text:
Comparative study of extraction yield and antioxidant property of sweet orange peels (Citrus Sinesis) essential oil

Article history: Received: September 25, 2019 Accepted: June 16, 2020 The research evaluates the extraction yield and antioxidant potentials of essential oil (EO) of sweet orange peels using pressurized liquid extraction (PLE), Soxhlet (Sox) and hydro distillation (HD). The extracts were investigated to find out the antioxidant properties using 2, 2 -diphenyl-1picryl-hydrazyl (DPPH) and 2, 2 azino-bis (3-ethylbenzothiazoline-6sulfonate) radical (ABTS•+). PLE and Soxhlet extracted essential oil showed additional polyphenol compounds and tannins using thin layer chromatogram (TLC) and chemical analyses, respectively. Hydrodistillation indicating a pure essential oil without identified tannins and polyphenols with the highest ABTS activity compared to other produced essential oils of PLE and Soxhlet. The major chemical constituents of the pure essential oil were identified by gas chromatography-mass spectrometry (GC-MS) and they include limonene (90.72%), myrcene (2.82%) and octanol acetate (1.24%). PLE had moderate high yield within short extraction time and the highest antioxidant (DPPH) and can be adjusted to individual materials to maximize the extraction yield and antioxidant property.

Introduction 
Citrus (Citrus spp) is an important fruit and one of the mostly cultivated crops with world production estimated at 115 million tons per year. In 2010, it was reported that the world citrus production is about 82 million tonnes with sweet oranges history of 61% (Alnaimy et al., 2017). Orange fruits have round, rough and green to yellow coloured skin. They are about 20-30 cm in length with a tough peels or skin known as epicarp (or flavedo) that acts as cover which protects the fruit from adverse effects from the environment. An orange peel comprises of epidermis and exocarp with irregular thin-walled cells, which enclose numerous glands or oil sacs (Farhat et al., 2011;Velazquez-Nunez et al., 2013). The oil in these sacs represents the citrus essential oil (EO) that represents secondary metabolites product in the citrus plant (Bousbia et al., 2009a). Citrus fruits have been discovered as excellent sources of essential oils, besides their use as flavouring agents. Citrus essential oil has * Corresponding author E-mail: oyebolabinjo@gmail.com gained relevance in the food industry due to its antimicrobial effects against both food bacteria and fungi (Rezzoug and Louka, 2009;Velazque-Nunez et al., 2013;Lago et al., 2014). EO is mostly present in peels, when compared to other parts, and it has got a wide application in food industries as additive, nutritious supplement and some other industrial applications (Maria et al., 2012). The main methods used to extract essential oil from plant material are distillation (hydro, steam and destructive), maceration and expression (Stahl-Biskup and Saez, 2002). However, in order to reduce the limitations associated with the main methods (reduce extraction time, cost of extraction and possibly improve the yield and quality of the extracts) new techniques, such as microwave-assisted extraction (MAE), pressurized liquid extraction (PLE), supercritical fluid extraction, and ultrasound-assisted extraction have also been developed (Wang and Weller, 2006). PLE is also known as accelerated solvent extraction (ASE). This method is widely used as an extraction technique for sample preparation to discover the presence of minor components in the extract. At higher extraction temperatures, it increases both solubility and mass conveyance rate of the analyte. It also decreases the viscosity and intermolecular forces of solvent, thereby improving extraction rate (Ibanez et al., 2003). The extraction of essential oil utilizing the ordinary extraction techniques that had been accounted for by Presti et al., (2005). Bousbia et al., (2009b) that the impediment is to be of lower essential oil yield and longer extraction time. Consequently, it is advantageous to enhance these impediments. The research is focused on extracting essential oil from sweet orange peels using some of the conventional extraction methods (Soxhlet and hydro distillation) with green extraction method (PLE). The antioxidant extract potentials using ABTS and DPPH, the purity (TLC Plate) and chemical constituents using GC-MS for the extracted essential oil were evaluated. The results were then compared to ascertain the best method of extraction in relation to the quality and purity of the extracted essential oil.

Raw Material Characterization
Harvested sweet oranges were purchased from fruit and vegetable market centre in Pirassununga, São Paulo, Brazil. Fruits at the same stage of ripeness were used for the research. The ripe fruits were processed at the Laboratory of High Pressure Technology and Natural Products, of the University of Sao Paulo (Pirassunuga SP, Brazil). These fruits were sorted and cleaned to remove foreign materials from the epicarp. The fruits were peeled with sterilized knife to remove epicarp or rind (flavedo or shell).

Pressurised Liquid Extraction (PLE)
PLE was performed using an ASE 150 accelerated solvent extraction system (Dionex, Sunnyvale, USA), in which the samples were packed inside a fixed bed and in a vertical position. The stainless-steel extractor with a capacity of 34 mL was filled with approximately 10 g of dried rind samples for each extraction process, with 5 g of diatomaceous earth (Thermo Scientific, Sunnyvale, USA), as adsorbent material, to disperse the vegetal matrix in the extraction cell. The diatomaceous allows a better contact with the solvent and clarifies the extract. Anhydrous ethanol was used as solvent because it is generally recognized as safe (GRAS), (FDA, 2013). A static time of 15 min in each cycle, purge time of 100 seconds, oven heat up time of 10 min, flush volume of 100% and pressure of 10 MPa were the fixed variables. The ethanol extract obtained by PLE was named crude extract, it was evaporated after the extraction and then prepared for analyses. The oven temperature (50-70 ºC) and static extraction cycles time (2-4) were varied in order to ensure that the mechanical and thermal equilibrium is guaranteed in the employed operating conditions.

Classical / Soxhlet Extraction
The soluble content of the essential oil extract was determined in triplicate by Soxhlet extraction using ethanol at 80 ºC for 3 h (12 extraction cycles time), followed by solvent removal at 35 ºC using a rotary evaporator (Yamato, Tokyo, JP). Approximately 10 g of dried orange peels were used for the Soxhlet extraction using methanol as solvent, which was carried out according to a method adopted from AOAC 2000.

Hydrodistillation Process
Dried milled peels 10 g were immersed in 250 mL of water and distilled for 10 h (40 extraction cycles time), using a Clevenger-type apparatus (Ebramhizadeh et al. 2009), which was found to be sufficient for completing the process. The extracted oil was collected and weighed via vial bottle. The extracted essential oils obtained were dried over anhydrous sodium sulfate and stored in a refrigerator prior to analysis (Chegini and Abbasipour, 2017).

Yield Calculation and Purification of Crude Extracts
The extracted weight of essential oil was determined using gravimetrical method. The extractable essential oil yield was determined as the percentage ratio of the extract mass to the mass of orange peels. The crude extracts obtained by PLE and Soxhlet were purified to eliminate tannins with a high degree of polymerization. The crude extract (100 mg) was diluted in 2.5 mL of methanol and 32.5 ml of chloroform using Lhuiller et al. (2007) standard method. The standard method without any modification was necessary in order not to remove other phenolic compounds in the extract. The diluted extract was stored at 4 ºC for 3h in the dark. The centrifugation of the extract was carried out (Excelsa II Model 26, Fanem, Sao Paulo, BR) at 4,000 rpm and 5 ºC for 10 minutes. The decanted extract was evaporated under nitrogen at room temperature in the dark and named purified methanol extract (Oliveira et al., 2014).

Trolox Equivalent Antioxidant Capacity (TEAC) Assay
The total antioxidant capacity was determined as 2, 2 azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) extracted essential oil according to the method described by Re et al. (1999). ABTS•+ values were determined by reacting ABTS solution (7mM) with K2S2O8 (2.45mM, final concentration) in the dark for 16h. The reading was taken between 700 to 734 nm with methanol. Thereafter, 0.2 mL of the essential oil was added to 2.0 mL ABTS•+ solution. The absorbance value was taken at 734 nm after 6 min. Trolox was used as a reference standard, and the results were expressed as mg of trolox equivalent (mg TE) by grams of extract.

Antioxidant by DPPH
The determination of sequestering capacity of the stable free radical 2, 2 -diphenyl-1-picryl-hydrazyl (DPPH) was based on the methodology of Brandi -Williams et al. (1995). Methanol solution of DPPH was prepared with absorbance between 0.700 at 515 nm. Thereafter, 0.4 mL aliquots of each extract diluted in methanol for the control were added to tubes containing 3.6 mL of this DPPH solution and measurements were performed in triplicates. The absorbance reading was taken after 2 h of incubation using a spectrophotometer (Biospectro SP 22, São Paulo, BR). The results were expressed as IC50 (µg/mg of extract) which is the amount of antioxidant required to cause 50% reduction of the initial concentration of DPPH (Equation 1). The value was calculated by plotting inhibition percentage against extract concentration (Sokmen et al., 2004).
where 50 is the radical scavenging activity (%), is the absorbance of control, and is the absorbance of test sample.

Thin Layer Chromatography
Thin layer chromatography (TLC) was carried out on the extracted oil to determine the authenticity of the oil. Polyphenolic compounds in essential oil extracts were ascertained by thin layer chromatography on TLC plates coated with Silica Gel G. The plates were cleaned and activated by heating at 150 ºC for 60 min to remove moisture. The Silica gel plates 60 F254 is the stationary phase which was eluted with commonly used solvent for designation and quantification of phenolic compounds, chloroform and ethyl acetate (70:30, v/v) as the mobile phase. The purified essential oil extract using chromatographic standard (40 -50 µL) for each essential oil obtained were injected to the plates and eluted with the mobile phase. The plate was placed in 20 mL of mobile phase solution in the developing chamber and allowed to rise by capillary movement until it reached a height of 10 cm from the point of spotting. The plate was dried and heated to visualize the bands that eluted with varied colouration prepared as described by Wagner and Bladt (2009). The image was captured under ultraviolet light (Boitton, model 2909, Porto Alegre, BR), patterns were recorded by camera and all visible spots were outlined with pencil.

Phytoconstituents composition using GC-MS
Gas chromatography coupled with mass spectrometry (GC-MS) is used to evaluate the constituent compounds in the essential oil. Hydrodistillation (HD) essential oil extracts were used for the phyto constituents based on the result from the thin layer chromatogram indicating its essential oil to be pure volatile oil without tanninsor polyphenolic compounds. Phytoconstituent composition of the hydrodistillation (HD) extracts was analysed by gas chromatography coupled with mass spectrometry (GC-MS) (QP 2010 Plus, Shimadzu, Tokyo, Japan) with auto sampler (AOC-5000, SWI, Tokyo, Japan). The compounds were separated on Rtx @ -5MS capillary column (30 m x 0.25 mm, film thickness 0.25 µm) (RESTEK, USA) with 5% diphenyl, 95% dimethylpolysiloxane as stationary phase. The injector and detector temperatures were 220 °C, the column temperature was held at 60 °C for 5 min (hold time compound in the column) and then was increased from 60 to 246 °C at 3 °C/min and was finally held at 246 °C for extraction time (taken from method). 1.0 μL of the sample was diluted in methanol (400 mg/L) and then injected by using the split mode (split ratio 1: 20). Helium was used as a carrier gas (extraction time (taken from method) mL/min). The MSD (EI mode) was operated at 70 eV and the scan range was set to 50 -500 m/z. The identification of volatile constituents was based on the comparison of their retention indices (RI), relative to the retention times of a homologous series of n-alkanes (C8 -C20), with those reported in the literature and their mass spectra with those of authentic compounds available in our laboratories or those listed in the NIST 08 mass spectral libraries. For accurate and reliable designation of the compounds, kovats retention index (KI) was determined for each compound identified according to Equation 2.
where Pz is the number of carbons in the alkane immediately preceding the analyte, RT(x) is the analyte retention time, and RT(Pz) is the retention time of the alkane immediately preceding the analyte.

Extraction Yield
The magnitude of extracted essential oil yield from sweet orange peels for PLE, hydrodistillation and Soxhlet ranged from 5.73 -53.4 % ( Table 1). The highest value 53.4% was recorded by Soxhlet with extraction time of 10 hours (40 cycles) and the lowest value of 5.73% by hydrodistillation with extraction time of 3 hours (12 cycles). Essential oil yield obtained in this study by hydrodistillation is low when compared to solvent extraction (PLE and Soxhlet). Similar trend was obtained by Ahsan et al. (2017) for extracting Jasminum sambac L essential oil using hydrodistillation and supercritical fluid extraction. The extract yield of PLE ranged from 21.6 -49.3 % with mean value of 26.75% and mean extraction time of 45 minutes (3 cycles) ( Table 1). The hydrodistillation with extraction time of 3 hours (12 cycles) had essential oil yields of 5.73% with closer value of 5.45% for lime peels (citrus latifolia Tanaka) using similar method as reported by Attisantos et al. (2005).
The yields obtained in this study were higher than those reported in literature. Mercy et al. (2015) reported an improved distillation method for extracting essential oil from peels of citrus sinesis and citrus reticulate with yield of 4.23% and 5.865% respectively. Franco-Vega et al. (2016) reported orange peels extract yields of 0.92 to 2.73 %. Megha and Mumtaj (2014) reported sweet lime with yields of 1.16% using microwave assisted hydrodistillation. Ahmad et al. (2006) accounted for essential oil yields varying from 0.30 to 1.21 % for four citrus varieties from Pakistan. Also, Kamal et al. (2011) reported that C. sinensis had the highest oil value yield of 0.24-1.07 % accompanied by C. reticulata with 0.30-0.50 % and the least C. paradisii with 0.20-0.40 %. There were significant variations in the yield of essential oils from our study in comparison with those of literature. Such variability could depend on several factors including climatic and environmental conditions, soil variations and season, geographical location, the stage of the vegetative cycle, and the method used to obtain the essential oil (Jing et al., 2014). In general, extraction yield obtained using the green extraction method and conventional methods in the study were higher than those in literature. However, the yield under Soxhlet was higher than those of PLE, although two methods (PLE and Soxhlet) were extracted with solvent and both showed polyphenolic compounds in addition to essential oil. PLE process is advantageous mainly due to the relatively short duration extraction time and it is more economical than the conventional methods (Soxhlet and hydrodistillation) used in this study.

Extracts Purification
The value of tannins was calculated gravimetrical and the value varies from 0.065 -0.084 g. PLE extracts ranged from 0.069 -0.084 g, Soxhlet had tannins with 0.065 g while in hydrodistillation, tannins were not recorded. The extracts from PLE and Soxhlet were yellowish in colour due to the presence of polyphenols and tannins. The highest value of 0.935 g was recorded by Soxhlet and the lowest value of 0.002 g by hydrodistillation. The hydrodistillation extract is an unadulterated unstable oil which is less dense and colourless which makes it diffuse effectively into the air. Obviously only volatile oil was extracted under hydrodistillation and the other two methods extracted volatile oil along with other polyphenolic compounds. The tannins identified denote the presence of other compounds with the volatile in the extracts (Table 1).

Antioxidants
Antioxidants carry out their functions in biological system either by preventing the production of free radicals or by negating free radicals produced (Oboh 2006). Due to the chemical complexity of the essential oil, several antioxidant parameters as typified by reducing property of ABTS and DPPH scavenging abilities were measured. Total antioxidant capacity (ABTS) of the essential oil expressed as Trolox equivalent antioxidant capacity (TEAC) had the value ranged from 11.45 to 11.74 mg TE/g of essential oil (Table 1). Hydrodistillation showed the highest activity of 11.74 mg TE/g and the lowest activity of 11.45 mg TE/g was recorded with PLE lowest temperature and lowest static cycles (PLE Test 1). For the DPPH method, the antioxidant activity of the EO extracts ranged from 15.27 mg/g to 56.13 mg/ g. The lowest temperature and the lowest static cycles (PLE Test 1) showed the highest antioxidant value of IC50 = 15.27 mg/g and hydrodistillation showed the lowest antioxidant value of IC50 = 56.13 mg/g. Prieto et al. (1999) reported that the smaller the IC50 values, the higher antioxidant activity of the plant extracts.

Thin Layer Chromatography
Thin layer chromatography (TLC) is a technique widely used for separating and purifying extracts, due to its simplicity and flexibility. Thin layer chromatography (TLC) was used to identify compounds in extracts as presented in Figure 1, numbered 1 to 5 for PLE, hydrodistillation (HD) and Soxhlet (Sox) on silica plate. The plate exhibited two narrow and intense blue bands for all the PLE and Soxhlet samples whilst the chromatogram in respect of hydro distillation exhibited none. The chromatogram with blue bands implied the presence of additional phenolic compounds with the volatile in the extracts obtained via PLE and Soxhlet. PLE can be used to detect minor compounds in the extract as shown using thin layer chromatogram and chemical analysis to determine polyphenols and tannins.

Identification and quantification of phytoconstituents from orange essential oil
Based on the results from the chemical and thin layer chromatography from this study, it was discovered that hydrodistillation consists of volatile compounds without any additional polyphenols. The essential oil without polyphenol essential oil from hydrodistillation was injected into GC-MS. Essential oils are natural complex mixtures of volatile compounds which had about ten to hundred constituents at different concentrations. The percent composition was computed from the area of the peaks of the gas chromatography (GC) in terms of the components having mass fractions equal to or greater than 0.01. The constituent was identified by GC-MS when there is a quality match of more than 80%. The individual constituent of the essential oil was identified via mass spectrometry and its identity confirmed in comparison with mass spectra of authentic standard based on the National Institute of standards and technology, (NIST, Gaithersburb, MD, USA) NIST 08 and NIST 08s libraries The result from the spectrograph showed 62 peaks which were found on the total ion chromatogram and mass spectra from the GC-MS which amounts to 100% of the entire concentration. Figure 2 shows the total ion chromatogram (TIC) of citrus essential oil peel of hydrodistillation from GC-MS. The result from retention characteristics and GC-MS analysis revealed the identification of 47 constituents in six groups: terpenes (95.13%), aldehydes (1.19%), alcohols (0.68%), esters (1.65%), oxide (0.1%) and ketone 0.05% from Noot ketone representing about 99.8% of the essential oil (Table 2). Total ion chromatogram (TIC) of essential oil of sweet orange using Rtx @ -5MS capillary column for peak identification In our study the major constituent groups in the citrus sinesis peels essential oil are terpenes (monoterpenes and sesquerpenes), while it also contains aromatic compounds (aldehydes, alcohols, esters, oxide and ketone). The essential oils are characterized by two or more major constituents at fairly high concentrations compared to other constituents present in small quantity. The findings from this research showed that the main constituents in the citrus sinesis peels were limonene (90.72%), myrcene (2.82%), octanol acetate (1.24%), nonanal (0.58%), sabinene (0.39%) and elemol (0.14 %). The constituents in essential oils are terpenes (monoterpenes and sesquerpenes), aromatic compounds (aldehyde, alcohol, phenol, methoxy derivatives), and terpenoids (isoprenoids) as reported by Bakkali et al., 2008.
The GCMS analysis revealed that limonene is the most abundant compound in the essential oil. The observed high levels of limonene in this study correlate with the reports of Khaoula et al. (2015), Ademosun et al. (2015) and Yousmel et al. (2015). The major constituent from Citrus sinensis was limonene of 90.72% using hydrodistillation essential oil extracts and was lower than the one reported by Rodriguez et al., (2011) of same sample with limonene of 97%. The limonene value of our study is higher than the value of Tunisian Citrus aurantium of 87.523% as reported by Khaoula et al. (2015). The result from the research showed that Citrus sinesis could be used as a source of limonene production. Limonene could be introduced in the nutritional, pharmaceutical and cosmetic fields as reported by Vivian et al., (2016). Moreover, other compounds such as myrcene (2.82%), octanol acetate (1.24%), nonanal (0.58%), sabinene (0.39%) and elemol (0.14%) were present at minimal level.

Conclusion
A comparison of extraction yield results of PLE, Soxhlet and hydrodistillation indicated that PLE process is advantageous mainly due to the relatively short extraction time. PLE extraction time, solvent and extraction temperature can be tailored to individual materials to maximize the extraction yield and antioxidant property. The TLC and chemical purification further showed the presence of minor additional compounds (polyphenol) with essential oil in PLE and Soxhlet. The result from the purified extracts and thin layer chromatogram showed that hydrodistillation extract is pure essential oil without additional polyphenol compounds. Hence, this necessitate further study to determine the constituents of polyphenol compounds in PLE and Soxhlet extracts. The result from GC-MS showed sweet orange essential oils as mixtures of many compounds which include terpenes, aldehydes, alcohols, terpenes oxide, ketone and esters.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Genetic evaluation of milk yield and milk composition of Saudi Aradi and Damascus goats

The aim of the present study was to evaluate the effects of various non-genetic factors on milk production traits and to estimates its' heritability and variance component in milk production trait among Saudi Aradi and Damascus goats. A total number of 213 (98 Aradi and 115 Damascus goats) was classified according to breed, season, year, and type of birth. Data were analyzed statistically. The variation in milk yield (MY) were moderate (32.80 %), while variations in fat (F %) and protein (P %) were low (fat 17.67 % and protein 19.75 %). The genetic groups had a significant (P≤0.05) effect on milk yield and highly significant on (F % and P %, (P≤0.01)). Damascus goats had higher than Aradi in milk yield, fat and protein. Milk yield and fat% during seasons were not significant differ. Milk protein% during Summer/Spring season (S2) had a significant higher (P≤0.01) than at during Winter/Autumn (S1). The highest fat% (P≤0.05) found in year 2009. Triplets had higher (P≤0.01) effect on milk yield than twins or single. However, there were no significant differences between type of births for protein% and fat%. Damascus had higher heritability estimates (0.45) than Aradi goats (0.29) for milk yield. Also, for protein%, the heritability estimates of Damascus had higher heritability estimates (0.69) than Aradi goats (0.37). But the heritability for fat % in Aradi (0.23) was similar to that in Damascus goats (0.22). In conclusions, the percentage of genetic variance component in Aradi goats is low so that the best programs for the genetic improvement in the Aradi goats could be upgraded through crossing with Damascus goats.

Introduction
Breeding programs depend on selecting does that have superior genetic to improve performances in dairy goats. The estimation of genetic and non-genetic factors affecting economically important traits is basics to select breeders for a certain traits. So, estimates the heritability of genetic and phenotypic traits are essential in developing efficient breeding schemes as well as in predicting breeding values accurately. Kendall et al. (2009) indicated that the breed affecting on level of milk production and there is variation in milk yield among different breeds and within the breed. The economic value of goat milk depends to a great extent on fat and protein contents as well as total milk yield (Montaldo and Manfredi, 2002). Several researchers have reported on the effects of breed on milk production and have also investigated milk yield traits in different breeds of goat. However milk production significantly varied in different breeds of goat and attributed it to the difference in genetic potentials of the animals (Zahraddeen et al., 2009). The exotic breeds like the Swiss breeds have much higher milk yields but lower milk composition than Indigenous goat breeds like the West African Dwarf and Red Sokoto (Park and Haenlein ., 2010). Scarcely, published estimates of genetic parameters for milk yield and content traits in goats, few multi-trait animal model-REML estimates of (co)variance matrices are available for milk yield and content traits (Analla et al., 1996;Gonçalves et al., 2002;Valencia et al., 2007). The present study was undertaken to evaluate the effects of various non-genetic factors on milk production traits (milk yield, fat % and protein %) and to estimates heritability and variance component for the same traits in Aradi and Damascus goats.

Location of study and animal management
Saudi goats (Aradi, A) was a native breed of Saudi Arabia and Syrian goats (Damascus, D) was carried out at Camel and Range Research Center (Al-Jouf province, Northern region of Saudi Arabia located at latitude of 29.97 °N and longitude of 40.21 °W and at 684 meters above sea level). All animals were raised under similar environmental, nutritional, and management conditions. The animals were housed in semishaded/open front barn and fed on a commercial concentrate and alfalfa hay. The amount of concentrate and hay were calculated according to the nutritional requirements for goats (NRC, 1981), which were dependent on animal ages and production status. Water, straw, salt and minerals supplemented in blocks were freely available to all animals.

Breeding plan of the genetic groups
This study used total number of 213 (98 Aradi and 115 Damascus goats) during the time period from 2008 to 2010 of the study. The measurement of milk production began after the 3 rd day post-partum to allow kids consuming colostrum. Milk yield was recorded by the ICAR (International Committee for Animal Recording) A4 method at 28-day intervals during the 240 days of lactation ICRPMA (1990). All goats were milked after weaning by an automatic milking system that can measure the milk yield automatically. Milk yields of the dams were measured using the alternate day system until weaning. In this system, goats were milked at 6 AM on the recording day. Then, kids were fed with dams' milk with the assistance of the stock person and suckled their dams until 6 AM the following day. Kids were isolated from their dams until the 6 PM milk recording. Sampling was started after weaning. Every 28 days, 200 ml milk samples were collected from the morning milk and immediately taken to the laboratory in an icebox. The samples were analyzed for fat by the Gerber method, and total protein was determined by the phenol titration method as described by James (1988).

Data collection for statistical analysis
Data were recorded for all animals during season 1 (winter / Autumn), season 2 (spring / summer), and years from 2008 to 2010.They were classified according to breed, season, year, and type of birth. Data were analyzed using PROC GLM procedure (SAS, 2008) for the leastsquares means and standard errors of the fixed factors. Heritability and genetic parameters were estimated with derivative-free restricted maximum likelihood (REML) procedures using the MTDFREML program of Boldman et al. (1995).

Statistical model used for data analysis
The following statistical model was used for data analysis:

Yijklm= μ + Bi +Sj+ Yk+ Tl+ eijklm
Where; Y ijklm : The record of MY, F % or P % measured on does had milked at k th year, i th breed, j th season and l th type of birth; μ: Overall mean when equal subclass numbers exists; Bi: effect of i th breed (i = 1 and 2); S j : Fixed effect of j th season (j = 1 and 2); Y k : Fixed effect of k th year (k = 1, 2and 3); T l : Fixed effect of l th type of birth (l = 1, 2, and 3); e ijklm : Random error particular to the ijklm th observation assumed to be independently and normally distributed with mean zero and variance of δ 2 e. The assumed model for heritability, genetic correlations and breeding values was: Where; y: a vector of observations, b: a vector of fixed effects with an incidence matrix X; u: a vector of random animal effects with incidence matrix Z; e: a vector of random residual effects with mean equals zero.

Results and Discussion
The obtained results articulated wide phenotypic variations in all traits of the milk yield, fat and protein %. The percentages of variation for milk yield were moderate (32.80 %) but the difference between minimum and maximum were high ranged from 54.24 to 479.00, while these variations for fat and protein % were low (fat 17.67 % and protein 19.75 %) but the difference between minimum and maximum were high ranged from 2.16 to 6.34 for fat % and from 1.46 to 5.64 for protein % (Table 1). The genetic groups had a significant (P≤0.05) effect on milk yield and highly significant on fat and protein % (P≤0.01). Damascus goats had higher than Aradi in milk yield, fat and protein ( Table 2). The importance of the various environmental effects on milk yield, fat% and protein% has been also examined in other studies. Rabasco et al. (1993) in the Spanish Verata goats found that birth type and production year were significant effects for milk yield. It was recorded that milk yield significantly affected by season of kidding, litter size and year (Valencia et al., 2002;.  and Boichard et al. (1989) for Alpine (0.29). It was higher than that reported by Ribeiro et al. (1998) for Saanen goats (0.09), Kominakis et al. (2000) for Skopelos dairy goats (0.14 ± 0.04), Torres-Vázquez et al. (2009) for Saanen goats from Mexico (0.17 ± 0.04), Pesce Delfino et al. (2011) for Maltese Goat (0.21 ± 0,04), Valencia et al. (2007) for Saanen (0.22±0.07), Rabasco et al. (1993) for Verta goats (0.24). It was lower than that reported by Kala and Prakash (1990) for Jamunapari goats (0.40), and Grossman et al. (1986) for Toggenburg goats, Saanen goats and Nubian goats (0.41, 0.44 and 0.45 respectively). The heritability estimates for milk yield of Damascus goats (0.45) are similar to the estimate of Mavrogenis et al. (1989) worked with the same breed in Cyprus goats (0.46) and Hermiz et al. (2002) for Iraqi local goats (0.46). It was higher than that found by Constantinou (1989) for Damascus Goats (0.35) and Kala and Prakash (1990) for Barbari goats (0.36). It was lower than that reported by Voutsina et al. (1990( for Alpine (0.54), Thilagor et al. (1992, Other estimates were reviewed for Pinto et al. (1993) for Anglo-Nubian (0.55), Badamana et al. (1990) for British Sannen (0.68), Banda (1992( for Malawi goat (0.68) andFaiz et al (1994) for Beetal and Nachi (0.68). The heritability estimates in our study for fat % (0.23 & 0.22) and for protein% (0.37 and 0.69) among Aradi and Damascus goats was within the range of values estimated by other authors which ranged (from 0.14 to 0.73) for different dairy goat populations (Analla et al., 1996;Andonov et al., 2007;Bömkes et al., 2004;Ilahi et al., 1998;Torres-Vázquez et al., 2009). Generally, increasing the percentage of environmental variance component indicates that the trait is highly affected by environmental conditions. The moderate percentage of genotypic variance gives hope for the possibility of improving the trait through different breeding programs. In this study, the percentage of genetic variance component in Aradi goats is low so that the best programs for the genetic improvement and upgrading in the Aradi goats could be done through crossing with Damascus goats which has superior genetic characteristics.
effect of amount of protein in the concentrates on hay intake and rate of passage diet digestibility and milk production in British Saanen goats", Animal Production, Vol. 51, pp. 333-342.  Bömkes, D., Hamann, H. and Distl, O. (2004), "Estimation of genetic parameters for test day records of milk performance traits in German Improved Fawn", Archive fur Tierzucht, Vol. 47, pp. 193-202. Constantinou, A. (1985), "Genetic and phenotypic parameters for some reproduction and milk production characters of the Damascus goat", Journal of Animal Breeding and


extfieldsofstudy: Chemistry Medicine

text:
Physical, Barrier, Mechanical, and Biodegradability Properties of Modified Starch Films with Nut By-Products Extracts

Starch-based films with phenolic extracts could replace the use of petroleum-based plastics. In this study, octenyl succinate starch (OSS) films with pecan nutshell extract (PSE) or hazelnut skin extract (HSE) were prepared. The water resistance, as well as the optical, physical, mechanical, and biodegradable properties of these films, were investigated. The PSE and HSE improved the water resistance (decreasing the solubility to 17% and increasing the contact angle to 96.80°) and UV-light barrier properties of the films. For PSE and HSE, as their concentrations increased, the film rigidity decreased since these extracts acted as plasticizers. Micrographs obtained by scanning electron microscopy (SEM) depicted a homogeneous surface as a result of extracts dispersion through the polymeric matrix and the interactions between the phenolic compounds (PC) of the extracts and the OSS. The phenolic extracts from nut by-products and octenyl succinic anhydride (OSA) starch could be used to develop films to replace the conventional plastics.

Introduction
Recently, native and modified starch has become a potential alternative for petroleum-based plastics to mitigate the environmental problems associated with these materials [1]. In this context, the food industry has been looking to use starch-based materials for the production of films that act as packaging materials or as edible coatings, thereby ensuring the quality of foods [2,3]. In addition, the biomedical industry is also interested in the implementation of starch-based materials for the development of scaffolds, wound dressings, hydrogels, and nanofibers, among others [4]. Most of the materials mentioned above are produced with native starch. However, the characteristics of native starch can be greatly improved by chemical modification, giving it greater value and expanding the field of application [5,6]. Chemical modification with octenyl succinic anhydride (OSA) (Figure 1) is one of the main modifications used in the food industry because of the emulsifying properties that octenyl succinate starch (OSS) presents [7]. Blue squares indicate the inclusion zone of the OSA groups. Adapted from Sweedman et al. [7].
In the specific case of film formation, the OSA group inclusion has been shown to provide greater stability, water resistance, and flexibility to the starch films [8,9]. Another highlight of the inclusion of OSA groups in the starch structure is that they improve the encapsulation capacity of bioactive compounds. They do this by providing amphiphilic characteristics to the starch [10]. It has been reported that the addition of phenolic extracts in starch-based films could undergo changes in certain properties of the films, such as optical, barrier, mechanical, antioxidant, and antimicrobial, among others. These changes can depend on the amount and nature of the compounds, as well as on their interaction with starch [11][12][13][14][15]. Nowadays, agricultural or food by-products are considered a rich source of phenolic compounds (PC) that are not commonly used [16]. In this way, the nut industry produces a large quantity of by-products (shells, skin, others) since nuts are more often consumed in their processed form [17]. For example, the shell is the main by-product generated from pecan nuts, reaching up to 50% of the total weight of the nut production [18]. In the case of hazelnut, around 2.5% of the kernel weight corresponds to the skin, a by-product obtained from the roasting and processing hazelnut products [19]. In this context, the use of these by-products could provide added value to these nuts. It may also prevent these by-products from being considered as waste and becoming a pollution problem. Both shell and skin by-products are potential sources of PC; however, the use of the phenolic extracts from pecan and hazelnut by-products in starch-based films has lacked research. Therefore, the objective of the present work was to evaluate the effect of pecan nutshell extract (PSE) and hazelnut skin extract (HSE), at a range of low concentrations, on the characteristics and properties of OSS-based films. The use of the PC-rich extracts from pecan nutshell and hazelnut skin could define an important future application for these by-products.

Materials
Pecan nut (Carya illinoinensis (Wangenh.) K. Koch) was obtained from Grupo Alta S.A de C.V., Hermosillo, Sonora, Mexico. Shells were ground in a hammer mill (Tomas Model 4 Willey-Mill) and sifted to a particle size less than 1 mm. Hazelnut (Carylus Avella L var. Giresun) skin was obtained at the local market from Ordu, Turkey. The skins were ground in a coffee grinder and sifted to a particle size between 0.85-0.50 mm.

Preparation and Characterization of Phenolics Extracts
Phenolic extraction was carried out using the method of Liu, et al. [20], with modifications. The sample was mixed with 60% aqueous ethanol solution in a 1:30 ratio (w/v) and was sonicated for 15 min (Branson 1510, Bransonic, Danbury, CT, USA). The mix was centrifuged 15 min at 5000× g and 4 • C, and the supernatant was obtained using a vacuum filtration system. The solvent was removed using a rotavapor system (Yamato RE400, BM 200, LabConco, Kansas, MO, USA) at 45 • C. The resulting extracts were freeze-dried (Freezone 4.5, LabConco, Kansas, MO, USA) and stored at −4 • C.
Characterization of phenolic extracts consisted of the determination of total phenols and flavonoids content using the Folin-Ciocalteu [21] and aluminum chloride assay [22], respectively. For total phenols content, 30 µL aliquots of the extracts were placed in a flat-bottom 96-well microplate. Briefly, 150 µL of Folin-Ciocalteu reagent (1:10 v/v) and 120 µL of 7.5% Na 2 CO 3 were added to the phenolic extract. After 30 min incubation, absorbance was measured at 750 nm using a microplate reader (Multiskan Ascent, Thermo Electron Corporation, Dreieich, Germany). For flavonoid content, 250 µL of the extract, 1000 µL of deionized water, and 75 µL of 5% NaNO 2 were mixed and kept for 5 min. Later, 75 µL of 10% AlCl 3 was added and kept for 1 min. Then, 500 µL of 1M NaOH and 600 µL of deionized water were added and mixed to stop the reaction. From this solution, 300 µL was transferred to a flat-bottom 96-well microplate. The absorbance was measured at 492 nm with a microplate reader. Finally, a standard curve of gallic acid (total phenols) and catechin (total flavonoids) was used to express the results as gallic acid equivalents per gram of extract (EAG/g), and catechin equivalents per gram of extract (CE/g).

Preparation of Octenyl Succinate Starch
Potato starch (Sigma-Aldrich, Saint Louis, MO, USA) esterification with OSA (Dixie Chemical Company, Pasadena, CA, USA) was done, following the procedure described by Ovando-Martinez, et al. [23]. The degree of substitution (DS) was determined with 1 H nuclear magnetic resonance, using a 400 MHz Bruker Avance III HD (Billerica, MA USA), according to Whitney, et al. [24]. The DS value of the modified starch (OSS) was 0.013.

Preparation of Films
Films were produced by the casting method described by Zamudio-Flores, et al. [25] and Li, Ye, Lei and Zhao [9], with modifications. The film components are shown in Table 1. The film-forming solution was mixed, with constant stirring (1000 rpm), on a heating plate at 95 • C for 10 min. The film-forming solution was cooled down to 60 • C, and 10 mL of the extract solution was added. Both solutions were mixed for 15 min with constant stirring (1000 rpm). The final solution was cast in square plastic plates and dried at 60 • C, for 4 h, in an air convection oven. The dried films were removed from the plate, and the thickness was measured at five points using a Mitutoyo 2416F micrometer (Mitutoyo America Corporation, Birmingham, AL). The films were stored in a 50 ± 3% relative humidity (RH) chamber until their analysis.

Water Resistance Properties
The moisture content was determined by measuring the weight loss of the films according to Nouri and Mohammadi Nafchi [26], with slight modifications. First, the initial weight of the films was recorded. Then, films were dried in an oven at 105 • C for 24 h, and finally, the final weight of dried films was recorded. On the other hand, the water solubility was measured in the dried films (2 × 3 cm, previously weighed), which were placed in a flask with water (80 mL) and stirred (125 rpm, 1 h) at 25 • C. The flask content was filtered with a steel mesh (45 microns). The solid material was dried at 65 • C in the oven overnight, and the final weight was recorded. The moisture content and solubility were calculated using the following equation.
The contact angle of a droplet of water was measured to determine the surface hydrophobicity of films using a Dynamic Contact Angle Analyzer by First Ten Angstroms 125 (First Ten Angstroms, Portsmouth, VA, USA) with a CCD camera, according to the ASTM-D7334-08 [27]. The contact angle was determined with Ten Angstroms 32 Video 2.0 Software (First Ten Angstroms 2000, Portsmouth, VA, USA).
To obtain the water vapor permeability of the films, the water dish method was followed [28]. Films were placed between two metal plates with inner diameters of 5.4 cm. The metal plates with the films were placed over Petri dishes containing 20 mL of water. The separation between the water and the films was 13 mm. Using parafilm, the metal plates and the Petri dishes were held and sealed. The whole assembly was weighed with a Mettler Toledo New Classic MF analytical balance (Model No. ML203E/03). Mass, temperature, and RH were recorded at 0, 1, 3, 6, 12, 24, 48, and 72 h. Water vapor permeability was calculated according to the ASTM-E96/E96M-15 [26].

Optical Properties
Optical properties, including color, transparency, and UV-vis absorption, were obtained according to Kanmani and Rhim [29], with modifications. Color parameters (L*, a*, b*) were obtained using a MacBeth Color Eye 7000 Spectrophotometer with Pro Palette 5.0 software. Films were placed on a standard white calibration surface (L* = 95.69, a*= −0.90, and b= 1.94). The following values were measured using the CIELAB scale: L*: 0 (black) to 100 (white), −a* (greenness) to +a* (redness), and −b* (blueness) to +b* (yellowness). The color difference (∆E) was obtained according to the equation below: where ∆L = L of octenyl succinate starch film -L of octenyl succinate starch film with the extract. ∆a* = a* of octenyl succinate starch film -a* of octenyl succinate starch film with the extract. ∆b* = b* of octenyl succinate starch film -b* of octenyl succinate starch film with the extract. Transparency and UV-vis absorption of films were obtained using a Hach DR/4000 UV-vis Spectrophotometer (Hach, Loveland, CO). A rectangular piece of film (1 × 4 cm) was placed in a quartz cell, and the transmittance (T) was measured at 280 and 660 nm. Similarly, the UV-vis absorption spectrum was obtained in a wavelength range of 200 to 800 nm.

Mechanical Properties
Puncture resistance was determined according to the ASTM-D7192-18 [30] method, using a TA-XT2i Texture Analyzer (Scarsdale, NY, USA), with a 2 mm diameter flat head stainless probe (TA-52, Texture Technologies Corp., Hamilton, MA, USA), and extensibility platform (TA-108N, Texture Technologies Corp.). The films were placed between two polycarbonate disks with an inner diameter of 15 mm. The puncture was performed at 33 mm/s speed. Values were obtained using a Texture Exponent 32 software (Texture Technologies Corporation, Hamilton, MA, USA).
Tear resistance was determined according to the ASTM-D1004-13 [31] method using an Instron 5545 tensile test machine with a 100 N load cell. Testing conditions were done at a 51 mm/min strain rate, with an initial grip separation of 25.4 mm. Results were obtained with Instron Bluehill 2.1 software (Instron, Norwood, MA, USA).
Tensile properties, tensile strength, elongation at break, and Young's modulus were determined according to the ASTM-D882-18 [32]. The equipment, testing conditions, and results were done, as mentioned in the tear resistance determination.

Biodegradability
The biodegradability test consisted of measuring the C-CO 2 release by the soil microbiota, according to Anderson and Simsek [33], with slight modifications. The carbon (C) content was obtained using a Primacs TOC Analyzer (Model CS22, Skalar Inc, Buford, GA, USA). In glass jars, films (400 mg) were placed in the center of 100 g of soil (pH: 6.8 and 10% moisture). Soil without a film was used as a blank. On the top of the soil, a cup with 1 M NaOH (20 mL) was placed. The closed jars were stored in darkness and were motionless until analysis. On the 15th, 26th, 41st, 63rd, 81th, 99th, 120th, and 145th day, the cups of NaOH were removed and replaced by new cups with fresh NaOH. The removed NaOH solution from the flask was mixed with 25% BaCl 2 (5 mL) and 0.1% phenolphthalein (20 µL) and then titrated with 1 M HCl to determine the CO 2 produced. To calculate the total amount of biodegradable material in the films, the following equation was used: where A: the amount of HCl spent in reagent blank (mL), B: the amount of HCl spent in the sample (mL), C: acid molarity of HCl (M), Eq.g C-CO 2 : equivalent gram C-CO 2 . Finally, the biodegradation percentage was calculated with the following equation:

Morphological and Structural Analysis
Surface and cross-section micrographs were obtained using a JEOL JSM-6490LV scanning electron microscope (JEOL USA, Peabody, MA, USA), with an accelerating voltage of 15 kV. The surface analysis consisted of placing the films on cylindrical aluminum mounts with XYZ conductive tape (Electron Microscopy Sciences, Hatfield, NJ, USA). In the case of the cross-section analysis, films were frozen with liquid nitrogen, fractured, and placed in aluminum mounts with carbon adhesive tabs (Ted Pella Inc., Redding, CA, USA). All films were coated with gold using a Cressington 108auto sputter coater (Ted Pella Inc., Redding, CA, USA).
Interactions between OSS and PSE or HSE on films were analyzed with Fourier Transform Infrared (FTIR) spectroscopy. Extracts and film spectra were obtained using an FTIR (Thermo Scientific, Nicolet 8700, Wilmington, DE, USA) between 4000 and 700 cm -1 wavenumber range and 4 cm −1 resolution with 128 scans.

Statistical Analysis
Analysis of variance (ANOVA) with Tuckey's test was used to determine significant differences (p < 0.05) between films using IBM SPSS Statistics 23 software (IBM Corp, Armonk, NY).

Characterization of Phenolic Extracts
Total phenols and flavonoids content were significantly different (p < 0.05) between phenolic extracts, being HSE, the one with the highest concentration (Table 2). In the case of phenols content in PSE, the values were higher than those reported by Kureck, et al. [35] (186.02-275.24 mg GAE/g of extract), and similar to those reported by Contini, et al. [36] (680.3 ± 11.6 mg GAE/g of extract). On the other hand, the flavonoid content corresponded to approximately 35% and 62% of the phenolic content for PSE and HSE, respectively. That agrees with the reported by de la Rosa, et al. [37] in pecan nutshells (30%-40%) and Taş and Gökmen [38] (60%) in hazelnut skins. These results indicated that both nut by-products were a potential source of PC, and the extraction method used was suitable to obtain them. Besides, the differences in the total phenols and flavonoids content could help to explain the differences observed in the properties of the OSS films after the addition of the extracts.

The Thickness and Water Barrier Properties
The film´s thickness varied from 0.087 mm to 0.091 mm without presenting significant differences (p >0.05). Among films, OSS films with 0.100% of PSE, 0.075%, and 0.100% of HSE were significantly (p < 0.05) thicker than the other films (Table 3). According to Prietto, et al. [39], and Knapp, dos Santos, Pilatti-Riccio, Deon, dos Santos and Pinto [15], the thickness increases by the solid material provided for the extracts. On the other hand, all films had a moisture content of approximately 30%, without any significant (p >0.05) effect caused by the addition of extracts. Similar behavior has been reported for cassava starch films with rosemary extract [13]. Likewise, Feng, et al. [14] reported that the addition of tea polyphenols to hydroxypropyl starch films did not affect the moisture of the films. In this study, all of the films presented a high moisture content, which could be related to the water absorption capacity provided by the OSA groups to the starch granules [23].
In the case of water solubility, the OSS films with 0.075 and 0.100% of PSE, 0.050, 0.075, and 0.100% of HSE presented a lower solubility of about from 17%-18%, compared to the OSS film (20.3%) ( Table 3). Similar results were reported in sweet potato OSA starch films with oregano essential oil, by Li, Ye, Lei and Zhao [9]. According to Ghasemlou, et al. [40], the hydrophobic compounds, present in the essential oils, reduce the solubility of corn starch films. Nisa, et al. [41] related the decrease of solubility with the structural strength of the films. Consequently, this is caused by the interactions between the PC present in the green tea extract and in the potato starch. Therefore, the changes in the solubility properties are carried out by the hydrophilic/hydrophobic nature of the PC present in the extracts, and to the interactions between the PC and the starch structure. The contact angle values for the OSS films are reported in Table 3. Values indicated that the addition of the PSE and HSE significantly (p < 0.05) increased this parameter. According to Vogler [42], the film surface is considered hydrophilic when it presents a contact angle <65 • , and hydrophobic when it presents a contact angle >65 • . In this study, the contact angle of the films gradually increased with the addition of the different concentrations of the extracts, and only the OSS and OSS with 0.025% of PSE films presented a hydrophilic surface. Increases in the contact angle of starch films, with the addition of yerba mate, rosemary, and propolis extracts, have been reported previously [12,13,43]. This could be related to the interaction between the hydroxyl groups of the PC and the starch, whereby the amount of free hydroxyl groups, which could interact with the water, is limited. In reaction, the hydrophobicity of the films increase [14]. For this reason, the solubility of the films with the increase of PSE and HSE concentration decreased, while their hydrophobicity increased.
Finally, the addition of both extracts did not cause significant changes (p > 0.05) in the water vapor permeability of OSS films, presenting values of 2.46-2.62 (g mm/h m 2 KPa) ( Table 3). Similar behavior was reported in thermoplastic starch films with a low concentration of terpenes [44], cassava starch films with propolis extracts [11], and rosemary extract [13]. These authors mentioned that the water vapor permeability could be influenced by factors, such as the starch botanical source, the hydrophilic/hydrophobic nature of the extract components, defects in the film's surface, and others. Then, the lack of change in this property could be related to the decrease of the solubility and the increase of the contact angle due to the interactions between the PC of the extracts with the structural network of the OSS.

Optical Properties
The OSS film was colorless, whereas the addition of both extracts imparted color to the OSS films, as shown in Table 4. The a* and b* values increased with the addition of both extracts, but the OSS films with HSE presented more significant differences (p < 0.05) than the OSS films with PSE. This could be related to the highest concentration of phenols and flavonoids present in this sample ( Table 2). Contrary to a* and b* values, the L* values decreased with the addition of PSE and HSE. On the other hand, the highest color difference (∆E) was observed in the OSS film with 0.100% of HSE, whereas the lowest values were observed in the OSS film with 0.025% of PSE.
Similar behavior was reported in corn starch film with essential oils [40], cassava starch film with yerba mate extract [12], and adzuki bean starch film with cocoa nibs extract [45]. These results indicated that the type and concentration of the extract has an important role in the color of films. The transparency of the films was measured in the UV and visible regions at 260 nm and 660 nm, respectively (Table 5). Film thickness is an important factor in these values. However, due to the lack of differences between the thickness of the films (Table 3), this is not considered a determining factor in these values. According to the method used, high transmittance indicates high transparency. As can be observed, the OSS film was transparent, and the addition of PSE and HSE decreased transparency. At higher extract concentrations, a decrease in the transparency of the film was observed. A decrease of the transmittance values at the visible region (T 600 nm ) of thermoplastic starch films with high concentrations of terpenes was reported [44]. In addition, Knapp, dos Santos, Pilatti-Riccio, Deon, dos Santos and Pinto [15] and Qin, et al. [46] observed a decrease in the transparency of cassava starch films containing yerba mate extract and Lycium ruthenicum Murr anthocyanins. Like what was seen for color properties, the HSE had a greater impact on the transparency in the UV region (T 280 nm ) than the PSE. Probably, this could be a consequence of the highest concentration of PC detected in HSE (Table 2). So, it indicated that a higher PC content resulted in a higher UV-light absorption or lower percentage transmittance. According to these results, the addition of HSE or PSE to the OSS films improved their UV light barrier property. This could be demonstrated by the absorption spectra depicted in Figure 2. The control film did not present absorption in the wavelength range of 200-800 nm, indicating that light completely passed through the film. However, the OSS films with HSE and PSE showed absorption peaks between the range of 250-300 nm, and the intensity of the peaks increased with increased extract concentration. Some authors reported similar results and concluded that PC and pigments present in different extracts are responsible for the improvement of the UV-barrier properties of films [13,15,29].

Mechanical Properties
The addition of PSE and HSE affected the mechanical properties of the films (Table 6). Puncture and tear resistance significantly (p >0.05) decreased with the addition of PSE. This indicated that PC from PSE acts as a plasticizing agent in the structure of the film, and decreases the film rigidity [11,47]. The rigidity of these films was lower than that reported in cassava starch films with propolis extract [11].
On the other hand, tensile strength decreased with the addition of both extracts, but there were no significant differences (p > 0.05). The same trend has been reported in cassava starch films with propolis extracts [11], and corn starch films with red cabbage anthocyanins [39]. In this study, tensile strength values were higher than what has been reported for other films [9,39,43], indicating these films are more resistant to tearing. The elongation at break is a parameter that indicates the plastic behavior of the material. All films showed an elongation at break around 30% without significant differences (p > 0.05). The OSS films without and with PSE or HSE had more plastic behavior than other films [11,45]. It was observed that, except in the case of OSS films with 0.025 and 0.050% of HSE, the addition of extracts significantly (p < 0.05) decreased Young´s modulus. High values of Young´s modulus indicates the rigidity of the films. In this case, a decrease in Young´s modulus indicated that the plasticizing effect of HSE and PSE was due to the structural discontinuities in the polymer matrix, which is caused by interactions between PC and starch [11].

Biodegradability
The biodegradability of the OSS films with PSE and HSE in the soil is depicted in Figure 3. The OSS films showed a higher percentage of film biodegradability compared to the OSS films with PSE or HSE. After 146 days in soil, the biodegradation value of the OSS film was around 81%, while the values for the OSS films with PSE or HSE were decreasing with the increase of the extract concentration.
The biodegradation values did not exceed 71%. These results were opposite to those reported by Medina Jaramillo, et al. [48] and Kim, Baek, Go and Song [45], in which the extracts accelerated the degradation of starch films. According to these authors, the moisture and soil microbiota activity and its composition play an important role during the degradation process of films. However, they associated such an increase in the biodegradability values to the low molecular weight of some PC, which makes them more susceptible to soil microbiota. Another parameter involved in the biodegradation of films is related to their solubility. The greater the solubility, the higher the degradation by the soil microbiota. According to this, the decrease in the biodegradability of OSS films with PSE or HSE could be due to their low solubility (Table 3). On the other hand, the PC in both extracts could help to increase the integrity of the OSS films. The PC could do this by providing greater structural stability to the starch, but also increases the resistance of films to the degradation by soil microbiota [13]. Although biodegradability values were slightly higher in OSS films with PSE or HSE, they represented an option to the replacement of petroleum-based plastics, as they are much more readily biodegradable than conventional plastics.

Morphology Analysis
Surface and cross-section micrographs of OSS films by SEM are shown in Figures 4 and 5. All films showed a similar surface, highlighting the absence of pores, holes, or cracks. This indicated that the extracts are dispersed throughout the structure of the starch and do not have any effect on the structural organization of the films [14,49]. However, some irregularities were observed with the presence of the extracts and the increase of concentration. This could indicate that maybe some solids or PCs were not completely dispersed in the starch network. The cross-section was similar in all films; however, irregularities were more evident in OSS films with PSE. These irregularities can influence the mechanical properties of films [43]. In this study, such irregularities could explain why OSS films with PSE presented differences in their mechanical properties compared with those OSS films with HSE (Table 6). In addition, the absence of layers in the cross-section images could indicate that extracts were dispersed homogeneously into the starch network. With respect to the surface topography, each of the films presented variable roughness (Figures 4 and 5). The root mean square (RMS) in the OSS film was between the range of 72.03 to 166.10 nm. For OSS films with PSE, the range was from 72.03 to 484.44 nm, and for OSS films with HSE, the range was from 60.80 to 124.85 nm. The variation in the RMS indicated that some films have regions with greater roughness than others. This agreed with that observed in the surface micrographs (Figures 6 and 7). The increase in RMS could also explain the increase in water resistance (hydrophobicity) of the films, specifically the contact angle and the water solubility (Table 3). This is because roughness allows for a greater amount of air to maintain contact with the film surface, which, consequently, reduces its interaction with water molecules [12,43].

Structural Analysis
Interactions between PC and OSS were identified by comparing the infrared spectrum of the extracts (PSE and HSE), OSS films, OSS films with PSE, and OSS films with HSE (Figures 6 and 7). The PSE and HSE presented typical bands associated with PC, such as O-H stretching (3300 cm −1 ), C-H stretching for the aliphatic compounds or aldehyde groups, CH 2 asymmetric stretching (2800-3000 cm −1 region), C=C stretching for aromatic rings (1600 cm −1 ) , CH 2 bending (1444 cm −1 ), and C-O and C-C bonds stretching (1000-1400 cm −1 region). These bands were similar to those reported by other authors [50,51]. In addition, both extracts showed bands close to 1510 cm −1 (C=C from aromatic rings) [14], and in the 900-700 cm −1 region, which could correspond to hydrogen atoms from aromatic rings [52]. The HSE presented more intense bands at the wavelength of 2800-3000 cm −1 . There was a small band seen at 1715 cm −1 , which could be related to the carbonyl [52] or C=O groups [45]. Also, there was a band close to 1271 cm -1 , corresponding to C-O stretching [13].
This could be due to the differences in the PCs and their concentration in each extract. The OSS films presented typical bands from the starch molecule at 3330 cm −1 (OH groups), 2936 cm −1 (C-H stretching from the ring methane hydrogen atoms), 920-1160 cm −1 (C-O stretching) [53], and 1150 cm −1 corresponding to the glycosidic bond [52]. The bands at 1724 cm −1 and 1572 cm −1 could correspond to the C=O of the ester carbonyl groups and asymmetric stretching for the carboxylate group (RCOO-), respectively, as a result of the OSA modification [53]. The spectra of OSS films with PSE or HSE were similar, without presenting new peaks. This indicated that extracts were dispersed through the polymer matrix formed, as described by Eskandarinia, Rafienia, Navid and Agheb [43] and Feng, Yu, Zhu, Zhou, Liu, Yang, Zhou, Gao, Bao and Chen [14]. Despite this, some bands presented a slight increase in intensity in the presence of extracts. This could be a consequence of the intramolecular interaction between PC and starch through hydrogen bonds [14,45,46]. Overall, some PC were dispersed along the polymer matrix, while others were interacting directly with the starch structure mainly by hydrogen bonds.

Conclusions
The addition of pecan nutshell or hazelnut skin extracts partially improved the resistance to water by decreasing the solubility and increasing the contact angle (hydrophobicity), improving the UV-light blocking properties, and acting as plasticizing agents. It was observed that the phenolic compounds from extracts were complexed in the octenyl succinate starch matrix by hydrogen bonding, resulting in homogenous films. This indicated the potential use of octenyl succinate starch and nut by-products extracts as materials for the development of active films with applications in the food industry, such as packing material and edible films for foods that need to be stored under certain humidity conditions or UV protection. In addition, due to the antioxidant and antimicrobial properties of the phenolic compounds, these films should be evaluated using these properties against food spoilage, microorganisms, or pathogens of medical relevance to extend the application area of these films.


s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
Biology of mango inflorescence gall midges Desineura amaramanjarae recently found in Sindh, Pakistan

Gall midges are major pests in all mango cultivated areas of the globe. A complex of gall midge’s damages mango in Pakistan. The Dasineura amaramanjarae Grover, 1965 (Diptera: Cecidomyiidae), one such midge pest that feeds on mango inflorescence, was first time recorded in Sindh during 2008. They damage many parts of the plant including the leaves, shoots, bark, pre and post-flowering shoot buds, inflorescence buds, axillaries, newly formed fruit and twigs. The biology of mango inflorescence Dasineura amaramanjarae, was studied. The results showed that the adult emerged between 8.00 am to 12.00 am and matting started soon after adult emergence, mostly recorded in the morning time. The female of Dasineura amaramanjarae lay their eggs (40-50) in 8-10 minutes’ interval. After hatching, the young larvae start their feeding, destroy the flower buds and also make a small tunnel in branches. The infested buds had red-coloured petals, the flower buds become dark, dry and fall off. The inflorescence gall midges were recorded from January to June, the activity of midges was at the peak in the month of March and decreased up to June. After full feeding, mature larvae leave the inflorescences and enter to the soil for pupation. The mean percent of infested inflorescence was determined at 50.21% and 65.71% respectively. In natural conditions the development from egg to adult completed in 373.00-448.00 hours respectively. These results illustrated the biological plasticity of Dasineura amaramanjarae . Consequences of these results to elaborate IPM strategies against the mango inflorescence gall midges are discussed.

Introduction
The mango cultivated in Indo-Pak subcontinent since 4000 years, is the most relished fruit in the world. Mango, Mangifera indica L. is grown in lowland tropical and sub-tropical areas throughout the world. Its cultivation expended due to the demands has been increased day by day. Pakistan stands fourth in the ranking of mango producing and exporting countries [1][2][3]. The production of mango is undermined by more than 250 insect pests in the world [4]. In Pakistan, about 200 species of insect pests attacking mango, have been reported [5,6,7,8].
Among these, about 20 species of gall midges are known worldwide related with different parts of mango plant including leaves, shoots, bark, inflorescence buds, pre and postflowering shoot buds, axillaries, flowers, newly formed fruit and twigs [9][10][11][12]. Dasineura amaramanjarae is one of the midge pests of mango which feeds on flower buds. Female lays eggs near the stamens, larvae feed inside the flower buds and fullgrown larvae drop to the soil for pupation. In the case of severe infestation, the reduction in yield can reach 60% to 100% [13,14]. Grover reported that the adults emerge from the soil and immediately pair at the start of the flowering season [15,16]. A single female lays 40-50 eggs which are hatched in 30-36 hours, depending upon the temperature and humidity. There are four larval instars and the second and third instars cause the most of the damage. Prasad study that the pupation and diapause take place in soil under the mango tree [17]. Several larvae have been recorded from a single flower but there is only one larva was found per gall. The attacked panicles become dry and fall due to slight pressure of winds thus the loss of flower and newly set fruits occur. Prasad noted the infestation of the pest on mango fruits and reported that it is an important pest of mango that causes 60-100% fruit losses to mango production due to fall of hollowed fruits in the soil [14]. Prasad and Grover, reported the midge caused heavy fruit losses due to premature fall of mango without completing their development [13,17]. The Grover estimated losses to 100% and all most all flowers are being damaged [18,19]. Besides inflorescences, leaves are also damaged by midges indicating a complex species of midges. A list of midges in India was also provided [4]. However, no particular information on mango midges exists in Pakistan especially in Sindh [8]. Therefore, to achieve optimal outbreaks of this pest, it is necessary to understand the biology of mango inflorescence gall midges Dasineura amaramanjarae . The results of this study will provide useful information for integrated pest management programs.

Observations method
The method of observations was as under.
The observations were taken randomly from 12 branches of mango tree (one-meter length) each from the top, middle and base) visually from four directions i.e. 3 branches from each direction. The total of 12 branches were observed from each replication with 36 branches from each variety. Both healthy and infested leaves/branch, number of healthy and infested inflorescence was observed to record infestation percentage of leaves and inflorescence. The mean number of galls/leaf infestation percentage and inflorescence infestation percentage were also recorded.

Biology of midges associated with mango
The Gall midges (Dasineura amaramanjarae ) are very minute. Fragile fly, conducting research wok on biological parameters is very difficult because of their peculiar behaviour. The leaf gall midge completed all its development from egg to adult within the galls while the inflorescences midges larvae fell (after completing development) on the ground for pupation, therefore, for the biology of Dasineura amaramanjarae the following techniques were used for the appropriate result of biological parameters.

Biology of gall midges (Dasineura amaramanjarae )
In this trial, the data of biological parameters of Dasineura amaramanjarae were recorded on a daily basis. The methodology is given as under: For conducting an experiment on the biology of Dasineura amaramanjarae , five infested twigs of (15.0 cm) were observed visually from garden of different varieties. The samples were taken randomly from different sides (i.e., east, north, west and south of mango tree) and from different strata (i.e., top, middle and lower of mango trees) of each variety and brought in the laboratory. To keep the collected twigs, live/fresh, the proximal end of each twig was kept in small jars separately by passing it through the small holes in the lids of the jar (7.5 cm) so as the end remain dipped in the water present in the jars. The jars were then placed in a large cage (60×60×60cm) in the laboratory at room temperature of (25±2 0C). Meanwhile, pots (3 in number) of the new and fresh mango seedlings (22.5cm) were placed in another cage of the same size. The newly emerged adult of Dasineura amaramanjarae (two males and one female) from the infested twigs were collected by aspirator and released them in the cages having seedling for about 10 -15 days to record fecundity of the Dasineura amaramanjarae . The seedlings kept in the cage were examined twice a day for any egg lying on the leaves. Since the egg marks i.e. pinkish-red spots observed sufficient in number on the leaves of the seedling they were replaced with new ones in the cage for further culture needed for the experimentation. The leaves of seedling were examined under a microscope to count the number of egg onto them. Thereafter the seedlings were shifted in the field and covered with muslin cloth. The observations were carried out on daily basis. Five galls from these seedlings were made open daily to examine larval growth under a microscope. The opened galls after examination were marked with a ball-point pen and discarded. In this way, five galls of the same cohort were opened, examined and discarded daily to record time span of each life stage. To record adult emergence and sex ratio the cohort was developed separately with the same procedure; however, none of the galls was opened/ discarded till adult emergence and the pots were kept in a tray containing water to count adult that died after their longevity became over. The experiments of biological parameters were kept to continue for four generations from March to October.

Biology of inflorescence Dasineura amaramanjarae
In this trial, the data of biology of the inflorescence of Dasineura amaramanjarae were taken on a daily basis for 5 months from February to June. The details of the methodology are as under: The 10 infested inflorescences of ~20cm were brought in the laboratory; the proximal end was dipped in water present in small jars (7.5 cm x 3 dia) through the hole in their lids, which then placed in big jars (40×15 cm). The fallen larvae were taken and placed in a small jar containing sterilized soil for pupation. After emergence of adults, one pair (male and female) were released onto newly brought inflorescence in small jars. Before releasing the adults, onto inflorescence, the inflorescence was thoroughly examined for any egg-laying prior in the field. In this way, 3 jars were used (one jar was considered on replication). The jars were covered with muslin cloth for proper ventilation. The moist blotting papers were kept in the bottom of jars to collect and count the fallen larvae, which completed their larval life, and were to transform themselves into pupae. The fallen larvae immediately transferred into small jar containing sterilized soil for pupation. Through this procedure, the data on egglaying, larval period, pupal duration, emergence of adult, adult longevity and sexratios were recorded on a daily bases.

Statistical analysis
The raw data of all the sampled sites from the mango field orchards of the two consecutive years were transferred in an electronic format in a spreadsheet layout (Microsoft excel, 2016). The data was finally analyzed to calculate important value indices from all the sampling sites. The data thus collected were subjected to statistical analysis using analysis of variances, and randomized complete block design (RCBD), by using software STATISTICS-8.1 with three replications. The difference among the treatments means was compared by LSD (Least Significance Difference) test.

Results and discussion
Studies carried out on the biology of inflorescence Dasineura amaramanjarae which included the time of adult emergence, number of eggs, hatching period, larval period first instar, 2 nd instar, 3 rd instar, 4 th instar, pupal period, (Adult emergence period), sex ratio, mortality age, survival percentage and total life cycle period. The Dasineura amaramanjarae was first time recorded from Sindh, Pakistan. It was found in all the areas surveyed in Sindh including commercial and non-commercial orchards mentioned above. The specimens were identified by the taxonomist. We examined hundreds of flower buds with a special magnifying glass and found more than 50 to 70% of total flowers infested with Dasineura amaramanjarae nourishing inside the buds, different numbers of the larvae feeding per flower were observed. They became red pinkish at final stage and pupated in the soil within 1-2 days and red-coloured adults emerged in 3-7 days which survived for 3-4 days. Females were larger, as compared to adult Males.

Number of eggs / female
After ovipositional, the egg hardens and may change color. Other color changes, especially darkening, are visible before hatch. Fig. 1 showed that there was a highly significant difference between months. The data further showed that the maximum number of eggs laid/female was recorded in the month of April followed by the month of March and the minimum number of an egg laid/female was recorded in the month of February.

Hatching period
In the biology of Dasineura amaramanjarae, there was a highly significant difference between months. The data in (Fig. 2) showed that the maximum period of hatching was found in the month January and minimum period of hatching was found in the month of April.

st Instar larvae
The major role of larvae in the life cycle is to feed and grow. Consequently, their structural and temporal patterns of development are related largely to the need to survive in particular environments (generally chosen by ovipositing females) and to acquire and use the food resources there. In many species, first instars differ from later instars, but in this case, the data given in (Fig. 1) showed that there was a highly significant difference between months. The maximum period of 1 st instar larvae was found in the month January and minimum period of 1 st instar larvae was found in the month of April.

nd Instar larvae
The data presented in (Fig. 1) showed that there was a highly significant difference between the months. The maximum period of 2 nd instar larvae was found in the month of January and February while the minimum period of 2 nd instar larvae was found in the month of April.

rd Instar larvae
The data (Fig. 1) showed that there was a highly significant difference between months. The maximum period of 3 rd instar larvae was found in the month of January and February while the minimum period was found in the month of April. 4 th Instar larvae Once feeding ends, the final-instar larva enters the pre-pupal stage. The data (Fig. 1, and 2), showed that there was a highly significant difference between months. The maximum period of 4 th instar larvae was found in the month April and minimum period of 4 th instar larvae was found in the month of March.

Pupa period (adult emergence period)
The data (Table 1, Fig. 2) showed that there was a highly significant difference between months. The maximum period of pupa was recorded in the month April and minimum period of pupa was recorded in the month of March respectively.

Adult longevity
The results showed that there was a significant difference between months ( Table 2, Fig. 1). The maximum adult longevity was found in the month of April and the minimum period of adult longevity was found in the month of March. There was a positive correlation between egg hatching and in total life period.

Total life cycle period
The data ( Table 2, Fig. 1) showed that there was a highly significant difference between time periods in different months. The maximum life cycle period was found in the month of April and the minimum life cycle period was recorded in the month of March. It means that there was an impact of temperature on the duration life cycle. This showed that when the temperature decreased the life cycle will be decreased when temperature increased the duration of the life cycle will be increased. followed an aggregated pattern of distribution numbers of the larvae were significantly higher on the southern side of the trees than all other horizontal sides and significantly less at 6m height of tree canopy [3]. The adults of Dasineura amaramanjarae emerged in February. Larvae fed inside the buds at the base of stamen and carpel. Numbers of the larvae feeding per flower in different ranges. They dropped on to the soil under the mango tree for pupation. Dasineura amaramanjarae was noted from February to April when flowers were available and were not found from May to January on of mango trees. During the inactive period, they were not recorded on malformed inflorescences on mango trees and flowers of other nearby areas of experimental orchard. Their pupae were found in the soil under a mango tree canopy. In a study, larvae of Dasineura amaramanjarae damaged mango trees by feeding on buds and flowers [22]. Numbers of larvae were observed in severe infestation. Although the emergence of adults varied in different years yet was synchronized with flowering on mango [23]. Larvae of Dasineura amaramanjarae overwintered in soil under mango trees [13, 15, 24, 25]. These components are essential for developing an IPM program.

Conclusion
In the light of this study as Dasineura amaramanjarae was newly found in Sindh region. The Dasineura amaramanjarae go through four distinct stages during their life cycle. Knowing the different stages of the gall midges life will help you prevent Dasineura amaramanjarae, and also help you select the right time, and practices for your needs, if you decide to use them. To understand the biology of Dasineura amaramanjarae is very essential for developing an IPM program. Our findings will deliver useful information to pest managers of mango orchards.