Growing evidence has shown that exercise can affect the gut microbiota. The effects of exercise frequency on the gut microbiota in elderly individuals are still largely unknown. In the present study, samples from 897 elderly and 1,589 adult individuals (18–60 years old) from the American Gut Project were screened. Microbial diversity and composition were analyzed by QIIME2, and microbial function was predicted by PICRUSt2. The outcomes were further analyzed by STAMP. The analysis showed that the α‐diversity of gut microbiota increased with increasing age, and regular exercise reshaped the alterations in microbial composition and function induced by aging. Moreover, the α‐diversity of gut microbiota was higher in overweight elderly individuals than in normoweight elderly individuals, and regular exercise significantly affected the microbial composition and function in overweight elderly individuals. In conclusion, we revealed that regular exercise benefits elderly individuals, especially overweight elderly individuals, by modulating the gut microbiota. This study investigated whether microbial diversity increases with increasing age. The gut microbiota composition of daily exercising elderly individuals approached that of adults (18–60 years old), and regular exercise resulted in an increased relative abundance of bacterial functional pathways. We also showed that overweight elderly individuals had increased microbial diversity and significant changes in the microbial composition. Exercise helps regulate body weight, as it is inversely associated with weight gain and contributes to weight loss  , in which 25,376 samples from the American Gut Project were deposited, including 19,988 samples collected from fecal specimens. Upon excluding the samples without basic information , from patients who received antibiotic treatment within 6 months, from patients with tumors, and from patients who went on a trip within the previous three months, the data of 3,795 samples remained. Of these, samples from 897 elderly individuals and 1,589 18‐ to 60‐year‐old adults (adults18‐60) with normoweight were included in the study.The sample data were obtained from the Sequence Read Archive (SRA) : the 18‐ to 30‐year‐old group , 31‐ to 40‐year‐old group , 41‐ to 50‐year‐old group , 51‐ to 60‐year‐old group , 61‐ to 70‐year‐old group , and over 70‐year‐old group , regular exercise group , occasional exercise group , rare exercise group , and never exercise group  , normoweight elderly (NE) group (18.5 < BMI<25) , and overweight elderly (OE) group (BMI > 25)  group  and the never or rare exercise in OE individuals (NROE) group . OE individuals who occasionally exercised 117) were excluded from the analysis  were used for two‐group comparisons. Welch's inverted confidence interval (CI) method was used for CI calculation. The Benjamini–Hochberg false discovery rate method was used to calculate adjusted p‐values.STAMP was used to calculate the level of significance  with a normal BMI (18.5 to 25) were analyzed in this study. The α‐diversity analysis showed that the OTU numbers were 164.3, 168.6, 181.6, 186.3, 181.0, and 191.7 (p < .0001), while the Shannon indices were 4.989, 5.083, 5.222, 5.309, 5.203, and 5.391 (p < .0001) in the 18‐ to 30‐year‐old group, 31‐ to 40‐year‐old group, 41‐ to 50‐year‐old group, 51‐ to 60‐year‐old group, 61‐ to 70‐year‐old group, and over the 70‐year‐old group, respectively  and 71.1% (p < .05), respectively, while the relative abundance of Actinobacteria was significantly decreased by 33.3% (p < .05) in the elderly individuals compared with the adults18‐60. At the family level, the relative abundances of Aerococcaceae, Bifidobacteriaceae, Lactobacillaceae, Pasteurellaceae, Prevotellaceae, Tissierellaceae, Turicibacteraceae, and Veillonellaceae were significantly increased by 70.1%, 64.6%, 60.6%, 46.8%, 27.7%, 55.8%, 45.8%, and 29.6%, respectively (p < .05), while the relative abundances of Barnesiellaceae, Christensenellaceae, Desulfovibrionaceae, Enterobacteriaceae, Odoribacteraceae, Oxalobacteraceae, Rikenellaceae, and Verrucomicrobiaceae were significantly decreased by 31.3%, 101.5%, 20.4%, 46.8%, 58.5%, 70.2%, 38.7%, and 75.8%, respectively (p < .05), in the elderly individuals compared with the adults18‐60.Since α‐diversity significantly changed with age, we detected the gut microbiota composition in elderly individuals and compared it with that of adults0 Figure . At the 3.2p > .05), while the Shannon indices were 5.478, 5.466, 5.489, 5.351, and 5.332, respectively , occasional (1–2 times/week), regular (3–5 times/week), and daily). The analysis showed that the OTU numbers were 194.9, 195.5, 196.3, 186.7, and 191.9 , including 1 vitamin‐related pathway (8‐amino‐7‐oxononanoate biosynthesis I), 12 nucleotide metabolism‐related pathways , guanosine nucleotides de novo biosynthesis II, pyrimidine deoxyribonucleotides de novo biosynthesis (E. coli), pyrimidine ribonucleotides de novo biosynthesis, purine ribonucleosides degradation, and purine nucleotides de novo biosynthesis II), 2 cell wall biosynthesis‐related pathways , 7 glucose metabolism‐related pathways (glycolysis II (from fructose 6‐phosphate), pentose phosphate pathway, ‐butanediol biosynthesis, pyruvate fermentation to propanoate I, glycolysis, β‐D‐glucuronosides degradation, and hexuronide and hexuronate degradation), and 2 amino acid metabolism‐related pathways (l‐glutamate and l‐glutamine biosynthesis and urea cycle). The relative abundances of 18 of those pathways were significantly higher, while the abundances of 5 of those pathways were significantly lower in the DRE group than in the NRE group. Therefore, these results suggest that regular exercise significantly modulated microbial function in elderly individuals.For analysis of microbial function, elderly individual samples were recombined into the daily or regular exercise (DRE) group and never or rare exercise (NRE) group Figure . Twenty‐3.3p < .001), while the Shannon indices were 5.054, 5.244, and 5.508 (p < .001) in the underweight elderly, NE, and OE groups, respectively , while the Shannon indices were 5.681 and 5.508 (p < .001) in the DROE and NROE groups, respectively , respectively, while the Bacteroidetes abundance was decreased by 6.7% in OE individual samples (p < .05) compared with (NE) individual samples. At the family level, the relative abundances of Verrucomicrobiaceae, S24‐7, Prevotellaceae, Oxalobacteraceae, and Lachnospiraceae were increased by 34.2%, 52.7%, 40.4%, 102.4%, and 15.1%, respectively, while the relative abundances of Enterobacteriaceae, Christensenellaceae, Barnesiellaceae, Bacteroidaceae, Porphyromonadaceae, and Peptococcaceae were decreased by 18.1%, 24.3%, 21.4%, 17.2%, 20.3%, and 31.9%, respectively, in the OE individuals (p < .05) compared with the NE individuals.Next, the abundances of gut microbiota constituents were detected at the phylum and family levels in OE individuals Figure . At the p > .05) compared with nonexercising OE individual samples. At the family level, the relative abundance of Turicibacteraceae was significantly increased, while the relative abundances of Pseudomonadaceae, Oxalobacteraceae, Odoribacteraceae, and Barnesiellaceae were significantly decreased in the frequently exercising OE individuals compared with nonexercising OE individuals.As shown in Figure 3.5Microbial functions were detected in OE individuals, and it was found that the relative abundances of 129 pathways were significantly changed Appendix . The rell‐methionine biosynthesis was significantly decreased in OE individuals and significantly increased in the frequent exercise group.Notably, 13 common pathways were identified Figure . Among t4The gut microbiota plays a major role in age‐related diseases. Moreover, changes in gut microbiota diversity have profound impacts on host metabolism. In the present study, we detected the effect of exercise on the gut microbiota in elderly individuals by using data obtained from the American Gut Project. The microbial α‐diversity increased with age in the normal BMI population, and the gut microbiota was restored by exercise in elderly individuals.A recent study showed that microbial α‐diversity is positively associated with age in populations such as the United States, the United Kingdom, and Colombia  than in the adults18‐60 , while the Bacteroidetes/Firmicutes ratio was not significantly different between the two groups. At the family level, the abundances of Desulfovibrionaceae and Enterobacteriaceae were significantly increased, while that of Bifidobacteriaceae was significantly decreased in elderly individuals. Actinobacteria species are key in maintaining gut homeostasis and play an important role in the treatment of gastrointestinal diseases and systemic diseases . Shangfei Jiang: Software (lead). Guankui Du: Conceptualization (lead); Formal analysis (lead); Funding acquisition (lead); Investigation (lead); Methodology (lead); Project administration (lead); Resources (lead); Writing – original draft (lead); Writing – review and editing (lead).None required.


Food waste management plays a central role in the circular economy. To our knowledge, only a few studies have investigated the use of unsold fruit and vegetables from supermarkets as a substitute source for the extraction of natural colorants. Thus, the aim of this paper was to suggest a green, tailored protocol that avoids the use of organic solvents for the recovery of betalains from unsold red beets for use as a food colorant. The recovery of such pigments was carried out by a tailored enzymatic mix, blended considering the polysaccharide composition of the beetroot cell wall; thus, it consisted of: cellulase (37%), xylanase (35%), and pectinase (28%). The enzyme-assisted extraction protocol was optimized, and the most suitable conditions (in terms of pigment yield and color attributes) for the recovery of betalains from unsold beets appeared to be: 25 U/g total dose of enzymatic mix, temperature 25 °C, and processing time 240 min. Color is a distinctive characteristic of food and beverages. It is considered a quality indicator that affects their appeal to consumers . During Beta vulgaris ssp.); whereas, among betaxanthins, the best-known are vulgaxanthin, present in yellow beets (Betavulgaris L.), and indicaxanthin, found in cactus pears (Opuntia ficusindica) [From a Circular Economy approach, food waste (including unsold vegetables) is considered a promising source of natural colorants, sweeteners, antioxidants, and preservatives . Among tsindica) ,9.Although betalains and anthocyanins perform similar functions, they have never been reported in the same plant, seeming to be mutually exclusive in the plant kingdom. Betalains can also be found in roots, fruit, and flowers . The fooHylocereus polyrhizus by the application of pectinase. Betalains have been recovered by various methods, such as diffusion-extraction, solid–liquid extraction, reverse osmosis, and ultrafiltration . BetalaiThis research suggests a tailored protocol, based on targeted substrate-enzymatic extraction, for the recovery of betalains from unsold red beets. The procedure consists of applying a specific enzymatic mix that was designed on the basis of the composition of fully ripened red beet cell walls. Moreover, the optimal conditions  for betalain recovery yield, as well as the colorimetric parameters of the extract, were identified.Beta vulgaris ssp.), devoid of visual blemishes and contaminations, was supplied by a local market . The raw material was washed, crushed in an HR 2068 blender , and stored at 2–4 °C for 30 mins before use for betalains extraction.Unsold beetroot  and pectin-liasic (PL) activity , cellul I.U./g, ), and xy I.U./g, ) were su I.U./g, .Beetroot samples were dried at 30 °C for 5 days before dry matter (DM) and moisture determination . pH and 620nm = 0.0047 * glucose concentration; R2 = 0.99) using a stock solution of glucose (50–250 μg).Total sugar content was analyzed by measuring absorbance at 620 nm, following the Anthrone method ,23,24. TThe amount of cellulose, hemicellulose (xylans), and pectin in the beetroot was examined following Lombardelli et al.  after acThe whole red beet samples (200 g) were washed, drained, and mixed (Phillip HR 2068 blender), and the resulting purée was used for the betalain extraction. The conditions of solid–liquid extraction were as follows: (i) 1:15 solid–liquid ratio between raw material and acetate buffer containing the multi-component enzyme mix ; (ii) extraction temperature of 25 or 45 °C, at pH 5.5 ± 0.1; (iii) varied extraction duration up to five hours, with samples taken at various processing times. The mixture was then filtered and the extracts were analyzed. Trials were conducted in triplicates.536nm and ε = 60,000 (molar extinction coefficient in Lmol−1cm−1); for Bx A485nm and ε = 48,000 (Lmol−1cm−1); DF = dilution factor; molecular weight (MW) = 550 g/mol (for Bt) or 339 g/mol (for Bx); i = path length (1 cm). Samples were diluted and measured in triplicate.The amount of betalain, in terms of betacyanins (Bt) and betaxanthins (Bx), was determined spectrophotometrically by visible spectra (λ = 340–700 nm). Bt and Bx concentrations were calculated as described by Wruss et al. : Bt  to red (+a*) axis, and b* indicates the chromaticity on a blue (−b*) to yellow (+b*) axis. The analyses were performed in triplicate, with five measurements in each sample unit. Extracts were evaluated throughout in terms of redness index (a*/b*), Chroma (C* = (a*2 + b*2)0.5), and hue angle (h° = (arctan b*/a*)). The ratio a*/b* was considered a good indicator of color loss in beetroot puree. The hue expresses the color nuance and the values are defined as follows: red–purple = 0°, yellow = 90°, bluish-green = 180°, and blue = 270°. The C* is a measure of chromaticity, which denotes the purity or saturation of the color; the higher the chroma values, the higher the perceived color intensity of the samples to the naked eye [Color measurements were performed using a CR-5 colorimeter  with a D65 illuminant and CIE aked eye .p < 0.01). A Tukey’s honestly significant difference (HSD) post-hoc test (p < 0.05) was carried out by the EXCEL® Add-in macro DSAASTAT program [p < 0.05.Data were analyzed for statistical significance using a two-way analysis of variance (ANOVA) and a one-way ANOVA to check for an effect of the single factors  or an interaction with extraction yield and color parameters ( program . A Pearswwb)) of red beets were consistent with those reported in the literature [wwb; DM 11 ± 1%wwb; pH 6.11 ± 0.01; TA 0.14 ± 0.03 (gcitric acid/100 gwwb); and total sugar content of 12 g/100 gwwb. These data demonstrate that the red beet used had the optimal ripening stage, which corresponds to the highest betalain amount [Unsold red beets, used in this study for the recovery of betalains, were first analyzed in terms of moisture, DM, pH, TA, total sugars, and cell-wall polysaccharide composition. The main chemico-physical parameters (in wet weight basis (terature  and weren amount .dwb)) was: cellulose 37% (4.0 ± 0.1 g/100gdwb), pectin 28% (3.1 ± 0.1 g/100gdwb), and hemicellulose 35% (3.7 ± 0.1 g/100gdwb). Cellulose was the predominant component, followed by hemicellulose and pectin. Zieminski et al. [The cell-wall polysaccharide composition  and pH) for maximizing the activity of all the enzymes forming the tailored mix, assessed by response surface methodology analysis, were: temperature between 45 and 60 °C and pH between 5 and 6, as demonstrated in a previous study . MoreoveNowadays, the recovery of stable natural pigments from food-waste-based matrixes is gaining attention, as described by Galanakis , Gupta ep < 0.01, data not shown). Moreover, data analyzed by means of one-way ANOVA  demonstrated that Bx and Bt recovery yields were significantly affected by the investigated factors , it is suggested that, at the lower tested dosages (10 and 18 U/g), the small amount of pectinase activity may be not adequate to break down the pectin network and release pigments. Both at 45 °C and at 25 °C, the minimal suitable total dosage was 25 U/g. More specifically, at 45 °C, the extraction yield ranged from 3.83 ± 0.01 to 8.89 ± 0.03 after 20 and 120 min for Bx, and from 6.03 ± 0.01 to 12.15 ± 0.03 after 20 and 120 min for Bt . FurtherWhen a higher enzyme-mix dosage (38 and 50 U/g) was used, the extraction yields decreased with respect to 25 U/g, probably due to the fast hydrolysis degree, which may have led to inhibition of the end-product ,37.Overall, the optimal extraction conditions for maximizing the recovery yield of Bt and Bx appeared to be 25 °C for 240 min with an enzymatic-mix dosage of 25 U/g.a*/b*, indicating the redness of the extract), chroma , and hue angle , considered to be the best descriptors of color, were used to characterize the different betalain extracts recovered by the tailored enzymatic mix  was revealed. A similar trend was observed at 25 °C, despite a*/b* not decreasing at longer extraction times  . At 45 °on times A–E. In pon times A–E.h° angle values were always lower for the extracts recovered at 25 °C than for those obtained at 45 °C. More specifically, at 45 °C, the hue decreased slightly as the extraction time increased, and then significantly increased with longer times (240 and 300 min) irrespective of the dosage used. At 25 °C, this parameter significantly decreased as the extraction time increased, and then remained constant  and after 240 min at 25 °C (7.96 ± 0.01) (Observed constant F–J. Cons ± 0.01) F–J. a*/b* and h°) could be explained by the aforementioned betacyanin degradation reactions, which resulted in a differential extraction of betacyanin vs. betaxanthin fractions , a gradual rise in this parameter was evident as extraction time increased. The extracts recovered using the dosage 25 U/g were characterized by similar values of C*, despite the processing time at 25 °C (C* = 70.30 ± 0.08 after 240 min) being 2 times longer than that at 45 °C (C* = 71.02 ± 0.78 after 120 min).Concerning In order to suggest a novel, green, and sustainable biotechnological approach for the recovery of betalains from unsold red beets, the effects of enzymatic-mix dosage, processing time, and temperature on extraction yield and color attributes were considered. Accordingly, a correlational analysis between both dependent variables (extraction yield and color attributes) was conducted. Taking into account that measured color is determined by the contribution of both betacyanins and betaxanthins, the variable “total betalain content” (Bt+Bx) was used in the statistical analysis.a*/b*, chroma, and hue angle always appeared correlated (negatively or positively) with the total betalain content, regardless of the temperature and mix total dosage used (a*/b* and C* showed the strongest positive correlations (0.9478 and 0.9802), whereas h° angle had the strongest negative correlation (−0.8823)  . AccordiOverall, the data reported in this paper, as well as the strong Pearson correlation coefficients, prove that the most suitable extraction conditions (in terms of yield and color attributes) for the recovery of betalains from unsold red beets are: a 25 U/g total dose of enzymatic mix; a temperature of 25 °C, and a processing time of 240 min.The novel and significant contribution of the current research is the demonstration that the recovery of betalain from unsold red beets can be improved by applying a specific enzymatic mix based on the polysaccharide composition of the red-beet cell wall. Although the pigment extraction at higher temperatures (45 °C) was faster, a comparable recovery yield of betacyanin and betaxanthin fractions was achieved at lower temperatures (25 °C). Moreover, the low-temperature enzyme-assisted extraction protocol allowed better preservation of the color attributes of the betalain extracts.Overall, the optimal conditions for the extraction of betalain from unsold red beets were: 25 U/g total dose of enzymatic mix, a temperature of 25 °C, and a processing time of 240 min. These results prove that betalain recovery could be a “green route” to coloring foodstuffs, giving much greater value to unsold red beets.


The intake of sugar-sweetened beverages (SSBs) has increased rapidly, but the effects of this habit on health and physical performance are unknown. This study assessed the effect of excessive SSB intake on biochemical, physical performance, and biochemical and cardiovascular parameters of physically active males.  Seventeen volunteers consumed a placebo drink  and an excessive SSB drink (eSSBd = Pd plus 300 g sucrose). In a blind randomized crossover study, the subjects were assigned to Pd or eSSBd groups for 15 days. After an interval of 7 days, subjects were reassigned to the other condition. 2max) (48.15 ± 10.42 vs. 40.98 ± 11.20), peak heart rate (186.64 ± 8.00 vs. 179.64 ± 6.28), total exercise time (15.02 ± 1.57 vs. 14.00 ± 2.18), and mechanical work (15.83 ± 4.53 vs. 13.68 ± 5.67) decreased after eSSBd intake . The rates of perceived exertion were higher (1.300 vs.1.661 slope and −0.7186 vs. −1.118 y-intercept) after eSSBd intake.  After eSSBd intake, there was an increase in weight (69.34 ± 13.71 vs. 70.62 ± 14.06), body mass index (24.49 ± 4.01 vs. 24.97 ± 4.13), waist circumference (75.33 ± 11.22 vs. 76.79 ± 11.51), VLDL (19.54 ± 9.50 vs. 25.52 ± 11.18), triglycerides (78.94 ± 23.79 vs. 114.77 ± 43.65), and peak systolic blood pressure (178.57 ± 26.56 vs. 200.71 ± 24.64). The cardiorespiratory response to exercise (VO The present study shows that 15 days of eSSBd intake may negatively modulate biochemical parameters associated with cardiovascular risk. In addition, this overintake can impair the physical performance and cardiovascular responses to physical exercise. Over the last 20–30 years, the world population has reduced water intake and the intake of many healthy foods, such as legumes, fruits, and vegetables. On the contrary, the intake of sugar-sweetened beverages (SSBs) and ultraprocessed foods has increased rapidly .Some studies have shown that excess carbohydrate intake can impair cardiovascular health , 3. AnotAn excessive intake of SSB is also a risk factor for cardiovascular diseases through biochemical alterations as SSB stimulates lipogenesis and increases triglycerides (TG) and total serum cholesterol levels , 7.A recent study has shown that 14 days of a sucrose-supplemented diet attenuated vascular function in young healthy individuals during leg exercise. This impairment resulted from reduced activity of endothelial nitric oxide (NO) synthase. It is well established that NO contributes to the physical performance through increased perfusion of oxygen and nutrients; therefore, SSB can decrease the exercise performance via a decrease in vascular flux .The relationship between exercise and excessive intake of SSB has been investigated, and studies have shown that exercise may prevent transient SSB-mediated microvascular endothelial dysfunction . In addiExcess sugar intake induced a decrease in physical activity in rats , and thePhysical performance is related to carbohydrate ingestion, and it was noted that there is metabolic flexibility; that is, the body prioritizes the use of carbohydrates as the predominant substrate during exercise , 16. In Furthermore, carbohydrate intake has long been understood to be important for good physical performance . NeverthConsidering the effects of excessive SSB intake on the cardiovascular system and the impact on physical performance, the present study has two hypotheses: excessive SSB intake worsens biochemical and cardiovascular parameters after exercise as well as reduces the physical performance.The volunteers were seventeen active healthy males with a mean age of 24.92 ± 4.34 years, mean weight of 75.62 ± 11.01 kg, mean height of 176 ± 0.11 cm, and mean body mass index (BMI) of 24.59 ± 3.98. The inclusion criteria were as follows: practicing physical activity regularly at least 3 times a week and being available to visit the laboratory for 4 morning meetings. We excluded volunteers with diabetes mellitus, hypertension, physical injury, or sedentary status. All participants signed a consent form. The study was approved by the ethics committee for studies with humans of the Federal University of Ouro Preto under approval number 56317816.3.0000.5150.https://www.random.org/lists/) and assigned into one of two experimental conditions: placebo (SP) or sucrose (SS). Each treatment was performed for 15 days, followed by 7 days of interval between conditions , perform physical activities normally, and avoid training modifications during the study. The drinks were delivered to volunteers daily for intake certification and routine guidelines. In order to avoid that the volunteers spent many days without contact with the researchers, the washout was fixed at seven days, so it was intended to avoid significant changes in the dietary routine and/or physical activity levels that could interfere in the research results.On the day of each experimental condition, the volunteers visited our laboratory in the morning. After 8 h of fasting, blood samples were collected in 9 mL tubes without the anticoagulant and centrifuged at 1080 G. The serum was fractionated into 1 mL aliquots across 3 microtubes and stored at −20°C.The concentrations of fasting blood glucose, total cholesterol, high-density lipoprotein (HDL), very-low-density lipoprotein (VLDL), and TG were assessed using the Bioclin® kit . All blood measurements were performed in triplicate.The biometric parameters were measured: weight, height, and fat percentage . Systoli2max was directly measured using a VO2000® analyzer . Total exercise time (TET), cardiovascular response, SBP, and DBP throughout the test were assessed. Ratings of perceived exertion (RPE) were checked at each stage using the Borg scale [In each of the laboratory visits, the volunteers were submitted to the Bruce incremental treadmill protocol until voluntary exhaustion . VO2max rg scale .p < 0.05.Data normality was tested using the D'Agostino–Pearson test. Data that were normally distributed were analyzed with a two-way repeated measure ANOVA followed by Bonferroni's multiple comparisons test. A regression was used when indicated. In results, data were expressed as mean ± standard deviation (SD), and the significance level was set at After 15 days of placebo intake, we did not observe differences in body weight shown in , body ma2max)  also induces a decrease in lipid oxidation . Hence, The decreased physical performance induced by excessive SSB intake could also be explained through carbohydrate-mediated uric acid metabolism. Simple carbohydrate intake could increase urate synthesis in the kidney , 41. BecThe limitations of the present study were that we did not investigate about mechanisms involved in the decrease in the physical performance, for example, the uric acid metabolism and the heart rate variability in volunteers undergoing in conditions, this variables must be explored in future studies. Other limitation was that were not recorded the food consumption by a 24-hour recording, future studies must do the dietary recall at least 3 times by week during time of experiments.The present study shows that only 15 days of excessive SSB intake may negatively modulate biochemical parameters associated with cardiovascular risk. In addition, this overintake can impair the physical performance and cardiovascular responses to physical exercise.


Allium sativum) is the second most important Allium crop that has been used as a vegetable and condiment from ancient times due to its characteristic flavor and taste. Although garlic is a sterile plant that reproduces vegetatively through cloves, garlic shows high biodiversity, as well as phenotypic plasticity and environmental adaptation capacity. To determine the possible mechanism underlying this phenomenon and to provide new genetic materials for the development of a novel garlic cultivar with useful agronomic traits, the metabolic profiles in the leaf tissue of 30 garlic accessions collected from different geographical regions, with a special focus on the Asian region, were investigated using LC/MS. In addition, the total saponin and fructan contents in the roots and cloves of the investigated garlic accessions were also evaluated. Total saponin and fructan contents did not separate the garlic accessions based on their geographical origin, implying that saponin and fructan contents were clone-specific and agroclimatic changes have affected the quantitative and qualitative levels of saponins in garlic over a long history of cultivation. Principal component analysis (PCA) and dendrogram clustering of the LC/MS-based metabolite profiling showed two major clusters. Specifically, many Japanese and Central Asia accessions were grouped in cluster I and showed high accumulations of flavonol glucosides, alliin, and methiin. On the other hand, garlic accessions grouped in cluster II exhibited a high accumulation of anthocyanin glucosides and amino acids. Although most of the accessions were not separated based on country of origin, the Central Asia accessions were clustered in one group, implying that these accessions exhibited distinct metabolic profiles. The present study provides useful information that can be used for germplasm selection and the development of new garlic varieties with beneficial biotic and abiotic stress-adaptive traits.Garlic ( Allium sativum L.) is one of the most economically important species of the genus Allium that has been used from ancient times as traditional medicine due to its nutritional, antimicrobial, and antioxidant properties, as well as a spice and herb to heighten the flavor of the dishes and health quotient [Garlic , with an estimated haploid size of 15.9 gigabase pairs, which is ~32 times greater than the rice (Oryza sativa) genome [The center of origin for garlic is considered to be the northwestern side of the Tien Shan Mountains in Central Asia due to the discovery of some fertile clones of a primitive garlic type in this area , leading) genome . Garlic ) genome ,12. Alth) genome , garlic ) genome ,15. For ) genome ,16. The ) genome ,18,19.Garlic cultivars vary not only in their fertility but also in their chemical compositions, including organosulfur compounds, saponins, phenolic compounds, and polysaccharides ,20,21. I−1 DW, and the average was 14.49 mg g−1 DW  among the 30 investigated garlic accessions (−1 DW), whereas 16 accessions  showed moderate levels ranging between 7.82 to 18.21 mg g−1 DW (Saponin contents in garlic root tissues varied between 3.36 to 32.18 mg gg g−1 DW . Garlic g g−1 DW . For exacessions ). All thcessions . On the g g−1 DW ). The hig g−1 DW A.−1 FW), whereas F189, SHA_Shanghi, Fs414, IND-IIT, F1-200-34, VNM_Mai Dinh, and GER556 displayed lower fructan contents ranging between 2.58 to 10.93 g inulin equivalent 100 g−1 FW  in the cloves of investigated garlic accessions, and the geographical distribution did not show any significant influence on the fructan profile . For exa0 g−1 FW . The remW  < 3, leaving 101 metabolites for further analysis . All ther > 0.95), whereas Fs422 showed negative or weak correlations with the other garlic accessions , whereas lower saponin contents were detected in the leaves (10.48 mg g−1 DW), indicating that there is a dynamic variance trend in total saponins between different organs [Allium ampeloprasum), whereas most of the Allium vegetables did not contain β-chlorogenin, implying that garlic has its specific saponin profiles and β-chlorogenin may be a suitable biomarker for garlic [Allium sativum L. var. Voghiera and named voghieroside A1/A2, B1/B2, D1/D2, and E1/E2 based on the aglycon structure [A. sativum L. var. Voghiera [Trichoderma harzianum and the pathogenic Botrytis cinerea, indicating that saponin plays a pivotal role in garlic’s defense against fungi pathogens [The average content of the total saponins in dry roots of the investigated garlic accessions was 14.49 mg gectively . On the ectively . These rs Allium , there it organs . In addir garlic . In thistructure . In addiVoghiera . The isoathogens . Thus, b−1 FW, followed by the Japanese accession JPN40 with 19.48 g 100 g−1 FW  in comparison with Japanese bulb onions, which was linked with drought stress tolerance and protection against fungal and pathogenic attacks [The average content of fructan in the cloves of all the investigated garlic accessions was 14.19 g inulin equivalent per 100 g FW, where the Central Asian accession Fs407 showed the highest fructan content in comparison with the other investigated garlic accessions with 23.0 g 100 g0 g−1 FW . On the W , and bulbs of 33 garlic accessions were managed at Saga University, Japan  until 2012, when the management of both collections was taken over by Yamaguchi University, Japan . To maintain these important garlic genetic resources, ten cloves from three to five bulbs of each accession were vegetatively propagated every year in October and harvested by the end of June. Then, the entire garlic plant  were hung and kept in a dry place with good air ventilation inside the Greenhouse. Out of these harvested plants, 30 accessions were used in this study. The 30 garlic accessions contained 7 clones from Central Asia, 6 clones from Japan, 4 clones from China, two clones from Thailand, one clone from Taiwan, one clone from India, one clone from Syria, two clones from Egypt, two clones from Turkey, one clone from Germany, one clone from Greece, and one clone from Peru . DetaileFor the metabolome analysis, garlic accessions were cultivated in an experimental field at the Institute of Vegetable and Floricultural Sciences, NARO, Mie, Japan . The garlic accessions were planted in the same way as at Yamaguchi University described above (the previous crop was plowed into the soil after growing Sorghum). CDU-S555 , Ecolong 413-100 , and Ecolong 413-140  were applied at 130, 360, and 130 kg/ha, respectively before planting. The field experiment was carried out for two successive seasons (2017–2018 and 2018–2019). Two months after planting, the youngest leaves, which were over 4 cm in length, were sampled. Then, the leaf tissues were immediately placed in 2 mL tubes, immersed in liquid nitrogen, and stored at −80°C for further analysis. The monthly mean temperature (°C), precipitation (mm), and accumulation of daylength (h) (minimum/maximum) were 16.4 °C (4.7 °C/29.2 °C), 191.6 mm (17.0 mm/648.0 mm), and 194.3 h (95.0 h/279.2 h) in 2017–2018, while they were 16.8°C (6.0 °C/28.6 °C), 108.6 mm (22.0 mm/342.5 mm), and 174.4 h (103.3 h/262.1 h) in 2018–2019, respectively .®, Rotavapor® R-3; Merck KGaA, Darmstadt, Germany) under reduced pressure at 50 °C and then partitioned between butanol (BuOH) and H2O (1:1). The BuOH layer was filtered and then concentrated under vacuum, causing a crude extraction of saponins. The total saponin content in the crude extract was determined spectrophotometrically at 473 nm using a 0.7% vanillin-60% H2SO4 reagent. The absorbance was measured three times against a blank at 473 nm using a U-2000 spectrophotometer . The major saponin compound in garlic, namely, β-chlorogenin [−1 DW root).The saponin contents were obtained from the root tissues of 26 garlic accessions during a doctoral study ,51 Tabl. After torogenin , was use−1 FW).The fructan contents were obtained from the cloves of 27 garlic accessions during a doctoral study ,51 Tabl. The fru®, Rotavapor® R-3;) coupled with a vacuum pump v-700  under reduced pressure at 10 ± 1 °C. The sample preparation process was automatically performed using a liquid-handling system , as previously described [−1 extraction solvent  using a multibead shocker  at 1000 rpm for 2 min. After the centrifugation, the extracts were diluted to 400 µg mL−1 in first season and 1000 µg mL−1 in the second season using an extraction solvent. Then, 250 µL of the extract was transferred to a 96-well plate, dried, redissolved in 500 and 250 µL of ultrapure water, in season one and season two, respectively, and filtered using MultiScreenHTS384 well . One and 1.5 µL of the solution extract at a final concentration of 20 and 100 µg mL−1, in season one and season two, respectively, was subjected to widely targeted metabolomics using LC/MS  in the multiple reaction monitoring mode; for detailed information, see The leaf tissues were freeze-dried using a rotary evaporator (Büchiescribed . Brieflywww.r-project.org (accessed on 2 January 2021)).The final data results were obtained by taking the average of the results of two successive seasons. The histogram, PCA, heatmap, and correlation analyses were carried out using R v.4.0.4 (


Metopolophium dirhodum  (Hemiptera: Aphididae) is one of the most common aphid pests of winter cereals. To facilitate accurate gene expression analyses with qRT-PCR assays, the expression stability of candidate reference genes under specific experimental conditions must be verified before they can be used to normalize target gene expression levels. In this study, 10 candidate reference genes in M. dirhodum were analyzed by qRT-PCR under various experimental conditions. Their expression stability was evaluated with delta Ct, BestKeeper, geNorm, and NormFinder methods, and the final stability ranking was determined with RefFinder. The results indicate that the most appropriate sets of internal controls were SDHB and RPL8 across geographic population; RPL8, Actin, and GAPDH across developmental stage; SDHB and NADH across body part; RPL8 and Actin across wing dimorphism and temperature; RPL4 and EF1A across starvation stress; AK and RPL4 across insecticide treatments; RPL8 and NADH across antibiotic treatments; RPL8, RPL4, Actin, and NADH across all samples. The results of this study provide useful insights for establishing a standardized qRT-PCR procedure for M. dirhodum and may be relevant for identifying appropriate reference genes for molecular analyses of related insects. Nevertheless, gene expression analyses are affected by many factors such as the quality of RNA samples, the efficiency of reverse transcription, and PCR efficiency3. For accurate comparisons of expression levels, the expression data of the genes of interest are normalized against the expression data for a reference gene4. Moreover, the reference gene compensates for the above-mentioned limitations5. Because housekeeping genes are related to ubiquitous and basic cellular functions, they are considered to be constitutively expressed under diverse conditions6. Housekeeping genes, including those encoding actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal protein, 18S ribosomal RNA, elongation factor 1α and heat shock proteins, have been extensively used as endogenous controls for normalizing real-time PCR data11. However, several studies have indicated that the expression levels of the reference genes vary under diverse conditions14. In fact, no single reference gene is appropriate for all experimental conditions. Therefore, evaluating and validating the stability of reference genes under different experimental conditions is critical.The quantitative analysis of target gene expression is an essential part of most molecular studies. Quantitative real-time PCR (qRT-PCR) is a powerful tool for quantifying gene expression, combining improvements in both sensitivity and specificity with efficient techniques for signal detection. It is useful for the quantitative data analysis required for research related to molecular medicine, biotechnology, microbiology, and diagnostics and has become the preferred method for quantifying mRNAHarmonia axyridis, Propylea japonica, Aphis craccivora Koch, Henosepilachna vigintioctomaculata, Chilo suppressalis, Galeruca daurica, Liriomyza trifolii, Coccinella septempunctata, Phenacoccus solenopsis, Lipaphis erysimi, Myzus persicae, Acyrthosiphon pisum, and Megoura viciae27.There have recently been several reports regarding reference genes for molecular research on insects, including bumblebee, Metopolophium dirhodum  (Hemiptera: Aphididae) is one of the most major aphid pests affecting winter wheat and other cereals worldwide31. Additionally, M. dirhodum, which was first detected in the 1970s, originated in the Holarctic and was subsequently introduced to South America and other regions33. The M. dirhodum nymphs and adults damage cereals by directly feeding on plants, which may result in grain yield losses of 27–30%34. Moreover, they damage crops by transmitting several viruses, especially the barley yellow dwarf virus35. This aphid has most often been detected in semi-arid regions in South America, South Africa, Australia, and New Zealand, where it damages cereals, including wheat, barley, rye, and oat. A previous study revealed that M. dirhodum is the most abundant aphid species on cereals in the continental climate of central Europe33. With the technical advances occurring in the post-genomic era, researchers may soon have additional options for studying M. dirhodum at the molecular level, which may contribute to the development of improved control measures. Thus, identifying suitable reference genes is important for analyzing the expression of functional genes and for evaluating the efficiency of target gene silencing via RNA interference.M. dirhodum. Specifically, we analyzed the following 10 candidate genes: Actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), NADH dehydrogenase (NADH), arginine kinase (AK), succinate dehydrogenase B (SDHB), ribosomal protein L8 (RPL8), 18S ribosomal RNA (18S), elongation factor 1α (EF1A), ribosomal protein L4 (RPL4), and heat shock protein 68 (HSP68). The effects of the following factors on reference gene expression were evaluated: geographic population, developmental stage, body part, wing dimorphism, temperature, starvation stress, and exposure to an insecticide or antibiotic. The results indicate that the best reference genes for analyzing M. dirhodum gene expression vary among conditions.The objective of this study was to identify and evaluate a suite of experimental condition-specific reference genes to normalize target gene expression in M. dirhodum sample sets, mRNA levels were measured for all genes. The gene expression levels varied considerably between Ct values of 12.70 (18S) and 30.88 (GAPDH)  and GAPDH , respectively. The least variable expression among all samples was observed for Actin  and RPL8 (21.10 ± 0.35). In contrast, HSP68 (24.82 ± 1.86) exhibited the most variable expression in all the tested samples.To evaluate the expression profiles of the selected candidate genes in all SDHB, RPL4, and RPL8 were identified as the most stable genes, whereas HSP68 and GAPDH were the least stable genes  was highly consistent among the four methods. Specifically, es Table . The RefP68 Fig. . On the P68 Fig. . MoreoveGAPDH and Actin as the most stable genes. In contrast, the most stable genes were RPL8 and RPL4 according to BestKeeper and Actin and RPL8 according to GeNorm. Regardless of the method, HSP68 was identified as the least stable gene  of SDHB, NADH, 18S, Actin, GAPDH, EF1A, AK, RPL8, RPL4, and HSP68  and Actin (26.79 ± 0.42) had the least variable expression levels, whereas HSP68 (24.82 ± 1.86) produced the most variable expression levels among the examined candidate reference genes  , which are the principal components of ribosomes, are one of the most highly conserved proteins in all life forms. Earlier research proved that RP-encoding genes are among the most stably expressed reference genes, and have been widely used to normalize gene expression levels in insect molecular investigations during the past 10 yearsActin, which encodes a major structural protein, is important for cell secretion, motility, cytoplasm flow, and cytoskeleton maintenance. Moreover, Actin is expressed at various levels in many cell types, and is considered the ideal reference gene for qRT-PCR, which may explain its frequent use26. For example, it has been used to study the effects of diet on B. odoriphaga gene expression50 and for investigating M. persicae gene expression in different tissues and in response to the temperature, photoperiod, and wing dimorphism26. However, in Helicoverpa armigera, Actin was revealed to be the least stable reference gene following temperature and photoperiod treatments51. In our study, Actin was identified as one of the most stable reference genes for analyzing developmental stages, temperature effects, and wing dimorphism 68. In the current study, HSP68 was the least stable gene for all conditions , China  in 2018. Other colonies were collected in Langfang (Hebei), China  and Guiyang (Guizhou), China  in 2018. The alive adults were collected in wheat leaves of different plants of these geographic locations and were taken back to the lab to establish population. All the wheat aphid populations were reared on Lunxuan 987 wheat seedlings in a thermostatic chamber maintained at 20 ± 2 °C and 60% relative humidity, with a 16-h light:8-h dark cycle.Our original Insects collected in Yinchuan (Ningxia), Langfang (Hebei), and Guiyang (Guizhou) in 2018 were examined to assess the effects of geography on gene expression. These three locations are separated by more than 1000 km. For each geographic population, three samples of 20 adults were selected, flash frozen in liquid nitrogen, and stored at − 80 °C until total RNA extraction.M. dirhodum samples of about 30 first-instar nymphs, 30 second-instar nymphs, 20 third-instar nymphs, 20 fourth-instar nymphs, and 20 adults were collected, flash frozen in liquid nitrogen, and stored at − 80 °C until total RNA extraction.Three M. dirhodum adults. These body parts as well as whole adult bodies were stored as described earlier.We used a dissection needle and a tweezer to separate the head, thorax, and abdomen from wingless M. dirhodum adults were collected, flash frozen in liquid nitrogen, and stored at − 80 °C until total RNA extraction.Three samples of 20 winged and wingless M. dirhodum were divided into five groups for a 24-h exposure to one of the following five temperatures: 4, 10, 15, 20, and 25 °C. For each temperature, three samples of 20 adults were collected, flash frozen in liquid nitrogen, and stored at − 80 °C until total RNA extraction. None of the temperature treatments were lethal to the aphids.Potted wheat seedlings infested with Adult aphids were placed on moistened filter paper in a Petri dish (9 cm diameter) with no food for a 32-h incubation in a thermostatic chamber . The control (satiated) group comprised aphids able to feed on wheat seedlings in the same conditions. For the control and treatment groups, three samples of 20 adults were collected, flash frozen in liquid nitrogen, and stored at − 80 °C until total RNA extraction. The mortality rate among the starved aphids was approximately 10%.M. dirhodum subjected to one of the following three insecticide treatments: imidacloprid (9.87 mg/L), thiamethoxam (122.00 mg/L), and beta-cypermethrin (17.28 mg/L). These concentrations were selected because a bioassay indicated they are 30% to the mortality of the population (LC30)  to produce five concentrations. Water (containing 0.1% Tween-80) was used as a control solution. Wheat leaves with M. dirhodum were immersed in the prepared solutions for 3–5 s and then placed on moistened filter paper in a Petri dish (9 cm diameter). The samples were incubated for 24 h at 20 ± 2 °C and 60% relative humidity, with a 16-h light:8-h dark cycle. For each concentration, the mortality rate based on three replicates of 30 aphids was calculated. Additionally, for the control and treatment groups, three samples of 20 adults were collected, flash frozen in liquid nitrogen, and stored at − 80 °C until total RNA extraction.The effects of insecticides on the stability of candidate reference genes were assessed in 0) Table . Aphids M. dirhodum adults were fed a 30% sucrose solution containing 50 µg/mL rifampicin or an antibiotic-free sucrose solution (control) (25 aphids per feeder) for 48 h72. For the control and treatment groups, three samples of 20 adults were collected, flash frozen in liquid nitrogen, and stored at − 80 °C until total RNA extraction.The Total RNA was extracted with Trizol according to the protocol for the TRNzol Universal Reagent . The ratio of the absorbance at 260 and 280 nm was 1.981–2.121, indicating the extracted RNA was pure. Next, 1 μg RNA was used as the template to synthesize first-strand cDNA with Oligo dT primers using the FastKing gDNA Dispelling RT SuperMix (Tiangen) following the manufacturer-recommended protocol. The synthesized cDNA was stored at − 20 °C.EF1A and 18S , 1 μL reverse primer (100 μM), 1 μL cDNA working solution, and 9.5 μL RNase-Free ddH20. The PCR program was as follows: 95 °C for 5 min; 40 cycles of 95 °C for 30 s and 60 °C for 30 s. For each treatment, standard curves were produced based on a fivefold dilution series of cDNA as a template according to the linear regression model. The fixed threshold in this study was set to 500 to obtain all the threshold cycle (Ct) values of tested candidate reference genes. The qRT-PCR analyses were completed with three biological replicates and three technical replicates.A qRT-PCR assay was completed with the Talent qPCR PreMix  and the CFX Connect Real-Time system . Details regarding the primers for 8S Table  have bee40, NormFinder73, and BestKeeper74 algorithms and the comparative delta Ct method75. Finally, we compared and ranked the tested candidate reference genes with the web-based RefFinder analytical tool (https://www.heartcure.com.au/for-researchers).The stability of the 10 candidate reference housekeeping genes was evaluated with the geNormSupplementary Table.


Opuntia xoconostle F.A.C. Weber in Diguet cv. Cuaresmeño (XC) and Opuntia matudae Scheinvar cv. Rosa (XR). The samples were frozen , lyophilized , and homogenized (size particle 500 μm) to get the xoconostle powder. Both cultivars (XC and XR) had a high content of carbohydrates characterized by soluble sugars (9.8 ± 0.7 and 29.9 ± 0.5 g/100 g dm) and dietary fiber (30.8 ± 0.7 and 36.8 ± 0.9 g/100 g dm), as well as lower proportions of organic acids, mainly citric acid (18.8 ± 0.0 and 13.6 ± 0.0 mg/100 g dm). These samples also had a high content of phenolic compounds (1580.3 ± 33.1 and 1068.5 ± 70.8 mg GAE/100 g dm), vitamin C (723.1 ± 16 and 320.2 ± 7.5 mg/100 g dm), and antioxidant activity ABTS·+ and DPPH· (between 1348.1 ± 74.0 and 3318.7 ± 178.8 µmol TE/100 g dm). Since xoconostle samples had a high content of dietary fiber, they were characterized by the capacity of water retention (water holding capacity 6.00 ± 0.1 and 5.5 ± 0.2 g H2O/g dm) and gel formation (swelling 5.2 ± 0.0 and 5.5 ± 0.0 g H2O/g dm), related with the retention of lipids and glucose in the food matrix similar to other foods. XR was characterized by a higher amount of dietary fiber, sugars and organic acids, while XC had higher phenols content and antioxidant properties, with higher values of functional properties. Then, our data suggest that both xoconostle cultivars in powder can be used as a functional ingredient for its fiber content and antioxidant properties, contributing with sensorial aspects as flavor and color. Therefore, these highly valued products can be used in the pharmaceutical and food industries.The objective of this study is to evaluate the nutritional composition, antioxidant properties, and functional characteristics of two cultivars of xoconostle  Opuntia embraces about 1500 species of cactus [Opuntia generally produces tender young part of the cactus stem, known as cladodes, frequently consumed as a vegetable in salads, or fruits named cactus pear, characterized by an abundant sweet pulp. The fruits of the species, commonly known as xoconostles, stand out by its acidic taste [Opuntia spp. cladodes and cactus pear fruits, perfect candidates for the production of health-promoting food and supplements.The genus f cactus , and it ic taste . Generalic taste , while fic taste ,7,8. TheThe xoconostle fruit grows from cactus paddles, and it is produced mainly in arid and semi-arid zones of Mexico, characterized by its hypocholesterolemic, hypoglycemic, and antihyperlipidemic properties ,2. Some Therefore, the objective of the present study is the evaluation of two cultivars of whole xoconostle  in powder as a potential source of dietary fiber and antioxidants, so that these fruits can be used as functional foods or as ingredients for nutritional supplements in the food or pharmaceutical industry.Opuntia xoconostle F.A.C. Weber in Diguet cv. Cuaresmeño (XC) and Opuntia matudae Scheinvar cv. Rosa (XR) (Two varieties of xoconostle were selected for this study: osa (XR) . SamplesThen, the fruits were divided into three batches and were washed, sliced vertically (0.5 cm thickness) and frozen to −32 °C during 48 h and lyophilized at 96 h, −55 ± 1 °C with a vacuum of 0.040 Mbar, using a freeze dryer . Samples were milled  and sieved to a particle size of 500 μm. The powder was used for further analysis. The expression of results is as dry matter (dm).Analyses of moisture (Method 925.09), total protein (Method 950.48), fat (Method 983.23), and ash (Method 930.05) were carried out by the official methods described by the Association of Official Analytical Chemists .g, and the supernatants were pooled and filtered. The resultant extract was reduced in volume by using a rotary vacuum evaporator  set to 70 °C to evaporate the ethanol. The concentrate was brought up to 5 mL with distilled water. Then, the samples were passed through a previously washed (5 mL of methanol followed by 5 mL of water) Sep-Pak C18 cartridge . Two milliliters of filtrate were mixed with 8 mL of acetonitrile to yield a total volume of 10 mL. Samples were filtered through a 0.45 μm Millipore membrane  before injection  into the HPLC. The HPLC was equipped with a PU II isocratic pumping system , a Rheodyne valve, and a differential refractometer R401 detector (Jasco). The chromatographic column used was a Luna 5l NH2 100 R . The mobile phase was acetonitrile:water (80:20). Operating conditions were a flow rate of 0.9 mL/min and ambient temperature. All chromatograms were processed using Cromanec XP software (Micronec). The resultant peak areas in the chromatograms were plotted against concentrations obtained from standards of D(+)-sucrose, D(−)-fructose, and D(+)-glucose in a concentration range of 0.1–2 mg/mL.Soluble sugars were determined by high performance liquid chromatography (HPLC), according to Sanchez-Mata et al. . Five huSoluble (SDF) and insoluble (IDF) dietary fiber were determined according to AOAC , an enzyg, 15 min; Thermo Scientific, Sorval RC-5, Waltham). The supernatant was removed and pooled to 25 mL with metaphosphoric acid (4.5%), these extracts were filtered through filter paper and then through a PVDF filter of 0.45 μm for injection on HPLC.The content of individual organic acids was quantified by high performance liquid chromatography (HPLC), based on Sánchez-Mata et al. . Five hu2SO4 (pH = 2.6) to determine organic acids. For the identification of ascorbic acid, the flow rate was 0.9 min/mL and 245 nm was set as the wavelength detection.An aliquot of extracts was subjected to reduction with L-cysteine to transform the dehydroascorbic acid (DHAA) in ascorbic acid (AA) and analyze the total vitamin C content. The instrument was a liquid chromatographer  equipped with an isocratic pump (PU II), an automatic injector . The column used was a Sphereclone ODS 250 × 4.60, 5 μm (Phenomenex), and a UV–visible detector (Thermo Separation Spectra Series UV100). Chromatograms were processed using Cromanec XP software (Micronec). The conditions for the equipment were flow rate 0.4 mL/min at 215 nm and mobile phase of 1.8 mM HThe organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm with commercial standards .The AA was quantified by comparison of the area of their peaks recorded at 245 nm with calibration curves obtained from the commercial standard of AA. The oxidized form of vitamin C (DHAA) was calculated by the difference between total vitamin C and AA.2O , stirring during 30 min, and centrifugation . Then, the supernatant was transferred to a flask of 25 mL, and the residue was re-extracted with 10 mL of acetone/H2O . Both supernatants were combined, and the flask was graduated to 50 mL with MeOH/H2O . The supernatants (methanolic and acetonic) were recovered, combined, and used to determine the total phenolic content and antioxidant capacity.The extraction of XC and XR were carried out by a modification of the method of Pérez-Jiménez et al. . This mev/v) and sodium carbonate (75 g/L). After adding 0.4 mL of sodium carbonate, samples were allowed to stand for 30 min at room temperature, and the absorbance reading was performed at 765 nm. Gallic acid was used as a reference standard, and the results were expressed as milligrams of gallic acid equivalents per 100 g of dried matter (mg GAE/100 g dm).Total phenolic content was estimated based on the Folin–Ciocalteau procedure . An aliqThe procedure used was based on the modification reported by Maieves et al. . BrieflyIn order to optimize the TPC result in powdered fruits, a new methodology was employed using Fast Blue BB (FBBB) reagent, which reacts more specifically with phenolic compounds, along with direct QUENCHER . The comm/v) followed by addition of 0.4 mL of 5% NaOH (m/v), prepared with distilled water, and 4 mL of distilled water. After 1 h of incubation in an orbital shaker, the tubes were centrifuged at 5016× g for 25 min. Supernatants were filtered by gravity, and the absorbance was measured in each centrifuge tube at 420 nm in a Lambda 25 UV–vis spectrophotometer (Perkin Elmer). Furthermore, a control containing only the sample (4.0 ± 0.5 mg) with 4.8 mL of water was also prepared, with the aim of measuring the natural non-phenolic interfering compounds in the medium. Results were expressed as milligrams of gallic acid equivalents per 100 g of dry matter (mg GAE/100 g dm) by means of a dose-response curve for different quantities of the standard (0–155 mg/L).Briefly, 4.0 ± 0.5 mg sample were weighed in centrifuge tubes, with a subsequent addition of FBBB reagent  was produced by reacting 7 mmol/L ABTS stock solution with 2.45 mmol/L potassium persulfate under dark conditions and room temperature for 16 h before use. The ABTS solution was diluted with deionized water to an absorbance of 0.70 ± 0.10 at 754 nm. Then, 20 µL of the extract of the sample was added to 980 µL of diluted ABTS solution; absorbance readings (754 nm) were taken in a microplate reader  after incubation for 7 min at room temperature. The antioxidant capacity was expressed as micromol of trolox equivalents per 100 g of dried matter (µmol TE/100 g dm).Antiradical capacity was measured according to Kuskoski et al. . BrieflyThe antiradical activity was measured with DPPH· radical as described by Morales and Jiménez-Pérez . An etha−1 (b/a) and chroma (C = (a∗2 + b∗2)1/2) parameters [Color was measured using a Hunter Lab colorimeter , using the D65 illuminant with an angle of observation of 10°. Color was recorded using the CIE–L∗a∗b∗ values, where L∗ indicates lightness , a∗ is the axis of chromaticity between green (−) to red (+), and b∗ the axis between blue (−) to yellow (+). Numerical values of L∗, a∗ and b∗ were used to obtain hue angle (h°) (h° = tgrameters .Water holding capacity (WHC), swelling (S), and oil holding capacity (OHC) were determined following the corresponding methodology for each determination according to Valencia and Román g, 15 min) and the pellet was separated from the supernatant. Then, glucose content in the supernatant was determined by the DNS  colorimetric method [To determine the glucose retention index (GRI), the modified methodology of Ou et al.  was applc method . The difp < 0.05, p < 0.01, and p < 0.0001 to establish the differences between the two study samples. For this analysis, the statistical software Software GraphPad Prism version 5.03 was used.The results obtained were expressed as the mean ± standard deviation of at least three determinations. Data were analyzed using analysis of variance (ANOVA) with a significance level The Cuaresmeño cultivar (XC) samples were characterized by a smaller size, weight (77.6 g), and polar diameter (5.8 cm), but with a larger equatorial diameter (4.9 cm) compared with Rosa cultivar (XR) , with statistically significant differences in the values obtained for the polar and equatorial diameters of each variety . ParametThe powder sample of whole fruits studied had a lower content of soluble sugars than xoconostle endocarp reported in other studies ,11. It cGlucose was the major soluble sugar in both samples with 6.8 ± 0.2 and 15.6 ± 0.2 g/100 g dm , followed by fructose and sucrose. The same tendency was reported in the endocarp in previous studies of the same cultivars . Fructosp < 0.01), and both were characterized by a higher amount of insoluble, than soluble fiber. The results of TDF for XC and XR samples were higher than those reported by Guzmán-Maldonado et al. [The XC fruits had a lower content of TDF compared to XR, , these samples can be considered as “source of dietary fiber” according to the classification established by Regulation of the European Parliament and Council (>3 g/100 g fm) . The recThe XC powder presented a higher content of organic acids , compared to the XR powder, that had 17.0 ± 0.0 mg/100 g dm , similarp < 0.0001) was found in the total content of this vitamin, evidenced by a higher content of total vitamin C (86.8 mg/100 g fm or 723.1 ± 16.0 mg/100 g dm) in XC fruit powder, compared to the XR fruits (30.9 mg/100 g fm or 320.2 ± 7.5 mg/100 g dm).With the methodology applied, it was possible to quantify the two active forms of vitamin C: ascorbic acid (AA) and dehydroascorbic acid (DHAA). According to the results obtained, a significant difference , and Medina [Fast Blue BB (FBBB) is a method developed by Medina , and it d Medina  in dry bp < 0.01) since the reaction between polyphenols and FBBB is the same, only varying the extraction methodology.Among the different methodologies used, the highest values obtained for both samples corresponded to the Q-FBBB method. It should be noted that the Quencher protocol places in direct contact the solid material of the sample and the reagent solutions, allowing the phenols to bind to the insoluble matter by taking advantage of surface reactions occurring at the solid–liquid interface. This approach avoids time-consuming solvent extraction steps of the classical protocols  and alloThe ratio calculated between different methods of evaluation of total polyphenols FBBB/Folin–Ciocalteu, was 0.9 to XC and 1.0 to XR. The lower ratio in XC suggests the presence of higher levels of non-phenolic reducing constituents, for example, ascorbic acid, erroneously measured with the Folin–Ciocalteu method as “total phenolic” . This faRelative Antioxidant Capacity Index (RACI) has been proposed by Petrović et al.  and Lópe+  and DPPH·  of the studied cultivars were higher and compared with other varieties of xoconostle [The values obtained for the antioxidant activity determined by ABTS·conostle , and simconostle . These dconostle .In addition, the fruits studied had similar values than those reported for dehydrated fruit such as apple, pear, papaya, mango, and blackberry (1244 to 3580 μmol TE/100 g dm) ,45.p < 0.0001). Therefore, a positive correlation of the antioxidant capacity with total phenols was found , although other compounds play an important role in antioxidant activity, i.e., ascorbic acid.As can be seen, the XC powder presented a higher content of total phenols measured by different methodologies, and therefore antioxidant activity was higher  of 5.2 ± 0.0 and 5.5 ± 0.0 g H2O/g dm, and an oil holding capacity (OHC) of 6.1 ± 0.3 and 3.4 ± 0.2 g of oil/g dm for XC and XR, respectively. The values obtained for WHC in the cultivars of the present study were similar to the artichoke flour evaluated by López et al. [2O/g dm) [2O/g dm, while OHC of both cultivars were higher in comparison with the study previously mentioned [This is the first report on the functional properties evaluated in xoconostle fruits powder. The powder samples analyzed presented significant differences between cultivars on these properties . The powz et al.  and applz et al.  but high2O/g dm) . Swellin2O/g dm)  in orang2O/g dm) , with vaentioned ,51.A series of different concentrations of glucose was used to evaluate the glucose retention capacity of the sample characterized by a high content of fiber. The results showed that the samples of powdered xoconostle could bind glucose in solutions of different concentrations, and, therefore, it could be suggested that available glucose in the small intestine could be kept at a lower concentration by the presence of dietary fibers . This beFinally, given the high nutritional potential of freeze-dried xoconostle fruits, as it has been explored for other food ingredients , furtherThe whole xoconostle fruits (cv. Cuaresmeño and Rosa), as a powder, without separate peel, and seeds (these parts considered as waste), can be used as a functional ingredient that provides dietary fiber, antioxidants, soluble sugars, and organic acids with great importance in the pharmaceutical and food industry.


Phenols, terpenoids, and alkaloids were produced by Spirulinaplatensis, Isochrysisgalbana, and Tetraselmissuecica, whereas tocopherols and carotenoids were produced by the four microalgae, except for phycocyanin and allophycocyanin, which were only produced by S. platensis and Porphyridiumcruentum. The findings demonstrate that the continuous cultivation of microalgae in photobioreactors is a convenient method of efficiently producing antioxidants.Microalgae, including cyanobacteria, represent a valuable source of natural compounds that have remarkable bioactive properties. Each microalga species produces a mixture of antioxidants with different amounts of each compound. Three aspects are important in the production of bioactive compounds: the microalga species, the medium composition including light supplied and the photobioreactor design, and operation characteristics. In this study, the antioxidant content and productivity performance of four microalgae were assessed in batch and continuous cultures. Biomass productivity by the four microalgae was substantially enhanced under continuous cultivation by 5.9 to 6.3 times in comparison with batch cultures. The energetic yield, under the experimental conditions studied, ranged from 0.03 to 0.041 g biomass kJ Microalgae are a promising group of microorganisms that produce novel compounds, known as secondary metabolites. Value-added microalgal products are associated with the health and cosmetics industries, unrestricted products produced for medical care, and the aquaculture industry ,2. Cure The microalgae are primary marine producers consumed by many other marine organisms throughout the food chain. Recent studies showed that some species of algae contain large amounts of antioxidants, such as vitamin E, pigments, and phenolic compounds. Natural antioxidants found in algae may play important roles against various diseases and aging processes by protecting the cells against oxidative damage, including DNA and protein oxidative damage, through high scavenging activity ,6. The o−1. Phycocyanin is a natural product with important productive and bioactive advantages for food quality and in the pharmaceutical and cosmetic industries. The use of PC has been recently reported as a natural colorant and an important antioxidant protector. The health and nutraceutical effects of PC include neuroprotective, anti-inflammatory, anticancer, and hepatoprotective properties, which are properties linked to the oxidative mechanism in the photosynthesis system in vivo [−1 d−1 and a CO2 consumption of 1.58 g L−1 d−1 with Spirulina platensis [Phycobiliproteins (PBPs) are photosynthetic pigments found in cyanobacteria, rhodophytes, cryophytes, and glaucophytes. PBPs include phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE). PC is a fluorescent protein with a prosthetic group (bilins) linked to cysteine residues and a water-soluble compound, with a molecular weight of 586.7 g mol in vivo . Santiag in vivo . The che in vivo . Other mlatensis .Two kinds of photobioreactors exist that produce microalgae biomass: open and closed photobioreactor designs. The dynamics of the cultures allow them to be produced either in batches or continuously, working under natural or artificial light irradiance. However, open systems have two disadvantages: contamination with bacteria and light supply. Additionally, the general cell density is low and only few microalgae species are suitable for this production mode due to contamination. Closed systems are an alternative to producing highly valuable compounds such as antioxidants. The control of all operational parameters is more easily accomplished in closed systems, but costs are high. One alternative to enhancing biomass density and production is operating photobioreactors during continuous production. One difficulty to be overcome is the low growth rate of microalgae that may cause complications in achieving continuous cultivation before washout of the cells . In the −1 for Spirulina platensis, Isochrysis galbana, Tetraselmis suecica, and Porphyridium cruentum, respectively. Biomass concentration and biomass productivity values are summarized in −1 for S. platensis, I. galbana, T. suecica, and P. cruentum, respectively . The biomass concentrations of the four microalgae increased as light intensity increased in continuous culture .The batch cultures reached maximum cell concentrations over nine days of cultivation; maximum cell concentration indicates the change from batch to continuous cultivation. The dilution rate, which corresponds to the growth rate in continuous culture, was fixed at 0.6 d culture . The bioectively , in comp−1 d−1), where X is biomass (g), V is working volume (L−1), and D is dilution rate (d−1)) obtained in terms of total light supplied (I0A (kJ d−1) energy supplied from light to a surface area per unit of time) is shown in −1 in batch culture was 0.34, 0.24, 0.2, and 0.16 g biomass L−1 d−1  of 1.04, 0.95, 0.83, and 0.75, respectively .Biomass productivity , except for alkaloids between I. galbana and T. suecica, where no significant differences were observed. Phenol, alkaloids, and terpenoids were more abundant in S. platensis than the others; these compounds accumulated during growth.Total phenols, alkaloids, and terpenoids content were measured in tivation . All comS. platensis was four times that of I. galbana and 1.75 times that of T. suecica , carotenoids, superoxide dismutase (SOD), and phycobiliproteins were recorded during batch production in the four microalgae . The totcruentum , which iS. platensis and P. cruentum showed no significant differences. Carotenoids, PC, APC, and SOD productivities were statistically different in all microalgae.The productivities of antioxidants in microalgae are shown in S. platensis, I. galbana, and T. suecica at different light intensities. Total phenols, alkaloids, and terpenoids concentrations were significantly different among the three microalgae  and allophycocyanin (APC) were found only in ntensity . PC concS. platensis was 7.0 times that by P. cruentum; APC production of S. platensis was 8.9 times that of P. cruemtum , appearcarotene . AnotherThe microalgae studied in this work exhibit reliance on antioxidant production on the growth conditions; however, strain species is an important factor. Antioxidant accumulation occurs along with cell growth, playing important roles in microalgal cells.−l d−1 under a light intensity of 700 μmol m−2 s−1 (32.95 × 10−3 kJ cm−2 h−1) [−3 kJ cm−2 h−1 (320 μmol m−2 s−1) light intensity used in the present work. Here, the biomass and PC productivities were 2 and 0.4 g L−1 d−1, respectively, which are 2.7 times the biomass and 3.2 times the PC productivity at 320 mmol m−2 s−1 compared to the values reported by Chen et al. [−2 s−1. Although PC content in S. platensis was 235 PC mg g−1 biomass at 132 μmol photons m−2 s−1 and 198 PC mg g−1 biomass produced at 320 μmol m−2 s−1, PC productivity at 320 μmol photons m−2 s−1 was 1.7 times the PC produced at 132 μmol m−2 s−1. Zheng et al. [−2 s−1 in batch culture, obtained 0.39 g L−1 d−1 biomass productivity for S. platensis [Leptolyngbya sp. to produce PC in continuous culture at 80 μmol photons m−2 s−1. Under these conditions, 86 mg of PC g−1 biomass was produced with a productivity of 78.8 mg L−1 d−1 [Phycocyanin has different biotechnological applications in cosmetics, food, and feed industries, analytical biochemistry, and therapeutic applications . Zheng em−2 h−1) , which i L−1 d−1 .Porphyridium cruentum produced another promising compound, sulfate-polysaccharide, which has either anti-inflammatory or antiallergic properties. α-tocopherol is the most abundant with the highest antioxidant activity in terrestrial plants; it functions in the electron transport reactions and cell membrane stabilization functions [−1 biomass of α- and γ-tocopherol, respectively, in P. cruentum [−1 biomass, respectively) in P. cruentum found in this study are in agreement with those reported by Durmaz et al. [T. suecica, which produces γ-tocopherol in considerable amounts, but also produced α-tocopherols. Spirulina maxima produces α- and γ-tocopherols [S. platensis in this study.Microalgae produce important antioxidants: alkaloids, terpenoids, phenols, tocopherols, carotenoids, phycobilins, and other pigments and enzymes, which are involved in active antioxidant treatments due to their anti-inflammatory, antibacterial, and anticancer, lipid peroxidation activities. unctions . Animalsunctions . Durmaz unctions  reportedcruentum . The amoz et al. . In addiopherols , but theP. cruentum, Rebollo et al. [−1 biomass, whereas Santiago et al. reported 4.6 mg g−1 biomass for β-carotene in T. suecica [Carotenoids and their derivative compounds bond to lipoproteins and play an important role as antioxidants during photosynthesis; they have been associated with potential protection against lipid peroxidation, DNA and protein oxidation, and in cancer prevention . Photosyo et al.  found 1. suecica .S. maxima, Nannochloropsis sp., T. suecica, Chaetoceros sp., Synechoccocus, and P. cruentum, with different amounts in each microalga. In T. suecica and P. cruentum, activities of 20.4 and 58 UA mg−1 protein were measured, respectively [−1 protein reported in this work, respectively. The culture conditions may affect the formation of antioxidant compounds, in particular SOD; this enzyme may be induced in response to the increase or demand to drive reactive oxygen species generated by an increase in photosynthesis activity.SOD has been found in ectively , in compS. platensis extracts showed vibrational signals related to some antioxidant molecules, such the signal at 2835 cm−1 assigned to the methylene group, at 1230 cm−1 to the phenyl group, the stretching at 1517 cm−1 assigned to the flavone phenyl ring, and the vibrational signals at 1612–1652 cm−1 assigned to the C=O ketone groups found in flavonoids.The FTIR spectra of 10, peptides (glutathione), phenols, and pigments [Microalgae extracts containing a mixture of antioxidants have the capacity to scavenge superoxide anions, hydrogen peroxides, and hydroxyl radicals . The antpigments . All thePorphyridium cruentum, Isochrysis galbana, Tetraselmis suecica, and Spirulina platensis were acquired from our own culture collection of microalgae. For I. galbana, T. suecica, and P. cruentum, medium “f” [S. platensis was grown on SOT standard medium [2 and diameter of 11.8 cm. Light  was supplied on the top of the bioreactor. The reactor was operated at a 30 ± 2 °C, agitation 200 rpm, and with an initial pH of 7.5 for Porphyridium cruentum, Isochrysis galbana, and Tetraselmis suecica, and pH 8.5 for Spirulina platensis. The illumination was supplied with white light LED lamps on the top of the reactor. The photobioreactor was covered with aluminum foil, assuming that light filled the entire volume of the culture. The bioreactor was connected to a reservoir containing fresh medium; the flow rate was controlled by a valve. The fresh medium was fed into the bioreactor and the broth exit valve was open at the same flow rate. Dilution rate (D) was achieved after at least three days without changes in the cell density in the bioreactor, then the culture was considered in continuous mode or in a steady state. Cells were harvested by centrifugation at 12,000× g for 10 min at 4 °C, then the collected biomass was kept at –20° C if not used immediately. , (I0A is defined as the energy from light supplied to a surface area per unit of time). The microalgae dium “f”  of Guilld medium . CultivaS. platensis, I. galbana, and T. suecica biomass (0.1–0.5 g) were suspended in 5.5 mL of 0.01 M phosphate buffer, pH 6.5, and 0.15M NaCl. The suspension was frozen at −20 °C, thawed and sonicated, and then left at 5 °C overnight. The suspension was centrifuged at 12,000× g for 15 min at 4 °C; the supernatant was considered crude extract. Total alkaloids was measured as follows: The crude extract was mixed with chloroform (1:1 v/v) and the chloroform phase was separated and concentrated. The concentrate was resuspended with 1 mL of chloroform, and 6.26 mL of H2SO4 (0.02N) was added; chloroform was eliminated. Some drops of methyl red indicator were added into the solution, then the mixture was titrated with NaOH (0.02 N). Each mL of NaOH (0.02 N) added was equivalent to 5.78 mg of alkaloids. Total phenols was measured as follows: 0.1 mL of the extract was mixed with 2.8 mL of deionized water, 2 mL of sodium carbonate (2%), and 0.1 mL of Folin–Ciocalteu reagent (50%). The mixture was incubated for 30 min at room temperature and the absorbance of the mixture was recorded at 750 nm in a spectrophotometer [2SO4) was added. The tubes were heated at 60 °C in a water bath for 1 h. After cooling on ice, the absorbance was measured at 473 nm. Saponin was used as the standard. For FTIR analysis, an FTIR spectrometer  was used; the sample was redissolved with a mixture of acetonitrile/methanol (70:30).otometer . Ferulic2 atmosphere, then it was sonicated. The mixture was incubated for 1 h at 70 °C in the dark, then the mixture was chilled on ice and 3.13 mL (NaCl 2%) was added and then extracted with n-hexane (1:1 v/v). The n-hexane was eliminated under N2. The concentrated extract was suspended in 0.7–1.0 mL of methanol and filtered through a 0.45 μm membrane, the extract was filtered and stored at −70 °C. The methanolic extract was used to measure tocopherols in HPLC [−1. A fluorescence detector using an excitation λ292, an emission wavelength of λ340, and a UV detector at λ340 were used to measure the tocopherols content [1% = 2500 absorption coefficient) of absorbance in a UV-Vis spectrophotometer [Biomass (0.1–0.15 g) was mixed with a 2 mL solution containing 60% ethanol, pyrogallol (6%), and 0.87 mL KOH (60%) under N in HPLC . Twenty  content . Commercotometer .g at 4 °C for 30 min. The supernatant was used for measurements or stored frozen. For SOD activity determination, the Bioxytech SOD-525 assay kit  was used. The reaction was followed at 525 nm at 30 °C for 1 min. The reaction contained 50 μg protein mL−1 of extract. Commercial SOD was used as the standard. Protein content was measured using Bio-Rad reagent  at 595 nm in a spectrophotometer [We mixed 0.05 to 0.1 g of biomass with 1 mL phosphate buffer , sonicated for 2 min, then centrifuged at 12,000× otometer .S. platensis and P. cruentum biomass (0.1–0.5 g) was suspended in 5.5 mL of potassium phosphate buffer 0.01 M, pH 6.5, and 0.15M NaCl. The suspension was frozen at −20 °C, thawed, and sonicated, then left at 5 °C overnight. The suspension was centrifuged at 12,000× g for 15 min at 4 °C. The supernatant contained phycocyanin–allophycocyanin extract. Phycocyanin and allophycocyanin concentrations were calculated according to Bennet and Bogard [−1): where OD = optical densityd Bogard  by measup < 0.05).GraphPad Prisma software version 8.4.3  was used for all analyses. For growth, the Gompertz model was used to calculate growth parameters. For biomass productivity at different light intensities, a linear regression analysis was applied and we compared the 4 strains. To compare the concentration and productivity of all strains at different light intensities, Tukey’s multiple comparison test was applied (Microalgae, including cyanobacteria, are manipulatable organisms that grow in photobioreactors, where the medium composition, light intensity, reactor design, reactor operation, metabolism, and microalga species are important factors affecting the production of compounds synthesized by the microalgae. In this work, well-known microalgae species that produce secondary metabolites were assayed for the production of different compounds that have remarkable antioxidant activity. We found that the microalgae produce a mixture of antioxidants in different amounts depending on the species, light supplied, and bioreactor operation. Phenols, alkaloids, terpenoids, tocopherols, carotenoid, PC, and APC content, including an enzyme, SOD, were produced by the microalgae tested. Continuous bioreactor operation substantially improved biomass and antioxidant productivity in comparison with batch cultivation in the same bioreactor under the same conditions, except that continuous cultivation produces the same amount of biomass and secondary metabolites every day over nine days of cultivation as achieved in batch cultures. Continuous cultivation drives the microalgal cell metabolism of biomass and metabolites synthesis in the bioreactor; the mathematical description of continuous cultivation is referred to as a steady state, where biochemical pathways constantly dynamically synthesize molecules. Antioxidants are produced naturally as a mixture. We deduced that the combination of continuous culture with the correct nutrient limitation will improve or enhance the production of a specific antioxidant, but this may limit the production of other antioxidants.


The effects on egg hatching and survival of root-knot nematodes (RKN) were also examined. The sensitivity to the product was tested by estimating mortality and survival of EPN infective juveniles (IJ) after 24-hr treatment with four different concentrations of product  compared with the control in a 96-well plate. There was no significant difference in survival of IJ with 4% product compared to the control. A twofold reduction in survival was observed when the EPN were exposed to the product at 7%. A 10.5% RKN egg hatching was observed when RKN were exposed to 3% ACS 5075 concentration compared to 100% hatching in the untreated. A significant (p < 0.05) reduction in juvenile survival was observed at 0.5% treatment, however, it dropped to 0 when 1% and above was used for M. javanica. Juveniles did not survive with all treatment concentrations in the case of M. incognita. The improvement in tomato plant growth and development were also observed when plants were treated with 1 and 3% ACS 5075. The product shows potential as a sustainable soil health alternative causing no harm to beneficial nematodes (EPN) at concentrations below 4%, while is promising against plant parasitic nematodes (PPN) and toward plant growth promotion.An organic product that consists of proprietary blend of fermentation and plant extracts with micronutrients (ACS 5075, Alltech Meloidogyne arenaria, M. incognita, M. javanica, and Meloidogyne hapla  are widely utilized as biocontrol agents in crop protection .Verticillium chlamydosporium, Hirsutella rhossiliensis, and Paecilomyces lilacinus , on the egg hatching and survival of the root-knot nematodes M. javanica and M. incognita, while studying its compatibility with EPN, and monitoring its effects on the growth of tomato seedlings ; Boyle, 2007], Steinernema feltiae (e-nema), Steinernema carpocapsae (e-nema), and Heterorhabditis bacteriophora (Andermatt Biocontrol UK).Four EPN strains of the family Galleria mellonella , sourced commercially from Live Foods Direct . Standard size petri dishes (100 × 15 mm) were inverted and the lids were lined with two sheets of Whatman filter paper. Five G. mellonella were placed onto the filter paper, the base was placed on top. Approximately 1 to 1.5 ml of a dense IJ suspension was applied to the filter paper until it was moist. The dish was lightly sealed with parafilm to prevent the filter paper drying out. Plates were then incubated in the dark, at 21°C for three to seven days until mortality occurred.Entomopathogenic nematodes were reared in Once insect mortality had occurred, nematode IJ were recovered using White traps . Freshly® Inc) dilutions were added in each well to obtain the required concentration. In case of 100% treatment concentration, the product was directly added without any dilutions and the juveniles were picked up individually by observing under the stereoscope using micropipettes. The concentrations of product tested were 0, 10, 20, 40, 60, 80, and 100% in a total volume of 200 μl in each well of the 96-well plate. The Experiments were conducted to determine the lethal concentration of the product for each EPN strain. Furthermore, concentrations within the range of 0, 2, 4, 6, 8, and 10% were used to determine the sub-lethal concentration (LC50) of the product for each EPN strain. All the experiments were conducted with 10 replicates for each treatment concentration and the untreated (control), which received water. Each set of experiments was carried out three times.All the treatment studies were conducted in 96-well plates. Freshly cultured EPN, not older than two weeks post emergence, were used for the study. EPN were kept in room temperature for 30 min before conducting the experiments to check their viability. The four EPN strains were concentrated by transferring the cultures into sterile and clean falcon tubes. The falcon tubes were kept upright, to allow the IJ to settle down at the bottom of the tube. Slowly, the supernatant was decanted to obtain approximately 10  IJ/100 μl. In all, 100 μl of water containing approximately 10 juveniles and 100 μl of the various product . The mortality of immotile juveniles was ensured by gently touching them with a needle. Another set of plates with the same concentration range was analyzed after 48-hr incubation. It was observed that the mortality level was similar to that of 24-hr incubation. Therefore, data recorded after 24-hr incubation were considered for statistical analysis. All these bioassays were repeated two more times.M. javanica and M. incognita were a kind offer from Dr E. A. Tzortzakakis, Hellenic Agricultural Organization – DEMETER, Greece. Three- to four-week-old tomato seedlings were freshly infected with five to seven egg masses/plant around the root, for rearing and increasing the nematode population for further studies. All the plants were maintained in a plant growth room at 32 ± 2°C. Organic pesticide was sprayed on the leaves and stem to avoid white flies, spider mites, and insect infection. Tomato roots were assessed for nematode galling damage approximately after a period of 8 to 12 weeks. Egg masses were excised from the roots with the help of sterile scalpel and forceps and were stored at 9°C to cease hatching before application of the actual treatment. All PPN work, in culturing and in experimentation, was carried out under strict quarantine and containment conditions.Tomato roots infected with Experiments were conducted to collect nematode inoculum by hatching juveniles from the egg masses collected from infected tomato roots. The protocol was a slightly modified version of the one followed by Five concentrations of product were prepared in 5 ml water, i.e., 0% (control), 0.5%, 1%, 2%, and 3% and placed in 35 mm sterile petri dishes. Five similarly sized egg masses were randomly picked up and placed in each petri dish containing respective treatment concentrations. Egg masses placed in sterile water were considered the control. These petri dishes were incubated in dark at room temperature and checked for hatching at regular intervals of 24 hr for three days. After three days of treatment, the suspensions in each treatment were thoroughly mixed and placed in a counting dish each, to record the number of juveniles hatched using Olympus Stereo Microscope (SZX7).Freshly hatched RKN juveniles (J2) obtained three days after incubating egg masses at room temperature were used to study the effect of product on juvenile mortality. The study was conducted in a 96-well plate. In total, 100 μl of inoculum containing approximately 10 juveniles and 100 μl of the product dilutions were added to each well to obtain the required concentrations, i.e., 0.5, 1, 2, and 3%, respectively. In total, 100 μl inoculum containing 10 juveniles with only water was used as control and 10 replicates were run for each concentration.After 24-hr incubation at 20 to 22°C, in the dark, mortality was calculated by counting the motile and immotile juveniles using Olympus Stereo Microscope (SZX7) and expressed as a percent relative to the control. The mortality of immotile juveniles was ensured by transferring them into separate petri dishes containing water without product and were observed after 24 hr. Mortality was further confirmed after that. Another set of plates with the same concentration range was analyzed after 48-hr incubation, and the mortality level was similar to that of 24-hr incubation. Therefore, data recorded after 24-hr incubation were considered for statistical analysis. All these experiments were repeated two more times.Solanum lycopersicum) seeds were germinated under environmentally controlled glasshouse conditions at 32 ± 2°C, 70 ± 10% relative humidity (RH) and natural day/night cycle. The seeds were first grown on garden soil with the following characteristics: pH-7.6 ± 0.02, electrical conductivity- 540 μs/cm, clay (%) 8 ± 0.02, silt (%) 19 ± 0.5, sand (%) 73 ± 3.9, Na 12.58 (mg Kg−1), P 2.8 (mg Kg−1), K 30.59 (mg Kg−1). The seedlings attained a height of around 6 to 10 cm before taken for treatments. They were carefully removed from the soil bed and were transplanted into the individual soil pots. Seedlings were grown in plastic pots with 1 kg soil mixture with a composition of soil: compost ratio being 1:1 ratio.Tomato  and number of leaves (NL) from the day of treatment (0th day) up to 30 days.Shoot height (SH) (cm) and number of leaves (NL) of plants were recorded. SH (cm) was measured using meter rule and NL by visual counting.a (Chla), and Chlorophyllb (Chlb)) was determined to study the effect of treatment on tomato plants  − (2.69 × OD at 645) × v/1,000 × weight of the sample taken in gm. Chlb content was calculated by: (22.9 × OD at 645) − (4.68 × OD at 663) × v/1,000 × weight of the sample taken in gm. TC content was calculated by: {/34.5) × } = mg chlorophyll g−1 FW , Chlorophyllo plants . Approxil g−1 FW .50) at which 50% mortality was monitored. The mean values obtained from the plant experiments were separated based on least significant differences (LSD) obtained from analysis of variance (ANOVA) using IBM SPSS version 23. The plant experiments were also repeated two more times.All the experiments were statistically designed and analyzed. The experiments were arranged in a completely randomized factorial design (CRD). Dose-response bioassays were performed in 96-well plates with 10 replicates for each treatment including control (only water with no product). These bioassays were repeated two more times. Mortality data were recorded manually for each treatment and were subjected to probit regression analysis using IBM SPSS version 23 . The sofThere were no statistically significant different results recorded among the experiments carried out over repeated times in any type of bioassay and the results were reproducible.S. feltiae (SB 12(1)) and S. feltiae (e-nema). In total, 100% mortality was noted at concentrations above 10%. In case of S. feltiae (SB 12(1)), survival was 100, 95.6, 50.2, 9.5 and 0%, for the control, 4, 7, 8, and 10% product concentrations, respectively (S. feltiae (e-nema)  and S. feltiae (e-nema), respectively.ACS 5075 product had a significant and strong effect on ectively , whereas(e-nema) . There wp-value < 0.05) effect on S. carpocapsae (e-nema) was observed with 100% mortality at the 20% concentration of ACS 5075. Survival was 100, 91.6, 51.4, 25.1, 19.8, and 0%, for control and 4, 7, 8, 10, and 20% of product, respectively (S. carpocapsae (e-nema) survived. The LC50 concentration of ACS 5075 was 6.79% for S. carpocapsae (e-nema).A significant  effect on H. bacteriophora was observed, 100% mortality was observed when H. bacteriophora was exposed to 40% ACS 5075. In all, 90.8, 75.9, 73.9, 63, 50.4, 16.2, and 3.1% of H. bacteriophora juveniles survived, when exposed to control, and 1, 3, 7, 15, 30, and 40% of product, respectively  followed by 1% (82.8%), 5% (77.3%), control (72.9%), and 7% (64.4%) treatment concentrations, respectively, however these values were not significantly different from control .p-value = 0.00) was observed in terms of number of leaves  in treated plants compared to ratios in the untreated (−1) and Chla/Chlb ratio (1.52). In the cases of the untreated and the 1, 5, and 7% treated plants, TC and Chla/Chlb ratios were 3.77, 3.98, 3.63, and 3.51 mg g−1 and 0.98, 1.32, 1.08, 1.18, respectively. There was a 1.35- and 1.06-fold increase in TC content in 3 and 1% treated plants when compared to the TC content in the control plants  content and the Chlntreated . Tomato l plants . Howeverlb ratio  in 5 andp-value = 0.02) effect on both egg hatching and juvenile mortality in both the species of RKN was noted. In the case of M. javanica, 9.6-fold and 31.6-fold reductions in egg hatching were observed with 0.5 and 1% ACS 5075, respectively, treated egg masses when compared to that of control after 72 hr of treatment. At concentrations above 1%, the hatching process completely ceased. Similarly, there was 100% survival in the untreated, but this declined to 10% in the 0.5% ACS 5075 treated juveniles and 0% in the case of all other concentrations above 0.5% after 24 hr of treatment  effect on both egg hatching as well as juvenile survival  of this product for several EPN strains, and tested the effectiveness of the product against PPN, at concentrations below that LC50 limit at which EPN are unaffected.In this work we have tested the efficacy of an Alltech® crop science soil health product against the RKN 50) of a proprietary blend of fermentation and plant extracts with micronutrients against all four strains of EPN have clearly shown 6.7, 6.79, and 15% of the product as the LC50 concentration for S. feltiae, S. carpocapsae, and H. bacteriophora, respectively. At concentrations 4% and below, there was no or very negligible IJ mortality and the survival percentage was similar to that of IJ under untreated conditions. From this point, product concentrations of 4% and below were considered safe for EPN. The presence of EPN in soil not only manages insect pests but also induces systemic resistance in plants, by which a plant can inherently attain resistance against PPN infestation  and in turn could enhance the overall plant growth  before emerging as male or female adults . The secM. incognita showed a very low motility and survival level. The combination of fermentation products and plant extracts may be potentially having these effects on juveniles, as a similar effect was observed while evaluating BioNem, another biological product, against M. incognita juveniles (The study on juvenile survival, as affected by the product, noted a 10-fold reduction in juvenile motility at the 0.5% treatment, and this was reduced to zero in concentrations 1% and above . Juveniluveniles .Sustainable PPN management is a huge challenge to growers worldwide due to their broad host range and vast crop losses . The majThere are several studies on various products in which these were found to cause high juvenile mortality due to the presence of antimicrobial, nematostatic, or plant/animal additive compounds . HoweverUnder the conditions of this study, we conclude that the Alltech proprietary blend of fermentation and plant extracts with micronutrients was found to be an efficient, environmentally safe, and promising approach for PPN management.