Establishment of Microbial Limit Test Methods for Two Hospital Preparations

[Objectives] This study was conducted to establish microbial limit test methods for traditional Chinese medicine preparations Yunpi Granules and Bupi Qiangli Paste. [Methods] According to General Rules of Part IV of Chinese Pharmacopoeia, applicability tests were conducted on microbial limit test methods for the above two traditional Chinese medicine preparations by the plate method. [Results] The established methods showed recovery values in the range of 0.5-2.0 for both experimental strains, and the control bacteria could be detected in the experimental group, but not in the negative control group. [Conclusions] The microbial limit test methods were reliable for the two traditional Chinese medicine preparations and could be used for quality control.

Applicability of Microbial Limit Test Method for Qingyan Zhisou Powder

[Objectives]This study was conducted to establish a microbial limit test method for Qingyan Zhisou Powder.[Methods]Applicability tests were carried out according to Microbial Limit Tests of Non-sterile Products,General Rules 1105-1107 of Part IV of Chinese Pharmacopoeia,2020.[Results]Qingyan Zhisou Powder had a strong bacteriostatic effect on Pseudomonas aeruginosa.After increasing the dilution ratio(1:40),the bacteriostatic effect was obviously eliminated.When the total number of aerobic microbes was determined by the test liquid dilution method(1:40),the recovery values were in the range of 50%-200%;and when using the test liquid(1:10)to determine the total number of mold and yeasts,the recovery ranged from 50%to 200%.Escherichia coli,Salmonella and bile-tolerant Gram-negative bacteria were detected by both the routine method and dilution method in experimental groups.[Conclusions]The microbial limit test of Qingyan Zhisou Powder adopted the test liquid dilution method,and the routine method could be used for the test of Escherichia coli,Salmonella and bile-tolerant Gram-negative bacteria.The applicability test of the method is feasible,with scientific and accurate results,and the method can effectively control the quality of the preparation and is recommended for popularization.

Methodology Validation of Microbial Limit Test of Bupi Qiangli Ointment

[Objective] This study was conducted to establish a microbial limit test method for Bupi Qiangli Ointment. [Method] The conventional method and medium dilution method were used for bacterial, mold and yeast counting in sample recovery test. [Result] The medium dilution method （1：10 test solution, 0.5 ml/plate） could effectively eliminate the inhibition effect of the Bupi Qiangli Ointment, and the recovery of Staphylococcus aureus was greater than 70% in the 3 batches of samples; and the conventional method exhibited the recoveries of E. coil, Bacillus subtilis, Candida albicans and Aspergillus greater than 70% in the 3 batches of samples. [Conclusion] Due to Bupi Qiangli Ointment has strongly antibacterial effect on Staphylococcus au- reus, the medium dilution method was used for bacterial counting, and the conventional method was used for mold and yeast counting; and the conventional method was used for controlled bacterium examination of E. coll.

Microbial Limit Test of Zhideke Granules

[Objectives]To test the microbial limit of Zhideke granules.[Methods]According to the relevant regulations on the testing of granules in Chinese Pharmacopoeia in 2015,the applicability of the methods for counting the total number of aerobic bacteria,molds and yeasts and objectionable organisms in Zhideke granules was studied.[Results]The total number of aerobic bacteria in Zhideke granules was determined by dilution plate method(1∶20,1 mL per dish),the total number of molds and yeasts was determined by conventional plate method(1∶10,1 mL per dish),and E.coli was detected by conventional method of TTB(1∶10 test solution,10-100 mL TSB).[Conclusions]The method was suitable for the microbial limit test of Zhideke granules.

Validation of Microbial Limit Test Methods of Tongmai Tangyanming Capsule

[ Objectives] This study was conducted to establish a method for microbial limit test of Tongmai Tangyanming Capsule. [ Methods] According to the Chinese Pharmacopoei 2015 edition of the four general rules, the microbiological limits of non-sterile products were verified. [ Results] Tongmai Tangyanming Capsule has a strong inhibitory effect on Bacillus subtilis and Candida albicans. The antimicrobial activity was significantly eliminated after increasing the diluent （ 1 ： 20） ; and the recoveries were in the range of 0.5 -2 when the total quantities of aerobic microbes were determined by dilution method （1：20）. When the total quantities of mould and yeast were determined by dilution method （1：20）, the recoveries were from 0.5 to 2, and Escherichia coli, cholate-tolerant Gram-negative bacteria and Salmonella can be detected by the test solution dilution method. [ Condusions] With the plate method, the total quantities of aerobic bacteria and the quantities of mould and yeast were examined by increasing diluent, and conventional method was used for examination of control bacteria including E. coli, cholatetolerant Gram-negative bacteria and Salmonella. This method has been proved effective for microbial limit test of Tongmai Tangyanming Capsule, and can effectively control the quality of the preparation. The method is accurate and reliable.

Validation of Microbial Limit Test Methods of Compound Gangbangui Capsule

[Objectives] This study was conducted to establish a method for microbial limit test of Compound Gangbangui Capsule. [Methods] According to the Chinese Pharmacopoei 2015 edition of the four general rules,the microbiological limits of non-sterile products were verified. [Results]Compound Gangbangui Capsule has a strong inhibitory effect on S. aureus,and the antimicrobial activity was significantly eliminated after increasing the diluent（ 1∶ 20）; and the recoveries were in the range of 0. 5-2. 0 when the total quantities of aerobic microbes were determined by dilution method（ 1∶ 20）. When the total quantities of mould and yeast were determined by conventional method（ 1∶ 20）,the recoveries were from 0. 5 to 2. 0,and Escherichia coli can be detected in experimental groups by the test solution dilution method. [Conclusions]With the plate method,the total quantities of aerobic bacteria and the quantities of mould and yeast were examined by increasing diluent and conventional method,respectively,and control bacteria,E. coli was examined by conventional method. This method has been proved effective for microbial limit test of Compound Gangbangui Capsule,and can effectively control the quality of the preparation. The method is accurate and reliable.

Manipulated precipitation regulated carbon and phosphorus limitations of microbial metabolisms in a temperate grassland on the Loess Plateau,China

Manipulated precipitation patterns can profoundly influence the metabolism of soil microorganisms.However,the responses of soil organic carbon(SOC)and nutrient turnover to microbial metabolic limitation under changing precipitation conditions remain unclear in semi-arid ecosystems.This study measured the potential activities of enzymes associated with carbon(C:β-1,4-glucosidase(BG)andβ-D-cellobiosidase(CBH)),nitrogen(N:β-1,4-N-acetylglucosaminidase(NAG)and L-leucine aminopeptidase(LAP))and phosphorus(P:alkaline phosphatase(AP))acquisition,to quantify soil microbial metabolic limitations using enzymatic stoichiometry,and then identify the implications for soil microbial metabolic limitations and carbon use efficiency(CUE)under decreased precipitation by 50%(DP)and increased precipitation by 50%(IP)in a temperate grassland.The results showed that soil C and P were the major elements limiting soil microbial metabolism in temperate grasslands.There was a strong positive dependence between microbial C and P limitations under manipulated precipitation.Microbial metabolism limitation was promoted by DP treatment but reversed by IP treatment.Moreover,CUE was inhibited by DP treatment but promoted by IP treatment.Soil microbial metabolism limitation was mainly regulated by soil moisture and soil C,N,and P stoichiometry,followed by available nutrients(i.e.,NO^(-)_(3),NH^(+)_(4),and dissolved organic C)and microbial biomass(i.e.,MBC and MBN).Overall,these findings highlight the potential role of changing precipitation in regulating ecosystem C turnover by limiting microbial metabolism and CUE in temperate grassland ecosystems.

Plant above-ground biomass and litter quality drive soil microbial metabolic limitations during vegetation restoration of subtropical forests

Changes in litter quality(carbon:nitrogen,C:N)and above-ground biomass(AGB)following vegetation restoration significantly impact soil physicochemical properties,yet their effects on soil microbial metabolic limitations remain unclear.We measured litter quality,AGB,soil physicochemical properties,and extracellular enzyme activity(EEA)along a vegetation restoration gradient(7,14,49,70 years,and nearly climax evergreen broadleaved forests)in southern China.We also evaluated soil microbial metabolic limitations by a vector analysis of the EEA.Results revealed the soil microbial metabolisms were co-limited by C and phosphorus(P).The microbial C limitation initially decreased(before 14 years)and then increased,while the microbial P limitation initially increased(before 49 years)and then decreased.Partial least squares path modeling(PLS-PM)showed that the microbial C limitation was mainly attributed to microbial C use efficiency induced by litter quality,suggesting that microorganisms may transfer cellular energy between microbial growth and Cacquiring enzyme production.The microbial P limitation was primarily correlated with AGB-driven change in soil elements and their stoichiometry,highlighting the importance of nutrient stoichiometry and balance in microbial metabolism.The shifts between microbial C and P limitations and the strong connections of plant–soil-microbe processes during vegetation restoration revealed here will provide us with helpful information for optimal management to achieve forest restoration success.

Long-term warming does not affect soil ecoenzyme activity and original microbial nutrient limitation on the Qinghai-Tibet Plateau

Microbes play an important role in the carbon cycle and nutrient flow of the soil ecosystem.However,the response of microbial activities to long-term warming over decades is poorly understood.To determine how warming changes ecoenzyme activity and microbial nutrient limitation,we conducted a long-term,21 years,experiment,on the Qinghai–Tibet Plateau.We selected typical grass-and shrub-covered plots,used fiberglass open-top chambers(OTCs)to raise the temperature,conducted soil sampling at different depths,studied the response of nutrient-acquiring enzyme activity and stoichiometry,and conducted vector analysis of stoichiometry.Our results showed that long-term warming did not have a notable effect on the activity of nutrient-acquiring enzymes or enzymatic stoichiometry.However,Spearman correlation analysis indicated a significant and positive correlation between ecoenzyme activity and the available nutrients and microbial biomass in soil.Vector analysis of stoichiometry showed phosphorus limitation for all soil microbes at different depths,regardless of whether the soil experienced warming.These changes in enzymatic stoichiometry and vector analysis suggested that microbial nutrient limitation was not alleviated substantially by long-term warming,and warming did not considerably affect the stratification of microbial nutrient limitation.Our research has also shown that long-term warming does not significantly change soil ecoenzyme activity and original microbial nutrient limitation at different soil depths within the OTUsʼimpact range.These results could help improve understanding of microbial thermal acclimation and response to future long-term global warming.

Exogenous nitrogen input skews estimates of microbial nitrogen use efficiency by ecoenzymatic stoichiometry

Background Ecoenzymatic stoichiometry models(EEST)are often used to evaluate microbial nutrient use efficiency,but the validity of these models under exogenous nitrogen(N)input has never been clarified.Here,we investigated the effects of long-term N addition(as urea)on microbial N use efficiency(NUE),compared EEST and^(18)O-labeling methods for determining NUE,and evaluated EEST’s theoretical assumption that the ratios of standard ecoenzymatic activities balance resource availability with microbial demand.Results We found that NUE estimated by EEST ranged from 0.94 to 0.98.In contrast,estimates of NUE by the^(18)O-labeling method ranged from 0.07 to 0.30.The large differences in NUE values estimated by the two methods may be because the sum ofβ-N-acetylglucosaminidase and leucine aminopeptidase activities in the EEST model was not limited to microbial N acquisition under exogenous N inputs,resulting in an overestimation of microbial NUE by EEST.In addition,the acquisition of carbon by N-acquiring enzymes also likely interferes with the evaluation of NUE by EEST.Conclusions Our results demonstrate that caution must be exercised when using EEST to evaluate NUE under exogenous N inputs that may skew standard enzyme assays.

Coupling changes of soil functional gene abundances and extracellular enzyme activities across the diagnostic horizons of agricultural Isohumosols

Soil functional microbial taxa and extracellular enzymes are involved in a variety of biogeochemical cycling processes.Although many studies have revealed the vertical change patterns of microbial communities along soil profile,the general understanding of the coupling changes in the functional gene abundances(FGAs)and extracellular enzyme activities(EEAs)in soil profiles is still limited,which hinders us from revealing soil ecosystem processes.Herein,we comparatively investigated the FGAs and EEAs in the diagnostic A,B,and C horizons of soil profiles obtained from two suborders of Isohumosols(Mollisols),Ustic and Udic Isohumosols,in Northeast China based on quantitative real-time polymerase chain reaction and standard fluorometric techniques,respectively.The distribution patterns of both FGAs and EEAs significantly distinguished by the two soil suborders and were also separated from A to C horizon.Additionally,the variations of EEAs and FGAs were greater in Udic Isohumosols compared to Ustic Isohumosols along soil profiles,and greater changes were observed in C horizon than in A horizon.Both FGAs and EEAs correspondently decreased along the soil profiles.However,when normalized by soil organic carbon,the specific EEAs significantly increased in deep soil horizons,suggesting that microorganisms will input more resources to the production of enzymes to ensure microbial nutrient requirements under resource scarcity.More importantly,we revealed that soil microbial nutrient demands were limited by carbon(C)and phosphorus(P),and the C and P limitations significantly increased along soil profiles with a greater C limitation observed in Ustic Isohumosols than in Udic Isohumosols.Overall,our findings provided solid evidence showing the links between FGAs,EEAs,and microbial nutrient limitations,which would be helpful for a better understanding of the ecosystem processes in soil profiles.

Shifts in chemical and microbiological properties belowground of invader Ageratina adenophora along an altitudinal gradient

Tropical mountain ecosystems are usually colonized by numerous invasive plant species and represent an ideal‘natural laboratory’to study the effects of altitude on plant invasion.The aim of this study was to investigate the soil chemical and microbiological properties along an altitudinal gradient on a mountain colonized by the invader Ageratina adenophora.Rhizosphere soil of A.adenophora was collected over an altitudinal gradient(1400–2400 m)in Ailao Shan,China.We determined soil organic carbon(C),nutrient contents,enzyme activities,bacterial community composition as well as C and nitrogen(N)contents of the plant roots.Ecoenzymatic stoichiometric indices were calculated to estimate the relative C,N or P limitations of the microbial community.There was a significant effect of altitude on soil organic C in the rhizosphere,and a turning point in these measured variables was detected at an altitude of 2000 m.At low elevations,the rapid growth of invasive plants depleted the deficient phosphorus(P)in tropical soils,leading to microbial P limitation;at high elevations,microbes invested more energy to obtain C from resistant litter,leading to microbial C limitation.Bacterial beta diversity and soil pH contributed most to the altitudinal differences in ecoenzymatic stoichiometry,and Proteobacteria and Acidobacteria were the dominant bacterial phyla that determined the nutrient uptake status of microorganisms.These results demonstrate how microbial nutrient acquisition belowground of A.adenophora along an altitudinal gradient,which could contribute to further knowledge about the effects of altitude on biological invasion.