Improved Growth of Lactobacillus bulgaricus and Streptococcus thermophilus as well as Increased Antioxidant Activity by Biotransforming Litchi Pericarp Polysaccharide with Aspergillus awamori

This study was conducted to increase the bioactivity of litchi pericarp polysaccharides (LPPs) biotransformed by Aspergillus awamori. Compared to the non-A. awamori-fermented LPP, the growth effects of A. awamori-fermented LPP on Lactobacillus bulgaricus and Streptococcus thermophilus were four and two times higher after 3 days of fermentation, respectively. Increased 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and DNA protection activity of litchi pericarp polysaccharides were also achieved after A. awamori fermentation. Moreover, the relative content of glucose and arabinose in LPP after fermentation decreased from 58.82% to 22.60% and from 18.82% to 10.09%, respectively, with a concomitant increase in the relative contents of galactose, rhamnose, xylose, and mannose. Furthermore, lower molecular weight polysaccharides were obtained after A. awamori fermentation. It can be concluded that A. awamori was effective in biotransforming LPP into a bioactive mixture with lower molecular weight polysaccharides and higher antioxidant activity and relative galactose content.


Introduction
Litchi (Litchi chinensis Sonn.) is a tropical-to-subtropical fruit, with a large amount of polysaccharides in its pericarp, pulp, or seed. As a major byproduct of litchi fruit, the pericarp accounts for approximately 16% of the whole fresh fruit and contains a signi�cant amount of polysaccharides. Due to the health bene�ts, plant polysaccharides have become a desirable supplement in the functional food industry [1]. In vitro studies have indicated that polysaccharides possess broad biological activities including antitumor [2], prebiotic activity [3], and antioxidant capability [4], along with immunostimulating and anti-in�ammatory properties [5]. Furthermore, the polysaccharide from Lentinus edodes, Tremella fuciformis, and Astragalus membranaceus could selectively enrich bene�cial bacterial species such as lactobacilli and bi�dobacteria in an in vivo study [6]. ese biological activities of polysaccharides are closely related to their structural characteristics, such as molecular weight and monosaccharide composition. Chen et al. [7] reported the structures of two polysaccharides (LSP I and LSP II) from litchi seeds, with monosaccharide composition of LSP I being mainly glucose (57.3%), galactose (29.7%), and mannose (6.5%) while LSP II consisting of glucose (19.5%), galactose (24.3%), fructose (38.2%), and mannose (10.7%). Kong et al. [8] investigated the polysaccharides present in litchi pulp and determined a polysaccharide composition consisting primarily of glucose, galactose, and arabinose. Yang et al. [9] identi�ed structural information on a polysaccharide from the litchi pericarp. e polysaccharide exhibited a molecular weight of 14 kDa with 65.6% mannose, 33.0% galactose and 1.4% arabinose, and 8.7% of (1-2)-glycosidic linkage, 83.3% of (1-3)-glycosidic linkage, and 8.0% of (1-6)-glycosidic linkage. e polysaccharide also exhibited high superoxide radical scavenging activity.
Previous in vitro studies indicated that low molecular weight polysaccharides or hydrolyzed oligosaccharides can improve prebiotic effect [10][11][12] and increase antioxidant activity [4,8], but more studies are needed to better understand the structure/activity relationship of these polysaccharides. Currently, biotransformation of plant byproducts by microorganisms or enzymatic engineering is an effective means of producing more bioactive compounds as it can avoid de�ciency in chemical modi�cation. Increased biological activity of polysaccharides from agricultural byproducts by enzyme treatment has been reported [13]. However, due to the substrate selectivity of enzyme, the heterogeneity in monosaccharide composition and the structural diversity of plant polysaccharides and bioconversion efficiency by puri-�ed enzyme have been con�rmed to be low. e combined treatment of several enzymes can partly resolve this problem [13] but it is difficult to choose the appropriate enzymes when the polysaccharide is not clearly characterized. erefore, an attention to the efficient bioconversion of polysaccharides by microorganisms will be paid as the microorganism contains a multifunctional enzyme system. Aspergillus awamori is a �lamentous fungus belonging to the Aspergillus genus and contains an abundance of hydrolytic enzymes including xylanase, pectinase, and -glucosidase [14]. us, application of A. awamori may be an effective enzyme source for production of low molecular weight polysaccharides. e objective of this present study was to investigate the structural characteristic related to antioxidant activity and the growth effect of litchi pericarp polysaccharides (LPPs) on Lactobacillus bulgaricus and Streptococcus thermophilus aer A. awamori fermentation.

Litchi Pericarp and Microorganism.
Fresh fruit of litchi (Litchi chinensis Sonn.) cv. Huaizhi were harvested from a commercial orchard in Guangzhou, China. e fruit were washed with distilled water and then peeled manually. e pericarp was collected, then dried outdoors, and �nally ground into powder.
e microorganism Aspergillus awamori GIM 3.4 was obtained from Guangdong Culture Collection Center, Guangzhou, China. e fungus was cultured for 3 days on potato dextrose agar (Guangdong Huankai Microbial Science & Technology Co.) at 30 ∘ C. A. awamori spores were collected by washing the agar surface with sterile distilled water containing 0.1% Tween 80. e spore suspension was adjusted to a concentration of ca. 10 6 cfu/mL using sterile distilled water and then used to biotransform LPP.

Extraction of Polysaccharides from Litchi Pericarp.
Polysaccharides were extracted from litchi pericarp according to the method of Kong et al. [8] with minor modi�cations. Brie�y, litchi pericarp (150 g) was immersed into distilled water (1000 mL) aer washing with 80% alcohol twice to remove monosaccharides and oligosaccharides. e extraction was repeated three times at 80 ∘ C for 4 h, and each extraction solution was �ltrated through Whatman No. 1 paper. e �ltrates were collected, combined, and then concentrated to 100 mL under vacuum. e proteins in the extract solution were removed using the sevag reagent, and the polysaccharides were then precipitated with four volumes of ethanol overnight at 4 ∘ C. e precipitate was collected by centrifugation at 10,000 g for 20 min, then washed successively with ethanol and ether, and �nally dried under vacuum at 65 ∘ C to obtain the crude polysaccharides.

Fermentation.
For the fermentation, polyurethane foam (3 g) was placed into �rlenmeyer �asks (250 mL) and then mixed with the modi�ed Czapek-Dox medium (10 mL) containing NaNO 3 (30 mg), K 2 HPO 4 (10 mg), KCl (5 mg), MgSO 4 ⋅7H 2 O (5 mg), FeSO 4 (0.1 mg), sucrose (0.3 g), LPP (0.3 g), and distilled water (10 mL). e medium was sterilized for 30 min at 121 ∘ C and then incubated with l mL of fresh A. awamori spore suspension at 28 ∘ C. e fermented products were collected by adding 50 mL of distilled water and holding the mixture fro 4 h at 70 ∘ C prior to �ltering. is extraction was repeated, and the �ltrates were collected, combined, and then concentrated under vacuum at 65 ∘ C. A control sample was prepared by incubating the fresh spore suspension (1 mL) with the previous culture medium but without LPP.

Molecular
Weight Determination of LPP. e molecular weight (MW) of LPP was determined by gel permeation chromatography (GPC) by the method of Yang et al. [15]. e analysis was performed using high-performance liquid chromatography (Waters 5215, Milford, MA) equipped with a TSKG-5000 PW xL gel column (7.8 × 300 mm) and a TSK G-3000 PW xL gel column (7.8 × 300 mm) coupling with a model 2414 refractive index detector and a Breeze GPC workstation. Samples were eluted with 20 mM KH 2 PO 4 (pH 6.0) at a �ow rate of 0.6 mL/min. e column temperature was maintained at 35 ∘ C, and the injection volume was 30 L. Dextrans with different molecular weights (4400, 9900, 21,400, 43,500, 124,000, 196,000, 277,000, and 845,000 Da) were employed as standards. e molecular weights of the fermented and nonfermented LPPs were obtained from the equation based on the elution volume of standard dextrans to their log molecular weights.

Analysis of Monosaccharide Compositions of LPP.
A gas chromatograph (GC-2010, Shimadzu, Shanghai, China) equipped with a RT�-5 capillary column and a �ame ionization detector was employed for the identi�cation of the monosaccharides in the fermented and nonfermented LPPs. e fermented and nonfermented LPPs (10 mg) were hydrolyzed for 6 h with 10 mL of 2 M tri�uoroacetic acid (TFA) at 120 ∘ C [16]. Aer hydrolysis, LPP was dried at 65 ∘ C using a rotary evaporator (RE52AA, Yarong Instrument Co., Shanghai, China). e released monosaccharides were derivatized with trimethylsilyl reagent according to the method of Guentas et al. [17]. Brie�y, the hydrolyzed products were mixed for 5 min with 2 mL pyridine, 0.4 mL hexamethyldisilazane, and 0.2 mL trimethylchlorosilane at 25 ∘ C. Aer centrifugation at 13,200 g for 15 min, the trimethylsilyl derivative (1 L) was analyzed by gas chromatography. e temperature program started at 130 ∘ C, held for 1 min, increased to 180 ∘ C at 2 ∘ C/min, held for 3 min, increased to 220 ∘ C at 10 ∘ C/min, and held for 3 min [18]. e gas chromatograph was run in the splitless mode. Inositol was used as the internal standard. e monosaccharides were identi�ed and quantitated by comparing retention times and peak areas of the standards, respectively.

Measurement of DPPH Radical
Scavenging Activity. e method described by Sánchez-Moreno et al. was used to assess the DPPH radical scavenging activity of the fermented and nonfermented LPPs [19]. Brie�y, an aliquot (0.1 mL) of these samples at 0.05, 0.1, 0.2, 0.5, and 1 mg/mL was mixed with 2.9 mL of 0.1 mM DPPH in methanol. e absorbance was measured at 517 nm aer 30 min at 25 ∘ C. e control was carried out with water instead of the tested sample while methanol instead of the DPPH solution was used for the blank. e DPPH radical scavenging activity (%) of the tested sample was calculated as [1 − (absorbance of sample − absorbance of blank)/absorbance of control)] × 100.

Protection
Effect against DNA Breakage. e protective abilities of the fermented and nonfermented LPP on supercoiled DNA damage were investigated by the method of Kang et al. [20]. Escherichia coli DH5a cells were transformed with pUC19 plasmid DNA and then grown overnight in the Luria-Bertani (LB) medium containing ampicillin (50 g/mL) at 37 ∘ C. Plasmid DNA was puri�ed using the UNI�-10 Plasmid Kit (Wuhan Sikete Science & Technology Development Co. Ltd., Wuhan, China). Evaluation of the protection activity against Fenton-reaction-mediated DNA breakage was conducted using supercoiled plasmid DNA [21]. Five microliters of 100 mM Tris-HCl buffer (pH 7.5), 2 L of 50 mM hydrogen peroxide, 10 L of 0.2 g/ L plasmid DNA, and 1 L of samples at 0.05, 0.1, 0.2, 0.5, and 1 mg/mL were mixed. e reaction was initiated by adding 2 L of 5 mM ferrous sulfate into the mixture. Aer 15 min at 30 ∘ C, the reaction was stopped by adding 10 L of stop solution containing 8 M urea, 50% sucrose, 50 mM EDTA, and 0.1% bromophenol blue and then separated by 1% agarose gel electrophoresis for 40 min under 120 V condition. e agarose gel was stained with 0.05% (w/v) ethidium bromide and then analyzed with an image analyzer (Image station 2000 R, Kodak, New York, USA).

Growth Effects of LPP on Lactobacillus bulgaricus and
Streptococcus thermophilus . e growth effects of the fermented and nonfermented LPPs on L. bulgaricus and S. thermophilus were conducted by the method of Yang et al. [3]. An aliquot (100 L) of L. bulgaricus or S. thermophilus cells in 10% (v/v) glycerol/water solution was pipetted to Man, Rogosa, and Sharp (MRS) agar plate containing 10 g/L protease peptone, 10 g/L beef extract, 5 g/L yeast extract, 1 g/L Tween 80, 2 g/L ammonium citrate, 5 g/L CH 3 COONa, 0.1 g/L MnSO 4 , 0.05 g/L MgSO 4 , 2 g/L K 2 HPO 4 , 20 g/L glucose, and 13 g/L agar, then inoculated at 37 ∘ C, and �nally incubated for 48 h. A single colony was then transferred to a tube containing 5 mL of sterilized MRS broth (121 ∘ C, 20 min) and then cultured for 24 h at 37 ∘ C by shaking at 130 rpm. To comparatively evaluate the growth effect, an aliquot (100 L) of L. bulgaricus or S. thermophilus cell suspension (starting concentration of 1 × 10 6 cfu/mL) was mixed with 2.5 mL of MRS broth and LPP samples at 0, 50, 100, and 200 g/mL. e mixture was incubated for 24 h at 37 ∘ C with shaking at 130 rpm. Aer dilution, the mixture (100 L) was added to MRS agar plate and then cultured at 37 ∘ C for 48 h. e �nal cell concentration was estimated by counting the colonies on the plate.

Statistical
Analysis. Data were expressed as mean ± standard deviations (SD) and then analyzed by OriginPro 8 (OriginLab Corporation, Massachusetts, USA). Graphs were made using Sigmaplot (Systat Soware Inc., San Joes. CA). One-way analysis of variance (ANOVA) and the Tukey's multiple comparisons were carried out to test for signi�cant differences between the means. Differences between the means at the 5% level were considered signi�cant.

Effect of A. awamori Fermentation on Molecular
Weight of LPP. e molecular weight of LPP aer fermentation was examined by high-performance gel permeation chromatography, with the equation �tting the standard curve of log MW = 49.5 − 7.75 V + 0.459 V 2 − 0.00938 V 3 and the correlation coefficient of 0.997 (where MW and V represented the molecular weight and the elution volume, resp.). As shown in Figure 1(a), the non-A. awamori-fermented LPPs displayed two peaks in the chromatogram, and their molecular weights were estimated to be 98.524 and 24.441 kDa, respectively. Aer 3 days of fermentation, one band with an estimated MW of 69.736 kDa was observed (Figure 1(b)), which indicated that LPP was degraded. e LPP was further degraded aer 6 days of fermentation, and two polysaccharides with lower MW of 66.96 and 9.844 kDa were obtained (Figure 1(c)). e presence of hydrolytic enzymes such as arabinofuranosidase, xylanase, and glucoamylase [22] produced by A. awamori may account for LPP degradation.

Effect of A. awamori Fermentation on Monosaccharide
Compositions of LPP. e monosaccharide pro�les of non-A. awamori-fermented and A. awamori-fermented LPPs aer T 1: e relative changes in molar percentages of monosaccharide in non-A. awamori-fermented LPP (LPP 0) and A. awamori-fermented LPP aer 3 (LPP 3) or 6 days (LPP 6). 3 and 6 days of fermentation are shown in Table 1. e non-A. awamori-fermented LPP consisted mainly of glucose (58.82%), arabinose (18.78%), galactose (9.99%), rhamnose (5.03%), mannose (3.79%), and xylose (2.49%). e result was different from the previous report by Yang et al. [9], who identi�ed mannose (65.6%), galactose (33%), and arabinose (1.4%) as the major monosaccharides of polysaccharides from litchi seed. In the present, the polysaccharide was from litchi pericarp, and it was precipitated by 80% alcohol, while in the previous report one polysaccharide fraction was puri�ed se�uentially by DEAE Sepharose fast-�ow column and Sephadex G-50 gel column, which may explain the different information of polysaccharide structure. Aer fermentation, the relative molar percentages of glucose and arabinose decreased rapidly while those of galactose, rhamnose, mannose, and xylose increased. A. awamori can produce a number of arabinanases [22] and glucoamylase [23] that may speci�cally release arabinose and glucose from plant polymers and then use the hydrolysates as a carbon source [24], which could account for the decreases in the relative percentages of arabinose and glucose content in the fermented LPP. e decreases in relative arabinose and glucose contents in the fermented LPP could also explain the increase in other monosaccharides.

Effect of A. awamori Fermentation on DPPH Radical
Scavenging Activity of LPP. Epidemiological evidence indicates an inverse correlation between the consumption of plant antioxidants and incidence of several chronic diseases [25]. e DPPH radical scavenging activity of LPP is presented in Figure 2. A dose-dependent effect existed in all three LPP samples. However, the DPPH radical scavenging activity of LPP was relatively low in the present study. For the non-A. awamori-fermented LPP, less than 10% of the DPPH radical scavenging activity was observed when a concentration of 1 mg/mL was used, which was consistent with the previous reports in the polysaccharides extracted from litchi seed and soy sauce lees, respectively [3,6]. Aer A. awamorifermentation, the LPP exhibited a marked increase in the DPPH radical scavenging activity. Aer 3 and 6 days of fermentation, the A. awamori-fermented LPP had a stronger DPPH radical scavenging activity than the non-A. awamorifermented LPP. When 1 mg/mL was employed, the DPPH radical scavenging activity of the A. awamori-fermented LPP increased from 9 24 ± 3% on the initial day to 17 47 ± 3 % on the third day and to 18 27 ± 27% on the sixth day, which suggested that the polysaccharide modi�cation in�uenced greatly the DPPH radical scavenging activity. Previous report of Kong et al. [8] indicated that three fractions of polysaccharides (LFP1, LFP2, and LFP3) from litchi pulp with decreasing molecular weight (LFP1 > LFP2 > LFP3) exhibited an increasing trend in antioxidant activity (LFP1 < LFP2 < LFP3). In the study, Gel permeation chromatography analysis further revealed that lower MW polysaccharides from litchi pericarp can be produced by fermentation, which can account for the increase in the DPPH radical scavenging activity aer fermentation. us, the increased DPPH radical scavenging activity of the polysaccharides from litchi pericarp can be achieved by biomodi�cation, which will be bene�cial to the functional food industry.

Effect of A. awamori on DNA Protection Effect of LPP.
In this study, as litchi pericarp polysaccharide exhibits a relatively low antioxidant activity using the DPPH method ( Figure 2), it is needed to use DNA protection activity to assess generally antioxidant activity of litchi pericarp polysaccharides modi�ed by microorganism. Plasmid DNA has three forms that can be separated by agarose gel electrophoresis. ese forms are the supercoiled circular form (S form), the open circular form (O form), and the linear form (L form), as shown in Figure 2 (Line A). e S form of DNA is damaged, resulting in from the O form to L form when exposed to reactive oxygen species. Some plant extracts were reported to exhibit the ability to protect DNA from the Fenton-reaction-mediated breakage [20,26,27]. In the study, the A. awamori-fermented and non-A. awamori-fermented LPPs were used to further investigate the DNA protection activity. As shown in Figure 3, the S form of DNA was degraded to produce the O form and L form in the absence of LPP (Line G). It was further degraded into smaller DNA fraction [28] as the band intensity present in Line G was much lighter than Line A (pUC18 without treatment with reactive oxygen species). Compared to Line G, the band of the S form of DNA present in Lines B, H, and M was much brighter. e brightness decreased with decreasing LPP concentration. us, the non-A. awamori-fermented LPP (Line B to F) and A. awamori-fermented LPP aer 3 days (Line H to L) and  6 days (Line M to Q) exhibited an increased DNA protection activity against supercoiled breakage aer fermentation, which further con�rmed that the fermentation enhanced the antioxidant activity of LPP ( Figure 2). Overall, the A. awamori-fermented LPP possessed higher DNA protection activity. A number of published papers have reported the enhanced DNA protection activity of the polysaccharides from macro fungi [29,30] and microorganisms [31,32].

Effect of A. awamori Fermentation on Growth
Effects of LPP on L. bulgaricus and S. thermophilus . e growth effect of non-A. awamori-fermented and A. awamori-fermented LPPs on L. bulgaricus is presented in Figure 4(a). LPP more signi�cantly promoted ( the growth of L. bulgaricus. A dose-dependent response was observed in the present study. Compared to the non-A. awamorifermented LPP, the fermented LPP at 50 g/mL aer 3 days of fermentation showed four times higher growth effect, but no signi�cant difference was observed between the non-A. awamori-fermented and A. awamori-fermented LPPs at between 50 and 100 g/mL aer 6 days of fermentation. Similarly, the A. awamori-fermented LPP aer 3 days of fermentation showed much higher growth effects on S. thermophilus compared with the non-A. awamori-fermented LPP (Figure 4(b)). However, the growth effect of the A. awamorifermented LPP aer 6 days of fermentation decreased. e present study demonstrated that the A. awamori-fermented LPP aer 3 days of fermentation with an MW of 69.736 kDa possessed the higher growth effects on L. bulgaricus and S. thermophilus compared to the non-A. awamori-fermented LPP (about 98.524 kDa) and the fermented LPP (about 9.844 Da) aer 6 days of fermentation. Previous studies have also indicated that low weight polysaccharides or hydrolyzed oligosaccharides can enhance these effects [10][11][12].
Large amount of microorganisms including �i�dobacteria, Lactobacilli, and Streptococcus densely populates in human gut. e microorganisms belonging to Lactobacilli genus are believed to be bene�cial to human health through improving absorption of nutrients, preventing colonisation of pathogens and stimulating immune response in humans [33]. us, we evaluated the growth effect of the Aspergillus awamori-fermented litchi pericarp polysaccharides on L. bulgaricus and S. thermophilus in an effort to understand the bene�cial ability of the biomodi�ed litchi polysaccharides. However, the relationship between biomodi�ed litchi pericarp polysaccharide and growth effects on L. bulgaricus and S. thermophilus needs to be further investigated.

Conclusions
e fermentation of LPP by A. awamori can degrade markedly polysaccharide with reduced molecular weight and increased contents of galactose, rhamnose, mannose, and xylose. Increases in growth effect of L. bulgaricus and S. thermophilus, DNA protection activity, and DPPH radical scavenging activity were obtained aer fermentation of LPP by A. awamori. is study provided evidence that litchi paricarp polysaccharide can be enzymatically modi�ed to increase its bioactivity. us, A. awamori fermentation of LPP can be served potentially as a means of increasing the utilization of this readily accessible waste material.

Con�ict of �nterests
e authors declare that there is no con�ict of interests.