on the release of azadirachtin, since it is unknown whether the highest hydrolysis of the neem seed ensures maximum concentration of azadirachtin.
Results of the enzyme concentration effect over hydrolysis of neem seeds could be observed in Figure 3. Reducing sugar release was used as indirect measure of neem seed hydrolysis.
Optimum filter paper unit (FPU) of the food-grade enzyme preparations for hydrolysis of neem seeds. (A) Crystalzyme PML-MX, (B) Cellulase 17600, (C) Crystalzyme Cran, and (D) Crystalzyme 100XL.
Reactions catalyzed by Crystalzyme PML-MX and Cellulase 17600 L presented the highest hydrolysis of the neem seed, probably due to the presence of cellulases in its composition. A total of 1.8072 (± 0.0021), 2.0635 (± 0.0689), 2.0493 (± 0.0521), and 2.0742 (± 0.0283) g L−1 reducing sugars were released when 0.5 (3.48 FPU), 1 (6.96 FPU), 2 (13.92 FPU), and 4 mL (27.84 FPU) of Crystalzyme PML-MX were used. On another hand, 1.8034 (± 0.0387), 2.0809 (± 0.0023), 1.9921 (± 0.0456), and 1.9383 (± 0.0781) g L−1 reducing sugars were released when 0.5 (2.80 FPU), 1 (5.60 FPU), 2 (11.2 FPU), and 4 mL (22.4 FPU) of Cellulase 17600 were used. These results suggest that an increasing of enzyme concentration in the reaction not necessarily imply higher hydrolysis of the neem seed. Therefore, it is advisable to use the lowest concentration in which the same results were obtained for each enzyme preparation. Crystalzyme Cran and Crystalzyme 100XL showed less hydrolysis of the neem seed after 18 h. Crystalzyme Cran released 1.0022 (± 0.0576) g L−1 with the highest concentration of enzyme used (4 mL, 9.52 FPU); however, these results were not statistically different from those found when using 1 (2.38 FPU) and 2 mL (4.76 FPU), since the equilibrium concentration of reducing sugars was 0.9323 (± 0.0786) and 0.9859 (± 0.0341), respectively. On the other hand, the samples hydrolyzed with Crystalzyme 100XL had a maximum reducing sugar release when 4 mL of enzyme (21.2 FPU) was used.
In order to determine the optimum enzyme concentration to neem seed hydrolysis, equilibrium concentration and release rate of the reducing sugars were evaluated. The analysis of the results showed that reducing sugar release rate was not statistically different when 1, 2 or 4 mL for both Crystalzyme PML-MX and Cellulase 17600 L were used. When Crystalzyme Cran was evaluated, the higher rates of release of reducing sugars were obtained with 2 and 4 mL. In contrast, the highest reduced sugar release rate for Crystalzyme 100XL was presented when 1 mL (5.30 FPU) of this enzyme was added. Therefore, based on the analysis of results, it was considered that the units of activity necessary to hydrolyze the neem seed are 6.96, 5.60, 4.76, and 5.3 FPU mL−1 for Crystalzyme PML-MX, Cellulase 17600 L, Crystalzyme Cran, and Crystalzyme 100XL, respectively.
After optimum conditions of pH, temperature and enzyme concentration were determined, and the kinetics of azadirachtin released was carried out but using fresh seed instead of dehydrated seed to minimize the risk of losses due to the dehydration process.
Evaluation of the azadirachtin release kinetics was conducted with 100 g of neem seed homogenized and adjusted at 1:10 (w v−1) ratio with phosphate buffer. Enzymatic hydrolysis was conducted under optimal conditions of each enzyme preparation, and azadirachtin were quantified at 0, 2, 4, 6, 12, 18, and 24 h by the HPLC technique, and the results were presented on base of the quantity of neem seed (dry base) used for the neem seed extract elaboration (Figure 4). The highest azadirachtin concentrations obtained in this study were 2.55 g kg−1 (2550 ppm) and 2.35 g kg−1 (2350 ppm) neem seed when Cellulase 17600 L and Crystalzyme PML-MX enzyme preparations were used, respectively. Both of this enzyme preparation include cellulases and pectinases and were statistically different (p < 0.05) from the enzymatic preparations, Crystalzyme Cran (pectinases) and Crystalzyme 100XL (pectinases and arabinases).
Azadirachtin release kinetics from neem seeds extracted with food-grade enzyme preparations.
Azadirachtin concentrations found in this study were higher than those reported by other authors when conventional methods such as extrusion, extraction with solvents (hexane, methanol), and aqueous extraction which reported concentrations of 1080, 565, 400, 150 ppm, respectively [33] and were similar to the obtaining by cold methanol extrusion [32]. However, the concentration obtained is lower than those reported with nonconventional technologies such as extraction with pressurized solvents, which reported concentrations of up to 9510 ppm of azadirachtin [34].
One of the advantages of the use of organic solvents in the extraction process is to improve the solubility of nonpolar components which increase their extraction from vegetable matrices. However, in the present study, only phosphate buffer was used, which could explain the differences of azadirachtin extraction with the pressurized solvent method [34]. Also, in [43], it was reported that in the tropical regions, there are lower concentrations of azadirachtin after extraction processes due to high temperatures, moisture, and storage conditions that could encourage the azadirachtin degradation.
Although the highest concentrations of azadirachtin were obtained with the enzymatic preparations, Cellulase 17600L and Crystalzyme PML-MX, also Crystalzyme Cran and Crystalzyme 100XL could be used to elaborate neem extracts because of their azadirachtin concentrations of 1540 and 1690 ppm, respectively, are similar to those contained in commercial acaricidal products elaborated based on neem, but with the use of solvents [32]. This result indicates an advantage and a possible solution to the indiscriminate use of synthetic pesticides and the organic solvents employed in several extractions of active biomolecules. On the other hand, after 18 h of enzymatic hydrolysis, there are no changes on azadirachtin release, which means it is time required to carry out the obtaining of neem seed extracts. In addition, the conditions identified in this study as necessary to neem seed hydrolysis are not extreme or aggressive and could be easily scalable.
The present study had the objective of generating an alternative solution to the multiple problems generated by the indiscriminate use of synthetic pesticides. The use of enzymes in the production of metabolites with biological interest has been widely studied in the last decade, due to their specificity and their effect on the cellulose substrate hydrolysis, minimizing the times of obtaining and the necessary costs for their implementation. In addition, its application allows reducing the use of solvents, normally employed in the extraction of neem oil. Besides, the conditions required for neem seed hydrolysis under optimal activity of the enzyme preparations can be easily standardized and scaled for its industrial production. In addition, it was found no affectations on azadirachtin concentration when temperatures of 45 and 50°C were used during enzymatic hydrolysis coinciding with that reported in [34]. However, more studies are needed to determine the stability of azadirachtin under different temperatures and pH conditions during storage.
Although the enzyme-assisted extraction allows the obtaining of neem extracts with higher azadirachtin concentrations than those obtained with conventional methods such as extrusion, cold extrusion, water maceration, and percolation with hexane [32, 33] and in similar concentrations to those obtained by extrusion with cold methanol [32], the amount of neem seeds used in this method was lower, and therefore, the yield was higher than obtained with all these methods. In next studies, components identified as repellents and/or acaricides such as salannin and nimbin should be analyzed. In addition, it is necessary to carry on shortterm and long-term in vitro and in vivo analysis of enzymatic neem extracts on pest with public health impact, defining the vehicle of application and the lethal concentrations for its implementation.
This study was funded by the Dirección General de Educación Superior Universitaria (DGESU/SEP) (Project DSA/103.5/14/7147).
All authors who participate in the elaboration of this manuscript have no conflict of interest to declare.
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