BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

A novel exopolysaccharide from deep-sea bacteriumZunongwangia profunda SM-A87: low-cost fermentation,moisture retention, and antioxidant activities

Mei-Ling Sun & Sheng-Bo Liu & Li-Ping Qiao & Xiu-Lan Chen & Xiuhua Pang & Mei Shi &Xi-Ying Zhang & Qi-Long Qin & Bai-Cheng Zhou & Yu-Zhong Zhang & Bin-Bin Xie

Received: 2 April 2014 /Revised: 9 May 2014 /Accepted: 15 May 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Many marine microorganisms can secreteexopolysaccharides (EPSs) which have important applica-tions in biotechnology. We have purified a novel EPSfrom deep-sea bacterium Zunongwangia profunda SM-A87, identified its glycosyl composition and linkage,and optimized its production to 8.9 g/l in previous studies.To reduce the fermentation cost, an economical fermenta-tion medium containing 60.9 % whey, 10 g/l soybeanmeal, and 2.9 % NaCl was developed. The EPS yield ofbatch fermentation in this medium reached 12.1±0.3 g/l.Fed-batch fermentation was conducted and led to an EPSyield of 17.2±0.4 g/l, which represents the highest EPSyield ever reported for a marine bacterium. The EPS wasextracted and it displayed good rheological properties,moisture-retention ability, and antioxidant activity. Partic-ularly, its moisture-retention ability is superior to that ofother marine bacterial EPSs reported to date. SM-A87EPS also showed high antioxidant activity. These resultssuggest that SM-A87 EPS has promising potentials inbiotechnology.

Keywords Zunongwangia profunda SM-A87 .

Exopolysaccharide . Economical medium . Fed-batchfermentation .Moisture-retention ability .Antioxidant activity

Introduction

Microbial exopolysaccharides (EPSs) are ubiquitous in natureand have wide applications in biotechnology (Weiner 1997).For example, xanthan gum, the most well-known microbialEPS, is commonly used as a thickener in both food and non-food industries due to its physical properties (Becker et al.1998). Recently, isolating and identifying newmicrobial EPSsthat may have novel applications such as emulsifiers andstabilizers have drawn people’s attention (Nichols et al.2005). Marine environment is a rich natural resource of EPSs,and several EPS-producingmarine microorganisms have beenisolated (Antón et al. 1988; Lee et al. 2001). It has becomewidely acknowledged that extremophilic microorganisms willoffer a valuable resource for exploitation in biotechnologicalprocesses (Nichols et al. 2005). For example, biotechnologicalapplication of EPSs secreted by thermophilic bacteria fromdeep sea has been reported (Bozzi et al. 1996a, b; Rougeauxet al. 1996). Deep sea, representing 75 % of the total volumeof the oceans and influenced by high pressure and low nutrientconcentration, offers a new source of novel EPSs with ex-ploitable properties (Poli et al. 2010). However, little has beenreported on biotechnological application of the EPSs pro-duced by psychrophilic or mesophilic bacteria isolated fromthe low-temperature environments of deep sea (Li et al. 2008).

For better applications of microbial EPSs in biotechnology,it is required to enhance their production and decrease thefermentation cost. Compared to normal batch fermentation,fed-batch fermentation can extend the period of EPS produc-tion and thus improve the EPS yield. With the fed-batch

M.<L. Sun : S.<B. Liu : L.<P. Qiao :X.<L. Chen :X. Pang :M. Shi :X.<Y. Zhang :Q.<L. Qin :Y.<Z. Zhang :B.<B. XieState Key Laboratory of Microbial Technology, ShandongUniversity, Jinan 250100, Shandong Province, China

M.<L. Sun : S.<B. Liu : L.<P. Qiao :X.<L. Chen :X. Pang :M. Shi :X.<Y. Zhang :Q.<L. Qin : B.<C. Zhou :Y.<Z. Zhang :B.<B. XieMarine Biotechnology Research Center, Shandong University,Jinan 250100, Shandong Province, China

X.<L. Chen :X. Pang :M. Shi :X.<Y. Zhang :Q.<L. Qin :Y.<Z. Zhang :B.<B. Xie (*)Collaborative Innovation Center of Deep Sea Biology, ShandongUniversity, Jinan 250100, Shandong Province, Chinae-mail: xbb@sdu.edu.cn

Appl Microbiol BiotechnolDOI 10.1007/s00253-014-5839-8

method, Kim et al. (2006) improved the EPS yield ofGanoderma resinaceum DG-6556 from 3.05 to 4.6 g/l. Themain factor affecting the economy of EPS production is thesubstrate cost. Thus, it is important to utilize inexpensive andrenewable carbon and nitrogen sources to produce EPSs.Cheese whey, containing 4.5–5 % lactose, is a by-product ofdairy industries and is produced in large amounts (Guimarãeset al. 2010). Proper disposal of cheese whey has long been aconcern to the dairy industry (Prazeres et al. 2013). Fialhoet al. (1999) utilized cheese whey to replace lactose for thelow-cost production of gellan gum by Sphingomonaspaucimobilis ATCC 31461. Soybean meal is a by-product insoybean oil production and can be utilized in bioprocesses forvalue addition (Lima et al. 2008). Gao et al. (2012) utilizedsoybean meal as nitrogen source to enhance the EPS produc-tion by Gomphidius rutilus, which also led to the cost reduc-tion of fermentation.

Moisture-retaining materials are widely used in food, cos-metic, and clinical industries. As a conventional material,glycerin has good moisture-absorption ability but poor mois-ture retention. Hyaluronic acid (HA), a kind of glycosamino-glycan, has been applied in pharmaceutical and cosmeticfields due to its excellent moisture-absorption and retentionabilities, but the high price and low production have limited itsapplication (Zhao et al. 2013). Konjac glucomannan (KGM),a heteropolysaccharide comprising glucose and mannose, hasa good moisture-retention ability but can be obtained onlyfrom plants (Yan et al. 2012). There are little reports aboutmoisture-retention ability of bacterial products.

Free-radical reactions of biological molecules, such aslipids, proteins (including enzymes), DNA, and RNA, areinvolved in human health issues, for example atherosclerosisand carcinogenesis. The reactions also lead to food deteriora-tion (Halliwell and Gutteridge 1999). So, it is significant todevelop and apply antioxidants to decrease unwanted oxidiz-ing reactions. Most reported natural antioxidants such aspolysaccharides are isolated from plants, fungi, and marinealgae (Kardošová and Machová 2006; Leung et al. 2009;Wang et al. 2008). There is no report on the antioxidantactivities of EPSs from deep-sea bacteria to date.

Zunongwangia profunda SM-A87, isolated from the deep-sea sediment, can secrete a large amount of EPS to form athick capsule around the cells (Liu et al. 2011b; Su et al.2012). SM-A87 EPS displays good rheological properties,potential in enhancing oil recovery, and strong capacity foradsorbing Cu(II) and Cd(II) (Li et al. 2011; Liu et al. 2011b;Zhou et al. 2009). In our previous study, we optimized thecultural conditions of SM-A87 by using response surfacemethodology (RSM) to improve the EPS yield (Liu et al.2011b). Under the optimal conditions, the EPS yield reachedup to 8.90 g/l with a broth viscosity of 6,551 mPa s, whichrepresents the highest EPS yield reported for a deep-sea mi-crobe (Liu et al. 2011b). In this study, we formulated an

economical fermentation medium composed of industrial ma-terials (whey, soybean meal, and NaCl) to lower the fermen-tation cost and conducted fed-batch fermentation to enhancethe EPS yield. The EPS was extracted and its rheologicalproperties were evaluated. In addition, the moisture-absorption and retention abilities and antioxidant activity werestudied for estimating the potential applications of SM-A87EPS in biotechnology.

Materials and methods

Strain and culture

The strain Z. profunda SM-A87 (CCTCC AB 206139T =DSM 18752) was isolated from the sediment sample obtainednear the southern Okinawa Trough at a water depth of1,245 m. It is a species of Bacteroidetes and proposed torepresent a new genus of Flavobacteriaceae (Qin et al.2007). The strain was routinely cultured at 30 °C for 3 dayson a marine medium composed of 10 g/l peptone (Oxoid,Basingstoke, England), 5 g/l yeast extract (Oxoid,Basingstoke, England), 15 g/l agar, and artificial sea waterprepared by 30 g/l sea salt (Sigma, Santa Clara, USA) with pHadjusted to 8.5 and then stored at 4 °C. For EPS production,strain SM-A87 was inoculated into an Erlenmeyer flask(250 ml) containing 50-ml liquid marine medium (pH 8.5)and incubated for 60 h at 15 °C, 200 rpm to logarithmic phase,which was used as starter culture. Then, a 2 % (v/v) inoculumof the starter culture was added to the flask (500 ml) contain-ing 100-ml fermentation medium, and the flask was incubatedat 9.8 °C, 200 rpm for 6 days. The fermentation medium forEPS production optimized previously was used as a controlmedium, which contained (g/l): lactose (Sinopharm, Shang-hai, China) 32.21, peptone 8.87, yeast extract 7.5, and sea salt30 (pH 8.5; Liu et al. 2011b). All chemicals used in this studywere of analytical reagent grade.

Determination of EPS yield and broth viscosity

To determine the EPS yield, samples taken from the fermen-tation broth were mixed with 2 volumes of cold absoluteethanol to precipitate the EPS and centrifuged for 10 min at10,000 rpm (Selbmann et al. 2002). To remove impurities oflow molecular weight, the precipitation was washed twice bycold absolute ethanol. The resulting precipitation was dis-solved in deionized water, and the EPS concentration of thesolution was determined by the phenol-sulfuric acid method(DuBois et al. 1956). The same procedure was done with thepreviousmedium optimized by RSM (Liu et al. 2011b), whichwas used as a control.

The viscosity of the samples was measured on a Brookfieldviscometer (model LVDV-II + P; Brookfield Engineering

Appl Microbiol Biotechnol

Laboratories, USA) at 25 °C. All assays were carried out withthe small sample adapter and the spindle S16.

Low-cost fermentation of the EPS

To find the optimal concentrations of whey, soybean meal,and NaCl for the EPS production of strain SM-A87, differentconcentrations of whey at 52.2, 56.0, 60.9, and 65.2 % (v/v);soybean meal at 5, 6, 7, 8, 9, 10, 11, and 12 g/l; and NaCl at2.8, 2.9, 3.0, 3.1, 3.2, 3.3, and 3.4 % were added in the controlfermentationmedium to replace lactose, peptone/yeast extract,and sea salt, respectively. Whey containing 57 g/l lactose waskindly provided by a local dairy food processing plant inJinan. Soybean meal was purchased from a local farm productprocessing factory in Jinan. NaCl was an analytical reagentpurchased from Sinopharm Chemical Reagent Company,China.

Fed-batch fermentation was performed to study the EPSyield in a stirred bioreactor with the newly developed medi-um. The bioreactor (BioFlo/CelliGen 310, Eppendorf, USA)was a 5-l (working volume) agitated bioreactor with two six-bladed turbine impellers. The stirred reactor was aeratedthrough a ring sparger. Dissolved oxygen level was set above30 % of air saturation and controlled by adjusting agitationspeed and aeration rate during fermentation. At the end offermentation, the agitation speed was around 800 rpm andaeration rate was around 1.0 vvm. During the fermentation,pH was controlled at 8.5 with 4-M NaOH and HCl solutions.The preparation of starter culture and the inoculum size wasthe same as those in flask fermentation. The medium wascomposed of whey, soybean meal, and NaCl, and their con-centrations were the optimal values for EPS production ac-cording to above test. The culture temperature was 20 °C onthe first day for rapid growth of the bacterium and then wasadjusted to 9.8 °C. Samples were taken every 24 h for analysesof EPS yield, broth viscosity, and residual lactose. On the thirdand fifth days, when the residual sugar concentration waslower than 15 g/l, a fed-batch process with feeding 500-mlconcentrated whey solution (lactose concentration of 285 g/l)into the reactor was conducted to increase the lactoseconcentration.

Extraction of the EPS

The EPS from fed-batch fermentation was extracted accordingto the method described by Liu et al. (2011b). This EPS wasused to perform analyses of rheological properties, moistureretention, and antioxidant activities.

Rheological properties of the EPS solution

The rheological properties of the EPS solution, including thechange of viscosity with different shear rates, thermal and pH

stability, and salt resistance, were analyzed by the methodsdescribed previously (Liu et al. 2011b).

Moisture-absorption and retention abilities of the EPS

Prior to the moisture-absorption test, the EPS from strain SM-A87 and the control samples, including HA, chitosan, sodiumalginate, and glycerol, were pulverized to 80 mesh and driedover P2O5 in vacuo for 24 h. Moisture-absorption abilities ofthe samples were measured at 25 °C under two differentrelative humidity levels (RH): 81 % (using a desiccator con-taining saturated ammonium sulfate) and 43 % (using adesiccator containing saturated potassium carbonate; Chenet al. 2003).

The vessels containing 0.5000-g dried samples were keptin the desiccator for 3, 6, 12, 24, 36, 48, 60, and 72 h and thenweighed again. The moisture-absorption ability was evaluatedby the percentage of weight increase of dry sample (Ra):

Ra %ð Þ ¼ W n−W 0ð Þ=W 0 � 100 ð1Þ

where W0 and Wn are the weights of a sample before puttinginto and after taking from the desiccator.

In the moisture-retention test, wet samples were preparedby keeping the EPS and the control samples in 43 % RH(using a desiccator containing saturated potassium carbonate)for 72 h and weighed. The vessels containing the wet sampleswere left for 3, 6, 12, 24, 36, 48, 60, and 72 h in a desiccatorwith allochroic silica gel at 25 °C and then weighed. Themoisture-retention ability was calculated as follows:

Rh %ð Þ ¼ Hn=H0ð Þ � 100 ð2Þ

where H0 and Hn are the weights of water in a sample beforeputting into and after taking from the desiccator.

Antioxidant activity of the EPS

SM-A87 EPS and HA were dissolved in deionized water atdifferent concentrations (0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 5.0, 7.5,and 10.0 mg/ml). The capacity for scavenging 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH·) was analyzed by modi-fied method (Braca et al. 2002). The DPPH·solution(100 μM) was prepared by dissolving DPPH in 50 % ethanol.Then, the DPPH·solution (2 ml) was mixed with 1 ml of EPSor HA solution. The absorbance of the mixtures was measuredat 525 nm after the mixtures were incubated at 25 °C in thedark for 40 min.

The hydroxyl radical (·OH) scavenging assay was carriedout according to the well-established protocol (Wang et al.2008). FeSO4 solution (9mM, 1ml) and salicylic acid-ethanol

Appl Microbiol Biotechnol

solution (9 mM, 1 ml) were mixed with 1-ml EPS or HAsolution. Then, 1 ml H2O2 solution at 8.8 mM was added tothe mixtures to start the reaction. The absorbance of themixtures was measured at 510 nm after the mixtures wereincubated at 37 °C for 30 min.

The capacity for scavenging superoxide anion (O2−·) was

measured with the pyrogallol autoxidation method (Kim et al.1995). In the assay, 0.4-ml pyrogallol solution (prepared with10 mM HCl) and 1-ml EPS or HA solution were mixed with4.5-ml Tris–HCl buffer (50 mM, pH 8.2). The mixtures wereincubated at 25 °C for 5 min, and then, 1 ml HCl (8 mM) wasadded to terminate the reaction. The absorbance of the mix-tures was measured at 320 nm.

The capacity (D) for scavenging free radical was calculatedas follows:

D ¼ 1− Ai−A j

� �=A0

� �� 100 ð3Þ

where Ai is the absorbance of the sample, Aj is backgroundabsorbance of the sample, and A0 is the absorbance of theblank control.

Results

Low-cost and fed-batch fermentation of the EPS

The medium used in the last study (Liu et al. 2011b) was usedas the control medium and a starting point to formulate aneconomical fermentation medium for the EPS production ofstrain SM-A87. Different concentrations of whey, soybeanmeal, and NaCl were used to replace lactose, peptone/yeastextract, and sea salt in the control medium, respectively. TheEPS production and broth viscosity were only slightly affectedwhen 52.2–65.2 % whey was added. At this range, a maxi-mum level of 7.5±0.1 g/l for EPS production and 6,359±161 mPa s for broth viscosity was reached with a wheyconcentration of 60.9 % (Fig. 1a). The effect of differentconcentrations of soybean meal on EPS production and brothviscosity was shown in Fig. 1b. A maximum level of 11.0±0.4 g/l for EPS production and 11,589±519 mPa s for brothviscosity was obtained when 10 g/l soybean meal was includ-ed in the fermentation medium. The effect of different con-centrations of NaCl as a substitute for sea salt on the EPSproduction of strain SM-A87 was also studied. A level of 9.0±0.1 g/l for EPS production and 6,807±553 mPa s for brothviscosity was reached at a concentration of 2.9 %, indicatingthat NaCl could be used to replace sea salt (Fig. 1c). Based onthe results, batch fermentation of strain SM-A87 in a mediumwith 60.9 % whey, 10 g/l soybean meal, and 2.9 % NaCl(pH 8.5) was conducted, and the resultant EPS yield (12.1±

0.3 g/l) and broth viscosity (11,659±361 mPa s) were 1.36-and 1.78-fold of those from the control medium, respectively.This medium was referred to the economical mediumhereafter.

Based on the batch fermentation result, fed-batch fermen-tation was accomplished in a 5-l agitated bioreactor with theeconomical medium. On the third and fifth days, when the

Fig. 1 Effects of concentrations of whey (a), soybean meal (b), andNaCl(c) on EPS production and broth viscosity of strain SM-A87. Differentconcentrations of whey, soybean meal, and NaCl were used to replacelactose, peptone/yeast extract, and sea salt in the control fermentationmedium, respectively. Data are from triplicate experiments (mean ± SD)

Appl Microbiol Biotechnol

residual sugar was below 15 g/l, concentrated whey was fedinto the reactor to increase the concentration of lactose. Asshown in Fig. 2, after 7-day fermentation, the EPS productionreached 17.2±0.4 g/l, with a broth viscosity of 20,755±140 mPa s, which were 1.42 and 1.78-fold of those of thebatch fermentation, respectively. Therefore, with the econom-ical medium and the fed-batch fermentation, the EPS produc-tion and broth viscosity were 1.93 and 3.17-fold of thosereported in the previous study (Liu et al. 2011b), respectively.

Rheological properties of the EPS

The rheological properties of SM-A87 EPS prepared from thecontrol medium have been studied (Liu et al. 2011b). In orderto analyze whether medium composition affected the rheolog-ical properties, the rheological properties of the EPS from theeconomical medium were investigated and compared withthose from the control medium. The effects of EPS concen-tration, pH, inorganic salt, shear rate, and temperature on theviscosity of the EPS solution were measured.

The viscosity of the EPS solution from the economicalmedium went up quickly as its concentration increased(Fig. 3a), similar to that from the control medium (Liu et al.2011b), suggesting that the EPS from the economical mediumstill retained the non-newtonian fluid property. In addition, itwas noticeable that the viscosity of the EPS solution from theeconomical medium was higher than that from the controlmedium when at the same concentration (Fig. 3a). For exam-ple, when the concentration was 1.2 %, the viscosity of theEPS solution from the economical medium was 3,057±9 mPa s, higher than that from the control medium (2,465±73mPa s; Fig. 3a). The effects of pH, inorganic salt, shear rate,and temperature on the viscosity of the EPS solution from theeconomical medium were all similar to those from the controlmedium (Figs. 3b, c and 4). These results indicated that thechange of medium composition did not affect the rheological

properties of the EPS solution of strain SM-A87 except anincrease in viscosity. Therefore, the newly developed medium

Fig. 2 Fed-batch fermentation of strain SM-A87 to produce EPS in a 5-lstirred bioreactor. The medium composition was 60.9 % whey, 10 g/lsoybean meal, and 2.9 % NaCl (pH 8.5). The culture temperature was20 °C on the first day and then was adjusted to 9.8 °C. On the third andfifth days, 500-ml concentrated whey solution (200 g/l) was fed into thereactor. Data are from triplicate experiments (mean ± SD)

Fig. 3 Effects of concentration (a), pH (b), and inorganic salts (c) on theviscosity of SM-A87 EPS solution. Viscosity was measured by Brook-field viscometer with the small sample adapter and spindle S16 at 25 °C.Data for the economical medium are from triplicate experiments (mean ±SD). Data for the control medium are from our previous study (Liu et al.2011b)

Appl Microbiol Biotechnol

not only reduced the fermentation cost and improved the EPSyield, but also improved the EPS quality.

Moisture-absorption and retention abilities of the EPS

To evaluate the potential in biotechnological applications ofSM-A87 EPS, the moisture-absorption and retention abilitiesof the EPS were measured and compared with those of HA,chitosan, sodium alginate, and glycerol, which were common-ly used in cosmetics. At 43 % RH, the rank for moisture-absorption rates of all tested samples was glycerol > HA >SM-A87 EPS > sodium alginate > chitosan (Fig. 5a). At 81 %RH, the rank was similar to that at 43%RH, and the moisture-absorption rate of SM-A87 EPS was very close to that ofsodium alginate (Fig. 5b). The data also indicated that themoisture-absorption rate increased with increasing RH. Forexample, the moisture-absorption rates of SM-A87 EPS were12.5±0.5 % at 43 % RH and 28.8±2 % at 81 % RH. It couldbe concluded that the moisture-absorption rate of SM-A87EPS was only inferior to HA. The moisture-retention rates ofmost tested materials were maintained at 73–77 % after theywere kept in 43 % RH for 72 h. Glycerol was an exceptionwith its rate decreased to 50.3±0.9 % after 72 h (Fig. 5c).These data indicated that SM-A87 EPS had a similar

moisture-retention ability to HA, sodium alginate, andchitosan.

Antioxidant activity of the EPS

Antioxidant activity of SM-A87 EPS was evaluated by exam-ining the capacity for scavenging free radicals. As shown inFig. 6, the capacity of SM-A87 EPS for scavenging freeradicals increased with its concentration. At a concentrationof 10.0 mg/ml, the DPPH·, ·OH, and O2

−·scavenging ratios ofSM-A87 EPS were 48.5±1.5 % (Fig. 6a), 58.7±0.8 %(Fig. 6b), and 27.2±0.6 % (Fig. 6c), respectively. In contrast,HA did not show obvious DPPH·scavenging capacity(Fig. 6a), and its scavenging ratios for·OH and O2

− ·at aconcentration of 10.0 mg/ml were 19.1±0.1 (Fig. 6b) and15.2±0.2 % (Fig. 6c), respectively. Thus, SM-A87 EPS pos-sessed better antioxidant activity than HA.

Discussion

Many bacteria in the marine environment can produce EPSswith novel structures (Nichols et al. 2005). To date, the highest

Fig. 4 Effects of shear rate and temperature on the viscosity of SM-A87EPS solution. a, b Effects of shear rate on the viscosity of the EPSsolution from the control medium (a) and the economical medium (b).c, d Effects of temperature on the viscosity of the EPS solution from thecontrol medium (c) and the economical medium (d). Viscosity was

measured by Brookfield viscometer with the small sample adapter andspindle S16 at 25 °C. Data for economical medium are from triplicateexperiments (mean ± SD). Data for the control medium are from ourprevious study (Liu et al. 2011)

Appl Microbiol Biotechnol

EPS production by marine bacteria is from stain Bacillussubtilis MSBN17, which can produce 13.42 g/l polysaccha-ride after optimizing culture conditions by RSM(Sathiyanarayanan et al. 2013). Since 1994, new bacteria havebeen searched from deep-sea environment characterized byextreme pressure and temperature conditions (Guezennecet al. 1994). The EPSs secreted by the bacteria growing inthis environment have drawn people’s attention due to theirnovel properties for biotechnological applications (Nicholset al. 2005).

Deep-sea bacterium Z. profunda SM-A87 can secrete alarge amount of EPS (Liu et al. 2011b; Su et al. 2012). In

the previous study, we optimized the fermentation conditionsof SM-A87 and obtained an EPS yield of 8.90 g/l with a brothviscosity of 6,551 mPa s (Liu et al. 2011b). Since SM-A87EPS showed good properties (Liu et al. 2011b) and biotech-nological potential (Li et al. 2011; Zhou et al. 2009), wefurther optimized the culture conditions to improve EPS pro-duction and decrease its fermentation cost in this study. Theproduction of EPSs depends on the cultivation media used inthe fermentation (Lima et al. 2008). The use of agro-industrialproducts as the substrates is common in the bioprocesses andis useful in biopolymer synthesis (Lima et al. 2008). In thisstudy, we formulated a new medium containing 60.9 % whey,10 g/l soybean meal, and 2.9 %NaCl.Whey is a by-product of

Fig. 5 Moisture-absorption ability at 43 % RH (a) and at 81 % (b) andretention ability (c) of SM-A87 EPS, HA, chitosan, sodium alginate, andglycerol. Data are from triplicate experiments (mean ± SD)

Fig. 6 The capacity for scavenging free radicals DPPH·(a), ·OH (b), andO2

−·(c) of SM-A87 EPS and HA. Data are from triplicate experiments(mean ± SD)

Appl Microbiol Biotechnol

dairy industries and represents a significant environmentalproblem due to its high output and organic matter content(Guimarães et al. 2010). Soybean meal is cheaper than pep-tone and yeast extract. Thus, this economical medium de-creased the fermentation cost. It also improved the EPS yieldto 12.1±0.3 g/l in batch fermentation and to 17.2±0.4 g/l infed-batch fermentation. To our knowledge, 17.2 g/l representsthe highest EPS production reported for a marine bacterium.

SM-A87 EPS fermented in the economical medium hasalmost the same rheological properties as that in the controlmedium, except an increase in the viscosity. Therefore, differ-ent from those reported before (Fialho et al. 1999; Zall 1992),change of medium composition had little effect on the rheo-logical properties of SM-A87 EPS.When the concentration ofSM-A87 EPS solution was as low as 1.0 %, it showed anextremely high and shear-dependent viscosity, which enablesthe EPS to regulate water balance and flow resistance (Chonget al. 2005). The high viscosity, pseudoplastic property, goodthermal and pH stability, and salt resistance of SM-A87 EPSsolution suggest that the EPS may have good potential inbiotechnological applications, such as a thickener in food, anoil-displacing agent in oil recovery (Nichols et al. 2005).

Polymers that have highly efficient water-absorption andretention abilities may be characterized chemically aselectrolytical, cross-linked, and polymeric (Buchholz andPeppas 1994). SM-A87 EPS is an acidic polysaccharide con-taining the glucuronic acid residue (Li et al. 2011). Theexistence of 3,4-linked glucopyranosyl residue suggests thatthe EPS is branched (Li et al. 2011), which contributes to theformation of a spacious network structure to keep watermolecules inside more easily (Chen et al. 2002). In addition,SM-A87 EPS contains fucose, which may also contribute tothe water-retention ability (Kurane and Nohata 2002). Inaccord with the insights into the relationship between thechemical characterization and function, SM-A87 EPSdisplayed similar moisture-absorption and retention abilitiesto HA. For HA production, the commonly used strain, Strep-tococcus zooepidemicus, produces only 6~7 g/l HA under theoptimal culture conditions with uneconomical substrates (Liuet al. 2011a). In contrast, SM-A87 EPS is higher in productionand lower in fermentation cost compared to HA. In addition,the moisture-retention ability of SM-A87 EPS is superior tothose of other marine bacterial EPSs to date. Since HA withdistinctive moisture-retention ability has shown various appli-cations in the cosmetic, biomedical, and food industries(Chong et al. 2005), SM-A87 EPS may have good potentialapplications in cosmetics and clinical medicine.

Oxidative reaction caused by reactive oxygen species mayinduce human tissue damage and many diseases such ascancer and inflammation (Leung et al. 2009). Our data indi-cated that SM-A87 EPS showed higher capacity for scaveng-ing DPPH·, ·OH, and O2

−·than HA, which was capable ofscavenging radicals (Sato et al. 1988). This is the first report

on the antioxidant activities of EPSs from deep-sea bacteria todate. Its high scavenging rates are contributed by abundanthydroxyl groups, which can donate electrons to make theradicals to a more stable form or react with the free radicalsto terminate the radical chain reaction (Leung et al. 2009). Themetal-chelating ability of SM-A87 EPS (Zhou et al. 2009)may also result in its high antioxidant activity by chelating thepro-oxidant metals (Pasanphan et al. 2010). This novel type ofEPS antioxidant could be a model for developing antioxidantadditives for foods to prevent oxidative damage to lipids andproteins, as well as for cosmetic and even medical products.

Acknowledgments This work was supported by the National NaturalScience Foundation of China (grants 31290231, 31025001, 91228210,31170055, 31270117, and 81271896), the Hi-Tech Research and Devel-opment Program of China (grants 2011AA090703, 2012AA092103, and2012AA092105), and the China Ocean Mineral Resources R & D Asso-ciation (COMRA) Special Foundation (grants DY125-15-T-05 andDY125-15-R-03).

Conflict of interest The authors have no conflict of interest.

References

Antón J, Meseguer I, Rodriguez-Valera F (1988) Production of an extra-cellular polysaccharide by Haloferax mediterranei. Appl EnvironMicrobiol 54(10):2381–2386

Becker A, Katzen F, Pühler A, Ielpi L (1998) Xanthan gum biosynthesisand application: a biochemical/genetic perspective. Appl MicrobiolBiotechnol 50(2):145–152

Bozzi L, Milas M, Rinaudo M (1996a) Characterization and solutionproperties of a new exopolysaccharide excreted by the bacteriumAlteromonas sp. strain 1644. Int J Biol Macromol 18(1):9–17

Bozzi L, Milas M, Rinaudo M (1996b) Solution and gel rheology of anew polysaccharide excreted by the bacterium Alteromonas sp.strain 1644. Int J Biol Macromol 18(1):83–91

Braca A, Sortino C, Politi M, Morelli I, Mendez J (2002) Antioxidantactivity of flavonoids from Licania licaniaeflora. J Ethnopharmacol79(3):379–381. doi:10.1016/S0378-8741(01)00413-5

Buchholz FL, Peppas NA (1994) Superabsorbent polymers: science andtechnology. American Chemical Society

Chen L, Du Y, Wu H, Xiao L (2002) Relationship between molecularstructure and moisture‐retention ability of carboxymethyl chitin andchitosan. J Appl Polym Sci 83(6):1233–1241

Chen L, Du Y, Zeng X (2003) Relationships between the molecularstructure and moisture-absorption and moisture-retention abilitiesof carboxymethyl chitosan. II. Effect of degree of deacetylationand carboxymethylation. Carbohydr Res 338(4):333–340

Chong BF, Blank LM, McLaughlin R, Nielsen LK (2005) Microbialhyaluronic acid production. Appl Microbiol Biotechnol 66(4):341–351

DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956)Colorimetric method for determination of sugars and related sub-stances. Anal Chem 28(3):350–356. doi:10.1021/ac60111a017

Fialho AM, Martins LO, Donval M-L, Leitão JH, Ridout MJ, Jay AJ,Morris VJ, Sá-Correia I (1999) Structures and properties of gellanpolymers produced by Sphingomonas paucimobilis ATCC 31461from lactose compared with those produced from glucose and fromcheese whey. Appl Environ Microbiol 65(6):2485–2491

Appl Microbiol Biotechnol

Gao C, Wang Z, Su T, Zhang J, Yang X (2012) Optimisation ofexopolysaccharide production by Gomphidius rutilus and its anti-oxidant activities in vitro. Carbohydr Polym 87(3):2299–2305

Guezennec JG, Pignet P, Raguenes G, Deslandes E, Lijour Y, Gentric E(1994) Preliminary chemical characterization of unusual eubacterialexopolysaccharides of deep-sea origin. Carbohydr Polym 24(4):287–294. doi:10.1016/0144-8617(94)90073-6

Guimarães PMR, Teixeira JA, Domingues L (2010) Fermentation oflactose to bio-ethanol by yeasts as part of integrated solutions forthe valorisation of cheese whey. Biotechnol Adv 28(3):375–384

Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medi-cine, vol 3. Oxford University Press, Oxford

Kardošová A, Machová E (2006) Antioxidant activity of medicinal plantpolysaccharides. Fitoterapia 77(5):367–373

Kim SJ, Han D, Moon KD, Rhee JS (1995) Measurement of superoxidedismutase-like activity of natural antioxidants. Biosci BiotechnolBiochem 59(5):822–826

Kim HM, Paik S, Ra KS, Koo KB, Yun JW, Choi JW (2006) Enhancedproduction of exopolysaccharides by fed-batch culture ofGanoderma resinaceum DG-6556. J Microbiol (Seoul, Korea)44(2):233

Kurane R, Nohata Y (2002) Microbial high-functional biopolymer. JBiosci Bioeng 93(4):440

Lee HK, Chun J, Moon EY, Ko S-H, Lee D-S, Lee HS, Bae KS (2001)Hahella chejuensis gen. nov., sp. nov., an extracellular-polysaccharide-producing marine bacterium. Int J Syst EvolMicrobiol 51(2):661–666

Leung PH, Zhao S, Ho KP, Wu JY (2009) Chemical properties andantioxidant activity of exopolysaccharides from mycelial culture ofCordyceps sinensis fungus Cs-HK1. Food Chem 114(4):1251–1256

Li WW, Zhou WZ, Zhang YZ, Wang J, Zhu XB (2008) Flocculationbehavior and mechanism of an exopolysaccharide from the deep-seapsychrophilic bacterium Pseudoalteromonas sp. SM9913.Bioresour Technol 99(15):6893–6899

Li H-p, W-g H, Y-z Z (2011) Rheological properties of aqueous solutionof new exopolysaccharide secreted by a deep-sea mesophilic bacte-rium. Carbohydr Polym 84(3):1117–1125. doi:10.1016/j.carbpol.2010.12.072

Lima LFO, Habu S, Gern JC, Nascimento BM, Parada J-L, Noseda MD,Goncalves AG, Nisha VR, Pandey A, Soccol VT (2008) Productionand characterization of the exopolysaccharides produced byAgaricus brasiliensis in submerged fermentation. Appl BiochemBiotechnol 151(2–3):283–294

Liu L, Liu Y, Li J, Du G, Chen J (2011a) Microbial production ofhyaluronic acid: current state, challenges, and perspectives. MicrobCell Factories 10(1):1–9

Liu SB, Qiao LP, He HL, Zhang Q, Chen XL, Zhou WZ, Zhou BC,Zhang YZ (2011b) Optimization of fermentation conditions andrheological properties of exopolysaccharide produced by deep-seabacterium Zunongwangia profunda SM-A87. PLoS One 6(11):e26825. doi:10.1371/journal.pone.0026825

Nichols CAM, Guezennec J, Bowman JP (2005) Bacterialexopolysaccharides from extreme marine environments with specialconsideration of the southern ocean, sea ice, and deep-sea hydro-thermal vents: a review. Mar Biotechnol 7(4):253–271

PasanphanW, Buettner GR, Chirachanchai S (2010) Chitosan gallate as anovel potential polysaccharide antioxidant: an EPR study.Carbohydr Res 345(1):132–140

Poli A, Anzelmo G, Nicolaus B (2010) Bacterial exopolysaccharidesfrom extreme marine habitats: production, characterization and bio-logical activities. Mar Drugs 8(6):1779–1802

Prazeres AR, Carvalho F, Rivas J, Patanita M, Dôres J (2013) Pretreatedcheese whey wastewater management by agricultural reuse: chem-ical characterization and response of tomato plants Lycopersiconesculentum Mill. under salinity conditions. Sci Total Environ 463:943–951

Qin QL, Zhao DL, Wang J, Chen XL, Dang HY, Li TG, Zhang YZ, GaoPJ (2007) Wangia profunda gen. nov., sp. nov., a novel marinebacterium of the family Flavobacteriaceae isolated from southernOkinawa Trough deep‐sea sediment. FEMS Microbiol Lett 271(1):53–58

Rougeaux H, Pichon R, Kervarec N, Raguenes GHC, Guezennec JG(1996) Novel bacterial exopolysaccharides from deep-sea hydro-thermal vents. Carbohydr Polym 31(4):237–242

Sathiyanarayanan G, Seghal Kiran G, Selvin J (2013) Synthesis of silvernanoparticles by polysaccharide bioflocculant produced from ma-rine Bacillus subtilisMSBN17. Colloids Surf B: Biointerfaces 102:13–20

Sato H, Takahashi T, Ide H, Fukushima T, TabataM, Sekine F, KobayashiK, Negishi M, Niwa Y (1988) Antioxidant activity of synovial fluid,hyaluronic acid, and two subcomponents of hyaluronic acid.Synovial fluid scavenging effect is enhanced in rheumatoid arthritispatients. Arthritis Rheum 31(1):63–71. doi:10.1002/art.1780310110

Selbmann L, Onofri S, Fenice M, Federici F, Petruccioli M (2002)Production and structural characterization of the exopolysaccharideof the Antarctic fungus Phoma herbarum CCFEE 5080. ResMicrobiol 153(9):585–592

SuH-N, Chen Z-H, Liu S-B, Qiao L-P, Chen X-L, He H-L, ZhaoX, ZhouB-C, Zhang Y-Z (2012) Characterization of bacterial polysaccharidecapsules and detection in the presence of deliquescent water byatomic forcemicroscopy. Appl EnvironMicrobiol 78(9):3476–3479

Wang J, Zhang Q, Zhang Z, Li Z (2008) Antioxidant activity of sulfatedpolysaccharide fractions extracted from Laminaria japonica. Int JBiol Macromol 42(2):127–132. doi:10.1016/j.ijbiomac.2007.10.003

Weiner RM (1997) Biopolymers from marine prokaryotes. TrendsBiotechnol 15(10):390–394

Yan H, Cai B, Cheng Y, Guo G, Li D, Yao X, Ni X, Phillips GO, Fang Y,Jiang F (2012) Mechanism of lowering water activity of konjacglucomannan and its derivatives. Food Hydrocoll 26(2):383–388

Zall RR (1992) Sources and composition of whey and permeate. In:Zadow JG (ed) Whey and lactose processing. Springer,Netherlands, pp 1–72

Zhao L, Fan F, Wang P, Jiang X (2013) Culture medium optimization of anew bacterial extracellular polysaccharide with excellent moistureretention activity. Appl Microbiol Biotechnol 97(7):2841–2850

Zhou W, Wang J, Shen B, Hou W, Zhang Y (2009) Biosorption ofcopper(II) and cadmium(II) by a novel exopolysaccharide secretedfrom deep-sea mesophilic bacterium. Colloids Surf B: Biointerfaces72(2):295–302. doi:10.1016/j.colsurfb.2009.04.018

Appl Microbiol Biotechnol