A VIS/NIR hyperspectral imaging system was used to classify three different degrees of freeze-damage in corn seeds. Using image processing methods, the hyperspectral image of the corn seed embryo was obtained first. To find a relatively better method for later imaging visualization, four different pretreatment methods , standard normal variation (SNV) and 5 points and 3 times smoothing (5-3 smoothing)), four wavelength selection algorithms , principal component analysis (PCA), X-loading and full-band method) and three different classification modeling methods , K-nearest neighbor (KNN) and support vector machine (SVM)) were applied to make a comparison. Next, the visualization images according to a mean spectrum to mean spectrum (M2M) and a mean spectrum to pixel spectrum (M2P) were compared in order to better represent the freeze damage to the seed embryos. It was concluded that the 5-3 smoothing method and SPA wavelength selection method applied to the modeling can improve the signal-to-noise ratio, classification accuracy of the model (more than 90%). The final classification results of the method M2P were better than the method M2M, which had fewer numbers of misclassified corn seed samples and the samples could be visualized well. Zea mays L.), one of the world’s three major food crops, is currently one of the most grown food crops in several parts of the world [Corn  recommended two kinds of seed viability measurement methods in 1995 , the eleTherefore, a key factor in current study is how to quickly and accurately determine the characteristic changes in the freeze-damage seeds and identify the freeze condition , which will provide guidance for the seed agricultural production. In particular, it is of more specific significance to study the frost damage status of the embryo.In recent studies, through the use of near-infrared spectroscopy technology, the vitality , internaAt present, the application of hyperspectral imaging technology in non-destructive testing of agricultural products has become more extensive. As a technology that combines the advantages of traditional image technology and spectral technology, it can obtain the spectral information of every pixel on the collected image, which can be used to effectively analyze the chemical composition index of each part of the seed and avoid the instability of experimental results. Another advantage of this technology is that it can test the seeds individually. Many research studies such as those on water content , hardnesAccording to the current research, no study has been conducted on the freeze damage of corn seed, although there are some studies using hyperspectral imaging techniques to study frozen grown crops ,37,38,39To summarize, the objectives of this study were to: (1) conduct an electrical conductivity test on corn seeds to consider if the seed is damaged or not; (2) obtain the corn seed embryo hyperspectral image, and assess the potential of applying hyperspectral imaging technology for the classification of different degrees of freeze damage to the corn seeds; (3) evaluate the models established by different spectral pretreatment methods, wavelength selection methods and modeling methods, and then compare and identify the optimal model among them; (4) visualize the classification results of two different methods (M2M and M2P), and to identify the optimal method.According to the experiment description, In order to get better classification results, the noise wavelengths (before 444.23 nm and after 985.37 nm) were excluded and the remaining 430 wavelengths were used for later modeling. The SPA, PCA and X-loading methods were applied to select feature wavelengths after applying the pretreatment methods  to the spectrums. The feature wavelength results for the three varieties are shown in appendixes From When the feature wavelengths were selected, the classification models were established. The accuracy results of the three varieties of corn seed are shown as From By counting the number of >80% classification accuracy results, the 5-3 smoothing and no pretreatment method had similar classification result, and in the meantime, the PLS-DA and SVM modeling methods had similar classification result.From By counting the number of >80% classification accuracies, all of the pretreatment methods had similar classification result, and in the meantime, the number of >80% classification accuracy results for the calibration set and validation set of the KNN method were fewer than for the other two classification modeling methods.From By counting the number of >80% classification accuracies, the conclusion was drawn that the classification accuracy results for the PLS-DA modeling method with the 5-3 smoothing pretreatment method had better results than any other pretreatment methods.A better model was found when using the 5-3 smoothing pretreatment method and the PLS-DA classification modeling method. To more clearly understand the classification results of the corn seed samples, the spectrum of each pixel in the embryo image was classified to realize the visualization of the corn embryo images.At first, the six images  of three different degrees of freeze-damage in corn seed were merged, From Now, how to identify the final category for the corn seeds was next step. The percentage of each category of each corn seed was calculated, and the results are shown in From Among numbers 15, 23, 37, 83, 117 and 167, It was found that numbers 83 and 167 were classified into two categories , shown in It was also found that the category 1 percentage of number 15, 23, 37 and 51 corn seeds were lower than 0.37, and they were not included in category 1; the category 2 percentage of number 86, 115, 116 and 117 corn seeds were lower than 0.32, and they were not included in category 2. From the above study the number 15, 23, 37 and 117 corn seeds were classified, but the 51, 86, 115 and 116 corn seeds did not have their own category, so the percentage of each category was compared shown in To compare the above results, the number of misclassified corn seed samples were counted. There were 17 corn seed samples misclassified by method M2M while eight corn seed samples were misclassified by method M2P. Meanwhile, none of the severely freeze-damaged samples were misclassified by method M2P. In some respects, it could be drawn that method M2P shown better results than method M2M.From Next, the percentage of each category of each corn seed was calculated, and the results are shown in From Among numbers 113, 121, 122 and 155, the number 122 was classified as categories 2 and 3, shown in It was also found that the category 2 percentage of numbers 83, 84, 98, 101 112, 113 and 114 corn seeds were lower than 0.239, and they were not included in category 2. The category 3 percentage of numbers 121 and 155 corn seeds were lower than 0.45, and they were not included in category 3. Numbers 113, 121 and 155 corn seeds were classified, but the 83, 84, 98, 101, 112 and 114 corn seeds did not have their own category, so the percentage of each category were compared and shown in To compare the above results, the number of misclassified corn seed samples are counted. There are six corn seed samples misclassified by method M2M while five corn seed samples are misclassified by method M2P. In some respects, it can be drawn that the effect of method M2P is similar to that of method M2M.From Next, the percentage of each category of each corn seed was calculated, and the results are shown in From Among numbers 39, 59 and 135, numbers 39 and 135 were classified to two categories , shown in It was also found that the category 1 percentage of numbers 22 and 59 corn seeds was lower than 0.38, and they were not included in category 1. The category 2 percentage of number 62 was lower than 0.385; the category 3 percentage of numbers 122, 127, 130 and 140 corn seeds were lower than 0.34, and they were not included in category 3. The number 59 corn seed was classified from the above study, but the 22, 62, 122, 127, 130 and 140 corn seeds did not have their own category. The percentage of each category was compared and shown in To compare the above results, the number of misclassified corn seed samples were counted. There were four corn seed samples misclassified by method M2M while one corn seed sample was misclassified by method M2P. Meanwhile, none of the severely freeze-damaged samples were misclassified by method M2P. In some respects, it could be drawn that method M2P had better results than method M2M.To summarize, the visualization results of three corn varieties using method M2M and method M2P were compared. By setting several category thresholds, and comparing the percentage value or subtracting the percentage from the threshold, method M2P could get fewer numbers of misclassified corn seed samples than in method M2M. In some respects, the method M2P had better results than the method M2M.Three fresh corn seed varieties  were collected from the Jiuquan Julong Tengfei Seed Industry Co., Ltd. in Gansu province, China, with a moisture content of about 30% before harvesting in 2017. In order to obtain different freezing-damage corn seeds, the seeds were dried in an oven until the moisture content was 18% and were put in plastic bags for later experiment. Then the seeds were placed in different freezing temperatures for different duration and eventually were divided into three categories . The frozen environment is shown in 180 corn samples  of Haoyu21; 180 corn samples  of Haihe78; and 144 corn samples  of Jindan10. The three varieties of corn samples were randomly divided into calibration sets and validation sets . Next, the seeds were stored in a refrigerator with a temperature of 4 °C to prevent moisture absorption. Before collecting the hyperspectral images, the moisture content was tested again to ensure all the samples had nearly the same moisture content. The final moisture content was controlled at 13%.Before the collection of hyperspectral images, an electrical conductivity test of corn seeds was conducted to check for the effects of the freeze damage on the corn seeds. Fifty corn seed samples for each treatment condition were weighed and placed in a 500 mL beaker containing 250 mL of deionized water. The conductivity was measured after the corn seed soaked for 8, 12, 16, 20 and 24 h.The hyperspectral data of the corn seeds were collected using a VIS/NIR hyperspectral imaging system . The system consisted of a CCD camera , an imaging spectrometer , a lens , line light sources  and its controller , sample stage  and a moving stage controller .The CCD camera, VIS/NIR imaging spectrometer, lens, line light source, sample stage, moving stage and 0.5 mm extension tube were all placed in a dark box. The moving stage was installed in the bottom of the dark box and connected with the moving stage controller. The sample stage was installed on the top of the moving stage and the corn seed was placed on the sample stage; above the sample stage, two line tungsten halogen light sources were installed on both side walls of the dark box, which provided illumination for the corn seeds. The two line light sources were symmetrically at 60º and installed at a height of 26 cm above the moving stage to provide a stable and uniform diffuse reflection light for the corn seeds. The CCD camera, VIS/NIR imaging spectrometer and lens were installed on the top surface of the dark box and were connected to each other vertically. Above the lens, there was a 0.5 mm extension tube between the lens and the VIS/NIR infrared spectrometer. After adding the extension tube, more clear corn seed hyperspectral images were obtained.The hyperspectral image acquisition software, Spectracube 2.75b , was used for image acquisition and correction. At last, the corn seed embryo was placed upside down on black sample stage with an object distance of 28.5 cm, with an exposure time of 1 ms and the moving stage speed of 2.6 mm/s. The hyperspectral images were acquired with a spectral range of 400–1000 nm which had a total of 477 wavelengths. cI is the relative reflectance intensity of each wavelength, 0I is the original reflectance intensity of the hyperspectral image, bI is the intensity of the dark current, which was obtained by turning off the light source and completely covering the lens with its cap, and wI is the reflectance intensity of the Teflon white surface , which was obtained under the same conditions as the raw image.In order to overcome the inhomogeneity of the light source intensity at each wavelength and the influence of the dark current of the acquisition sensor, the collected hyperspectral images required black and white correction according to Equation (1).The hyperspectral images were analyzed using the ENVI 4.6.1 software  and MATLAB R2017b .After the hyperspectral images were obtained, the feature image, i.e., the embryo image was extracted for later processing. At first, the average spectrums of 10 pixel × 10 pixel regions of interest (ROI) in the endosperm and embryo were compared, and the gray image in the wavelength of 500 nm was selected as a segment image to separate the embryo (the brighter region) from the whole corn seed . Finally, the final embryo-binary image was obtained.After the embryo-binary image was obtained, it was masked with the original hyperspectral image and the average spectrum of corn seed embryo hyperspectral image was pretreated for spectral feature extraction. Since the spectrometer had low response and noise at the edge of the spectral region, the middle 31 to 460 wavelength spectrum was selected for analysis.In this paper, multiplicative scatter correction (MSC), standard normal variation (SNV) and 5 points and 3 times smoothing (5-3 smoothing) pretreatment methods were applied ,44.The MSC method is used to correct the scattering of each sample’s spectrum, get the desired spectrum, and remove the undesirable scatter effect which can improve the signal-to-noise ratio .The SNV method is commonly used to eliminate spectral errors among samples due to different solid particle sizes, scattering, or measurement path lengths. It converts the data mean to 0 and the standard deviation to 1 and is generally used for scatter correction and to remove the slope variation from the spectra . The smoothing method can effectively remove high-frequency noise which may be produced by instrument noise, random errors, etc., which can keep the original useful signal information and improve the signal-to-noise ratio at the same time . In thisIt is well known that a hyperspectral image has a large amount of spectral data and the whole spectral wavelength contains much noise and irrelevant information. According to the introduction, it is necessary to apply some algorithms , principal component analysis (PCA) and other methods were applied) which have faster speed in data computation and higher accuracy to obtain important wavelengths and establish the classification model .SPA is a forward selection algorithm that looks for variable combinations with minimal redundancy information in the variable matrix to minimize collinearity among the variables. Therefore, the SPA is used to extract the characteristic wavelengths during spectral data analysis processing .PCA is a commonly used dimension reduction mapping method, which maps original features with strong original correlation into a set of new features. Each new feature constructed is a linear combination of the original features. Each new feature is not related to each other .X-loading method uses spectral variables for partial least-squares modeling, and the wavelengths are extracted based on the absolute value of the regression coefficients for each wavelength. As the absolute value of the regression coefficient gets larger, the greater the influence of these wavelengths on the modeling, that is, the final extracted feature wavelength .In this paper, the partial least squares-discriminant analysis (PLS-DA), K-nearest neighbor (KNN) and support vector machine (SVM) models were established for classification of different frozen corn seeds.The PLS-DA is based on the partial least squares (PLS) technique that is a commonly used in multivariate statistical analysis methods . The maiKNN classifies by measuring the distance (the distance can be Euclidean distance or Manhattan distance) between different feature values . If a sa(1) Calculating the distance d (this paper Euclidean distance was applied as d) between the test data and each set of training data; (2) Sorting the distance increasingly; (3) Selecting the K points with the smallest distance; (4) Calculating the frequency f of each category of the K points; and (5) The category with the highest frequency among the K points is the predictive category for the test data. K is optimized by using ten-fold cross validation during the model development and updating stages.SVM is an important classification method and has many unique advantages in solving small sample sets, nonlinear and high-dimensional pattern recognition problems . It estaTo analyze the classification accuracy results, there were three aspects can be considered: (1) the results of different pretreatment methods at full-band treatment; (2) the results of different wavelength selection algorithms with the same pretreatment method; and (3) the results of different classification methods with the same pretreatment method and the same wavelength selection algorithm. The >80% classification accuracy results of both the calibration sets and the validation sets could be observed clearly by a pink 80% line among the three corn varieties.When the PLS-DA, KNN, and SVM methods were applied to establish the classification models, the robust classification model was selected to achieve the visualization of classification results of the corn seeds. The average spectrum and the pixel spectrum are explained. When the ROI of a hyperspectral image is extracted, it contains many pixels, and each pixel corresponds to a spectrum; in this paper, the average spectrum refers to the average spectrum of the seed embryo ROI, the pixel spectrum refers to the spectrum of each pixel in the seed embryo ROI. In this paper, the visualization images were obtained in Method M2M and Method M2P. Method M2M: the classification results were obtained from the above study and each corn seed was corresponding with the classification value. Method M2P: the spectrum of each pixel of the embryo images was classified, and each pixel of the corn seed was corresponding with the classification value. Mean-spectrum to mean-spectrum (M2M) means that both of the calibration and validation sets were the mean spectrums of the ROI; Mean-spectrum to pixel-spectrum (M2P) means that the calibration sets were the mean spectrums of the ROI while the validation sets were the spectrum of each pixel in the seed embryo ROI.It is known that the surface of the corn seed sample is not very flat where the height of the embryo edge is higher than the height of the embryo inside, thus, in this paper, 1~3 thresholds for each category were set to classify the different degrees of freeze-damage in corn seed after the percentage of each category was calculated.Conductivity test is a general method to distinguish different degrees of freeze-damage in corn seed. The more serious the freeze damage is, the higher its conductivity. In this feasibility attempt to classify different degrees of freeze-damage in corn seed with hyperspectral imaging technology, four different pretreatment methods , four wavelength selection algorithms  and three different classification modeling methods  were applied to find a relatively better method for the three different corn seed varieties. In order to better represent the freeze damage of the seed embryos, comparisons were made between method M2M and method M2P.(1)By using related image preprocessing methods on the gray image of corn seeds at 500 nm wavelength, the final embryo hyperspectral images can be clearly obtained to achieve the following classification of different degrees of freeze-damaged corn seed.(2)The 5-3 smoothing pretreatment method has higher classification accuracy than the other pretreatment methods from the results of different pretreatment methods at full-band treatment. The classification accuracy almost reaches 90%, maybe because the pretreatment has the advantages of keeping the signal from the original spectrums and improving the signal-to-noise ratio.(3)The classification accuracies of the full-band, SPA, PCA and X-loading algorithms can be sorted as follows: the classification accuracies of the full-band method are higher than those of the SPA and PCA methods, and the X-loading method has the lowest classification accuracy from the results of different wavelength selection algorithms with the same preprocessing method. Maybe this is because a great deal of information about the corn seeds can be got in the full band situation, but in some way, it necessary to find several wavelengths to reduce the modeling time and improve the efficiency, so the SPA algorithm could be a good choice.(4)The classification accuracies of the PLS-DA, KNN and SVM methods can be sorted as follows: the PLS-DA modeling method has the best classification accuracy compared to the SVM and KNN modeling methods, while the KNN modeling method has the lowest classification accuracy.(5)By setting several category thresholds, and comparing the percentage value or subtracting with method M2P, fewer numbers of misclassified corn seed samples can be obtained. In some respects, method M2P has better results than method M2M.The following conclusions are drawn from this study:Based on the above several conclusions, it is feasible that the hyperspectral imaging technology used to establish classification models for the embryos of corn seeds with different degrees of freeze damage. The smoothing method and wavelength selection method can be applied to modeling to improve the signal-to-noise ratio, classification efficiency and result accuracy of the model, although good modeling results can be obtained in the full-band case. The method M2P allowed visualization of the classification result of each embryo pixel and the final classification result is better than the method M2M.


In the original article, there was an error. There were two spelling mistakes in the article.Discussion and Conclusion, last sentence of Paragraph 8:A correction has been made to “Recognition of the characteristics of these patients with the T10191C mutation will help us improve the clinical understanding of LS or Leigh-like syndrome.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.In the original article, we had not obtained the heteroplasmy levels of the proband and her aunt from the gene sequencing company at the time of manuscript publication.Through follow-up research, we make the following addendum:By detecting the proband's and her aunt's blood sample, we found the heteroplasmy level of the proband is 33.2% (forward percent) or 38.2% (reverse percent) respectively, the heteroplasmy level of her aunt is 4.4% (forward percent) or 4.8% (reverse percent), respectively.


R2) was 0.91, in the first-derivative treatment equation R2 = 0.95 and in the second-derivative treatment R2 = 0.96. The estimation accuracy was based on RPD  and RER  of the external validation. RPD and RER values were of 2.75 and 9.00 for the treatment without derivative, 3.41 and 11.79 with first-derivative, and 3.10 and 11.03 with second-derivative. With the acid detergent residue spectrum the wavelengths were identified and associated with the ADF contained in the sample. The results showed a great potential of NIRS for predicting ADF content in turnip greens and turnip tops.Standard wet chemistry analytical techniques currently used to determine plant fibre constituents are costly, time-consuming and destructive. In this paper the potential of near-infrared reflectance spectroscopy (NIRS) to analyse the contents of acid detergent fibre (ADF) in turnip greens and turnip tops has been assessed. Three calibration equations were developed: in the equation without mathematical treatment the coefficient of determination ( Brassica constitute one of the economically most important plant groups in the world. They are valuable sources of roots, stems, leaves, shoots and inflorescences, as well as of oils, condiments and forage for nutrition or industrial use [Brassica has been related to human health with regard to the reduction in the risk of suffering from certain types of chronic diseases, such as cardiovascular problems and different types of cancer [Brassica, four species, Brassica oleracea, Brassica rapa, Brassica napus and Brassica juncea, are the crops with a horticultural use. Brassica rapa L. subsp. rapa, commonly known as turnip, is one of the oldest crops used for human consumption. It was the first species of Brassica domesticated by humans thousands of years ago, and it was already cited in Sanskrit literature under the name of Siddharta, which proves the antiqueness of its cultivation [The plants of the genus rial use . Dependif cancer ,3. Withitivation . In the tivation . Tradititivation . One drativation . Neutraltivation . Severaltivation ,10. In ttivation ,12,13.Brassica were carried out by Panford, Williams and Man [Brassica species have been performed by Font, Del Río, Fernández and De Haro-Bailón [Standard wet chemistry analytical techniques currently used to determine plant fibre constituents (as those described above) are costly, time-consuming and destructive. Additionally, they need specialized workers for their application. During the last 40 years technology based on near-infrared reflectance spectroscopy (NIRS) has become one of the most attractive analytical techniques that is routinely used to estimate numerous quality components in agriculture and food research, since analysis can be carried out at a low cost, with an important saving of time, and without using hazardous chemicals. Moreover, NIRS is a non-destructive technique which requires minimal or zero sample preparation ,18,19,20 and Man  and Mich and Man . More reo-Bailón , Font eto-Bailón , Dimov, o-Bailón  and Witto-Bailón . Lately,o-Bailón , in ordeo-Bailón , or to so-Bailón . Brassica rapa (turnip greens and turnip tops), in order to employ them as a tool for a fast and non-destructive analysis in the screening of germplasm and in the selection of genotypes of the highest quality with respect to this component.This work has aimed to develop and validate NIRS calibration equations for the determination of acid detergent fibre (ADF) in aerial edible parts of Brassica rapa L. subsp. rapa were grown on the Institute for Sustainable Agriculture experimental farm in Córdoba,  in a random block design with three replications. The climate is a typical Mediterranean one with a mean rainfall of 650 mm and deep loamy-sandy soil classified as Typic Xerofluent.During the seasons 2013–2014 and 2014–2015, a set of five varieties of Brassica rapa L. subsp. rapa varieties came from the Brassica Germplasm Bank at the Biological Mission of Galicia (Pontevedra), where they had been characterized by their agronomic characteristics and their aptitude for the production of turnip greens and turnip tops. During each agricultural season, and at the optimal consumption moment, samples of turnip greens (4–5 leaves per plant) and of turnip tops  were harvested from the plants selected for each of the varieties studied  equipment. The lyophilized samples were ground in an IKA-Labortechnik®  model A10 mill for 20 s and stored in desiccators up to the moment of being analysed by the reference method or scanned in the NIRS equipment.The  studied . In tota®, Barcelona, Spain) machine. 0.5 g of lyophilized sample was weighed in glass filtering crucibles (porosity 2). This was digested for one hour in 100 mL of hot cetyl-methyl-ammonium bromide in an acid medium (sulphuric acid) and then filtered to obtain the residue considered as being the acid detergent fibre of the sample. Next, the residue was washed with hot water and acetone and dried in a stove at 110 °C for 90 min. Then it was stored in desiccators for 30 min to temper the crucibles and prevent the sample from becoming moist, after which the sample was weighed. The acid detergent residue (ADR) remaining after digestion was removed from the crucibles and stored to obtain the NIRS spectrum from the pure residue.The ADF content was determined following the procedures described by Goering and Van Soest  in a DosThe acid detergent fibre of the sample was calculated according to Equation (1):®, Inc., Silver Spring, MD, USA) near-infrared spectrophotometer in the reflectance mode. One spectrum was recorded for each sample. The samples were placed in a round capsule 3 cm in diameter made of quartz glass and anodized aluminium to prevent interferences in their absorption. From each sample, reflectance spectra in the wavelength range of 400–2500 nm, at 2 nm intervals, were obtained. Collection of spectral data and their chemometric analysis was conducted with the WinISI II v1,50 software . Sample spectra were recorded with a Model 6500 (Foss-NIRSystemsn = 26) and was used to evaluate the prediction capacity of each of the equations developed. The external validation set samples were selected by taking the list of samples ordered on the basis of their H values, choosing 1 of each of the five samples on the list [The spectral outliers were detected by a principal component analysis (PCA) applied to the whole set of the population based on the calculation of the Mahalanobis distance (H) ,34. In athe list . In thisthe list . To devethe list ,27.R2) which shows the percentage of the variability in the ADF concentrations explained by the regression equation, and the standard error of calibration (SEC), which is the standard error in the residuals for the calibration set. It should be noted that the standard error in the calibration only advises one of the fitting of the reference values to the regression line, so that it cannot be considered as being an adequate statistic for assessing the validity of the calibration equation obtained [The spectrum correction procedure SNV-DT was applied. The latter provides the WinISI software for the elimination of dispersion due to the effects caused by the differences in particle size or the variation in length, halfway between the dispersion of the samples and fitting the baseline . The treobtained .To evaluate the prediction capacity of the calibration equations, two validation models were used, permitting the establishment of a comparison  between the true value (obtained by the reference method) and the estimated one (obtained by NIRS).2cv), the standard error of cross validation (SECV), which represents the standard error of the residuals for the cross validation set; and the statistic (RPD) [cv is a statistic which permits the evaluation of the SECV in terms of the standard deviation of the reference data for the population being studied [i = laboratory reference value for the sample; n = number of samples, k = number of wavelengths used in an equation; SD = standard deviation of the chemical data.A cross validation was made based solely on the data employed at the calibration stage, in order to calculate the optimal number of terms in the regression. The algorithm selects different calibration and validation sets within the whole population considered, making with each selection a simulation of the regression algorithm ,35. Fina studied :(2)RPD=n = 26 in our work) were evaluated. The external validation statistics include: the coefficient of determination of validation (r2ev), the standard error of prediction (SEP), the RPDev (which is the ratio SD/SEP), and the RER [i = laboratory reference value for the sample; n = number of samples, k = number of wavelengths used in an equation; SD = standard deviation of the chemical data.The calibration equations selected with samples which did not intervene in the calibration  and high RPDev and RER values. Those equations in which RPD is higher than 3 were considered to have an excellent prediction ability, those with RPDs of between 2 and 3 allowed approximate predictions to be made, and those whose RPD was between 1.5 and 2 could only be used for classification purposes in groups with a high-medium-low content. Similarly, the RER values obtained with the different calibration equations with a good prediction capacity should be over 10 [The RPD and RER statistics permitted a comparison of the performance of the model through populations with different standard deviations . The bes over 10 ,40.The standard error of laboratory (SEL) for the ADF analysis was determined and compared with the SEP for all the equations. To obtain the total error of the reference method (SEL), 10 samples of turnip tops and turnip greens were selected and analysed in duplicate at different times and by different analysts. The statistical ratio SEP/SEL permitted the NIRS error to be related to the error in the reference method.Brassica rapa were analysed (63 turnip greens and 71 turnip tops) by the Goering and Van Soest method [t-test, showed significant differences between the means. (p < 0.001). A collection of 134 samples of Brassica rapa was a good source of fibre with high concentrations in some samples (21.91%) and lower concentrations in others (8.55%). The differences in the ADF content between turnip greens and turnip tops samples can be explained by the fact the turnip greens are formed by young leaves and the turnip tops by flower stems with a higher content of fibre. Therefore, we can conclude that the maturity of plants and the increase in structural carbohydrates lead to higher accumulation of fibre amounts in turnip tops when compared to turnip greens. These results highlight that Brassica rapa were 23.50% [B. rapa widely used in human nutrition) have found values of 11.20% [Brassica their values ranged from 5.33% (B. carinata) to 16.31% (B. juncea) [The variability in the ADF content in the samples analysed in this work was similar to that published in others studies, in which ADF values present in leaves of e 23.50% ; in crude 23.50% . Previouf 11.20%  and 14.6f 11.20% . In rapef 11.20% ; and in  juncea) . The statistical data describing the calibration and validation sets are shown in Brassica rapa samples in the range of 400 to 2500 nm; The principal component analysis was carried out to locate any possible spectral outliers from the calibration set . Figure Brassica rapa, B. rapa and ADR, 2H and 2278 nm related to groups CH and CH2 associated with the structural polysaccharides of the plants, 2468 nm related to groups CH, CH2 and C-N-C associated with proteins [With the aim of identifying wavelengths and associate spectrum bands with the ADF contained in the sample, the acid detergent residue spectrum (ADR), R2 = 0.91) and the equations with treatments with derivative the value of R2 = 0.95 in the first derivative  and a value of R2 = 0.96 in the second one , with both values being very similar to each other  and 3.33 . In both equations the RPDcv values were higher than 3, proving the ability of the calibration equations to be used for diagnosis and investigation purposes [On the basis of the statistics obtained in the cross validation, the final calibration equations for the ADF content were selected on the premise of maximizing the purposes .The two derivatization treatments  successfully optimized the model getting some optimal results in the statistics values. Both two models were valid for calibration. In the study of the profile of fatty acids in milk calibration equations, the first derivative and the second derivative were developed,  and , and both treatments were valid to be used in the characterization of the fat content in milk . Other ar2ev = 0.87 was obtained, which led to lower RPDev and RER values than those of the treatments with derivatives. In treatments  and  the same high value in the coefficients of determination of the prediction, (r2ev = 0.91) was obtained. The RPDev values were very similar to each other, 3.41 in the first derivative and 3.10 in the second one. Those results were very similar to those of RPDcv obtained in the cross validation  and they confirmed the excellent ability to predict ADF content by using both the equations developed with treatments  and  [Once the equations were obtained and the cross validation was performed, a second evaluation of the equations was made by using the samples not included in the calibration  for the prediction of the ADF content. 5, 5, 1) ,40. Fina5, 5, 1) ,40.Brassica rapa have been described in the bibliography up to now. However, some equations developed for the ADF content in leaves of woody species have been reported with higher values than those described (RPDev = 5.3), possibly due to the heterogeneity in the samples selected for the development of the calibration, in which different woody species collected on different dates were included [ve values of 2.9 [No calibration equations of the ADF content in green parts of included . ADF cals of 2.9 , and oths of 2.9 .Brassica, the values obtained in the calibration equations in our work are the highest described to the moment for ADF content, compared to the equations developed for ADF content in seeds found in the literature. To summarized, calibrations in intact Brassica napus seeds were described, with RPDcv values of: 2.13 and 2.20 (in a volume of 10 mL of seed) and values of 1.91 and 2.34 (in a volume of 1 mL of seed) [B. napus seeds, with RPDev of 2.2 and RER of 10.03 [Regarding calibrations within the genus of seed) ,29; thesof seed) . As for of 10.03 .Brassica species [To evaluate the precision of the equations the reference method error (SEL) was calculated and was related to the SEP. The SEL value obtained was of 0.25. The SEP/SEL ratio shown in the ADF was of 4.56 in the treatment 0, 0, 1, 1, which indicates a poor precision, and values of 3.48 and 3.72 were obtained for the treatments of 1, 4, 4, 1 and 2, 5, 5, 2, respectively, which reveal a good precision; those values are similar to the ones obtained by other authors in other  species .Brassica species and for dietary fibre in high-fat cereal products [Panels a, b, and c of products .Brassica rapa  were the molecular associations most frequently used in modelling the equation. Shape and positioning the bands presented by the different loadings very closely resembled those reported by Font et al. [Brassicas, in which effects due to CH groups of lipids and OH groups of water were the most important in the model. Recent NIRS calibrations for fibre fractions in intact seeds of Brassica napus also showed a significant contribution to the model of the CH, OH and NH groups in aromatic and protein regions [The study of the MPLS loadings of the ADF equation developed in this study suggests that OH groups of water, CH and CHt et al.  for oils regions . The results obtained in the present work, both in the cross-validation and in the external validation confirm the reliability and potential of the calibration equations developed with treatments  and  to predict accurately and precisely the ADF content in turnip greens and turnip tops. In addition, both calibration equations  displayed the same ability prediction of the ADF content in samples of turnip greens and turnip tops.As a conclusion, the accurate predictions provided by the NIR equations developed in this work confirm that NIR technology could be very useful for the rapid evaluation of the ADF content in turnip greens and turnip tops. Furthermore, this technique allows us to save considerable time and money in comparison to the standard methods of analysis, making it possible to conduct large numbers of analyses for ADF content in a short time.


AimNinety percent of knee ligament injuries involve the medial collateral ligament (MCL) and the anterior cruciate ligament (ACL) of the knee joint. Matrix metalloproteinases (MMPs) are a large group of calcium- and zinc-dependent endopeptidases responsible for cleaving and rebuilding various connective tissue components. Previous studies showed that MMP-2 and 9 have a significant effect on the healing process of injured ligaments. Therefore, the aim of this study was to evaluate for the first time in literature the expression and localization of MMP-2 and 9 in the epiligament (EL) and the ligament tissue of the MCL and the ACL of the human knee joint in order to assess their role in ligament healing.Materials and methodsFor the present study, we used histological material from the mid-portion of the MCL and the ACL of 14 knee joints from fresh cadavers. For the purpose of the immunohistochemical analysis, we used primary polyclonal antibodies against MMP-2 and 9. The obtained results were evaluated semi-quantitatively through ImageJ.ResultsImmunoreactivity for MMP-2 was predominantly positive (2+) in the EL of the MCL and remained mostly negative (0) in the ligament tissue. The expression of MMP-9 was mostly low-positive (1+) in the EL of the MCL and almost entirely negative (0) in the ligament tissue. In the EL of the ACL, the immunohistochemical expression of MMP-2 was predominantly low-positive (1+) and that of the MMP-9 was read as mostly low-positive (1+). Expression of the two enzymes in the ligament tissue was similar to the MCL.ConclusionThe present study is the first comparison of the expression of the aforementioned MMPs in the EL tissue of the MCL and the ACL in human knees, which may play a key role in physiological and pathophysiological processes such as tissue healing and repair and basement membrane degradation. The medial collateral ligament (MCL) of the knee joint is one of the most commonly injured ligaments of the knee -2. NinetThe thin connective tissue layer enveloping the ligament was first described as the ‘epiligament’ (EL) by Bray et al. . In 2017Matrix metalloproteinases (MMPs), also known as matrixins, are a large group of calcium- and zinc-dependent endopeptidases responsible for cleaving and rebuilding connective tissue components such as collagen, elastin, gelatin, and casein -21. ThesTherefore, the aim of this study was to evaluate for the first time in literature the expression and localization of MMP-2 and 9 in the EL and the ligament tissue of the MCL and ACL in the human knee joint in order to assess their role in ligament healing.Tissue preparationFor the present study, we used 14 knee joints from fresh cadavers from the autopsy material available at the Department of Anatomy, Histology and Embryology at the Medical University of Sofia. Eight specimens were female and six were male with a mean age at death of 64.5 years old. No medical or surgical history for trauma of the knees of the cadavers was available. The study was approved by the Medical Legal Office, the Local Ethics Committee, and the Institutional Review Board (No. 4866).After skin incision, the underlying subcutaneous tissue was dissected to expose the MCL of the knee joint. The MCL and the external surface of the surrounding EL were precisely dissected, the points of insertion of the ligament were excised, and the mid portion of the MCL was immediately fixed in 10% formalin solution  for 24 hours and were then dehydrated in increasing concentrations of ethanol  . After opening the capsule of the knee joint, the mid portion of the ACL was processed and fixed in the previously described way. Next, ethanol was removed using cedar oil until the samples became translucent. The samples were then rinsed in xylene  and embedded in paraffin .Immunohistochemistry protocoln = 28, 14 of each ligament) was cut on a microtome  into 5 µm thick sections which were mounted on slides previously coated with chrome-gelatin. Next, we randomly selected 10 slides per paraffin block, thus obtaining a total number of 140 slides of each ligament. Sections were deparaffinized, rehydrated with ethanol  , and washed in 0.1 M phosphate buffer , pH 7.4, at room temperature. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (H2O2) for 10 minutes at room temperature. The sections were rinsed in phosphate-buffered saline (PBS)  and nonspecific binding sites were blocked with Super Block  for five minutes. Primary rabbit anti-human polyclonal antibodies against MMP-2  and MMP-9  at a dilution 1:500 were added and the sections were incubated overnight at 4ºC, rinsed in PBS , and incubated with biotinylated goat anti-rabbit immunoglobulin G (IgG)  for 10 minutes at room temperature. Sections were rinsed as before and incubated with streptavidin-HRP  for 10 minutes at room temperature. Antibody binding was visualized using 3,3′-diaminobenzidine (DAB)  as chromogen for 10 minutes. Sections were counterstained with hematoxylin , dehydrated in increasing concentrations of ethanol  , cleared in xylene , and cover-slipped with Canada balsam . Sections used as controls were incubated in the way previously described, but omitting the primary or secondary antibody. All controls were negative. The immunohistochemical staining of all sections was conducted under the same conditions. Photomicrographs of representative fields of the immunohistochemical staining were obtained using an Olympus CX 21 microscope fitted with an Olympus C5050Z digital camera .Each paraffin block , positive (2+), low positive (1+), and negative (0). The immunohistochemical expression in the EL and the ligament tissue of the MCL and the ACL were presented as percentages of the respective scores as calculated by the IHC Profiler.Immunohistochemical analysis of MMP-2 and -9 expression in the MCLThe immunohistochemical analysis of the expression of MMP-2 and 9 revealed a heterogeneous distribution of the enzymatic activity of the two enzymes in the EL and in the ligament tissue of the MCL. The positive staining for MMP-2 was distributed along the entire thickness and was best expressed in the adventitia of blood vessels in the EL, as well as in the perivascular zone Figures -1b.The semi-quantitative analysis revealed positive (2+) expression in 70% of visual fields and low-positive (1+) expression in the remaining 30%. Perhaps an interesting finding was that some fibroblasts in the ligament tissue also stained positive for MMP-2. Immunoreactivity was calculated as negative (0) in 76% of visual fields and as low positive (+) in the remaining 24%. The enzymatic activity of MMP-9 in the EL was read as low positive (+) in 63% of visual fields, positive (2+) in 28%, and negative (0) in 9% of visual fields. As in the case of MMP-2, staining was best expressed in the adventitia of blood vessels and in the perivascular zone Figures -2b.In the ligament tissue, the expression of MMP-9 was calculated as negative (0) in 88% of visual fields and low positive (1+) in 12% of visual fields, where it was observed mostly in the fibroblasts and in the adventitia of the few blood vessels.Immunohistochemical analysis of MMP-2 and -9 expression in the ACLIn the EL of the ACL, we observed MMP-2 expression in fibroblasts, fat tissue cells, and the adventitia of blood vessels Figures -3b.In 74% of visual fields, immunoreactivity was read as low positive (1+), while in the remaining 26%, it was calculated as positive (2+). In the ligament tissue, enzymatic activity was predominantly negative . In certain areas, it was calculated as low positive (1+) . This low-positive reaction was observed in some fibroblasts located close to the border between the EL and the ligament tissue. The distribution of the immunoreactivity of MMP-9 was similar to that of MMP-2. In the EL, it was reported in the adventitia of blood vessels and the perivascular space and in the adipose tissue cells Figures -4b.The semi-quantitative analysis revealed a low-positive reaction (1+) in 83% of visual fields and negative reaction (0) in 17% of visual fields. In the ligament tissue, enzymatic activity was read as negative (0) in 94% of visual fields and low positive (1+) in the remaining 6% of visual fields. This enzymatic expression in the ligament tissue was only noted in individual fibroblasts located close to the border between the ligament tissue and the EL.The MMPs are a family of calcium- and zinc-dependent endopeptidases which play a key role in the breakdown and removal of extracellular matrix molecules from the tissue, a process that takes place during developmental stages such as growth and morphogenesis , 24. MMPThe MMP family members have been classified into different subgroups with close characteristics. The classification recognizes collagenases , gelatinases (MMP-2 and 9), stromelysins , elastases , and membrane type MMPs , and a group of unnamed members . GelatinAfter comparing the expression of different MMPs in the MCL and the ACL, Zhou et al.  concludeIn the present study, we compared the immunohistochemical expression of MMP-2 and 9 in the EL and ligament tissue of the MCL and the ACL. Our data supported the findings of Zhang et al. , who repLimitations of the current study existed and should be outlined. The visual quantification of immunohistochemical images is associated with significant inter- and intra-observer variation. We attempted to resolve this issue by using the IHC Profiler plugin for ImageJ software, which eliminates inter-observer visual perception bias. In fact, 88.6% of scoring determined by the IHC Profiler coincides with blinded manual scoring determined by trained pathologists  .This study represents the first report on the immunohistochemical localization and distribution of the enzymes MMP-2 and 9 in the EL of the MCL and the ACL in humans. Furthermore, we demonstrated and compared the enzymatic activity of MMP-2 and 9. These findings confirm our previous observations in rat model and are thus a further piece of evidence of the important role of the enzymes of the MMP group for the normal function of the MCL and the ACL and their role for the difference in the healing potential between the two ligaments.


Titanium dioxide nanotubes/hydroxyapatite nanocomposites were produced on a titanium alloy (Ti6Al4V/TNT/HA) and studied as a biocompatible coating for an implant surface modification. As a novel approach for this type of nanocomposite fabrication, the atomic layer deposition (ALD) method with an extremely low number of cycles was used to enrich titania nanotubes (TNT) with a very thin hydroxyapatite coating. X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used for determination of the structure and the surface morphology of the fabricated nanocoatings. The biointegration activity of the layers was estimated based on fibroblasts’ proliferation on the TNT/HA surface. The antibacterial activity was determined by analyzing the ability of the layers to inhibit bacterial colonization and biofilm formation. Mechanical properties of the Ti6Al4V/TNT/HA samples were estimated by measuring the hardness, Young’s module, and susceptibility to scratching. The results revealed that the nanoporous titanium alloy coatings enriched with a very thin hydroxyapatite layer may be a promising way to achieve the desired balance between biofunctional and biomechanical properties of modern implants.  The growing demand for bone implants, which could replace worn bone elements or support their regeneration, is closely related to the active lifestyle of contemporary people, as well as the aging of societies. Numerous groups in the world have carried out intensive studies on the development of new implant technologies and have been searching for novel biomaterials for implant production, with great progress . The aimTitanium and its alloys are biomaterials widely used in the implants intended for orthopaedics, dentistry, maxillofacial surgery, as well as in laryngology and cardiology ,3. This 10(PO4)6(OH)2) on the implant surface seems to be highly beneficial from a biological point of view because HA is the most stable form of calcium phosphate .,47.33,47t formed . The fab3/E2 ratios  compared to the uncoated Ti6Al4V/TNT system. For the Ti6Al4V/TNT15/HA system, the situation is reversed, i.e., the deposition of the hydroxyapatite reduced these ratios by 7% and 33%, respectively. The H/E and H3/E2 ratios influence the wear resistance of a material, either in bulk or in coating form. The first ratio characterizes the resistance of the material to elastic deformation, and the second ratio allows estimation of the ability of the material to dissipate energy upon plastic deformation during loading. The HA coating deposited on the Ti6Al4V/TNT5 improved the elasticity index and the resistance to plastic deformation, which suggests a better tribological performance of the HA coated surface in medical applications. The scratch test of the nanocomposites based on the HA-coated TNT surface did not give information about the adhesion of HA nanocrystals to TNT. The calculated critical forces were rather related to the loss of adhesion of the entire TNT/HA nanocoating to the Ti6Al4V substrate rather than to the loss of adhesion of HA to the nanotubes. SEM images of the surfaces of the Ti6Al4V/TNT5/HA–Ti6Al4V/TNT30/HA samples proved that the packing density of the HA nanocrystals on the surface of the TNT layers increases as the diameter of the nanotubes increases. Also, nanoindentation results highlight clear differences between the nanocrystalline HA coatings deposited on the surface of the TNT layers produced at different potentials. The HA coatings present heterogeneous morphology with discontinuities, which is a result of the distinct nanotubular architecture of the TNT layer on which the HA coating is formed a. Fabric2 nanocoatings enriched with the hydroxyapatite. Additionally, fibroblasts are considered to be the most common cells in connective tissue, one of the main components of peri-implant soft tissue, which is key to the formation of the peri-implant mucosal seal and helps to prevent epithelial ingrowth [2) and the highest roughness (Sa = 0.135 µm) among the studied Ti6Al4V/TNT/HA samples. The surface roughness of biomaterials has a significant impact on the possibility of creating a proper connection between the implant and the bone. The influence of the implant surface roughness on the adhesion of cells and their proliferation has also been proven. A surface with a high roughness improves the adhesion of cells and tissues to the implant and provides improved primary stabilization between the implant and bone [2 nanotubes covered with a hydroxyapatite compared to an untreated titanium surface [2 nanotubes showed higher cell density, more live cells, and better spreading of the mouse osteoblastic cell line, MC3T3-E1, as compared with an untreated titanium plate surface [The mechanical properties of biomaterials used in the construction of implants should go hand in hand with their bioactivity. Therefore, studies on the ability of proliferation of fibroblast cells on the surface of the produced nanocomposites and on their potential antibacterial activity were carried out. Since mouse connective tissue fibroblasts, including L929 cells, are recommended as the standard model for biocompatibility testing , we chosingrowth . The resand bone ,53. Analand bone . Filopodand bone ,56. Our  surface . Similar surface . Taken t surface . 2 nanotubes, nanowires, nanofibers), metal ions/nanoparticles (mainly silver and copper), graphene oxide thin film, or covalently bounded antibiotics enhanced antimicrobial activity of titanium implants [2 nanotubes formed on the Ti6Al4V alloy possessed in vitro anti-biofilm properties as tested on the S. aureus model [S. aureus tested by the agar diffusion method. Although the choice of the method used was not the best one due to the lack of standardization in the reading of growth inhibition zones, the antimicrobial effect of the hybrid microspheres was noticeable and it was shown that CMCS/Ag-HA was more efficient than HA [Streptococcus mutans and nonpathogenic Staphylococcus carnosus adhesion in a human cavity, indicating the presence of saliva as a factor strongly determining the adhesion of S. mutans, but not S. carnosus, to HA [Biofilm-associated infections of inserted and implanted biomaterials, such as intravascular catheters, prosthetic valves, orthopedic implants, vascular grafts, etc., remain a serious medical problem, which threaten the lives of patients. Therefore, the estimation of the antibacterial activity of the produced TNT/HA coatings, which could be applied in modifications of Ti6Al4V implant surfaces to limit potential infection development, was important for us. Earlier works revealed that the nanoarchitecture of the surface (e.g., TiOimplants ,64,65,66 than HA . Similars, to HA . Colonizs, to HA .2 nanotubes on a titanium alloy in an attempt to produce novel nanocomposites with optimized biocompatible, antibacterial, and mechanical properties for applications in medical implants. The results of our work revealed that the use of TiO2 nanotubes as a link system between the titanium alloy and a thin layer of hydroxyapatite nanocrystals seems to be an excellent solution to inhibit the delamination of the hydroxyapatite layer. Their nanoporous architecture led to a much stronger connection with hydroxyapatite than if the hydroxyapatite was deposited directly on the surface of the titanium alloy. However, one condition had to be fulfilled - the hydroxyapatite layer must be extremely thin. Such a thin layer can be provided by the method used in our experiment, i.e., the atomic layer deposition of CaCO3 films on the Ti6Al4V substrate followed by their conversion to hydroxyapatite nanocrystals by wet treatment in dilute phosphate solution. ALD was exploited to add hydroxyapatite on top of TiOThe results of our work indicate the promising properties of the Ti6Al4V/TNT5/HA as a bio-active nanocomposite. This system showed the best adhesion and proliferation of fibroblast cells, in comparison to the other studied nanocomposites, and simultaneously good antibacterial activity. Moreover, this system was characterized by high surface roughness, an appropriate elasticity index, and resistance to plastic deformation, which suggests optimal tribological performance of the surface for medical applications.


Lysozyme is a type of enzymatic protein found in a wide range of organisms. Among the many applications of lysozyme, the antibacterial activity features caused by the hydrolysis of 1–4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine of gram-positive bacteria are beneficial in the food industry, medicine, trade, and pharmacology. Studies have indicated that specific modifications of lysozyme cause oligomerization of the enzyme, and the resulting dimer, which also undergoes changes in physicochemical properties, shows greater total antibacterial activity. Among these modifications, thermo-chemical methods are one of the most important groups. In this study, the microwave method of the enzyme heating with the associated process of enzyme oxidation was used as a novel thermo-chemical method to induce lysozyme oligomerization. The research shows that using this new method can produce enzymatic preparations composed of approximately 58.9% oligomers, including 33.5% dimer and 25.4% trimer under a hydrogen peroxide concentration of 4% and pH of 8. The maximum percentage of lysozyme dimer of 39.4% was obtained at pH 6.0 with the addition of 2% oxidant. In addition, as a result of the modification process, the hydrolytic activity and surface hydrophobicity of the enzyme were changed. Lysozyme is a small molecule enzyme 14 400 Da). Its antibacterial activity is the original natural mechanism for defense against bacteria in humans, animals, and plants and is a large component of the eggs. The action of lysozyme is based on hydrolysis of the β-1, 4-glycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine, which present in the cell walls of many bacteria. Unfortunately, the antibacterial action of the enzyme only applies to gram-positive bacteria. Gram-negative bacteria are resistant to its effects 4 400 Da.. Many stinterferon alpha and gamma  as well as interleukin-2 (Il-2) and interleukin-6 (Il-6) by human lymphocytes [To date, several original methods have been developed to modify lysozyme. Research has focused mainly on thermal and chemical modifications of the enzyme, in low-, medium- and high-temperature ranges. Membrane techniques have also been used for this purpose , 7–9. Thphocytes –13.Seeking more effective ways to modify lysozyme, the purpose of this work was to introduce a new method of lysozyme modification based on the use of microwave field interaction with chemical processes and to evaluate selected physicochemical properties of the obtained preparations. Our previous research indicates that such a modification system can provide a very good result.The experimental material was lysozyme monomer isolated from chicken egg white with a hydrolytic activity of 21 252 U/mg, as produced by the Belovo company .The modification was carried out in two stages: A-modified by a microwave field and B-chemical modified by oxidation.For both modifications, lysozyme was prepared as a 5% (w/V) water solution. The solution was adjusted to a pH of 4.0, 6.0, and 8.0 using 1 M NaOH or 1 M HCl, respectively and then 15 ml of each solution was placed in sealed conical flask, numbered containers (5 replicates for each pH level).A-modified by microwave field:The modification by a microwave field was carried out in a microwave device  under the following conditions:microwave power—270 watts.time of modification—3 minutes.After this process, the samples were immediately cooled in ice water.B-The chemical modification was carried out immediately after the microwave modification. This process consisted of oxidation of the previously modified enzyme with hydrogen peroxide at the following concentration in the samples: 0%, 1%, 2%, 3%, and 4%. This process lasted 24 hours, after which the samples were frozen and lyophilized in a Labconco freeze dryer (USA). The operation conditions of the lyophilization were 0.022 mbar, -54°C and drying to obtain samples with a water content of 7–8%.The obtained preparations were subjected to analysis of the base chemical properties, the degree of enzyme oligomerization, hydrolytic activity and changes in the surface hydrophobicity of the enzyme.According to a previously published method , 14, theAfter the electrophoresis process, the gels were fixed in a solution of acetic acid, methanol, and water for 1 hour and stained with 0.025% Coomassie brilliant blue R solution for 20 hours. Finally, the gels were discolored in diluted acetic acid until a transparent background obtained.The amounts of oligomer in individual samples were calculated with the TotalLab Quant software .Micrococcus lysodeikticus  cells at 450 nm. The activity presented as the rate of decrease in absorbance per minute (Δ abs/min).Lysozyme hydrolytic activity was determined by the use of the spectrophotometric method, which based on the phenomenon of bacterial cell wall lysis by the enzymes , 16. TheThe hydrophobicity of the modified lysozyme was determined according to the research . This meThe test results were analyzed using the STATISTICA software for correlation, dependent regression analysis and one-way analysis of variance (ANOVA).The method of lysozyme modification presented in this work is a combination of thermophysical and chemical processes. For the thermophysical process, we chose the energy of the microwave field. Generally, the use of microwaves in chemical technology is not new and is even widely applied. There are also a few examples of the using of microwaves as an auxiliary factor in enzyme chemistry with precise control of the application parameters . The novAfter modification carried out in accordance with the procedure given in paragraph 3.2, we obtained preparations of modified lysozyme  that wer showed that the microwave modification alone gave good results. At pH 4 and 6, a composition of more than 17% dimer was obtained (sample 1 and 6), and when the pH was 8, the composition was approximately 18% dimer and 6% trimer, producing over 24% oligomers in total (sample 6). However, when the oxidation modification was added, a significant increase in the resulting level of oligomers was observed  of the equation approaching 1 means the goodness of fit of the formula is high.The coefficient (8.3767) of this formula suggested that HP. mirabilis in their thermochemical modification experiments. In addition, the cluster of hydrophobic side chain formed out a hydrophobic patch of solvent accessible surface area that lead to modified lysozyme interactions with bacteria membranes and penetration of bacteria [Furthermore, our research showed that in addition to intensive oligomerization, the microwave and oxidative modification of lysozyme also changed the hydrolytic activity and hydrophobic surface of the enzyme , Fig 2. bacteria , 26, 27.bacteria . And theIn our research presented in this work, we do not analyze the effects of the preparations on different types of bacteria, but we can expect that the effects will be similar to those described by Ibrahim  and othe2O2. is the amount we recommend. The optimal treatment conditions still need to be explored in future studies. Evaluation of the complete antimicrobial effect of the microwave-modified lysozyme, separate studies are being conducted now. These results of which will be the subject of our next work.In conclusion, taking into account the results of research and their analysis in relation to the current state of knowledge, we can state that to obtain the best antibacterial effect in this study, i.e., the largest effect of enzyme dimerization, the more suitable for application lysozyme modification conditions by this new method is an environment with a pH of 6.0 and an addition of 4% oxidant. Although dimerization is positive correlates with the concentration of hydrogen dioxide. However, the higher amount of oxidants can pose difficult applications in the food and pharmaceutical industries and safety threat to consumers. Hence, 4% of the concentration of HThis study indicates that microwave thermal modification of lysozyme can increase the formation of oligomers in comparison to conventional physical heating, improving the enzyme’s hydrophobicity by the use of this novel heat source for physicochemical modification; in addition, the method has the advantage of improving the modified lysozyme powder with faster and easier operation. Three minutes of heating can achieve a good result. The solubility of preparations reached 100%, making them easier to use than the preparations from conventional thermal methods with a solubility of 50–80%. The mixing method was very simple and compact. This method requires fewer technological devices, common chemicals, and a suitable device for manufacturing a microwave field.The next steps of research should address the microwave processing time of the microwave modification and microbiological tests. This investigation described the relationship between microwave modification and antimicrobial activity in the mixer modification of lysozyme.S1 Data(XLSX)Click here for additional data file.


The results show that adding nano-CaCO3 to PP could improve its mechanical properties and cell structure. The thermal stability and crystallinity enhances with increase of nano-CaCO3. As a bubble nucleating agent, adding nano-CaCO3 to PP improves the cell structure in both the parallel sections and vertical sections. The mechanical properties increase first and then decrease with increase of nano-CaCO3. The mechanical properties are affected by the cell structure, as well. The mechanical properties and cell structure are optimum when the content of nano-CaCO3 is 6 wt %.Using supercritical nitrogen as the physical foaming agent, microcellular polypropylene (PP) nanocomposites were prepared in microcellular injection molding. The main purpose of this work is to study effects of content of nano-CaCO Microcellular foam polymers are new materials with a high impact strength, high insulation properties, and low electrical conductivity. However, there are some disadvantages to using microcellular foam products, such as a decrease of the tensile strength, bad surface quality, and an uneven distribution of cells.Polypropylene (PP) has been widely used in many fields, such as in electrical equipment, furniture, and automobile interiors. In the automobile interiors, reducing the amounts of polymer is an important way to lightweight. Also, microcellular foam is an advanced technology used to reduce the amount of polymer used. Microcellular foam can make the polymer create a cell structure, which has a cell diameter less than 10 μm and a cell density larger than 103, multiwalled carbon nanotube, and so on. Nano-clays are widely used because of their good economic benefits. For example, Hwang added the montmorillonite into the polybutylene terephthalate (PBT), and the results show that the addition of montmorillonite improved the thermal stability of the microcellular injection molded PBT/clay nanocomposites, and the optimum content of organoclay was 1.0 wt % [3, a common additive, there are many studies about the influence of nano-CaCO3 on the microcellular polymer. X shi et al. found that, with addition of nano-CaCO3 into a microcellular PLA/PBAT blend, the cells present a significant increase in the cell uniformity and cell density [3 on the microcellular PP, and a batch process was used. The results show that PP/5 wt % nano-CaCO3 exhibits an optimum cell structure [3/PP in a batch process, too [3 foams using an extrusion foaming process, and results showed that the nano-CaCO3 affected the cell sizes significantly [For improving the performance and cell morphology of microcellular foam products, adding nano-filler as nucleation agents to the polymer was widely used by the researchers. On the one hand, the nano-filler additives could improve the distribution of the cells. The fine cells distribution, cell size, and cell density greatly affected the mechanical properties. Marsavina et al. found that the fracture toughness increased with increase of the foam density, and the yield strength improved because of a higher foam density ,2. On th1.0 wt % . Polylac1.0 wt % . Xie et 1.0 wt % . Layered1.0 wt % ,8. Regar1.0 wt % . The low1.0 wt % . As for  density . Ding et density . Ding altructure . Huang iess, too . Yu inveficantly . In this3 content on the crystallization behavior, cell structure, and mechanical properties of the microcellular foamed PP. The cells of different sections and layers are discussed, respectively.Therefore, the nano-fillers affect the crystallization behavior, mechanical properties, and cell structure badly. However, there are no detailed studies about the cell structure and the effect of the cell structure on mechanical properties. In this work, we reported experimental studies to systematically investigate the effects of the nano-CaCO3 with a mean particle diameter of 60–80 nm was supplied by Changshan Jinxiong Co., Ltd. . The PP-g-mah (1.2% grafting) that was used as a compatilizer was provided by Dongyuan Ziheng Plastics Co., Ltd. . The industrial N2 (99% purity) that was used as a foaming agent was provided by Wuhan XiangYun Industry Co., Ltd. .Polypropylene (K8303) with a melt flow index of 2 g/10 min was supplied by Sinopec Beijing Yanshan Co., Ltd. . The nano-CaCOThe microcellular injection molding process is shown in 3 contents of the composites were 2, 4, 6, 8, and 10 wt %, respectively. The 5 wt % PP-g-mah was added into all of the composites. The materials were dried for 24 h at 80 °C. The temperature of the barrel of the extruder was 190–190–190–200–200 °C.The nano-CaCO2 was controlled by a microcellular foaming console, . The supercritical fluid N2 (17 MPa) was injected into the barrel when the composites were plasticized. The injection time of SCF-N2 was 3 s, and the content of SCF-N2 was 0.5%. The nano-composites and SCF-N2 changed to the single phase melt after the mixing function of the screw. The injection pressure of the injection machine was 80 MPa and the back pressure was 10 MPa. The nano-composites foamed after the melt was injected in the mold, because of the decrease in the pressure and temperature. The samples were completed after cooling for 20 s.As shown in 2.A DSC8500  was used for the differential scanning calorimeter (DSC) analysis, and the analysis was done using two heating cycles over a temperature range of 40–200 °C in the heating/cooling rate of 10 °C/min. The tests were performed in an atmosphere of N2.A thermogravimetric analysis (TGA) was performed with a STA2500 , in the temperature range of 40–800 °C, at the heating speed of 10 °C/min in an atmosphere of NA JSM-IT300  scanning electron microscope (SEM) was used to characterize the cell structure and the distribution of the cells. The flexural samples were immersed in liquid nitrogen for 3 h. The flexural samples were fractured, and the fractured surface was coated with a gold layer before SEM testing. The parallel and vertical sections (10 mm × 4 mm) were taken from the flexural samples, as shown in The average cell diameter could be calculated with the following equation:n is the number of cells. The density of the cell can be calculated with the following equation:M is the magnification of SEM, and A is the area of the picture.As for the parallel section, the transition layer cells that nucleated and grew at the filling time deformed, and turned into an irregular shape. The mean ratio of length–diameter of the cells was used to describe the degree of deformation. The length and diameter of a cell are shown in As shown in An electromechanical universal test machine, CMT6104,  was used to measure the tensile properties and flexural properties. The method for the tensile tests was ISO 527-1:1993, and the crosshead speed was 50 mm/min. The method for the flexural tests was ISO 178:2001, and the speed was 2 mm/min. The impact strength (IZOD) was obtained according to ISO 180:2000. The values of all of the mechanical properties were calculated using the average values of five specimens.3. The reason is that, as a nucleating agent, nano-CaCO3 reduced the degree of supercooling. With the addition of nano-CaCO3, the main method of nucleating the nanocomposites was heterogenous nucleation. As for the melt curves, the melt peak temperature had no obvious change with increase of nano-CaCO3. When the content of nano-CaCO3 was 4, 6, and 8%, a tiny peak existed around 154 °C, and it was a fusion peak of 3 could promote 3 addition also promoted crystallization. The results of the DSC are shown in the c) can be calculated by the following equation:m), crystallization temperature (Tc), heat of fusion (Hm), and crystallization (Xc) of the nanocomposites are compared in the m and Tc change have been discussed above. The Hm and crystallinity increased with increase of nano-CaCO3. As a nucleating agent, the addition of the nano-CaCO3 improved the efficiency of crystal, and provided more nucleating sites. For the nano-CaCO3 with more than 6 wt %, the increment of crystallinity decreases, as shown in 3 conglomerating, the efficiency of the nucleating agent declines. The crystallinity affects the mechanical properties. So, the addition of nano-CaCO3 could improve the material’s hardness and elastic modulus [Crystallization . However, if the differential thermal gravity (DTG) increased with the increase of nano-CaCO3, it implied that the thermal stability increased with the increase of nano-CaCO3. At 550 °C, the polymer matrix almost completed its decomposition, and the residue was nano-CaCO3. This indicated that the content of nano-CaCO3 of the composites is almost same as the formula. Nano-CaCO3 started to decompose into CO2 and CaO at around 600 °C.3, it is obvious that the mechanical properties will change. The microcellular injection molding processing also has an effect on the mechanical properties. It is meaningful to investigate how the addition of nano-CaCO3 and the microcellular foam processing affect the mechanical properties of the samples.With the addition of nano-CaCO3, and decreased as the content of nano-CaCO3 was over 6 wt %. When the content of nano-CaCO3 was 6 wt %, the yield strength was 17.5 MPa, and the elongation at break was 134%. The tensile stain–stress curves of the solid composites and foamed composites were totally different, as shown in the 3 could improve the mechanical properties of solid polymer [3 was 6 wt %, the decrement was the smallest. With the addition of nano-CaCO3, the cell structure and distribution improved, because of the heterogeneous nucleation. The fine cell structure was advantageous for improving the yield strength. It can be concluded that both the cell structure and nano-CaCO3 affected the yield strength. As the content of nano-CaCO3 was over 6 wt %, the nano-CaCO3 conglomerated. The improvement of nano-CaCO3 on the tensile properties decreased.As shown in  polymer . The ten3 could improve the flexural strength of both the solid and foamed composites. The flexural strength increased first, and then decreased, with the addition of nano-CaCO3. The improvement of the flexural strength solid composites means that the rigidity of nano-CaCO3 could improve the stiffness. However, the flexural strength of the foamed composites was smaller than that of the solid composites. The decrement of the flexural strength was the smallest, because the fine cell structure could improve the flexural strength. As the content of nano-CaCO3 was 8 and 10 wt %, the nano-CaCO3 conglomerated and cell structure got worse, so the flexural strength of the foamed composites decreased.As shown in 3. With the addition of nano-CaCO3, the change of impact strength of the foamed composites was more obvious. As shown in 3 was 6 wt %. It is evident that more cells absorb more impact energy. When the content of nano-CaCO3 was over 6 wt %, the efficiency of nucleation declined and there was less cell generating, so the impact strength decreased. When the content of nano-CaCO3 was 10 wt %, the impact strength of the samples was even smaller than those without the addition of nano-CaCO3.As shown in 3.As discussed above, it is evident that there are some effects of the microcellular structure on the mechanical properties. It is important to investigate the microcellular structure of the microcellular foam PP nanocomposites with different contents of nano-CaCO3 affected the cell structure and distribution because there were more heterogeneous nucleations. The cells of vertical and parallel sections were investigated. The results showed that both the vertical and parallel sections were divided into three layers (two transition layers and one core layer), as shown in As the nucleating agent, the addition of nano-CaCOFrom From (1) Core layer of Vertical Section3 are shown in 3), from 3, the diameters of the cells decreased, and there were more cells generating. Research indicates that the cells nucleate in the boundary between the polymer matrix and the nano-CaCO3, and as the homogeneous nucleation agent, nano-CaCO3 can provide many nucleating sites [3 is 2 wt % and 4 wt %, as shown in 3 is 6 wt %, the average diameter of the samples was the smallest, the cell density of the samples was the biggest, and the distribution of the cells was also great, because the major way of nucleation was heterogeneous nucleation, and the cells nucleated and grew evenly, as shown in 3, the cell structure got worse, as shown in 3 conglomerated as the nano-CaCO3 was too much for a certain amount of compatilizer. It caused the uneven distribution of cells and it was easier for the cells to coalesce. The big and uneven distribution of the cells and nano-CaCO3 may cause the mechanical properties to get worse, as discussed above.The cell structure of the core layer of the vertical sections with different contents of nano-CaCOng sites . There i3 was bad. There were many large-sized cells with diameters larger than 100 μm; the amount of cells where the diameters were from 10 μm to 60 μm is large. The range of the cell size distribution was large. With the addition of nano-CaCO3, the cell distribution improved. The number of large cells decreased, and the cells had a concentrated distribution, as shown in 3 was the optimum, as shown in 3 was more than 6 wt %, the cell distribution got worse, as shown in The cell size distribution described the cells’ proportion in different ranges of cell size. As shown in The average cell diameter and cell density can be calculated with Image Pro Plus. The results of the average cell diameter and cell density are shown in 3 and increased when the content of nano-CaCO3 was more than 6 wt %. The cell density increased with the addition of nano-CaCO3 and decreased when the content of nano-CaCO3 was more than 6 wt %. The best cell structure was found, with an average diameter of 34 μm and cell density of 5.18 × 109 cells/cm3, when the content of nano-CaCO3 was 6 wt %. The reason for the cell structure changing in this way is that as the nucleating agent, the nano-CaCO3 can affect the cells’ nucleating and their distribution.The average diameter of the cells decreased with the addition of nano-CaCO3. It was opposite to the change of the cell density, because more cells mean that less gas is separated out. The content of the foamed agent was same in this work, so more cells helped in reducing the density of the foamed composites. When the amount of nano-CaCO3 was over 6 wt %, there were less polymers for foaming, and the addition of nano-CaCO3 did improve the mass of composites, so the density increased.As shown in the (2) Transition Layer of Vertical Section3 is shown in The cell structure of the bottom transition layer of PP with different contents of nano-CaCO3 changes from 2 wt % to 10 wt %. The cell sizes of the transition layer were smaller to those of the core layer, because the cooling speed of the transition layer was high and took less time to grow the cells. As shown in There were some big cells from the transition layer of the vertical section of the neat PP, as shown in 3 is different than the others.From 3, as shown in The cell size was more scattered in the transition layer of the neat PP, as shown in 3, the crystallization of PP was homogeneous. The time taken for the homogeneous crystallization was longer than for the heterogeneous crystallization, and there was more time for cells to grow. The way in which the cells were nucleated was homogeneous nucleation, the cells were unevenly distributed. In addition, the low melt strength led to the cells’ coalescence. With the addition of nano-CaCO3, the average cell diameter decreased, and there was no evident difference in the average cell diameter with the further addition of nano-CaCO3. The temperature of the transition layer was lower than the core layer, and cells did not have enough time to grow up. The melt strength increased because of the addition of nano-CaCO3. There were less cells that experienced coalescence, so the cell diameters were the similar. The way in which the cell density changed is similar to that of the core layer.As shown in (1) Core Layer of Parallel Section3, as shown in 3, the cells were more regular and the cells were more dense. When the content of nano-CaCO3 was 6 wt%, the cell density was biggest and the cell sizes were the smallest. Firstly, the addition of nano-CaCO3, as the nucleating agent of the cells, improved the cell structure. Also, the addition of nano-CaCO3 improved the melt strength, which led to less cells coalescence. So, the cells were more regular and intensive.It can be found that the cells were irregular and unevenly distributed without addition of nano-CaCO3, there were more cells with sizes from 100 to 120 μm, as shown in As shown in 3 was similar to that in the vertical section, as shown in 3, and increased when the content of nano-CaCO3 was more than 6 wt %; and the trend of cell density change was opposite. When the content of nano-CaCO3 was 6 wt %, the cell density was at its biggest (0.267 × 108 cells/cm3), and the average cell diameter was the smallest (100 μm). The main reason is that nano-CaCO3 improved the nucleation of cells and improved the melt strength. There were less cells coalescence and more well-distributed cells.As for the way in which the average cell diameter and cell density change, the addition of nano-CaCO(2) Transition Layer of Parallel SectionAs shown in The cells that nucleated and grew up in the period of filling deformed under the shear stress. The cells changed to an ellipse and the deformation of the cells was along the melt flow. The average ratio of length–diameter of the transition layer cell can be used to characterize the deformation to some degree. The large ratio of length–diameter means that the deformation is large. This paper did not investigate the average cell diameter of the transition layer of the parallel sections. It is meaningless to investigate the average cell diameter because of deformation. The results of the average ratio of length–diameter and cell density are shown in 3 was 2 wt %, the average ratio of length–diameter increased compared with the one without the addition of nano-CaCO3, because without addition of nano-CaCO3, the cell density was smaller, the cell walls were bigger, and the ability of resistance to deformation was bigger. With the continuous addition of nano-CaCO3, the melt strength increased and the ability of resistance to deformation improved. The deformation of the transition layer cell decreased and the average ratio of length–diameter decreased, too.When the content of nano-CaCO3 content on the crystallization behavior, mechanical properties, and cell structure of microcellular foam PP nanocomposites were investigated. The thermal stability and crystallinity increases with the increase of the nano-CaCO3. The mechanical properties increased first and then decreased with addition of nano-CaCO3, because of the synergistic effect of the cell structure and nano-CaCO3. The cell structures of the parallel and vertical sections were used to characterize the cell structure of the whole sample. Both the cells of parallel and vertical sections were divided into three parts (two transition layers and one core layer). The addition of nano-CaCO3 could improve the cell structure. When the content of nano-CaCO3 was 6 wt %, the cell structures both in the parallel and vertical sections were the best.The effects of the nano-CaCO3 can improve the crystallization behavior, mechanical properties, and cell structure of microcellular foam PP. When the content of nano-CaCO3 was 6 wt %, the mechanical properties and cell structure were optimum.In conclusion, the addition of nano-CaCO