Probiotics may influence one‐carbon (C1) metabolism, neurotransmitters, liver function markers, or behavior.n = 22) received Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 (109 or 1010 colony‐forming units per day) or vehicle for 10 weeks. The controls, Flinders Resistant Line rats , only received vehicle. C1‐related metabolites were measured in plasma, urine, and different tissues. Monoamine concentrations were measured in plasma, hippocampus, and prefrontal cortex. Vehicle‐treated FSL rats had higher plasma concentrations of betaine, choline, and dimethylglycine, but lower plasma homocysteine and liver S‐adenosylmethionine (SAM) than FRLs. FSL rats receiving high‐dose probiotics had lower plasma betaine and higher liver SAM compared to vehicle‐treated FSL rats. FSLs had higher concentrations of norepinephrine, dopamine, and serotonin than FRLs across various brain regions. Probiotics decreased plasma dopamine in FSLs in a dose‐dependent manner. There were no detectable changes in liver function markers or behavior.Male adult Flinders Sensitive Line rats (model of depression, FSL; Probiotics reduced the flow of methyl groups via betaine, increased liver SAM, and decreased plasma dopamine and norepinephrine. Since these changes in methylation and catecholamine pathways are known to be involved in several diseases, future investigation of the effect of probiotics is warranted. Deficiency of methyl donors such as folate or choline may cause fatty liverMoreover, several lactic acid bacterial strainsLactobacillus helveticus R0052 and Bifidobacterium longum R0175) at two different doses can alter one‐carbon (C1) metabolism, monoamine neurotransmitters, liver function, and depressive‐like behavior in a rat model of depression.Intervention with probiotics may affect nutrient bioavailability, metabolism, or utilization in the host, as gut bacteria and host seem to compete for nutrients. We hypothesized that the exposure to probiotics (22.1n = 22) and Flinders Resistant Line  rats were obtained from the breeding colony maintained at Aarhus University, Denmark . The FSL rat is a validated genetic animal model of depression. FRL rats are used as controls to ascertain the depressive‐like phenotype of FSL rats.p = 0.004) and weighed less (p = 0.030) than FRL rats. The lower weight is part of the characteristic phenotype of the FSL animalsHealthy male adult Flinders Sensitive Line FSL, n =  and Flin2.2n = 7); probiotics at a low dose of 109 colony‐forming units per day (CFU d−1) (n = 7); or probiotics at a 10‐fold higher dose of 1010 CFU d−1 (n = 8). FRL rats (n = 8) only received vehicle  to control for the depressive‐like phenotype.FSL rats were randomly assigned to one of three groups: vehicle (L. helveticus R0052 and B. longum R0175) and excipients . The vehicle formulation was of identical taste and consisted of the same excipients without any active cultures. The probiotic and vehicle solutions were freshly prepared just before administration by dissolving the respective weight of the powder in tap water . Rats received the intervention dose dissolved in a total volume of 0.3 mL administered via syringe‐feeding The commercial probiotic formulation Probio'Stick  was used. This product contained a mixture of freeze‐dried lactic acid bacteria , hippocampi, and prefrontal cortices were quickly removed, snap‐frozen in pre‐cooled isopentane, and stored at −80 °C. Hippocampal and prefrontal cortex tissue was extracted from both hemispheres. To prevent hemispheric bias, half of the SAM, S‐adenosylhomocysteine (SAH), and monoamine measurements were conducted in the left hemisphere and half in the right one.Rats were decapitated after 10 weeks of starting the treatment. Decapitation took place between 2 and 5 p.m. over 2 consecutive days and was conducted in a random order. To minimize stress, rats were housed in an adjacent room and were brought singly into the decapitation room by an experimenter free of blood scent. Urine and fecal boli were collected 2 d before decapitation and immediately frozen at −80 °C until further analyses. After decapitation, trunk blood was collected in EDTA‐coated tubes and immediately centrifuged at 3000 × 2.42O mg−1 feces) for betaine, choline, and dimethylglycine assays, or in 1 n acetic acid for SAM and SAH assays (10 μL 1 n acetic acid mg−1 feces). After centrifugation at 10,000 × g for 10 min, the supernatants were used for the assays.Supporting Information Table 2 shows the types of tissues collected, the preservation method, and the markers measured in the respective tissues. Frozen fecal samples were thawed on ice, weighed, and extracted either in distilled water (10 μL Hn acetic acid (10 μL 1 n acetic acid mg−1 tissue) using blades. After centrifugation at 10,000 × g for 10 min, the supernatants were collected and used for SAM and SAH assays. Aliquots of frozen EDTA plasma and urine samples were thawed and used immediately for measurements of the biomarkers (Supporting Information Table 2).Snap‐frozen livers, hippocampi, and prefrontal cortices were thawed on ice. Liver tissues were washed with cold PBS to remove possible blood contaminations. Tissues were then weighed and homogenized in 1 n perchloric acid (5 μL mg−1 tissue) and centrifuged at 10,000 × g at 4 °C for 10 min. The supernatant was collected, filtered using Costar Spin‐X , and used for measurements of monoamines. For plasma samples, 20 μL perchloric acid was added to 180 μL of plasma, followed by centrifugation and supernatant collection as described above.Frozen brain tissue samples were sonicated  in cold 0.2 The concentrations of C1‐related metabolites were measured at the Department of Clinical Chemistry, Saarland University Hospital, Germany by using established methods on an Acquity Ultra Performance LC system coupled to a MicroMass Quattro Premier XE tandem quadrupole mass spectrometer (UPLC‐MS/MS) . Plasma concentrations of total homocysteine and cystathionine were measured using an in‐house gas‐chromatography mass spectrometry method. The concentrations of betaine, choline, and dimethylglycine were measured in EDTA plasma, urine, and fecal water extracts by using labelled internal standards and acetonitrile precipitation as described previously.13C5‐SAH and 2H3‐SAM).−1 for SAM and 150 nmol L−1 for SAH). All markers measured in tissues are expressed as nmol g−1 tissue. Plasma liver markers, alanine transaminase (ALT), and aspartate transaminase (AST), were measured using routine automated methods. Supporting Information Table 3 shows information on method performances such as between‐day coefficient of variations and limits of detections.Concentrations of SAM and SAH in the acidified extracts of liver, brain, and fecal samples were measured using established methods that depend on the use of labelled isotopes as internal standards (−1 (in plasma) or nmol g−1 tissue.Concentrations of serotonin (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA), dopamine, 3,4‐dihydroxyphenylacetic acid (DOPAC), and norepinephrine were measured in plasma samples and tissue extracts of hippocampus and prefrontal cortex at Translational Neuropsychiatry Unit, Aarhus University, Denmark by using established methods as described elsewhere.2.5Six weeks after starting the intervention, standard tests for cognition, anxiety, locomotion, and depression were conducted over the following 3 weeks to characterize the behavioral phenotype of the animals. All behavioral procedures were performed by the same experimenter in the active phase of the animal under dim red light between 7 a.m. and 1 p.m. Animals were habituated to the behavioral rooms 1 h before testing. Additional habituation phases to the arenas were performed before the Novel Object Recognition (duration: 1 h) and Social Interaction (duration: 20 min) test 24 h before testing. All arenas were thoroughly cleaned between each trial using 85% ethanol. Tests were scored by an observer blinded to treatment. All behavioral tests were performed according to established protocols in our facility and all animals underwent the behavioral tests in the following order: Novel Object Recognition Test,2.6Tables α = 0.05, power = 80%).The primary outcomes of the study were differences in methylation markers  between intervention groups. The secondary outcomes were differences in monoamines, liver markers, and behavioral tests. Interventions with the same outcomes were not available to predict the study power, thus the present study is explorative in nature and sample size was based on similar animal intervention studies.p < 0.05), log‐transformed variables were created. If these log‐transformed variables were then able to meet the parametric assumptions , they were kept as log‐transformed variables throughout the statistical analysis; if the assumptions were still violated upon log‐transformation, the variable was left untransformed. The following variables were log‐transformed: plasma betaine, plasma norepinephrine, plasma 5‐HIAA, hippocampal DOPAC, prefrontal cortex SAH, prefrontal cortex DOPAC, prefrontal cortex dopamine, prefrontal cortex 5‐HIAA, and urinary dimethylglycine. Log‐transformation did not affect the overall interpretation of the data, as no p‐values changed from a nonsignificant level to a significant/trend level or vice versa. Bodyweight was included as a covariate in preliminary analyses, but was never significant. Therefore, metabolite concentrations were not corrected for bodyweight.All statistics were performed using IBM SPSS 24.0 . Assumptions of normality and homogeneity of variances were tested using Shapiro‐Wilk test and Levene's test, respectively, with both strain and intervention included in the factor list. For variables violating normality or homogeneity of variances  were considered statistically significant and p‐values between 0.05 and 0.10 were considered to indicate a tendency.Results are expressed as mean ± standard deviation (SD). Two separate one‐way analysis of variance (ANOVA) tests were used to study differences in concentrations of the biomarkers between FSL and FRL rats and between the three intervention groups. A significant ANOVA was followed by post‐hoc pairwise comparison of probiotic treatment with vehicle using Dunnett's procedure to correct for multiple comparisons. 3Supporting Information Figure 2 shows the growth curve of the rats according to the strain and the intervention type. In FSL rats, weight and weight gain (from baseline to week 10 of the intervention) did not differ between intervention groups at any time point. There were no significant differences in water or food intake between the two strains or the three intervention groups (data not shown).3.1−1; F = 16.93, p = 0.001) and dimethylglycine  = 6.92, p = 0.021). Concentrations of plasma choline tended to be higher in FSL than FRL rats  = 3.90, p = 0.070). FSL rats had lower concentrations of plasma homocysteine  = 12.17, p = 0.004).FSL and FRL rats showed no differences in plasma concentrations of cystathionine Table . Compare−1 tissue; F = 4.44, p = 0.055). SAM concentrations in hippocampal extracts were approximately 2‐fold lower than in liver extracts, but did not differ between the strains. Similarly, hippocampal SAH was unchanged between FSL and FRL rats. In the prefrontal cortex, FSL rats had higher concentrations of SAM than FRLs  = 6.32, p = 0.026), but the strains did not differ in SAH or SAM/SAH ratio.Concentrations of SAH in liver tissue extracts did not differ significantly between the two strains, whereas SAM in liver extracts tended to be lower in FSL than in FRL rats  = 14.53, p = 0.002) and 200% higher urinary excretion of dimethylglycine  = 11.84, p = 0.004) than FRL rats (Supporting Information Table 4). The concentrations of betaine, choline, and dimethylglycine in stool extracts were very low, suggesting that the dietary nutrients, choline and betaine, had been utilized and dimethylglycine was not present or produced by the gut bacteria in a significant amount. The concentrations of SAM in stool extracts were remarkably high (in mmol L−1 range) but did not differ between the strains (Supporting Information Table 4). In contrast, the molar concentrations of SAH in stool extracts were very low compared with SAM, which prevented the analysis of SAH and SAM in the same run .The urinary excretion of betaine did not differ significantly between the strains. FSL rats showed 56% lower urinary excretion of choline  = 3.54, p = 0.050; post‐hoc: p = 0.042) and a lower betaine/choline ratio  = 4.00, p = 0.036; post‐hoc: p = 0.030). Concentrations of SAM in liver extracts were significantly higher in FSL rats treated with high‐dose probiotics as compared with vehicle  = 6.66, p = 0.006; post‐hoc: p = 0.017). Concentrations of SAM in hippocampal or prefrontal cortex samples did not differ significantly between intervention groups. Urinary and stool excretions of the metabolites did not show significant differences between the three interventions  = 4.41, p = 0.056)  = 14.77, p = 0.002), dopamine  = 15.81, p = 0.002), and 5‐HT  = 8.51, p = 0.012). The strains did not differ in plasma 5‐HIAA. Plasma DOPAC/dopamine and 5‐HIAA/5‐HT ratios, commonly used as indicators for dopamine and serotonin turnover, respectively, did not differ between the strains.Plasma concentrations of norepinephrine tended to be higher in FSL than FRL rats  = 3.73, p = 0.075), but they did not differ in hippocampal DOPAC, dopamine, 5‐HT, or 5‐HIAA  = 18.10, p = 0.001)  = 12.17, p < 0.001). 5‐HT concentrations in the prefrontal cortex were higher in FSLs compared to FRLs  = 12.55, p = 0.004). There were no differences in 5‐HIAA or serotonin turnover.FSL rats tended to have higher concentrations of norepinephrine in the hippocampus than FRLs  = 5.30, p = 0.015; post‐hoc: p = 0.033). Compared to vehicle, FSLs receiving low‐dose probiotics had lower plasma concentrations of dopamine  = 6.73, p = 0.006; post‐hoc: p = 0.027. Compared to vehicle, FSLs receiving high‐dose probiotics also had lower plasma concentrations of dopamine . The DOPAC/dopamine ratio was higher in the high‐dose probiotic group than in the vehicle group  = 3.73, p = 0.043; post‐hoc: p = 0.036). There were no significant differences in hippocampal or prefrontal cortex neurotransmitters between intervention groups.Differences in monoamine concentrations according to the intervention are shown in Supporting Information Table 7 provides the results of a stepwise multiple linear regression analysis to predict concentrations of monoamines (dependent variables) in FSL rats. Remarkably, 84% of the variability in plasma dopamine between FSL rats was explained by a model containing the following independent variables: treatment, plasma dimethylglycine, cystathionine, taurine, and hippocampal SAM. Different C1 metabolites were significant determinants for plasma serotonin, 5‐HIAA, and prefrontal cortex dopamine.3.5F = 3.77, p = 0.042; post‐hoc: p = 0.024).Concentrations of plasma ALT and AST did not differ between strains Table  or inter3.6F = 9.40, p = 0.009). Moreover, FSL rats were more immobile  = 13.50, p = 0.003) and swam less  = 10.13, p = 0.007) in the pre‐Forced Swim Test than FRL rats. The same differences in immobility and swimming behavior were observed in the test session 24 h later (Supporting Information Table 5). These results have been reported previously and confirm the validity of the depression model. FSL and FRL rats did not differ in nonspatial memory, spatial memory, anxiety, or social behavior. There were no significant differences between the intervention groups.Vehicle‐treated FSL rats moved a greater distance in the Open Field than FRL rats . Second, plasma dopamine was elevated in the depressed compared with the control rats, which was lowered by probiotics in a dose‐dependent manner without changes in the dopamine catabolite DOPAC. The influence of probiotics on two interrelated biochemical pathways involved in mood disorders  could be driven by interrelated or independent mechanisms as discussed below.Our study provided novel evidence for probiotics influencing two main metabolic pathways in the host. First, depressed rats had lower liver concentrations of the methyl donor SAM than control rats, which was increased by administration of 4.110 CFU d−1) reduced the metabolic dependency of the rats on betaine and choline as methyl donors, and increased liver SAM to levels comparable with control animals. Vehicle‐treated FSL rats had higher SAM concentrations in the prefrontal cortex than FRL rats, potentially indicating higher requirements for SAM. Probiotics did not affect SAM in the brain but increased SAM in the host's liver. The liver is the main SAM‐producing organ, but it remains unclear whether the increase in liver SAM is due to the rats’ own synthesis.Higher plasma concentrations of betaine, choline, and dimethylglycine in addition to lower plasma homocysteine in vehicle‐treated FSL rats compared with FRL rats suggest enhanced homocysteine remethylation and SAM production via the betaine‐homocysteine methyltransferase (BHMT) pathway . Treatment with probiotics (10−1 stool wet weight) suggests that bacteria‐driven fermentation processes in the gut could constitute a significant, yet underestimated source of SAM for the host . Alternatively, lowered methyl group flow through the BHMT pathway and increased liver SAM in animals treated with high‐dose probiotics could be mediated by saving tetrahydrobiopterin (BH4) and SAM due to lowering dopamine as discussed below.Lactic acid bacteria in fermented Cheonggukjang have been shown to produce SAM in vitro,4.2Compared to FRL rats, FSLs had higher plasma and brain  concentrations of norepinephrine. Dopamine, norepinephrine's precursor, was increased only in plasma. This distinct increase has been described previously,Bifidobacterium infantis reversed the stress‐induced decrease of norepinephrine  in the brainstem of Sprague‐Dawley rats.L. helveticus R0052 and B. longum R0175  attenuated the stress‐induced increase of plasma norepinephrine  in mice.Compared to vehicle, probiotics were associated with a dose‐dependent decline in plasma dopamine and a reduction in plasma norepinephrine (only in the low‐dose group). Previous studies reported heterogeneous effects of probiotics on catecholamines, likely due to using different animal models, tissues, and bacterial strains. l‐dopa, or enhanced degradation via COMT (SAM dependent) or via monooxygenase (MAO) to norepinephrine  could represent target organs for probiotics. Lowering of plasma dopamine could be linked to enhanced gastrointestinal motility.e Figure . Howeverl‐dopa, tyrosine, phenylalanine, or more dopamine excreted in urine. The effects on plasma dopamine were stronger than on norepinephrine and showed dose‐dependency, suggesting that dopamine is likely to be closer to an upstream pathway  where probiotics would interfere with catecholamine metabolism.Low plasma dopamine, associated with lower plasma norepinephrine in the low‐dose probiotics group only, was unlikely due to enhanced metabolism to DOPAC or norepinephrine (via MAO). Instead, dopamine lowering in the probiotic groups is likely due to less dopamine synthesis from l‐amino acid decarboxylase (AADC/B6 dependent). Inhibition of AADC by probiotics is very unlikely, because probiotics specifically lowered dopamine but not serotonin.Elevated serotonin in plasma and prefrontal cortex of FSL has been reported before.4.3Lactobacillus strains produce SAM in vitroL. helveticus is also able to produce C1 units and metabolize phenylalanine and tyrosine via transamination and dehydrogenation pathways,Elevated dopamine and serotonin in FSL rats could challenge C1 metabolism in at least two ways. High dopamine input may deplete SAM . Moreover, production of serotonin from tryptophan (via tryptophan hydroxylase) and dopamine from phenylalanine (via tyrosine and tyrosine hydroxylase) requires tetrahydrobiopterin (BH4)  or behavior . The literature is not consistent about effects of probiotics on these health outcomes. A slight decrease in plasma ALT, along with increased liver SAM after probiotic treatment, may be indicative of protective effects of probiotics on the liver. Probiotics did not significantly affect behavior in the Forced Swim Test, although mean immobility values were slightly lower in the probiotic group. The lack of changes in behavioral tests is consistent with unchanged monoamine neurotransmitters in the brain, as these outcomes often coincide.4.5The limitations of the current study deserve mentioning. First, the relatively low sample size may have limited the study power to detect some differences. Furthermore, such differences may have been attenuated by the methyl donors present in the standard diet. However, since the metabolites were independently measured in several biological matrices, and the independent comparisons of the strain and the intervention effects supported the hypothesis, it is very unlikely that the results were due to chance.Taken together, probiotic intervention was associated with higher liver SAM and attenuated metabolic flow of methyl groups from betaine to dimethylglycine via BHMT. In addition, probiotics lowered plasma concentrations of dopamine and norepinephrine, but did not influence brain monoamines. Future studies need to investigate whether the increase of SAM was due to bacterial metabolism by using the methyl donors available in the host diet. Future studies may investigate whether administration of probiotics together with selected nutrients may serve as supportive treatment for C1 metabolism–related diseases. Furthermore, the health impacts of lowering extraneuronal dopamine and norepinephrine by probiotics deserve further investigations. At this early stage, no direct extrapolation to humans can be made.5‐HIAA5‐hydroxyindoleacetic acidAADCl‐amino acid decarboxylasearomatic ALTalanine transaminaseASTaspartate transaminaseBH4tetrahydrobiopterinBHMTbetaine‐homocysteine methyltransferaseCFUcolony‐forming unitsCOMTcatechol‐O‐methyltransferaseDOPAC3,4‐dihydroxyphenylacetic acidFRLFlinders Resistant LineFSLFlinders Sensitive LineMAOmonoaminoxidasePNMTN‐methyltransferasephenylethanolamine SAHS‐adenosylhomocysteineSAMS‐adenosylmethionineThe authors declare no conflict of interest.Supporting Information Figure 1. Experimental design. Animals were allowed to acclimatize for 2 weeks (week −2 to week 0) before the start of the intervention (probiotics/vehicle) from week 0 to week 10. Behavioral tests were conducted from week 6 to week 9. After 10 weeks of intervention, animals were euthanized and tissue was collected. CFU, colony‐forming units; FRL, Flinders Resistant Line; FSL, Flinders Sensitive Line. * Vehicle treatment consisted of xylitol, maize‐derived maltodextrin, plum flavor, and malic acid; low dose = 109 CFU d−1; high dose = 1010 CFU d−1.Supplemental Figure 2. Weekly growth curves of FSL and FRL rats assigned to intervention groups from baseline to week 10 and weight increase between baseline and week 10. Vehicle‐treated FSL rats weighed significantly less than FRL rats at baseline (p = 0.030). The weight differences were maintained at all weeks until the end of the study. The increase in weight between baseline and week 10 did not differ significantly between vehicle‐treated FSL and FRL rats (p = 0.187), or among FSL rats between the treatment arms (p = 0.793). Vehicle treatment consisted of xylitol, maize‐derived maltodextrin, plum flavor, and malic acid; low dose = 109 CFU/d; high dose = 1010 CFU/d.Supplemental Figure 3. Summary of the proposed effect of probiotics on host C1‐metabolism. The liver is the main SAM‐synthesizing organ. Probiotics that are able to use and/or synthesize methyl donors might be a source of SAM that could support the host's needs. 5‐MTHF, 5‐methyltetrahydrofolate; BHMT, betaine‐homocysteine methyl transferase; SAM, S‐adenosylmethionine; SAH, S‐adenosylhomocysteine; THF, tetrahydrofolate.Click here for additional data file.1. Main nutritional values and composition of nutrients directly related to one‐carbon metabolism in the standard diet (Altromin 1324)SUPPLEMENTAL TABLE SUPPLEMENTAL TABLE 2. Biosamples collected from the rats; storage and extraction conditions and markers measured in each tissue matrix.SUPPLEMENTAL TABLE 3. Performance of the methods used in the present paper according to the matrix used for method validation.SUPPLEMENTAL TABLE 4. Concentrations of C1‐metabolites and related compounds excreted in urine and stool extracts from Flinders Resistant Line (FRL) rats and Flinders Sensitive Line (FSL) rats treated with vehicleSUPPLEMENTAL TABLE 5. Results of mood‐related behavioural tests performed after 6–9 weeks of intervention with probiotics or vehicle.Lactobacillus helveticus R0052 genome.SUPPLEMENTAL TABLE 6. Genes coding for enzymes related to C1‐metabolism are present in SUPPLEMENTAL TABLE 7. Stepwise multiple linear regression analysis applied to find predictors of plasma and prefrontal cortexClick here for additional data file.


Ascites’ neutrophil function tests were repeated after incubation with autologous plasma. Ascites’ neutrophils showed an impaired PA and OBA (median blood PA 98.1% (86.8–99.8) vs. ascites’ PA 50.5% (0.4–97.3), p < 0.0001; median blood OBA 98.7% (27.5–100) vs. ascites’ OBA 27.5% (0.3–96.7), p < 0.0001). Patients with non-cirrhotic ascites showed higher PA but equally suppressed OBA. Ascites’ neutrophil function could be partially restored after incubation with autologous plasma (median increase PA: 22.5% (−49.7 – +93.2), p = 0.002; OBA: 22.8% (−10.4 – +48.8), p = 0.002). Ascites’ neutrophils of patients with cirrhosis are functionally impaired, but could be partially restored after incubation with plasma. Further investigations are needed to identify the factors in ascites that are associated with neutrophils’ function.Systemic immune cell dysfunction is a typical feature of liver diseases and increases the risk of bacterial infection, especially spontaneous bacterial peritonitis. We evaluated functional properties of neutrophil granulocytes in blood and ascites of patients both with and without decompensated cirrhosis. We collected blood and ascites samples from 63 patients with cirrhosis and eight without cirrhosis. Phagocytosis activity (PA) and oxidative burst activity (OBA) were evaluated after  Patients with end-stage liver diseases typically express features of a dysfunctional immune system that are associated with a suppressed response of peripheral blood neutrophils to invading pathogens1245679101113Studies that specifically address peritoneal host defence mechanisms in decompensated cirrhosis cases are few and far between. The phagocytosis and oxidative burst capacity of peritoneal macrophages has been found to be severely impaired1416Due to this lack, we were interested in whether neutrophils in ascites of patients with decompensated cirrhosis show a higher degree of functional impairment, not only compared to their blood counterparts, but also to ascites’ neutrophils derived from patients with non-cirrhotic ascites.E. coli bacteria. Phagocytic rate and oxidative burst rate were determined as the percentage of active neutrophils in relation to the total number of viable neutrophils. Phagocytosis could be determined in 62 out of 63 blood samples and in 60 out of 63 ascites samples from patients with cirrhosis. Oxidative burst was measurable in all (63/63) blood samples and in 62 out of 63 ascites samples.Neutrophil function was determined by flow cytometry after stimulation with inactivated and opsonised The median ascites’ phagocytic rate was 50.5% (range 0.4–97.3), compared to 98.1%  in blood neutrophils. The median ascites’ oxidative burst rate was 27.5% (range 0.3–96.7), compared to 98.7%  in blood see . The ascThe median phagocytic rate of neutrophils in non-cirrhotic ascites was 83.5% (range 14.1–95.4), 33% higher than in ascites’ neutrophils of patients with cirrhosis (p = 0.038) see . The medThe blood neutrophils of patients without cirrhosis showed a median phagocytosis rate of 99% (97.2–99.8), 1.8% lower than in the cirrhosis group (p = 0.049). The median oxidative burst rate, with a median of 98.5% (62.7–100) was not different in blood neutrophils in cirrhosis  compared to patients with cirrhosis.We further assessed whether patient characteristics, as well as markers of liver disease severity, correlated with neutrophil function in ascites. Although male patients exhibited higher phagocytosis and oxidative burst activity than their female counterparts, the difference did not reach statistical significance  vs. 35.7% (range 0.4–97.3) in females, p = 0.065; median oxidative burst rates in males was 28.8% (range 0.3–96.7) vs. 9.7% (range 0.5–89.2) in females, p = 0.18). In addition, for all other parameters, including age, body weight, Child-Pugh score, model of end-stage liver disease (MELD) score, white blood cell count (WBC), C-reactive protein and medical treatment, no clear correlation with the level of ascites’ neutrophil function could be found see .The ascites’ leukocyte count showed a weak association with the oxidative burst rate  but not with the phagocytic rate  in ascites’ neutrophils see . In patiAdded to this, the ascites’ protein content showed neither an association with the phagocytic rate  nor with the oxidative burst rate  see .There was no clear correlation between functional properties of blood neutrophils and baseline parameters such as age, body weight, Child-Pugh score, serum protein and albumin . Althougex vivo via incubation with autologous plasma.We artificially modulated environmental conditions for ascites’ neutrophils ex vivo plasma incubation, with a median net increase of 22.8% (range −10.4–48.8) (median oxidative burst rate 28.8% (range 0.3–86) vs. 51.6% (range 1.6–94.7), p = 0.002)  (from 60.5% (range 4.1–94.8) to 85.7% (range 21.9–99.2), p = 0.002) see  after sa002) see , it alsoA total of five patients did not show any response to plasma incubation, but actually presented a worsening of phagocytosis (n = 2) and oxidative burst (n = 3); only one of these suffered from non-cirrhotic ascites.of the peritoneal cavity to bacterial infections. We therefore evaluated phagocytosis and oxidative burst activity in both ascites and blood neutrophils derived from patients with and without cirrhosis.Immune paralysis in general, and neutrophil dysfunction in particular, are common phenomena in chronic liver diseases and increase the risk of infectious complications192021In a significant cohort of 63 patients with decompensated cirrhosis, we showed for the first time that, when compared with peripheral blood neutrophils, ascites’ neutrophils were severely dysfunctional, showing a reduction of phagocytosis and oxidative burst rates of approximately 50% and 70% respectively. So far, only one studyIn comparison to previous reports, the functional properties of blood neutrophils in cirrhosis were found to be less depressed in our cohort, which might be, however, partly explained by a relative underrepresentation of patients with heavily impaired liver function18An intriguing finding of our study was the high degree of variability of ascites’ neutrophil function, ranging from 0.4% to 97.3% for phagocytosis and from 0.3% to 96.7% for oxidative burst. This result was in contrast to the results obtained from the blood-derived neutrophils, in which the activity rates were more homogeneous, ranging from 86.8% to 99.8% (phagocytosis) and from 27.5% to 100% (oxidative burst). This variability of ascites’ neutrophil activity could not be explained by certain patient characteristics, such as liver function, inflammatory parameters or age, although it is known that activation, migration and response to bacteria of circulating neutrophils depends on the degree of liver insufficiency, systemic inflammation and age1824ex vivo experiments, we were not able to directly measure these factors. However, certain results may indicate the presence or absence of such stimulating or inhibiting factors.We therefore hypothesised that peritoneal cavity- and/or ascite-specific factors must exist, and are responsible for the observed site-specific differences in neutrophil function. Appropriate neutrophil activation is closely regulated by a number of stimulatory, but also inhibitory, factors such as immunoglobulins, complement factors, especially C3b, cytokines  and endotoxin levels26E. coli bacteria, factors associated with the opsonic capacity of ascites fluid could not be studied in our test system. In contrast to the phagocytic rate, the leukocyte count in ascites had a weak, yet still significant, impact on the oxidative burst rate, being lower when the leucocyte count was high, thereby confirming the results of Nieto et al.et al.in vitro restores neutrophil function. Lebrun et al.Staphylococcus aureus cells. All functional neutrophil properties were significantly more favourable in samples derived from malignant ascites, as compared to those obtained from ascites of patients with cirrhosis. In addition, it was effectively demonstrated that a disequilibrium between inhibitory and stimulatory signals most likely contributes to the high variability of ascites’ neutrophil function. If the ascites of patients with cirrhosis were diluted with saline, their neutrophil function was partially restored. In contrast, when cirrhotic ascites were diluted with their counterparts derived from malignant ascites, their function was significantly improved. It has to be pointed out, however, that there was a lack of direct evidence for environmental factors affecting neutrophil functions. Our results may, however, stimulate further research that would elucidate the functional mechanisms that are potentially involved in neutrophil activation and migration within the micro-milieu of cirrhotic ascites.We were able to stratify our results according to the ascites’ protein content and leucocyte count as well as the cause of ascites (cirrhotic vs. non-cirrhotic), all of which are well-known predictors of SBP risk141622In conclusion, this study was able to show, for the first time, that neutrophil function in ascites is severely impaired. This may explain the high susceptibility to spontaneous bacterial peritonitis in cirrhotic patients. The dysfunction of ascites’ neutrophils may be partially restored after incubation with autologous plasma. The high functional variability observed in neutrophils derived from ascites suggests that ascites’ neutrophil function is dependent on both stimulatory and inhibitory factors. Further studies are needed to clarify the individual factors involved in ascites’ neutrophil activity, as these may become potentially interesting targets for SBP treatment and prevention.in vitro tests for phagocytosis and oxidative burst. Patients with ascites but without cirrhosis (n = 8) served as controls. All patients with ascites, who had been admitted to our hospital, were considered for participation in the study. In line with international clinical practice guidelinesBetween August 2014 and May 2015, ascites, fluid and corresponding blood samples were consecutively collected from 63 patients with decompensated cirrhosis at the Section of Hepatology, University Hospital Leipzig, for the purpose of evaluating the ascites’ neutrophil function by performing 3) in 11 out of 63 patients (17.5%) with cirrhosis. At baseline, the following clinical and laboratory data was collected: cause of ascites, etiology of liver cirrhosis, drug history, sex, age, liver and renal function test, platelet count, white blood cell count, serum sodium, haemoglobin, C-reactive protein, serum albumin and protein content. Ascites samples were characterised by their total leukocyte count (via automated cell counter) and their protein content was defined according to institutional standards. SBP was diagnosed in patients with cirrhosis if the ascites’ leukocyte count was elevated above 500/mm3 The patients’ characteristics are summarised in An initial fraction of 50 mL of ascitic fluid was used for routine laboratory analysis, while 40 mL of ascitic fluid was obtained for tests related to the study. Venous blood samples were collected under standard aseptic conditions, using a 0.8 mm Multifly-Safety needle  immediately after paracentesis, and decanted into heparinised tubes (18 mL) and EDTA tubes (5.7 mL). All ascites and blood samples were processed within four hours after sample collection, under pyrogen-free conditions. For antibody staining, 2 mL of ascitic fluid was poured into Eppendorf tubes (1 mL each). In preparation for flow cytometry and neutrophil function tests, the remaining 38 mL of ascitic fluid was centrifuged in a 50 mL tube at 500 × g for five minutes at room temperature. The neutrophil-containing pellet was re-suspended with 1.5 mL of ascites’ supernatant and used for the neutrophil function tests. The remaining ascites; supernatant was stored at −80 °C. The heparinised blood was processed for neutrophil function tests without prior preparation. The following steps were the same for blood and ascitic fluid (hereafter referred to as test substances).Ascites samples were collected after careful skin disinfection and under ultrasound guidance. For local anaesthesia, 5–10 mL of Xylocaine (1%) was injected, and afterwards, a 6 French paracentesis cannula  was inserted into the peritoneum. Each test substance was analysed by flow cytometry using FACS CantoII DiVa software , in order to identify neutrophils and perform neutrophil function tests see . As flowE. coli bacteria; samples were subsequently analysed by flow cytometry (see E. coli bacteria) were added to the test tubes and incubated in a shaking water bath at 37 °C for 10 minutes. The control tube remained on ice. Phagocytosis was stopped after 10 minutes with 100 μL of quenching suspension. All samples were washed twice in 3 mL of washing solution, before being centrifuged at 500 × g for 5 minutes. The supernatant was discarded. The remaining pellet was then re-suspended in 2 mL of erythrocyte lysis buffer and subsequently incubated in darkness at room temperature for 20 minutes. After centrifugation at 500 × g for 5 minutes, the supernatant containing the lysed erythrocytes was again discarded. In order to identify viable neutrophils, the pellet was re-suspended in 200 μL of DNA staining solution (propidium iodide (PI)). Flow cytometry analysis was performed within one hour. During this period, samples remained on ice in darkness.The phagocytosis test was performed using the Phagotest test kit , which contains fluorescein isothiocyanate (FITC)-labelled, opsonised and inactivated etry see . In brieE. coli bacteria and was used to quantify neutrophil oxidative burst  for the positive control were added to the respective tubes containing 100 μL of the test substance, and incubated at 37 °C in a shaking water bath for 10 minutes. Thereafter, 20 μL of the burst substrate (DHR123) was added to each tube, before they were incubated again at 37 °C in a shaking water bath for 10 minutes. The next steps, consisting of erythrocyte lysis and DNA staining, corresponded to the phagocytosis test and were described previously.Phagoburst  contains unlabelled, opsonised urst see . For samin vitro and subsequently repeated the neutrophil function tests in the last 19 patients . For this purpose, patients’ ascites’ neutrophils were incubated with autologous plasma. Heparinised blood was centrifuged at 2000 × g at room temperature for 5 minutes. 300 μL of the resulting plasma (supernatant) was mixed with 300 μL of the re-suspended ascites pellet. Following this, the phagocytosis and oxidative burst tests were performed as previously described.We hypothesised that environmental factors play a major role with respect to neutrophil function in ascitic fluid. Accordingly, we modified the environmental conditions Statistical analysis was performed using SPSS 22 software . Categorical variables were displayed as percentages or frequencies, and continuous variables as mean ± standard deviation or median and range, as appropriate. A two-sided p-value of < 0.05 was considered statistically significant. Comparison of unpaired samples was performed by Mann-Whitney U test in the case of continuous data and by Chi-square test for discrete data. Paired samples were compared by Wilcoxon signed rank test. For correlation analysis, the Spearman-Rho coefficient was calculated and a correlation coefficient of r > 0.5 was considered relevant.How to cite this article: Engelmann, C. et al. Ascites’ neutrophil function is significantly impaired in patients with decompensated cirrhosis but can be restored by autologous plasma incubation. Sci. Rep.6, 37926; doi: 10.1038/srep37926 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Motion Induced Blindness (MIB) is a well-established visual phenomenon whereby highly salient targets disappear when viewed against a moving background mask. No research has yet explored whether contracting and expanding optic flow can also trigger target disappearance. We explored MIB using mask speeds corresponding to driving at 35, 50, 65 and 80 km/h in simulated forward (expansion) and backward (contraction) motion as well as 2-D radial movement, random, and static mask motion types. Participants (n = 18) viewed MIB targets against masks with different movement types, speed, and target locations. To understand the relationship between saccades, pupil response and perceptual disappearance, we ran two additional eye-tracking experiments (n = 19). Target disappearance increased significantly with faster mask speeds and upper visual field target presentation. Simulated optic flow and 2-D radial movement caused comparable disappearance, and all moving masks caused significantly more disappearance than a static mask. Saccades could not entirely account for differences between conditions, suggesting that self-motion optic flow does cause MIB in an artificial setting. Pupil analyses implied that MIB disappearance induced by optic flow is not subjectively salient, potentially explaining why MIB is not noticed during driving. Potential implications of MIB for driving safety and Head-Up-Display (HUD) technologies are discussed. MIB is one of many bistable perceptual phenomena2 used with increasing popularity to investigate the mechanisms of perceptual organisation5. These bistable phenomena allow subjects to experience varying phenomenology while receiving physically constant visual input, thus, they can help delineate the neural correlates of consciousness by dissociating physical stimuli from conscious perception6.Motion Induced Blindness (MIB) is a visual phenomenon whereby highly salient visual targets become temporarily invisible despite their ongoing physical presence in one’s visual field, when viewed against the background of a global moving mask8. While it has been suggested that MIB may happen in the real world1, no research has yet explored MIB using parameters that approximate those experienced in real world movement. To date only two predominantly anecdotal studies have explored whether MIB may occur in the real world. Shimojo9 used a mirror ball to create a moving mask of bright spots across a room, and was able to cause the perceptual disappearance of a live person in their interactive museum display. Another demonstration of MIB in real life was reported by Inoue, Yagi and Kikuchi10, where they induced MIB by superimposing a target over the optic flow of a movie, filmed from the driver’s point of view while travelling in a car. Perceptual disappearance was induced much more often while travelling in forward motion compared to viewing the target over a still frame. Our own pilot studies have also informally replicated this finding. This evidence suggests that the optical flow experienced in forward self motion can induce MIB in situations where a stable image is projected to the retina over a moving background. In the current study we aimed to lay the groundwork toward establishing the critical parameters of potential MIB phenomena in much more natural situations, focussing on variations of mask speed, trajectory of mask movement and target location.MIB has been extensively studied under stimulus parameters designed to optimise perceptual disappearances1. Using this display, various stimulus parameters have been studied16, among which are the effect of target location21, mask speed22, and cues of mask depth23.The most frequently used display for demonstrating MIB consists of three yellow targets and a blue mask of rotating crosses, originally introduced by Bonneh and colleagues24. A more frequent disappearance of upper left targets has been replicated several times24. However, research finding a significant upper left bias generally uses the original three target triangular display1, and finds a significant bias only in relation to the lower central target24. There has not yet been a controlled comparison of targets presented in all four quadrants of the screen to determine the effects of visual quadrant on disappearance, despite evidence suggesting that upper visual field may exhibit an effect over target disappearance. The target location effect is an important feature of MIB due to its potential relationship with the neural mechanisms of attention17. Indeed, Bonneh and colleagues1 interpreted the top-left bias as the result of processing the global mask at the expense of the local target, mediated by the right-hemisphere dominance of visuospatial attention.In terms of target location, a number of studies have reported greater disappearance when a target is located in the upper left quadrant of the visual display, compared to other tested locations6. While it has been suggested that it is the temporal frequency rather than retinal speed of the mask underlying MIB25, trajectory of motion has shown to be equally influential21. As yet, no study has explored variations in the speed of masks that contract or expand in a way that occurs during self-motion.It has been shown that mask speed influences MIB, with increased speeds leading to both an increased rate and duration of target disappearance19 used a three dimensional (3-D) moving mask that mimicked the experience of real world movement and found that significantly more disappearances occurred when masks were perceived to be convex compared to concave, an effect restricted to targets presented in the left field of vision. This and related findings regarding depth ordered masks23, suggests that the 3-D interpretation of the visual scene experienced during self-motion is likely to influence MIB, prompting a need for further research on how optic flow (with its implicit depth cues) may influence target disappearances.Several studies have investigated the effects of other variations in mask composition and movement. In particular, Rosenthal and colleaguesWe aim therefore to explore whether mask properties modelled on the real world are conducive to MIB, focussing on mask speeds that would typically occur in a driving situation, and the expanding and contracting optic flow experienced in (simulated) forward and backward self-motion. We also aim to explore the effect of target location on disappearance, to investigate the previously identified upper left bias for disappearance and to test the hypothesis that MIB is mediated by the right-hemisphere dominant attentional mechanism.26. It was found that MIB produced larger pupillary responses than target re-appearance, suggesting disappearance is a more subjectively salient and surprising event to the brain29. Previous research however, has not manipulated mask type to examine if such an interpretation of pupillary response as a proxy of subjective saliency holds across different stimulus conditions. Therefore we will also investigate the saliency of target disappearance and reappearance in our stimuli, measured by phasic pupil response, as well as the overall saliency of the different movement patterns by looking at tonic  pupil dilation for each mask condition.Finally, while it is possible that MIB might be occurring in real-life settings, it has not yet been reported. A reason for this may be that the saliency of MIB phenomenon is so low that we do not notice it in everyday life. To estimate the saliency of MIB, phasic pupillary responses have recently been investigated during MIB eventsn = 18; Experiment 2: n = 19, see Method) comparing masks simulating forward (expansion) and backward (contraction) self motion with a static stimulus  = 5.65, p < 0.001, r = 0.82) and contraction (t(16) = 5.45, p < 0.001, r = 0.81). Mean rate of target disappearance was also significantly lower than during both expansion (t(16) = 4.53, p = 0.001, r = 0.75) and contraction  = 5.27, p < 0.001, r = 0.80). Post-hoc paired-samples t-tests for Experiment 2  = 6.01, p < 0.001, r = 0.82), and contraction (t(18) = 6.57, p < 0.001, r = 0.84). Mean rate of target disappearance during a static condition was also significantly lower than during both expansion (t(18) = 7.50, p < 0.001, r = 0.87) and contraction (t(18) = 9.70, p < 0.001, r = 0.92)  and Experiment 2 (p = 0.42). Likewise, mean rate of disappearance for expansion and contraction were not significantly different from each other in Experiment 1 (p = 0.94) and Experiment 2 (p = 0.87).Post-hoc paired-samples t-tests for Experiment 1 , by also analysing all saccades (micro-saccades + macro-saccades). Figure We recorded eye movements and detected saccades with an offline velocity-based saccade detection algorithm32, we quantified how much of the percentage of target disappearance in each trial can be accounted for by the number of saccades, alongside our main factors of interest of mask motion types and target position (see the details of LME in Methods). The significance of each factor was assessed with a likelihood ratio test32.Using linear mixed effect (LME) modelingχ2(1) = 40.71, p < 0.001; Experiment 2: χ2(1) = 96.06, p < 0.001), while taking account of the significant effects of saccades (Experiment 2: χ2(3) = 25.31, p < 0.001). Note the effect size of mask type, measured as χ2 value32, is much larger than the effect of saccades. Thus we conclude that the effects of motion mask type on target disappearance cannot entirely be explained by the saccades in each condition. In addition, we found that the vertical position of the target also influenced the percentage of MIB disappearance (Experiment 1: χ2(1) = 5.32, p = 0.022; Experiment 2: χ2(1) = 25.30, p < 0.001) while the horizontal position did not (Experiment 1: χ2(1) = 0.95, p = 0.033; Experiment 2: χ2(1) = 0.024, p = 0.61). See Table LME and likelihood ratio tests confirmed the significant effects of mask type  and expansion  did not overlap with the 95% confidence interval of static . The results of these regression analyses were not significantly changed when performed after combining both macro and micro saccades  = 3.59, p = 0.01, r = 0.66). Mean percentage of 35 km/h was also lower than that of 50 km/h (t(17) = 2.69, p = 0.09, r = 0.55) and 80 km/h (t(17) = 2.90, p = 0.06, r = 0.58), although differences fell short of significance after the Bonferroni corrections applied for multiple comparisons. Similar effects were found in post-hoc tests for mean rate, where number of target disappearances for the lowest speed level of 35 km/h were significantly less than 80 km/h (t(17) = 3.08, p = 0.04, r = 0.60) and 65 km/h (t(17) = 3.22, p = 0.03, r = 0.62). Mean rate of 35 km/h was also lower than of 50 km/h (t(17) = 2.57, p = 0.12, r = 0.53), although the difference fell short of significance after the Bonferroni corrections. We also found that the mean percentage of target disappearance showed linear dependency on the mask speed  = 8.63, p = 0.009, r = .58) but neither quadratic (p = 0.08) nor cubic (p = 0.80) dependency was significant, although mean percentage of target disappearance does appear to level off at higher speeds. The same effect was found for mean rate of target disappearance, with a significant linear dependency across conditions  = 10.30, p = 0.005, r = 0.61), but neither quadratic (p = 0.18) nor cubic dependency (p = 0.49).In Experiment 3, we examined the effects of the simulated speed of forward self-motion, comparing masks that simulated moving at 35 km/h, 50 km/h, 65 km/h and 80 km/h. There was a significant main effect of speed on mean MIB percentage and mean MIB rate of target disappearance (see Table χ2(1) = 14.58, p = 0.002), while vertical (χ2(1) = 3.50, p = 0.061) and horizontal (χ2(1) = 2.87, p = 0.091) position did not have a significant influence. See Table We again performed a linear-mixed effect analysis, investigating the influence of mask type and target location on mean percentage of target disappearance. The analysis showed that mask type had a significant influence  to investigate whether MIB still occurred as strongly when mask dots moved at a constant speed compared to a more natural accelerating speed in our previous experiments.Experiment 4 had two main purposes: (1) to compare our optic flow conditions to a condition with random motion that lacks any coherent motion structure, which has previously been shown to be important in MIBt(18) = 5.46, p < 0.001, r = 0.79), 2-D radial expansion (t(18) = 5.10, p < 0.001, r = 0.77) and 2-D radial contraction conditions (t(18) = 4.80, p < 0.001, r = 0.75). Likewise, mean rate of target disappearance was significantly lower in the static condition compared to 2-D random (t(18) = 4.96, p = 0.001, r = 0.76), 2-D radial expansion (t(18) = 4.72, p = 0.001, r = 0.74) and 2-D radial contraction (t(18) = 4.95, p = 0.001, r = 0.72). However, in both rate and percentage, 2-D random, 2-D radial expansion and 2-D radial contraction were not significantly different from each other.Again, there was a significant main effect of the type of mask movement on percentage Fig.  and meanp = 0.50) or mean rate of disappearance (p = 0.50).As the mean percentage and rate of disappearance in Experiment 4 appeared to be lower than that of Experiments 1–3, we also compared the data of Experiment 4 to the 35 km/h condition of Experiment 3, which had a mask speed of 3.8°/sec around the target, which closely matched the 3.4°/sec in Experiment 4. This specific comparison between these mask types was necessary because the dot speeds around the location of the target were quite different  in Experiment 1–3. In this comparison no difference in MIB between conditions was found for either mean percentage (t(13) = 3.58, p = 0.003, r = 0.70) and 2-D radial expansion (t(13) = 4.12, p = 0.001, r = 0.75). Disappearance length in the 2-D radial contraction condition was also longer than in static (t(13) = 2.44, p = 0.030, r = 0.56), although the difference fell short of significance after Bonferroni corrections.Mean and median duration of individual disappearance episodes were significantly different between mask conditions in Experiment 4 Table . Post-hoχ2(3) = 9.62, p = 0.047). However the effect size was much smaller than the highly significant influence of mask type (χ2(1) = 68.85, p < 0.001). Vertical position of the target (χ2(1) = 8.13, p = 0.004), but not horizontal position (χ2(1) = 0.005, p = 0.75) accounted partly for the percentage of MIB disappearance. See Table The linear-mixed effect analysis showed that saccades overall did influence percentage of MIB disappearance  across mask motion types.Recent research33 in Fig. et al.26, and we did not find any significant differences between reappearance conditions in either Experiment 2 or 4. Our results suggest that perceptual disappearances were subjectively more salient than reappearances, in support of Kloosterman et al.26, regardless of mask types.In terms of phasic pupil responses, target disappearances induced the largest pupil response in the static condition compared to the contraction and expansion conditions in Experiment 2  = 6.04, p = 0.001). More specifically, in Experiment 4, average pupil diameter remained comparable to the static condition for contraction (t(18) = −0.18, p = 0.86, r = 0.04), but increased significantly for the expansion condition (t(18) = −2.44, p = 0.03, r = −0.50). In Experiment 2 tonic pupil diameter was significantly greater in the static condition, compared to expansion (t(18) = 3.43, p = 0.003, r = 0.63) but not contraction (t(18) = 0.65, p = 0.52, r = 0.15). There were no significant differences in phasic or tonic responses dependent on target location, for both disappearance and reappearance (data not shown). We present a potential interpretation of these pupil responses within a predictive coding framework below.To further test if subjective saliency of the different motion types also modulated the overall tonic (i.e. baseline) pupil diameter, we also compared tonic pupil responses across Experiments 2 and 4, and across motion types Fig. . Interes21, but none have used a contracting or expanding optic flow analogous to when walking or driving, despite cues of self-motion being essential to safe navigation34. The present study showed that optic flow patterns, similar to those experienced when driving a car in a realistic speed range, can reliably elicit MIB.In the current study we tested whether stimulus parameters modelled on optic flow simulating self-motion are able to induce MIB. Various other motion patterns have been used in the past25, it is the first time that optic flow has been shown to elicit MIB. While claims that MIB can occur real world cannot be made based on this finding alone, it nevertheless functions as a necessary first step in that direction.While mask movement was conducive to target disappearance, disappearance was not affected by variations in the direction of movement that we tested within Experiments 1, 2 and 4, with optic flow expansion and contraction causing comparable target disappearance. While this effect may be expected based on the reliability of MIB occurring with a variety of mask parameters20. Therefore, it is possible that a stronger disappearance effect within simulated forward and backward motion could have been observed if we were to equate dot density around the target in these conditions. Further research addressing inconsistencies between mask movement and dot density around the target will be needed to explore this possibility.Expanding and contracting optic flow were shown to induce significantly more disappearance than radial expansion and contraction patterns with constant speed in Experiment 4, but this difference disappeared once we controlled for mask speed differences between Experiments 1 and 2 vs 4. However, the similar level of disappearance is still surprising, given that the dot density of the mask around the peripheral target within expanding and contracting optic flow was around 9-times lower, compared to 2-D radial expansion and contraction. Lower mask density necessarily results in less competition between the mask and target, causing less target disappearance5. Specifically, there was a significant positive linear dependency, with upper mask speeds eliciting significantly more disappearances than the lowest of 35 km/h in Experiment 3. While overall target disappearance  did increase as a function of mask speed, the differences between the three upper speeds were not significant, suggesting that there may be a plateau effect of increased target disappearance that is reached at around 50 km/h . On the other hand, a bias for the left visual field was not significant in any experiment.To summarise the location biases in MIB, Fig. 19. Past research has taken the increased disappearance of upper left targets to suggest a possible neural mechanism underlying MIB1, such as spatial attention, which also shows strong visual field asymmetries38. However, most studies that report such a bias presented three targets in a triangular display with two targets in the upper field and one centred in the lower field, and found significant results only between the upper left and lower central target24. If we interpret this in light of our current findings, in which the upper field bias was much stronger, it is possible that the upper left bias in MIB builds largely on a strong bias to the upper field overall.While not significant, there was a trend for target disappearance to be greatest for presentations in the upper left quadrant of the screen which is consistent with previous research39, where object recognition in natural scenes is particularly important for self-motion, hunting and general survival40. This lower visual field advantage has been reported in a range of visual tasks, including target detection and discrimination41, contrast sensitivity43, and the perception of illusory contours44. The present results extend this list to include MIB, as less target disappearance in the lower visual field may be explained as a consequence of greater visual acuity.The upper field bias of perceptual disappearance is also compatible with previous research that suggested a greater visual acuity exists for the lower visual field31. However, the magnitude of the effect was small compared to the influence of the mask, in both Experiment 2 and 4, and it did not explain our main findings, which is consistent with the observation that saccades do not eliminate MIB entirely45 and MIB occurs even with a moving target1. This is also evident from our scatter plot analyses  saccades per minute  saccades per minute in the optic flow mask conditions. Although around 80 saccades per minute typically occur while driving – more than twice as frequent as in our experiments – there is great variation in this according to the type of road and environment29, allowing us to investigate whether there were any differences in the saliency of MIB across mask conditions.While our study overall implies that MIB may occur during actual self-motion, such as when driving, we know of no spontaneous reports of MIB during driving. Why is that so? One possibility is that MIB phenomena are subtle so that not many people notice them, regardless of the functional significance of object disappearance. As we did not ask participants how salient their MIB experiences were, we used phasic pupil responses as a proxy for subjective salience. Phasic pupil dilation is known to increase in response to surprising, novel or unexpected stimuli26. We further found that in the optic flow conditions, target disappearances in the static mask condition induced larger pupil dilations than both the expansion and contraction conditions. This suggests that disappearances in the static condition were more salient, which might be explained by the fact that target disappearances were much less frequent in this condition. Conversely, and importantly, this means that target disappearances are less salient during optic flow stimulation. Reappearances did not show differences in terms of pupil size between mask conditions, probably because every disappearance is predictably followed by a reappearance after a relatively short period, making them less surprising. Interestingly, the differences in pupil responses between static and moving mask conditions were not significant in Experiment 4, perhaps because the target was embedded in a mask with higher dot densities, making the disappearance less salient. Overall, the pattern of pupillary responses we observed appears consistent with an interpretation that they signal the subjective saliency or surprise of events29. Importantly for our discussion here, disappearances are less salient in optic flow conditions than those in a static scene, which may explain why people seldom notice MIB during driving.We found that phasic pupil responses were larger in response to target disappearances than reappearances, suggesting that disappearances were more salient, consistent with previous researchWhen analysing the tonic pupil diameter (i.e. the average pupil diameter per mask condition), our results were again consistent with the phasic saliency account, though this time concerning a less-transient surprise measure that was dependent on motion mask type. Specifically, compared to the static mask condition, tonic pupil size was smaller during 3-D expanding optic flow, which is a condition that is frequently experienced during forward self-motion. Consistent with this idea, the opposite effect was found in Experiment 4, where average pupil size increased significantly for the 2-D radial expansion condition, where dots moved in an unnaturally constant speed across the screen - a situation we do not encounter in our environment.48. The visual system does not have good internal models for unnatural 2-D expansion motion, which therefore produces greater surprise and an increase in subjective saliency.These opposite effects of the tonic pupil diameter on the background motion pattern can be explained if we assume that they are dependent on the subjective amount of surprise. Frequently experienced 3-D expansion (and contraction) caused by self-motion are well internalized in the visual system, causing little surprise17. Further research on MIB is necessary to understand how surprise-related pupillary responses are modulated by the allocation of top-down attention and subjective awareness of perceptual events and by requirement of reports51. For example, one could quantify the threshold contrast increment of a target52. Such a probe task can be performed during a driving task without any explicit report on the target visibility itself51, as a function of concurrently measured pupil diameter as a proxy of subjective disappearance of the target. If indeed the threshold increases during the phasic increase of pupil dilation under the no-report condition, it has a strong implication for the existence of unreported MIB during real-life driving.If realistic patterns of motion are indeed less salient, and thus attract less attention, it could mean that more attention is available to be directed toward other elements in the visual display, such as the targets. If there is indeed more attention paid to the targets embedded in realistic motion types, more target disappearance should result53. Additionally, the mask was based on a tunnel, yet driving through tunnels in reality is usually brief and infrequently experienced.While providing support for future studies on the occurrence of MIB in the natural world, our research was still undertaken in an artificial setting. The experiment was conducted on a computer display and some aspects of the display itself were also inconsistent with the type of motion perceived in daily life. Specifically, the mask dots did not increase in size as they approached the periphery, as would occur in normal optic flow movement6, percentages of disappearance were smaller and mean disappearance episode lengths were shorter. This is because we intentionally avoided optimising the task design to increase target disappearance. Our original research objective was to investigate if MIB could occur under naturalistic self-motion induced optic flow, and thus we did not attempt to optimise the parameters for disappearances . The task also had a lower density of dots around the target as a result of the expanding motion, which reduces competition between the mask and target, causing less target disappearance20. Similarly, compared to previous studies1, we found a relatively high rate of Troxler fading within the static mask, a control condition for MIB. Troxler fading is known to increase with lower luminance contrast as used in our study6. Indeed, when compared with previous research utilising masks also not designed to optimise MIB8 our results were comparable or stronger in effect. Our results thus provide a conservative estimate of the potential occurrence of MIB in real-life settings.Compared to previous research56. Additionally, items that need to be monitored, such as brake lights of cars ahead, overtaking cars or GPS devices attached to the windscreen, reside mainly in (upper) peripheral quadrants, which we found prone to perceptual disappearance.Collectively, our findings show that optic flow simulating self-motion successfully elicits MIB. If found to occur in the real world, MIB may hold implications for driving safety, where the most common point of gaze is at the centre of the expanding optic flow of the approaching road57, during times of danger or hazard46, when multi-tasking59 and when experiencing fatigue60. Given that fewer saccades and longer fixations result in greater target disappearance, MIB may be more likely to occur during hazardous situations or driver fatigue - two of the most influential factors relating to traffic accidents61.While drivers may often saccade to check mirrors, scan for other cars and so forth, there are also situations in which fixations lengthen and saccade frequency decreases. These situations include driving on rural as opposed to urban roadsFuture research should attempt to measure the occurrence of MIB directly in conditions that approximate the statistics of the real world experience in a closer fashion, especially given its potential impact on driving safety. Examples of this may be tasks that measure brightness increments and reaction time in MIB, or apply the probe technique used in binocular rivalry to measure MIB indirectly, where the detection of a probe is expected to be impaired during target disappearance. Tasks such as these will hold stronger implications for driving safety, where MIB may play a role in the failure to detect crucial objects or changes in appearance, such as the brake lights of a car travelling in front. Such research could utilise a virtual reality environment to directly test the hypothesis of MIB occurring under driving circumstances, which would allow for stronger conclusions of the real-world implications than can be drawn from our stimuli.62, or increasingly in commercially available cars with dashboard information projected onto the windscreen63.If confirmed to exist under more realistic visual settings, MIB may also hold implications for the development of Head-Up Display (HUD) technology. Using this technology crucial information is projected onto the windscreen or visor in the form of stable symbols and text, through which the moving background of road or land is viewed. Such technology can be found, for example, in the use of F-35 Fighter Jet helmets which have been developed to project flight information onto the visor viewed by the pilot over the moving backgroundM = 24, SD = 9; 15 females) and 19 participants for Experiments 2 and 4 .Ethics approval was obtained from the Monash University Human Research Ethics Committee, and informed consent was obtained from all participants in the form of written permission prior to participation. Experiments were carried out in accordance with with the relevant guidelines and regulations. Thirty-seven participants were recruited at Monash University, all meeting the requirement of normal or corrected-to-normal vision. There was no overlap between the 18 participants for Experiments 1 and 3  and OpenGL, with the PsychToolbox extension2 (31 Cd/m2), and white mask dots at 83 Cd/m2 (169 Cd/m2), each with a radius of 0.05°  radius, surrounded by a thin exclusion boundary of 0.34° (0.25°), through which no mask dots travelled. The disc was located 10.75° (9.32°) degrees diagonal distance from the central fixation cluster, 6.36° (4.61°) vertical and 8.70° (8.10°) horizontal, positioned in either the upper left, upper right, lower left or lower right quadrant of the screen in each trial.Within each 60-second trial, one catch episode occurred in which the target physically disappeared from the display for a short period of time. The luminance of the target was linearly ramped off over 1.5 seconds, and stayed grey  for 1–4 seconds , then linearly ramped up to white  over another 1.5 seconds. The total duration of the catch episode therefore varied randomly from 4–7 seconds across trials. Each catch episode started randomly between 10 seconds and 50 seconds after the onset of each trial, to appear indistinguishable from perceptual disappearances. Participants were not told of the catch, but only encouraged to report all perceived disappearances. Accordingly, if the participants did not report physical disappearance of the catch appropriately, we regarded that they had not paid attention to the task in that trial. Debriefing with a number of participants revealed that they were surprised to hear of the catch disappearances, as they had not been able to tell the catch from the genuine perceptual disappearances. While three participants failed to record the catch on one or more trials, analyses with these participants excluded produced no significant difference to results. Their data was therefore included for analysis.45, which might to some degree explain the effects of our experimental manipulation of motion stimuli.In Experiments 2 and 4, we also recorded participant fixation locations using an eye tracker. One purpose of eye tracking was to exclude participants who looked directly at the peripheral target, failing to fixate on the central point at any time. Fixations were defined as any duration between macro-saccades (<1 deg). If the fixation duration on the peripheral target per 60-second trial was longer than one second, the participant was considered for exclusion. Only three participants met this exclusion criterion on at least one trial. As including or excluding them did not significantly alter the results, we included their data for analysis. The other purpose of eye tracking was to quantify the influence of eye movements on MIBWe summarise the purpose of each experiment as follows.In Experiments 1 and 2, three different types of mask were presented–expansion (simulated forward motion); contraction (simulated backwards motion), and a static still frame in which there was no dot movement. The simulated movement speed was 65 km/h for both expansion and contraction. The four different target locations were balanced and randomised over trials. Both Experiments 1 and 2 consisted of 12 × 1-minute trials, presenting each possible combination of mask (3 types) and target location (4 quadrants). Eye-movements were recorded during Experiment 2.In Experiment 3, only forward motion was simulated, resulting in an expanding motion pattern. Four different movement speeds were simulated: 35 km/h, 50 km/h, 65 km/h and 80 km/h. Experiment 3 consisted of a total of 16 × 1-minute trials, presenting each possible combination of speed and target location.Experiment 4 had two purposes: (1) to compare coherent radial expansion/contraction motion conditions to a condition with 2D random motion and (2) to investigate whether MIB still occurred as strongly when the mask dots moved at a constant speed on a 2D plane, unlike those that moved in an accelerating speed as in the optic flow conditions in 3D space (as in Experiment 1–3). In Experiment 4, instead of a central cluster, the fixation point was explicitly presented as a white cross with a size of 0.50° × 0.50°  for each experiment. The mean percentage, rate or duration of disappearance per condition were the three dependent variables (DV). For comparison, all analyses were also run using the median instead of the mean. The results did not change in a significant way , NumberOfSaccades , and the target location (LeftRight and UpDown) as DV MaskType + NumberOfSaccades + LeftRight + UpDown + (1| SubjectID). Subject variability was taken into account as a random effect. NumberOfSaccades was only entered when available (Experiment 2 and 4), in which case we included it in the unrestricted model as an interaction with MaskType.To examine how much of the observed MIB disappearance  could be explained by the frequency of saccades, which are expected to reduce MIB, and the location of the target, we used the linear mixed effect model (LME) in Matlab (ver 2016a). Likelihood ratio tests were performed through a 68. A constant of 1 was added to all data when performing the transformation, due to the dataset containing 0 values66.Assumptions for all tests were met, with the exception of violated normality in several conditions across experiments. Unless otherwise stated a square root data transformation was performed for all ANOVA analyses, as small sample size (<30) meant the Central Limit Theorem was not applicable in correcting normality67. Alternate transformations  also failed to correct normality in this variable. The violation in the static mask control condition was due to the majority of participants recording disappearance scores of zero for this condition, as expected. Given that negative values were not possible in our study, this necessarily led to a skewed distribution in the static condition. As the violated static condition was serving as a control for comparison only within our ANOVA analyses, the square root transformation was retained and parametric tests run despite the violation. However, non-parametric Friedman’s tests corroborated our findings in all cases. All graphical representations of data were created using within-subjects error bars displaying +/−1 SE, as recommended by Cousineau69.Despite the transformation, normality was occasionally still violated in the static mask condition across Experiments 1, 2 and 4, with skewness Z-Scores exceeding the alpha = 0.05 cutoff of 1.9630. Saccades with an amplitude larger or smaller than 1 degree of visual angle were categorised as macro-saccades or micro-saccades, respectively.Eye movements were recorded binocularly using a Tobii TX-300 Eyetracker  at a sampling rate of 300 Hz, controlled through the software package T2T (http://psy.cns.sissa.it/t2t/About_T2T.htmlhttp://psy.cns.sissa.it/t2t/About_T2T.html). Saccades and fixations were detected offline using a velocity-based algorithmet al.26. Briefly, to obtain phasic pupillary responses, we took excerpts from the pupil trace from −1 second to +1.5 seconds around the time of a reported disappearance or reappearance. The pupil diameter over the first 400 ms of each excerpt was subtracted from the signal, and the result was then divided by the mean pupil diameter over the entire experiment . This was done per participant separately. Results were then analysed over participants, with multiple comparisons corrected with a False Discovery Rate (FDR) 0.05. To estimate the sustained, baseline tonic level of pupillary diameter for each motion stimulus, we calculated the mean pupil diameter for each of the movement conditions separately, without normalisation.Pupil diameter was also recorded by the Tobii TX-300 Eyetracker. We analysed the left eye and followed procedures by Kloosterman Video LegendSupplementary Video S1Supplementary Video S2


BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for  Amylase hydrolyzes the starch molecules in dextrin, maltose, and finally glucose units. The enzymes are of industrial importance due to their commercial applications in starch liquefaction, paper, desizing of textile fabrics, preparing starch coatings of paints, removing wallpaper, brewing industry, sugar induction by the production of sugar syrups, pharmaceuticals, and preparing cold water dispersible laundry. They can be produced by micro- and macroorganisms . MicrobiRecently, starch saccharification, the major application of amylase, has completely replaced chemical utilization with amylase enzyme hydrolysis. Saccharification is performed at elevated temperature and thermophilic microorganisms could be most promising candidates for amylase production because these strains will produce thermostable amylase. This is why, still, search for novel microbial strains is continued to fulfill industrial demands of enzymes. In addition, amylase is supplemented in local detergents due to high alkaline pH stability required for industries . MoreoveBacillus genus is well known and famous for alpha-amylase production and several Bacillus strains such as B. stearothermophilus, B. subtilis, B. cereus, B. licheniformis, and B. amyloliquefaciens are isolated and screened for amylase production [ Bacillus strains are involved in raw starch degradations [ Bacillus sp. BCC 01-50 for extracellular amylase production using molasses as a cost-effective carbon source. Crude amylase applicability showed great potential for its application in detergent industry and saccharification of starchy materials. Results obtained herein are more promising for the inexpensive production of amylase by using agroindustrial residue as cost-effective carbon source for biotechnological applications.oduction . Some Baadations –9. Stilladations –16. The  Bacillus sp. BCC-01-50 was selected on the basis of the large clear zone for further study to evaluate its fermentability in agroindustrial residue and characterization of the crude enzyme for its biotechnological applications. The strain was incubated in LB medium containing 10 g/L of tryptone, 5 g/L of yeast extract, and 10 g/L of sodium chloride for 24 h at 37°C in shaking incubator. After 24 h, 10 (% v/v) culture was inoculated in the fermentation medium , and culture was incubated at 37°C for 120 h in shaking incubator. Samples were collected at a regular interval of 12 h, and growth (OD) was measured by a spectrophotometer. The culture broth was centrifuged at 12,100g for 10 min to collect supernatant and was used for biochemical analysis of α-amylase activity, total protein, and total sugars. All media were sterilized at 121°C, 15 lb/inch2 pressure for 20 minutes prior to inoculation.Bacterial strains previously isolated, identified, and stored in our laboratory were screened for amylase production in starch agar medium. Medium contained soluble starch 20 g/L, peptone 20 g/L, and agar 20 g/L, then culture was spread, and plates were incubated for 48 h. After 48 h, incubation plates were flooded with Gram's iodine solution to observe clear zone that was indication of starch hydrolysis. Straing for 10 min at 10°C) and the broth was used for biochemical analysis.Effect of various carbon sources was checked by using glucose, date syrup, molasses, fructose, starch, lactose, galactose, and sucrose with w/v concentration (20 g/L) for microbial growth and enzyme production. 250 mL Erlenmeyer Flasks with 50 mL fermentation medium were inoculated with 10 (% v/v) seed culture and inoculated at 37°C for 60 h. In another set of experiment, different concentrations of molasses were also tested. At the end of fermentation, culture broth was collected by centrifugation  concentration. Amylase titer was determined as described above. In a separate set of experiment, the effect of beef extract concentrations on amylase yield and bacterial growth was determined.g for 10 min at 10°C).Amylase activity was checked at different initial pH values ranges 5.0–11.0 and temperature range 30–60°C. 250 mL flask containing 50 mL of enzyme production medium was inoculated with 10 (% v/v) inoculum from 24 h old seeds culture and was incubated in shaking incubator at 37°C for 60 h, while, in case of temperature optimization, pH was adjusted to 8.0 and other conditions remained unchanged. Amylase activity was determined from culture broth obtained after centrifugation  and 8.0 pH was adjusted. After incorporation of seed culture, flasks were incubated in shaking incubator for 60 h at 50°C. After 60 h incubation, culture filtrate was collected and amylase activity was tested.Optimum pH requirement is very important for an enzyme-substrate reaction, so broader pH range 5.0–12.0 was investigated. The pH values were obtained with the addition to different buffer solutions: sodium acetate buffer, pH 5.0; phosphate buffer, pH 6.0–8.0; and glycine–NaOH buffer, pH 9.0–12.0. The reaction mixture was incubated at optimum temperature and activity was determined by DNS method. pH stability was determined by incubating enzyme in the above-mentioned buffers at 65°C for 1 h and, in terms of temperature and temperature stability after incubating enzyme at a different temperature range (30–95°C) for 1 h, residual amylase activity was measured under assay conditions.α-amylase. We observed that crude amylase is efficient for starch hydrolysis in a short time and suitable for local detergent formulations and as starch degrading enzyme.Amylase plays vital role in starch processing and in local detergent industries. Applicability of bacterial amylase was checked by determination of released glucose from starch hydrolysis during cloth washing. Experimental design was used as proposed by Shukla and Singh  and sameThe washout was used to estimate the reducing sugar with modified Anthrone's method reported by Shukla and Singh . Same meA is amount of glucose released after reaction (g/mL) in a washout and B is amount of starch (μg/mL) applied for staining of clean cotton cloth piece.The starch amount was monitored by multiplying the glucose amount with 0.9 as reported by Shukla and Singh . WashingA1 was the concentration of reducing sugars in clear solution (supernatant) after enzymatic hydrolysis reaction and A0 was the concentration of raw starch used in the reaction.Starch hydrolysis % was measured using the following equation proposed by Liu and Xu, and Hasjim et al. , 20:(2)R1.0 mL of sample and an equal amount of substrate (1.0% w/v soluble starch) were mixed thoroughly and test tubes were incubated at 37°C for 15 min in water bath. After 10 min, reaction was stopped by addition of 2.0 mL of DNS reagent and tubes were kept in boiling water bath for 5 min. Tubes were cooled at room temperature and absorbance was measured at 540 nm against substrate and enzyme blank. The blanks were prepared by replacing sample and substrate with the same amount of water. All other reagents were added in the same concentration.p < 0.05.Data were expressed as means ± standard deviation (SD) for triple determinations. The differences among means were assessed via least significant difference (LSD) tests. The significance level was set at  Bacillus sp. BCC 01-50 and cell forming unit (CFU/mL) are shown in  Aspergillus niger BTM-26 and Bacillus amyloliquefaciens released maximum enzyme concentration after 48 h in submerged fermentation [In our previous studies, molasses, a brown liquid obtained from sugar industry, has been applied for alkaline phosphatase –13 and pentation .α-amylase from too expensive pure sugars, but agroindustrial residues could be attractive carbon sources for the production of enzymes and other valuable products to reduce the cost of fermentation medium because more than 60% of cost belongs to carbon sources. So in present research study, for agroindustrial residue, molasses was utilized as a cost-effective carbon source and molasses medium secreted highest α-amylase yield. Abdullah et al. [The effects of different carbon sources  were investigated on growth and enzyme production results are shown in h et al.  have uti Bacillus sp. and amylase yield. All tested organic nitrogen sources supported bacterial growth, but higher amylase titer was obtained when organic nitrogen sources were used as compared to inorganic nitrogen sources. All tested nitrogen sources supported bacterial growth, but higher amylase titer was obtained when organic nitrogen sources were used as compared to inorganic nitrogen sources. Our results are supported and in agreement with Qureshi et al. [Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were tested as a nitrogen source. Results shown in i et al.  for amyli et al. . In anot Bacillus sp. in minimal medium containing 30 g/L molasses and 15 g/L beef extract incubated at 37°C for 60 h in shaking conditions (150 rpm). Microbial growth and amylase production were noted highest at pH 8.0, which indicates alkaliphilic nature of strain. Moreover, no significant reduction was observed in microbial growth and amylase titer in alkaline pH. Microbial growth and amylase production were noted highest at pH 8.0, which indicates alkaliphilic nature of strain. Literature supports optimum alkaline pH for amylase production from Bacillus cereus under solid state fermentation [ Bacillus sp. (30–60°C) grown in fermentation medium containing 30 g/L molasses and 15 g/L beef extract pH 8.0 which was incubated for 60 h. Enzyme concentration increased with incubation temperature and maximum amylase yield was noted at 50°C; further increase of temperature reduced amylase titer due to decreased microbial growth at elevated temperature. Enzyme concentration increased with incubation temperature and maximum amylase yield was noted at 50°C; further increase of temperature reduced amylase titer due to decreased microbial growth at elevated temperature. Similarly, Vijayaraghavan et al. and Asad et al. [ Bacillus cereus IND4 and Bacillus sp. WA21 at 45°C on starch agar medium, whereas Abdullah et al. [ Bacillus sp. thermophilic strain is growth-dependent.entation . In contentation  have obtd et al.  obtained Bacillus sp. thermophilic strain is growth-dependent. Our results are in line with literature for enzyme production from various strains in terms of inoculum size. The size of inoculum and age of inoculum play a significant role in the rate of fermentation. Therefore, optimization of inoculum age should always be considered as important fermentation parameter. Bacillus methylotrophicus strain P11-2 [ B. methylotrophicus amylase was stable for 1 h at pH 6.0–9.0, with the reduction in the residual activities at pH 4.0 and 5.0. Amylase was thermostable with optimum temperature for activity at 65°C. The results are comparable with an amylase from Nocardiopsis sp. where the similar profile is reported by Stamford et al. [ Bacillus sp. BCC 01-50 was active in the temperature range of 30–95°C, the optimum being at 65°C; results are shown in Crude amylase was characterized in terms of pH, temperature, pH stability, and thermostability. in P11-2 . The B. d et al. . The resα-amylase in washing process and are summarized in Tables α-amylase produced from Bacillus sp. BCC 01-50 towards different raw starch sources including wheat, potato and corn starches, and soluble starch was investigated by monitoring the extent of hydrolysis of various starch material in a short duration of time; results are depicted in α-amylase produced from Bacillus sp. BCC 01-50 towards different raw starch sources including wheat, potato and corn starches, and soluble starch was investigated and compared with previous studies. Full comparison with other amylase producing bacterial species is not possible due to the different reaction conditions, different strains, and fermentation conditions [ Bacillus species. 50 mg of starch was mixed with 2 mL of amylase enzyme and reaction mixture.The starch stained cotton fabric pieces were washed with water, detergents, crude enzyme, and detergent plus enzyme. The efficiency was determined from washout on the basis of the removal of starch content and efficiency can be expressed as the higher the starch content, the higher the efficiency. The washing efficiency was very low in tap water but improved with the addition of detergent and enzyme alone plus detergent. In our study, washing efficiency was significantly improved, when detergent plus crude enzyme was used. All the results of washing efficiency have been proved for the efficient use of nditions , 28. Yan Bacillus sp. BCC 01-50 using molasses in shake flasks as a batch culture technique. Highest amylase titer of ~4500 U/mL was obtained when 30 g/L of molasses was used. Our study suggests that molasses could be potentially very cost-effective energy source for commercial amylase production and that utilization of molasses not only reduces the cost of fermentation medium but also helps to reduce pollution problem due to agroindustrial residues. Moreover, crude amylase applicability showed great potential for its application in detergent industry and saccharification of starchy materials. Results obtained herein are more promising for the inexpensive production of amylase by using agroindustrial residue as cost-effective carbon source for biotechnological applications.Increasing amylase demand, high nutrient cost, and environmental pollution have compelled utilizing agroindustrial residues as inexpensive nutrients for enzyme production, so the present study has been taken up with a view to explore the possible use of agroindustrial residues as nutrient source for the production of amylase through submerged fermentation conditions because the use of inexpensive carbon and nitrogen sources can reduce the cost of production. In the present study, cultural conditions were optimized for amylase production from