Enterococcus faecium in tyramine production and its response to fermentation temperature in a traditional Korean fermented soybean paste, Cheonggukjang. Tyramine content was detected in retail Cheonggukjang products at high concentrations exceeding the recommended limit up to a factor of 14. All retail Cheonggukjang products contained Enterococcus spp. at concentrations of at least 6 Log CFU/g. Upon isolation of Enterococcus strains, approximately 93% (157 strains) produced tyramine at over 100 µg/mL. The strains that produced the highest concentrations of tyramine (301.14–315.29 μg/mL) were identified as E. faecium through 16S rRNA sequencing. The results indicate that E. faecium is one of the major contributing factors to high tyramine content in Cheonggukjang. During fermentation, tyramine content in Cheonggukjang groups co-inoculated with E. faecium strains was highest at 45 °C, followed by 37 °C and 25 °C. The tyramine content of most Cheonggukjang groups continually increased as fermentation progressed, except groups fermented at 25 °C. At 45 °C, the tyramine content occasionally exceeded the recommended limit within 3 days of fermentation. The results suggest that lowering fermentation temperature and shortening duration may reduce the tyramine content of Cheonggukjang, thereby reducing the safety risks that may arise when consuming food with high tyramine concentrations.The study evaluated the role of  Cheonggukjang is a traditional Korean soybean paste produced by fermenting soybeans with Bacillus subtilis. Traditional methods of Cheonggukjang production utilize rice straw added to steamed soybeans for a short fermentation period of approximately 2–3 days, while starter cultures are used instead of rice straw for modern methods of production [Cheonggukjang is a process involving microbial enzymatic proteolysis resulting in uniquely characteristic savory aromatic and flavor properties [Cheonggukjang has been reported to be associated with numerous benefits such as antioxidative, antihypertensive, thrombolytic, and antimicrobial properties [Cheonggukjang, potentially hazardous biogenic amines (BAs) may be produced during fermentation of the proteinous food rich in precursor amino acids.oduction ,2. Fermeoperties ,5. Howevβ-phenylethylamine, 100 mg/kg for histamine, and 100–800 mg/kg for tyramine in foods.The majority of BAs are formed through the reductive amination of ketones and aldehydes, as well as the decarboxylation of amino acids by microbially produced enzymes . Though Cheonggukjang have shown that tyramine in particular has been detected in high concentrations up to 1913.51, 251.66, and 905.0 mg/kg, respectively. Ibe, et al. [Enterococcus faecium may be largely responsible for the BA content of Miso (a Japanese fermented soybean paste). Notably, numerous studies have reported that Enterococcus spp. possess the tyrosine decarboxylase gene (tdc) [E. faecium in Cheonggukjang, while a previous study by Jeon, et al. [Enterococcus spp. isolated from Cheonggukjang exhibited tyramine production at concentrations of at least 351.59 μg/mL. Taken together, the previous reports imply that E. faecium may also be responsible for the BA content of Cheonggukjang. Meanwhile, the growth of Enterococcus spp. has been reported to occur at temperatures ranging from 10 °C up to 45 °C that overlap with Cheonggukjang fermentation temperatures ranging from 25 to 50 °C [E. faecium growth and tyramine production during the fermentation of contaminated Cheonggukjang products. Furthermore, a previous study reported that tyramine content increases in fermented soybeans as fermentation progresses [E. faecium strains may be affected by incubation conditions such as temperature and time. Therefore, the current study assessed the safety risk of BAs (particularly tyramine) in Cheonggukjang, clarified the microorganism responsible for tyramine accumulation, and evaluated the effect of fermentation temperature/duration on E. faecium growth and subsequent tyramine production in the food.Previous studies by Ko, et al. , Jeon, e, et al.  suggestene (tdc) ,24. Morene (tdc)  confirmeto 50 °C ,28,29,30ogresses , the proCheonggukjang products were purchased from various retail markets in the Republic of Korea and stored at 4 °C until further experimentation. Within a day of storage, the BA content of Cheonggukjang products was measured, followed by physicochemical and microbial analyses.Six representative, but different Cheonggukjang, the physicochemical properties of Cheonggukjang samples (retail Cheonggukjang products purchased and Cheonggukjang groups fermented in this study) were measured as described below. Samples weighing 10 g using an analytical balance  were homogenized with 90 mL of distilled water using a stomacher . The pH of the homogenates was measured using a pH meter , while salinity was measured using the procedure described by the Association of Official Analytical Chemists  [To investigate the influencing factors such as pH, salinity, and water activity on BA content in  960.29) . The watCheonggukjang samples was conducted using Plate Count Agar ; de Man, Rogosa, and Sharpe  agar; and m-Enterococcus Agar  for the enumeration of total mesophilic viable bacteria, lactic acid bacteria, and Enterococcus spp., respectively. Samples weighing 10 g were homogenized with 90 mL of sterile 0.1% peptone saline using a stomacher. The homogenates were 10-fold serially diluted with 0.1% peptone saline up to 10−5, and 100 μL of each dilution was spread on PCA, MRS agar, and m-EA in duplicates. Incubation conditions were set according to the manufacturer’s instructions: PCA at 37 °C for 24 h and m-EA at 37 °C for 48 h under aerobic condition; MRS agar at 37 °C for 48 h under anaerobic condition. Anaerobic condition was achieved using an anaerobic chamber  containing an atmosphere of 95% nitrogen and 5% hydrogen. After incubation, the bacterial concentrations of the Cheonggukjang samples were calculated by enumerating the colony-forming units (CFU) on the plates of respective media with approximately 10 to 300 colonies [The analysis of the microbial community in colonies  and adjuEnterococcus strains were isolated from retail Cheonggukjang products according to the method described by Mareková, et al. [v/v).A total of 169 , et al. , with miEnterococcus strains that displayed the highest tyramine production were further investigated through sequence analysis of 16S rRNA gene amplified with the universal bacterial primer pair 518F (5′-CCAGCAGCCGCGGTAATACG-3′) and 805R (5′-GACTACCAGGGTATCTAAT-3′) . The identities of sequences were determined using the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information .The identities (at species level) of the individual E. faecium, several temperatures were set for in situ Cheonggukjang fermentation experiments. The temperature for Cheonggukjang fermentation (intermediate-temperature group) was determined based upon previous studies in which 37 °C was reported as the temperature commonly used for Cheonggukjang production [Cheonggukjang fermentation were utilized for the low and high temperature groups, respectively [To investigate the effect of fermentation temperature on tyramine production by oduction ,35,36. Iectively ,30.Glycine max Merrill) were purchased from a retail market in the Republic of Korea. The soybeans were soaked in distilled water at 4 °C for 12 h, and subsequently drained for 1 h. Approximately 200 g of soybeans were adjusted to a final salinity of 2.40% according to the salinity of Cheonggukjang outlined in the 9th revision of the Korean food composition table [B. subtilis KCTC 3135  and 4 Log CFU/g of E. faecium KCCM 12118  or E. faecium CJE 216 (strain isolated from Cheonggukjang and selected owing to both strong tyramine production and tdc gene expression). The control group (without any E. faecium strains) was inoculated with only B. subtilis KCTC 3135 to a final concentration of 6 Log CFU/g. The sizes of inocula were selected with consideration of the cell count of each microorganism in Cheonggukjang products determined in our previous study [White soybeans  were homogenized by vortex  and stored at 4 °C for 2 h. The mixture was then centrifuged at 3000× g for 10 min at 4 °C , and the supernatant was collected. Upon resuspension of the pellet with 20 mL of 0.4 M perchloric acid, the mixture was stored at 4 °C for 2 h and centrifuged again at 3000× g at 4 °C for 10 min. The supernatant was combined with the previously collected supernatant and adjusted to a final volume of 50 mL with 0.4 M perchloric acid. Then, the extract was filtered through Whatman paper No. 1 .Quantification of the BA content of , et al. . Five grEnterococcus spp. based on Marcobal, et al. [g at 4 °C for 10 min. The extract was filtered through Whatman paper No. 1.The bacterial production of BAs was measured using the procedures described by Eerola, et al. , modifie, et al. . A loopfβ-phenylethylamine hydrochloride, putrescine dihydrochloride, cadaverine dihydrochloride, histamine dihydrochloride, tyramine hydrochloride, spermidine trihydrochloride, and spermine tetrahydrochloride . Internal standard solution with the same concentrations was prepared using 1,7-diaminoheptane (Sigma-Aldrich).Standard solutions with concentrations of 0, 10, 50, 100, and 1000 ppm were prepared for tryptamine, g for 5 min. After filtration using 0.2 μm pore-size filters (Millipore), the filtered supernatant was kept at 4 °C until further analysis using HPLC.Derivatization of BAs was conducted according to the method described by Eerola, et al. . One mil18 4 μm column  held at 40 °C was utilized. The mobile phases were 0.1 M ammonium acetate dissolved in deionized water  and acetonitrile  adjusted to a flow rate of 1 mL/min with a linear gradient starting from 50% of solvent B reaching 90% by 19 min. A 10 μL sample was injected and monitored at 254 nm. The limits of detection were approximately 0.1 μg/mL for all BAs in standard solutions and bacterial cultures, and about 0.1 mg/kg for all BAs in food matrices [Chromatographic separation of BAs was conducted according to the method previously developed by Eerola, et al.  and modimatrices .tdc) involved RNA extraction from bacterial cultures (for in vitro experiments) and Cheonggukjang samples  with a Ribo-Ex Total RNA isolation solution . The extraction was conducted according to the manufacturer’s instructions with minor modifications as follows. To prepare bacterial culture for in vitro gene expression analysis, a loopful (10 µL) of glycerol stock of each enterococcal strain was inoculated in 5 mL of MRS broth supplemented with 0.5% L-histidine monohydrochloride monohydrate, L-tyrosine disodium salt hydrate, L-ornithine monohydrochloride, L-lysine monohydrochloride (pH 5.8), and 0.0005% pyridoxal-HCl  and incubated at 37 °C for 48 h. Approximately 100 μL of the broth culture was then transferred to another tube containing 5 mL of the same medium and incubated under the same conditions. As for Cheonggukjang samples, 10 g of Cheonggukjang were gently mixed with 40 mL of phosphate buffer in a sterile bag, and the liquid part was collected. Subsequently, 3 mL of the bacterial culture or all liquid part of the mixture were immediately transferred into a 50 mL conical tube and centrifuged at 10,000× g at 4 °C for 5 min. After removing the supernatant, the pellet was suspended with 7 mL of phosphate buffer and centrifuged under the same conditions. Then, the pellet was homogenized with 800 μL of Ribo-Ex reagent in a bacterial lysing tube  using a Precellys 24 homogenizer  with two cycles for 30 s at 6800 rpm, pausing for 90 s between cycles. Approximately 200 μL of chloroform were added to the lysate, vortexed, and centrifuged at 10,000× g for 1 min. Approximately 400 μL of the supernatant were mixed with 600 μL of chilled absolute ethanol. The mixture was reacted at −70 °C for 15 min and purified with a Nucleospin RNA kit  according to the manufacturer’s instructions. The quality of the extracted RNA was evaluated using a NanoDrop 1000 spectrophotometer .Expression analysis of tyrosine decarboxylase gene  containing reverse transcriptase, RNase inhibitor, oligo (dT) primers, random primers, and deoxynucleoside triphosphates (dNTPs) was used to synthesize cDNA from 1 μL of extracted RNA according to the manufacturer’s instructions. Reverse transcription was conducted under the following conditions: 37 °C for 15 min, 50 °C for 5 min, and 98 °C for 5 min. After the reaction, the resulting cDNA was stored at −70 °C until quantitative PCR analysis.q-tdc F (5′-AGACCAAGTAATTCCAGTGCC-3′) and q-tdc R (5′-CACCGACTACACCTAAGATTGG-3′) primers were used for the quantitation of tdc gene expression by E. faecium. The primers for reference genes including q-gap F (5′-ATACGACACAACTCAAGGACG-3′) and q-gap R (5′-GATATCTACGCCTAGTTCGCC-3′) [tufA-RT F (5′-TACACGCCACTACGCTCAC-3′) and tufA-RT R (5′-AGCTCCGTCCATTTGAGCAG-3′) [tdc gene expression. The efficiency of each set of primers for reverse transcription quantitative polymerase chain reaction (RT-qPCR) was determined by the following equation: E = 10 S) values of serial dilutions of cDNA obtained from reverse-transcription of RNA from E. faecium KCCM 12118.As designed by Kang, et al. , q-tdc FCGCC-3′) , along wGCAG-3′)  were usetdc gene expression in bacterial cultures and Cheonggukjang samples, 5 μL of a 10-fold diluted cDNA were added to 15 μL of a master mix containing 10 μL of Power SYBR Green PCR Master Mix , 3 μL of RNase free water, and 1 μL of each primer . Subsequently, thermal cycling was conducted using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems) with the thermal cycling conditions programmed as follows: initial denaturation at 95 °C for 10 min; 40 cycles at 95 °C for 15 s (denaturation step), and 60 °C for 60 s . Annealing and elongation conditions for primer tufA-RT were set at 55 °C for 60 s. Melting curve analysis was conducted using the RT-PCR system to confirm the specificity and to analyze the amplified products. Ct values were detected when the emissions from fluorescence exceeded the fixed threshold automatically determined by thermocycler software. Relative expression of tdc genes was further calculated by the 2−(ΔΔct) method, normalized to the expression levels detected in E. faecium KCCM 12118  or Cheonggukjang groups fermented at 37 °C , and expressed as n-fold differences to compare gene expression levels in different bacterial cultures and Cheonggukjang samples.For the RT-qPCR analysis of p) value of <0.05 were considered statistically significant.Data were presented as means and standard deviations of duplicates or triplicates. All measurements on retail products were performed in triplicates, while the other experiments were conducted in duplicate. The significance of differences was determined by one-way analysis of variance (ANOVA) with Fisher’s pairwise comparison module of the Minitab statistical software, version 17 , and differences with probability  in Cheonggukjang . The results were similar to the study conducted by Lee, et al. [Cheonggukjang to be 7.0 ± 0.8. Jeon, et al. [Cheonggukjang to be pH 6.07 ± 0.72 (range of pH 4.62–8.14) and pH 7.21 ± 0.59 (range of pH 5.89–7.95), respectively. Such differences in the pH of the Cheonggukjang products may be owing to different fermentation conditions [Cheonggukjang products ranged from 1.95 to 9.36% with an average salinity of 5.16 ± 2.78%. In comparison, Ko, et al. [Cheonggukjang to be 2.12 ± 1.66% (0.12–11.51%), 1.56 ± 1.19% (0.10–5.33%), and 3.51 ± 2.45 (1.64–8.39%), respectively. Though the salinity of the Cheonggukjang products was found to vary substantially, Ko, et al. [Cheonggukjang salinities may be traced to the production process, as some methods utilize the addition of different amounts of salt to preserve the fermented soybean product. The water activity of retail Cheonggukjang products ranged from 0.919 to 0.973 with an average of 0.951 ± 0.019. In a previous study by Kim, et al. [Cheonggukjang products analyzed in the current study were mostly similar to the values reported in previous studies. Although the results of the current study did not show any correlation between physicochemical properties and BA content  based on linear regression analyses (data not shown), it is noteworthy that the ranges of the physicochemical parameters were within the specific conditions for the growth of E. faecium, which are as follows: pH, from 4 to 10 [Physicochemical and microbial properties as well as BA content in retail ggukjang . Table 1, et al. , which r, et al.  and Yoo,, et al.  also repnditions  and/or fnditions . The sal, et al. , Jeon, e, et al. , and Kan, et al.  reported, et al.  suggeste, et al. , the ave 4 to 10 ; salinit 4 to 10 ; water a 0.12–11.%, 1.56 ±Cheonggukjang products. The number of total mesophilic viable bacteria ranged from 8.54 to 9.81 Log CFU/g, with an average of 9.27 ± 0.45 Log CFU/g. Comparatively, Ko, et al. [Cheonggukjang products to be 7.50 ± 1.01 Log CFU/g (5.30–9.98 Log CFU/g) and 9.65 ± 0.77 Log CFU/g (8.23–11.66 Log CFU/g), respectively. The wide range of total mesophilic viable bacteria may result from an insufficient standardization of Cheonggukjang manufacturing processes such as different fermentation materials and conditions [Enterococcus spp. were detected at concentrations of 6.64–7.99 Log CFU/g, with an average of 7.17 ± 0.49 Log CFU/g. The number of lactic acid bacteria was found to be approximately 6.66–8.12 Log CFU/g, with an average of 7.09 ± 0.58 Log CFU/g . Evaluation of β-phenylethylamine content in two Cheonggukjang products revealed that concentrations exceeded the recommended limit of 30 mg/kg, with one product exceeding the limit by a factor of approximately 3. Though the histamine content of all Cheonggukjang products was found to be below the recommended limit of 100 mg/kg, the tyramine content of three products exceeded the 100 mg/kg limit by factors of 2, 9, and 14, respectively. Other studies have reported similarly high concentrations of tyramine in Cheonggukjang. Ko, et al. [Cheonggukjang products, exceeding the recommended limit by factors of 19 and 9, respectively. Furthermore, Cho, et al. [Cheonggukjang products contained high concentrations of tyramine, which exceeded the recommended limit by up to factors of 5, 5, and 3, respectively. Altogether, as β-phenylethylamine and tyramine content of several Cheonggukjang products exceeded the recommended limits, overconsumption of such fermented soybean products may occasionally result in adverse effects on the body. Moreover, Cheonggukjang was found to contain other BAs enhancing the toxicity of β-phenylethylamine and tyramine. Therefore, further research remains necessary for precautionary measures and remedial methods to reduce the BA content of Cheonggukjang to ensure the safety of the fermented soybean food.lalanine . The higectively . A previ, et al. , Han, et, et al. , and JeoBacillus, Clostridium, Enterobacteriaceae, enterococci, Lactobacillus, and Pseudomonas, are capable of producing the decarboxylases responsible for the conversion of amino acids into BAs [Enterococcus spp. have been suggested to be responsible for tyramine accumulation in Chinese and Japanese fermented soybean products [Cheonggukjang products using an MRS broth-based assay medium. As shown in β-phenylethylamine, putrescine, cadaverine, spermidine, and spermine was observed at concentrations lower than 10 µg/mL. Histamine production by 168 of the 169 strains was detected at quantities lower than 2 µg/mL; however, only one strain was capable of producing histamine at 96.06 µg/mL. Though tyramine production ranged from ND (not detected) to 315.29 µg/mL, 157 strains (about 93%) produced over 100 µg/mL. Through 16S rRNA sequencing, the seven strains  that produced the highest levels of tyramine  products may partially result from tyramine production by E. faecium. Jeon, et al. [Enterococcus spp. exhibited strong production of tyramine ranging from 0.41 μg/mL to 351.59 μg/mL in assay media. The author also found tyramine-producing Bacillus spp. (up to 123.08 μg/mL) and suggested that the species is one of the major tyramine producers in Cheonggukjang along with Enterococcus species based on the in situ fermentation experiment. Consequently, the present results suggest that Enterococcus spp. (particularly E. faecium) may be largely responsible for high tyramine concentrations in Cheonggukjang.Microbial decarboxylation of free amino acids is one of the main causing factors in the production of BAs, and various microorganisms, including into BAs ,61,62. Cproducts ,53, the , et al.  suggeste, et al.  demonstr, et al. , high leq-tdc (for the quantitation of tdc gene expression) along with q-gap and tufA-RT  was calculated to be 100.71%, 94.84%, and 95.03%, respectively. An amplification efficiency between 90 and 110% indicates that the results of gene expression obtained using RT-qPCR are reproducible [In the current study, the efficiency of primer sets oducible .tdc gene expression by the seven E. faecium strains  with the highest tyramine production in vitro as described in the previous section . Among the strains, E. faecium strain CJE 216 showed the highest expression level of tdc gene . Each group was further divided into three groups based on fermentation temperatures of 25 °C, 37 °C, and 45 °C . As shown in Cheonggukjang were measured at 24-hour intervals for 3 days of fermentation. The pH of all Cheonggukjang groups was lowest on day 2, with progressively lower pH depending on the fermentation temperature, independent of which inoculum was used. The pH on day 2 of Cheonggukjang fermentation at 25 °C, 37 °C, and 45 °C ranged from pH 6.34 to 6.36, pH 5.90 to 6.09, and pH 5.49 to 5.88, respectively (Cheonggukjang groups fermented at 45 °C (high-temperature group) resulted in a lower pH than other groups fermented at 37 °C and 25 °C , regardless of inoculum, the BA content was expected to be detected at the highest concentration among all Cheonggukjang groups. As for water activity, all Cheonggukjang groups remained within 0.95–0.97 during fermentation  increased by 1.63 Log CFU/g, 3.52 Log CFU/g, and 4.06 Log CFU/g after 3 days of fermentation at 25 °C, 37 °C, and 45 °C, respectively (Cheonggukjang co-inoculated with E. faecium CJE 216 at 4 Log CFU/g (and B. subtilis KCTC 3135), enterococcal count increased at all fermentation temperatures of 25 °C, 37 °C, and 45 °C by 3.48 Log CFU/g, 4.78 Log CFU/g, and 4.80 Log CFU/g, respectively, by day 3. Enterococcus spp. were not detected in the control group for the duration of the fermentation period. The results displayed progressively higher enterococcal counts that increased alongside rising fermentation temperatures with the highest enterococcal counts in Cheonggukjang fermented at 45 °C (high-temperature group). The findings were comparable to a previous study by Morandi, et al. [E. faecium growth as the reported generation time at 25 °C was nearly two times longer than at 37 °C. E. faecium has been reported to display active growth within the temperature range of 37–53 °C, with an optimal growth temperature of 42.7 °C [E. faecium growth, thereby increasing the potential for high tyramine production during Cheonggukjang fermentation.The enterococcal count in ectively . In Cheo, et al. , which s 42.7 °C ,70. The Cheonggukjang co-inoculated with either E. faecium KCCM 12118 or E. faecium CJE 216, along with B. subtilis KCTC 3135, was measured during fermentation, as seen in E. faecium was detected at concentrations that did not exceed 10 mg/kg in all fermentation conditions  during the fermentation period, and consequently exceeded the recommended limit for consumption. Jeon, et al. [Enterococcus spp. during soybean fermentation at 37 °C. The report demonstrated that tyramine concentrations continued to increase as fermentation progressed. Given the results, safety precautions regarding the limitation of fermentation duration and temperature appear to be necessary as extended periods of fermentation as well as high fermentation temperatures may increase tyramine content in Cheonggukjang beyond the recommended safe limit for consumption. Besides, the results showing a lower tyramine content in Cheonggukjang during fermentation at lower temperatures coincide with the results in the previous section that displayed a reduction in enterococcal count alongside a decrease in fermentation temperature. Taken together, the present study indicates that lower fermentation temperatures inhibit enterococcal growth, thereby limiting acid production and maintaining low levels of tyramine in Cheonggukjang. Therefore, utilizing lower temperatures for Cheonggukjang fermentation may reduce the risks associated with Enterococcus growth and tyramine accumulation.The tyramine content of nditions a. In conectively b. As forentation c. All Chnd 45 °C b,c. The , et al. , E. faec, et al. . In the , et al.  also reptdc gene expression by tyramine-producing E. faecium strains were detected and quantified during fermentation of Cheonggukjang at 25 °C, 37 °C, and 45 °C. As Cheonggukjang is mostly fermented at 37 °C, the tdc gene expression detected in Cheonggukjang groups fermented at 45 °C was normalized to that detected in the corresponding groups fermented at 37 °C according to the E. faecium strains used as inoculants. In Cheonggukjang fermented at 25 °C, tyramine content continuously remained at concentrations lower than 10 mg/kg, and tdc gene expression by E. faecium KCCM 12118 and E. faecium CJE 216 was not detected in all Cheonggukjang groups. In contrast, the highest tdc gene expression by E. faecium KCCM 12118 was detected in Cheonggukjang fermented at 45 °C and was upregulated in the range of 1.90- to 7.15-fold throughout Cheonggukjang fermentation, compared with that in Cheonggukjang fermented at 37 °C . Nonetheless, the variation in detected tdc gene expression levels during fermentation did not necessarily reflect the tyramine content observed for Cheonggukjang. After one day of fermentation, the Cheonggukjang group with E. faecium CJE 216 fermented at 37 °C contained a lower tyramine content than at 45 °C; however, tdc gene expression was slightly higher at 37 °C as described right above. The results showed that there may be differences between gene expression level and enzyme activity (and products thereof). Glanemann, et al. [E. faecium strains in an assay medium appeared to be insignificantly related to tdc gene expression level (data not shown). Therefore, utilizing HPLC analysis appears to be essential for the quantification of BA content and/or bacterial BA production in food samples [Cheonggukjang at high temperatures results in increased tdc gene expression and tyramine production. Therefore, low-temperature fermentation appears to be necessary to minimize both tdc gene expression and tyramine production by Enterococcus spp. and thereby ensure the safety of fermented soybean products.The changes in at 37 °C a–c, left, et al.  reported, et al. , while t samples ,73,74. W samples ,31. In tCheonggukjang, diagnosed the microbial causative agent  responsible for high tyramine levels, and evaluated the impact of fermentation temperature on enterococcal growth (as well as acid production and tdc gene expression) and tyramine production. Of the retail Cheonggukjang examined, half of the products contained tyramine content that exceeded the recommended limit for safe consumption by up to a factor of approximately 14. E. faecium strains isolated from the retail Cheonggukjang products were highly capable of producing tyramine in assay media, which indicated that the species is principally, or at least partly, responsible for tyramine accumulation in the food.The current study assessed the safety risk of tyramine in Cheonggukjang groups co-inoculated with B. subtilis (used as an inoculant to ferment soybeans) and E. faecium (either isolated in this study or designated previously as the type strain) strains was highest at 45 °C, followed by 37 °C and 25 °C. On the other hand, the control group inoculated with only B. subtilis strain (without any E. faecium inoculants) had the lowest tyramine content at all fermentation temperatures, which supported the notion that E. faecium may be a key producer of tyramine in Cheonggukjang. Another implication of the results was that a lower fermentation temperature leads to a lower tyramine content below the recommended limit in Cheonggukjang, even though the tyramine content continually increases during fermentation. Therefore, low temperatures and a short fermentation duration may reduce the accumulation of tyramine caused by E. faecium growth in Cheonggukjang, thereby reducing the safety risks associated with consuming food with high BA concentrations.During in situ fermentation at different temperatures, the tyramine content of


The sensitivities of kits I‐IV detected using 142 nasopharyngeal swab specimens from COVID‐19 patients were 91.55%, 81.69%, 80.28%, and 90.85%, respectively, while they were 92.68%, 85.37%, 82.93%, and 93.90%, respectively, for the 82 sputum samples. The specificity of each kit was 100.00% (77/77). The total expected detection rate using sputum samples was 88.59% (691/780) higher than 86.15% (672/780) of nasopharyngeal swabs. Comparison of nasopharyngeal swab and sputum samples from the same COVID‐19 patient led to the detection of ORF1ab and N genes in 16 (100%) sputum samples; only ORF1ab and N genes were detected in 12 (75%) and 14 (87.5%) nasopharyngeal swab specimens, respectively. In conclusion, comparison of commercial COVID‐19 RT‐PCR kits should be performed before using a new batch of such kits in routine diagnostics.We compared the sensitivity and specificity of four commercial coronavirus disease (COVID‐19) diagnostic kits using real‐time reverse transcription–polymerase chain reaction (RT‐PCR). Kits I‐IV approved by the State Drug Administration of China were selected, and the detection targets were ORF1ab gene and N gene. Specificity was evaluated by detecting other respiratory viruses. The sensitivity and batch effect of each kit were evaluated by testing 10‐fold dilutions of RNA. Clinical application was verified by testing nasopharyngeal swab and sputum specimens from COVID‐19 patients. Among the 78 cases infected by other respiratory viruses, no amplification curve was observed using these four COVID‐19 RT‐PCR kits. The minimum detection limits of kits I‐IV were 10 Suspected COVID‐19 cases with low RNA concentration or at the initial stages of the disease should be examined using different COVID‐19 kits or sampling sputum specimens to a feasible extent and comparison of commercial COVID‐19 RT‐PCR kits should be performed prior to using new batches of the kits in routine diagnostics. The manufacturers included Beijing Applied Biological Technologies Co., Ltd; Beijing Kinghawk Pharmaceutical Co., Ltd; Beijing NaGene Diagnosis Reagent Co., Ltd; and Coyote Bioscience Co., Ltd . All the kits were suitable for ABI 7500 real‐time PCR system, and the detection targets were ORF1ab gene, N gene, and ribonucleoprotein (RNP).2.2Clinical specimens, including nasopharyngeal swab and sputum specimens, were collected from confirmed or excluded COVID‐19 patients experiencing acute respiratory tract infection. The criteria for confirmed COVID‐19 cases included the following: (a) already‐confirmed cases of COVID‐19; (b) nasopharyngeal swab and sputum specimens positive for SARS‐CoV‐2 nucleic acids upon detection by RT‐PCR tests performed at the Beijing Center for Disease Prevention and Control (BJCDC); and (c) cases that obtained a fraction of the RNA sequence of SARS‐CoV‐2 by PCR amplifications and gene sequencing. Exclusion of cases was based on both clinical evidence and RT‐PCR results. This study was approved by the Ethics Committee at BJCDC.2.3™ KingFisher™ Flex Magnetic Particle Processors . Approximately 60 µL of total nucleic acid eluates was recovered in nuclease‐free tubes and tested immediately.Total nucleic acids (RNA and DNA) were extracted from the clinical specimens using Thermo Scientific2.4Nasopharyngeal swab specimens containing other respiratory viruses, including influenza virus A, influenza virus B, respiratory syncytial virus, parainfluenza virus, human adenovirus, human rhinovirus, other HCoVs , human metapneumovirus, and human bocavirus, were also collected to identify the specificity of the assessed kits. A commercial RT‐PCR kit  was used to detect these viruses.2.56) of nucleic acid eluates from a COVID‐19 patient (tC = 17) were prepared in duplicate. The concentrations of nucleic acid eluates (10−1 to 10−6 dilutions) were detected by digital PCR. Three batches  of the four kits were selected to evaluate sensitivity and batch effects. Sensitivity was evaluated by testing all serial dilutions with the kits. Further, batch effects were evaluated using linear regression analyses. R2 values of the linear regression analyses were obtained using SPSS software, version 20.0: IBM, Amonk, NY. All charts were made using Origin software, version 9.0: OriginLab, Northampton, Ma.Tenfold serial dilutions , 3 cases of respiratory syncytial virus, 3 cases of parainfluenza virus, 3 cases of human adenovirus, 3 cases of human rhinovirus, 3 cases of human metapneumovirus, and 3 cases of human bocavirus. No amplification curve was observed for the samples tested using the four RT‐PCR kits.3.2−1 to 10−6 dilutions) were 27 230 copies/mL, 5399 copies/mL, 1395 copies/mL, 437 copies/mL, 10 copies/mL, and 1 copies/mL, respectively, as detected by digital PCR. The minimum detection limits of the ORF1ab gene and N gene targets of kit I (10−5 dilution of ORF1ab gene and 10−6 dilution of N gene), kit II (10−5 dilution of ORF1ab gene and 10−5 dilution of N gene), kit III (10−5 dilution of ORF1ab gene and 10−5 dilution of N gene), and kit IV  are shown in Figure Ten fold serial dilutions of nucleic acid eluates of the COVID‐19 case were tested in duplicate to evaluate the sensitivity of the kits. The concentrations of the nucleic acid eluates  kit I: ORF1ab gene, 0.9933; and N gene, 0.9869; (b) kit II: ORF1ab gene, 0.9696; and N gene, 0.9848; (c) kit III: ORF1ab gene, 0.9548; and N gene, 0.9707; and (d) kit IV: ORF1ab gene, 0.9791, and N gene. Additionally, for the four kits, the standard deviations of R2 values were as follows: ORF1ab gene, 0.0039, 0.0259, 0.051, and 0.0199; N gene, 0.0146, 0.053, 0.0088, and 0.0347, respectively.Batch effect was evaluated by testing the detection difference between three batches of the assessed kits, and the data were analyzed by linear correlation analysis using SPSS software, version 20.0. The 3.3A total of 301 nasopharyngeal swab and sputum specimens were collected between January and March 30, 2020, including 224 and 77 specimens of confirmed and excluded cases, respectively. Among kits I to IV, the positive detection rates of the specimens from the confirmed COVID‐19 patients were 91.96% (206/224), 83.04% (186/224), 81.25% (182/224), and 91.96% (206/224), respectively, and the negative detection rates of the specimens from the excluded COVID‐19 patients were 100.00% (77/77).3.4: 85.70‐95.56), 81.69% (95% CI: 74.33‐87.68), 80.28% (95% CI: 72.78‐86.48), and 90.85% (95% CI: 84.85‐95.04), respectively. The negative detection numbers of 47 nasopharyngeal swab specimens from the excluded COVID‐19 patients using kits I to IV were 47, and the specificities of kits I to IV were all 100.00% (95% CI: 92.45‐100.00).The types and corresponding patients of the 189 specimens are shown in Table : 0.92‐0.98) for kit I, 0.95 (95% CI: 0.91‐0.98) for kit IV, 0.91 (95% CI: 0.86‐0.95) for kit II, and 0.90 (95% CI: 0.85‐0.94) for kit III. The consistencies of kits I and IV were strong, with κ values 0.84 (95% CI: 0.80‐0.89) and 0.83 (95% CI: 0.79‐0.88), respectively. The consistencies of kits II and III were high, with κ values 0.69 (95% CI: 0.64‐0.74) and 0.67 (95% CI: 0.62‐0.72), respectively.The area under the curve values of the four kits for nasopharyngeal swab specimens were all >0.9, including 0.96 (95% CI3.5: 84.75‐97.27), 85.37% (95% CI: 75.83‐92.20), 82.93% (95% CI: 73.02‐90.34), and 93.90% (95% CI: 86.34‐97.99), respectively. Further, the detection numbers of the specimens from the excluded COVID‐19 patients using kits I‐IV were all 30, and the specificities of the kits were all 100% (95% CI: 88.43‐100.00).Among the 301 specimens, 112 were sputum samples, including 82 specimens from confirmed COVID‐19 patients and 30 specimens from excluded COVID‐19 patients, as shown in Table : 0.92‐0.99) for kit IV, 0.96 (95% CI: 0.91‐0.99) for kit I, 0.93 (95% CI: 0.86‐0.97) for kit II, and 0.92 (95% CI: 0.85‐0.96) for kit III. The consistencies of kits I and IV were strong, with κ values 0.87 (95% CI: 0.82‐0.92) and 0.89 (95% CI: 0.85‐0.94), respectively. Further, the consistencies of kits II and III were high, with κ values 0.76 (95% CI: 0.69‐0.82) and 0.72 (95% CI: 0.66‐0.79), respectively.The area under the curve values of the four kits for sputum specimens were all >0.9, including 0.97 (95% CI3.6Direct standardization was performed using SPSS software, version 20.0, to select sample type between the nasopharyngeal swab and sputum specimens for SARS‐CoV‐2 detection. The expected detection numbers of all the specimens are shown in Table tC values of kits I and IV are shown in Table S1. The head‐to‐head comparison of sputum and nasopharyngeal swab specimens collected from the same COVID‐19 patient is shown in Figure tC values of 12 sputum specimens tested for ORFlab and N genes were higher than those of the nasopharyngeal swab specimens.Further, nasopharyngeal swab and sputum specimens were collected from 16 COVID‐19 patients between January and March 30, 2020, and used for head‐to‐head comparisons to show whether different types of specimens from the same patient would affect the testing capabilities of the kits. The clinical information and 4COVID‐19 outbreaks have become a global public health concern.This study showed that detection rates in the sputum specimens from the lower respiratory tract of COVID‐19 patients using kits I to IV were higher than those in the nasopharyngeal swab specimens from the upper respiratory tract of the patients. Several studies have shown that the sampling quality of specimens obtained from the upper respiratory tract cannot be guaranteed, and specimens with low RNA concentration from the initial stage of COVID‐19 cases also lead to false‐negative detection.According to the manufacturers’ instructions of the different RT‐PCR kits for COVID‐19 diagnosis available commercially, minimum detection limits are 100‐1000 copies/mL, and a few kits could reach 20 copies/mL.Additionally, the results of batch effects among the four kits showed that the abilities to detect the same gene target were slightly different between different batches of the same kit, and overall batch effects were slightly different between the tested commercial kits. Although there might be differences in the raw materials and production lines employed in producing COVID‐19 diagnostic kits, manufacturers should ramp‐up supervision to ensure product quality from batch to batch, which may result in only a slight detection difference within an allowable error range. Further, criteria of comparison should be formulated and comparisons should be made to confirm the sensitivity and specificity of the kits prior to using new batches.In conclusion, the sensitivities and batch effects of the assessed kits were slightly different for different targets, and sputum specimens were more applicable for SARS‐CoV‐2 detection than nasopharyngeal swab specimens. Therefore, these data suggest that suspected COVID‐19 cases with low RNA concentration or at the initial stages of the disease should be examined using different COVID‐19 kits or sampling sputum specimens to a feasible extent and that comparison of commercial COVID‐19 RT‐PCR kits should be performed prior to using new batches of the kits in routine diagnostics. Additionally, with the increasing number of commercial COVID‐19 kits, it is necessary for researchers to share information such as multi‐center kit comparison methods and the detection abilities of various commercial RT‐PCR diagnostic kits among different specimens.Table S1Click here for additional data file.


Cerulenin and ketoconazole were used to inhibit fatty acids and the sterol biosynthesis pathway, respectively, in order to enhance β-carotene production by diverting metabolic pool towards the mevalonate pathway. All three variables used in screening experiments were found to be significant for the production of β-carotene. The synergistic effect of the C/N ratio, cerulenin, and ketoconazole was further evaluated and optimized for superior β-carotene production using central composite design of RSM. Our results found that the synergistic combination of C/N ratios, cerulenin, and ketoconazole at different concentrations affected the β-carotene productions significantly. The optimal production medium (std. order 11) composed of C/N 25, 10 μg/mL cerulenin, and 150 mg/L ketoconazole, producing maximum β-carotene of 4.26 mg/L (0.43 mg/g) which was 157% greater in comparison to unoptimized medium . So, it was concluded that metabolic flux had been successfully redirected towards the mevalonate pathway for enhanced β-carotene production in CBS 277.49.Carotenoids produced by microbial sources are of industrial and medicinal importance due to their antioxidant and anticancer properties. In the current study, optimization of  Phaffia rhodozyma, Dunaliella salina, and Blakeslea trispora [Carotenoids are well known bioactive compounds and high-value nutritional molecules that play a defensive role against chronic diseases due to their antioxidant potential . In the trispora . The chetrispora . Carotentrispora . Howeverβ-Carotene is an orange-yellow lipophilic carotene and known as provitamin A. It has high market value due to its diverse biological functions and extensive uses in pharmaceutical, cosmetics, food, and textile industries [β-carotene is the key factor in the prevention of pharynx oral and larynx cancer, which was confirmed by a high intake of food rich in β-carotene resulting in the reduction of risk for throat and oral cancers by about 50% [dustries . Human ibout 50% .Blakeslea trispora, Phycomyces blakesleeanus, and Mucor circinelloides, have been reported for carotene biosynthesis of which β-carotene is the principal carotenoid [B. trispora and P. blakesleeanus are well known for the highest production of carotenoid. However, a few drawbacks are linked with the industrial utilization of these two fungi. Shaking cultivation of B. trispora was associated with declined accumulation of carotenoid, so surface cultivation must be performed, and the optimum growth temperature for these zygomycetes should never be above 25°C [M. circinelloides could be simpler as compared to the complicated fermentation process used for the production of β-carotene in B. trispora [. M. circinelloides is considered a true “microbial factory” due to its intrinsic potential to produce enzymes, organic acids, essential amino acids, food pigments, ethanol, chitosan, polyphenols, and microbial lipids [β-Carotene production in M. circinelloides occurs naturally, and the carotenoid biosynthesis in this fungus is well studied owing to the accessibility of genetic tools that can help in the development of hyper β-carotene-producing strain. Furthermore, the dimorphic nature of M. circinelloides aids in biotechnological production [M. circinelloides [Commonly three zygomycetes, rotenoid . The maiove 25°C . Hence, trispora . M. circl lipids , 13. β-Coduction . Being aelloides .β-carotene in terms of both overall productivity and relative yield of β-carotene in association with cell growth [β-carotene production, such as environmental conditions [Cost reduction is one of the crucial factors in the fermentation scale-up process for any bioactive compound production. Hence, industrial production of carotenoid would only be feasible if production cost could be minimized by using suitable carbon sources and increasing the product yield . The devl growth . Apart fnditions , the usenditions , applicanditions , 19, andnditions , 20 in vM. circinelloides strain CBS 277.49 as a dominant producer of β-carotene than M. circinelloides strain WJ11 [β-carotene by screening different carbon sources and chemical modulators to divert metabolic flux of acetyl-CoA and FPP from fatty acid synthesis (FAS) and sterol synthesis pathways towards the mevalonate pathway. Based on a well-established fact that acetyl-CoA is a shared precursor for terpenoid and fatty acid biosynthesis [β-carotene by using an ergosterol inhibitor  optimization of medium components by screening different carbon sources and C/N ratios and (2) redirection of metabolic flux towards the mevalonate pathway by using inhibitors of the FAS and sterol pathway, individually or in combination. Many studies have been conducted for the enrichment of β-carotene in different microorganisms. To date, an optimization study of β-carotene biosynthesis in Mucor using statistical analysis has not been studied extensively and required further comprehensive exploration. So, to the best of our knowledge, this is the first study to optimize the production of β-carotene in CBS 277.49 by diverting metabolic flux towards the mevalonate pathway from the FAS and ergosterol synthesis pathway using inhibitors of the mentioned pathways.Our previous finding indicated ain WJ11 . So, in ynthesis , it was nhibitor . To confM. circinelloides strain CBS 277.49 was used in the present study [μL spore suspension (nearly 107 spores/mL) into 150 mL of Kendrick and Ratledge (K&R) medium. This medium contained 30 g glucose, 7 g KH2PO4, 3.3 g diammonium tartrate, 2 g Na2HPO4, 1.5 g yeast extracts, 1.5 g MgSO4·H2O, 0.1 g of CaCl2·2H2O, 8 mg FeC13·6H2O, 1 mg ZnSO4·7H2O, 0.1 mg of MnSO4·5H20, 0.1 mg CuSO4·5H2O, and 0.1 mg Co(NO3)2·6H2O in one liter of distilled water and was stored in 500 mL baffled flasks to support aeration and incubated for 24 h in a shaking incubator at 28°C and 150 rpm. Then, 10% (v/v) of this overnight grown culture was inoculated in 150 mL modified K&R media, which contained 80 g of glucose and 2 g/L diammonium tartrate held in a 1000 mL baffled flask for subsequent fermentation under the continuous light. All experiments were repeated three times.nt study . Seed cuβ-carotene by CBS 277.49.In the first part of this study, different screening experiments were performed to achieve optimized medium concentration to produce β-carotene production and growth of CBS 277.49. The carbon sources used for this study were fructose, glucose, glycerol, maltose, and molasses at a concentration of 80 g/L with other medium components. The culture of CBS 277.49 was grown for 72 h under continuous light at 28°C and 150 rpm. After 72 h, fungal biomass was harvested and analyzed for cell dry weight (CDW), total lipid %, and β-carotene concentration for all carbon sources.The fungal strain was cultivated on five carbon sources for enriched w/w). The effect of different C/N ratios on microbial cell growth, lipid %, and β-carotene production was investigated. The control flask did not contain any inorganic nitrogen source.The optimal C/N ratio was determined in 1000 mL baffled flask holding 150 ml K&R media. Inorganic salt ammonium tartrate dibasic served as a nitrogen source in our study. Carbon sources were kept constant at 80 g/L while the nitrogen source was adjusted to make C/N ratios of 5, 10, 20, 40, and 80  and Design Expert Software  in a 1000 mL baffled flask holding 150 mL K&R media with the variable of factors as presented in w/w; cerulenin, 20-50 μg/mL; and ketoconazole, 10-200 mg/L) and cultivated for 72 h  response surface and contour plots were created to visualize the trend of the maximum responses and interaction outcome of significant variables on the responses.Statistical investigation using ANOVA with the DOE software was conducted to estimate the optimal factors for the maximum production of Biomass was collected on a dried and preweighed filter paper by filtration through a Buchner funnel using reduced pressure and washed thrice with distilled water. Then, after freezing at -80°C, the biomass was freeze-dried. The weight of the biomass was gravimetrically calculated.v/v), and after drying using nitrogen, the weight of the tube with lipid was measured. Total lipid % in CBS 277.49 was calculated using the following equation:Total lipids were extracted as defined by Folch et al., with slight modifications . Briefly mycelia .β-carotene was done according to our previous investigation [μL of hexane, followed by a vortexing step. This extraction step was repeated until the mycelia powder became colorless. Extracts were then collected in a 50 mL falcon tube, mixed, and partitioned in an equal volume of 10% diethyl ether. 2 mL of distilled water was introduced to help the separation of two layers followed by centrifugation at 3200 rpm and 4°C for 8 min. The fraction of petroleum ether was dried under nitrogen.Extraction of tigation . Brieflyβ-carotene for samples and standard purchased from Sigma. Dried extracts were resuspended in 800 μL tetrahydrofuran added with butylated hydroxytoluene (100 μg/mL). The samples were then subjected to analysis by HPLC immediately. 10 μL of samples were analyzed on the infinity Lab Pro shell C18 column . 96% of methanol and 100% methyl-terc-butyl ether were used as mobile phases listed as A and B, respectively. The following gradient of these two solvents was used for the analysis of samples: min/solvent A%/solvent B% was 0/99/1, 8/60/40, 13/46/54, 15/0/100, 18/0/100, 21/99/1, and 25/99/1). The flow rate was set as 1 mL/min. The temperature of the column thermostat was adjusted as 35°C, and the diode array detector was used with a detection wavelength of 450 nm .High-performance liquid chromatography (HPLC) was conducted for μL of methanol and analyzed using methanol : water  as a mobile phase flowing at a rate of 1 ml/min under isocratic conditions by HPLC . Ergosterol was visualized using a diode array detector at 280 nm channel. The standard of ergosterol was bought from Sigma .Ergosterol was extracted according to the previous investigation . In shorp < 0.05 was considered statistically significant.All the experiments were carried out in triplicate to get the average value of the data and error bars by ± standard deviation. All statistical analyses were performed using a two-way ANOVA with multiple comparison tests. These calculations were done by using the GraphPad Prism software for Windows . β-carotene production to a different extent. As shown in β-carotene 1.68 mg/L (0.17 mg/g) and biomass (9.70 g/L). Fructose was found as a good source for the growth of CBS 277.49, but the production of β-carotene was lower than that in glucose, almost 0.8-fold less than that in glucose, i.e., 1.42 mg/L (0.14 mg/g), so glucose was chosen as a carbon source for further experiments. It was also observed that M. circinelloides grew well while using maltose as a carbon source but produced a lesser amount of β-carotene. However, glycerol and molasses were not found as good carbon sources for β-carotene production by strain CBS 277.49  was found at a C/N ratio of 20 with biomass of 9.93 g/L. The increasing C/N ratio resulted in the lower nitrogen content in the media. According to Ratledge, limitation of the nitrogen in the media increased the cascade of reactions leading to the formation of acetyl-CoA which then led to a more active fatty acid and terpenoid pathway [β-carotene production as shown in Another most critical parameter that influences the development of microbial lipids and carotenoid is the mass balance of carbon and nitrogen sources, in particular cultivation medium improvement . The caroduction . The max pathway . This reβ-carotene was observed, suggesting that both low and high nitrogen source concentrations could have a negative effect on carotenoid production [β-carotene production in Rhodotorula graminis RC04 [R. glutinis [β-carotene in Yarrowia lipolytica [However, with further increase in the C/N ratio to 40 and 80, decrease in cellular oduction . Ferrao nis RC04 . Braunwaglutinis . Similarpolytica . More repolytica .Mycoplasma species and to investigate the effect of cerulenin on growth [Cerulenin is known as a potent antifungal compound that can block fatty acid synthesis in many fungal and yeast strains . Cerulenn growth .μg/mL to achieve enhanced β-carotene production without having an adverse effect on cell growth. It was used in very little concentration as it is a potent antifungal drug and can affect fungal cell growth beyond certain levels. The amount of β-carotene produced by CBS 277.49 varied widely, depending upon the concentration of cerulenin added, as shown in β-carotene accumulation was confirmed by the decline in total lipid production in the cell. As the cerulenin concentration increased, a gradually decreasing trend in lipid production was observed, which corresponded to increased concentration of β-carotene, as presented in β-carotene 3.23 mg/L (0.31 mg/g) was obtained at a concentration of 40 μg/mL, with a decline in total lipid production up to 10% from 14% by CBS 277.49. Further increasing the concentration of cerulenin was found to inhibit cell growth, and even a decline in β-carotene accumulation was also observed, which might be attributed to the cytotoxic effect of the high dose of cerulenin and partial inhibition of HMG-CoA synthase activity [β-carotene accumulation at high cerulenin dose (80 and 100 μg/mL) could also be explained as feedback inhibition at the metabolic level; i.e., high concentration of cerulenin might cause a decline in lipid production (up to 6.3%) in CBS 277.49, resulting in excess of free carotenoid which might inhibit the carotenogenic enzyme, causing a decrease in the de novo synthesis of β-carotene [β-carotene production by an optimal concentration of cerulenin (40 μg/mL) as compared to the control flask (1.68 mg/L) in which no cerulenin was added. These results concluded that the acetyl-CoA pool was successfully redirected towards the mevalonate pathway from the FAS pathway in CBS 277.49 by using cerulenin.In the current study, cerulenin was added in different concentrations such as 5, 10, 20, 40, 80, and 100 Phaffia rhodozyma, termed as MK19 [Mucor racemosus and found that a concentration of 0.8 μg/mL was effective in the complete inhibition of morphogenesis in M. racemosus [β-carotene accumulation was inversely correlated with total lipid percentage in CBS 277.49 by redirecting metabolic flux of acetyl-CoA in CBS 277.49. This was according to Miao et al. who also reported an inverse correlation between FAS and the mevalonate pathway [Miao et al. reported the use of another fatty acid inhibitor, triclosan, and found a two-fold increment in astaxanthin production by a mutant strain of  as MK19 . Ito et acemosus . Our fin pathway .M. circinelloides [β-carotene synthesis. Hence, we speculated that enhanced production of β-carotene could be achieved by redistribution of FPP from sterol towards the carotenoid biosynthesis pathway by using ketoconazole in CBS 277.49. To test this hypothesis, ketoconazole was used in different concentrations based on the previous literature [Mucor spp. were reported to produce 10.82 μg/mg of ergosterol [It has been described in the literature that carotenoid and ergosterol are part of the highly conserved mevalonate pathway in elloides . These tterature . Since tterature . Mucor sgosterol .μg/mg in CBS 277.49, as shown in β-carotene in CBS 277.49 by diverting FPP towards carotenoid synthesis. The optimal production of β-carotene corresponding to 3.45 mg/L (0.35 mg/g) was achieved at 150 mg/L ketoconazole, accompanied by a reduction of ergosterol concentration (4.16 μg/mg) without having negative effect on growth. This value corresponded to a 105% increment in β-carotene production in comparison to the control culture (1.68 mg/L) in which no ketoconazole was added. It was observed that further increasing the concentration (200 mg/L) has slightly affected the growth and β-carotene production of Mucor  is shown in In the present study, the synergistic effect of the C/N ratio, cerulenin, and ketoconazole was checked and optimized for superior mization . It creamization . The conβ-carotene productions by CBS 277.49  was achieved at std. order 11 with the combination of C/N 25, 10 μg/mL cerulenin, and 150 mg/L ketoconazole. On the other hand, other combinations of factors were shown to be lower than those of individual supplementation of the C/N ratio, cerulenin, and ketoconazole which achieved maximum production of 3.29 (0.31 mg/g), 3.23 (0.31 mg/g), and 3.45 mg/L (0.35 mg/g) at C/N 20, 40 μg/mL, and 150 mg/L, respectively. Similarly, the interaction of factors that were visualized by the three-dimensional (3D) response surface also recorded the same pattern where minimal β-carotene production range between 1.5 and nearly 3.0 mg/L was observed .The optimum levels of the variables for β-carotene production by CBS 277.49, further experiments were carried out, under the estimated suggested conditions created by the software and compared to the std. order 11 (maximum production of β-carotene), KCN , and control (standard K&R medium)  were comparable to the predicted values proposed by the model. Hence, this experiment validated the model, and CCD can be used as an optimization approach to enhance the production of β-carotene in CBS 277.49.To validate the predicted value of  medium) . The resβ-carotene production under the suggested condition was approximately 76.80% higher as compared to the control, it was significantly lower than that of the std. order 11, which achieved nearly 4.26 mg/L (0.43 mg/g) of β-carotene production . This observation was different than the individual supplementation of cerulenin, where moderate concentration (40 μg/mL) of cerulenin was needed to obtain maximum β-carotene production. However, when the cerulenin was omitted from the mixture (KCN), the β-carotene production dropped noticeably as compared to the std. order 11, signifying the role of cerulenin in the mixture to achieve maximum β-carotene production by CBS 277.49 of β-carotene was produced as compared to control, which only showed 1.68 mg/L (0.17 mg/g)of β-carotene production. The result suggested that RSM has the benefit of recognizing the most significant medium components and their optimal levels and was useful for functioning the fermentation process towards the accumulation of the desired metabolite. Furthermore, it was also suggested that being a model strain of carotenogenesis and valuable lipid production, M. circinelloides can be used as a promising zygomycete after medium optimization and genetic improvement for the biotechnological production of β-carotene and lipid.The present study optimized the medium components for attaining high


Low muscle strength is a powerful predictor of negative health‐related events and a key component of sarcopenia. The lack of normative values for muscle strength across ages hampers the practical appraisal of this parameter. The aim of the present study was to produce normative values for upper and lower extremity muscle strength across a wide spectrum of ages, in a large sample of community‐dwellers recruited in the Longevity check‐up (Lookup) 7+ project.Lookup 7+ is an ongoing project that started in June 2015 and conducted in unconventional settings  across Italy with the aim of fostering the adoption of healthy lifestyles in the general population. Candidate participants are eligible for enrolment if they are 18+ years and provide written informed consent. Upper and lower extremity muscle strength is assessed by handgrip strength and five‐repetition chair‐stand [5 × sit‐to‐stand (STS)] tests, respectively. Cross‐sectional centile and normative values for handgrip strength and 5 × STS tests from age 18 to 80+ years were generated for the two genders. Smoothed normative curves for the two tests were constructed for men and women using the lambda‐mu‐sigma method.From 1 June 2015 to 30 May 2019, 11 448 participants were enrolled. The mean age of participants was 55.6 years , and 6382 (56%) were women. Normative values for handgrip strength and the 5 × STS test, both absolute and normalized by body mass index, were obtained for men and women, stratified by age groups. Values of upper and lower extremity muscle strength across ages identified three periods in life: an increase to peak in young age and early adulthood (18–24 years), preservation through midlife (25–44 years), and a decline from midlife onwards (45+ years).Our study established age‐specific and gender‐specific percentile reference values for handgrip strength and the 5 × STS test. The normative curves generated can be used to interpret the assessment of muscle strength in everyday practice for the early detection of individuals with or at risk of sarcopenia. During the last two decades, sarcopenia has received increasing attention by researchers and clinicians, such that it is now recognized as a ‘true’ disease with its specific International Classification of Diseases (ICD)‐10 code.Muscle strength remains substantially constant during the first decades of adulthood, starts declining at middle age (45–50 years), and drops remarkably in late life.The aim of the present study was to produce cross‐sectional centile values for handgrip strength and 5 × STS tests across ages, using a large sample of community‐dwellers enrolled across Italy in the Longevity Check‐up 7+ (Lookup 7+) project.Data used in the present study are from the Lookup 7+ project, an initiative promoted by the Department of Geriatrics of the Fondazione Policlinico ‘Agostino Gemelli’ IRCCS at the Università Cattolica del Sacro Cuore .Between 1 June 2015 and 30 May 2019, 11 448 persons were enrolled in different Italian cities adhering to the national Lookup 7+ project. Candidate participants were considered to be eligible for enrolment if they were at least 18 years old and provided written informed consent. Exclusion criteria were self‐reported pregnancy, inability to perform the physical performance tests (e.g. wheel‐chaired), or inability to give written informed consent. Analyses of the present study were conducted in 11 331 persons for handgrip strength analysis (after excluding 117 participants with missing handgrip strength measurement data) and 11 164 participants for 5 × STS analysis (after excluding 284 people with missing 5 × STS test measurement data). The general characteristics of the excluded participants did not differ from those with complete data for the variables of interest.2).All persons who accepted to participate in the study were offered individual assessment consisting of a brief questionnaire and the objective measurement of several parameters. In particular, the Lookup 7+ visit was structured to collect the following information and data: informed consent, lifestyle habits , and measurement of body mass, height, blood pressure, blood glucose, total cholesterol levels, and muscle strength.Handgrip strength and 5 × STS tests were conducted according to the EWGSOP2 recommendations.Lower extremity muscle strength was assessed via the 5 × STS test. Participants were asked to stand up from a chair with their arms folded across the chest five consecutive times as quickly as possible. A standard armless chair, 43–47 cm in height, was used. The chair's height was chosen to allow participants placing their feet comfortably on the floor and to minimize the influence of chair's seat height on the test results.All analyses were performed using the SPSS software version 11.0 . Continuous variables are shown as mean ± standard deviation (SD), categorical variables as frequencies by absolute value and percentage of the total. Descriptive statistics were run to describe demographic and main clinical characteristics of the study population according to age groups and gender.Smoothed percentile curves for handgrip strength and 5 × STS test values, both absolute and normalized by BMI, were constructed for men and women separately using the lambda‐mu‐sigma (LMS) method .The mean age of participants included in the analyses was 55.6 years , and 6382 (56%) were women. The general characteristics of the study sample according to age and gender are summarized in Table Normative values for handgrip strength (kg) in men and women, stratified by age groups, are shown in Tables Normative values for the 5 × STS test(s) in men and women, stratified by age groups, are shown in Tables Centile values for handgrip strength and the 5 × STS test indicate three periods in life: an increase to peak in early adulthood 18–24 years), maintenance through midlife (25–44 years), and decline from midlife onwards (45+ years). To help translate these data in clinical practice, ad hoc charts  and medications. However, it may reasonably be excluded that acute and/or severe illnesses were present at the time of evaluation. Indeed, candidate participants had to reach exhibition places and shopping centres, which might have selected a relatively healthy and functionally competent population. Although efforts were made to enrol a heterogeneous population, no detailed information was collected on the sociodemographic characteristics of participants. Hence, potential selection biases cannot be ruled out. It should however be considered that the unconventional setting in which the investigation was carried out required the assessment be completed within a reasonable amount of time. Finally, the study sample only included Caucasian persons; thus, our findings and charts may not be applicable to other ethnic groups.Apart from these limitations, the Lookup 7+ project offered the unique opportunity to measure upper and lower extremity muscle strength in a large and relatively unselected population across a wide age spectrum. The normative curves and centile values generated by our study may greatly facilitate the practical appraisal of muscle strength and allow the timely identification of people with or at risk for sarcopenia.Steering Committee: F. Landi and R. Bernabei.Coordination: E. Marzetti, R. Calvani, and L. Mariotti.Writing Panel: A. M. Martone, E. D'Angelo, E. Serafini, A. Picca, V. Palmieri, M. Tosato, P. Abete, A. Cherubini, F. Corica, and S. Salini.Field investigators: M. Bianco, D. Biscotti, V. Brandi, M. Broccatelli, L. Catalano, F. Ciciarello, M. D'Elia, M. T. Fuga, E. Ortolani, G. Savera, and M. B. Zazzara.Participants: A. Almohani, F. Benvenuto, M. Bianchi, G. Bramato, R. Capitani, C. Celesti, G. Colloca, F. Cuccaro, L. Damato, E. Di Murru, S. Fabrizi, D. Fusco, A. Galliani, V. Galluzzo, S. Gervasi, R. Quarta, G. Modica, R. Monti, S. Orvieto, C. Pais, C. Pantanelli, S. Rocchi, D. Ronconi, E. Rota, F. Sollazzo, and M. Tritto.Carni Sostenibili, Danone Italia, Errekappa, Ferrarini, Italfarmaco, Italia Longeva, IVSI, Laborest, Yakult, Marche Region, Nutricia, ProAction, Serenissima, and Novartis supported the Lookup 7+ project. The study was also partly supported by intramural research grants from the Università Cattolica del Sacro Cuore (D3.2 2013 and D3.2 2015) and by the non‐profit research foundation ‘Centro Studi Achille e Linda Lorenzon’.All of the authors participated in the conceiving, design, and writing of the manuscript. All authors read and approved the final manuscript.None to disclose.Figure S1. Reference percentiles of handgrip strength normalized by body mass index for men aged 18 to 80+ years. The 5th, 25th, 50th, 75th, and 95th percentiles are shown in black, red, green, blue and purple, respectively.Click here for additional data file.Figure S2. Reference percentiles of handgrip strength normalized by body mass index for women aged 18 to 80+ years. The 5th, 25th, 50th, 75th, and 95th percentiles are shown in black, red, green, blue and purple, respectively.Click here for additional data file.Figure S3. Reference percentiles of the 5‐repetition chair‐stand test normalized by body mass index for men aged 18 to 80+ years. The 5th, 25th, 50th, 75th, and 95th percentiles are shown in black, red, green, blue and purple, respectively.Click here for additional data file.Figure S4. Reference percentiles of the 5‐repetition chair‐stand test normalized by body mass index for women aged 18 to 80+ years. The 5th, 25th, 50th, 75th, and 95th percentiles are shown in black, red, green, blue and purple, respectively.Click here for additional data file.Table S1. Normative values for handgrip strength normalized by body mass index in men, stratified by age.Click here for additional data file.Table S2. Normative values for handgrip strength normalized by body mass index in women, stratified by age.Click here for additional data file.Table S3. Normative values for the 5‐repetition chair‐stand test normalized by body mass index in men, stratified by age.Click here for additional data file.Table S4. Normative values for the 5‐repetition chair‐stand test normalized by body mass index in women, stratified by age.Click here for additional data file.


Lactobacillus sp. bacteria (12 strains) and S. cerevisiae yeast (6 strains) towards mycotoxins, such as aflatoxin B1, deoxynivalenol, fumonisins, T-2 toxin and zearalenone, which pose as frequent feed contamination. The experiment involved analysing changes in concentration of mycotoxins in PBS solutions, after 6, 12 and 24 h of incubation with monocultures of tested microorganisms, measured by high-performance liquid chromatography (HPLC). We found that all strains detoxified the mycotoxins, with the highest reduction in concentration observed for the fumonisin B1 and B2 mixture, ranging between 62 and 77% for bacterial strains and 67–74% for yeast. By contrast, deoxynivalenol was the most resistant mycotoxin: its concentration was reduced by 19–39% by Lactobacillus sp. strains and 22–43% by yeast after 24 h of incubation. High detoxification rates for aflatoxin B1, T-2 toxin and zearalenone were also observed, with concentration reduced on average by 60%, 61% and 57% by Lactobacillus, respectively, and 65%, 69% and 52% by yeast, respectively. The greatest extent of reduction in the concentration for all mycotoxins was observed after 6 h of incubation; however, a decrease in concentration was noted even after 24 h of incubation. Thus, the tested microorganisms can potentially be used as additives to decrease the concentrations of toxins in animal feed.The aim of the following research was to determine the detoxification properties of probiotic  Ascomycota phylum. The most common source of food and feed contamination are mycotoxins produced by the fungi Aspergillus, Penicillium and Fusarium genera [Alternaria, Chaetomium, Cladosporium, Claviceps, Diplodia, Myrothecium, Monascus, Phoma, Phomopsis, Pithomyces, Trichoderma and Stachybotrys [1 is most frequently found in feed in the European Union (> 98% of tested samples); however, DON (~ 90% of tested samples) and ZEN (~ 70% of tested samples) are often detected as well. The presence of FUM and OTA, on the other hand, are more sparsely observed [Mycotoxins are secondary metabolites with low molecular mass ~ 700 Da), which are synthesised by filamentous fungi, belonging mostly to the m genera –3. Otherhybotrys . Aflatoxhybotrys , 6. AFB100 Da, whFusarium sp.). Likewise, under favourable growth conditions of temperature and humidity, mycotoxin-producing fungi, such as Aspergillus sp. and Penicillium sp., are also found in food and feed that are stored [Plants are contaminated with mycotoxins, synthesised by filamentous fungi, most frequently at the time of cultivation in the fields  2017/625 of the European Parliament and of the Council of 15 March 2017, regarding official controls and other official activities performed to ensure the application of food and feed law, rules on animal health and welfare, plant health and plant protection products will come into effect on 14th December 2019. According to this regulation, Member States are obliged to establish multiannual plans and carry out food and feed controls, to ensure safety in the agri-food chain, as well as animal welfare and health, thereby providing safe food [The process of detoxification or mycotoxin removal is complicated, especially because of heat stability of these compounds, and their breakdown into toxic products. Nevertheless, detoxification may be accomplished by application of the following methods: physical , chemical , and biological . For bioLactobacillus, Bifidobacterium, as well as Enterococcus faecium, and the yeasts Saccharomyces cerevisiae and Saccharomyces boulardii have been shown to have mycotoxin detoxification properties [In 1998, El-Nazami performed pioneering in vitro studies on the binding properties of mycotoxin by lactic acid bacteria, which has initiated a systematic search for microorganisms having specific abilities to adsorb mycotoxins . Among ooperties .Lactobacillus sp. and S. cerevisiae towards mycotoxins, which often contaminate feed for livestock animals. This study was part of one of the stages of strain selection for designing synbiotic preparations for poultry and swine.The aim of our study was to determine detoxification properties of probiotic strains of Lactobacillus genus and strains of the yeast S. cerevisiae, which are deposited in the Łódź Collection of Pure Cultures 105 of Institute of Fermentation Technology and Microbiology at Technical University of Łódź  and one S. cerevisiae (ŁOCK 0119) strain have been documented in patent application no. 422603 [The biological material included potential probiotic bacteria of dź Table . Five La. 422603 .Table 2SThe detoxification activity of bacteria and yeast was determined against five mycotoxins, namely aflatoxin B1, fumonisin mixture of fumonisin B1 and B2 (FUM), T-2 toxin, zearalenone and deoxynivalenol Table . MycotoxLactobacillus sp. were cultivated in de Man, Rogosa, and Sharpe (MRS) broth  at 37 °C, while S. cerevisiae was grown at 30 °C, in yeast extract–peptone–glucose broth . Both bacteria and yeast cultures were grown in normal oxygen conditions for 24 h. After 24 h of incubation, monocultures of the analysed strains, in three repetitions, were centrifuged at relative centrifugal force (RCF) 3468×g for 10 min . Subsequently, the supernatants were removed and the bacteria and yeast biomass were washed three times with PBS buffer to remove any residual culture medium. The cell pellets were again centrifuged under the same conditions. Ten millilitres of mycotoxin solutions were added to the prepared samples, with a defined concentration of 100 μg/ml for each mycotoxin. These samples were further incubated in normal oxygen conditions for 24 h at 37 °C or 30 °C for lactic acid bacteria and yeast, respectively. After 6, 12 and 24 h of incubation, 2 ml of each sample was collected, centrifuged at RCF of 3468×g for 10 min, and the supernatants were filtered with PTFE syringe filters with 0.22-μm-diameter pores . As a positive control sample, a solution of analysed mycotoxin in PBS was used, and bacterial or yeast suspension served as negative control sample.The prepared samples were subjected to high-performance liquid chromatography (HPLC) analysis, the parameters of which are presented in Table p < 0.05 . A comparative Duncan test was carried out at a significance level of p > 0.05 .The results presented here constitute the arithmetic mean of values from three repetitions, with standard deviation. All statistical analyses were carried out using the one-way ANOVA test, with a significance level of Lactobacillus genus were characterised by their diverse ability to detoxify aflatoxin B1. In merely after 6 h of incubation, a statistically significant reduction of AFB1 concentration was noticed, ranging from 35.33 to 79.65 μg/ml  compared to the initial mycotoxin concentration of 100 μg/ml. In subsequent hours, further reduction in AFB1 concentration was observed. After 24 h of incubation, the concentration of mycotoxin in the samples was 28.96–55.80 μg/ml . Therefore, there was a reduction of between 44 and 71%  compared to the initial concentration of the mycotoxin.Bacteria belonging to the S. cerevisiae showed detoxification activity similar to that of analysed strains of Lactobacillus sp. After 6 h of incubation, AFB1 concentrations were statistically significantly reduced by 47–66% (average 58%) and ranged from 33.64 to 53.27 μg/ml. In subsequent hours of incubation, the concentration of the mycotoxin further decreased, and after 24 h, the mycotoxin concentrations were 32.48–4.45 μg/ml (average reduction of AFB1 by 65%)  concentrations were significantly reduced after 6 h of incubation in the presence of the bacteria monoculture, varying between 78.39 and 94.24 μg/ml , indicating that these bacteria have the ability to decrease DON concentration by an average of 16%. Further decrease in DON concentration in the subsequent incubation hours was also observed. After 24 h, DON concentrations ranged between 60.63 and 80.72 μg/ml , thereby showing a reduction of 19–39% .S. cerevisiae strains used in the analysis also demonstrated the ability to reduce the concentration of DON in suspension. After 6 h, significant reductions of DON concentrations were observed, ranging between 12 and 22%  of the initial concentration of DON. After a further 6 h, the concentrations were 65.57–80.81 μg/ml . After 24 h of incubation, the DON concentrations were reduced by 22–40%  relative to the initial concentration of mycotoxin (Table Lactobacillus genus detoxified the mixture of fumonisin B1 (FB1) and B2 (FB2) mycotoxins (FUMs). After 6 h of incubation, the FUM concentration was reduced by 36–64%  compared to the initial concentration of the mycotoxin mixture. Subsequent incubation resulted in a further significant decrease in FUM concentration, which after 24 h of incubation was reduced by an average of 70% and ranged from 23.08 to 38.42 μg/ml .Bacteria belonging to S. cerevisiae also reduced the concentrations of FUM, significantly reducing them by 29–60%  to 40.15–70.57 μg/ml . In subsequent hours of incubation, the concentrations of mycotoxin further declined. Finally, after 24 h, the FUM concentration ranged between 25.53 and 32.57 μg/ml, demonstrating an average reduction of 72% of the initial concentration . Continued incubation caused a further decrease in T-2 concentration, as a result of which after 24 h of incubation the concentrations of mycotoxin were 31.09–50.10 μg/ml . This shift in concentration values indicated a reduction of 50–69%  in relation to the initial quantity of T-2.After 6 h of incubation, significant reduction of T-2 toxin concentrations by monocultures of analysed S. cerevisiae strains subjected to the analysis were characterised by diverse T-2 detoxification activity. After 6-h incubation, a statistically significant decrease in the concentration of T-2 to level of 46.92–54.98 μg/ml  was observed. In subsequent hours of incubation, T-2 concentration further decreased. The concentration of the mycotoxin after 24 h of incubation reduced by 60–63%  of the initial concentration and ranged between 37.36 and 40.40 μg/ml   detoxification. After only 6 h of incubation, a statistically significant reduction of ZEN concentration was observed, which ranged between 28 and 59%  relative to the initial mycotoxin concentration of 100 μg/ml. In subsequent hours of incubation, a further reduction in the mycotoxin concentration was noted. After 24 h of incubation, the concentration of ZEN was 27.39–60.05 μg/ml . Therefore, a reduction of 40–73%  in relation to the initial concentration of the ZEN was observed in monocultures incubated with Lactobacillus sp.S. cerevisiae also demonstrated the ability to reduce the concentration of ZEN, and after 6 h of incubation, a significant decrease of 24–42% (average 34%) to level of 57.64–75.60 μg/ml  was observed. In subsequent hours of incubation, the concentration of the mycotoxin further reduced, and after 24 h, ZEN concentrations ranged between 41.88 and 55.84 μg/ml  (Table n) Table .Table 9R1, DON, FUM, T-2, ZEN) detoxification properties of Lactobacillus sp. and S. cerevisiae were strain-dependent. Both bacteria and yeast strains, subjected to analysis, showed the highest detoxification activity towards FUM. The most resistant mycotoxin was DON.Based on these results, we concluded that mycotoxin  should be maintained, including crop rotation, cultivation of resistant plants, ploughing, irrigation, chemical and biological control of plant diseases and proper use of chemicals (e.g. fungicides) , 34, 35.Lactobacillus sp. and S. cerevisiae, are used in case of mycotoxin contamination of food and fodder [Lactobacillus sp. are able to bind mycotoxins mostly to cell wall peptidoglycans, polysaccharides and teichoic acid, primarily through hydrophobic interactions, whereas S. cerevisiae are bind toxic metabolites of filamentous fungi to the cell wall. In addition, microorganisms biodegrade mycotoxins that prevent adsorption of these components inside the intestines on animals that feed on the food [Among other mycotoxin detoxification methods, microorganisms, inter alia probiotic strains of d fodder . Lactobathe food , 37, 38.1, DON, FUM, T-2, ZEN in PBS solution by 12 strains of bacteria from Lactobacillus genus, and 6 S. cerevisiae strains.In this article, in vitro results demonstrated the ability to reduce the concentration of mycotoxins: AFB1 concentration in PBS solution was reduced by the tested strains of bacteria to varying degrees, on average 49.44% after the first 6 h of incubation, by another 4.82% during the next 6 h of incubation, and maintained within a range of 44.20–71.04 μg/ml (average reduction of 59.97%) after 24 h of incubation. The greatest amount of AFB1 was bound by bacteria strains after 6 h of incubation, suggesting that the adsorption of the mycotoxin by Lactobacillus is an immediate process. Haskard et al. (2001) tested the ability of eight Lactobacillus strains to bind AFB1 to bacterial surfaces using ELISA; their results were similar to ours [The AFB to ours .1 binding assays with ELISA, confirmed results obtained by Haskard et al. (2001), and observed that live cells of Lactobacillus casei Shirota were more efficient in binding mycotoxin, than heat-treated organisms [1 by Lactobacillus sp. In these studies, AFB1 was bound by the tested strains of bacteria in the range of 14–49%, 20.88–59.44% and 0–85%, respectively [Lactobacillus strains to AFB1 could be the result of differences in cell wall structure, especially in terms of teichoic acid and peptidoglycan content [1 by Lactobacillus sp. is a rapid process, which was also confirmed in research conducted by Kumar et al.(2018), as well as in our studies [S. cerevisiae strains varied in their reduction of AFB1 concentration , which is consistent with the results obtained by Pizzolitto et al. (2012a) and Poloni et al. (2017) [1 detoxification, by bacteria and yeast, was also observed in the results of studies by Pizzolitto et al. (2011) [1 was similar to that of the tested Lactobacillus, and what is also known in the case of yeast is that the cell wall components are also responsible for the binding of the AFB1 mycotoxin [Liew et al. (2018), who also performed AFBrganisms . Hernandectively –43. The  studies . Moreove. (2017) , 47. Str. (2011) . The detycotoxin .Lactobacillus isolated from the intestinal content of monogastric animals showed higher binding activity towards DON, than did strains derived from plant silages. After 6 h of incubation, DON concentration was reduced by the bacterial strains tested by 5.76–21.64% ; after 12 h, it was reduced by an average of another 7.39%; and after 24 h, the concentration of DON was reduced by 19.28–39.37%  of the initial concentration of the mycotoxin. Niederkorn et al. (2007) showed low detoxification activity of 137 Lactobacillus strains, which was also observed in the strains used in our study [Lactobacillus rhamnosus RC007, based on their results, they concluded that Lact. rhamnosus contributed to counteract toxic effect of DON and helped to maintain healthy gastrointestinal tract of pigs [S. cerevisiae were demonstrated, decreasing the concentration of DON by 18.01%, on average, after 6 h of incubation, by 8.80% after another 6 h and an average of 33.03% of the initial concentration after 24 h of incubation. These results are in line with results obtained by Campagnollo et al. (2015) [On the basis of the results obtained, we conclude that strains of ur study . Similar of pigs –52. In o. (2015) . Based o. (2015) , 53.Lactobacillus strains analysed reduced FB1 and FB2 mixture concentrations by the most significant amounts among all mycotoxins tested. The FUM concentrations decreased on average by over 51.23% after 6 h of incubation, and a further 8.61% after 12 h. After 24 h of incubation, the concentrations were maintained at an average level of 29.53 μg/ml . The greatest reduction in concentration occurred after 6 h, while the FUM binding process was observed up to 24 h of incubation, which is comparable to the results obtained by Abbès et al. (2016) [Lactobacillus is rapid, as they observed the binding after 1 h of incubation [1 removal (61.7%) by Lact. plantarum MYS6, after 4 h of incubation; furthermore, they observed that tested strain suppressed fumonisin biosynthesis [Lactobacillus sp., which was dependent on the strain tested and mycotoxin type, coincided with the results obtained by Niderkorn et al. (2007), Abbès et al. (2016) and Zhao et al. (2016) [1 detoxification might not only be a matter of adsorption, but also the result of biodegradation to other compounds ; however, this was not observed for all analysed Lactobacillus sp. strains in their study [S. cerevisiae strains reducing FUM concentration was also shown, and the results agree with those of Pizzolitto et al. (2012b) [1 binding properties of S. cerevisiae yeast (strain RC016), observed that ability of binding the mycotoxin increases along with concentration [The 12 . (2016) . Zhao etcubation . In factcubation . Deepthiynthesis . In our . (2016) , 54, 55.ir study . In our  (2012b) . Furtherntration . Our resLactobacillus and S. cerevisiae to a high degree, where reduction exceeded a mean of 60% after 24 h of incubation. After 6 h of incubation, the Lactobacillus strains reduced the concentration of T-2 by 26.68–51.75% (average 42.64%), and after 24 h, it remained at a level of 31.09–50.10 μg/ml (average 39.01 μg/ml). Bacterial strains used in our study showed higher T-2 binding affinity than shown by the four strains analysed by Zou et al. (2012) [S. cerevisiae strains show significantly stronger detoxification properties (average T-2 binding after 24 h of incubation was 69.18%) than the tested bacterial strains (average decontamination rate after 24 h was 61.45%). Zou et al. (2015) obtained a similar level of T-2 detoxification by S. cerevisiae strains in their studies and found the yeast’s reduction of the concentration of T-2 toxin was also the result of mycotoxin binding to the yeast cell wall [The concentrations of T-2 toxin were decreased by the tested strains of Lactobacillus strains to bind ZEN was similar to that observed in the case of AFB1 and the detoxification level of the mycotoxin was mean of 56.90% after 24 h of incubation. Our results showed that the greatest reduction in ZEN concentrations, by 27.55–58.79% , occurred after 6 h of incubation. The ZEN concentrations were reduced further by an average of 8.17% after the next 6 h of incubation, finally reaching an average level of 43.10 μg/ml after 24 h . The capability of Lactobacillus strains to decrease ZEN concentration was also found by Zhao et al. (2015); ZEN binding activities on the cell walls of the 27 tested Lact. plantarum strains were weaker compared to those of the strains we used in our study [Lactobacillus is an immediate process, which is consistent with the results we obtained [Lactobacillus sp. to reduce ZEN concentration [S. cerevisiae strains reduced ZEN concentration by 24.40–42.36% (average 34.45%) after 6 h of incubation, with an additional 11.59% after 12 h and an average 5.82% after 24 h. Similar results were obtained by Keller et al. (2015); they concluded that at the beginning, large amounts of ZEN is bound to the yeast cell wall due to the higher concentration of the mycotoxin in the environment [The ability of ur study . Furtherobtained . Niderkontration –65. We fironment . The ZENironment , 67.Lactobacillus and S. cerevisiae to lower the concentrations of the mycotoxins AFB1, DON, FUM, T-2 and ZEN. We found that the detoxification of these compounds is a rapid process. To a great extent, the concentration of the toxins decreases within 6 h of incubation, and can last up to 24 h. Nevertheless, the differences in concentrations were not so significant at a prolonged period of 24-h incubation.Our data allowed us to observe the potential of Lactobacillus and S. cerevisiae strains analysed showed detoxification properties towards mycotoxins  in this in vitro study. Therefore, these strains, after further investigation, could be used as food and feed additives to detoxify contaminating mycotoxins, which pose potential threats to human and animal health.The