Sarcophaga crassipalpis Macquart, 1839 (Diptera: Sarcophagidae) is a flesh fly species of medical, veterinary, and forensic importance. It is often used in the laboratory for several biological studies. In the current research, we investigate its life history under changing temperatures ranging from 15.7 to 31.1 °C, with an average of 24.55 °C, and the relative humidity ranges from 31.4 to 82.8% and at six fixed temperatures of 15, 20, 25, 30, 32, and then 35 °C. Also, pteridine from the head was used to assess adult age grading. Our results revealed that the life history and development rate of S. crassipalpis under changing temperatures were very close to those observed at a fixed temperature of 25 °C. The longest and shortest growing times were found at a low rearing temperature of 15 °C and higher temperature of 32 °C, respectively. The pattern of pteridine increase differed depending on the temperature. By employing a changing temperature model in the current study, we aim to provide information on the life history of S. crassipalpis that will be useful in future research.Sarcophaga crassipalpis Macquart 1839 (Diptera: Sarcophagidae) reared under variable temperatures ranging from 15.7 to 31.1 °C, with an average of 24.55 °C and relative humidity ranges from 31.4 to 82.8% and at six fixed temperatures of 15, 20, 25, 30, 32, and then 35 °C. Moreover, pteridine from the head was used to assess adult age grading. Our results allowed us to provide three development models: the isomorphen chart, the isomegalen chart, and the thermal summation models. The time taken for S. crassipalpis to complete its development from larviposition to adult emergence at constant temperatures of 15, 20, 25, 30, 32, and 35 °C was 1256.3 ± 124.2, 698.6 ± 15.1, 481.8 ± 35.7, 366.0 ± 13.5, and 295.8 ± 20.5 h, respectively, except 35 °C, where all pupae were unable to attain adulthood. They lasted 485.8 ± 5.4 h under variable temperatures. The minimum developmental limit (D0) temperature and the thermal summation constant (K) of S. crassipalpis were 9.31 ± 0.55 °C and 7290.0 ± 388.4 degree hours, respectively. The increase in pteridine content exhibited variations across different temperatures. There was quite a considerable distinction in the pteridine contents of male and female S. crassipalpis at 15 °C (p = 0.0075) and 25 °C (p = 0.0213). At 32 °C and variable temperatures, the pteridine content between female and male S. crassipalpis was not statistically divergent. However, temperature and gender remain the main factors influencing the pteridine content in the head of S. crassipalpis. We aim to provide detailed developmental data on S. crassipalpis that can be used as a valuable resource for future research and PMI estimation.Flesh flies (Diptera: Sarcophagidae) are regarded as significant in medical and veterinary entomology, and their development models can be utilized as considerable markers to ascertain the minimum postmortem interval (PMImin). In this research, we explored the growth cycle and larval body length of  Sarcophaga crassipalpis Macquart, 1839 (Diptera: Sarcophagidae) is a hemisynanthropic Sarcophagidae species of medical and veterinary significance. Since 2003, its life cycle has been studied [S. crassipalpis under varying temperature conditions remain poorly understood. Little research has been performed on the life history of this species, including its development under non-diapause conditions [S. crassipalpis with different development patterns in populations from different geographical locations in the USA, China, and Turkey. studied ,5. It is studied ,7,8,9,10 studied , as well studied ,13,14. T studied . Since i studied ,16,17,18nditions ,20, consnditions , reprodunditions ,23 and fnditions , eclosionditions ,26, as wSarcophagidae family exhibit a slow growth rate at low temperatures and a fast growth rate at high temperatures, as evidenced by the development patterns exhibited by Sarcophaga dux Thomson, 1869 (Diptera: Sarcophagidae) [Sarcophaga peregrina  (Diptera: Sarcophagidae) [Sarcophaga (Liopygia) argyrostoma Robineau-Desvoidy, 1830 [Sarcophaga ruficornis  [Wohlfahrtia nubia Wiedemann, 1830 (Diptera: Sarcophagidae) [Sarcophaga africa had a slow life cycle, with a 1.66-day difference between the rainy and winter seasons and a rapid one during the summer [S. peregrina had a longer development and a lower pupariation rate and emergence rate than those observed at constant temperatures [Most of the species belonging to the hagidae) , Sarcophhagidae) , Sarcophdy, 1830 , Sarcophs, 1794) , and Wohhagidae) . Sarcophe summer . Shang eeratures . Protophormia terraenovae, Robineau-Desvoidy, 1830  developed quicker at a higher changing temperature and slower at the lowest temperature range [S. argyrostoma (Diptera: Sarcophagidae) and Lucilia illustris   and lower development of Calliphora vicina (Robineau-Desvoidy) and Calliphora vomitoria   [Aldrichina grahami   had a cold thermal preference with a low thermal threshold of 3.41 ± 0.48 °C [Sarcophaga haemorrhoidalis  (Diptera: Sarcophagidae) had a maximal thermal preference of 30 °C [Lygus Hesperus (Hemiptera: Miridae) observed at moderate and high fluctuating temperatures of 22 °C and 29 °C, respectively. Mironidis [Helicoverpa armigera (Lepidoptera: Noctuidae) were decreased at variable temperatures ranging from 17.5 to 32.5 °C. One of the reasons for these various effects is that fluctuating temperatures may delay the development process at all stages, with survival and longevity gradually decreasing as temperatures decrease and increase [Some discrepancy was observed in the growth rates of insects maintained under controlled laboratory conditions with a constant temperature and those subjected to variable temperatures in their natural habitat. For instance, it has been reported that re range . Niederehoridae) , whereas 0.48 °C . Byrd anof 30 °C . Brent aof 30 °C  found noironidis  observedincrease . It is vincrease . ConsequC. vicina (Robineau-Desvoidy)  [Musca domestica Linnaeus, (Diptera: Muscidae) [Boettcherisca peregrina (Robineau-Desvoidy) [Lucilia sericata (Meigen)  [Chrysomya megacephala (Fabricius)  [Pteridines are heterocyclic substances derived from the pyrimidine-pyrazine and are 2-amino-4-hydroxy derivatives that accumulated in the eyes of adult flies over time . They arhoridae) , Musca duscidae) , Boettchesvoidy) , Luciliahoridae) , and Chrhoridae) , have beS. crassipalpis were obtained at six fixed and variable temperatures. In addition, the pteridine content in the head of S. crassipalpis was quantified and analyzed at the same variable temperature conditions and three constant temperatures  to assess adult age grading. The findings have significant implications for utilizing this species in estimating the minimum postmortem intervals during forensic examinations.In this research, the baseline developmental data for S. crassipalpis were captured on pork bait using Nylon nets for trapping flies in Xi Hu Park in Changsha City , Hunan Province, China, in September and October 2021. Before raising, they were anesthetized at −20 °C for 1–2 min and identified under Zeiss AxioCam 208 color microscopy using morphological keys by a medicolegal entomologist expert [S. crassipalpis were cultivated in a rearing nylon box and placed in an artificial climate cage . The weather conditions were 25.0 °C temperature, 70% relative humidity, and 12:12 h of light/dark photoperiod cycles. The flies were fed a diet consisting of water and milk powder and were reared to the fifth generation before starting the experiments. Before the commencement of the study, the total number of adult S. crassipalpis was maintained at 1000–2000 specimens. Wild adults of t expert  and conft expert . The spet expert . Adult SSix artificial climate incubators  were set at fixed temperatures of 15 °C, 20 °C 25 °C, 30 °C, 32 °C, and then 35 °C, with 70% RH and a photoperiod 12:12 h L/D cycle, while the flies did not undergo eclosion at a temperature of 35 °C. As in the variable temperature conditions the rearing occurs outside, the variable temperatures were determined based on meteorological conditions in Hunan Province, Southeast China. A GPS temperature and humidity data logger  was used to record the minimum, average, and maximum ambient temperatures of 15.7 °C, 24.55 °C, and 31.1 °C, respectively, during September and October 2022. The relative minimum and maximum humidity ranged from 31.4% to 82.8% .S. crassipalpis were collected. Each batch containing 400–500 larvae was reared in a plastic bowl with a relative quantity of pig lungs [Approximately 50 g of pig lungs were put into Petri dishes and introduced into a fly-rearing box to stimulate larviposition. After 2 h, approximately 2000–3000 larvae deposited by gravid female ig lungs . The bowig lungs . Larvae Larvae were monitored every 8 h from the first instar to wandering and every 12 h from pupation to adult eclosion. Fresh pieces of pork lungs were provided 1 to 3 times per day based on consumption . Eight oS. crassipalpis were collected at three constant  and variable (15.7 °C to 31.1 °C) temperatures when approximately 50% of the pupae emerged into adults and were designated as zero days. Eight adults  were then sampled every 48 h for fourteen days. The samples were preserved in 2 mL centrifuge tubes at −80 °C for the subsequent analysis. A global number of 32 adult males and 32 adult females were collected per group. The experiment was repeated with three biological replicates.Adult g for 5 min in 4 °C; then, 150 μL of the supernatant was loaded into a 96-well cell culture plate  and covered with aluminium foil. The fluorescence intensity was gauged instantly after stimulation. The pteridine fluorescence was gauged in an EnSpire® Multimode Plate Reader  at a transmitter frequency of 482 nanometers with the agitation set at 360–450 nanometers. The capacity of the released light was expressed in the relative fluorescence units (RFU) [Pteridine extraction and fluorescence analysis were performed using the modified methods previously described ,60,61,62ts (RFU) .D0 and thermal accumulated constant K, respectively [GraphPad Prism 9.4.1 and IBM SPSS Statistics 26 were used to perform the data analysis; One-way ANOVA examined the consequence of the temperature on the total duration of the life story . Nonlineectively . S. crassipalpis was defined based on the methods prior described [t-tests and two-way ANOVA were utilized to analyze differences in the pteridine levels between different temperatures and sexes.The age of the adults of escribed ,49. The escribed  without escribed . Using Iescribed . Paired S. crassipalpis was observed under variable temperatures ranging from 15.7 to 31.1 °C (mean: 24.55 °C)   and at 64.55 °C) .p-value inferior to 0.05 demonstrated a considerable distinction between the means of total developmental durations for constant vs. variable temperatures . However, comparisons between each group of constants vs. variable temperatures (VT) showed varying results. According to Tukey’s multiple comparison test and one-way ANOVA at alpha = 0.05, there was no statistical significance between the total developmental duration at 25 °C vs. VT . In contrast, significant differences were found between total developmental durations at 15 °C and VT , 20 °C and VT , 30 °C and VT , and 32 °C vs. variable temperature   .S. crassipalpis (p < 0.0001), at 25 °C and 20 °C , at 25 °C and 30 °C , and at 25 °C and 32 °C   .The isomorphen diagram was designed based on the time of all developmental phases from larviposition to adult emergence (x-axis) vs. different fixed temperatures (y-axis), where each line characterizes larval body changes, and the distances between lines describe the developmental stages . An incrS. crassipalpis from larviposition to adult emergence (x-axis) and accumulated degree hours (y-axis), six thermal summation patterns were plotted using linear regression examination (R2) of all thermal accumulated models was ≥0.90, demonstrating that all data matched relatively similarly to the linear models (D0) and thermal summation constants (K) were estimated to be 9.31 ± 0.55 °C and 7290.0 ± 388.4 degree hours, respectively. In addition, the thermal requirements (K) were 293.0 ± 58.1, 355.5 ± 13.9, 904.2 ± 100.7, 774.4 ± 56.8, and 5066.0 ± 272.4 degree hours at the first, second, and third immature stages, as well as wandering and pupal stages, respectively  with the elapse of time (T), with larval body length as the dependent variable and time after larviposition as the independent variable. The coefficient of determination (R2), F value, and p-value suggest that the models have a good correlation with the data. The equations in T) and larval body length (L) by using time after larviposition as the dependent factor and the larval body length as the independent factor.The equations in The Isomegalen diagram  was consp = 0.0019, t = 4.847, df = 7), 15 °C against 32 °C , 15 °C against VT , 25 °C against 32 °C , and 32 °C against VT . However, there was no considerable distinction between 25 °C and VT   .An increase in the pteridine content was observed over time following eclosion, as illustrated in S. crassipaplis at 15 °C  and 25 °C . Nevertheless, there was no statistical variation in the pteridine level between males and females at 32 °C  and under variable temperatures   .S crassipalpis exhibited a linear regression that correlated with the rising log10 of ADD and, therefore, with time after eclosion , the index of determination of all regression patterns was R2 ≥ 0.700, indicating that all data fit relatively well to the linear models . However, the development was slower under constant temperatures of 15 °C (1256.3 ± 124.2 h) and 20 °C (698.6 ± 15.1) than under variable temperature conditions (485.8 ± 5.4 h), while development was relatively long under variable temperatures compared with the developmental duration under 30 °C (366.0 ± 13.5 h) and 32 °C (295.8 ± 20.5), with significant differences. These data indicated that the mean temperature of variable temperatures was nearer to the optimal constant temperature. In a comparison to the effect of variable temperatures vs. constant temperatures on S. crassipalpis, it appeared that, under the low constant temperature groups, S. crassipalpis grew more slowly, moderately under the optimum temperature and greater under high temperatures.The investigational results showed that the variable temperature group generated a moderated life cycle duration and a moderated rate in comparison with the control group of a fixed temperature. Under variable temperatures, the total developmental duration of S. crassipalpis, the present research findings were discussed with the available literature on other taxa. In the current study, we found that variable temperatures can influence the development rates and durations of S. crassipalpis. Analogously, Dadour et al. [Hydrotaea rostrata (Diptera: Muscidae) had slower developmental rates under winter conditions than under a summer regime. Clarkson et al. [P. terraenovae  developed more quickly in the early immature stages under fixed temperatures than under fluctuating temperatures.Since there is not yet a published study on the consequence of variable temperatures on the lifespan of r et al.  found thn et al.  reportedS. argyrostoma and L. illustris developed more rapidly than C. vicina and C. vomitoria. In 2013, Warren and Anderson [P. terraenovae and found that its growth was accelerating at 4–28 °C higher fluctuating temperatures, reasonable at 9–23 °C changing temperatures, and slowest at 16 °C stable temperatures. Interestingly, this discrepancy in development rate is thought to have been caused by the rate summation effect, as temperature variations above the mean tend to raise the rate relatively more than they can lower it. Recently, Chen et al. [A. grahami  under stable and changing temperatures. They found that A. grahami developed slowly under changing temperatures ranging from 6 to 20 °C, according to the natural meteorological conditions, than under stable temperatures ranging from 8 to 36 °C. These correlated with our findings in the present study. Sert et al. [S. argyrostoma (Diptera: Sarcophagidae) and found a similar intrapuparial development with a non-significant difference between adult emergence times at fixed temperatures (25 °C) and variable temperature conditions. Although this result concerned exclusively the intrapuparial stage, it was similar to the observation made on the growth time of S. crassipalpis under variable temperatures vs. 25 °C in the current research.Similarly, an investigation on the development of larvae of different forensic flies was conducted by Niederegger et al.  under naAnderson  conducten et al.  investigt et al.  analyzedA thorough understanding of insect development parameters under fluctuating temperatures can aid in addressing various challenges, including accelerated, delayed, or unchanged developmental rates . The resS. crassipalpis [S. crassipalpis developed successfully at 15 to 32 °C and ceased to accomplish its growth at 35 °C, similar to the thermal conditions chosen by Bulut et al. [S. crassipalpis in Turkey. In contrast, the developmental rate of Turkey’s colony was lower at 32 °C for specimens fed bovine minced meat, bovine tongue, and chicken heart, and not all pupae reached adult emergence when fed minced meat [Several studies on various diapause parameters in insects have been conducted on sipalpis ,75,76,77sipalpis ,19. In ot et al.  to analyced meat . This isced meat .S. crassipalpis in the present study at 15 °C is shorter than those fed with bovine tongues and bovine minced meat in Turkey [S. crassipalpis in low temperature conditions can be attributed to its cold tolerance capacity. Chen et al. [The growth time of the immature stages of n Turkey . Howevern Turkey . Interesn Turkey , except n Turkey . The sucn et al.  found thn et al. .S. crassipalpis varied among the temperatures [In the current investigation, the developmental period of the pupal phase decreased with the growing temperatures, similar to the study carried out by Bulut et al. . Howevereratures . These deratures .S. crassipalpis to were 30, 32, and 35 °C. Although the larvae accomplished their development at 30 °C and 32 °C with total growing durations of 366 h and 295.8 h, respectively, unfortunately, all pupae ceased to reach the adult phase at 35 °C. This suggests that the longer exposure time at this temperature resulted in insufficient development of muscle contraction motor patterns for successful adult emergence [S. crassipalpis can complete their developmental time at 30 °C, with lower average developmental times to achieve the developmental cycle than at 32 °C and at 35 °C, where all larvae failed to reach the pupal phase. These findings suggested that 32 °C could be close to the upper growing threshold temperature for S. crassipalpis. These observations supported those made by Nassu et al. [Microcerella halli (Engel 1913) (Diptera: Sarcophagidae) raised under diverse constant temperatures. They found that larvae of M. halli raised at 35 °C attained the pupal phase much later than other groups but never reached the adult stage. Contrary, Joseph et al. [S. crassipalpis but did not affect the larvae and pupae. In our study, the higher constant temperatures we exposed mergence . Similarmergence  found thu et al.  on the dh et al.  discoverS. crassipalpis from the immature phases to adult emergence. These findings supported those of Bulut et al., who discovered that temperature and tissue type had a significant impact on the immature stage and pupal survival, as well as the adult weight of S. crassipalpis [Our study found that temperature significantly impacted the development period of sipalpis . Also, Csipalpis  noted thsipalpis . Researcsipalpis . Temperasipalpis .S. crassipalpis is a reliable technique for determining the age of adult necrophagous species for the PMI estimation. In the current study, there is a logical correlation between the time after eclosion, thus the age of the adult S. crassipalpis, and the pteridine intensity, as well as between an increased pteridine concentration and different temperatures. Previous studies have proven a positive interaction between the pteridine content and the age of necrophagous flies, including Stomoxy calcitrant [Glossina morsitrans [Cochliomyia hominivorax [Chrysomya bezziana [Lucilia sericata [Aldrichina grahami [Musca domestica [Chrysomya megacephala [Cochliomyia macellaria, Phormia regina [Boettcherisca peregrina [Calliphora vicina [The pteridine content in the heads of lcitrant ,84, Glosrsitrans , Cochlioinivorax , Chrysombezziana , Luciliasericata ,86, Aldr grahami , Musca domestica , Chrysomacephala , Cochlioa regina , Boettcheregrina , and Cala vicina .B. peregrina, Stomoxys calcitrans, and M. domestica as well [C. vicina. We additionally observed that the head capsule had no impact on the pteridine levels, which is in agreement with the findings of Bernhardt et al. [Our findings indicate that temperature is a crucial factor in the increase of pteridine. The pattern of this increase varied across different temperatures, suggesting that the metabolic activity of pteridine increased at higher temperatures. This is coherent with the results of Zhu et al. , who fou as well ,51,84. C as well  found a t et al. , who fouS. crassipalpis at different constant and variable temperatures. This corresponds to the conclusion of Zhu et al. [B. peregrina (Diptera: Sarcophagidae) raised in different constant temperatures. In our study, there was a considerable variation in the content of pteridine between female and male S. crassipalpis at 15 °C and 25 °C. Nevertheless, we found no statistical distinction in the pteridine content between female and male S. crassipalpis at 32 °C and variable temperatures. This contrasts with previous research, which showed that male flies exhibited higher pteridine levels than female flies  [S. crassipalpis eyes and increasing temperature may be crucial factors in our findings  ,88. We sfindings . The lowk et al.  also disn levels . For a mn levels .Our results allowed us to generate the following development patterns, including isomorphen diagram , isomegaAll of these models could be used to estimate the PMImin; however, the biology of the insects and the climatic parameters under which they are dynamic should be considered . In addiS. crassipalpis at variable and constant temperatures in China for the minimum postmortem interval estimation (PMImin). Our findings showed that the life history and development rate of S. crassipalpis under variable temperatures were very close to those observed at a fixed temperature of 25 °C. The longest and shortest growing times were found at a low rearing temperature of 15 °C and higher temperature of 32 °C, respectively. However, all pupae failed to emerge at 35 °C, suggesting that the upper developmental threshold temperature for S. crassipalpis may be close to this temperature. The pattern of pteridine increase varied across different temperatures. In addition, most of the total developmental durations exhibited significant differences between variable and constant temperatures. By utilizing a fluctuating temperature model in our study, we aimed to provide detailed developmental data on S. crassipalpis that can serve as a valuable resource for future research.This research offers the initial experimental laboratory information on the lifespan of


This suggests that there are significant genetic differences between amaranths in terms of biochemical and agro-morphological characteristics. The findings of the current work support plant breeders to investigate the genetic potential of the amaranth germplasm, notably in biochemical parameters. High colorants enrich genotypes that can be selected for extracting natural colorants to use in food processing industries.Twenty vegetable amaranth (VA) genotypes were evaluated to assess the variability, associations, path coefficient, and principal component analysis (PCA) of morpho-chemical traits. The genotypes exhibited adequate antioxidant colorants, phytochemicals, and antiradical capacity with significant variations across genotypes. Genetic parameters revealed selection criteria for the majority of the traits for improving amaranth foliage yield (FY). Based on correlation coefficient, stem weight, stem base diameter, root weight, plant height, and shoot weight for significant development of FY of VA. Observing significant genotypic correlation with high positive direct effects on FY, the path coefficient (PC) of root weight, stem base diameter, stem weight, and shoot weight could contribute to the noteworthy development of FY of VA. The genotypes AA5, AA6, AA8, AA10, AA11, AA19, and AA20 might be selected for high FY, antioxidant colorants, and antiradical phytochemicals to utilize as colorants and antiradical rich superior high yielding cultivars. The first PC accounted for 37.8% of the variances, which implies a larger proportion of variable information explained by PC1. The features that contributed more to PC1 were FY, SW, STW, RW, and PH, whereas Chl  Amaranth compared to other vegetables, is a low-priced source because of its high yield and low production cost. Sometimes, it is considered a poor's man vegetable. It contains high-quality and quantity protein with essential amino acids, like lysine and methionine, carotenoids, and dietary fiber ,2. Vegeta, β-cyanins, chlorophyll b, β-xanthins, and total chlorophyll [It contains plentiful antiradical colorant pigments like chlorophyll orophyll  which isorophyll . The colAmaranthus [4 dicotyledonous plant with over 70 species, 17 of which yield edible leaves and 3 of which yield food grains [Amaranth is the most significant vegetable grown in Bangladesh and the Indian subcontinent. Amaranth is the crop of the Amaranthaceae family, with chromosomal numbers 2n = 32, 34, under the genus aranthus . VA leavaranthus . VA is ad grains . Althougd grains . VA is ad grains .The intensity of genetic diversity in a crop plant measures its genetic dynamism. Plant breeding is based on selection, which can only be done efficiently if desirable features are present. Consequently, the achievement of breeding is entirely dependent on variety. Exploiting the hereditary variation of available germplasm to select high-yielding genotypes along with high colorant pigments and antiradical phytochemicals that may produce a sufficient yield along with these nutraceuticals is the key tactics used by plant scientists to tackle the difficulty of achieving a higher yield .Variability emerges owing to environmental and genetic factors. Additive and non-additive variability are the two types of variability. The phenotypic variance has been divergent into the environmental and genotypic variance to provide sufficient knowledge of the variations pattern. The intensity of diversity in any feature is critical for crop enhancement through breeding. Genetic diversity determines the achievement of any crop development operation . We hypo22.1−1.The test site is situated in the center of Madupur Tract, with a latitude of approximately 24° 23′, a longitude of 90° 08′, and an average elevation of 8.4 m above sea level. The soil conditions were slightly acidic (pH 6.4), low in organic matter (0.87%), total N of 0.09%, and exchangeable K of 0.13 cmol kg2.2Seeds of 20 genotypes of VA were collected from the Genetics and Plant Breeding Department in Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) Salna, Gazipur-1706, Bangladesh. The study was executed using 20 promising genotypes of VA . The study was executed at BSMRAU with three replications in a randomized complete block design (RCBD). Three replications were executed by dividing the experimental field into three blocks. Each block was also subdivided into twenty experimental plots/experimental units. The experimental unit was 1 m × 1 m. The row-to-row and plant-to-plant spacings were 25 cm and 5 cm, respectively.2.3The experimental site was located at a lower altitude with well irrigation facilities. The experimental field's ground was plowed with a tractor-drawn disc plow and then harrowed. The land was thoroughly prepared by plowing and cross-plowing three to four times, then laddering to achieve good tilt conditions. Stubbles and weeds were cleared and the area was ultimately prepared by applying a recommended base dose of fertilizers. Recommended chemical fertilizer and compost were applied. In a final land preparation, 10 t/ha compost was applied. Urea, TSP (triple super phosphate), murate of potash (MP), and gypsum were applied @, 200, 100, 150, and 30 kg/ha, respectively.2.4During the cropping time, necessary intercultural operations were performed to ensure the plants' healthy growth and development. Weeding and hoeing were done every seven days intervals. Irrigation was scheduled every 5–7 days intervals.2.5After 30 days of seed sowing, data were randomly collected from 10 plants for each trait of all replications. Without any loss of roots, plants were uprooted from the soil. At the base of the stem (base of ground level), the plants were cut into separate shoots.2.5.12.5.1.1It was measured in centimeters (cm) from the base to the peak of the stem.2.5.1.2It was measured in millimeters using digital slide calipers at the base of the stem.2.5.1.3Ten plants were randomly selected and all leaves were physically counted. The average number was obtained by dividing the total number by 10.2.5.1.4The shoot weights (stem + leaves) of ten randomly chosen plants were measured in grams and divided by ten to get the average.2.5.1.5The root weights of ten randomly chosen plants were measured in grams which were then divided by ten to get the average.2.5.1.6The stem weights of ten randomly chosen plants were measured in grams which were then divided by ten to get the average.2.5.1.72 area and weighted by balance.To calculate FY, leaves of all plants were picked from a 1 m2.5.22.5.2.1a (Chl a), total chlorophylls, and chlorophyll b (Chl b) were estimated using a spectrophotometer  at 663 and 646 nm from acetonic (80%) extracts of fresh amaranth leaves using the Lichtenthaler and Wellburn's method [Chlorophyll s method .2.5.2.22 mol−1, while β-xanthins had a mean molar extinction coefficient of 48 × 106 cm2 mol−1. For β-cyanins and β-xanthins, the data were represented as equivalent to indicaxanthin ng/g fresh weight (FW).β-cyanins and β-xanthins were extracted from fresh amaranth leaves using 80% methanol containing 50 mM ascorbic acid ,38. At 52.5.2.33H6O and to complete the extraction process it was centrifuged for 3–4 min at 10,000 rpm. The supernatant was removed and brought to a volume of 20 mL in a volumetric flask, where the absorbance at 510 and 480 nm was measured with a spectrophotometer .We followed the previously described procedure for extracting and estimating β-carotene . A freshThe β-carotene content was calculated by the following formula:β-carotene content = 7.6 (Abs. at 480) – 1.49 (Abs. at 510) (1000 fresh weight of leaf taken)/final volume.2.5.2.436H2O (0.1 mL), 0.1 mL CH3CO2K (1 M), and 2.8 mL distilled water were added to estimate TF. For TP estimation, in a 20 mL test tube, standard solutions for leaf extract (50 L), 1 mL Folin-Ciocalteu reagent (1:4 with deionized water), and 1 mL Na2CO3 solution (10% wt/vol) were mixed, and allowed to stand at room temperature for 90 min in the dark. A spectrophotometer was utilized to estimate absorbance at 415 and 760 nm. TF and TP were determined using a gallic acid standard curve. The data were expressed as gallic acid equivalent (μg/g DW).TF and TP were measured using the aluminum chloride colorimetric technique  and the 2.62b). The formula followed by Lush [Replication-wise means data of a trait was obtained by averaging the sample mean ,44. The  by Lush  was used by Lush  was used by Lush  was used by Lush  was used by Lush  in the R by Lush . The pri by Lush ,64.3Zea mays [Oryza sativa [Cocos nucifera [Abelmoschus esculenthus [All examined traits exhibited noteworthy deviations for ANOVA which specified a wide divergence in the traits among accessions. Previous studies also recorded similar variations in VA , Zea mayZea mays , Oryza sa sativa , Cocos nnucifera ,78, Abelulenthus ,80, Mungulenthus ,82 and bulenthus  genotype3.1The intensity of variation across the genotypes in respect of FY, related morphological traits, bioactive colorant pigments, and antiradical phytochemicals were studied and their grand mean value, standard error, CV, and least significance difference at 1% have been shown in 3.1.1The substantial differences in genotype were demonstrated in plant height. The plant height of twenty VA genotypes varied from 15.55 cm to 29.02 cm, with a mean performance of 20.44 cm . The max3.1.2. [SBD differed from 4.25 mm to 8.92 mm along with a mean value of 5.59 mm . The max.  found a 3.1.3. [The mean performance of L/P of 20 VA genotypes ranged from 6.33 to 11.33, with a mean performance of 8.82 . Genotyp.  reported3.1.4Shoot weight varied progressively in different amaranth genotypes which range varied from 3.97 g to 15.32 g with a mean performance of 7.44 g . The ext3.1.5Genotypes diverged in root weight ranging from 0.20 g to 1.16 g along with an average value of 0.54 g . Across 3.1.6The highly noteworthy deviation in genotypes was confirmed indicating a huge divergence in stem weight. The stem weight of twenty VA genotypes diverged from 1.06 g to 7.15 g with a mean performance was 3.05 g . The gen3.23.2.1a displayed noteworthy differences in genotypes, indicating a huge spectrum of divergence among genotypes. Chlorophyll a differed from 0.40 to 0.63 mg/g FW with a mean performance of 0.53 mg/g FW  . Across 3.2.92 ranged from 120 to 1132.5 g which is greater than the current results. The chromosomal constitution of genotypes may significantly influence the deviations between the two results. Furthermore, factors like management processes, environmental conditions, and geographical locations may contribute to the differences in β-xanthins content. Based on foliage yield and related morphological traits, colorant pigments, and antiradical phytochemicals 7 genotypes i.e., AA5, AA6, AA8, AA10, AA11, AA19, and AA20 can be selected for high foliage yield, bioactive colorant pigments and antiradical phytochemicals for use as colorant pigments and antiradical enrich preferable high yielding cultivars. The genotypes containing high colorant pigments may select to extract environment-friendly natural colorants for food processing industries.The noteworthy deviation in genotypes was confirmed indicating a broad spectrum of variation for foliage yield across the accessions. The mean foliage yield of 20 VA genotypes ranged from 9.44 to 72.29 g with a mean performance was 31.78 g . The hig3.32b, GA, and GAPM are presented in In a crop improvement program, variability is fundamental for selecting improved desirable genotypes. In crop improvement programs, the selection of superior genotypes entirely depends on the variability of genotypes . The ext2p) and genotypic variance (σ2g) recorded for all the traits were very close or similar indicating the absence of environmental influence on the expression of these traits, i.e., additive genes played a significant role in the expression of these traits. So, the selection of these traits could significantly contribute to the quality and foliage yield of amaranth. In the current study, β-cyanins displayed the maximum GCV and PCV there after β-xanthins, total flavonoids, TP, and FY, signifying that the parameters under study had an extensive divergence and had a greater scope of selection for improving amaranth genotypes. Plant height, shoot weight, stem weight, SBD, and L/P showed moderate GCV and PCV, and these parameters may be selected to improve the examined amaranth genotypes. Low GCV and PCV were noted in root weight, chlorophyll a, chlorophyll b, total chlorophyll, and β-carotene. The remaining traits had parallel GCV and PCV, indicating a superiority of the effects of the additive gene for these traits, i.e., the expression of these features was a minor consequence of environment or most of the phenotypic variance was genetic in nature, facilitating more opportunity for amaranth development via selection. For all of the features tested, PCV was to some extent larger than GCV. This suggests that the environment governed the phenotype to some extent. Among all attributes, GCV and PCV of TP were both high thereafter β-cyanins, stem weight, and FY per m2mentioning the existence of a broad divergence for the features studied and had a larger selection window for amaranth genotype development. The lowest PCV and GCV values were noted for chlorophyll a, β-xanthins, β-carotene, and total chlorophyll. When it comes to determining the heritable fraction of variation, variability alone isn't very helpful. The intensity of achievement awaited from a selection is measured by the trait's heritability and genetic progress. Heritability is a term that is commonly used to describe the intensity to which a character can be passed from generation to generation. Information about a character's heritability is significant since it suggests the feasibility and extent of improvement via selection [Phenotypic variance (σelection . High heelection .2 (97.72), root weight (95.27), and shoot weight (85.81) whereas, the lowest GAPM was noted for L/P (24.28), chlorophyll a (26.16) and β-xanthins (27.87). TF All the traits showed high heritability along with high to moderate GAPM. So, all traits may be selected for the economical and practical enhancement of FY of VA. According to Bhargava et al. [Amaranthus sp. has a high heritability value in terms of yield per hectare. Lohithaswa et al. [Amaranthus spp both at the phenotypic and genotypic levels.β-Cyanins, β-xanthins, TP, and showed 100% heritability indicating the absence of environmental influence in the expression of these traits, i.e., these traits are fully additive in nature. We know that qualitative traits are controlled by mono or oligogenes and have very minor or no environmental influence on their expression. For this reason, all the qualitative traits studied in the experiment had high heritability (᷉⁓ 100%). Hence, the selection of these quality traits could noteworthy improve the food values of our daily diet by developing high nutritious amaranth variety. All the traits had high heritability values and were moderate for L/P. The highest GAPM was noted for TP (222.16) thereafter β-cyanins (113.94), stem weight (109.31), FY per ma et al. , the yiea et al.  found th3.4g) were larger than the phenotypic correlation coefficients (rp) in the current study, representing that the viewed relationships among the various traits were due to hereditary causes and under the impact of the environment, the phenotypic expression of correlations is reduced. Estimates of rg and rp between traits are put in g and rp between yield and its related characters were estimated and the findings are put in g = 0.73** and rp = 0.73**), SBD (rp = 0.59** and rg = 0.61**), L/P (rp = 0.26* and rg = 0.27*), shoot weight (rp = 0.93** and rg = 0.93**), root weight (rp = 0.85** and rg = 0.86**), stem weight (rp = 0.86** and rg = 0.86**). Alternatively, it was negatively correlated with β-cyanins (rp = −0.31* and rg = −0.31*), β-xanthins (rp = −0.43** and rg = −0.43**) and total polyphenols (rp = −0.49** and rg = −0.49**). The correlation coefficient revealed that the genotype selected on plant height, leaves/plant, shoot weight, SBD, root weight, and stem weight was directly associated with the FY of amaranth genotypes.Yield is the ultimate product that is influenced by several interconnected quantitative characteristics. Consequently, yield selection may be ineffective unless other yield components that impact it directly or indirectly are included. When selection pressure is applied to improve a character that is linked strongly to yield, it has an impact on several other connected characters. Therefore, understanding traits and yields and their correlationships are a guide for plant breeders to make improvements in selection concerning a fair understanding of the participation of genetic and non-genetic elements in generating the association . The gen3.52 was treated as a resultant (dependent) variable whereas, plant height, leaves/plant, shoot weight, SBD, root weight, stem weight, chlorophyll a, chlorophyll b, total chlorophyll, β-cyanins, β-xanthins, β-carotene, TF, and TP were causal (independent) variables. Assessment of the direct and indirect effect of PC analysis for VA is put in 2 was obtained by β-cyanins (−0.39) and thereafter plant height (−0.09). The highest indirect positive effect on FY was confirmed in total chlorophyll (18.09) thereafter chlorophyll b (17.79) and chlorophyll a (11.05) which could be in consideration for the prospective trial program. In any plant breeding, it is very difficult to fully understand the characteristics of all yield components. The residual effect allows an accurate explanation of the pattern of interaction of other possible components of yield that were not included in the investigation of the dependent variables. The residual effect was 0.01084, prevailing the contribution of component characters on FY per m2 was 98.92% by the 14 characters studied in path analysis, the rest of 1.08% was the contribution of other factors that were not included in the dependent variable investigation.Path coefficient analysis is used in any plant breeding program to persuade the mood of the correlations between yield and its contributing traits that might be utilized as a selection criterion to improve crop production . The pur3.6The heatmap analysis constructed double dendrograms, the dendrogram in the horizontal direction, an arrangement that represents the amaranth accessions, and the 2nd dendrogram in the vertical direction representing traits . The acc3.7viz., FY, SW, STW, RW, and PH  and the vector loadings for each agronomic character and PC were eternized , Fig. 3.4a, β-cyanins, total chlorophyll, chlorophyll b, with extensive variations across genotypes. VA is the only basis of colorant pigments. Taken together of all genetic parameters, genotypes can be chosen on β-cyanins, plant height, β-xanthins, leaves per plant, TF, shoot weight, SBD, root weight, TP, stem weight, chlorophyll a, β-carotene, chlorophyll b, total chlorophyll, which ultimately suggestively advance the FY of amaranth. However, a correlation study exposed that the progress of FY of VA can be accomplished by selecting root weight, shoot weight, stem weight, plant height, and stem base diameter. Taken together, high direct effects and significant correlation indicated that direct selection through root weight, SBD, stem weight and shoot weight would significantly raise the FY of amaranth genotypes. Seven genotypes  may be selected for high FY, bioactive colorant pigments, and antiradical phytochemicals for use as colorant pigments and antiradical enrich preferably high yielding cultivars. Moreover, PCA gave optimum information for the evaluation of genetic diversity among the genotypes and also aids in identifying the genotypes to be further analyzed at the genetic level. The genotypes containing high colorant pigments may select to extract environment-friendly natural colorants for food processing industries. We also can recommend genotypes with moderate FYs and antiradical pigments and phytochemicals for parent materials in future breeding programs.The ANOVA of all attributes examined was significant and showed wide deviations among the 20 genotypes of the VA. All accessions had adequate bioactive compounds, such as antiradical phytochemicals like TP, total flavonoid compounds, β-carotene; and colorant pigments like β-xanthins, chlorophyll Conceived and designed the experiments and Performed the experiments: Nishat Jahan and Umakanta Sarker.Analyzed and interpreted the data: Umakanta Sarker, Nishat Jahan, Mohammad Golam Azam, Daoud Ali, Sezai Ercisli, Mohammad Mehfuz Hasan Saikat, Md. Motaher Hossain and Kirill S. Golokhvast.Contributed reagents, materials, analysis tools or data: Umakanta Sarker, Nishat Jahan and Mohammad Golam Azam.Wrote the paper: Nishat Jahan, Umakanta Sarker, Mohammad Golam Azam, Daoud Ali, Sezai Ercisli, Mohammad Mehfuz Hasan Saikat, Md. Motaher Hossain and Kirill S. Golokhvast.Data included in article/supplementary material/referenced in article.No additional information is available for this paper.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Protaetia brevitarsis Lewis larvae, which is a saprophagous pest prevalent in China. In this study, comparing the whole-genome sequence of Protaetia brevitarsis Lewis larvae with the Antimicrobial Peptide Database (APD3) led to the identification of nine peptide templates that were potentially AMPs. Next, based on the peptide templates, 16 truncated sequences were predicted to the AMPs by bioinformatics software and then underwent structural and physicochemical property analysis. Thereafter, candidate small-molecule AMPs were artificially synthesized and their minimal inhibitory concentration (MIC) values were assessed. A candidate peptide, designated FD10, exhibited strong antimicrobial activity against both bacteria and fungi comprising Escherichia coli (MIC: 8 μg/mL), Pseudomonas aeruginosa (MIC: 8 μg/mL), Bacillus thuringiensis (MIC: 8 μg/mL), Staphylococcus aureus (MIC: 16 μg/mL), and Candida albicans (MIC: 16 μg/mL). Additionally, two other candidate peptides, designated FD12 and FD15, exhibited antimicrobial activity against both E. coli (MIC: both 32 μg/mL) and S. aureus (MIC: both 16 μg/mL). Moreover, FD10, FD12, and FD15 killed almost all E. coli and S. aureus cells within 1 h, and the hemolytic effect of FD10 (0.31%) and FD12 (0.40%) was lower than that of ampicillin (0.52%). These findings indicate that FD12, FD15, and especially FD10 are promising AMPs for therapeutic application. This study promoted the development of antibacterial drugs and provided a theoretical basis for promoting the practical application of antimicrobial peptides in the Protaetia brevitarsis Lewis larvae.Antimicrobial peptides (AMPs) are widely recognized as promising natural antimicrobial agents. Insects, as the group of animals with the largest population, have great potential as a source of AMPs. Thus, it is worthwhile to investigate potential novel AMPs from  HoweverAntimicrobial peptides (AMPs) are a potential alternative to antibiotics due to their broad-spectrum antimicrobial activities and low drug resistance , and theInsect AMPs are small cationic molecules with various biological activities, and they are an essential component of the insect innate immune system . AccordiProtaetia brevitarsis Lewis (Coleoptera: Scarabaeidae) is an important agricultural pest distributed in most areas of China. Its larvae are saprophytic, which means that they are exposed to many different pathogenic microorganisms in the living environment . By comparing the whole-genome sequence with the APD, it was found that some P. brevitarsis Lewis gene sequences corresponded to AMP sequences. To obtain highly active small-molecular-weight AMPs, truncation, amino acid substitutions, and N-terminal acetylation were used to modify the AMP sequences. Thereafter, their antimicrobial activity, hemolytic effects, cytotoxicity, and stability were studied in vitro. This study could provide ideas for the design of small-molecule AMPs and contribute to solutions for the issue of antibiotic resistance. To increased awareness of this study, it was illustrated with the strategistic diagram  CMCC(B) 26003 and Pseudomonas aeruginosa (P. aeruginosa) CMCC(B)10104, and the fungal strain Candida albicans  CMCC(F) 98001, were purchased from Beijing Kezhan Biotechnology Co., Ltd. 2019, China. The bacterial strains Bacillus thuringiensis and Escherichia coli were obtained from our laboratory. Mouse RAW 264.7 macrophages were purchased from Shanghai Yaji Biotechnology Co., Ltd. 2022, China.The bacterial strains 2.2.Mueller–Hinton broth (MHB), Mueller–Hinton agar (MHA), nutrient agar (NA), and Sabouraud dextrose agar (SDA) were obtained from BEIJING AOBOXING BIO-TECH Co., Ltd. 2019, China. Trypsin was obtained from Nanjing Beyotime Biotechnology Co., Ltd., 2021. 3--2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich, 2021 (China).2.3.Staphylococcus aureus CMCC(B) 26003, B. thuringiensis, E. coli, and P. aeruginosa CMCC(B)10104 were cultured in NA medium and C. albicans CMCC(F) 98001 was cultured in SDA medium. They were incubated in a constant-temperature  incubator for 16–20 h. Thereafter, single colonies were picked and incubated in 50 mL MHB at 160 rpm/min at the optimal growth temperature . Microbial suspensions of 1 × 106 CFU/mL were then prepared (prepared .2.4.Protaetia brevitarsis Lewis larvae (GenBank accession number: CM014285.1) was compared to the AMP sequences in APD3.The whole-genome sequence of 2.5.The template peptide was used to screen out the truncated sequences, and then the antimicrobial region within the predicted peptide was screened using the random forest classifier calculation method in CAMP2 could be added to the end of the peptide  to preserve its functional activity  and -NHactivity . The 16 2.6.6 CFU/mL microbial suspension 10104, B. thuringiensis, S. aureus CMCC(B) 26003, or C. albicans CMCC(F) 98001) was evenly applied to dishes containing 20 mL NA (bacteria) or SDA (fungi). After the microbial solution solidified, six holes per dish were punched with a sterile pipette tip of 6 mm in diameter, with each hole spaced >20 mm apart and > 10 mm apart from the edge of the dish. The holes were marked with (+), (−), and (1–16), representing the positive control (ampicillin), negative control (deionized water), and treatment groups (16 candidate peptides dissolved in sterile water), respectively. Next, 55 μL of 1 mg/mL control/treatment was added to each hole and incubated in a constant-temperature  incubator for 16–18 h 10104, B. thuringiensis, S. aureus CMCC(B) 26003, or C. albicans CMCC(F) 98001). The plates were incubated  for 16–18 h. Ampicillin was used as the positive control. The MIC was defined as the lowest peptide concentration where no visible growth occurred , which were selected based on physicochemical properties and antimicrobial effects (determined by the agar punch method), were evaluated using a micro-broth dilution method . Serial occurred . The exp2.8.Six AMPs , selected based on antimicrobial activity and the peptide modifications, were processed by Heliquest2.9.E. coli and S. aureus CMCC(B) 26003. Therefore, the follow-up experiments involved these two bacteria, along with the three AMPs with the best antimicrobial effects . The dynamic antimicrobial effects were analyzed by measuring the effects of the AMPs on bacterial growth over 10 h. First, 100 μL microbial solution (1.0 × 106 CFU/mL) was added to the wells of a 96-well culture plate, and then 100 μL AMP (at 1× and 1/4× MIC) was added and the mixtures were cultured at 37°C (600) was measured every hour for 10 h using a microplate reader  had inhibitory effects against  at 37°C . 100 μL e reader . The exp2.10.S. aureus CMCC(B) 26003 and E. coli) in vitro over time, based on the number of viable bacteria left at various time points after AMP exposure. Briefly, AMP at 2× MIC was added to MHB containing 106 CFU/mL S. aureus CMCC(B) 26003 or E. coli and the mixture was then incubated at 37°C and 100 rpm. At various time points, a sample was taken, serially diluted, and plated on MHA for colony counting . Sterile water with 1 × 106 CFU/mL microbial suspension was used as the blank control. After culturing at 37°C for 16–18 h, colony counting was conducted  26003 (1 × 106 CFU/mL) were incubated in MHB with various final physiological concentrations of serum salts . 50 μL of these E. coli or S. aureus CMCC(B) 26003 suspensions and 50 μL of AMP  were added to a 96-well plate and incubated at 37°C for 16–18 h ( 16–18 h . The MIC2.13.v/v). Thereafter, 100 μL of this solution was added to a 96-well plate containing an equal volume of AMP (1–256 μg/mL) and incubated at 37°C for 1 h. The positive control was 100 μL 1% Triton X-100 and 100 μL 8% erythrocytes, and the negative control was 100 μL phosphate-buffered saline PBS and 100 μL 8% erythrocytes. Next, each mixture was centrifuged at 1,200 g for 15 min, and 100 μL of the supernatant was transferred to a clean 96-well plate to measure the light absorption value at 576 nm with a microplate reader. The percent hemolysis was calculated using the following formula: Percent hemolysis = [(A–A0)/(A+–A0)] × 100%, where A, A+, and A0 represent the absorbance of the peptide sample and positive and negative controls, respectively  and incubated for 24 h. After another 24 h incubation with Amp and FD10, FD12, FD15 (1 to 256 μg/mL), MTT solution was added and incubated for 4 h. Followed the removing of the MTT, dimethyl sulfoxide (DMSO) (150 μL/well) was added and the absorbance was measured at 570 nm. Untreated cells were used as a control. The cell survival rate was calculated using the following formula: survival rate (%) = (Abs570 nm of treated sample/Abs570 nm of control) × 100% (The cytotoxicity of AMPs toward mouse RAW 264.7 macrophages was determined by MTT assays . Cells w) × 100% . The cyt2.14.n” represents the number of samples. One-way analysis or two-way analysis of variance (ANOVA) in SPSS software was used to analyze significant differences between groups. p < 0.05 indicated that the differences were statistically significant. GraphPad Prism 8.0  was used for statistical computing and plotting.All data are presented as mean ± standard deviation (SD), with “3.3.1.Protaetia brevitarsis Lewis genomic sequence to known AMP sequences in a database, nine aligned sequences were identified and used as template peptides  (After comparing the AP00208) .2PHYRE3.2.2) could be set up, and -NH2 could be added to the FD16 terminal in order to preserve the integrity of functional activity.Based on the nine template peptides, key truncated sequences were identified , and 16 3.3.The physicochemical properties of the 16 candidate peptides were clearly and concisely compared . The net3.4.E. coli, P. aeruginosa CMCC(B)10104, B. thuringiensis, S. aureus CMCC(B) 26003, and C. albicans CMCC(F) 98001) were compared to the effects of ampicillin (positive control) and sterile water (negative control). The candidate peptides differed in their inhibitory action against the five microbes, based on the size of the inhibition circles formed  26003, with inhibition circle diameters of 10.12 and 9.47 mm, respectively (P. aeruginosa CMCC(B)10104, B. thuringiensis, and S. aureus CMCC(B) 26003, and it was the only candidate peptide with activity against fungi  98001), indicating excellent inhibitory effects. FD12 was comparable to ampicillin against B. thuringiensis and S. aureus CMCC(B) 26003, indicating antimicrobial activity against Gram-positive bacteria. FD14 and FD15 were both much less effective than ampicillin.Five peptides  generated inhibition circles, with the growth of the five microbes being prevented in these zones (the remaining candidate peptides did not form inhibition circles). FD4 was effective against ectively . However3.5.E. coli and P. aeruginosa CMCC(B)10104 was as low as 8 μg/mL, while those against Gram-positive B. thuringiensis and S. aureus CMCC(B) 26003 were 8 and 16 μg/mL, respectively, and that against C. albicans CMCC(F) 98001 was 16 μg/mL. FD12 and FD15 also had antimicrobial activity against S. aureus CMCC(B) 26003 with a MIC of 16 μg/mL each and against E. coli with a MIC of 32 μg/mL each  were selected for further antimicrobial activity analysis. FD10 had an antimicrobial effect on all five microbes at a low MIC; the MIC against Gram-negative bacteria /mL each .3.6.Heliquest was used to calculate the helix properties of six AMPs . 3.7.600 of the microbial solutions were essentially unchanged until 1 h, indicating slow microbe proliferation (E. coli and S. aureus CMCC(B) 26003 growth was almost inhibited at 10 h .In the dynamic antimicrobial analysis, the ODferation . At 1× ME. coli growth after 1 h and increased growth after 2 h, while the AMPs at 1/4× MIC led to E. coli growth only after 3–4 h, and after 4 h, E. coli continued to grow. FD10 had a significantly higher inhibitory effect against E. coli than the other AMPs (S. aureus CMCC(B) 26003 growth at around 2 h, while AMPs at 1/4× MIC led to slow growth after 3 h (S. aureus CMCC(B) 26003 began to increase more rapidly.Ampicillin at 1/4× MIC led to slow her AMPs . Similarfter 3 h , and aftThis indicates that, compared to ampicillin, the AMPs exhibited good microbial inhibition and broad antimicrobial activity even at concentrations below the MIC.3.8.E. coli was exposed to them, with FD10 having the steepest curve and the fastest decline at any given time point, indicating that it was the most effective at killing E. coli, followed by FD12 and then FD15 (E. coli and S. aureus CMCC(B) 26003) was most rapid before 0.5 h, with a large reduction in the number of colonies, and the rate of bactericidal action gradually decreased with time until the curves plateaued. Similarly, all three AMPs were bactericidal immediately after S. aureus CMCC(B) 26003 was exposed to them, with FD10 and FD12 causing a more rapid decline in S. aureus CMCC(B) 26003 than FD15, indicating that they were better than FD15 at killing S. aureus CMCC(B) 26003 (S. aureus CMCC(B) 26003 decreased and the number of colonies began to increase, but the bacteria grew more slowly in the FD10 group.All three tested AMPs  were bactericidal immediately after hen FD15 . The bacB) 26003 . By 10 h3.9.After being treated with trypsin for 1 h, the antimicrobial activity of the three AMPs was greatly reduced. The antimicrobial activity was less than half of the original activity, with >60% of the bacteria surviving . This sh3.10.E. coli and S. aureus CMCC(B) 26003 were minimally affected, or even promoted, by the presence of certain monovalent (K+) and trivalent (Fe3+) cations, while they were slightly decreased in the presence of Na+ or Mg2+. Among FD10, FD12, and FD15, FD10 was the most stable in the presence of salt; when certain salt ions were present, it maintained or increased its antimicrobial activity.The salt sensitivity of antimicrobial agents is generally tested by the addition of physiological concentrations of various salts . The ant3.11.The percentage of mouse erythrocyte hemolysis after FD10 and FD12 treatment (at 256 μg/mL) were 0.31 and 0.40%, respectively, which were lower than that of ampicillin (0.52%), while the rate after FD15 treatment was 0.55%, which was higher than that of ampicillin . The sur4.P. brevitarsis Lewis larvae.With the overuse of traditional antibiotics for animal and human health, a large number of super-resistant bacteria have emerged, which seriously threatens animal and human health and the development of industries such as the aquaculture industry. Therefore, the exploration and development of new antimicrobial drugs have become a hot research area. Due to the broad-spectrum antimicrobial properties of AMPs and their unique antimicrobial mechanisms, it is difficult for bacteria to become resistant to AMPs, and AMPs are one of the most potent agents to replace antibiotics. At present, there are many studies on insect AMPs, but few studies on the AMPs of 2) had an obvious antimicrobial effect but FD16 (STLHLVLRLR) had no effect without -NH2. Although C-terminal acetylation is not a requirement for antimicrobial activity, it can increase the charge of peptides and enhance their interaction with microbial membranes. In contrast, adding amino acid residues or truncating sequences at the N-terminus makes it more difficult for the AMP to insert into the membrane, which reduces the AMP’s activity  26003, which is resistant to antibiotics (E. coli and S. aureus CMCC(B) 26003 before 0.5 h in the colony numbers. The best antimicrobial activity was observed for FD10. The designed peptides exert a similar rapid antimicrobial activity as what has been observed for naturally produced antimicrobial peptides. This rapid activity has been linked to the lower resistance development potential against AMPs  26003 in a short time. The findings indicate that FD10 in particular may be a novel promising antimicrobial agent. This study provides a basis for the design of small-molecule AMPs and enriches AMP resources.In summary, 16 small-molecule potential AMPs were designed in this study. Among them, FD10 had the best antimicrobial activity, especially regarding antifungal activity. FD12 and FD15 also exhibited activity against The original contributions presented in the study are included in the article/The experiments were approved by the Institutional Animal Care and Use Committee of the Northeast Agricultural University (SRM-11) in 2022 (China).QF, DC, RL, and CS contributed to the conception and design of the study. QF and JS organized the database. QF performed the statistical analysis and wrote the first draft of the manuscript. DC and XL wrote sections of the manuscript. DC, RL, and HL reviewed and edited the manuscript. QF and DC contributed equally to this work. All authors contributed to the manuscript revision and read and approved the submitted version of the manuscript.This research was funded by Heilongjiang Provincial National Science Foundation (LH2020C007), China from 2020-07-01 to 2023-07-01.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Table salt, rock salt, and iodized salts  are commonly used in many areas, such as medicine, cooking, industry, or personal care. Common fried, salty, and spicy foods contain an excess of added salt, which has detrimental health effects, especially on the kidneys. Our research aims to enhance the inherent saltiness of these three salts, which would reduce intake and thereby minimize salt's health hazards. We invented a water‐based 2–6 μm of Mid‐Infrared Generating Atomizer (MIRGA), which, when applied to salts, caused changes in the salts' chemistry and enhanced saltiness, thus allowing the reduction of salt intake by 25%–30%. This easy‐to‐use technology did not show any side effects. MIRGA was found to have enhanced the saltiness, thus allowing the reduction of salt intake by 25%–30%. MIRGA is safe, portable, highly economical, unique in the mid‐IR laser technology, and possesses vast research scope in other areas of food science. MIRGA is a highly economical, easy‐to‐use, and safe technology in which a water‐based solution is contained in a pocket‐sized 20‐mL capacity atomizer. During spraying over the salt packets from a distance of 0.25–0.50 m, 2–6 μm mid‐infrared is generated which causes chemical bond alteration in the salt molecules. This resulted in 25%–30% increased saltiness, thereby the salt use for cooking can be cut down by 25%–30% than the usual. Iodized salt is enriched with iodine salts and its intake is recommended to overcome/avoid iodine deficiency McNeil, . Out of 22.1under‐patent no.: 401387) is a 20‐mL pocket‐sized atomizer containing inorganic water‐based solution. During spraying, depending on pressure (vary with the user) applied to plunger, every spraying is designed to generate 2–6 μm of mid‐IR. Design of the MIRGA and the estimation of emitted 2–6 μm of mid‐IR  and the remaining were numbered from 1 to 12. Corresponding to the packet number, 1 to 12 MIRGA sprayings were applied from 0.25‐ to 0.50‐m distance over the packets as one or two sprayings on one or both sides. The control packet received no spraying. Twelve sprayings had earlier been found to cause a complete loss of saltiness in the samples.The 13 packets were individually subjected to evaluation by a sensory expert panel , Fourier‐transform infrared spectroscopy (FTIR), powder X‐ray diffraction (PXRD), and transmission electron microscopy (TEM), and their details are as follows:GC–MS: Agilent technologies, 7820 GC system, 5977E MSD, Column DB‐5, Over temp 100–270°C, Detector MS, Flow rate 1.2, Carrier gas Helium.FTIR: IR AFFINITY I – FTIR Spectrophotometer, FTIR 7600, Shimadzu.PXRD: D8 Advance (Bruker). Cu Kα radiation. High‐speed wide‐angle Lynx eye detector. Small‐angle scan for mesoporous materials can be collected from 2‐theta = 0.5°.TEM: Model JEM‐2100; Make: JEOL; HT—200 kV. Sample preparation procedure: A small amount (~1 mg) of powdered sample was dispersed in the Isopropanol solvent, and the dispersion was then loaded over the TEM grid (lacey carbon‐coated copper grid—300 mesh) for analysis. Sample‐loaded dry grids were used for TEM analysis, and images were captured from different regions at 200 kV acceleration voltage.2.3The acceptability index used by the employed sensory expert panel was the hedonic scale with a 9‐point nominal structure: 1—Dislike extremely, 2—Dislike very much, 3—Dislike moderately, 4—Dislike slightly, 5—Neither like nor dislike, 6—Like slightly, 7—Like moderately, 8—Like very much, 9—Like extremely Everitt, . The spr33.1Comparing the sensory scores of control sample with rest of the sprayed samples, four spraying samples of table salt, rock salt, and iodized salt have been scored ‘8’ . Excessive number of sprayings beyond 4 has caused a gradual reduction in the sensory score. Also, the four sprayed samples has significantly reduced moisture than the control. Increased sensory qualities and reduced moisture enhanced the saltiness preferably.Table 3.2Raw data and detailed interpretations for each salt are available, respectively, in Supplementary Data D1 and Supplementary Text T1.3.2.1Control: numerous peaks with major ones at around 11.8, 12.8, 13.4, 14.3, 14.7, 15.7, 16.2, 16.6, 17.4, 17.9, 18.1, 19.2, 19.7, 20.3, and 21.0 min. Four sprayed samples: compared to control, peaks are missing at around 11.8, 12.8, 14.3, 16.6, 17.4, 17.9, 18.1, and 19.2 min. There are additional major peaks at 13.7 and 17.1 min. It is suggested that the missing peaks and additional peaks give rise to the enhanced properties of the four sprayed samples. Twelve sprayed samples: relative to the GC–MS pattern of the control sample, the 12 sprayed samples show peaks at 7.9, 9.8, and 15.5 min and appear to be missing at 11.8, 12.8, 14.3, 15.7, and 19.2 min. These apparent differences in the GC–MS pattern are related to the strong, more pungent, undesirable taste  content in the sample. This peak is increased in 12 sprayed samples by 28% than control and four sprayed samples. Peak at 1635 cm−1 indicates the distinct presence of scissor peak of a water molecule (–OH stretch), this peak is decreased in four sprayed samples by 3%; however, in 12 sprayed samples, an increase of this peak by 36% can be noticed, which caused an unpleasant taste. Peak at 1018 cm−1 arises from the presence of silicates (SO32−), which decreased by 1% in four sprayed samples and increased by 26% in 12 sprayed samples, which also caused an unpleasant taste  might have broken down to medium chain  during spraying. When the rock salt sample was sprayed 10 times, there were found the major peak of pentadecen‐1‐ol acetate and 13‐octadecenal. These both could be the by‐products of long‐chain fatty acid degradation and transformation Figure .−1 indicates the presence of water content  in the control. This peak decreased for 2‐fold in the four sprayed samples and 2.7‐fold in the 10 sprayed samples. Peak at 2924 cm−1 indicates the presence of bicarbonates; this peak is increased by 3% in the four sprayed samples and 136% in the 10 sprayed samples, which caused unpleasant taste. Peak at 1627 cm−1 originates from fluorides; this peak decreased by 46% and 48% in the four and the 10 sprayed samples, respectively. Peak at 1481 cm−1 comes from carbonates, and decreased by 35% and 33% in the 4 and the 10 sprayed samples, respectively. Peak at 1018 cm−1 arises from silicates, and increased five times and seven times in the 4 and the 10 sprayed samples, respectively bis), 10.6 min , and 11.8 min (n‐Hexadecanoic acid). It is suggested that these additional peaks may be related to the increased saltiness of the four sprayed samples relative to the control sample. Twelve sprayed samples: There are additional key peaks at 15.5 and 16.2 min compared to the control sample. These additional peaks are due to compounds that alter the taste of the 12‐time sprayed sample, making it pungent and undesirable .Control: it shows only one prominent peak located at 32.3°. Visible minor peaks are located at 46.0°, 57.1°, 66.7°, and 75.7°. The present peaks indicate the presence of cubic structure, close to the mineral halite , causing alteration of physicochemical characters, hence enhancement of saltiness . For patients with high blood urea nitrogen, high creatinine, and renal diseases, MIRGA‐sprayed salt with various degrees of saltiness can be manufactured and suitably advocated.Madhu Mathi: Data curation ; investigation ; visualization ; writing – original draft .Conceptualization ; methodology ; project administration ; resources ; supervision ; validation ; writing – review and editing . This study received no specific funding.under‐patent no.: 401387) which is a major material employed in this study.In accordance with the journal's policy and our ethical obligation as researchers, we, the authors, are reporting that we together are the inventors and patentee of Indian patent for MIRGA (Appendix S1Click here for additional data file.Data S1Click here for additional data file.


In order to reveal the succession of the microbial community structure and flavor components during the pickling process, this study used PacBio Sequel to sequence the full length of 16S rRNA  and ITS  genes, and detected flavor components simultaneously, including organic acids, volatile flavor components (VFC), monosaccharides, and amino acids. Eleven phyla and 148 genera were identified in the bacterial community, and 2 phyla and 60 genera in the fungal community. During the four stages of pickling, the dominant bacterial genera were Leuconostoc, Lactobacillus, Leuconostoc, and Lactobacillus, while the dominant fungal genera were Aspergillus, Kazachstania, Debaryomyces, and Debaryomyces, respectively. There were 32 main flavor components . Correlation heat mapping and bidirectional orthogonal partial least squares (O2PLS) analysis showed that the flora having close relation to flavor components included 14 genera of bacteria  and 3 genera of fungi . This study provides detailed data regarding the microbial community and flavor components during the salt‐reducing pickling process of zhacai, which can be used as a reference for the development and improvement of salt‐reducing pickling methods.The salt‐reducing pickling method has been applied to the industrial production of  This study investigated a whole process of the salt‐reducing pickling of zhacai. The microbial community structure during the four stages was analyzed by using the PacBio Sequel platform to sequence full‐length 16S rRNA and ITS genes, the flavor components were measured , the correlations between microbial communities and flavor components were analyzed using correlation heat mapping and O2PLS modeling, the core functional flora were inferred through integrated correlation analysis, and the predicted functions of the microbial communities after using PICRUSt2 analysis were highlighted. Zhacai is produced in three major areas in China, Fuling in Chongqing, Yuyao in Zhejiang, and Xiapu in Fujian. Among them, Fuling is well known as the “hometown of zhacai,” because it is the origin of Brassica juncea varieties as it is located at the intersection of Yangtze River and Wujiang River, and has a unique climate, soil, and other geographical environments which are especially suitable for growing Brassica juncea ” because the process includes pressing treatment utilizing wood to expel the brine from the tuber  salt solution in a cement pool for about 7, 20–30 days, and 4–6 months, respectively. At the end of each pickling stage, the tuber is taken out and pressed to remove the brine, and then transferred to the next pickling stage   for use by other microorganisms, or converted into organic acids. Monosaccharides and organic acids produce sweet and sour tastes, respectively. The free amino acids produced by degradation of proteins produce umami, sweet, or bitter taste. Microorganisms can also metabolize and produce volatile flavor components (VFCs), such as esters, aldehydes, and alcohols, that are the main components of zhacai's aroma  has been used to study microbial diversity. It is capable of full‐length sequencing 16S rRNA genes (1400 bp) in bacteria and the ITS gene (1300 bp) in fungi with 99% accuracy  salt solutions, respectively, during the four stages of the pickling process. The acidity of the tubers was controlled at 3.0, 6.0, 8.0, and 10.0 g·kg−1 for the four stages, respectively. At the end of the first three stages, the tuber was taken out of the brine, pressed until most water flows out, and then transferred to the next stage. The pickling times of the four stages were 7, 20, 37, and 36 days, respectively , salt, nitrite, and organic acid concentrations of tuber samples were determined by previously established methods . Methodological details including standard reagents and standard curves can be found in Appendix Zhacai sample (50 mg) was weighed into a 2.0‐mL screw cap glass vial, and 700 μL 80% ethanol was added. The vial was shaken for 2 h at 50°C, then diluted with 700 μL water, centrifuged at 10000 rpm for 3 min, and the supernatant was recovered and adjusted to an appropriate dilution for injection. Ion chromatography used a Dionex CarboPac PA10 column  at 30°C and a 20 μL injection volume. Mobile phase A was water and B was 100 mmol·L−1 NaOH. The elution gradient (flow rate 0.5 mL·min−1) was as follows: 0–30 min mobile phase B increased from 2.5% to 20%, 30.1–45 min held at 40% B, and 45.1–60 min held at 2.5% B.−1), V is volume of sample extract (mL), F is dilution factor, and M is mass of sample (mg).The concentration of monosaccharides in 2.2.3zhacai sample (5 g) was placed in a 30‐mL SPME glass vial containing 10 mL of water saturated with salt. Internal standard (10 μL of 100 μg·mL−1 ethyl decanoate in absolute ethanol) was added and the vial was sealed. SPME extraction fiber  was inserted into the headspace bottle and VFCs were extracted for 40 min in a 60°C water bath. The fiber was then inserted into the sample inlet (250°C) and desorbed for 5 min. GC conditions were as follows: 40°C (0 min) held for 3 min, 5°C min−1 to 100°C then held for 2 min, and 10°C min−1 to 250°C then held for 1 min; splitless injection; and helium (purity 99.999%) carrier gas flow 1.0 mL·min−1. The mass spectrometer was operated in electron ionization mode (EI) at 250°C and 70 eV, and mass scanning range 35–400 m/z. All analyses were conducted in triplicate. The Retention index value of VFCs was determined using a set of n‐alkanes  standard with the same parameters. Analytes were identified using the NIST 17 library and RI value, and concentrations were calculated using ethyl decanoate as internal standard as follows:−1), ρ is mass concentration of internal standard (μg·mL−1), V is volume of internal standard (mL), Ai is peak area of the analyte, A is peak area of the internal standard, and m is mass of tuber sample (g). The odor activity value (OAV) of each compound was obtained by dividing the concentration by its corresponding odor threshold. The odor threshold comes from the book compiled by Van Gemert and Nettenbreijer (VFCs were determined by solid‐phase microextraction (SPME) and GC–MS  using a DB‐5MS capillary column . Homogenized nbreijer , and the2.2.4zhacai were determined by ultrahigh‐performance liquid chromatography  coupled with a triple quadrupole mass spectrometer  using a C18 column . Method details including standard reagents and standard curves are given in Appendix Zhacai sample (50 mg) was weighed into a 2‐mL Eppendorf tube, 400 μL 10% formic acid in methanol‐ddH2O  was added, vortexed for 30 s, and centrifuged at 12000 rpm for 5 min at 4°C. Ten microliters of supernatant was then diluted with 990 μL 10% formic acid in methanol‐ddH2O  and vortexed for 30 s. The diluted sample (100 μL) was mixed with 100 μL isotopic internal standard (100 ppb), vortexed for 30 s, and filtered through a 0.22 μm membrane into a microvial.The concentrations of 22 amino acids in −1 at 0–8.5 min, and increasing from 0.3 to 0.4 mL·min−1 over 8.5–12.5 min. The gradient elution program was as follows: 0–6.5 min mobile phase B increased from 10% to 30%; 6.5–7 min phase B 30% to 100%; 7–8 min phase B held at 100%; 8–8.5 min phase B 100%–10%; and 8.5–12.5 min phase B held at 10%. Mass spectrometry conditions were as follows: electrospray ionization (ESI) source, positive ionization mode, ion source temperature 500°C, ion source voltage 5500 V, collision gas 6 psi, air curtain gas 30 psi, and atomization and auxiliary gases 50 psi. The taste activity value (TAV) of each compound was obtained by dividing the concentration by its corresponding taste threshold.Chromatography conditions were as follows: injection volume 5 μL, column temperature 40°C, mobile phase A 10% methanol in water (with 0.1% formic acid), mobile phase B 50% methanol in water (with 0.1% formic acid), flow rate 0.3 mL·min2.3zhacai brine by centrifuging at 7000 g for 15 min. The particles were washed with 1x TE buffer and microbial macrogenomic DNA was extracted using a DNeasy mericon food kit  was used to filter chimeras in the sequences following quality control to obtain high‐quality sequences. The high‐quality sequences were clustered at 97% similarity level using the “cluster_size” module, yielding the amplicon sequence variant (ASV) and operational taxonomic unit (OTU) table. Singleton OTUs  and their representative sequence ASVs were removed from the OTU table. The high‐quality sequences were processed and analyzed further using QIIME2      was used to predict the functions of bacterial and fungal communities while figures were generated on the R package. Some data analysis was also performed on the website (https://www.genescloud.cn/) of Shanghai Personal Biotechnology Co., Ltd.Cutadapt v.2.3 was used to cut the primer fragment (set at −0 to 10) and discard the unmatched primer sequence Martin, . The “fa2.4p values among bacteria, fungi, and flavor components and to plot heat maps. O2PLS models were constructed in SIMCA 14.1 to analyze correlations among bacteria, fungi, and flavor components. Genera with variable importance in projection (VIP) predictive values greater than 1.0 and significant positive correlations with more than three flavor components were considered to be the core functional flora.The bacterial and fungal genera with the highest relative abundance greater than 1% and the average relative abundances greater than 0.1% and major flavor components  were selected for correlation analysis. The Hmisc package in R was used to calculate Pearson's correlation coefficients and 2.5https://www.ncbi.nlm.nih.gov/sra), with the registration numbers PRJNA725670 and PRJNA725681, respectively.Sequences of the bacterial 16S rRNA gene and fungal ITS gene were submitted to the Sequence Read Archive (SRA) database of the National Center for Biotechnology Information (NCBI)  salt solutions during the four stages, respectively. The salt penetrates into each tuber and the tuber is also dehydrated by salt, therefore the salt concentrations within the tubers gradually increase at each stage, in the ranges 13.85–20.75, 22.41–31.17, 35.81–66.71, and 70.49–87.15 g·kg−1, respectively. The nitrite concentrations were below 1.30 mg·kg−1 during the whole process, which is lower than the 4 mg·kg−1 standard applied to “green food” , were identified in the Cruciferae family and contains characteristic volatile compound isothiocyanates. Eight isothiocyanates were detected from the raw tuber samples in this study, designated as E1‐E8, namely allyl, 2‐phenylethyl, 3‐butenyl, 3‐(methylthio) propyl, butyl, isobutyl, 3‐methylbutyl, and pentyl isothiocyanate. Among them, the concentrations of E1, E2, E3, and E5 were 16612.3, 2919.3, 313.6, and 340.0 μg·kg−1, respectively, with an OAV greater than 1, indicating that they were the predominant aroma components of raw tubers, showing a strong pungent aroma. The aldehydes ranked second in the amount of VFCs in the raw mustard tubers. Aldehydes with OAV values greater than 1 included A8 ‐2,4‐decadienal), A5 , A9 ‐ 2,6‐nonadienal), A7 , and A6 ((E)‐2‐octenal), and their aromas are fruity (oily cucumber/melon/citrus), green sweet floral hyacinth, green fatty dry cucumber, waxy aldehydic rose, and fresh cucumber, respectively . Other components with OAV values greater than 1 were C2 (linolenic acid) and N2 (benzenepropanenitrile), and their aromas are faint fatty and powerful nasturtium, respectively.The mustard tuber belongs to −1, to 135.5, 614.2, 167.6, 197.3, 73.2, 165.9, and 77.4 μg·kg−1, respectively. This indicated that significant amounts of these compounds were produced during the pickling process, which changed the composition of aroma components of the tuber samples and made the aroma softer and richer.During the pickling process, the concentrations of isothiocyanates decreased significantly, and the strong pungent aroma became lighter. The other esters (E9–E13), aldehydes, alcohols, ketones, acids, nitriles, and other components increased from 0, 115.0, 0, 6.9, 29.3, 49.9, and 29.4 μg·kg−1, but its odor threshold was as low as 0.1 μg·kg−1 .Although the concentration of isothiocyanates present decreased significantly, it was still the category with the highest concentration of VFCs in the final pickled product (S46), and the OAVs of E1 and E3 were greater than 1. The other ester component E10 (octanoic acid ethyl ester) also showed an OAV greater than 1, its aromas have been described as fruity wine, waxy, and sweet apricot. The second category was still the aldehydes, and there were seven aldehydes with an OAV exceeding 1, namely A5, A7, A9, A1 , A6, A11 , and A4 ‐2,4‐heptadienal). The aromas of A1, A11, and A4 have been described as fresh green fatty aldehydic grass, oil and green grass, and fatty green oily aldehydic vegetable, respectively. The concentration of O4 (dimethyl trisulfide) reached 28.90 μg·kg3.1.3zhacai samples. The complete dataset is shown in Table In total, 21 amino acids were detected within the −1 in raw tuber to 1091.9, 1360.1, 539.9, 351.1, and 849.9 mg·kg −1 by the end of the process. In the final pickling sample (S46), the TAV of glutamate, alanine, lysine, and arginine reached 3.6, 2.3, 1.7, and 1.1, respectively. These data indicate that a large amount of flavoring amino acids are produced in the pickling process, which probably contributes to zhacai's overall flavor. Glutamate is an important umami taste amino acid. Alanine elicits a sweet and umami taste and is widely used as a flavor enhancer in soy sauce, fish sauce, and other condiments, to make the taste richer and more umami and intense  in the raw tuber was as high as 1350.7 mg·kg −1, and its concentration showed an increasing trend during pickling, reaching 2767.5 mg·kg −1 in the final pickled sample (S46). γ‐Aminobutyric acid is not a flavoring amino acid, but it has important physiological functions.During the pickling process, the concentration of five major flavoring amino acids, glutamate, alanine, arginine, valine, and lysine, increased from 164.8, 477.4, 214.1, 215.8, and 218.6 mg·kg 3.2Tables t‐tests showed significant differences in the bacterial Chao 1 indexes between stages 3 and 4 , and in the bacterial Shannon index between stages 1 and 4 . Significant differences were also detected in the fungal Shannon index between stages 1 and 3  and between stages 1 and 4 , and in the Simpson index between stages 1 and 3  and stages 1 and 4 . This suggests that the significantly reduced bacterial and fungal diversity observed in the fourth stage of processing may have been due to the high salt concentration (100 g·L−1). Figure Figure Leuconostoc (40.26%), Psychrobacter (10.50%), Lactobacillus (6.23%), Stenotrophomonas (6.19%), Klebsiella (5.29%), Ralstonia (5.04%), Acinetobacter (2.91%), Lactococcus (1.68%), Pelomonas (1.61%), Pseudoalteromonas (1.26%), Devosia (1.19%), Weissella (1.14%), and Sphingobacterium (1.09%). In the second stage, the abundance of Firmicutes increased (increasing to 64.86%), Proteobacteria decreased (down to 33.38%), and Bacteroidetes maintained its previous level (1.23%). At the genus level, Lactobacillus proliferated rapidly, becoming the dominant genus (abundance 43.47%) and the abundance of Leuconostoc decreased to 15.78%, ranking second. The other major genera included Ralstonia (4.72%), Stenotrophomonas (3.31%), Klebsiella (3.29%), Psychrobacter (3.04%), Erwinia (2.46%), Pediococcus (2.32%), Acinetobacter (1.93%), Pelomonas (1.53%), Citrobacter (1.32%), and Providencia (1.05%). In the third stage, Firmicutes (67.21%) and Proteobacteria (31.05%) were still the dominant phyla, followed by Bacteroidetes (1.26%). The dominant genera changed significantly as salt concentrations increased to 80 g·L−1. Leuconostoc became the dominant genus (24.75%) followed by salt‐tolerant Staphylococcus (18.06%), in addition to Lactobacillus (15.55%), Halomonas (9.57%), Psychrobacter (8.29%), Pediococcus (6.05%), Stenotrophomonas (3.49%), and Ralstonia (1.30%). In the fourth stage, the salt concentration increased further (100 g·L−1) and Firmicutes (70.05%) and Proteobacteria (28.99%) remained the dominant phyla. Lactobacillus (50.94%) became the dominant genus again, and the salt‐tolerant genera proliferated, which included Halomonas (8.67%), Chromohalobacter (6.63%), Staphylococcus (2.38%), Alkalibacterium (2.26%), Pseudoalteromonas (1.61%), Salinivibrio (1.26%), and Halanaerobium (1.02%). The other genera present included Weissella (6.04%), Aerococcus (3.12%), Psychrobacter (2.63%), Ralstonia (2.16%), Arcobacter (1.69%), and Leuconostoc (1.19%).The succession pattern of bacterial community structure is shown in Figure Aspergillus (51.24%), Malassezia (6.28%), Hannaella (5.01%), Cystofilobasidium (3.91%), Debaryomyces (3.12%), Alternaria (2.70%), and Filobasidium (1.19%). In the second stage, the abundance of Ascomycota increased sharply (92.66%), whereas Basidiomycota levels decreased (2.75%). At the genus level, Kazachstania proliferated rapidly and became the dominant genus (60.12%), followed by Aspergillus (16.37%) and then Debaryomyces (14.45%). In the third stage, Ascomycota (91.33%) remained the dominant phylum, followed by Basidiomycota (5.38%). At the genus level, Debaryomyces proliferated rapidly and became the dominant genus (47.19%), followed by Kazachstania (21.64%), Aspergillus (19.15%), Cystofilobasidium (2.76%), and Starmerella (1.66%). In the fourth stage, Ascomycota (98.37%) was still the dominant phylum and Debaryomyces remained the dominant genus (47.82%), followed by Kazachstania (17.76%), Aspergillus (3.70%), and Starmerella (28.37%).The succession pattern of fungal community structure is shown in Figure Linear discriminant analysis effect size (LEfSe) can be used to identify species with significant differences and a sense of biomarker between groups. Figure 3.3During the four pickling stages, there were 38 bacterial genera and 12 fungal genera with the highest relative abundance measured at greater than 1% and their average relative abundances calculated at greater than 0.1%, and 32 main flavor components . These data were subjected to correlation analysis.3.3.1Figure Marinomonas, Pectobacterium, Lactococcus, Clostridium, Serratia, Acinetobacter, Klebsiella, Devosia, Acidovorax, Leuconostoc, Stenotrophomonas, and Sphingobacterium, and was also positively correlated with fungal genera Aspergillus, Malassezia, and Filobasidium. Ester E10 was significantly positively correlated with Chromohalobacter, Halanaerobium, Tetragenococcus, and Halomonas.The isothiocyanates  showed a significantly positive correlation with bacterial genera Chromohalobacter, Halanaerobium, and Tetragenococcus. A8 was significantly positively correlated with the bacterial genera Klebsiella, Devosia, Acidovorax, Leuconostoc, Stenotrophomonas, Sphingobacterium, Rhodobacter, and Erwinia, and with the fungal genera Acremonium and Aspergillus.Some aldehydes  were positively correlated with Pectobacterium, Lactococcus, Clostridium, Serratia, Acinetobacter, Klebsiella, Devosia, Acidovorax, Leuconostoc, Stenotrophomonas, and Sphingobacterium, and positively correlated with the fungal genera Aspergillus, Malassezia, and Filobasidium. Galactose was significantly positively correlated with the bacterial genera Chromohalobacter, Halanaerobium, Tetragenococcus, and Alkalibacterium.Monosaccharides (glucose and fructose) were significantly positively correlated with the bacterial genera Chromohalobacter, Halanaerobium, Tetragenococcus, Halomonas, and Alkalibacterium, and with the fungi genus Debaryomyces.The five amino acids were significantly positively correlated with the bacterial genera 3.3.22X (cum), R2Y (cum), and Q2 (cum) used for evaluating the validity of the O2PLS model were 0.996, 0.966, and 0.538, respectively. All were greater than 0.5, and the first two were closer to 1, which indicated that the fitting effect of the model was good, its prediction was statistically significant  and Y representing the flavor component data. The parameters R3.3.3Leuconostoc, Clostridium, Devosia, Lactococcus, Pectobacterium, Sphingobacterium, Serratia, Stenotrophomonas, Halanaerobium, Tetragenococcus, Chromohalobacter, Klebsiella, Acidovorax, and Acinetobacter. Three genera of fungi also met the above criteria, Filobasidium, Malassezia, and Aspergillus. These 17 genera are inferred to be core functional flora in the fermentation process and are probably closely related to the formation of major flavor components.Based on the microbial community structure analysis, correlation heat mapping and O2PLS modeling, 14 genera of bacteria with VIP predictive values greater than 1.0 and significant positive correlations with at least three flavor components were identified: 3.4According to the KEGG pathway prediction by PICRUSt2, both bacterial and fungal communities included several pathways relating to fatty acid metabolism Figure .4zhacai. The microbial community structure during the four stages was analyzed using the PacBio Sequel platform to sequence full‐length 16S rRNA and ITS genes, the flavor components were measured , the correlations between microbial communities and flavor components were analyzed using correlation heat mapping and O2PLS modeling, the core functional flora was inferred through integrated correlation analysis, and the predicted functions of the microbial communities after using PICRUSt2 analysis were highlighted.This study investigated the whole process of the salt‐reducing pickling of zhacai includes pickling the tubers three times in a high concentration of salt solution (brine). On the third occasion, the salt concentration is the highest at around 140–160 g·L−1 and the immersion time is longer at around 4–6 months. The high salt concentrations inhibit the lactic acid fermentation of the microorganisms and make the acidity increase slowly, ensuring that the microorganisms have a longer time to produce the various flavor components , yet the flavor of the final pickling product (zhacai) is equivalent to that of the traditional method. The pressing treatment plays an important role in the salt‐reducing method. It not only helps to expel water from inside tuber and promote the penetration of salt into tuber but also facilitates the outflow of various components inside tuber for microbial metabolism and utilization, accelerating the transformation reactions of chemical components. Thus, although the pickling time is shortened, microorganisms still can complete metabolic transformation of various components and produce large amount of flavor components. Therefore, the addition of pressing operation is necessary for the salt‐reducing pickling process.The traditional pickling process for Leuconostoc, Lactobacillus, Leuconostoc, and Lactobacillus, and the dominant fungal genera were Aspergillus, Kazachstania, Debaryomyces, and Debaryomyces, respectively, whereas in all three pickling stages of traditional method, the most dominant bacterial genus is Lactobacillus, and dominant fungal genus cannot be determined according to a previous report , and other adverse conditions, with remarkable abilities to produce endo‐ and exopeptidases to degrade proteins into free amino acids, as well as lipases to hydrolyze fat into fatty acids, and thus contribute to flavors  fructose is catalyzed by hexokinase to produce fructose‐6‐phosphate, which is then degraded through glycolysis. (2) Some microorganisms, typically the zhacai include isothiocyanates, aldehydes, and dimethyl trisulfide. The VFCs in traditional pickling zhacai have been investigated, and Lin and Hua . Glucosinolates and endogenous myrosinase are located in different locations within the tuber cells, and extrusion or pressing can break cells, facilitate contact, and enable hydrolysis of the glucosinolates  expressed as the dominant component, which was consistent with previous studies  and benzenepropanenitrile (N2), which are probably also the degrading products of glucosinolates owing to acidic brine conditions, whereas consuming a large amount of nitriles probably exert negative effects on health, such as affecting thyroid function, hepatotoxicity, and nephrotoxicity  and linolenic acid (C2) were detected in the raw tubers, and their oxidation reaction probably produce hexanal (A1) and (E)‐2‐heptenal (A3). Secondly, there is the metabolic activity of microorganisms. PICRUSt2 function prediction  in tuber samples increased significantly during the pickling process. Microorganisms can produce GABA through the GABA shunt pathway Figure , the alp5zhacai were elucidated using the PacBio Sequel platform. In the bacterial community, the average number of nonsingletons per sample was 4989.47 ± 1087.24 and the sequencing length was 1400 bp. Eleven phyla and 148 genera were identified. In the fungal community, the average number of nonsingletons per sample was 5811.65 ± 2104.19 and the sequencing length was 1200 bp. Two phyla and 60 genera were identified. During the four stages of pickling, the dominant bacterial genera were Leuconostoc, Lactobacillus, Leuconostoc, and Lactobacillus, while the dominant fungal genera were Aspergillus, Kazachstania, Debaryomyces, and Debaryomyces. The simultaneous development of flavor components in zhacai was also demonstrated. There were 32 main flavor components . Correlation heat mapping and O2PLS analysis showed that the flora playing key roles in the formation of flavor components included 14 genera of bacteria , and 3 genera of fungi . The bacteria and fungi probably produce zhacai flavor components through the metabolic activity of saccharides, glucosinolates, fatty acids, and amino acids.The succession patterns of microbial communities during the salt‐reducing pickling process of The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.This study does not involve any human or animal testing.Appendix S1.Click here for additional data file.