s2fieldsofstudy:
Agricultural And Food Sciences
Biology

text:
BIOCHEMICAL AND HISTOLOGICAL INDICATORS OF BLOOD AND M. LONGISSIMUS DORSI OF YOUNG BULLS OF KAZAKH WHITE-HEADED BREED OF DIFFERENT GENOTYPES BY THE CAPN1 AND GH GENES

The use of molecular genetic markers for productivity is one of the modern approaches in breeding beef cattle. The article presents the results of the studies on the influence of genotypes by the calpain (CAPN1) and growth hormone (GH) genes on live weight, fatty acid composition of blood plasma, quantitative and qualitative indicators of meat of young cattle of Kazakh white-headed breed. It was established that animals of the homozygous geno-types СС–CAPN1 and VV-GH had a larger live weight and its average daily gain on the 240th and 365th days. Their superiority over the GG and LL genotypes was 13.8% (P<0.05) and 6.9%; 14.6% (P<0.05) and 6.9%, respectively. The carriers of the desirable alleles in the homozygous state had the sum of unsaturated fatty acids higher by 6.3% and 7.1%, respectively, than the young bulls, in which genotypes they were absent. The predominance of unsaturated fatty acids over saturated fatty acids also determined lower values of lipid me-tabolism direction index (0.92 and 1.00) in the animals with the desirable genotype. Muscle tissue of animals of the СС and VV genotypes was characterized by a higher level of protein, fat by 0.28–2.13 abs. per cent, energy value, and the quantity of muscle fibers with a smaller diameter per unit area compared to the GG and LL genotypes by on average 8.7% and 25.5% (P<0.01). Due to the greater number of interfiber fat inclusions, muscle tissue of the desirable genotypes received higher marbling scores. The obtained data testify to the prospects of replication of animals, which genotype has the desirable alleles for further improvement of traits of meat productivity of Kazakh white-headed breed. Funding: The study was financed by the grant of the Russian Foundation for Basic Research (Project No.19–316–90061/19). FOR CITATION: Selionova M. I., Plakhtyukova V. R. Biochemical and histological indicators of blood and m. longissimus dorsi of young bulls of Kazakh white-headed breed of different genotypes by the CAPN1 and GH genes. Theory and practice of meat processing. 2020; 5(2): 20–25. DOI: https://doi.org/10.21323/2414–438X–2020–5–2–20–25. Marina I. Selionova*, Viktoria R. Plakhtyukova Russian State Agrarian University — Moscow Timiryazev Agricultural Academy, Moscow, Russia DOI: https://doi.org/10.21323/2414–438X–2020–5–2–20–25

Introduction
One of the main tasks in selective breeding is selection and breeding of animals with targeted economic traits. This result can be achieved by using genetic markers for productive traits. The use of genetic markers in beef cattle selection in combination with the main zootechnic methods for animal breeding make it possible to significantly accelerate the process of creation of herds with genetically preset potential of high productivity [1,2,3].
The calpain gene (CAPN1) can be considered one of the candidate genes that are directly associated with meat tenderness and marbling. The mechanism of its action consists in the fact that the calpain system creates conditions for uniform distribution of intramuscular fat between fibers on the basis of calcium-dependent cysteine protease due to decomposition of the so-called Z-discs of skeletal muscles and weakening of bonds between muscle fibers, which ensures the so-called marbling and corresponding meat tenderness and juiciness [4,5].
The biological effect of the growth hormone gene (GH) consists in regula-tion and stimulation of many metabolic processes. The hormone (somatotropin) controlled by GH is produced by the anterior lobe of the hypophysis and plays a key role in carbohydrate and lipid metabolism [6,7,8].
It is known that lipids are very active in terms of metabolism; they act not only as a source of energy but also have more significant functions -structural and regulatory. Lipids take part in the metabolic processes mainly as free saturated and unsaturated fatty acids. It is also necessary to note that fatty acids not only play a key role in the vital activities of the body and formation of productivity, but several of them act as signal molecules involved in the process of expression of individual genes; that is, the development of fat in fatty tissue of ruminants is operated and modulated by certain genes [9].
Several studies by foreign and Russian scientists revealed that the C allele of the CAPN1 gene and the V allele of the GH gene are desirable for selection regarding an increase in quantitative and qualitative indicators of beef cattle productivity [10,11,12,13,14,15,16].
Another aspect in assessment of the action of certain genes with regard to farm animal productivity is a complex analysis of quantitative and qualitative indicators of final products. For beef cattle, these indicators are an amount of produced meat, its nutritional properties, energy value, quality and safety, consumer properties, which are also determined by chemical, biochemical and microstructural indicators [17,18].
In this connection, the study of the fatty acid composition of blood plasma, meat productivity of beef cattle of Kazakh white-headed breed of different genotypes by the calpain and growth hormone genes, including biochemical and microstructural analysis of muscle tissue is of scientific and practical interest.
The above mentioned was a substantiation of the aim of the present work: to study the biochemical and histological indicators of blood and m. longissimus dorsi of young bulls of Kazakh white-headed breed of different genotypes by the CAPN1 and GH genes.

Materials and methods
The studies were carried out in the laboratory of immunogenetics and DNA technologies, laboratory of veterinary medicine and laboratory of morphology and quality of products of the All-Russian Research Institute of Sheep and Goat Breeding (VNIIOK) -the branch of the Federal State Budgetary Scientific Institution North Caucasian Agrarian Center (North Caucasian FNAC). The object of the research were young bulls of Kazakh white-headed breed (n=93) of SPK Kolhoz Gigant (Blagodarensky district, Stavropol Krai).
The fatty acid (FA) content in blood plasma was determined by the method of gas-liquid chromatography in a form of methyl esters on a gas chromatograph «Cristal 200» with a capillary column HP-FFAP 50 m, 0.32 mm, 0.5 pm (USA). Fatty acid methyl esters were obtained using the method by Morrison and Smith. FAs were identified using the standards of Sigma and Fluka. Quantitative determination of FAs was carried out with the use of Chromatec Analytic software [19]. The lipid saturation index -a ratio between the sum of saturated fatty acids and the sum of unsaturated fatty acids, was used as an integral indicator that also characterizes the direction of lipid metabolism.
Analysis of the level of total lipids, cholesterol and glucose in blood was carried out according to the methods given in «Methods of veterinary clinical laboratory diagnostics: reference book» [20].
Dynamics of live weight of young bulls of different genotypes was deter-mined by their weighing at birth, on the 240 th and 365 th days. An average daily gain was calculated by the difference in the values and period of measurement.
During control slaughter of three animals of each genotype at the age of 12 months, samples of m. longissimus dorsi were taken for chemical analysis and histological investigations. An amount of moisture was measured according to GOST 9793-2016 «Meat and meat products. Methods for determination of moisture content» [21]. A meat sample was minced in a homogenizer, thoroughly mixed, dried in a drying oven at a temperature of 103±2 °C until constant air-dry mass. The mass fraction of moisture was determined by the difference between the sample weight before and after drying and expressed in percent. An amount of fat was determined according to GOST 23042-2015 «Meat and meat products. Methods of fat determination» [22], by extraction with petroleum ether from a minced and dried sample in the Soxhlet apparatus with the following removal of the solvent and drying of fat until the constant weight. An amount of protein was determined according to GOST 25011-2017 «Meat and meat products. Protein determination methods» [23] using the Kjeldahl method by an amount of nitrogen formed in min-eralization of organic substances in a minced and dried sample and its conversion to protein using the coefficient of 6.25. Caloricity was determined by calculation using V. M. Aleksandrov's equation: where C is caloricity, kcal; D, A, F -an amount of dry matter, ash and fat, respectively.
For histological investigations, the samples were fixed in the 10% solution of neutral formalin and then densified by pouring into gelatin. Sections with a thickness of 7-8 µm were obtained on a freezing microtome. Structural components of muscle tissue were revealed by the methods of staining using Carazzi's hematoxylin with sudan III according to Ehrlich. The muscle bundle area, the quantity of muscle fibers in a bundle, a diameter of a muscle fiber and bundle, a ratio of muscle and connective tissues were determined according to GOST 19496-2013 «Meat and meat products. The method of histological investigation» [24].
The obtained data were subjected to biometrical processing using Microsoft Office and BIOSTAT. Based on the mean values and standard errors, significance of difference between the mean values was calculated using the Student test.

Results and discussion
As a result of the performed genotyping, it was established that the frequency of the CC, GG, CG genotypes of the CAPN1 gene was 0.06, 0.81 and 0.13, respectively; the frequency of the VV, LL, LV genotypes of the GH gene was 0.31, 0.51 and 0.18, respectively. The revealed polymorphism allowed dividing animals according to the established genotypes and studying the biochemical composition of blood plasma in the young bulls with medium indicators of productivity according to a corresponding genotype, as well as performing chemical analysis and histological investigations of m. longissimus dorsi.
It was established that the young bulls of Kazakh whiteheaded breed of the homozygous СС and VV genotypes of the CAPN1 and GH genes had 6.3% and 7.1% higher sum of unsaturated fatty acids in blood plasma, respectively, compared to the homozygous GG and LL variants. Predominance of unsaturated fatty acids over saturated fatty acids also determined the lower values of the index of lipid metabolism direction in the СС and VV genotypes: 0.92 and 1.00 versus 1.04 and 1.13 in the GG and LL genotypes ( Table 1 and Table 2).
Comparative analysis of the content of total lipids, cholesterol and glucose in blood revealed that the total amount of lipids and glucose in blood was lower by on average 11.2% with on average 12.8% higher cholesterol level in the СС and VV homozygotes and the CG and LV heterozygotes of the CAPN1 and GH genes compared to the GG and LL homozygotes (Table 2). Therefore, the obtained data indicate that the intensity of lipid metabolism in youngsters of Kazakh white-headed breed depended on a genotype by the CAPN1 and GH genes. It can be assumed that the carriers of the desirable C and V alleles of the CAPN1 and GH genes in the homo-and heterozygote state used the energy components of blood with higher intensity upon realization of the biosynthetic processes.
The revealed regularities were reflected in the value of live weight and average daily gains in different periods of ontogenesis. The carriers of the C allele of CAPN1 and the V allele of GH were superior at birth, weaning and 12 months of age. With that, the largest differences were found between the animals that were homozygous by these alleles and the young bulls, in which genotype they were absent. The difference on the 240 th and 365 th days was 13.8% (Р < 0.05) and 6.9%; 14.6% (Р < 0.05) and 6.9%, respectively ( Table 3).
Comparison of the indicators of chemical analysis of m. longissimus dorsi revealed that the CC and VV genotypes were superior to other genotypes in the protein and fat content by 0.28-2.13 abs. percent.
Different protein and fat content in meat from animals of different genotypes was reflected in its caloricity. For example, the highest level of caloricity was observed in m. longissimus dorsi of the homozygous CC genotype of CAPN1 and the VV genotype of GH (118.9 and 122.5 kcal, respectively), which was 8.2% and 9.3% (Р<0.05) higher than the similar indicator in meat of the homozygous GG and LL genotypes or on average by 8.7% (Table 4).     The muscle fiber diameter was lower by 4.23 µm or 11.9% and by 6.82 µm or 16.8%, respectively, in the animals with the desirable genotype at high significance (Р<0.001). Desirable genotypes also had an advantage in terms of the value of the marbling coefficient: the difference was 12.6% in the CAPN1 gene, 15.1% in the GH gene. At the same time, the content of connective tissue was higher by 2.13 and 3.34 abs. percent in the carriers of the undesirable G and L alleles in the homozygous state.

Conclusion
As a result of DNA typing, the frequency of desirable genotypes by the CAPN1 and GH genes among youngsters of Kazakh white-headed breed was established.
The carriers of the desirable СС and VV genotypes in the CAPN1 and GH genes had the largest live weight at the age of 8 and 12 months. Their superiority over the GG and LL genotypes was 13.8% and 6.9%, 14.6% and 6.9%, respectively.
The intensity of lipid metabolism in the young bulls of Kazakh white-headed breed depended on a genotype in the CAPN1 and GH genes. The carriers of the desirable alleles (С and V) in the homo-and heterozygote state in the CAPN1 and GH genes used the energy components of blood with higher intensity upon realization of the biosynthetic processes, which is indicated by their superiority in terms of the sum of saturated and unsaturated fatty acids and the index of lipid metabolism direction.
Muscle tissue of the animals of the СС and VV genotypes was characterized by the highest content of protein, fat and, respectively, energy value compared to the GG and LL genotypes. Meat from the young bulls of the desired genotypes had higher scores for marbling due to a larger quantity of interfiber fat inclusions.
Therefore, genotyping of youngsters of Kazakh whiteheaded breed by the CAPN1 and GH genes, selection of animals with desirable genotypes and their replication through the targeted selection of parent pairs will allow not only increasing meat productivity of a herd, but also will facilitate production of high quality beef.


s2fieldsofstudy:
Agricultural And Food Sciences
Chemistry

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

text:
Effects of drying methods on the structure and emulsifying capacity of egg yolk lecithin

The purpose of this study was to investigate the effects of different drying methods, including ultrasonic vacuum drying, vacuum drying, vacuum freeze-drying, hot-air drying and spray drying, on the structure and emulsifying capacity of egg yolk lecithin based on Raman spectra. The results showed that ultrasonic vacuum drying and spray drying can induce the vibration of C–N bonds in the polar O–C–C–N head skeleton of egg yolk lecithin. The shift of the peak attributed to the C–N bond from 717 cm to 774 and 772 cm indicated that the vibration of some C–N bonds in the O–C–C–N skeleton had transformed from gauche to trans. Ultrasonic vacuum drying exerted the most intense effect on the C–C skeleton of egg yolk lecithin, with the greatest vibration peaks at 1062 cm, 1128 cm, and 1097 cm in the Raman spectra of egg yolks. Specifically, it relieved gauche vibration and strengthened trans vibration in the C–C skeleton. Hence, the Igauche/Itrans ratio of the egg yolk lecithin processed through ultrasonic vacuum drying decreased. Ultrasonic vacuum drying and spray drying decreased the I2850/I2878 ratio of the vibration peak of C–H bonds in the lipid chains of egg yolk lecithin. The weakening of the symmetric stretching vibration of the C–H bond and the strengthening of antisymmetric stretching vibration indicated that orderliness among the molecular chains of lipid bilayer membranes had increased, whereas liquidity had decreased. The emulsifying capacities were highly significantly different among various egg yolk lecithin samples, in which the highest emulsifying capacity (49.58 m/g) was shown for the egg yolk lecithin prepared through vacuum freeze-drying, and ultrasonic vacuum drying produced the lowest emulsifying capacity (14.77 m/g). This study demonstrated that ultrasonic vacuum drying and spray drying drastically affected the structure of egg yolk lecithin. The appropriate drying method can be selected based on sample volume and production situation.

Introduction 
The yolk accounts for 28%-29% of the total mass of an egg. Solids contribute 50% of the total mass of egg yolks, and 32% of the total mass of egg yolk solids is attributed to lipids. Phospholipids, such as lecithin, account for 30% of the total egg yolk lipids [1] . Egg yolk lecithin is a composite phospholipid, and its major functional constituent is phosphatidylcholine (PC), which accounts for 70%-80% of the total egg yolk lecithin [2] . Egg yolk lecithin possesses high nutritional value and has been called "the gold for brain strengthening and a great nutrient for health" by British scientists [3] . It releases choline after digestion by the human body. Choline reaches the brain through blood circulation. It can prevent intellectual deterioration and enhance memory. Moreover, it facilitates hepatocyte regeneration, increases the plasma protein content, promotes metabolism, and boosts immunity. In addition, it is a natural surfactant and has numerous critical physiological functions. Egg yolk lecithin is applied as a pharmaceutical accessory. For example, it is used as an emulsifier in fat emulsion injections. It has also been utilized to produce liposomes for targeted drug delivery [4] . Egg yolk lecithin is mainly extracted from egg yolk powder. Hence, drying methods for the production of egg yolk powder must ensure that the structure of egg yolk lecithin is maintained in the final product. The emulsifying capacity is one of the key functional properties for egg yolk lecithin and is closely related to its structure which is influenced by different processing methods [4] . Moreover, the suitable detection method should be applied to analyze the quality of dried egg yolk powder.
Infrared spectroscopy and Raman spectroscopy are common methods used to test the structures of materials. Although these methods function through different mechanisms, both acquire spectra on the basis of molecular vibration and can complement each other. Infrared spectroscopy is mainly employed to identify groups of molecules, whereas Raman spectroscopy is mainly applied to observe the skeletons of molecules [5,6] . Infrared spectrometry requires grinding solid samples with KBr and complicated sample preprocessing procedures.
By contrast, Raman spectroscopy is a nondestructive detection method that does not require sample preprocessing. Herrero announced that the protein structure in muscle food could be precisely monitored by Raman spectroscopy [7] . Czaja et al. verified that gluten structure could be well characterized by Raman spectroscopy [8] . Therefore, Raman spectroscopy could be widely used as a convenient, rapid, and accurate method for the structural characterization of samples.
Existing studies on egg yolk lecithin mainly focused on the extraction technologies and the functions of egg yolk lecithin. Liu et al. [9] found that a high voltage pulse electric field could improve PC extraction efficiency without drastically affecting the detection of the molecular structure of the PC through mid-infrared spectroscopy. Palacios et al. [10] demonstrated that the egg yolk lecithin extraction efficiencies of the non-deoiling and deoiling techniques with ethyl alcohol as a solvent were 23.9% and 13.5%, respectively.
Asomaning et al. [11] optimized the enzymatic extraction of egg yolk lecithin by introducing transesterification. Akashi et al. [12] discovered that the addition of egg yolk lecithin to enteral formulations could protect emulsifiers from gastric acid damage, promote lipid adsorption, and ameliorate diarrhea. Nevertheless, the effects of drying methods on the structure of egg yolk lecithin and the effects of structural modifications on emulsifying capacity have not yet been reported.
In the lecithin industry, spray drying is the current commercial method for drying egg yolk lecithin, but egg yolk lecithin is easily oxidized or denatured during drying and the quality of the dried product is usually unsatisfactory. Thus, some alternative drying methods with low pressure and low temperature could be considered. In this study, egg yolk powder was processed from liquid egg yolk through five different drying methods including ultrasonic vacuum drying, vacuum drying, vacuum freeze-drying, hot-air drying and spray drying. Egg yolk lecithin was extracted from egg yolk powder samples prepared through different methods and the emulsifying capacities were quantified. The effects of drying methods on the polar O-C-C-N + head skeleton, C-C skeleton, and lipid bilayer membrane liquidity of egg yolk lecithin were investigated through laser Raman spectrometry.
The relationship between the emulsifying capacity and the structure of egg yolk lecithin was discussed.

Test materials
Fresh eggs were procured from a market in Luoyang, China. Standard egg yolk lecithin samples (purity>99.3%) were purchased from Shanghai Yuan Ye Biotechnique Co., Ltd. The analytical agent SDS was obtained from Luoyang AokeHuabo Instrument Distribution. Peanut oil (Lu Hua 5S pressing peanut oil) was acquired from a supermarket in Luoyang City.

Processing of egg yolk powder
The processing of egg yolk powder is illustrated in Figure 1.

Drying methods
After the removal of sugar impurities, the liquid egg yolk samples were dried with five different methods, cooled, and ground. The powders were passed through a 120 mesh sieve to obtain the final egg yolk powder samples. The moisture content of dried egg yolk powder samples was less than 5%. The drying methods were performed under the following conditions: Ultrasonic vacuum drying [13] : An ultrasonic system was installed into a vacuum dryer (DZF-6050, Yiheng Scientific Instrument Company, Shanghai, China) to achieve an ultrasonic vacuum drying device. The ultrasonic system was self-fabricated by Henan University of Science and Technology and mainly consisted of an ultrasound receiver, an ultrasonic transducer, and an ultrasonic generator. The schematic of the ultrasonic vacuum dryer is demonstrated in Figure 2, and detailed information and parameters of the ultrasonic system and the ultrasonic vacuum dryer could be seen in the literature [13] . Liquid egg yolk (500 g) was poured into the material tank of the ultrasonic system. The energy density of the ultrasonic wave, ultrasonic frequency, vacuum pressure, drying temperature, ultrasonication time, and drying time were set as 2 W/g, 28 kHz, 0.01 MPa, 50°C, 3 h, and 3 h, respectively. Vacuum drying [14] : Liquid egg yolk was poured into a disk to form a layer with a thickness of 4 mm. The disk was then placed into a vacuum drying oven (DZF-6050, Yiheng Scientific Instrument Company, Shanghai, China) with the vacuum pressure, drying temperature, and drying time set as 0.01 MPa, 50°C, and 7 h, respectively.
Vacuum freeze-drying [15] : Liquid egg yolk was poured into a disk and then covered with a perforated piece of thin plastic film. The liquid was frozen for 1h under −30°C. The egg yolk was dried in a vacuum freeze-dryer (LGJ-10D, Sihuan Scientific Instrument Company, Beijing, China) after complete freezing. The thickness of the material layer, temperature, drying room pressure, and drying time were 4 mm, −60°C, 30 Pa, and 24 h, respectively.
Hot-air drying: Liquid egg yolk was poured into a disk to form a layer with a thickness of 4 mm and then dried for 24 h in a blast-drying oven (DHG-9420A, Yiheng Scientific Instrument Company, Shanghai, China) at the constant temperature of 60°C .
Spray drying [16] : Liquid egg yolk was poured into a constant-temperature water bath and preheated to 25°C before drying in a spray-drier (SP-1500, Shunyi Experimental Apparatus Company, Shanghai, China).
The spraying flow rate was 500 mL/h. Inlet temperature and outlet temperature were held at 120°C and 90°C , respectively.
During the drying processes, the sample was taken out and weighed every 20 min, and the drying ended until the sequential two mass readings are approximately the same, with the corresponding moisture content of around 5% for the dried sample. All drying experiments were performed in triplicates.

Raman spectra of standard egg yolk lecithin samples and egg yolk powder
A confocal microscopic Raman spectrometer (XploRA ONE, HORIBA JOBIN YVON S.A.S, France) was applied to acquire the spectra of the egg yolk lecithin samples and egg yolk powder. The spectrometer was equipped with a 50×objective telephoto lens, 785 nm laser, and CCD detector. The spectrometer was operated with the laser power, spectral resolution, integral time, integration number, and spectral scanning range of 50%, 4 cm −1 , 20 s, 2 and 200-3500 cm −1 , respectively [17] .
In each experiment, 200 points were selected randomly for scanning. The Raman spectra of standard egg yolk lecithin samples and egg yolk powder were recorded under the above conditions. Peaks attributable to egg yolk lecithin in the spectra of egg yolk powder dried through different methods and fresh liquid egg yolk were indexed to the characteristic peaks of standard egg yolk lecithin. The Raman spectra of fresh liquid egg yolk were used as the control group to analyze the effects of drying methods on the structure of egg yolk lecithin.

Extraction of egg yolk lecithin
Referring to previous studies [18] , egg yolk lecithin was extracted by ethanol. Egg yolk powder of 30 g was weighed into a 250 mL beaker with the addition of 95% ethanol of 4 times volume, was stirred with a homogenizer (Angni Instrument Company, Shanghai, China) at a speed of 500 r/min for 15 min at 60°C water bath, and was centrifuged at 3600 g for 8 min, and then the supernatant was poured out. The precipitate was extracted again using the same method described above. The supernatant was combined and placed in a refrigerator at 4°C for 12 h to precipitate a neutral fat.
After filtration, the filtrate was concentrated by a rotary evaporator (Yarong Biochemical Instrument Company, Shanghai, China) to obtain egg yolk lecithin.

Emulsifying capacity test of egg yolk lecithin
This test was performed according to the references of Marefati et al. [19] and Ishii [20] . In this test, 30 mL of distilled water was combined with 10 mL of peanut oil, into which 300 mg of egg yolk lecithin was added. The mixture was then homogenized for 1 min at 16000 r/min. Subsequently, 0.2 mL of the homogenate was collected from the bottom of the container by using a transfer pipette and mixed with 40 mL of 0.1% SDS. The optical density of the mixture was quantified at 500 nm by using a UV-visible spectrometer (Shimadzu, Japan) with a 0.1% SDS solution as the blank control and three parallel samples. Emulsifying capacity was calculated as follows: where, EAI is the emulsifying capacity; n is the dilution ratio (200 in this experiment); ρ is the mass concentration of egg yolk lecithin (g/mL); φ is the volume fraction of oil (1/4 in this experiment), and A 0 is the optical density of homogenized samples at 500 nm. 2.2.6 Spectral preprocessing and data analysis All Raman spectra of 200 points were recorded and presented as the mean value. Data analysis was carried out with Origin 8.5 software. The statistical analysis including analysis of variance and multiple comparisons was carried out using SAS 6.0 software.

Raman spectra of standard egg yolk lecithin samples
The Raman spectra of standard egg yolk lecithin samples are shown in Figure 3.  Table 1 provides the affiliations of peaks in the Raman spectra of the standard egg yolk lecithin samples shown in Figure 3.  [15] Notes: ν is stretching vibration; νsym is symmetric stretching vibration; νasym is asymmetric stretching vibration; δd is deformation vibration; ρt is torsional vibration; δS is shear vibration; ρW is rocking vibration.
Egg yolk lecithin is composed of glycerin, choline, phosphoric acid, and saturated and unsaturated fatty acids. The molecular structure of egg yolk lecithin can be divided into three regions on the basis of spectral features: the hydrophobic chain consisting of C-H stretching modes, CH 2 deformation, CH 2 twisting, and C-C stretching modes, the interfacial region containing C=O stretching, and the polar headgroup region comprising of a band of PO 2 − asymmetric stretching and C-N stretching [15,26] .
As shown in Figure 3 and Table 1, C-N stretching vibration occurred at 717 cm −1 .
The vibration of the carbonyl group in the interfacial region gave a weak band at 1736 cm −1 .
The stretching vibration region of the C-C skeleton was observed in the 1000-1200 cm −1 region. In this region, peaks at 1062 cm −1 and 1128 cm −1 were ascribed to the vibration peaks of the trans conformation of the bilayer C-C skeleton, whereas the peak at 1096 cm −1 was attributed to the stretching vibration peak of the gauche conformation of the C-C skeleton and PO 2 − [23] .
The deformation peak of CH2 was located at 1439 cm −1 , and the peak at 1298 cm −1 belonged to the twisting mode of CH 2 .
The stretching vibration mode of C-H was located in the 2800-3000 cm −1 region. The vibration peaks at 2850 and 2882 cm −1 were contributed by the symmetric and asymmetric stretching vibrations of methylenes, respectively. The peak at 2932 cm −1 was ascribed to the symmetrical stretching vibration of terminal methyls. The peak at 3035 cm −1 was attributed to the asymmetric stretching vibration of choline methyl [27] .

Raman spectra of egg yolk powder and fresh liquid egg yolk
The Raman spectra of egg yolk powder and fresh liquid egg yolk samples are shown in Figure 4.   Figure 4 illustrates that the spectra of egg yolk powder and fresh liquid egg yolk samples presented the characteristic peaks of standard egg yolk lecithin samples. In addition, the peak intensity and positions in the Raman spectra of egg yolk powder prepared through various drying methods were different from those in the Raman spectra of fresh liquid egg yolk samples.

Effects of drying methods on the polar O-C-C-N + head skeleton of egg yolk lecithin
The symmetric stretching vibration of the choline head base C-N is sensitive to the conformation of the O-C-C-N + skeleton. When O-C-C-N + is in its gauche conformation, the vibration peak of C-N appears at 717 cm −1 . When O-C-C-N + is in its trans conformation, the vibration peak of C-N moves to 770 cm −1 [28,29] . The orientation of the polar head changed from the parallel membrane plane to the vertical membrane plane when the conformation of O-C-C-N + transformed from gauche to trans. Therefore, the effects of the drying method on the polar headgroup region of egg yolk lecithin can be determined based on the vibration peak position of C-N.
As illustrated in Figure 5, ultrasonic vacuum drying and spray drying drastically affected the conformation of the polar headgroup region of egg yolk lecithin. A vibration peak was developed at 774 cm −1 in the spectra of the sample prepared through ultrasonic vacuum drying. An intense vibration peak was observed at 772 cm −1 in the spectra of the spray-dried sample. This result confirms that these drying methods modified the conformation of the O-C-C-N + skeleton in egg yolk lecithin from gauche to trans. By contrast, no vibration peaks were observed near 770 cm −1 in the spectra of the samples prepared through vacuum freeze-drying, vacuum drying, and hot-air drying. These results indicate that these methods negligibly affected the conformation of the O-C-C-N + skeleton.  Figure 5 Raman spectra of the stretching vibration of C-N The massive energy exerted by ultrasonic propagation during ultrasonic vacuum drying reduced the orderliness of lecithin molecules and the liquidity of the bilayer membrane [30] . Molecules in the substance vibrated under the action of ultrasonic waves. Vibration frequency is equal to ultrasonic frequency. Molecular structures are intensively damaged by high frequency [31,32] . Therefore, ultrasonic vacuum drying influenced the structure of egg yolk lecithin.
Similar to ultrasonic vacuum drying, spray drying drastically influenced the structure of egg yolk lecithin. Under the high inlet temperature (120°C) of spray drying, materials acquired sufficiently high energy that stimulated and accelerated molecular vibration [33] . By contrast, the structure of molecules is hardly changed under the low temperatures of vacuum freeze-drying, vacuum drying, and hot-air drying due to their inadequate energy.

Effects of drying methods on the conformation of the C-C skeleton
The stretching vibration of the C-C skeleton was located in the1000-1200 cm −1 region ( Figure 6). In this region, the vibration of C-C was highly sensitive to the conformation of chain molecules.
The hydrocarbon chain can exhibit various conformations because of the rotation of C atoms in the chain.
System energy is minimized when the chain is in a trans configuration and the skeleton of the hydrocarbon chain is on a zigzag plane. A certain amount of energy is required to form various gauche conformations that then result in the different torsions of the hydrocarbon chain [34] . In the stretching vibration region of the C-C skeleton, the vibration peaks at 1062 cm −1 and 1128 cm −1 were contributed by the bilayer C-C skeleton of egg yolk lecithin, whereas the peak at 1097 cm −1 was ascribed to the vibration peak of the C-C skeleton in gauche conformation [26] . The orderliness of the C-C chain can be acquired by comparing the strengths of the trans and gauche spectra. High I gauche /I trans ratios indicate low lecithin molecule order and high liquidity. Otherwise, the orderliness of lecithin molecules had increased and liquidity had decreased [35] . The I gauche /I trans ratios of the samples are listed in Table 2.    Table 2, the I 1096 /I 1062 and I 1096 /I 1128 ratios of powdered egg yolk samples prepared through ultrasonic vacuum drying were lower than those of fresh liquid egg yolk and samples prepared through other drying methods. Moreover, as presented in Figure 6, the vibration peaks at 1062, 1128, and 1097 cm −1 in the Raman spectra of egg yolks prepared through ultrasonic vacuum drying were stronger than those in the spectra of fresh liquid egg yolk and samples dried through other methods, indicating that ultrasonic vacuum drying showed the most intense effect on the C-C skeleton. These results indicate that the conformation of the C-C skeleton was drastically affected by ultrasonic energy to change the order of the internal structure of a molecule while it was negligibly influenced by other drying methods.

Effects of drying methods on the bilayer membrane liquidity of egg yolk lecithin
Peaks within the 2800-3000 cm −1 region in the Raman spectra of egg yolk lecithin were attributed to the stretching vibration of some C-H bonds in the lipid chain ( Figure 7). The symmetric stretching and antisymmetric stretching vibrations of the C-H bond manifested as two peaks at 2850 cm −1 and 2878 cm −1 , respectively. The vibration modes of these two peaks drastically influenced the lattice accumulation of the lipid bilayers and conformation between chains. The degree of order among the molecular chains of egg yolk lecithin could be inferred based on the ratio of the intensities of the two peaks at 2850 cm −1 and 2878 cm −1 . The I 2850 /I 2878 ratio is negatively correlated with the degree of order among the molecular chains of lecithin but is positively correlated with the liquidity of the lipid bilayer membrane [36,37] . The I 2850 /I 2878 ratios are shown in Table 3.   As presented in Table 3, that I 2850 /I 2878 ratios of egg yolk samples processed through ultrasonic vacuum drying and spray drying were 1.00 and 1.02, respectively, and were lower than those of fresh liquid egg yolk and samples dried through other methods. The liquidity of the lipid bilayer membrane was considerably influenced by ultrasonic vacuum drying and spray drying but was slightly affected by other drying methods.

Effects of drying methods on the emulsifying capacity of egg yolk lecithin
Egg yolk lecithin was extracted with 95% ethanol from egg yolk powder samples prepared through different methods and the emulsifying capacities were quantified using the method described above. The results are shown in Figure 8, and different letters in the figure indicate that their differences are significant at the level of α=0.05. The effects of different drying methods on the emulsifying capacity of egg yolk lecithin were analyzed by variance analysis and multiple comparisons, as shown in Tables 4 and 5.
It has been suggested in Table 4 and Table 5 that the effects of different drying methods on the emulsifying capacity of egg yolk lecithin were highly significant.   As shown in Figure 8, the highest emulsifying capacity of 49.58 m 2 /g was exhibited by the egg yolk lecithin sample prepared through vacuum freeze-drying, followed by that of samples processed through vacuum drying, hot-air drying, spray drying, and ultrasonic vacuum drying. The lowest emulsifying capacity of 14.77 m 2 /g was obtained by the egg yolk lecithin sample dried through ultrasonic vacuum drying. The structures of lecithin were altered by different drying methods and drying conditions, which led to the change of emulsifying capacity.
In this study, ultrasonic vacuum drying and spray drying altered the membrane orientation of the polar head region of egg yolk lecithin from parallel to vertical. This alteration exposed the hydrophobic groups of egg yolk lecithin and consequently weakened the hydrophilicity and emulsifying capacity of egg yolk lecithin [38,39] . Meanwhile, the combined effects of ultrasonic wave and high temperature decreased the liquidity of the lipid bilayer membrane [40,41] . This reduction was accompanied by increased lipophilicity and reduced emulsifying capacity [42] .
Egg yolks begin to solidify at 65°C . The temperature of spray drying exceeded the solidification temperature of egg yolks and reached 120°C , under which emulsifying capacity drastically decreased [43,44] . In actual operation, egg yolk lecithin prepared through ultrasonic vacuum drying was partially insoluble in the oil-water mixture. The precipitation of particles and the rapid formation of different liquid layers after homogenization were indicative of extremely low emulsifying capacity [42,45] . The egg yolk lecithin prepared through vacuum freeze-drying exhibited the highest emulsifying capacity mainly because molecular vibration is insensitive to low temperature. The temperature of vacuum drying and hot-air drying was 50°C , which was lower than the temperature required for egg yolk solidification. Therefore, the egg yolk lecithin samples prepared through vacuum drying and hot-air drying presented moderate emulsifying capacity. Vacuum drying was quicker than hot-air drying and slightly degraded the structure of egg yolk lecithin. Therefore, the emulsifying capacity of samples processed through hot-air drying was slightly higher than that of samples prepared through vacuum drying.

Conclusions
Ultrasonic vacuum drying, vacuum drying, vacuum freeze-drying, hot-air drying, and spray drying influenced the structure of egg yolk lecithin at different degrees. Among others, the greatest influence was observed on the structure of egg yolk lecithin dried by ultrasonic vacuum, which can change its conformation, making the inter-chain order increased, and the fluidity of the membrane bilayer reduced. The influence of spray drying on the structure of egg yolk lecithin is less than that of the ultrasonic vacuum drying, with the little effect observed for other drying methods. The emulsifying capacity of egg yolk lecithin is closely related to its structure. The effects of drying methods on the emulsifying capacity of egg yolk lecithin are highly significant. The egg yolk lecithin sample prepared through vacuum freeze-drying presented the highest emulsifying capacity, followed by the samples processed through vacuum drying, hot-air drying, spray drying, and ultrasonic vacuum drying, respectively.


text:
Selection between and within half-sibling progenies of Ilex paraguariensis for adventitious rooting of mini-cuttings

This study aimed to evaluate half-sibling progenies for developing breeding strategies and to select mate clones with adventitious root competence, required for vegetative propagation by mini-cuttings. For rooting, single-bud mini-cuttings of 2 cm length were treated with indolebutyric acid at 2000 mg L-1. At 60 days of cultivation, the mini-cuttings were evaluated for percentages of survival and rooting, number and mean length of the three largest roots, and number of rooted mini-cuttings per mini-stump. These data were analyzed considering the mixed model described for a completely randomized design, an environment, half-sibling progenies, and a single plant per plot, by using the SELEGEN-REML/BLUP software. The 50 genotypes with the highest number of rooted mini-cuttings were selected. The genetic gain was 48.41% for the number of rooted mini-cuttings per mini-stump. Selection for adventitious rooting based on the number of rooted mini-cuttings can be used in mate breeding programs for vegetative propagation by mini-cuttings.

INTRODUCTION
, belonging to the Aquifoliaceae family and native to the mixed ombrophilous forest (Dartora et al. 2013, Bergottini et al. 2015, Barbosa et al. 2020), presents socioeconomic, environmental, and cultural prominence in the southern areas of Brazil, Paraguay, Argentina, and Uruguay (Schmitt et al. 2018). Its leaves and thin branches are the main products marketed, mainly as raw material for the manufacture of teas and beverages (Santin et al. 2019). Mate is consumed in the form of energy drinks in California, and sold in combination with other herbs, as an energetic or weight loss tea, in Europe (Bracesco et al. 2011). Cardozo Junior and Morand (2016) reported an increase in consumption of products derived from mate in several countries such as Italy, Spain, United States, France, Korea, Australia, Japan, Germany, Russia, and Syria. At present, this species is gaining importance in the pharmaceutical, therapeutic, and nutraceutical industries (Dallabrida et al. 2016), because of its antioxidant, anti-inflammatory, antimutagenic, and hypolipidemic activities (Bracesco et al. 2011).
The demand of the mate market can be met by guaranteeing the continuous production of raw material. One of the factors directly influencing yield and quality of the produced raw materials is the genetic origin and good development of seedlings used for the establishment of new plantations. Thus, the production of seedlings can D Gazzana et al. be considered as one of the most important factors and has direct effects on the productivity and quality of the final product in the mate chain (Santin et al. 2015). However, the expansion of the commercial mate plantations has been limited by the difficulties related to its propagation, which can be seminal (seedlings) or clonal (plantlet production). Seedling production is limited by low germination rate, mostly caused by embryo immaturity and dormancy of the tegument (Wendling and Santin 2015), and by using non-improved seeds that result in low-quality seedings (Pimentel et al. 2017). Thus, an alternative to assist in the multiplication of the species is the direct selection for characteristics related to propagation of improved populations or elite individuals, in order to explore the potential for cloning these genotypes. In addition, the vegetative propagation of superior individuals allows the multiplication of the best genotypes and the production of plants genetically identical to the stock one, contributing to the uniformity of populations (Wendling and Santin 2015). That is, genetic improvement is indispensable to obtain gains in productivity, homogeneity, and quality of the produced plantlet, through cloning of genetically superior individuals (Brondani et al. 2018).
Single-bud mini-cutting is an alternative technique for the production of high-quality plantlets of mate (Pimentel et al. 2020), but cloning genetically superior genotypes is still a challenge, because of the low rooting rate of vegetative propagules (Wendling and Santin 2015). Adventitious rooting formation is crucial for mini-cutting survival and is a critical and essential process for vegetative propagation. Adventitious rooting is a complex, genetically controlled, and environmentally affected process, involving phytohormone reprograming and differentiation of specific cells of its base, with coordinated resource reallocation of the entire mini-cutting (Druege et al. 2019). The formation of adventitious roots is an especially complex process in woody species (Negishi et al. 2014), which is genotype-dependent and is regulated by multiple endogenous factors, such as genetic characteristics and propagule quality, including phytohormones, and environmental factors (Zhang et al. 2016). Thus, identifying the factors that alter the formation of roots is important for the success or failure of plantlet production via minicuttings, which depends on adventitious rooting (Cunha et al. 2009).
Breeding for vegetative propagation involves few possibilities for genetic recombination by sexual reproduction and many generations of selection for different traits and vegetative propagation (Bisognin 2011). Genetic tests assist in the selection of genotypes with greater competence for mini-cutting rooting, among which, progeny tests allow the estimation of genetic parameters between and within families and assessment of gains through selection. However, the genetic gains resulting from the use of this selection strategy depend on the heritability of the vegetative propagation-associated traits (Oliveira et al. 2015) used for the identification of superior genotypes for cloning. Although adventitious rooting competence is essential for vegetative propagation of mate, information on the use of any associated trait in the selection of superior genotypes for plantlet production by mini-cuttings is lacking. This information is still more relevant for mate, since it is considered a recalcitrant species for vegetative propagation, because its ability to form adventitious roots in vegetative propagules is very restricted, and the selection of superior genotypes may enable the vegetative propagation of this species.
Since mini-cuttings is a highly feasible technique for vegetative propagation of native tree species, especially when propagules are produced in mini-stumps of seedlings or young plantlets, because mini-cuttings are more responsive for adventitious rooting (Dias et al. 2012), as shown for mate (Pimentel et al. 2019, Pimentel et al. 2020, this study aimed to evaluate halfsibling progenies for developing breeding strategies and to select mate clones with adventitious root competence, required for vegetative propagation by mini-cuttings.

MATERIAL AND METHODS
The experiments were performed in a climatized greenhouse and in a humidity chamber at the Center of Plant Breeding and Vegetative Propagation (MPVP) of the Plant Science Department of the Federal University of Santa Maria (UFSM), Santa Maria (RS), Brazil. The mini-clonal garden was established in a closed soilless cultivation system by using seedlings of seminal origin of six mate half-sibling progenies, named 17SM1, 17SM3, 17SM4, 17SM7, 17SMLZ, and 17SMJS. The stock plants are centenary native individuals selected for seedling production and marketing, based upon progeny uniformity, seedling quality, leaf and branches yield, and commercial quality of established plantations. In the clonal mini-garden, 50 seedlings (genotypes) of each progeny were planted in a row, with 10 cm spacing between seedlings and rows.
The nutrient solution, consisting of macro-and micronutrients, was supplied twice a day for 15 min at a flow rate of 8.67 L min -1 ; the solution contained the following macronutrients (mmol L -1 ): 6.13 potassium nitrate, 10.0 calcium nitrate, 16.78 magnesium sulfate, 1.44 ammonium nitrate, 1.39 potassium monophosphate, and 1.68 iron. The micronutrients were added to the nutrient solution (7.5 mL) as a previously prepared mixture containing (mmol L -1 ) 0.15 sodium molybdate, 0.89 boric acid, 1.25 copper sulfate, 1.23 manganese sulfate, and 0.28 zinc sulfate. The nutrient solution was distributed in the cultivation bed by using a submerged motor pump, equipped with a timer, until the substrate formed by coarse sand was completely soaked. The pH of the nutrient solution was maintained between 5.5 and 5.8 and the electrical conductivity at 1.5 dS m -1 ; both were weekly adjusted.
After a period of adaptation of the seedlings, the plants were drastically pruned to promote the overcoming of the apical dominance of the mini-stumps and induce the growth and development of lateral shoots. In each mini-stump, two pairs of leaves were retained to guarantee their photosynthetic activity. The produced shoots were collected and sectioned to singlebud mini-cuttings of 2 cm length, with leaf and area reduced to 50% of the original surface. The mini-cuttings were treated with hydroalcoholic solution (1:1 v/v) of indolebutyric acid at 2000 mg L -1 ; they were grown in 100-well polyethylene trays containing an equal proportion of commercial substrate of pine bark-based medium, vermiculite, and coarse-grained sand (Pimentel et al. 2017). The trays were kept in a humidity chamber with a relative humidity of approximately 85%, provided by automated nebulization 8 times a day for 1 min. During each collection, the number of mini-cuttings produced per mini-stump was recorded to obtain the productivity of the mini-cuttings per mini-stump.
At 60 days of cultivation in the humidity chamber, mini-cuttings were evaluated for the percentages of survival and rooting (over total mini-cuttings), number of roots, mean length of the three largest roots (cm), and the number of mini-cuttings rooted per mini-stump. Mini-cuttings were considered rooted when they had at least one adventitious root of 0.1 cm or more in length. Four consecutive collections were performed in December of 2017 and February, May, and September of 2018, in periods ranging from 75 to 120 days, according to the availability of shoots in the mini-stumps.
The experiment had a completely random design, with 4 replications (collection times) and number of mini-cuttings ranging from 1 to 43, depending on the mini-cutting productivity per mini-stump. The assumption of data normality and homogeneity of variance were verified using the Shapiro-Wilk test (p > 0.05) and Bartlett test (p > 0.05), respectively, by using Action Stat software. The percentage, number, and length data that did not meet the assumptions of normal error and homogeneity of variances were transformed using Box-Cox and √ (x + 0.5), respectively. Subsequently, Action software was used to perform Pearson's correlation analysis among the evaluated traits, and the significance was verified using Student's t-test, at 5% probability of error.
The estimation of the variance components and the prediction of genetic values were performed using the procedure of maximum restricted likelihood/best impartial linear predictors (REML/BLUP), by using the SELEGEN-REML/BLUP (Statistical System and Computerized Genetic Selection) software (Resende 2016). For the evaluation of the individuals, the characters were analyzed using the mixed model, described for a completely randomized design, an environment, half-sibling progenies, and single plant per plot. The selection was performed between and within progenies for the 50 best evaluated genotypes for adventitious rooting competence, since this trait has greater importance for the mass production of plantlets by mini-cuttings, considering the number of rooted mini-cuttings per mini-stump. Resende and Barbosa (2005) stated that the best 50 genotypes are those having an adequate size to contain superior plants, when general selection is desired. According to the authors, when the number of genotypes is increased to above 50, many mean individuals are added, which contributes little to the selection. The selection gain in percentage with the best 50 genotypes was calculated using the formula: G (%) = MM -MO MO * 100, where G (%) is the selection gain in percentage; MM, improved mean; and MO, original mean observed.

RESULTS AND DISCUSSION
In this study, significant (p < 0.05) and positive correlations were noted between the mini-cutting survival from mate half-sibling progenies and the rooting percentage (0.61; Table 1). This positive relationship was also observed in the rooting of cuttings of Averrhoa carambola L. (Bastos et al. 2005) and in Cabralea canjerana clones (Burin et al. 2020). This result can be explained by the fact that rooted mini-cuttings have greater capacity for absorbing water and nutrients from the substrate, which reduces the mortality over the period of cultivation in a humidity chamber. In addition, mini-cuttings with a greater number of roots have higher survival rates, since a well-formed root system facilitates greater initial plantlet growth and plant development when transplanted in the field, providing better fixation and reducing mortality (Dias et al. 2012, Timm et al. 2015).
Significant and positive correlation was also observed between the number of rooted mini-cuttings per mini-stump and the number of mini-cuttings produced, the percentages of survival and rooting, the number of roots, and the mean length of the D Gazzana et al. three largest roots (Table 1), indicating a linear relationship between the productivity of mini-cuttings and adventitious rooting competence in mate half-sibling progenies. The mini-stumps capable of producing vigorous shoots could also contribute to the greater productivity of mini-cuttings per mini-stump, which in turn generates clonal seedlings with faster growth, both in a humidity chamber and greenhouse. This greater vigor of mini-stumps may be attributed to the higher amount of carbohydrate reserve and co-factors of adventitious rooting that maximize the rhizogenic process (Dias et al. 2011). Although the size of minicuttings was standardized during preparation, the greater interval between collections facilitated the greater accumulation of reserves in the collected shoots, thereby accelerating the development and rooting of the mini-cuttings. Paiva and Gomes (1995) indicated that reserves are indispensable for the survival of the propagule until it forms roots and develops. At appropriate levels, reserves are responsible for increasing the photosynthetic area and favoring root formation, since most of the reserves are transferred to the base of the mini-cuttings, contributing to the induction and formation of the root primordium (Paiva and Gomes 1995). These results are relevant, since the balance between productivity and rooting of mini-cuttings is extremely important for the production of vigorous and high-quality plantlets.
Since mini-cutting productivity is associated with adventitious rooting competence in mate half-sibling progenies, the greater the number of mini-cuttings rooted per mini-stump, the greater would be the percentage of rooting and the number and length of roots. Similar results have been reported in herbaceous mini-cuttings of Prunus persica (Timm et al. 2015) and mini-cuttings of C. canjerana (Burin et al. 2018), where high correlations were found between the number and length of roots. Considering the linear correlation among evaluated traits, the number of rooted mini-cuttings per mini-stump can be used to identify mate genotypes with high adventitious rooting competence within and between half-sibling progenies, which would facilitate and simplify the selection process of superior clones for vegetative propagation by mini-cuttings. These results are in agreement with those observed in C. canjerana, suggesting that the selection of clones should be performed based on the number of rooted mini-cuttings during the period of vegetative growth (Burin et al. 2018).
The REML/BLUP analysis procedure enabled the estimation of the genetic parameters by their genotypic values and predicted gains, being effective in identifying superior genotypes, as found by Bezerra et al. (2020). The estimates of individual heritability coefficients in the strict sense (h 2 ) were higher than those obtained in individuals within the progeny (h 2 ad), for all evaluated traits (Table 2). In Eucalyptus camaldulensis, higher heritability estimates were observed for progeny mean than for individuals within progeny, suggesting the selection of the best progenies and genotypes within them (Azevedo et al. 2015). However, these results indicate that the selection must be performed based on the genotypes, regardless of their progeny, for the percentage of survival and rooting of mini-cuttings, number of roots, mean length of the three largest roots, and number of rooted mini-cuttings. This result is very important, since this study aimed to identify genotypes with high competence for adventitious rooting, to develop mate clones for vegetative propagation by mini-cuttings.
Among the evaluated traits, the number of produced mini-cuttings, percentages of survival and rooting, and mean length of the three largest roots showed heritability estimations of low magnitude (Resende 2016), which resulted in low gain from selection (Table 2). However, heritability estimations considered by Resende and Barbosa (2005) of moderate to high magnitude were found for the number of roots and number of rooted mini-cuttings. These results indicate that the selection should be made for the number of rooted mini-cuttings, an easily measured trait, with the possibility of satisfactory genotype selection gains, both between and within mate half-sibling progenies propagated by mini-cuttings. This is because the expected progress of selection depends directly on heritability (Resende 2016, Matos Filho et al. 2019, and moderate to high estimates of heritability favor the initial selection among and within the best progenies (Carias et al. 2016). Individual heritability estimates in the narrow sense of Table 1. Pearson's correlation matrix among number of produced mini-cuttings per mini-stump (CN), percentage of mini-cutting survival (S, %), percentage of rooting (R, %), number of roots per mini-cutting (RN), mean length of the three largest roots (RL, in cm), and number of rooted mini-cuttings (CNR) of four consecutive collections in mate half-sibling progenies 0.00 * 0.61 ** RN 0.15 ** 0.24 ** 0.61 ** RL (cm) 0.02 * 0.09 * 0.50 ** 0.67 ** CNR 0.63 ** 0.39 ** 0.67 ** 0.53 ** 0.34 ** moderate (0.15 < h 2 < 0.50) to high (h 2 > 0.50) magnitudes have already been observed for the production of leaf mass (Simeão et al. 2002), and those of high magnitude were noted for seedling height and diameter of mate progenies (Costa et al. 2005).
The individual additive genetic variation coefficient (CV gi) quantifies the magnitude of genetic variation available for selection ); therefore, the higher its value, the more appropriate is the trait for selection. In this study, the highest CV gi values were obtained for the mean length of the three largest roots and number of rooted minicuttings ( Table 2). Considering that CV gi values above 10% are sufficient for effective selection (Resende 2016), only the percentage of survival could not be considered in the selection of mate genotypes and progenies. These results indicated a high magnitude of genetic variation available for selection (Pimentel et al. 2014), enabling significant genetic gains from selection between and within mate progenies based on adventitious rooting competence. According to Bezerra et al. (2020) this shows the existence of variability that can be exploited by selection and, consequently, the possibility of identifying superior genotypes for these traits. The CV gi values between 25.9% and 41.6% were observed for rooting traits of P. persica cuttings . CV gi values between 10.4% and 17.6% resulted in genetic gains from selection of 4.2% and 7.2%, respectively, for height and diameter of mate seedlings (Costa et al. 2009).
The selection accuracy (Acgen) is directly proportional to the heritability of the respective trait, which refers to the correlation between the predicted and true genetic values of the individuals (Resende andDuarte 2007, Lopes et al. 2018). As expected, one of the highest values of Acgen was obtained for the number of rooted mini-cuttings (Table 2), also indicating that the selection for rooting competence of mini-cuttings should be performed based on this trait. These results are in agreement with those of Burin et al. (2018), who performed the selection of C. canjerana clones based on the number of rooted mini-cuttings over three successive collections, highlighting the feasibility of using this trait to identify genotypes with high competence for adventitious rooting of mini-cuttings.
The REML/BLUP procedure allows the prediction of genetic values (g) and, consequently, ranking of the best genotypes for a given trait (Resende 2016), in this case, for the number of rooted mini-cuttings per mini-stump. Following this order, the 50 genotypes with the highest genotypic values were selected, revealing that progenies 17SM1, 17SM3, 17SM4, 17SM7, 17SMLZ, and 17SMJS had at least five selected genotypes each, and progenies 17SM3 and 17SMLZ contributed to the largest number (11) of selected genotypes (Table 3). Selection of the number of rooted mini-cuttings within the progenies resulted in selection intensities ranging from 9.80% to 22.90%, corresponding to an intensity of selection of 17.86%, considering all evaluated individuals. This selection intensity can be considered to be adequate for maintaining sufficient genetic variability for the selection of other traits. Higher selection intensities (1.26% and 2.14%) were used for Pinus caribaea var. hondurensis in different sources of clonal seed orchard (Sampaio et al. 2000), and, for E. camaldulensis, selection intensity was 15.15% among progenies and 6.67% within progenies (Azevedo et al. 2015).
The results of this study showed that the selection of genotypes based on the number of rooted mini-cuttings was important to identify those with high adventitious rooting competence during vegetative growth, since this trait varied between and within half-sibling progenies of mate. As expected, the highest genetic gain was observed for the number of rooted minicuttings (48.41%), because it was the directly selected trait (Table 3). The selection of the 50 genotypes with high adventitious rooting competence increased the original observed mean from 6.44 (Table 2) to 9.97 rooted mini-cuttings in four consecutive collections, equivalent to an increase of 54.81%. The highest values of indirect genetic gain of selection were observed for the number of roots and the mean length of the three largest roots (Table 3). These results were also expected, as the rooting traits showed the highest values of linear correlation with the number of rooted mini-cuttings.
The results of this study showed that Pearson correlation estimations and genetic parameters associated with the prediction of genetic values allowed the identification of the best trait to be used in the selection and order the genotypes based on their genetic value. The number of rooted mini-cuttings was the most suitable trait for the selection of genotypes with high competence for adventitious rooting between and within mate half-sibling progenies. The genetic gains provided with the selection between and within the progenies can be considered satisfactory as well as more efficient for choosing the best genotypes. Both the direct genetic gain for the number of rooted mini-cuttings and the indirect gains, mainly for the number of roots and the mean length of the three largest roots, can be considered to be of high magnitude. This is an important result, since a new plantlet must have a well-formed root system to increase its capacity to absorb water and nutrients, enabling good establishment and development in the field. The best clones from selected progenies should result in uniform growth and development, and high yield and quality of leaves and branches, positively impacting farmer profits and quality of industrial products. The breeding strategy developed in this study enabled the selection of mate genotypes with high competence of adventitious rooting within all evaluated half-sibling progenies and maintained variability for selecting other traits to develop new clones for vegetative propagation. The mass production of plantlets by using mini-cuttings requires, in addition to adventitious rooting, a well-formed and developed root system associated with high morphological and physiological quality of the produced plantlets. Therefore, selection for adventitious rooting competence based on the number of rooted mini-cuttings can be used in the breeding programs of mate for vegetative propagation by mini-cuttings.

ACKNOWLEDGMENTS
This research was partially financed by the Brazilian Federal Agencies "Conselho Nacional de Desenvolvimento Científico e Tecnológico" (CNPq), and "Coordenação de Aperfeiçoamento de Pessoal de Nível Superior" (CAPES), finance code 001. Table 3. Number of selected genotypes and mean of mate half-sibling progenies, with the respective mean of all selected genotypes, original mean, and percentage genetic gain for the number of mini-cuttings produced by mini-stump (CN), percentage of survival of mini-cuttings (S%), rooting percentage (R%), number of roots (RN), mean length of the three largest roots (RL cm), and number of rooted mini-cuttings (CNR) collected at different times throughout the year and evaluated at 60 days of cultivation in a humidity chamber


extfieldsofstudy: Medicine Chemistry

text:
Chinese Propolis: Ultrasound‐assisted enhanced ethanolic extraction, volatile components analysis, antioxidant and antibacterial activity comparison

Abstract This study was aimed to enhance the extraction yield of propolis samples using ultrasound technology, analyze the volatile compounds, and compare the antioxidant and antimicrobial effect of propolis extracts of different areas. Four propolis samples were collected from different regions of China, namely: Linqing, Shandong Province (LSP); Yingchun, Heilongjiang Province (YHP); Changge, Henan Province (CHP); and Raohe, Heilongjiang Province (RHP). The ultrasound extracts of CHP and RHP showed a higher total phenolic content (TPC) of 201.78 ± 4.60 mgGAE/g and 166.071 ± 1.53 mgGAE/g, total flavonoid content (TFC) of 519.77 ± 29.90 and 341.227 ± 10.82 mg quercetin/g respectively, as well as high antioxidant and antibacterial activity. Conventional extraction showed 15%–20% lower yield for TPC ranging from 72.02 ± 1.99 to 155.95 ± 3.69 mg GAE/g, TFC ranges from 129.675 ± 6.82 to 412.83 ± 12.14 mg quercetin/g, with lower antibacterial activity. The antioxidant activity of propolis extracts was determined by assays of reducing power, DPPH*, FRAP*, TEAC*, hydroxyl radical scavenging activity and superoxide anion scavenging activity. Collectively, the antioxidant activities of extracts from CHP and RHP were higher than those of the other two extracts(YHP and LSP). All the extracts showed high antimicrobial activity on Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis, but no effect on Escherichia coli. A total of 150 compounds in propolis were detected by GC/MS. Terpenes (RHP 34%, YHP 5%, LSP 18%, and CHP 12%) and alcohols (RHP 12%, YHP 13%, LSP 12%, and CHP 10%) showed the highest relative content among all other extracts.

| INTRODUC TI ON
Propolis is a material collected by bees from the sap of trees and mixed with their saliva to seal and sterilize the hive (Reis et al., 2019).
Many studies have indicated a particular interest in the antioxidant activities of propolis using various assays. These assays are mainly spectrophotometric, which include free radical scavenging (Pobiega et al., 2019), ferric reducing antioxidant power (Ahmed et al., 2017;Gülçin et al., 2010), and electrochemical methods (Rebiai et al., 2011). A lot of results have described the antimicrobial and antibacterial activity of propolis extracts, noting that phenolic compounds, terpenoids and nonaromatic acids are primarily responsible for the inhibiting microorganism growth (Bayram et al., 2017;Martinotti & Ranzato, 2015;Yuan et al., 2020).
The essential oils extracted from propolis exhibited antimicrobial activity mostly against gram-positive bacteria and fungi (Franchin et al., 2016). The primary polyphenol (flavonoid) content in propolis fluctuates qualitatively and quantitatively depending on the plant's environment and ecology (Dantas Silva et al., 2017). Headspace solid-phase microextraction (HS-SPME) has been proposed as a dependable tool for the analysis of volatile organic compounds (Mohtar et al., 2018), because it removes many drawbacks: (a) organic extraction, (b) higher cost and (c) prolonged extraction time.
In this study, we focus on enhanced extraction of propolis compound using a novel technology to attain a higher yield at the cost of relatively less energy and time. Ultrasound wave-mediated energized medium (solvent) indorsed easy recovery of the active compounds from the samples. Ultrasound-assisted extraction was done from propolis to analyze the volatile compounds using HS-SPME and GC-MS to evaluate their antioxidant and antibacterial activities, in vitro. Results of this study prove that the proposed technology may be economically and efficiently superior for the industrial use in the manufacturing of functional foods. Gallic acid, vanillin, quercetin, ethanol, methanol, HPLC- China. Escherichia coli (DH5α) was prepared and stored at −20ºC.

| Initial processing
Propolis samples after collection were crushed using a chilled mortar at freezing temperature, then sieved using a sieve size of 40 mesh and kept at −20°C once powdered. A pulverized propolis sample (5 g) was added to 100 ml of 95% ethanol and placed in an ultrasonic device (Jiangsu Jiangda Wukesong Biological Technology Co., Ltd., Jiangsu, China) at 220 W and 40 kHz for 30 min. After three extractions, the extract was subjected to suction filtration and filtered to a volume of 300 ml to obtain the extract for further analysis. After ultrasound extraction, the mixtures were centrifuged for 10 min at 1,644 g (TGL-16M High-speed Desktop Refrigerated Centrifuge, Changsha Xiangyi Centrifuge Instrument Co., Ltd.). The supernatant was filtered using vacuum filtration and ethanol was added up to 250 ml. Supernatants of different extracts were divided into two parts: one part was prepared for detection and the other part was concentrated using a rotary evaporator at 40°C (R-210 BUCHI Labortechnik AG, Flawil, Switzerland). Subsequently, samples were shifted to a vacuumdrying oven to remove any residual solvent (DZF-6050 Shanghai Yiheng Scientific Instrument Co., Ltd.). All measurements were carried out in triplicate.

| Determination of Total Phenolic Content (TPC)
TPC was determined by the Folin-Ciocalteu assay (Hernandez Zarate et al., 2018). Samples were mixed with diluted Folin-Ciocalteu solution and 500 ml of 20% Na 2 CO 3 solution, mixture was kept in the dark for 1 hr at room temperature and centrifuged at 872.1 g for 10 min, and absorption was measured at 750 nm using UV spectrophotomerer (Cary 100, Agilent Technologies, Santa Clara CA, USA). Gallic acid at various concentrations was used as a standard to construct a standard curve. TPC was denoted as mg of Gallic acid equivalent (GAE)/grams of propolis extract using a calibration curve of 50-250 mg of gallic acid/mL.

| Determination of Total Flavonoid Content (TFC)
Total flavonoid content was determined using colorimetric method based on aluminum chloride complex formation (Hernandez Zarate et al., 2018). Briefly, 0.5 ml of 5% AlCl 3 mixed with 2 ml of extracts in 25 ml volumetric flask distilled water was used to adjust the volume.
Mixture was left in the dark at room temperature for 30 min, and absorption was measured at 425 nm using UV spectrophotometer.
Quercetin was used as reference to plot a standard curve. Results were expressed as mg of quercetin of propolis extract.

| Antioxidant assays
2.5.1 | DPPH scavenging assay 0.2 ml of DPPH solution (0.1 mM) was mixed with Propolis extracts at various concentrations (0-5 mg/ml) using vortex. The reference compound (Trolox) was used to draw a standard curve. Samples were then incubated in the dark for 30 min at room temperature, and absorbance was measured at 517 nm using UV-spectrophotometer (Wen et al., 2020). The equation used for DPPH scavenging activity was as follows: Where: Abs 0 = absorbance of the control sample; Abs 1 = absorbance in the presence of samples tested.

| Ferric reducing antioxidant power (FRAP)
All reagents were prepared according to  with some modifications. FRAP reagent was prepared by adding 25 ml of 300 mM acetate buffer (pH 3.6) with 2.5 ml of 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) solution (in 40 mM HCl) and 2.5 ml of 20 mM FeCl 3 .6H 2 O solution. The newly prepared reagent was heated at 37°C. The FRAP was done using the method of (Benzie & Strain, 1996). Crude extracts of propolis were dissolved in ethanol (95%) with a concentration of 50 mg/ml and diluted to 100, 80, 60, 40, 20, and 0 µg/ml. Aliquots (100 µg/ml) of newly prepared samples were mixed with 0.5 ml of FRAP reagent. Ferrous sulfate ion concentration (10-100 µM) was used as the standard, and the absorbance was measured at 593 nm. Results were expressed as µM ferrous sulfate per gram of propolis extract.

| Total Equivalent Antioxidant Capacity (TEAC)
The method used to determine the total antioxidant capacity was ABTS by consulting the method of Osés et al. (2020). Trolox at different concentrations was used as the standard. ABTS cations preparation includes oxidization of ABTS solution in water with the treatment of potassium supersulphate (molar ratio 1:0.35) and kept in the dark for 12-16 hr. Subsequently, the solution was diluted by adding 0.1 M potassium phosphate buffer (pH 7.4) to provide the absorbance of 0.70 ± 0.02 at 734 nm. The sample (200 µl) was added to 1.8 ml of the reagent, mixed and incubated at room temperature.
After every 10 min, absorbance was read using a spectrophotometer.
Distilled water instead of the sample was used to measure the absorbance value Abs 0 , whereas distilled water without H 2 O 2 was used to measure the absorbance value Abs 2 . The equation used for hydroxyl radical scavenging rate was as follows: 2.5.6 | Superoxide anion scavenging activity

| Bacterial Growth Inhibition determination assay
A sterilized spatula was used to drop and coat 120 µl of bacterial suspension uniformly. Filter paper soaked with antimicrobial propolis extract was pasted on the plate containing bacteria on agar medium.
Each plate was pasted three times. Two plates were cultured in a constant temperature incubator at 37ºC for 24 hr to determine the size of the bacteriostatic zone (Kalogeropoulos et al., 2009). The size of the inhibited area was measured (mm).

| Determination of volatile compounds by HS-SPME GC-MS
Analysis of the volatile compounds in propolis was done using headspace solid-phase microextraction (HS-SPME) combined with GC-MS. HS-SPME was done using a manual holder and fiber (050/30 µm DVB/CAR/PDMS, 24Ga). In a 15 ml flat bottom head vial, 0.5 g of sample was placed and sealed with a magnetic crimp cap and PTFE/ silicone septa (Supelco). A thermostatic bath was used to heat the sample at 60°C for 10 min. The SPME device was introduced into the sealed vial by manually diffusing the septum and then displayed to the headspace extraction for 30 min. The SPME fiber was quickly inserted into the GC injector.
The GC-MS analysis was performed according to the method of (Kasiotis et al., 2017) with some modifications. An Agilent 6,890 GC connected to 5973N MSD mass spectrometer framework supplied with an HP5-MS slender section (60 m × 0.25 mm i.d., 0.32 µm film thickness) was used. Analysis of the sample was done with the column held initially at 40°C for 2 min, inclined at a rate of 10-60° and then contained for 5 min. After 2 min the temperature was straightly stepped up to 280°C with a 3°C/min heating ramp and holding at 280°C for 10 min. The carrier gas was Helium with a 1.0 ml/min flow rate. The infusion was performed in pulse splitless mode, and at injector temperature 250°C. Full scans EI (Electron Impact) spectra were recorded from 30 to 450 m/z (mass/charge) with two scans per second. The ionization voltage was 70 ev with 230°C temperature and the ionization source for EI-MS in positive mode.
The component identity in the extracts was referred to by the correlation of their maintenance records and mass spectra with those stored on the PC library and with those distributed in the literature. NIST05 library sources were utilized to match the identified compounds.

| Statistical analysis
The data were expressed as the mean ± standard deviation (SD), analyzed by one-way analysis of variance (ANOVA) and Tukey's test was used for comparison of the means using Minitab 18 (Minitab Inc., State College, PA, USA). Origin Pro software (2018) was used for graphical presentation. All experiments were performed in triplicate.

| Total phenolic and flavonoid contents of various extracts of propolis samples
According to the results in Table 1, TPC and TFC varied from 89.343 to 201.786 mg GAE/g of and 166.227 to 519.769 mg quercetin/g of ultrasound-assisted extracts respectively for propolis in different areas of China with significant difference (p < .05). In comparison with conventional extraction, TPC varied from 72.02 ± 1.99 to 155.95 ± 3.69 mg GAE/g,TFC varied from 129.675 ± 6.82 to 412.83 ± 12.14 mg GAE/g, that is, 15%-20% lower than the ultrasound-assisted extraction. Molnár et al. (2017) ) and Haile and Lin (2011) reported that TPC of propolis of different regions of Europe (Hungary and Bulgaria) and China (Zhejiang, Hubei, Hebei) varies approximately from 200 to 300 mg GAE/g, while TFC content reported is lower than that in these locations of China and TPC is higher or comparable with these values. (Kumazawa et al., 2004) reported that the TPC in propolis extract from Shandong, China was 433.8 ± 1.7 mg GAE/g, which is higher than the present study results. The reason could be due to climate change, sea- 3.2 | DPPH free radical scavenging activity of various Propolis sample extracts 2,2-diphenyl-1-picrylhydrazyl has been broadly used to determine the free radical scavenging activity of multiple samples (Benhanifia et al., 2013). In Figure 1(a), all extracts of propolis samples showed significant DPPH scavenging activity. Figure 1(a) shows that the order of action of DPPH free radical scavenging activity is CHP > RHP > LSP > YHP. The samples with more DPPH activity have higher phenolic contents. Phenols in the ethanolic extracts of propolis are for the antioxidant activity of propolis because of their biological properties, which is responsible in scavenging reactive oxygen species (Osés et al., 2020). YHP and LSP have higher phenolic content as compared to other samples. Phaniendra, Jestadi, and Periyasamy (2015) reported that propolis samples from China and Brazil have vigorous DPPH free radical scavenging activity ranging from 98.80 ± 1.0% to 70.50 ± 1.2%. Similar results were also published by (Hatano et al., 2012), which demonstrate that Chinese propolis has more vigorous DPPH free radical activity than Brazilian propolis.

| Reducing power activity of different propolis extracts of samples
Various studies have shown that Chinese propolis has high reducing activity of phenolic compounds. Greek, Polish, and Slovenian propolis have high reducing power (Yang et al., 2011). In this study, propolis samples collected from different areas of China showed varying reducing power in varying dilutions. The sample with the highest reducing power activity was CHP; the sample with the lowest reducing power was YHP. The order of activity of all the samples, as shown in Figure 1

| Ferric Reducing Antioxidant Power (FRAP) of propolis extracts
The antioxidant power of propolis samples from different regions of China was determined by using ferric, reducing antioxidant power (FRAP). Figure 1(

| TEAC of Ultrasound-assisted extracts of Chinese propolis from different areas
The Propolis antioxidant effect was determined by Trolox equivalent antioxidant capacity (TEAC) per gram of propolis sample.
According to the results shown in Figure 1

| Hydroxyl radical scavenging activity of samples extract
The extracted samples hydroxyl scavenging activity was reviewed using the Fenton reaction, and the trend is shown in Figure 1(e).

| Superoxide anion scavenging activity of extract of propolis of different areas
The developing of pathophysiological conditions and redox cell signalling is significantly completed by production of superoxide radical species. In most of the biological reaction medium, these species are comparatively unreactive. O2•− is also a precursor of many strong oxidants. Figure 1(

| Antibacterial activity of different Chinese propolis extract
The ultrasound-assisted extract of propolis was used to determine the antibacterial activity of propolis. Our previous research revealed that the antibacterial activity of Chinese propolis increased by using ultrasound to extract propolis. Moreover, the extraction of flavonoids and polyphenols yield also increased . The results of the antibacterial assay F I G U R E 1 (a). DPPH activity of ultrasound-assisted extract of propolis of different areas. Means that do not share a letter are significantly different. Figure 1(b). Reducing power rate of ultrasound-assisted extract of propolis of different areas. Means that do not share a letter are significantly different. Figure 1(c) FRAP assay of ultrasound-assisted extract of propolis of different areas. Means that do not share a letter are significantly different. Figure   little or no effect on gram-negative bacteria (E. coli). In contrast extract from conventional procedure showed antimicrobial acitivity but the effect was lower than ultrasound-assisted extracts; that is, the highest activity was from YHP: Staphylococcus aureus 17.59 ± 0.21 mm and Listeria monocytogenes 17.12 ± 0.37 mm to 0 mm for E.coli as both ultrasound-assisted and conventional extraction did not show any effect on E.coli (Table 2). Gram-negative bacteria have a complex chemical structure, and their flexible cell wall contains polysaccharide; these characteristics account for its toxicity in microorganisms. Another main reason for the resistance against propolis extract is the presence of the multidrug obstruction pumps, which expels the entrance of external poisons over the external layer (Tukmechi et al., 2010). Likewise, this bacterial group has a higher lipid ration than that detected in gram-positive bacteria (Revilla et al. (2017). Mohdaly et al. (2015) also reported

| Analysis of volatile compounds in raw propolis by HS-SPME GC-MS
The chemical composition of volatile compounds depends mainly on the flora and collection site (Wojtyczka et al., 2013). Previously, Salatino, Fernandes-Silva, Righi, and Salatino (2011) determined 99 compounds from propolis samples collected from Italy by using HS-SPME GC-MS. In our study, a total of 150 compounds were identified in the propolis samples collected from different regions of China.
According to the results (Table 3), LSP contains 58 kinds of substances, accounting for 38% of the total peak area. CHP comprises 51 substances, accounting for 34.0% of the total peak area. RHP contains 74 elements, accounting for 49.3% of the total peak area.
YHP contains 41 kinds of substances, accounting for 27.3% of the total peak area.
For acid substances, LSP contains the highest content of acid materials, accounting for 11.51% of the total peak area. CHP, RHP, and YHP have 7.96%, 3.25%, and 6.35% acid relative contents, respectively. The variability in acid materials may be related to regional and climatic differences and the plants growing in each region.
Linqing, Shandong Province contains the highest content of esters, accounting for 3.25% of the total peak area, followed by YHP, where the content of esters accounted for 1.81% of the total peak area. CHP and RHP have 1.1% and 1.04% of relative ester contents TA B L E 2 Antibacterial activity of ultrasound-assisted and conventional extracts of different Chinese Propolis

TA B L E 3 (Continued)
of peak area, respectively. Although the content of the esters is relatively low, they are essential for the flavor, thus they were analyzed under the same detection conditions to obtain variable results.
Alcohol is the main contributors to propolis flavors. The content of alcohol in propolis in different regions is relatively high.
A very low content of olefins was found in the samples. The relative content of olefins in CHP was 3.77% of the total peak area. The relative content of olefins in LSP was 1.88%.
The aromatic substances in propolis contain bactericidal and bacteriostatic effects. The relative proportion of aromatic substances in LSP, CHP, RHP, and YHP accounted for 10.38%, 2.73%, 6.72%, and 3.42% of the total peak area, respectively. Aldehydes are also one of the active compounds in propolis, but the proportion and type are relatively small. The aldehydes in LSP, CHP, RHP, and YHP accounted for 2.96%, 2.44%, 2.89%, and 6.16%, respectively.

| CON CLUS ION
It can be concluded from the explained results that the polyphenolic compounds in propolis chiefly vary according to their botanical origin and environmental ecology of the plant.
Ultrasound-assisted extraction can increase the yield of TFC and TPC and thus may strongly enhance the antimicrobial activity of these extracts. Chinese propolis has high antioxidant and antimicrobial activity; variability in antioxidant activity of Chinese propolis is strictly related to variability in different amounts and kinds of phenolic compounds. Chinese propolis also contains much higher flavonoids than the other chemical substances such as alcohols, terpenes, aromatic acids, ketones, hydrocarbons, aliphatic acids, and their esters, thus they are very active as antioxidant agents.
Chinese propolis may be used in natural antibacterial agents when extracted by ultrasound-assisted extraction, as it is time-saving and increases the extraction yield. Further studies about its uses and functional efficacy are warranted.

ACK N OWLED G M ENT
Sincere appreciation to the National Key R&D Program of

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.