Antibacterial mechanism of Ginkgo biloba leaf extract when applied to Shewanella putrefaciens and Saprophytic staphylococcus

Nannan Zhang,Weiqing Lan,Qian Wang,Xiaohong Sun,Jing Xie,**

aCollege of Food Science&Technology,Shanghai Ocean University,Shanghai 201306,China

bShanghai Engineering Research Center of Aquatic Product Processing and Preservation,Shanghai 201306,China

Keywords:Ginkgo biloba leaf extracts(GBLE)Shewanella putrefaciens Saprophytic staphylococcus Antibacterial mechanism

A B S T R A C T The antimicrobial mechanism of Ginkgo biloba leaf extracts(GBLE)when applied to predominant spoilage bacteria(Shewanella putrefaciens and Saprophytic staphylococcus)on refrigerated pomfret and minimal inhibitory concentrations(MICs)were measured by the plate counting method.GBLE at MIC and 2MIC were prepared in tryptic soy broth(TSB)medium and equivalent amounts of sterile distilled water were used in place of GBLE as a control group.The impact of GBLE on the growth of bacteria,the permeability of cell membrane,and cell wall were also investigated by growth curve of bacteria,alkaline phosphates activity(AKP),and electrical conductivity.A scanning electron microscope(SEM)was used to study the effects of GBLE on the cellular structure of S.putrefaciens and S.staphylococcus.The results showed that the MICs of GBLE when applied to S.putrefaciens and S.staphylococcus were 100 mg/mL,the inhibitory rates of MIC and 2MIC concentrations of GBLE when applied to S.putrefaciens were 36.11%and 100%,while 27.78%and 62.22%for S.staphylococcus.Meanwhile,GBLE inhibited the growth of S.putrefaciens and S.staphylococcus until the number of cells at 2MIC values decreased to 0 and 4.29 log CFU/mL,respectively,after 24h.The electrical conductivity of bacteria increased with GBLE treatment,which was followed by an increased leakage of AKP.The SEM revealed that the structure of bacterial cells was destroyed and the bacteria began to be adhere to each other.The inhibition effect of GBLE when applied to S.putrefaciens and S.staphylococcus was related to the damage of cell membrane and cell wall.It was also revealed that GBLE damages the morphology of bacteria and had stronger effects on the cell membrane of S.putrefaciens than that of S.staphylococcus.

1.Introduction

Food,no matter the source,is always subject to spoilage,mainly due to microorganisms,particularly bacteria(Patra,Das,&Baek,2015).Pseudomonas,Aeromnas,Shewanella,Staphylococcusare common organisms cultured from freshwater fish(He,Guo,Song,Li,&Zhang,2017;Kačániová et al.,2017;Yano et al.,2015;Zhao,Zhu,Ye,Ge,&Li,2016).S.saprophyticushas been identified as a major cause of spoilage in pomfret.Specific spoilage organisms(SSO)participate in the spoilage process at its onset.It is known thatShewanella balticais identified to be an SSO for temperate fish species(Gu,Fu,Wang,&Lin,2013;T.;Li,Li,&Hu,2013;Zhao et al.,2016;Zhu,Zhao,Feng,&Gao,2016).The growth of SSO is a matter of great concern.He,Guo,Song,Li,&Zhang.(2017)reported that 1%Chitosan combined with 0.6%Nisin had significant inhibitory effects on the growth and biofilm formation ofS.putrefaciensandShewanellaalgae inP.crocea.Studies have shown that plants and their byproducts are rich sources of phenolic substances possessing antimicrobial and antioxidant capabilities.Siwe,Krause,van Vuuren,Tantoh,&Olivier.(2014)examined the antibacterial activity ofAlchorneafloribundaextract when applied toS.saprophyticusthrough a micro-dilution assay and found that the minimum inhibitory concentration(MIC)value was 63μg/mL.Al-Zoreky,&Al-Taher.(2015)studied the activity of extracts fromspathe(date palm)applied toS saprophyticusthrough an agar diffusion assay and the inhibition zones and the minimum inhibitory concentration(MIC)were 31 mm and 3 mg/mL,respectively.Plant extracts have shown considerable promise for a range of applications in the food industry.

Ginkgo bilobais a unique tree species in China and a living fossil that has existed for 200 million years(Boonkaew&Camper,2005;López-Gutiérrez,Romero-González,Vidal,&Frenich,2016;Zhang,Wang,Tao,&Fang,2016).Ginkgo bilobaleaf extract contains 24%flavonoids,6%terpene trilactones,and less than 5 ppm of ginkgolic acids(van Beek&Montoro,2009),which belong to a group of natural antioxidants(Boonkaew&Camper,2005;Goh&Barlow,2002).Previous studies indicated that extract from yellow and greenGinkgo(Ginkgo biloba)leaves have in vitro model systems with strong antioxidant activity.Joanna,Ewa,Magdalena,&,Dominik.(2014).Demonstrated that extracts ofGinkgo bilobaleaves had stabilizing effects on lipids and cholesterol in pork meatballs over 21 days of refrigerated storage.Other studies,such as Xie,Hettiarachchy,Jane,&,Johnson.(2003), reported that GBLE and sodium ethylenediaminetetraacetic acid(EDTA)effectively inhibited the growth ofListeria monocytogenes,demonstrating the antibacterial capacity of GBLE.Q.U.Li,Yong-Mei,Song,Yan,&Han-Wei. (2014)studied the antibacterial activity of GBLE onStaphylococcus aureusandEscherichia coli,measured the antimicrobial circle diameter and indicated that GBLE showed potential for development and utilization as a natural antibacterial agent and antiseptic(Sati,Pandey,&Palni,2012).mentioned that GBLE displayed antibacterial activities,with minimum inhibitory concentrations(MIC)reported to range between 300-400 and 400-600μg/mL for Grampositive and Gram-negative bacteria,and between 500-600 and 700-900μg/mL for actinomycetes and fungi,respectively.It has been demonstrated that GBLE antibacterial activity is dependant on the strain examined and on their growth phase,GBLE concentration,pH,temperature,and the composition of the medium(Sati et al.,2012).Although the chemical compositions and antimicrobial properties of GBLE have been studied,there is little work reported on the mechanism that prevents spoiling bacteria from growing in aquatic products.

In this study,S.putrefaciensandS.staphylococcuswere isolated from previously spoiled pomfret that were stored at 4°C.The aim was to investigate the spoiling potential ofS.putrefaciensandS.staphylococcusbased MIC,inhibitory rate,the growth curve of bacteria,and permeability of cell membrane measurements,AKP assay,and SEM observation.This research suggests that GBLE should be used as a natural bacteriostatic agent for aquatic product storage.

2.Materials and methods

2.1.Materials

The fresh,pest-free,and fan-shapedGinkgo bilobaleaves were collected in main loop around Shanghai Ocean University,Shanghai,in August 2016.These leaves were cleaned,dried,and crushed into powder for use.10 gGinkgo bilobaleaf powder was added with 500mL 60%ethanol by an ultrasonic-assisted method and the conditions were extracted after 30 min at 60°C.The ethanol extract was centrifuged with 6000 r/min at 4°C for 20 min.Then,the filtrates were rotary-evaporated to remove ethanol and water and concentrated to a final volume of filtrates to 20 mL.Therefore,the concentration of GBLE was measured the absorbance at 510nm and compared with rutin concentration.S.putrefaciensandS.saprophyticuswere separated and purified from spoiled pomfret,identified,and preserved by the Shanghai Engineering Research Center of Aquatic Product Processing and Preservation.

2.2.Culture preparation

Strains ofS.putrefaciensandS.saprophyticuswere activated and inoculated into TSB(Sinopharm Chemical Reagent Co.,Ltd.)medium,cultivated with shaking with 150 rpm at 37°C for 7h,to produce a final cell concentration of about 106-107CFU/mL.

2.3.Determination of minimal inhibitory concentrations(MICs)

The antimicrobial effects of GBLE onS.putrefaciensandS.saprophyticuswere determined byanalyzing MICs via the method of plate counting(Liu et al.,2016).Serial dilutions of GBLE were prepared to obtain the final concentrations of 150,125,100,75,50,and 25 mg/mL in TSB mediums.The same amountof sterile distilled water was used as the control.SuspendedS.putrefaciensandS.saprophyticuswere inoculated to different concentrations of GBLE and mixed well to obtain a mixture with a final microbial content of 106CFU/mL.100μL of the mixture was diluted 10-fold with sterile physiological saline in 1.5 mL centrifuge tube,then 100μL of the appropriate dilution was uniformly coated on the TSA plates and incubated at 37°C for 24h,after which the colonies were counted(0h GBLE treatment).At the same time,the mixtures were placed in shaker and incubated at 37°C for 24h.After that,the bacterial suspensions were diluted,coated,incubated and counted one more time(24 h GBLE treatment).The MICs were determined to be the lowest concentrations of GBLE at which the growth of tested bacteria was inhibited completely.

2.4.Determination of inhibitory rate

MIC and 2MIC concentrations of GBLE were added to sterile 96-well plates.Each well was inoculated with 100μL of prepared bacteria suspended to the final concentration of 106CFU/mL,then mixed well and cultured in a microplate oscillator at 37°C for 12h.Then the 96-well plates were measured by a Microplate Reader at 600 nm value.The inhibitory rate was defined as formula(1)(du Toit&Rautenbach,2000):

where ODRis absorbance value of a control well,OD is absorbance value of a sample well,and ODBis absorbance value of a blank well.

2.5.Growth curves of bacteria

TheS.putrefaciensandS.saprophyticussuspensions were added to GBLE at the concentration of MIC and 2MIC,and an equivalent amount of bacterial suspension without GBLE was used as control group.The bacteria were cultured at 37°C in a 150 r/min mixer for 24h.The bacteria growth was indexed by measuring the number of colonies on the plate surfaces every 2 h.

2.6.Cell membrane permeability assay

The effects of GBLE on cell membrane permeability ofS.putrefaciensandS.saprophyticuswere characterized by changes in electric conductivity.The prepared test bacteria suspensions were mixed with GBLE to final concentrations of MIC and 2MIC,and the control group was maintained without GBLE.The samples were mixed well and incubated in a 150 r/min mixer for 12h at 37°C,after which the conductivity was measured every 2 h with an electrical conductivity meter(Tao,Qian,&Xie,2011).

2.7.The activity of alkaline phosphatase(AKP)

AKP activity was determined using an AKP kit(Nanjing Jiancheng Bioengineering Institute,Nanjing,China)according to manufacturer instructions(Wang et al.,2017).S.putrefaciensandS.saprophyticuswere incubated in broth and collected in the logarithmic phase.100μL of bacterial suspension was mixed with GBLE solution at the MIC and 2MIC concentrations,along with a group without GBLE as the control.The mixtures were incubated in a shaking incubator at 150 r/min and 37°C for 12 h.Then the samples were centrifuged at 3500 r/min for 10 min each 2h.The supernatant was used to detect AKP activity.

2.8.Scanning electron microscopy(SEM)

SEM was carried out as previously reported with some modifications(Bajpai,Sharma,&Baek,2013).The 1%prepared bacterial solutions ofS.putrefaciensandS.saprophyticuswere inoculated into the GBLE solution at the concentrations of MIC and 2MIC.The inoculations were incubated at 37°C mixing at 50 r/min for 12 h and bacterial cells were collected by centrifugation at 8000 r/min for 5 minat 4°C.The bacteria precipitations were fixed in 2.5%glutaraldehyde solution at 4°C for 10 h.The fixed bacteria cells were washed three times with phosphate buffer solution(PBS)followed by serial dehydration with 30%,50%,70%,90%,and 100%ethanol solutions at 15 min intervals.After freeze-drying for 12 h,the cells were covered through cathodic spraying and then observed via SEM.

2.9.Statistical analysis

The experiment followed a completely randomized design(n=3).Data were expressed as mean±standard deviation(SD).The Duncan test and One-Way analysis of variance(ANOVA)were used for multiple comparisons by the SPSS 17.0 software package.Significant differences were those whereP＜0.05.

3.Results

3.1.Minimal inhibitory concentration(MIC)

MIC of GBLE was measured via plate counting as shown in Table 1.Total viable counts(TVC)decreased with an increase in GBLE concentration and both MICs of GBLE applied to the two bacteria were 100mg/mL.TVC ofS.staphylococcus(7.02)were smaller than that ofS.putrefaciens(7.28)at MIC,which indicating thatS.staphylococcuswas more susceptible to GBLE thanS.putrefaciens.The inhibitory effect of GBLE onS.putrefaciensandS.saprophyticusalso increased with increasing concentrations of GBLE.This result is similar to the research of Kumar,Gautam,&,Atri.(2015),since the MIC of both exo-and intra-cellular polysaccharides fractions ranged from 60 to 100mg/mL.

3.2.Inhibitory rates

As shown in Table 2,the inhibitory rates of MIC and 2MIC of GBLE applied toS.putrefacienswere 36.11%and 100%,respectively,while inhibitory rates ofS.saprophyticuswere27.78%and 62.22%.In contrast,the antibacterial effect of GBLE onS.putrefacienswas stronger thanS.saprophyticus,indicating thatS.putrefacienswas more sensitive to GBLE thanS.saprophyticus.

3.3.Growth curve of bacterial

To further confirm the antibacterial activity of GBLE when applied toS.putrefaciensandS.saprophyticus,the cell growth in different concentrations of GBLE were plotted over time.Bacteria,at initial concentrations of 106-107CFU/mL,were incubated in GBLE for 24h prior to plating to determine viability.As shown in Fig.1,S.putrefaciensgrowth curve of GBLE treatment group changed significantly.S.putrefaciensgrowth was completely inhibited after 24h incubation at the concentration of 2 MIC.The CK group increased to 9.82 log CFU/mL when cultured for 24h and the MIC group was 5.80 log CFU/mL.ForS.saprophyticus,the colony number in CK group increased to 9.03 log CFU/mL after cultured for 24h.At the same time,the colony number increased to 7.31 log CFU/mL in MIC group,while the colony number of 2MIC group decreased to 4.29 log CFU/mL.

Table 2 Inhibitory rates of GBLE on Shewanella putrefaciens and Saprophytic staphylococcus.

Table 1 Effects of GBLE on microbial growth of Shewanella putrefaciens and Saprophytic staphylococcus at 37°C for 0 and 24h.

Fig.1.Growth curve of Shewanella putrefaciens(a)and Staphylococcus saprophyticus(b)treated with GBLE.

3.4.The cell membrane permeability

The electrical conductivity of samples is shown in Fig.2.A slight increase in the electric conductivity of the control bacteria was found,likely due to the autolysis and death of bacterial cells(Y.Zhang et al.,2017).Compared to the control,the electric conductivity of the suspension increased immediately after the addition of GBLE.It was found that the electric conductivity forS.putrefaciensbegan to increase after MIC and 2MIC GBLE treatment for 2 h,becoming stable afterwards.GBLE with 2MIC was more effective than with MIC.Similarly,the conductivity values ofS.saprophyticustreated by GBLE at MIC and 2MIC concentrations increased significantly at 2 h.However,the electric conductivities of all samples forS.saprophyticusalmost decreased after 2 h.It might be attributed to the stress response of mycelium;whose cells consume the substance.The result indicated a positive correlation between the increase in GBLE concentration and the damage of cell membrane.

3.5.The activity of alkaline phosphatase(AKP)

Fig.2.Effects of GBLE on electric conductivity of Shewanella putrefaciens(a)and Staphylococcus saprophyticus(b).

The cell wall plays an important role in maintaining normal growth by isolating intracellular enzymes and macromolecular substances.The AKP effused from the cell when the cell cytoderm was destroyed(Tang et al.,2017;Xu,Liu,Hu,&Gao,2016).The concentration of AKP in the cell suspension could reflect the integrity of the bacterial cell wall(Wang et al.,2017).The AKP activity significantly increased in the suspension of all tested strains treated with GBLE(Fig.3).Treatments plateaued after 2h forS.putrefaciensand after 6h forS.saprophyticus.The AKP activity of bacterial cells increased with increased GBLE concentration from 1 MIC to 2 MIC.These results were similar to those of black pepper petroleum ether extract applied toListeria monocytogenesandSalmonella typhimurium(Tang et al.,2017).

3.6.Scanning electron microscope(SEM)

SEM can detect the antibacterial mechanism of GBLE when applied to bacteria by observing changes in its morphology.Cell membranes maintain material and energy balance,which are important for maintaining normal bacterial activities(W.R.Li et al.,2010).Fig.4 shows thatS.putrefacienscells without GBLE treatment exhibit regular short-bar-shaped morphology,with a smooth and intact cell surface.However,broken and ruptured cells were found inS.putrefacienstreated with GBLE at MIC(ii)and 2 MIC values(iii)and only severely wrinkled,coarse outer surfaces and clumped cells were observed.Furthermore,the damaging effect of 2 MIC(iii)on cell walls was stronger than that of MIC(ii),which had a smooth surface and a relatively intact outer layer of bacteria.Similarly,the normalS.saprophyticuscells(i)were round,spherical,and had smooth,grape-clustered surfaces which were uniform in size and distribution.After treating GBLE for 6h at MIC and 2 MIC concentrations,theS.putrefacienscells became deformed,shriveled,pitted,adhered to each other,and parts of the cell were broken and shrunk to a smaller size.These results indicated that GBLE can cause damage toS.putrefaciensandS.saprophyticuscells.These findings were consistent with those concerning ε-poly-lysine applied toEscherichia coliandStaphylococcus aureus(Li et al.,2014).

4.Discussion

4.1.MIC

This study analyzed the antibacterial activity of GBLE when applied toS.putrefaciensandS.saprophyticus.It has also been reported that flavonoids are major components of GBLE and responsible for its antioxidant and antibacterial activity(Lan et al.,2018).Similarly,an inhibitory effect on the growth of bacteria was observed inAsplenium nidus(Jarial et al.,2016).To date,only few studies have investigated the effects of GBLE on antibacterial activity.Based on the results,the higher GBLE concentration was needed to inhibit the two bacterial strains.Therefore,optimization of the GBLE extracting technique is needed to further investigate and improve the antibacterial activity.

4.2.Inhibitory rates

With the increase of GBLE concentration,the inhibitory rates were increased.Therefore,it concluded that GBLE can significantly slow down reproduction and delay cell growth into the logarithmic phase,thereby inhibiting the growth of cells(Diao et al.,2018).GBLE was used to further study the growth ofS.putrefaciensandS.saprophyticus.

4.3.Growth curve

The results showed that GBLE exhibited the strongest inhibition on the growth of tested strains.Increased bacteria at MIC forS.saprophyticusmight be influenced by bacterial contamination during the experiment.GBLE at low concentrations did not have efficient effect on bacterial growth,generally.In contrast,the antibacterial effect of GBLE toS.putrefacienswas stronger thanS.saprophyticus.Results suggested that GBLE could not only delay the growth cycle of cells but lead to the death of cells(Tang et al.,2017).

Fig.3.Effects of Ginkgo biloba leaf extracts on the cell wall of Shewanella putrefaciens(a)and Staphylococcus saprophyticus(b).

Fig.4.SEM images of(a)Shewanella putrefaciens(×40 000),(b)Staphylococcus saprophyticus(×40 000)cells without GBLE(i)and treated with MIC(ii)and 2MIC(iii)GBLE.

4.4.The cell membrane permeability

The membrane permeability was significantly dependent on the concentration of GBLE.We also found a higher electric conductivity with treatment compared to the control.Along with increased reaction time,the number of ions in the culture decreased,resulting in lower electric conductivity and changed membrane permeability.Changes in electric conductivity betweenS.putrefaciensandS.saprophyticusmight be attributed to differences in cell membrane components and structure(Tao,Qian,&Xie,2011).GBLE,an effective antibacterial,has a broad spectrum of both gram-positive and gram-negative bacteria,though specific effects on each pathogen could vary across different strains.

4.5.AKP activity

The activity of AKP treated with GBLE increased significantly when compared to CK.The result indicated that the permeability and integrity of bacterial cell wall had been changed by GBLE treatment.The protection of cell wall was lost,killing the bacteria(Wang et al.,2017).In this study,GBLE caused different antibacterial activity levels in the two bacteria strains,perhaps due to the differences in the structure of cell wall between Gram-positive(S.saprophyticus)and Gram-negative bacteria(S.putrefaciens).The production and support role of the cell wall disappeared as morphology of bacterial cells changed,affecting the cell membrane and organelles,leading to cell death(Tang et al.,2017).GBLE probably induced cell wall damage,leading to the leakage of AKP from the cells,and a loss of cytoderm integrity(Diao et al.,2018).

4.6.SEM

The morphological changes of bacterial cells might have occurred due to the effect of GBLE on the permeability and integrity of membranes,thereby resulting in the lysis of bacterial cell wall followed by the loss of intracellular dense materials on the surface of treated cells(Hameed et al.,2016).This supported the electric conductivity,AKP activity and membrane structure assay results.

5.Conclusions

Our present study showed that GBLE was an effective antibacterial forS.putrefaciens and S.saprophyticus.The antibacterial activity increased with increasing GBLE concentration and treatment time.The MICs of GBLE when applied toS.putrefaciens and S.saprophyticuswere all 100 mg/mL.Growth curve assays showed that GBLE not only delay the growth ofS.putrefaciens and S.saprophyticus butkill the cells.GBLE affected the structure of the membrane,causing lysis of cells and cell membrane damage,eventuallyleading to cell death.The antibacterial components of GBLE destroyed the cell membrane by changing the permeability,thereby allowing antibacterial agents into the bacteria cell.SEM results further confirmed the disruption of cell membrane caused byGBLE.Further research should be carried out to fully elucidate the antibacterial mechanism of GBLE,such as its effects on DNA and protein synthesis,other food related bacteria,and their interactions with food ingredients.This study provides an approach for developing convenient and efficient antimicrobial agents in the food or pharmaceutical industries,which is of great significance to food security.

Conflicts of interest

We declare that we have no conflict of interest.

Acknowledgement

The study was financially supported by China Agriculture Research System(CARS-47-G26),Shanghai promote agriculture by applying scientific & technological advances projects(2015No.4-12),Ability promotion project of Shanghai Municipal Science and Technology Commission Engineering Center(16DZ2280300).